Human Fetal Lung Fibroblast Cells Research Articles

IN VITRO ANTIOXIDANT ACTIVITIES OF THE FERMENTED MARINE MICROALGA PAVLOVA LUTHERI (HAPTOPHYTA) WITH THE YEAST HANSENULA POLYMORPHA(1).

Microalgae are major primary producers of organic matter in aquatic environments through their photosynthetic activities. Fermented microalga (Pavlova lutheri Butcher) preparation (FMP) is the product of yeast fermentation by Hansenula polymorpha. It was tested for the antioxidant activities including lipid peroxidation inhibitory activity, free-radical-scavenging activity, inhibition of reactive oxygen species (ROS) on mouse macrophages (RAW264.7 cell), and inhibited myeloperoxidase (MPO) activity in human myeloid cells (HL60). FMP exhibited the highest antioxidant activity on free-radical scavenging, inhibitory intracellular ROS, and inhibited MPO activity. MTT [3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide] assay showed no cytotoxicity in mouse macrophages (RAW264.7 cell), human myeloid cells (HL60), and human fetal lung fibroblast cell line (MRC-5). Furthermore, the antioxidative mechanism of FMP was evaluated by protein expression levels of antioxidant enzyme (superoxide dismutase [SOD] and glutathione [GSH]) using Western blot. The results obtained in the present study indicated that FMP is a potential source of natural antioxidant.

Structural and In Vitro Biological Studies of Organotin(IV) Precursors; Selective Inhibitory Activity Against Human Breast Cancer Cells, Positive to Estrogen Receptors

Crystals of Ph3SnCl (1) were grown from a methanol/acetonitrile solution. Compounds [Ph3SnOH]n (2) and [(Ph2Sn)4Cl2O2(OH)2] (3) were crystallized from diethyl ether/methanol/acetonitrile and hot acetone/water solutions respectively, of the white precipitation, formed by adding KOH to solutions of 1 and [Ph2SnCl2] in 1 : 1 and 1 : 2 molar ratios respectively. Complex 1 was characterized by X-ray crystallography. X-ray structure determination of compounds 2 and 3 confirmed the previously reported identities. The molecular structure of 1, reported here, is a new polymorphic form of the known one for Ph3SnCl. Four independent [Ph3SnCl] molecules constitute the crystal structure of 1. The moieties are packed in two pairs in a tail-to-tail arrangement. Complexes 1–3 were evaluated for their in vitro cytotoxic activity (cell viability) against human cancer cell lines: HeLa (human cervical), MCF-7 (breast, estrogen receptor (ER) positive), MDA-MB-231 (breast, ER negative), A549 (lung), Caki-1 (kidney carcinoma), 786-O (renal adenocarcinoma), K1 (thyroid carcinoma), and the normal human lung cell line MRC-5 (normal human fetal lung fibroblast cells) versus, the normal immortalized human mammary gland epithelial cell line MTSV17 with a sulforhodamine B (SRB) assay. The results show potent cytotoxic activity of the complexes against all cell lines used, which was superior to that of cisplatin (CDDP). Compounds 1–3 showed higher activity against breast cancer cells MCF-7 (ER positive) than against of MDA-MB-231 (ER negative). These findings prompted us to search for possible interaction of these complexes with other cellular elements of fundamental importance in cell proliferation. The influence of these complexes 1–3 upon the catalytic peroxidation of linoleic acid to hydroperoxylinoleic acid by the enzyme lipoxygenase (LOX), as well as their binding affinity towards calf thymus-DNA, were kinetically and theoretically studied.

Large-scale methylation domains mark a functional subset of neuronally expressed genes

DNA methylation is essential for embryonic and neuronal differentiation, but the function of most genomic DNA methylation marks is poorly understood. Generally the human genome is highly methylated (>70%) except for CpG islands and gene promoters. However, it was recently shown that the IMR90 human fetal lung fibroblast cells have large regions of the genome with partially methylated domains (PMDs, <70% average methylation), in contrast to the rest of the genome which is in highly methylated domains (HMDs, >70% average methylation). Using bisulfite conversion followed by high-throughput sequencing (MethylC-seq), we discovered that human SH-SY5Y neuronal cells also contain PMDs. We developed a novel hidden Markov model (HMM) to computationally map the genomic locations of PMDs in both cell types and found that autosomal PMDs can be >9 Mb in length and cover 41% of the IMR90 genome and 19% of the SH-SY5Y genome. Genomic regions marked by cell line specific PMDs contain genes that are expressed in a tissue-specific manner, with PMDs being a mark of repressed transcription. Genes contained within N-HMDs (neuronal HMDs, defined as a PMD in IMR90 but HMD in SH-SY5Y) were significantly enriched for calcium signaling, synaptic transmission, and neuron differentiation functions. Autism candidate genes were enriched within PMDs and the largest PMD observed in SH-SY5Y cells marked a 10 Mb cluster of cadherin genes with strong genetic association to autism. Our results suggest that these large-scale methylation domain maps could be relevant to interpreting and directing future investigations into the elusive etiology of autism.

Synthesis, structural characterization and biological studies of novel mixed ligand Ag(I) complexes with triphenylphosphine and aspirin or salicylic acid

Two new mixed ligand silver(I) complexes of formulae {[Ag(tpp)3(asp)](dmf)} (1) (aspH=o-acetylsalicylic acid and tpp=triphenylphosphine) and [Ag(tpp)2(o-Hbza)] (2) (o-HbzaH=o-hydroxy-benzoic acid) were synthesized and characterized by elemental analyses, spectroscopic techniques and X-ray crystallography at ambient conditions. Three phosphorus and one carboxylic oxygen atoms from a de-protonated aspirin ligand in complex 1 and two phosphorus and two carboxylic oxygen atoms from a chelating o-Hbza anion in complex 2 form a tetrahedral geometry around Ag(I) ions in both complexes.Complexes 1 and 2 and the silver(I) nitrate, tpp, aspNa and o-HbzaH were tested for their in vitro cytotoxic activity against leiomyosarcoma cells (LMS), human breast adenocarcinoma cells (MCF-7) and normal human fetal lung fibroblasts (MRC-5) cells with Thiazolyl Blue Tetrazolium Bromide (MTT) assay. For both cell lines 1 and 2 were found to be more active than cisplatin. Additionally, 1 and 2 exhibit lower activity on cell growth proliferation of MRC-5 cells. The type of LMS cell death caused by 1 and 2 were evaluated in vitro by use of flow cytometry assay. The results show that at concentrations of 1.5 and 1.9μΜ of complex 1, 44.1% and 69.4%, respectively of LMS cells undergo programmed cell death (apoptosis). When LMS cells were treated with 1.6 and 2.3μM of 2, LMS cells death was by 29.6% and 81.3%, respectively apoptotic. Finally, the influence of the complexes 1 and 2, upon the catalytic peroxidation of linoleic acid to hydroperoxylinoleic acid by the enzyme lipoxygenase (LOX) was kinetically and theoretically studied. The binding of 1 and 2 towards LOX was also investigated by Saturation Transfer Difference (STD) 1H NMR experiments.

Synthesis, structural characterization and biological studies of the triphenyltin(IV) complex with 2-thiobarbituric acid

The reaction between 2-thiobarbituric acid (H 2TBA), which was treated with an equimolar amount of potassium hydroxide, in a water with triphenytin chloride in methanol, results in the formation of the {[Ph 3Sn(O-HTBA)]} n ( 1) complex. Crystals of the hydrated 1 with formula {[Ph 3Sn(O-HTBA)]·0.7(H 2O)} n were growth from methanol/acetonitrile solution, of the white precipitation, filtered off, from the reaction. The crystal structure of complex 1 has been determined by X-ray diffraction at 120 K. Complex 1 is polymeric. The geometry around the tin(IV) ions is trigonal bi-pyramidal with coordination to three C atoms from phenyl groups and one O atom from a de-protonated HTBA ligand. Complex 1 and the already known [(n-Bu) 3Sn(O-HTBA)·H 2O] ( 2) were evaluated for their in vitro cytotoxic activity (cell viability) against human cancer cell lines: HeLa (cervical), OAW-42 (ovarian), MCF-7 (breast, ER positive), MDA-MB-231 (breast, ER negative), A549 (lung), Caki-1 (renal) and additionally, the normal human lung cell line MRC-5 (normal human fetal lung fibroblast cells) and normal immortalized human mammary gland epithelial cell line MTSV17 with a Trypan Blue assay. Moreover complex 1 was evaluated for its in vitro cell growth proliferation activity against leiomyosarcoma cells (LMS), MCF-7 and MRC-5 cells with a Thiazolyl Blue Tetrazolium Bromide (MTT) assay. The type of cell death caused by complexes 1 and 2 was also evaluated by use of flow cytometry assay. The results showed that these compounds mediate a strong cytotoxic response to normal and cancer cell lines tested through apoptosis and induce cell cycle arrest in S phase of the cell cycle, suggesting DNA intercalation (direct or indirect) with the complexes. Finally, the influence of these complexes 1 and 2 upon the catalytic peroxidation of linoleic acid to hydroperoxylinoleic acid by the enzyme lipoxygenase (LOX) was kinetically and theoretically studied.

Biphasic modulation of airway mesenchymal cell proliferation by hogbarn dust extract

Workers in concentrated animal feeding operations (CAFOs) often develop chronic inflammatory lung disease. Previous studies have characterized effects of an aqueous hogbarn dust extract (HDE) on inflammation responses of airway epithelial cells. We hypothesized that HDE would also exhibit disease‐relevant effects on airway mesenchymal cells. In both primary isolates of human airway smooth muscle (HASM) cells and the HFL1 human fetal lung fibroblast cell line, HDE at low concentrations stimulated proliferation assessed by [3H]thymidine incorporation, with maximal stimulation at 1%. HDE stimulation was blocked by the EGF receptor tyrosine kinase inhibitor AG1478, implicating EGF receptor involvement, as shown previously for some HDE effects on epithelial cells. At higher concentrations, HDE stimulation decreased, with no stimulation and often significant inhibition induced by HDE at 5% or higher. The stimulatory and inhibitory activities were found in different gel filtration fractions of HDE, though the specific mediators remain unknown. HDE modulation of airway mesenchymal cell proliferation may contribute to airway remodeling in hogbarn workers. Supported by NIH Grant OH008539 to DJR.

Mitochondrial dysfunction and transactivation of p53-dependent apoptotic genes in BaP-treated human fetal lung fibroblasts

Benzo(a)pyrene (BaP) has been shown to be an inducer of apoptosis. However, mechanisms involved in BaP-induced mitochondrial dysfunction are not well-known. In this study, human fetal lung fibroblasts cells were treated with BaP (8, 16, 32, 64 and 128 μM) for 4 and 12 h. Cell viability, intracellular level of reactive oxygen species (ROS), total antioxidant capacity (T-AOC), mitochondrial membrane potential (ΔΨ(m)) and cytochrome c release were determined. Changes in transcriptional levels of p53-dependent apoptotic genes (p53, APAF1, CASPASE3, CASPASE9, NOXA and PUMA) were measured. At time point of 4 h, BaP induced the intracellular ROS generation in 64 (p < .05) and 128 μM BaP groups (p < .01) but decreased the T-AOC activities in 32, 64 (p < .05 for both) and 128 μM BaP groups (p < .01). At time point of 12 h, ΔΨ(m) significantly decreased in ≥32 μM BaP groups (p < .05 for all). Amount of mitochondrial cytochrome c significantly increased in 128 μM BaP group (p < .01). Transcriptional levels of CASPASE3, CASPASE9, APAF1 and PUMA were up-regulated in all BaP groups (p < .05 for all) and in ≥32 μM groups for NOXA (p < .05). But only in 16 μM BaP group a relatively little expression of p53 mRNA was observed (p < .05). The results indicate that in the earlier period BaP promoted the generation of excessive ROS and subsequently the mitochondrial depolarization, whereas transactivations of the p53-dependent apoptotic genes were significantly induced at the later period.

Bleomycin induces upregulation of lysyl oxidase in cultured human fetal lung fibroblasts

To investigate the mechanism of bleomycin (BLM)-induced pulmonary fibrosis. Cultured human fetal lung fibroblast (HLF) cells were exposed to bleomycin (BLM) at 0-30 microg/mL for 24 h. Western blot analysis was used to detect lysyl oxidase (LO) protein expression. Real-time RT-PCR was used to detect LO mRNA level. LO catalytic activity was measured using diaminopentane as a substrate and Amplex red as a hydrogen peroxide probe. Copper (Cu) concentration was detected by flame atomic absorption spectrophotometry. Exposure of HLF cells to BLM at 10 microg/mL and 30 microg/mL increased LO catalytic activity to 130% and 158% of the control in the conditioned media. The expression of LO mRNA was increased to 5.5-fold of the control in HLF cells exposure to BLM at 3 microg/mL. BLM at 3 microg/mL also increased the expression of 46 kDa preproLO, 50 kDa proLO and 32 kDa mature LO to 219%, 130%, and 135% of the control, respectively. The Cu concentrations in conditioned media of cultured HLF cells exposed to BLM (10 and 30 microg/mL) were increased significantly to 1.48 and 2.46-fold of the control, respectively. Bleomycin induces upregulation of LO in cultured human fetal lung fibroblasts, which may be the mechanism of bleomycin-induced pulmonary fibrosis.

Expression of caveolin-1 and extracellular matrix induced by transforming growth factor beta1 in human fetal lung fibroblasts

To investigate the expressions of caveolin-1, collagen-I and alpha-SMA in human fetal lung fibroblasts induced by transforming growth factor beta(1) (TGF-beta(1)). Human fetal lung fibroblasts (HFLF) were cultured in vitro, and exposed to TGF-beta(1) with different final concentrations (2, 5, 10 microg/L). Cells without TGF-beta(1) exposure served as the control. Caveolin-1 mRNA and protein, collagen-I and alpha-SMA proteins in cell (at least 5 x 10(8)) lysates, were detected at different points after the treatment of TGF-beta(1), by RT-PCR, Western blot and Immunohistochemistry. Each experiment was repeated 3 times. One-way analysis of variance (ANOVA) was used to compare data among groups, and linear regression was established for correlation analysis. TGF-beta(1) reduced caveolin-1 mRNA and protein expressions in a dose- and time-dependent manner, measured by integral optical density. Compared with the control group (1.22 +/- 0.12 and 1.45 +/- 0.06), caveolin-1 mRNA and protein expressions (0.59 +/- 0.06 and 0.53 +/- 0.04) were significantly reduced, with statistical significance (F = 29.279 and F = 95.786, P < 0.01), in cells exposed to TGF-beta(1) (5 microg/L) for 12 h. The collagen-I and alpha-SMA protein expressions (1.35 +/- 0.09 and 0.75 +/- 0.06) measured by integral optical density increased after stimulation, peaked at 24 h in 5 microg/L TGF-beta(1) group. At that time point, the collagen-I and alpha-SMA protein expressions were 4.2 and 4.8 times of the control group (0.28 +/- 0.04 and 0.18 +/- 0.04), F = 81.221 and F = 65.477, P < 0.01. Caveolin-1 expression had a negative correlation with collagen-I (r = -0.923, P < 0.05) and alpha-SMA protein expression (r = -0.793, P < 0.05). The reduced caveolin-1, which was negatively correlated with expressions of collagen-I and alpha-SMA proteins in HFLF cells exposed to TGF-beta(1), may be related to the development and progress of pulmonary fibrosis.

Thalidomide has a therapeutic effect on interstitial lung fibrosis: evidence from in vitro and in vivo studies

The objective of this study was to investigate the effects of thalidomide (THD) on interstitial lung fibrosis (ILF). In vitro, human fetal lung fibroblast (HFL-F) to myofibroblast (MF) trans-differentiation was induced by transforming growth factor (TGF)-beta1. The effects of THD on trans-differentiation process or differentiated MF were evaluated by measuring hydroxyproline (HYP) content by alkaline hydrolysis colorimetry, alpha-smooth muscle actin (alpha-SMA) protein by Western blot and alpha-SMA and pro-collagen III mRNA expressions by semi-quantitative reverse transcription-polymerase chain reaction; in vivo, a mouse model of ILF was generated by daily subcutaneous injection of bleomycin (BLM) in female C3H mice. Gastric perfusion of THD began 1 week prior to injection and lasted for 8 weeks. Lung specimens were harvested at different time-points (1, 4, 6 and 8 weeks) for pathology and immunohistochemistry examination. The HYP content, alpha-SMA and pro-collagen III mRNA expressions were also assessed. THD inhibited the up-regulation of HYP protein, pro-collagen III mRNA and alpha-SMA protein induced by TGF-beta1 in HFL-F cells, and additionally inhibited pro-collagen III mRNA expression on trans-differentiated MF. THD reduced HYP synthesis in the lung tissues of BLM-treated mice at week 4, and slightly reduced the numbers of alpha-SMA-positive cells. THD had an effect on ILF models both in vitro and in vivo.

Poly(N‐isopropylacrylamide) surface‐grafted chitosan membranes as a new substrate for cell sheet engineering and manipulation

The immobilization of poly(N-isopropylacrylamide) (PNIPAAm) on chitosan membranes was performed in order to render membranes with thermo-responsive surface properties. The aim was to create membranes suitable for cell culture and in which confluent cell sheets can be recovered by simply lowering the temperature. The chitosan membranes were immersed in a solution of the monomer that was polymerized via radical initiation. The composition of the polymerization reaction solvent, which was a mixture of a chitosan non-solvent (isopropanol) and a solvent (water), provided a tight control over the chitosan membranes swelling capability. The different swelling ratio, obtained at different solvent composition of the reaction mixture, drives simultaneously the monomer solubility and diffusion into the polymeric matrix, the polymerization reaction rate, as well as the eventual chain transfer to the side substituents of the pyranosyl groups of chitosan. A combined analysis of the modified membranes chemistry by proton nuclear magnetic resonance ((1)H-NMR), Fourier transform spectroscopy with attenuated total reflection (FTIR-ATR) and X-ray photoelectron spectroscopy (XPS) showed that it was possible to control the chitosan modification yield and depth in the solvent composition range between 75% and 100% of isopropanol. Plasma treatment was also applied to the original chitosan membranes in order to improve cell adhesion and proliferation. Chitosan membranes, which had been previously subjected to oxygen plasma treatment, were then modified by means of the previously described methodology. A human fetal lung fibroblast cell line was cultured until confluence on the plasma-treated thermo-responsive chitosan membranes and cell sheets were harvested lowering the temperature.

Genetic manipulation of stromal cell-derived factor-1 attests the pivotal role of the autocrine SDF-1-CXCR4 pathway in the aggressiveness of breast cancer cells

Stromal cell-derived factor-1 (SDF-1), via its receptor CXCR4, has been implicated in metastasis of cancer, including breast cancer. Exogenous SDF-1 is known to regulate locomotion, chemotaxis and adhesion. The knowledge regarding the effect of autocrine SDF-1 on breast cancer cells is not available. The current study evaluated the effects of SDF-1 on the biological behaviour of breast cancer cells by genetically modifying the expression of SDF-1 in breast cancer cells. Two human breast cancer cell lines (MDA-MB-231 and MDA-MB-435s) and a human fetal lung fibroblast cell line (MRC5) were used. The expression of SDF-1 and the SDF-1 receptor, CXCR4 in the cell lines were studied. Expression cassettes of human SDF-1 and hammerhead ribozyme transgenes specifically targeting human SDF-1 were constructed and used to over-express SDF-1 or to knockout the expression of SDF-1 in cancer cells, respectively. Invasiveness, migration and growth of the genetically modified cells were assessed. SDF-1 was expressed in wild-type human breast cancer cell line MDA-MB-435s and fibroblast cell line MRC5, but not in MDA-MB-231 cell line. In contrast, CXCR4 expression was observed in all three cell lines tested. The ability of invasion and migration was significantly reduced in SDF-1 knockout MDA-MB-435s cells, compared with wild-type and vector control cells (p<0.01). On the other hand, SDF-1 transfected MDA-MB-231epsilonSDF1+/+ cells that stably expressed SDF-1 showed a different behaviour from MDA-MB-231SDF1+/- (plasmid control) and wild-type MDA-MB-231 cells, both being SDF-1 negative. MDA-MB-231epsilonSDF1+/+ cells displayed a higher degree of invasiveness and migration, compared with wild-type and MDA-MB-231SDF+/- cells (p<0.01). Furthermore, SDF1-knockout MDA-MB-435s cells showed a slower growth rate over a 7-day period compared with the respective control and wild-type MDA-MB-435s cells. In contrast, the growth of the SDF-1 transfected MDA-MB-231SDF1+/+ cells was markedly enhanced when compared with wild-type and vector control cells. Breast cancer cell lines, when equipped with the autocrine SDF-1-CXCR4 signal pathway, display aggressive behaviour, including an increase in invasiveness, migration together with faster growth. SDF-1, together with its receptor CXCR4 may provide important information for predicting the aggressive nature and constitute important therapeutic targets in human breast cancer.

Tumor-specific expression detected with the use of an expression vector driven by human telomerase reverse transcriptase gene promoter

To construct the tumor-specific expression vector driven by human telomerase reserve transcriptase gene promoter, we amplified a fragment of the enhanced green fluorescent protein (EGFP) gene from the pEGFP-N1 plasmid and cloned it into the multiple cloning site of the pLNCX vector, then named the recombinant as pLNCX-EGFP. The fragment of human telomerase reverse transcriptase gene promoter (hTERT) was amplified from the human genome with the use of human telomerase reserve transcriptase gene-specific primers and cloned into the pLNCX-EGFP vector, from which the cytomegalovirus promoter had previously been removed through the use of restriction enzymes, in sense orientation relative to the green fluorescent protein coding sequence. Then the expression vector pLNT-EGFP--under the control of the human telomerase reserve transcriptase gene promoter, which contains green fluorescent protein reporter gene-was successfully constructed. To detect the transcriptional activity of the human telomerase reserve transcriptase gene promoter, we conducted transient transfection of this specific expression vector into human lung fibroblast (HLF) cell lines with high telomerase activity and normal human fetal lung fibroblast (WI38) cell lines without telomerase activity. The results of transient transfection showed that the pLNT-EGFP vector strongly expressed the green fluorescent protein reporter gene in telomerase-positive cells but not in telomerase-negative cells.

L-Carnosine reduces telomere damage and shortening rate in cultured normal fibroblasts

Telomere is the repetitive DNA sequence at the end of chromosomes, which shortens progressively with cell division and limits the replicative potential of normal human somatic cells. l-Carnosine, a naturally occurring dipeptide, has been reported to delay the replicative senescence, and extend the lifespan of cultured human diploid fibroblasts. In this work, we studied the effect of carnosine on the telomeric DNA of cultured human fetal lung fibroblast cells. Cells continuously grown in 20mM carnosine exhibited a slower telomere shortening rate and extended lifespan in population doublings. When kept in a long-term nonproliferating state, they accumulated much less damages in the telomeric DNA when cultured in the presence of carnosine. We suggest that the reduction in telomere shortening rate and damages in telomeric DNA made an important contribution to the life-extension effect of carnosine.

Glycyrrhizin and related compounds down-regulate production of inflammatory chemokines IL-8 and eotaxin 1 in a human lung fibroblast cell line

Glycyrrhizin (GL) is known to have various immunomodulating activities and has long been used clinically as an anti-allergic and anti-hepatitis agent. While the potency of GL against lung inflammatory diseases has been expected, the effect of GL on the lung has been poorly understood. Lung fibroblasts are known as a potent producer of inflammatory chemokines, IL-8 and eotaxin 1, by which neutrophils and eosinophils are strongly attracted during inflammation. Therefore, we studied the effects of GL on the production of these chemokines using a human fetal lung fibroblast cell line, HFL-1, stimulated with TNF-α and IL-4. Moreover, we examined the structure–activity relationships of GL to explore more beneficial compounds. 18α,β-GL inhibited IL-8 dose-dependently and inhibited eotaxin 1 slightly. 18α,β-Glycyrrhetic acid (GA) did not inhibit IL-8 but inhibited eotaxin 1. The effect of 18α,β-glycyrrhetic acid monoglucuronide (MGA) resembled that of 18α,β-GL but was weaker. Both 3β-[(2- O-β- d-glucopyranuronosyl-β- d-glucopyranuronosyl)oxy]-18β-11-deoxo-olean-12-en-30-oic acid (11-deoxo-GL) and 3β-[(2- O-β- d-glucopyranuronosyl-β- d-glucopyranuronosyl)oxy]-olean-11,13,(18)-dien-30-oic acid (hetero-GL) exhibited inhibitory activity with significant cytotoxicity. 3β-[(2- O-β- d-Glucopyranuronosyl-β- d-glucopyranuronosyl)oxy]-18β-olean-9,12-dien-30-oic acid (homo-GL) did not have cytotoxicity but its activity was mild like that of 18α,β-GL. 3β-[(2- O-β- d-Glucopyranuronosyl-β- d-glucopyranuronosyl)oxy]-olean-11,13(18)-dien-30-ol (hetero-30-OH-GL) and 3β-[(2- O-β- d-glucopyranuronosyl-β- d-glucopyranuronosyl)oxy]-18β-olean-9,12-dien-30-ol (homo-30-OH-GL) showed potent inhibitory effects, at concentrations lower than 18α,β-GL with no significant cytotoxicity. These results suggest that GL-related compounds are effective in reducing chemokine production and that GL-modified compounds including hetero-30-OH-GL and homo-30-OH-GL appear most beneficial in view of their inhibitory capacity with less cytotoxicity.

Histamine and Serotonin Stimulate Eotaxin Production by a Human Lung Fibroblast Cell Line

Histamine and serotonin are important inflammatory mediators in the pathophysiology of asthma, and asthmatic patients have higher plasma histamine and serotonin levels than nonasthmatic control subjects. Eotaxin, a potent eosinophil-specific chemotactic factor, is increased in the lower respiratory tract of allergic patients. Recently, lung fibroblasts have been reported to produce eotaxin and are suggested to be the major cellular source of eotaxin. We postulated that lung fibroblasts might release eotaxin in response to histamine or serotonin. To test this hypothesis, we evaluated the potential of histamine or serotonin to induce the release of eotaxin by the human fetal lung fibroblast cell line, HFL-1. HFL-1 released eotaxin in response to histamine and serotonin in a dose- and time-dependent manner (p < 0.05). Histamine or serotonin treatment of HFL-1 augmented the expression of eotaxin mRNA. Eosinophil chemotactic activity by HFL-1 supernatant fluids was inhibited by anti-human eotaxin-neutralizing antibody. These findings lead to the hypothesis that lung-fibroblast-derived eotaxin may in part be responsible for the eosinophil infiltration observed in allergic disease of the airways.

INFLAMMATORY CYTOKINES MODULATE EOTAXIN RELEASE BY HUMAN LUNG FIBROBLAST CELL LINE

Eotaxin, a potent eosinophil-specific chemotactic factor, is increased in the lower respiratory tract of asthma patients. Recently, lung fibroblasts have been reported to produce eotaxin and their activation can be modulated by inflammatory cytokines. To test the hypothesis that inflammatory cytokines modulate the eotaxin release from lung fibroblasts, we investigated the potential of interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), or interferon-gamma (IFN-gamma) to induce the release of eotaxin and eotaxin mRNA by the human fetal lung fibroblast cell line, HFL-1, was evaluated. HFL-1 released eotaxin constitutively without stimulation, but IL-1beta or TNF-alpha stimulated eotaxin release in a dose- and time-dependent manner. IL-1beta or TNF-alpha treatment of HFL-1 also resulted in the augmented expression of eotaxin mRNA. Although IFN-gamma alone had negligible effect on eotaxin release and mRNA expression, IFN-gamma induced a significant, concentration-dependent attenuation of eotaxin release and eotaxin mRNA expression from HFL-1 stimulated with IL-1beta or TNF-alpha. These findings are consistent with the concept that lung fibroblast-derived eotaxin may in part be responsible for the eosinophil infiltration observed in the airways of asthmatic patients and that network of cytokines may modulate the eosinophil recruitment to the airways by stimulation of fibroblasts to release eotaxin.

Novel non-peptide lead structures for bradykinin B2-receptor antagonists

A series of new non-peptide Bradykinin (BK) B2-receptor antagonists is reported. These new leads belong to a larger set including both commercially or otherwise available potential candidates found by proprietary database searches using 3D-pharmacophore models as query, and several bis-benzamidino compounds selected from our tryptase-like protease inhibitor library on the basis of topological considerations, derived from the same models. Some of these compounds show functional competitive antagonistic activity, inhibiting in vitro the BK-induced contraction of isolated guinea-pig ileum (GPI) and rat uterus with a pA2 up to 5.3 and 7.0, respectively. They display also binding affinity (IC50 up to 0.56 μM) to the BK B2-receptor in radioligand binding assays on GPI membrane preparations and on human IMR-90 fetal lung fibroblast cells expressing this receptor subtype. Furthermore, the results with the commercially available compounds, in some cases developed as therapeutics, show that the used 3D-pharmacophore model allows to predict to some certainty possible side actions of potential drugs.

Age-dependent changes in DNA polymerase fidelity and proofreading activity during cellular aging

DNA polymerase α and the 3′→5′ exonuclease involved in the proofreading of DNA synthesis were isolated from human diploid fetal lung fibroblast (TIG-1) cells at various population doubling levels (PDL). The final PDL of the TIG-1 cells used in these experiments was 70. The fidelity of DNA polymerase α remained high until late passage and fell suddenly just before the end of the life span between 65 and 69 PDL. The activities of the 3′→5′ exonuclease related to proofreading remained unchanged from 21 to 61 PDL, but the activity decreased rapidly in more aged cells. The 3′→5′ exonuclease activity at 69 PDL was about 50% of that in TIG cells at 21 PDL. In vitro DNA synthesis by DNA polymerase α from TIG-1 cells harvested at 69 PDL showed the amount of non-complementary nucleotides incorporated to be decreased by the addition of the 3′→5′ exonuclease from the same cells. However, not all errors were edited out since the ratio of DNA polymerase activity to 3′→5′ exonuclease activity was adjusted to reflect that in vivo and the infidelity of DNA synthesis by error-prone DNA polymerase α from aged cells was improved by the addition of the highly active 3′→5′ exonuclease from cells at 41 PDL. These results suggested that the mutation frequency rises just before the end of the life span of TIG-1 cells.

D3/1 - Meta-analysis in chemical toxicity is more often abused than used (for)

To construct the tumor-specific expression vector driven by human telomerase reserve transcriptase gene promoter, we amplified a fragment of the enhanced green fluorescent protein (EGFP) gene from the pEGFP-N1 plasmid and cloned it into the multiple cloning site of the pLNCX vector, then named the recombinant as pLNCX-EGFP. The fragment of human telomerase reverse transcriptase gene promoter (hTERT) was amplified from the human genome with the use of human telomerase reserve transcriptase gene-specific primers and cloned into the pLNCX-EGFP vector, from which the cytomegalovirus promoter had previously been removed through the use of restriction enzymes, in sense orientation relative to the green fluorescent protein coding sequence. Then the expression vector pLNT-EGFP—under the control of the human telomerase reserve transcriptase gene promoter, which contains green fluorescent protein reporter gene—was successfully constructed. To detect the transcriptional activity of the human telomerase reserve transcriptase gene promoter, we conducted transient transfection of this specific expression vector into human lung fibroblast (HLF) cell lines with high telomerase activity and normal human fetal lung fibroblast (WI38) cell lines without telomerase activity. The results of transient transfection showed that the pLNT-EGFP vector strongly expressed the green fluorescent protein reporter gene in telomerase-positive cells but not in telomerase-negative cells.