SV40 Cells Research Articles

Rutaecarpine alleviates inflammation and fibrosis by targeting CK2α in diabetic nephropathy

Diabetic nephropathy (DN) is one of the serious microvascular complications of diabetes mellitus. During the progression of DN, the proliferation of glomerular mesangial cells (GMCs) leads to the deposition of excessive extracellular matrix (ECM) in the mesangial region, eventually resulting in glomerulosclerosis. Rutaecarpine (Rut), an alkaloid found in the traditional Chinese medicinal herb Fructus Evodiae (Euodia rutaecarpa (Juss.) Benth.), has many biological activities. However, its mechanism of action in DN remains unknown. This study used db/db mice and high glucose (HG)-treated mouse mesangial cells (SV40 MES-13) to evaluate the protective effects of Rut and underlying mechanisms on GMCs in DN. We found that Rut alleviated urinary albumin and renal function and significantly relieved renal pathological damage. In addition, Rut decreased the ECM production, and renal inflammation and suppressed the activation of TGF-β1/Smad3 and NF-κB signaling pathways in vitro and in vivo. Protein kinase CK2α (CK2α) was identified as the target of Rut by target prediction, molecular docking, and cellular thermal shift assay (CETSA), and surface plasmon resonance (SPR). Furthermore, Rut could not continue to play a protective role in HG-treated SV40 cells after silencing CK2α. In summary, this study is the first to find that Rut can suppress ECM production and inflammation in HG-treated SV40 cells by inhibiting the activation of TGF-β1/Smad3 and NF-κB signaling pathways and targeting CK2α. Thus, Rut can potentially become a novel treatment option for DN.

Establishment of immortalized Egyptian Rousettus bat cell lines.

The Egyptian Rousettus bat (Rousettus aegyptiacus) is a common fruit bat species that is distributed mainly in Africa and the Middle East. Bats serve as reservoir hosts for numerous pathogens. Human activities, such as hunting bats for food, managing vermin, and causing habitat loss, elevate the likelihood of transmission of bat pathogens to humans and other animals. Consequently, bat cell lines play a crucial role as research materials for investigating viral pathogens. However, the inherent limitation of finite cell division in primary cells necessitates the use of immortalized cells derived from various bat tissues. Herein, we successfully established six fibroblast cell lines derived from an infant bat heart and lungs and an elderly bat heart. Three of the six cell lines, called K4DT cells, were transduced by a combination of cell cycle regulators, mutant cyclin-dependent kinase 4, cyclin D1, and human telomerase reverse transcriptase. The other three cell lines, named SV40 cells, were transfected with simian virus 40 large T antigen. Transgene protein expression was detected in the transduced cells. All three K4DT cell lines and one lung-derived SV40 cell line were virtually immortalized and nearly maintained the normal diploid karyotypes. However, the two other heart-derived SV40 cell lines had aberrant karyotypes and the young bat-derived cell line stopped proliferating at approximately 40 population doublings. These bat cell lines are valuable for studying pathogen genomics and biology.

Characterization of Common Minke Whale (Balaenoptera Acutorostrata) Cell Lines Immortalized with the Expression of Cell Cycle Regulators.

Primary cultured cells cannot proliferate infinite. The overcoming of this limit can be classified as immortalization. Bypass of p16 senescence protein induces efficient immortalization various types of mammalians is previously reported. However, the Cetacea species is not known. Here, that common minke whale-derived cells can be immortalized with a combination of human genes, mutant cyclin-dependent kinase 4 (CDK4R24C ), cyclin D1, and Telomerase Reverse Transcriptase (TERT)is reported. These results indicate that the function of cell cycle regulators in premature senescence is evolutionarily conserved. This study describes the conserved roles of cell cycle regulators in the immortalization of cells from humans to Cetacea species. Furthermore, using RNA-seq based on next-generation sequencing, the gene expression profiles of immortalized cells are compared with parental cells as well as those immortalized with SV40 large T antigen, which is once a popular method for cellular immortalization. The profiling results show that newly established common minke-whale-derived immortaliozed cells have completely different profiles from SV40 cells. This result indicates that the expression of mutant CDK4, cyclin D1, and TERT enables to establish immortalized cell lines with different biological nature from SV40 expressing cells.

Leptin Induces MMP-1 Expression Through the RhoA/ERK1/2/NF-κB Axis in Human Intervertebral Disc Cartilage Endplate-Derived Stem Cells.

Intervertebral disc (IVD) degeneration, associated with aging, may cause low back pain and disability, with obesity as a significant risk factor. In a prior study, we found a positive correlation between IVD degeneration and levels of matrix metalloproteinase-1 (MMP-1) and leptin. Yet, the interaction between MMP-1 and leptin in IVD degeneration is unclear. Our research seeks to explore leptin's influence on MMP-1 expression and the underlying mechanisms in human intervertebral disc cartilage endplate-derived stem cells, specifically SV40 cells. The mRNA and protein expression in leptin-stimulated SV40 cells were assessed using RT-real-time PCR and Western blotting or ELISA, respectively. We examined leptin-mediated RhoA activation through a GTP-bound RhoA pull-down assay. Furthermore, the phosphorylation levels of mitogen-activated protein kinases and AKT in leptin-stimulated SV40 cells were analyzed using Western blotting. The activation of NF-κB by leptin was investigated by assessing phosphorylation of IKKα/β, IκBα, and NF-κB p65, along with the nuclear translocation of NF-κB p65. To understand the underlying mechanism behind leptin-mediated MMP-1 expression, we employed specific inhibitors. Leptin triggered the mRNA and protein expression of MMP-1 in SV40 cells. In-depth mechanistic investigations uncovered that leptin heightened RhoA activity, promoted ERK1/2 phosphorylation, and increased NF-κB activity. However, leptin did not induce phosphorylation of JNK1/2, p38, or AKT. When we inhibited RhoA, ERK1/2, and NF-κB, it resulted in a decrease in MMP-1 expression. Conversely, inhibition of reactive oxygen species and NADPH oxidase did not yield the same outcome. Additionally, inhibiting RhoA or ERK1/2 led to a reduction in leptin-induced NF-κB activation. Moreover, inhibiting RhoA also decreased leptin-mediated ERK1/2 phosphorylation. These results indicated that leptin induced MMP-1 expression in SV40 cells through the RhoA/ERK1/2/NF-κB axis. This study provided the pathogenic role of leptin and suggested the potential therapeutic target for IVD degeneration.

MES SV40 Cells Are Sensitive to Lipopolysaccharide, Peptidoglycan, and Poly I:C Expressing IL-36 Cytokines.

Mesangial cells (MC) maintain the architecture and cellular communication and indirectly join in the glomerular filtration rate for the correct functioning of the glomerulus. Consequently, these cells are activated constantly in response to changes in the intraglomerular environment due to a metabolic imbalance or infection. IL-36, a member of the IL-1 family, is a cytokine that initiates and maintains inflammation in different tissues in acute and chronic pathologies, including the skin, lungs, and intestines. In the kidney, IL-36 has been described in the development of tubulointerstitial lesions, the production of an inflammatory environment, and is associated with metabolic and mesangioproliferative disorders. The participation of IL-36 in functional dysregulation and the consequent generation of the inflammatory environment by MCs in the presence of microbial stimulation is not yet elucidated. In this work, the MES SV40 cell cultures were stimulated with classical pathogen-associated molecular patterns (PAMPs), mimicking an infection by negative and positive bacteria as well as a viral infection. Lipopolysaccharide (LPS), peptidoglycan (PGN) microbial wall components, and a viral mimic poly I:C were used, and the mRNA and protein expression of the IL-36 members were assessed. We observed a differential and dose-dependent IL-36 mRNA and protein expression under LPS, PGN, and poly I:C stimulation. IL-36β was only found when the cells were treated with LPS, while IL-36α and IL-36γ were favored by PGN and poly I:C stimulation. We suggest that the microbial components participate in the activation of MCs, leading them to the production of IL-36, in which a specific member may participate in the origin and maintenance of inflammation in the glomerular environment that is associated with infections.

Sclareol ameliorates hyperglycemia-induced renal injury through inhibiting the MAPK/NF-κB signaling pathway.

Diabetic nephropathy (DN) represents the most serious complication of diabetes. Previous studies have shown that the activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) are linked to inflammation in the development of DN. Sclareol, a natural diterpene compound, has beneficial effects on inflammation. Thus, we hypothesized that sclareol might prevent DN via anti-inflammatory actions. This study aimed to investigate the actions of sclareol in the progression of DN, and explored the related molecular mechanism. Sclareol treatment significantly alleviated renal dysfunction, fibrosis, and inflammatory cytokine levels in a dose-dependent manner in diabetic mice. Moreover, sclareol inhibited the activations of MAPKs and NF-κB in diabetic kidney tissues. The therapeutic effects of sclareol were confirmed under high levels of glucose in SV40 cells, and sclareol prevented high glucose-induced fibrosis and inflammatory responses, which was largely driven by MAPKs and NF-κB inhibitions. In particular, MAPKs inhibitors mixture could suppress the NF-κB pathway and release of inflammatory cytokines that sclareol was involved in. In conclusion, sclareol has benefits for diabetes-induced renal dysfunction, which was partially associated with amelioration of fibrosis and inflammation via mediation of the MAPK/NF-κB signaling pathway. Sclareol may be a promising agent for preventing the progression of DN.

The role of green peas protein hydrolysate in TGF/SMAD signaling to prevent renal fibrosis

ObjectivesThe protein of green peas (Pisum sativum) hydrolyzed by bromelain (PHGPB) shows an effective renal fibrosis therapy by improving renal function parameters in the CKD rats model. The study aimed to determine anti-fibrotic properties effects of PHGPB and LERGDT on TGF-β1 level/SMAD 2,3,4 and 7 gene expressions in a fibrosis renal cell line model, glucose-induced MES13 SV40 cells. MethodsPHGPB was obtained using simple hydrolysis methods, LERGDT peptide was synthesized using Merryfield method, Chronic kidney model cells by glucose-induced MES13 SV40 cells. SMADs gene expression was counted using qRT-PCR, and TGF-β1 levels were measured by the ELISA method. ResultsThe result showed that treatment of 20 mM glucose induction in mesangial cells (kidney sclerosis model) increased the gene expression of SMAD2, SMAD3, and SMAD4, and decreased the expression of SMAD7 genes. Provision of PHGPB and LERGDT to kidney sclerosis cells models decreased the TGF-β1 level and gene expression of SMAD2 and SMAD4 genes at all concentrations, SMAD3 with 25 µg/mL PHGPB and LERGDT treatment. Gene expression of the SMAD7 increased after administration of PHGPB and LERGDT samples in all concentrations. ConclusionThe provision of PHGPB and LERGDT suppressed the expression of SMAD 2, 3, and 4 genes, yet increased the SMAD 7 gene. PHGPB and LERGDT have promising anti-fibrotic effects on TGF-β1 level/SMAD2, SMAD3, SMAD4, and SMAD7 gene expression in glucose-induced MES13 SV40 cells.

Glutinol inhibits the proliferation of human ovarian cancer cells via PI3K/AKT signaling pathway


 
 
 
 Purpose: To investigate the anticancer effect of glutinol on OVACAR3 human ovarian cancer cells, and to elucidate the underlying molecular mechanisms.
 Methods: Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cell cycle distribution, reactive oxygen species (ROS) and mitochondrial membrane potential were measured via flow cytometry, while protein expression levels were determined with western blotting assay.
 Results: Glutinol exerted significant growth-inhibitory effects on human OVACAR3 cells, but interestingly, it exhibited comparatively lower cytotoxic effects against normal SV40 cells. The IC50 of glutinol against human OVACAR3 cells was 6 μM, while the IC50 against normal SV40 cells was 60 μM. Flow cytometric analysis showed an increase in population of OVACAR3 cells in G2/M phase from 4.02 % in control to 29.05 % on treatment with 12 μM glutinol, suggestive of G2/M phase arrest. The G2/M arrest of OVACAR3 cells was also accompanied by suppression of cyclin B1. It was also found that increases in ROS levels and decreases in MMP activities contributed to the glutinol-induced antiproliferative effects on human OVACAR3 cells. Moreover, glutinol deactivated the PI3K/AKT signaling pathway in OVACAR-3 ovarian cancer cells.
 Conclusion: Glutinol exerted potent anticancer effects against human ovarian cancer. Thus, it might be of potential benefit in the treatment of ovarian cancer.
 
 
 

The transcriptome of wild-type and immortalized corneal epithelial cells

Cellular immortalization enables indefinite expansion of cultured cells. However, the process of cell immortalization sometimes changes the original nature of primary cells. In this study, we performed expression profiling of poly A-tailed RNA from primary and immortalized corneal epithelial cells expressing Simian virus 40 large T antigen (SV40) or the combination of mutant cyclin-dependent kinase 4 (CDK4), cyclin D1, and telomere reverse transcriptase (TERT). Furthermore, we studied the expression profile of SV40 cells cultured in medium with or without serum. The profiling of whole expression pattern revealed that immortalized corneal epithelial cells with SV40 showed a distinct expression pattern from wild-type cells regardless of the presence or absence of serum, while corneal epithelial cells with combinatorial expression showed an expression pattern relatively closer to that of wild-type cells.

STING-Mediated Autophagy Is Protective against H2O2-Induced Cell Death.

Stimulator of interferon genes (STING)-mediated type-I interferon signaling is a well characterized instigator of the innate immune response following bacterial or viral infections in the periphery. Emerging evidence has recently linked STING to various neuropathological conditions, however, both protective and deleterious effects of the pathway have been reported. Elevated oxidative stress, such as neuroinflammation, is a feature of a number of neuropathologies, therefore, this study investigated the role of the STING pathway in cell death induced by elevated oxidative stress. Here, we report that the H2O2-induced activation of the STING pathway is protective against cell death in wildtype (WT) MEFSV40 cells as compared to STING−/− MEF SV40 cells. This protective effect of STING can be attributed, in part, to an increase in autophagy flux with an increased LC3II/I ratio identified in H2O2-treated WT cells as compared to STING−/− cells. STING−/− cells also exhibited impaired autophagic flux as indicated by p62, LC3-II and LAMP2 accumulation following H2O2 treatment, suggestive of an impairment at the autophagosome-lysosomal fusion step. This indicates a previously unrecognized role for STING in maintaining efficient autophagy flux and protecting against H2O2-induced cell death. This finding supports a multifaceted role for the STING pathway in the underlying cellular mechanisms contributing to the pathogenesis of neurological disorders.

Liraglutide protects against high-fat diet-induced kidney injury by ameliorating apoptosis.

BackgroundObesity is associated with the development and progression of chronic kidney disease. Emerging evidence suggests that glucagon-like peptide-1 receptor agonist could reduce renal damage and albuminuria. Sirtuin 1 (SIRT1) was considered as a crucial regulator in metabolism-related kidney disease. Herein, the role of SIRT1 in liraglutide-ameliorated high-fat diet (HFD)-induced kidney injury was illustrated.MethodsMale C57BL/6 mice were fed HFD for 20 weeks to induce kidney injury that was then treated with liraglutide for 8 weeks to estimate its protective effect on the kidney. Also, the mechanism of the drug in SV40 MES 13 (SV40) mouse mesangial cells was elucidated.ResultsLiraglutide treatment ameliorated HFD-induced metabolic disorders, including hyperglycemia, increasing body weight, and insulin resistance. In addition, kidney weight, urine albumin-to-creatinine, and kidney morphological changes such as vacuolated tubules, glomerulomegaly, thickened glomerular basement membrane, and tubulointerstitial fibrosis were also significantly ameliorated. Furthermore, apoptotic cells and apoptosis markers were downregulated in the kidney of liraglutide-treated mice. In addition, the expression of SIRT1 protein was upregulated, whereas thioredoxin-interacting protein (TXNIP), which serves as a mediator of oxidative stress and apoptosis in metabolism disease, was downregulated by liraglutide. In SV40 cells, the effect of liraglutide on reversing the upregulation of cleaved caspase-3 induced by high glucose (30 mM) was hampered when SIRT1 was knocked down; also, the downregulation of TXNIP by liraglutide was blocked.ConclusionsLiraglutide might have a beneficial effect on metabolism-related kidney damage by inhibiting apoptosis via activation of SIRT1 and suppression of TXNIP pathway.

Potential Role of IL-39 in the Development of Gvhd

Allogeneic hematopoietic stem cell transplantation (allo-HCT) is an effective means by which to treat a wide variety of diseases resulting from hematological dysfunction. However, the development of graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality post transplantation. The IL-12 family of cytokines is comprised of IL-12, IL-23, IL-27, IL-35, and potentially IL-39. IL-12 family members are unique in that each cytokine and cognate receptor is comprised of heterodimers in which either the a or b subunit is shared among the others. IL-12 (p35+p40) and IL-23 (p19+p40) have well documented proinflammatory functions responsible for Th1 differentiation and Th17 stabilization, respectively, and play critical roles in GVHD development. IL-12R and IL-23R share a β-chain (IL-12Rβ1) yet use distinct α-chains to mediate their respective receptor signaling. While both IL-12R and IL-23R are widely implicated in inflammatory disorders, the role of IL-12Rβ1 in this context remains much less defined. We therefore studied the impact of eliminating the common IL-12Rβ1 chain or the unique IL-23Rα chain in T cells on GVHD using murine models of allogeneic bone marrow transplantation (BMT). In agreement with previous publications, we found a pathogenic role for IL-23Rα on donor T cells in aGVHD. Strikingly, a similar effect was not seen for IL-12Rβ1 (Figure 1A, B). These data suggest that that IL-23Rα contributes to GVHD pathogenesis via a pathway independent of IL-12Rβ1. To confirm that functional differences existed between T cells deficient for IL-23Rα or IL-12Rβ1 in GVHD, we assessed cytokine profiles of these T cells in target organs 14 days post-BMT. We found that, while production of IFNγ and IL-17 in the spleen was similarly decreased in both cohorts, GM-CSF production by CD4+ T cells was reduced exclusively in T cells deficient for IL-23Rα. Further, a significant reduction of IFNγ and GM-CSF in target organs, such as the liver and gut, was only observed in T cells deficient for IL-23Rα. The newest member of the IL-12 family, IL-39, was recently shown to contribute to SLE pathogenesis; this cytokine has been described to be composed of IL-23p19 and EBI3. Given the cognate receptor for IL-39 includes IL-23Rα and gp130, we hypothesized that IL-39 may play a role in aGVHD as this would explain why IL-12Rβ1 is dispensable. To validate that p19 and EBI3 can form a heterodimer, we transfected SV40 cells with vectors containing control, IL-23p19, EBI3 or both cDNAs. We detected IL-39 heterodimers only in the supernatant of cells transfected with both IL-23p19 and EBI3 via ELISA (Figure 1C). Furthermore, we observed significantly increased levels of IL-39 in allogeneic recipients at day 14 post BMT compared to naïve mice or recipients of BM alone in two models of aGVHD (Figure 1D). This may implicate IL-39 in the GVHD development. Taken together, our studies indicate that IL-23Rα plays an essential role, whereas IL-12Rβ1 is dispensable, for donor T cells to induce aGVHD. Our proposed model is that in the absence of IL-12Rβ1, IL-39 could transmit IL-23Rα signaling, hypothetically by forming a heterodimer with gp130. This new finding indicates that IL-23Rα and IL-39 are potential therapeutic targets for controlling aGVHD in the clinic. Figure 1. Effect of IL-12R 𝛃 1 in aGVHD and the potential role of IL-39 Lethally irradiated BALB/c mice were transplanted with 5x106 TCD-BM alone or plus 1x106 purified T cells from WT B6, IL-12R𝛃1KO, or IL-23RαKO mice. Survival (A) and body weight loss (B) are shown. Supernatant from SV40 cells transiently transfected with vectors containing control, IL-23p19, EBI3 or both cDNAs. p19 and EBI3 heterodimers were detected via ELISA (C). Serum was collected from naïve or lethally irradiated mice transplanted with 5x106 TCD-BM alone or plus 1x106 purified T cells. Formation of p19 and EBI3 heterodimers were tested in serum at 14 days post BMT via ELISA (D). Disclosures No relevant conflicts of interest to declare.

Comparison of antiproliferative effect of epigallocatechin gallate when loaded into cationic solid lipid nanoparticles against different cell lines

Several therapeutic properties have been attributed to epigallocatechin gallate (EGCG), a phytopharmaceutical polyphenol with antioxidant and antiproliferative activity. EGCG is, however, very prone to oxidation in aqueous solutions which changes its bioactive properties. Its loading in nanoparticles has been proposed to reduce its degradation while increasing its in vivo efficacy. The aim of this study was to compare the antiproliferative effect of EGCG before and after its loading in solid lipid nanoparticles (SLNs), against five different cell lines (Caco-2, HepG2, MCF-7, SV-80 and Y-79). EGCG produced concentration- and time-dependent antiproliferative effect, with efficacy dependent on the cell line. The order of potency was: MCF-7>SV-80>HepG2>Y-79>Caco-2, for 24 h exposure (MCF-7 IC50=58.60 ± 3.29 µg/mL; Caco-2 IC50>500.00 µg/mL). To the best of our knowledge this is the first study reporting EGCG antiproliferative effect in SV-80 and Y-79 cells. DDAB-SLN physicochemical properties (size ∼134 nm; PI∼0.179; ZP ∼+28mV) were only slightly modified with EGCG loading (EGCG-DDAB-SLN: ∼144 nm; PI∼0.160; ZP ∼+26mV). EGCG loading in SLN, only slightly increases the EGCG antiproliferative effect in MCF-7 and SV-80 cells. SLN exhibited intrinsic toxicity, attributed to the surfactant used in its production. From the obtained results, the biocompatibility of blank SLN must be also considered when testing the efficacy of loaded phytopharmaceutics.

Girinimbine Inhibits the Proliferation of Human Ovarian Cancer Cells In Vitro via the Phosphatidylinositol-3-Kinase (PI3K)/Akt and the Mammalian Target of Rapamycin (mTOR) and Wnt/β-Catenin Signaling Pathways.

BackgroundWorldwide, ovarian cancer is increasing in prevalence and has a high mortality rate. Girinimbine is a carbazole alkaloid isolated from Murraya koenigii (the curry tree) and is used in Chinese herbal medicine. The aim of this study was to evaluate the effects of girinimbine on cell proliferation, cell migration, and apoptosis in human ovarian cancer cells in vitro.Material/MethodsA human ovarian cancer cell line panel, which included SKOV3 cells and the SV40 immortalized normal human ovarian cell line, were treated with increasing doses of girinimbine. Cell proliferation was evaluated using the MTT assay. Confocal immunofluorescence using 4′,6-diamidino-2-phenylindole (DAPI), Annexin-V, and propidium iodide (PI) were used to measure cell apoptosis. Cell migration and invasion were determined by transwell assays. Protein expression was determined by Western blot.ResultsGirinimbine inhibited SKOV3 ovarian cancer cell proliferation in a dose-dependent manner. The half maximal inhibitory concentration (IC50) of grinimbine was 15 μM for the SKOV3 cells, and 120 μM for the SV40 cells. Grinimbine treatment resulted in apoptosis of SKOV3 cells, from 2.2% in untreated cells to 58.8% at a dose of 30 μM, which was associated with an increase in the Bax/Bcl-2 ratio. Girinimbine inhibited cell migration and invasion of the SKOV3 cancer cells in vitro and inhibited the PI3K/AKT/mTOR and Wnt/β-catenin signaling pathways.ConclusionsGirinimbine, a carbazole alkaloid used in Chinese herbal medicine, inhibited the proliferation and cell migration of human ovarian cancer cells in vitro, in a dose-dependent manner, via the PI3K/Akt/mTOR and Wnt/β-catenin signaling pathways.

Difference in Susceptibility of 3T3 and 3T3-SV40 Cells to Invasion by Opportunistic Pathogens Serratia grimesii

Cells isolated from a body are resistant to bacterial invasion but lose the resistance upon immortalization and transformation. In this work, interaction of immortalized 3T3 fibroblasts and their in vitro transformed analog 3T3-SV40 cells with opportunistic bacteria Serratia grimesii was studied in a conventional medium and after incubating the cells with an antioxidant N-acetylcysteine (NAC). The 3T3 cells were shown to be approximately twice less sensitive to S. grimesii infection than similar but virus-transformed 3T3- SV40 cells. Incubation of 3T3 cells with 10 and 20 mM NAC enhanced the invasion 1.6 and 2.5-fold, respectively. Under the same conditions, the invasion of 3T3-SV40 cells by the bacteria was enhanced 2.1 and 2.4- fold. These results show that 3T3 cells are more resistant to invasion by S. grimesii than 3T3-SV40 cells, and the difference is preserved after the cells are exposed to 10 and 20 mM NAC. Among the genes which expression is known to be increased by NAC, a special role plays E-cadherin shown to interact with surface proteins (invasins) of pathogenic bacteria. Incubation of 3T3 and 3T3-SV40 cells with NAC resulted in an increased expression of E-cadherin, which correlates with the increased sensitivity of these cells to invasion. Confocal fluorescence microscopy revealed, for the first time, colocalization of S. grimesii with E-cadherin of 3T3 and 3T3-SV40 cells indicating that E-cadherin can be involved in the penetration of S. grimesii into eukaryotic cells.

EGFR inhibition attenuates diabetic nephropathy through decreasing ROS and endoplasmic reticulum stress.

Diabetic nephropathy (DN) is a progressive kidney disease due to glomerular capillary damage in diabetic patients. Endoplasmic reticulum (ER) stress caused by reactive oxygen species (ROS) is associated with DN progression. Epidermal growth factor receptor (EGFR) mediates oxidative stress and damage of cardiomyocytes in diabetic mice. Here we demonstrated that AG1478, a specific inhibitor of EGFR, blocked EGFR and AKT phosphorylation in diabetic mice. Oxidative stress and ER stress markers were eliminated after AG1478 administration. AG1478 decreased pro-fibrotic genes TGF-β and collagen IV. Furthermore, we found that high glucose (HG) induced oxidative stress and ER stress, and subsequently increased ATF4 and CHOP. These changes were eliminated by either AG1478 or ROS scavenger N-acetyl-L-cysteine (NAC) administration. These results were confirmed by knock-down approaches in renal mesangial SV40 cells. However, AG1478, not NAC, reversed HG induced EGFR and AKT phosphorylation. These results suggest that EGFR/AKT/ROS/ER stress signaling plays an essential role in DN development and inhibiting EGFR may serve as a potential therapeutic strategy in diabetic kidney diseases.

Juxtanuclear Drebrin-Enriched Zone.

Drebrin E contributes to remodeling of the actin cytoskeleton and formation of cell processes. Therefore, its role in cell migration was studied in prototypes of motile cells with prominent lamellipodia such as murine B16F1 melanoma and Swiss 3T3 cells and in human SV80 fibroblasts. Confocal microscopy revealed absence of drebrin from the tips of lamellipodia but enrichment in the tail of the cells, in retraction zones and in a specific juxtanuclear actin filament compartment, named "drebrin-enriched zone." A similar subset of juxtanuclear actin filaments is characterized by the actin-binding protein SWAP-70, but drebrin and SWAP-70 localized to different compartments, suggesting the existence of novel distinct subdomains within the actin filament system. In cells overexpressing drebrin-EGFP, numerous long, branched cell processes were formed which slowly retracted and extended. However, in stable transfectants containing lower amounts of the fusion protein, drebrin-EGFP was recruited to the same sites as the endogenous protein during cell migration, i.e., to retracting membrane domains and into the juxtanuclear drebrin-enriched zone. In the leading edges of SV80 cells, characterized by pronounced actin microspikes, drebrin was concentrated along posterior portions of the microspikes, together with tropomyosin, with which it competes for actin binding. Drebrin knockdown by siRNA did not impact forward migration or ruffling. Taken together, these findings suggest that during cell migration drebrin is involved in retraction processes but not in lamellipodia formation. The novel, sizable juxtanuclear drebrin-enriched zone remains to be characterized in detail with respect to its molecular assembly and functions.

Novel Epidermal Growth Factor Receptor Inhibitor Attenuates Angiotensin II-Induced Kidney Fibrosis.

Chronic activation of renin-angiotensin system (RAS) greatly contributes to renal fibrosis and accelerates the progression of chronic kidney disease; however, the underlying molecular mechanism is poorly understood. Angiotensin II (Ang II), the central component of RAS, is a key regulator of renal fibrogenic destruction. Here we show that epidermal growth factor receptor (EGFR) plays an important role in Ang II-induced renal fibrosis. Inhibition of EGFR activation by novel small molecules or by short hairpin RNA knockdown in Ang II-treated SV40 mesangial cells in vitro suppresses protein kinase B and extracellular signal-related kinase signaling pathways and transforming growth factor-β/Sma- and Mad-related protein activation, and abolishes the accumulation of fibrotic markers such as connective tissue growth factor, collagen IV. The transactivation of EGFR by Ang II in SV40 cells depends on the phosphorylation of proto-oncogene tyrosine-protein kinase Src (c-Src) kinase. Further validation in vivo demonstrates that EGFR small molecule inhibitor successfully attenuates renal fibrosis and kidney dysfunction in a mouse model induced by Ang II infusion. These findings indicate a crucial role of EGFR in Ang II-dependent renal deterioration, and reveal EGFR inhibition as a new therapeutic strategy for preventing progression of chronic renal diseases.

Cytotoxicity evaluation of self-etching dentine bonding agents in a cell culture perfusion condition

The aim of this study was to evaluate the cytotoxicity of three dentine bonding agents (G-Bond, Clearfil S(3) Bond and Clearfil SE Bond X) in cell-culture perfusion. In this experiment, 8×10(4) TCPC SV40 cells (bovine-pulp-derived cells transfected with simian virus 40 large T-antigen) in MEM-alpha media, 20%FCS were seeded on mesh in a 6-well plate and incubated at 37 °C with 5% CO(2). After 2 days, the mesh inserts were transferred to a 24-well plate and incubated in MEM-alpha media, 20%FCS with 50 μg/ml of ascorbic acid at 37 °C with 5% CO(2) for 14 days. The test materials were applied to the dentine discs of dentine barrier models. Three-dimensional cell cultures in mesh and perfusion conditions were generated in this experiment. Each material, as well as the negative control (President) and the positive control (vitrebond), was tested in 5 models with the tests repeated in triplicate. The MTT assay was used to determine cell viability after the diffusion of leachable toxicity from the tested materials through the dentine discs. The cell survival rate with G-Bond and Clearfil S(3) Bond was 113.03 and 90.98 percent, respectively, whereas that with Clearfil SE Bond X was 111.83 percent. All three dentine bonding agents had no toxicity compared with the negative control group (P>.05). All three self-etching dentine-bonding agents are nontoxic. Pulp damage caused by these three bonding agents is unlikely to occur in the clinic.

PS2-60 Biological characterization of feline interferon-NU: a new member of the Type I interferon family

The cytotoxic effect of lymphotoxin (LT) and its modulation by interferon (IFN) was quantitatively assessed in uninfected and vesicular stomatitis virus (VSV)-infected cultured cells. Preparations of human LT, which were depleted of IFN, had a significant cytotoxic effect on VSV-infected HeLa, SV-80, WISH, and Vero cells. IFN, most notably IFN-γ, further potentiated destruction of the infected cells by these LT preparations, when applied on the cells at sub-antiviral IFN concentrations. In contrast, no cytotoxic effect could be observed in any of the examined cells, when applying LT, IFN, or their combination, in the absence of viral infection. Infected cells in which VSV replication was suppressed by treatment with antiviral concentrations of IFN also resisted destruction by LT. These findings indicate that LT cytotoxicity can be selectively directed against virus-infected cells and that IFN can augment this cell-killing mechanism when failing to exert an antiviral effect.