Achieving Long-Wavelength Electroluminescence Using Two-Coordinate Gold(I) Complexes: Overcoming the Energy Gap Law.

Two-coordinate coinage metal complexes have emerged as promising emitters for highly efficient organic light-emitting devices (OLEDs). However, achieving efficient long-wavelength electroluminescence emission from these complexes remains as a daunting challenge. To address this challenge, molecular design strategies aimed at bolstering the photoluminescence quantum yield (Φ) of Au(I) complex emitters in low-energy emission regions are investigated. By varying amido ligands, a series of two-coordinate Au(I) complexes is developed that exhibit photoluminescence peak wavelengths over a broad range of 533-750 nm. These complexes, in particular, maintain Φ values up to 10% even in the near-infrared emission region, overcoming the constraints imposed by an energy gap. Quantum chemical calculations and photophysical analyses reveal the action of radiative control, which serves to overcome the energy gap law, becomes more pronounced as the overlap between hole and electron distributions (Sr (r)) in the excited state increases. It is further elucidated that Sr (r) increases with the distance between the hole-distribution centroid and the nitrogen atom in an amido ligand. Finally, multilayer OLEDs involving the Au(I) complex emitters exhibit performances beyond the borderline of the electroluminescence wavelength-external quantum efficiency space set by previous devices of coinage metal complexes.

High-performance blue OLED using multiresonance thermally activated delayed fluorescence host materials containing silicon atoms.

We report three highly efficient multiresonance thermally activated delayed fluorescence blue-emitter host materials that include 5,9-dioxa-13b-boranaphtho[3,2,1-de]anthracene (DOBNA) and tetraphenylsilyl groups. The host materials doped with the conventional N7,N7,N13,N13,5,9,11,15-octaphenyl-5,9,11,15-tetrahydro-5,9,11,15-tetraaza-19b,20b-diboradinaphtho[3,2,1-de:1',2',3'-jk]pentacene-7,13-diamine (ν-DABNA) blue emitter exhibit a high photoluminescence quantum yield greater than 0.82, a high horizontal orientation greater than 88%, and a short photoluminescence decay time of 0.96-1.93 µs. Among devices fabricated using six synthesized compounds, the device with (4-(2,12-di-tert-butyl-5,9-dioxa-13b-boranaphtho[3,2,1-de]anthracen-7-yl)phenyl)triphenylsilane (TDBA-Si) shows high external quantum efficiency values of 36.2/35.0/31.3% at maximum luminance/500 cd m-2/1,000 cd m-2. This high performance is attributed to fast energy transfer from the host to the dopant. Other factors possibly contributing to the high performance are a T1 excited-state contribution, inhibition of aggregation by the bulky tetraphenylsilyl groups, high horizontal orientation, and high thermal stability. We achieve a high efficiency greater than 30% and a small roll-off value of 4.9% at 1,000 cd m-2 using the TDBA-Si host material.

Phosphorescent Ir(III) Complexes for Biolabeling and Biosensing.

Cyclometalated Ir(III) complexes exhibit strong phosphorescence emission with lifetime of submicroseconds to several microseconds at room temperature. Their synthetic versatility enables broad control of physical properties, such as charge and lipophilicity, as well as emission colors. These favorable properties have motivated the use of Ir(III) complexes in luminescent bioimaging applications. This review examines the recent progress in the development of phosphorescent biolabels and sensors based on Ir(III) complexes. It begins with a brief introduction about the basic principles of the syntheses and photophysical processes of cyclometalated Ir(III) complexes. Focus is placed on illustrating the broad imaging utility of Ir(III) complexes. Phosphorescent labels illuminating intracellular organelles, including mitochondria, lysosomes, and cell membranes, are summarized. Ir(III) complexes capable of visualization of tumor spheroids and parasites are also introduced. Facile chemical modification of the cyclometalating ligands endows the Ir(III) complexes with strong sensing ability. Sensors of temperature, pH, CO2, metal ions, anions, biosulfur species, reactive oxygen species, peptides, and viscosity have recently been added to the molecular imaging tools. This diverse utility demonstrates the potential of phosphorescent Ir(III) complexes toward bioimaging applications.

Cerebral angiography using transauricular access in a rabbit model: a new technique.

BACKGROUND: Cerebral angiography in a rabbit model is widely used in the field of interventional radiology. Conventionally, the femoral artery is used for cerebral angiography in radiology departments. However, angiographic studies require surgical cutdown of the femoral artery, which is technically difficult. PURPOSE: To evaluate a new cerebral angiography technique involving a transauricular approach in a rabbit model. MATERIAL AND METHODS: In each of 10 rabbits, central auricular arteries were punctured in the right or left ear with a 20-gauge i.v. catheter. A microcatheter (2.0 F) with a 0.016-inch guide wire was introduced through the i.v. catheter and advanced to the aortic arch. The microcatheter and guide wire were advanced selectively into cerebral arteries and angiography was performed. RESULTS: Central auricular arteries were successfully punctured with 20-gauge i.v. catheters. After approaching the aortic arch, microcatheter tips and guide wires were advanced manually to cerebral arteries on both sides. Difficulties in selecting the carotid arteries were resolved by using a looping technique within the cardiac chamber. Microcatheter loops within the cardiac chamber disappeared or remained during artery superselection. CONCLUSION: Transauricular cerebral angiography appears to be a feasible technique for brain or carotid intervention studies in rabbits. In addition, vertebral angiography using a transauricular approach is possible using the looping technique. Selection of carotid or vertebral arteries on each side was not difficult when the microcatheter and guide wire were looped within the cardiac chamber. The ear chosen for the initial puncture does not appear to be important.

Tetracycline Analogs Inhibit Osteoclast Differentiation by Suppressing MMP-9-Mediated Histone H3 Cleavage.

Osteoporosis is a common disorder of bone remodeling, caused by the imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Recently, we reported that matrix metalloproteinase-9 (MMP-9)-dependent histone H3 proteolysis is a key event for proficient osteoclast formation. Although it has been reported that several MMP-9 inhibitors, such as tetracycline and its derivatives, show an inhibitory effect on osteoclastogenesis, the molecular mechanisms for this are not fully understood. Here we show that tetracycline analogs, especially tigecycline and minocycline, inhibit osteoclast formation by blocking MMP-9-mediated histone H3 tail cleavage. Our molecular docking approach found that tigecycline and minocycline are the most potent inhibitors of MMP-9. We also observed that both inhibitors significantly inhibited H3 tail cleavage by MMP-9 in vitro. These compounds inhibited receptor activator of nuclear factor kappaB ligand (RANKL)-induced osteoclast formation by blocking the NFATc1 signaling pathway. Furthermore, MMP-9-mediated H3 tail cleavage during osteoclast differentiation was selectively blocked by these compounds. Treatment with both tigecycline and minocycline rescued the osteoporotic phenotype induced by prednisolone in a zebrafish osteoporosis model. Our findings demonstrate that the tetracycline analogs suppress osteoclastogenesis via MMP-9-mediated H3 tail cleavage, and suggest that MMP-9 inhibition could offer a new strategy for the treatment of glucocorticoid-induced osteoporosis.

S-Nitrosoglutathione loaded poly(lactic-co-glycolic acid) microparticles for prolonged nitric oxide release and enhanced healing of methicillin-resistant Staphylococcus aureus-infected wounds.

Methicillin-resistant Staphylococcus aureus (MRSA)-infected wounds have become a significant clinical issue worldwide. Recently, nitric oxide (NO) has emerged as a potent antibacterial agent against MRSA infections and a wound-healing enhancer. Nevertheless, clinical applications of NO have been largely restricted by its gaseous state and short half-life. In this study, our aim was to develop S-nitrosoglutathione (GSNO, an endogenous NO donor)-loaded poly(lactic-co-glycolic acid) [PLGA] microparticles (GSNO-MPs) that release NO over a prolonged period, to accelerate the healing of MRSA-infected wounds with less frequent dosing. GSNO was successfully encapsulated into PLGA microparticles by a solid-in-oil-in-water emulsion solvent evaporation method. Scanning electron microscopy and X-ray diffraction analyses confirmed the successful fabrication of GSNO-MPs. The latter released NO in a prolonged manner over 7 days and exerted a remarkable antibacterial activity against MRSA in a concentration- and time-dependent manner. Moreover, GSNO-MPs had good antibacterial efficacy and were found to accelerate wound healing in a mouse model of MRSA-infected wounds. Therefore, NO-releasing MPs devised in this study may be a promising option for the treatment of cutaneous wounds infected by drug-resistant bacteria such as MRSA.

Acetylshikonin induces apoptosis of hepatitis B virus X protein-expressing human hepatocellular carcinoma cells via endoplasmic reticulum stress.

Since it has been known that shikonin derived from a medicinal plant possesses anti-cancer activity, we wonder whether acetylshikonin (ASK), a derivate of shikonin, can be used to treat hepatocellular carcinoma cells expressing hepatitis B virus X protein (HBX), an oncoprotein from hepatitis B virus. When ASK was added to Hep3B cells stably expressing HBX, it induced apoptosis in a dose-dependent manner. ASK induced upregulation and export of Nur77 to the cytoplasm and activation of JNK. Likewise, suppression of Nur77 and JNK inactivation protected the cells from ASK-induced apoptosis, indicating that Nur77 upregulation and JNK activation were required for ASK-mediated apoptosis. Furthermore, ASK increased the expression of Bip and ubiquitination levels of cellular proteins, features of endoplasmic reticulum (ER) stress, via the production of reactive oxygen species in a dose-dependent manner. Suppression of reactive oxygen species with N-acetylcysteine reduced levels of Bip protein and ubiquitination levels of cellular proteins during ASK treatment, leading to protection of cells from apoptosis. Cycloheximide treatment reduced ASK-induced ER stress, suggesting that protein synthesis is involved in ASK-induced ER stress. Moreover, we showed using salubrinal, an ER stress inhibitor that reactive oxygen species production, JNK activation, and Nur77 upregulation and its translocation to cytoplasm are necessary for ER-induced stress. Interestingly, we found that JNK inactivation suppresses ASK-induced ER stress, whereas Nur77 siRNA treatment does not, indicating that JNK is required for ASK-induced ER stress. Accordingly, we report that ASK induces ER stress, which is prerequisite for apoptosis of HBX-expressing hepatocellular carcinoma cells.

SIRT1 sensitizes hepatocellular carcinoma cells expressing hepatitis B virus X protein to oxidative stress-induced apoptosis.

We previously showed that SIRT1 deacetylase inhibits proliferation of hepatocellular carcinoma cells expressing hepatitis B virus (HBV) X protein (HBX), by destabilization of ß-catenin. Here, we report another role for SIRT1 in HBX-mediated resistance to oxidative stress. Ectopic expression and enhanced activity of SIRT1 sensitize Hep3B cells stably expressing HBX to oxidative stress-induced apoptosis. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for sensitization of oxidation-mediated apoptosis. Furthermore, ectopic expression of SIRT1 and treatment with resveratrol (a SIRT1 activator) attenuated JNK phosphorylation, which is a prerequisite for resistance to oxidative stress-induced apoptosis. Conversely, suppression of SIRT1 activity with nicotinamide inhibited the effect of resveratrol on JNK phosphorylation, leading to restoration of resistance to oxidation-induced apoptosis. Taken together, these results suggest that up-regulation of SIRT1 under oxidative stress may be a therapeutic strategy for treatment of hepatocellular carcinoma cells related to HBV through inhibition of JNK activation.

Whole transcriptome analyses of six thoroughbred horses before and after exercise using RNA-Seq.

BACKGROUND: Thoroughbred horses are the most expensive domestic animals, and their running ability and knowledge about their muscle-related diseases are important in animal genetics. While the horse reference genome is available, there has been no large-scale functional annotation of the genome using expressed genes derived from transcriptomes. RESULTS: We present a large-scale analysis of whole transcriptome data. We sequenced the whole mRNA from the blood and muscle tissues of six thoroughbred horses before and after exercise. By comparing current genome annotations, we identified 32,361 unigene clusters spanning 51.83 Mb that contained 11,933 (36.87%) annotated genes. More than 60% (20,428) of the unigene clusters did not match any current equine gene model. We also identified 189,973 single nucleotide variations (SNVs) from the sequences aligned against the horse reference genome. Most SNVs (171,558 SNVs; 90.31%) were novel when compared with over 1.1 million equine SNPs from two SNP databases. Using differential expression analysis, we further identified a number of exercise-regulated genes: 62 up-regulated and 80 down-regulated genes in the blood, and 878 up-regulated and 285 down-regulated genes in the muscle. Six of 28 previously-known exercise-related genes were over-expressed in the muscle after exercise. Among the differentially expressed genes, there were 91 transcription factor-encoding genes, which included 56 functionally unknown transcription factor candidates that are probably associated with an early regulatory exercise mechanism. In addition, we found interesting RNA expression patterns where different alternative splicing forms of the same gene showed reversed expressions before and after exercising. CONCLUSION: The first sequencing-based horse transcriptome data, extensive analyses results, deferentially expressed genes before and after exercise, and candidate genes that are related to the exercise are provided in this study.

Hepatitis B virus X (HBX) protein upregulates ß-catenin in a human hepatic cell line by sequestering SIRT1 deacetylase.

Hepatitis B virus X (HBX) protein has been reported to induce upregulation of ß-catenin, a known proto-oncogene, in p53-knockout and p53-mutant hepatic cell lines both in a GSK-3ß-dependent manner and via interaction with adenomatous polyposis coli, which results in protection from ß-catenin degradation. In this study, we describe a novel mechanism for HBX-mediated upregulation of ß-catenin. We observed that HBX interacts with SIRT1, a class III histone deacetylase. Furthermore, the presence of HBX attenuated the interaction between SIRT1 and ß-catenin, leading to protection of ß-catenin from the inhibitory action of SIRT1. Reduction of SIRT1 with siRNA or suppression of SIRT1 activity with nicotinamide upregulated ß-catenin protein levels. In contrast, enhancement of SIRT1 activity with resveratrol reduced ß-catenin protein levels. Furthermore, in Hep3B cells stably expressing HBX, overexpression of SIRT1 or treatment with resveratrol enhanced sensitivity to doxorubicin-induced apoptosis, indicating that upregulation of SIRT1 could be a therapeutic strategy for HBV-related hepatocellular carcinoma. Based on these results, we propose that HBX upregulates ß-catenin by sequestering SIRT1, which leads to anticancer drug treatment resistance.

Hepatitis B virus X protein inhibits extracellular IFN-α-mediated signal transduction by downregulation of type I IFN receptor.

We have previously shown that hepatitis B virus (HBV) protein X (HBX), a regulatory protein of HBV, activates Stat1, leading to type I interferon (IFN) production. Type I IFN secreted from HBX-expressing hepatic cells enforces antiviral signals through its binding to the cognate type I IFN receptor. We therefore investigated how cells handle this detrimental situation. Interestingly, compared to Chang cells stably expressing an empty vector (Chang-Vec), Chang cells stably expressing HBX (Chang-HBX) showed lower levels of IFN-α receptor 1 (IFNAR1) protein, a subunit of type I IFN receptor. The levels of IFNAR1 transcripts detected in Chang-HBX cells were lower than the levels in Chang-Vec cells, indicating that HBX regulates IFNAR1 at the transcriptional level. Moreover, we observed that HBX induced the translocation of IFNAR1 to the cytoplasm. Consistent with these observations, HBX also downregulated Tyk2, which is required for the stable expression of IFNAR1 on the cell surface. Eventually, Chang-HBX cells consistently maintained a lower level of IFNAR1 expression and displayed no proper response to IFN-α, while Chang-Vec cells exhibited a proper response to IFN-α treatment. Taken together, we propose that HBX downregulates IFNAR1, leading to the avoidance of extracellular IFN-α signal transduction.

Up-regulation of Foxo4 mediated by hepatitis B virus X protein confers resistance to oxidative stress-induced cell death.

The hepatitis B virus X (HBX) protein, a regulatory protein of the hepatitis B virus (HBV), has been shown to generate reactive oxygen species (ROS) in human liver cell lines; however, the mechanism by which cells protect themselves under this oxidative stress is poorly understood. Here, we show that HBX induces the up-regulation of Forkhead box class O 4 (Foxo4) not only in Chang cells stably expressing HBX (Chang-HBX) but also in primary hepatic tissues from HBX-transgenic mice. HBX also increased ROS, but reduction of the abundance of ROS using N-acetylcystein (NAC) diminished the levels of Foxo4. Elevated Foxo4 was also detected in nuclei of Chang-HBX cells but not in Chang cells stably expressing the vector (Chang-Vec), suggesting that HBX activates the transcriptional activity of Foxo4. When we examined whether HBX bypasses JNK signaling that targets Foxo4, we found that the activity of JNK but not of ERK is required for the up-regulation of Foxo4 even in the presence of HBX. Furthermore, the reduction of Foxo4 levels using siRNA or a JNK inhibitor rendered Chang-HBX cells sensitive to apoptosis under oxidative stress, suggesting that up-regulation of Foxo4 mediated by HBX enhances resistances to oxidative stress-induced cell death. Accordingly, we propose that Foxo4 may be a useful target for suppression in the treatment of HBV-associated hepatocellular carcinoma cells.

Pancreatic adenocarcinoma up-regulated factor (PAUF) enhances the expression of ß-catenin, leading to a rapid proliferation of pancreatic cells.

It is not yet understood how the enhanced expression of pancreatic adenocarcinoma up-regulated factor (PAUF; a novel oncogene identified in our recent studies), contributes to the oncogenesis of pancreatic cells. We herein report that PAUF up-regulates the expression and transcriptional activity of ß-catenin while the suppression of PAUF by shRNA down-regulates ß-catenin. The induction of b-catenin by PAUF is mediated by the activities of Akt and GSK-3ß, but inhibition of downstream ERK does not reduce ß-catenin expression. To test whether PAUF emulates either the Wnt3a-mediated or the protein kinase A-mediated signaling pathway for the stabilization of ß-catenin, we examined the phosphorylation status of ß-catenin in the presence of PAUF compared with that of ß-catenin during treatment with Wnt3a or dibutyryl cAMP, a cell permeable cyclic AMP analogue. PAUF expression induces phosphorylation at Ser-33/37/Thr-41 and Ser-675 of ß-catenin but no phosphorylation at Ser-45, indicating that a unique phosphorylation pattern of b-catenin is caused by PAUF. Finally, the expression of PAUF up-regulates both cyclin-D1 and c-Jun, target genes of ß-catenin, leading to a rapid proliferation of pancreatic cells; conversely decreased PAUF expression (by shRNA) results in the reduced proliferation of pancreatic cells. Treatment with hexachlorophene (an inhibitor of ß-catenin) reduces the proliferation of pancreatic cells despite the presence of PAUF. Taken together, we propose that PAUF can up-regulate and stabilize ß-catenin via a novel pattern of phosphorylation, thereby contributing to the rapid proliferation of pancreatic cancer cells.

Reovirus infection induces apoptosis of TRAIL-resistant gastric cancer cells by down-regulation of Akt activation.

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been used to treat a variety of cancer cells. However, since some gastric cancer cells are resistant to TRAIL, we explored whether reovirus induces cytolysis in TRAIL-resistant gastric cancer cells. We found that TRAIL-resistant SNU-216 gastric cancer cells were susceptible to apoptosis by reovirus infection. Furthermore, co-treatment with reovirus and TRAIL accelerated apoptosis of SNU-216 cells by down-regulation of Akt activation as assessed by a very low activation of Akt in TRAIL-sensitive SNU-668 gastric cancer cells. Inhibition of Akt signaling with wortmannin or suppression of Akt expression with sh-Akt lentivirus promoted reovirus-mediated apoptosis of SNU-216 gastric cancer cells. Reovirus infection also down-regulates the activation of signaling molecules such as Ras and ERK involved in cell proliferation and survival but not the activation of p38 MAPK involved in cellular stress. In addition, the co-treatment with reovirus and TRAIL resulted in cleavage of caspase-8, caspase-9 and Bid, leading to a decrease in the mitochondrial membrane potential, indicating that reovirus may utilize the mitochondrial intrinsic apoptotic pathway in TRAIL-resistant SNU-216 gastric cancer cells. Accordingly, we first demonstrate that reovirus infection down-regulates Akt activation, leading to apoptosis of TRAIL-resistant gastric cancer cells.

Inhibition of GSK-3beta enhances reovirus-induced apoptosis in colon cancer cells.

Reovirus functions as an oncolytic agent for many types of cancer including colon cancer. Although most studies have emphasized the role of activated Ras signaling in enhancing reoviral oncolysis in susceptible cells, we note that many colon cancers also display elevated beta-catenin. Thus, it is possible that enhanced beta-catenin may augment reoviral susceptibility in colon cancer cells. To explore this hypothesis, HEK293 cells were treated with the glycogen synthase kinase (GSK)-3beta inhibitor LiCl, thereby inducing beta-catenin, followed by reoviral infection. Co-administration with LiCl indeed enhanced cell death compared to reovirus infection alone, but this was not associated with elevated reoviral replication. Similarly, HEK293 cells expressing the Frizzled-1 receptor in Wnt3a-conditioned medium also showed reovirus replication equivalent to that in cells in control medium, further suggesting that up-regulation of beta-catenin does not enhance the replication of reovirus. Instead, we observed that inhibition of GSK-3beta with LiCl decreased reovirus-induced NF-kappaB activation, leading to accelerated apoptosis via caspase 8 activation. We further found that colon cancer HCT116 cells were sensitized to apoptosis by co-treatment with reovirus and a GSK-3beta inhibitor, AR-A014418. Finally, we identified that inhibition of NF-kappaB sensitized apoptosis of HEK293 or HCT 116 cells during reovirus infection. Taken together, we propose that inhibition of GSK-3beta sensitizes reovirus-induced apoptosis of colon cancer cells by down-regulation of NF-kappaB activity, offering a potentially improved therapeutic strategy for the treatment of colon cancer.

STP-C, an oncoprotein of herpesvirus saimiri augments the activation of NF-kappaB through ubiquitination of TRAF6.

Herpesvirus saimiri (HVS), a member of the gamma-herpesvirus family, encodes an oncoprotein called Saimiri Transforming Protein (STP) which is required for lymphoma induction in non-human primates. Previous study has shown that STP-C, an oncoprotein of HVS, activates NF-kappaB signaling pathway. However, the detailed mechanism of STP-C-mediated NF-kappaB activation has not been reported yet. We first report that STP-C interacts with TRAF6 protein in vivo and in vitro and further investigation shows that Glu(12) residue of STP-C is critical for binding to TRAF6. Introduction of ubiquitin together with STP-C augments NF-kappaB activity compared to that of STP-C expression alone. STP-C expression further induces ubiquitination of endogenous TRAF6. In addition, either a deubiquitination enzyme, CYLD or a dominant negative E2-conjugation enzyme reduced NF-kappaB activity in spite of the presence of STP-C, supporting that the interaction between STP-C and TRAF6 induces ubiquitination of TRAF6. NF-kappaB activation by STP-C through the ubiquitinated TRAF6 causes the increased production of IL-8, an inflammatory chemokine and the enhanced expression of costimulatory molecule ICAM, which might ultimately contribute cellular transformation by the exposure of HVS-infected cells with inflammatory microenvironment and chronic activation.

STP-A11, an oncoprotein of Herpesvirus saimiri augments both NF-kappaB and AP-1 transcription activity through TRAF6.

Herpesvirus saimiri (HVS), a member of the gamma-herpesvirus family, encodes an oncoprotein called Saimiri Transforming Protein (STP) which is required for lymphoma induction in non-human primates. However, a detailed mechanism of STP-A11-induced oncogenesis has not been revealed yet. We first report that STP-A11 oncoprotein interacts with TNF-alpha receptor-associated factor (TRAF) 6 in vivo and in vitro. Mutagenesis analysis of the TRAF6-binding motif (10)PQENDE(15) in STP-A11 reveals that Glu (E)(12) residue is critical for binding to TRAF6 and NF-kappaB activation. Interestingly, co-expression of E12A mutant, lack of TRAF6 binding, with cellular Src (Src) results in decreased transcriptional activity of Stat3 and AP-1, a novel target of STP-A11 compared to that of wild type. Furthermore, the presence of STP-A11 enhances the association of TRAF6 with Src and induces the translocation of both TRAF6 and Src to a nonionic detergent-insoluble fraction. Taken together, these studies suggest that STP-A11 oncoprotein up-regulates both NF-kappaB and AP-1 transcription activity through TRAF6, which would ultimately contribute cellular transformation.

Activation of non-canonical NF-kappaB pathway mediated by STP-A11, an oncoprotein of Herpesvirus saimiri.

Although Saimiri Transforming Protein (STP)-A11, an oncoprotein of Herpesvirus saimiri, has been known to activate NF-kappaB signaling pathway, the detailed mechanism has not been reported yet. We herein report that STP-A11 activates non-canonical NF-kappaB pathway, resulting in p100 processing to p52. In addition, translocation of p52 protein (NF-kappaB2) into the nucleus is observed by the expression of STP-A11. STP-A11-mediated processing of p100 to p52 protein requires proteosome-mediated proteolysis because MG132 treatment clearly blocked p52 production in spite of the expression of STP-A11. Analysis of STP-A11 mutants to activate NF-kappaB2 pathway discloses the requirement of TRAF6-binding site not Src-binding site for STP-A11-mediated NF-kappaB2 pathway. Blockage of STP-A11-mediated p52 production using siRNA against p52 enhanced a chemotherapeutic drug-mediated cell death, suggesting that p52 production induced by the expression of STP-A11 would contribute to cellular transformation, which results from a resistance to cell death.

Serially passaged human nasal epithelial cell monolayer for in vitro drug transport studies.

PURPOSE: To evaluate the feasibility of using a serially passaged culture of human nasal epithelial cell monolayers on a permeable support for in vitro drug transport studies. The optimum conditions for passaged culture as well as the correlation between the transepithelial electrical resistance (TEER) value and drug permeability (Papp) were evaluated. METHODS: Fresh human nasal epithelial cells were collected from normal inferior turbinates and were subcultured repeatedly in serum-free bronchial epithelial cell growth media (BEGM) in petri dishes. The subcultured cells of each passage were seeded onto permeable supports at 5 x 10(5) cells/cm2 and grown in Dulbecco's modified Eagle medium (DMEM). Morphologic characteristics were observed by scanning electron microscopy (SEM). To verify the formation of tight junctions, actin staining and transmission electron microscopy (TEM) studies were conducted. In the drug transport study, [14C]mannitol and budesonide were selected as the paracellular and the transcellular route markers, respectively. RESULTS: Serially passaged cells were successfully cultured on a permeable support and showed significantly high TEER values up to passage 4. After 14 days of seeding, SEM showed microvilli, and protrusions of cilia and mucin granules were detected by TEM. The paracellular marker [14C]mannitol showed a nearly constant permeability coefficient (Papp) when the TEER value exceeded 500 omega x cm2 regardless of the passage number. However, as expected, budesonide showed a higher permeability coefficient compared to [14C]mannitol and was less affected by the TEER value. CONCLUSIONS: Human nasal epithelial cell monolayers were successfully subcultured on a permeable support up to passage 4. These cell culture methods may be useful in high-throughput screening of in vitro nasal transport studies of various drugs.