Biomedical subjects

S C Shen

Publications and source records attributed to S C Shen.

At least 19 recordsLinked to original sources

PubMed2006

Lipopolysaccharide plus 12-o-tetradecanoylphorbol 13-acetate induction of migration and invasion of glioma cells in vitro and in vivo: Differential inhibitory effects of flavonoids.

In an earlier study, we reported that nitric oxide is involved in lipopolysaccharide plus 12-o-tetradecanoylphorbol 13-acetate-induced malignant transformation via increases in metalloproteinase 9 enzyme activity and inducible nitric oxide synthase gene expression in rat glioma C6 cells, however the mechanism has remained undefined. Lipopolysaccharide plus 12-o-tetradecanoylphorbol 13-acetate, but not lipopolysaccharide or 12-o-tetradecanoylphorbol 13-acetate alone, induced transformation in glioma C6 cells (but not in human glioblastoma cells GBM-8401 cells) without affecting their viability. An increase in inducible nitric oxide synthase protein expression, nitric oxide production, and metalloproteinase 9 enzyme activity is identified lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate-treated C6 cells, however lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate and 12-o-tetradecanoylphorbol 13-acetate (but not lipopolysaccharide) addition shows the similar inductive pattern on metalloproteinase 9 enzyme activity without affecting inducible nitric oxide synthase protein expression and nitric oxide production in GBM-8401 cells. Treatment of C6 cells with lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate increases the expression of phosphorylated extracellular regulated protein kinases and Jun N-terminal kinases, but not p38, proteins, and an addition of the extracellular regulated protein kinases inhibitor PD98059 or Jun N-terminal kinases inhibitors SP600125, but not the p38 inhibitor SB203580, significantly blocked lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate-induced inducible nitric oxide synthase protein expression and metalloproteinase 9 enzyme activity accompanied by blocking morphological transformation in C6 cells. Among 19 structurally related flavonoids, kaempferol and wogonin exhibit significant inhibitory effects on lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate-induced morphological transformation and colony formation, and attenuation of inducible nitric oxide synthase, phosphorylated extracellular regulated protein kinases protein expression, and metalloproteinase 9 enzyme activity was observed. 2'-OH flavone at a dose of 100 microM inhibition of lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate-induced events via apoptosis induction is identified. Furthermore, lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate, but not lipopolysaccharide or 12-o-tetradecanoylphorbol 13-acetate, induces tumoral invasion and migration in vitro and in vivo, and those are blocked by kaempferol and wogonin addition. These data suggest that combination of lipopolysaccharide and 12-o-tetradecanoylphorbol 13-acetate promotes tumoral progression via activating metalloproteinase 9 enzyme activity and inducible nitric oxide synthase gene expression, which is located downstream of mitogen-activated protein kinases activation, in rat glioma cells C6. Kaempferol and wogonin exhibit effective inhibitory effects on lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate-induced events, and thus possess the potential for further development.

Animals↗

PubMed2004

CO2 adsorption over Si-MCM-41 materials having basic sites created by postmodification with La2O3.

Moderate basic sites could be created onto mesoporous Si-MCM-41 materials by postsynthesis modification with highly dispersed La2O3. The La2O3-modified MCM-41 materials (designated here as LaM) have been characterized by Fourier transform infrared spectroscopy, temperature-programmed desorption, X-ray photoelectron spectroscopy, X-ray diffraction (XRD), and N2 adsorption/desorption and have been tested as model adsorbents for CO2 adsorption. XRD and N2 adsorption results showed that all LaM materials still maintained their uniform hexagonal mesoporous structure even after postsynthesis modification with La2O3 loading up to 20 wt %. Although the surface area, pore size, and pore volume of LaM materials decreased with increasing La2O3 loading, their capacity for CO2 storage could be significantly improved when La2O3 loading was increased from 0 to 10 wt %. Unidentate and bidentate carbonates have been identified by in situ FTIR as the two types of CO2 species adsorbed on LaM surface. The LaM material also possesses good thermal stability, allowing the model adsorbent to be regenerated at high temperature and recyclable.

Adsorption↗

PubMed2001

Transdermal drug delivery enhanced and controlled by erbium:YAG laser: a comparative study of lipophilic and hydrophilic drugs.

The influence of an erbium:YAG laser on the transdermal delivery of drugs across skin was studied in vitro. Indomethacin and nalbuphine, which have the same molecular weight, were selected as model lipophilic and hydrophilic drugs, respectively, to compare skin permeation by laser treatment. The results indicate a significant increase in the permeation of indomethacin and nalbuphine across skin pretreated with an erbium:YAG laser. The laser had a greater effect on the permeation of hydrophilic molecules which usually possess low permeability. The laser intensity and its spot size were found to play an important role in controlling transdermal delivery of drugs. Permeation of the hydrophilic drug increased following an increase of laser energy. On the other hand, a different result was observed for the lipophilic drug transported across laser-treated skin. The stratum corneum (SC) layer in skin could be partly ablated by the erbium:YAG laser. The barrier function of the SC may also be modulated by a lower intensity of the laser without affecting the viability and structure of the epidermis/dermis as determined by histological observations. However, ultrastructural alteration of the epidermis/dermis may be caused by laser treatment. Use of an erbium:YAG laser is a good method for enhancing transdermal absorption of both lipophilic and hydrophilic drugs, because it allows precise control of SC removal, and this ablation of SC can be reversible to the original normal status.

Administration, Cutaneous↗

PubMed2001

Wogonin, baicalin, and baicalein inhibition of inducible nitric oxide synthase and cyclooxygenase-2 gene expressions induced by nitric oxide synthase inhibitors and lipopolysaccharide.

We previously reported that oroxylin A, a polyphenolic compound, was a potent inhibitor of lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In the present study, three oroxylin A structurally related polyphenols isolated from the Chinese herb Huang Qui, namely baicalin, baicalein, and wogonin, were examined for their effects on LPS-induced nitric oxide (NO) production and iNOS and COX-2 gene expressions in RAW 264.7 macrophages. The results indicated that these three polyphenolic compounds inhibited LPS-induced NO production in a concentration-dependent manner without a notable cytotoxic effect on these cells. The decrease in NO production was in parallel with the inhibition by these polyphenolic compounds of LPS-induced iNOS gene expression. However, these three compounds did not directly affect iNOS enzyme activity. In addition, wogonin, but not baicalin or baicalein, inhibited LPS-induced prostaglandin E2 (PGE2) production and COX-2 gene expression without affecting COX-2 enzyme activity. Furthermore, N-nitro-L-arginine (NLA) and N-nitro-L-arginine methyl ester (L-NAME) pretreatment enhanced LPS-induced iNOS (but not COX-2) protein expression, which was inhibited by these three polyphenolic compounds. Wogonin, but not baicalin or baicalein, similarly inhibited PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS-co-treated RAW 264.7 cells. These results indicated that co-treatment with NOS inhibitors and polyphenolic compounds such as wogonin effectively blocks acute production of NO and, at the same time, inhibits expression of iNOS and COX-2 genes.

Animals↗

PubMed2001

Overexpression of interleukin-6 in human basal cell carcinoma cell lines increases anti-apoptotic activity and tumorigenic potency.

Interleukin-6 (IL-6) is a pleiotropic cytokine that is capable of modulating the diverse functions of cells such as acute phase responses and inflammation. Excessive or insufficient production of IL-6 may contribute to certain diseases of the skin. The aim of this study was to investigate the possible role of IL-6 in the tumorigenesis of basal cell carcinoma (BCC). Initially, we transfected IL-6 expression vector, under the control of a CMV promoter, into human BCC cells and successfully obtained IL-6-overexpressing clones (BCC/IL-6-c1 and BCC/IL-6-c2) and a mixture (BCC/IL-6). DNA synthesis assay determined using (3)H-thymidine pulse incorporation revealed that IL-6-expressing BCC cells exhibited a much higher DNA synthesis rate than the neo control or parental BCC cells. We also detected a greater abundance of IL-6-expressing cell colonies formed in soft agar than in the vector control cells. Furthermore, BCC/IL-6 cells, but not vector control cells, were resistant to UV and photodynamic therapy (PDT)-induced apoptosis, as confirmed using DNA fragmentation and morphologic change analyses. Immunoblot analysis showed that Mcl-1, an anti-apoptotic protein, was specifically up-regulated IL-6 transfectants but not in the control cells. Transient transfection of IL-6 transfectants with antisense mcl-1 greatly enhanced their apoptosis frequency by UV treatment. In tumorigenesis assay, IL-6 transfected clones formed tumors in nude mice more rapidly than the control cells. These tumors appeared to be highly vascularized using pathological examination. Supportive of this finding, we found that IL-6 transfected cells expressed elevated levels of two angiogenic factors, cyclooxygenase (Cox)-2 and vascular endothelial growth factor (VEGF). These results suggest that overexpression of IL-6 enhances the tumorigenic activity of BCC cells by both suppressing apoptosis and actively promoting angiogenesis.

Aminolevulinic Acid↗

PubMed2001

Inhibition of nitric oxide synthase inhibitors and lipopolysaccharide induced inducible NOS and cyclooxygenase-2 gene expressions by rutin, quercetin, and quercetin pentaacetate in RAW 264.7 macrophages.

Several natural flavonoids have been demonstrated to perform some beneficial biological activities, however, higher-effective concentrations and poor-absorptive efficacy in body of flavonoids blocked their practical applications. In the present study, we provided evidences to demonstrate that flavonoids rutin, quercetin, and its acetylated product quercetin pentaacetate were able to be used with nitric oxide synthase (NOS) inhibitors (N-nitro-L-arginine (NLA) or N-nitro-L-arginine methyl ester (L-NAME)) in treatment of lipopolysaccharide (LPS) induced nitric oxide (NO) and prostaglandin E2 (PGE2) productions, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expressions in a mouse macrophage cell line (RAW 264.7). The results showed that rutin, quercetin, and quercetin pentaacetate-inhibited LPS-induced NO production in a concentration-dependent manner without obvious cytotoxic effect on cells by MTT assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide as an indicator. Decrease of NO production by flavonoids was consistent with the inhibition on LPS-induced iNOS gene expression by western blotting. However, these compounds were unable to block iNOS enzyme activity by direct and indirect measurement on iNOS enzyme activity. Quercetin pentaacetate showed the obvious inhibition on LPS-induced PGE2 production and COX-2 gene expression and the inhibition was not result of suppression on COX-2 enzyme activity. Previous study demonstrated that decrease of NO production by L-arginine analogs effectively stimulated LPS-induced iNOS gene expression, and proposed that stimulatory effects on iNOS protein by NOS inhibitors might be harmful in treating sepsis. In this study, NLA or L-NAME treatment stimulated significantly on LPS-induced iNOS (but not COX-2) protein in RAW 264.7 cells which was inhibited by these three compounds. Quercetin pentaacetate, but not quercetin and rutin, showed the strong inhibitory activity on PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS co-treated RAW 264.7 cells. These results indicated that combinatorial treatment of L-arginine analogs and flavonoid derivates, such as quercetin pentaacetate, effectively inhibited LPS-induced NO and PGE2 productions, at the same time, inhibited enhanced expressions of iNOS and COX-2 genes.

Animals↗

PubMed2001

Alternative activation of extracellular signal-regulated protein kinases in curcumin and arsenite-induced HSP70 gene expression in human colorectal carcinoma cells.

We have investigated the regulation mechanism of chemical stress-induced HSP70 gene expression in human colorectal carcinoma cells (COLO205 and HT29). Our data show that chemical treatments including sodium arsenite and curcumin, induced significant synthesis of HSP70 and its mRNA. The induced HSP70 gene expression appears to be increased at the transcriptional level. The increase in HSP70 gene expression by both chemicals is associated with an increase in HSF binding to HSE and induction of HSF1 di- or trimerization. Phosphorylation and activation of extracellular signal-regulated proteins (ERK1/2) were detected in sodium arsenite-treated COLO205 and HT29 cells, and the free radical scavenger N-acetyl-L-cysteine (NAC) was able to inhibit this ERK1/2 activation and HSP70 gene expression. MAPK blockade by the specific MEK1 inhibitor (PD98059) decreased the ability of sodium arsenite to increase HSP70 gene expression in a dose-dependent manner along with dephosphorylation of ERK1/2 proteins. In contrast to arsenite treatment, activation of ERK1/2 was not detected in curcumin-treated colorectal carcinoma cells, and NAC and PD98059 did not show any inhibitory effect on HSP70 gene expression induced by curcumin. Overexpression of a dominant negative mutant of mitogen-activated protein kinase kinase kinase 1 (MEKK1-DN) prevents arsenite-induced ERK1/2 phosphorylation and HSP70 protein synthesis. These results indicated that the ERK signaling pathway can participate in HSP70 gene expression induced by the prooxidant sodium arsenite, but not by the antioxidant curcumin.

Acetylcysteine↗

PubMed2001

Application of selective implantation in Al0.5Ga0.5As/In0.25Ga0.75As/GaAs pseudomorphic single quantum wire structures.

A pseudomorphic Al0.5Ga0.5As/In0.25Ga0.75As/GaAs asymmetric quantum wire (QWR) structure was grown on GaAs V-grooved substrate by low pressure metal organic vapor phase epitaxy. The formation of crescent shaped QWRs at the bottom of the V-grooves was confirmed by both transmission electron microscopy and photoluminescence (PL) spectra. The temperature dependence of PL spectra demonstrated a fast decrease of the sidewall quantum well PL intensity with increasing temperature, which originates from relaxation of carriers from well to wire region. The self-aligned dual implantation technique was successfully used to selectively disable the adjacent quantum structures. Decrease of the PL intensity of QWR at 8 K was observed after selective implantation, which resulted from a decreased number of carriers relaxed from adjacent quantum structures.

Aluminum↗

PubMed2000

Facilitated wound healing by activation of the Transglutaminase 1 gene.

Transglutaminase 1 (TGase 1) is a Ca(2+)-dependent enzyme which catalyzes epsilon-(gamma-glutamyl)lysine cross-linking of substrate proteins such as involucrin and loricrin to generate the cornified envelope at the cell periphery of the stratum corneum. We have shown that disruption of the TGase 1 gene in mice results in neonatal lethality, absence of the cornified envelope, and impaired skin barrier function. Based on the importance of TGase 1 in epidermal morphogenesis, we have now assessed its role in wound healing. In neonatal mouse skin, TGase 1 mRNA as well as keratin 6alpha was induced in the epidermis at the wound edges as early as 2 hours after injury and that expression continued in the migrating epidermis until completion of re-epithelialization. The TGase 1 enzyme co-localized on the plasma membrane of migrating keratinocytes with involucrin, but not with loricrin, which suggests the premature assembly of the cornified envelope. Similar injuries to TGase 1 knockout mouse skins grafted on athymic nude mice showed substantial delays in wound healing concomitant with sustained K6alpha mRNA induction. From these results, we suggest that activation of the TGase 1gene is essential for facilitated repair of skin injury.

Animals↗

PubMed2000

A promoter region binding protein and DNA gyrase regulate anaerobic transcription of nifLA in Enterobacter cloacae.

Our work provides evidence that a sequence characteristic of FNR binding sites, when interacted with by a trans-acting factor, activates anaerobic transcription of the nifLA operon in Enterobacter cloacae. DNA gyrase activity has been found to be important for the anaerobic transcription of the nifLA promoter. Our results suggest that anaerobic regulation of the nifLA operon is mediated through the control of the promoter region-binding trans-acting factor at the transcriptional level, while DNA supercoiling functions in providing a topological requirement for the activation of transcription.

Anaerobiosis↗

PubMed2000

[Determination of inositol by chromatography].

Inositol was acetylated to inositol hexacetate and determined by GC. The content of inositol was got. The gas chromatographic conditions were as follows: 2 m x 3 mm i.d. column packed with 10% SE-30/201 Diatoport PAW(60-80 mesh) was kept at 240 degrees C. The temperature of both injector and FID was 260 degrees C. The flowrate of N2 carrier gas was 40 mL/min. The sample was analyzed by this method with a coefficient of variation of 0.15% and a recovery of 98.2%-104.3%.

Chromatography, Gas↗

PubMed2000

[Determination of residual acetone in soybean phospholipids by headspace gas chromatography].

Soybean phospholipids have many functions and alimentary actions. In our country, powder soybean phospholipids are generally got by extraction with acetone, followed by vacuum drying. There may be some residual acetone present in the soybean phospholipids, which is harmful to health. So, we must know residual acetone content in the soybean phospholipids. However we have not found a method to determine the residual acetone in the soybean phospholipids. In this paper, headspace GC was used to determine residual acetone in powder soybean phospholipids. The headspace bottle was glass with a volume of 15 milliliters. Certain amounts of water, ammonium sulfate, and sample were added into the bottle. The mixture was made into a brei as soon as possible. The bottle was put into a water bath at 40 degrees C for an hour. The GC column was a 2 m x 3 mm i.d. stainless steel tube packed with GDX-103 stationary phase. Temperatures of both injector and detector were kept at 120 degrees C. Column temperature was 160 degrees C. Injection volume was 1 mL. External standard method was used for quantitation. The RSD was 1.2%. The recoveries in the range of 25.0 micrograms/g-100 micrograms/g were 98.4%-104%.

Acetone↗

PubMed1999

Transglutaminase type 1 and its cross-linking activity are concentrated at adherens junctions in simple epithelial cells.

Transglutaminase type 1 was identified as a tyrosine-phosphorylated protein from the isolated junctional fraction of the mouse liver. This enzyme was reported to be involved in the covalent cross-linking of proteins in keratinocytes, but its expression and activity in other cell types have not been examined. Northern blotting revealed that transglutaminase type 1 was expressed in large amounts in epithelial tissues (lung, liver, and kidney), which was also confirmed by immunoblotting with antibodies raised against mouse recombinant protein. Immunoblotting of the isolated junctional fraction revealed that transglutaminase type 1 was concentrated in the fraction not only as a 97-kDa form but also as forms of various molecular masses cross-linked to other proteins. In agreement with this finding, endogenous transglutaminase type 1 was immunofluorescently colocalized with E-cadherin in cultured simple epithelial cells. In the liver and kidney, immunoelectron microscopy revealed that transglutaminase type 1 was concentrated, albeit not exclusively, at cadherin-based adherens junctions. Furthermore, by in vitro and in vivo labeling, transglutaminase cross-linking activity was also shown to be concentrated at intercellular junctions of simple epithelial cells. These findings suggested that the formation of covalently cross-linked multimolecular complexes by transglutaminase type 1 is an important mechanism for maintenance of the structural integrity of simple epithelial cells, especially at cadherin-based adherens junctions.

3T3 Cells↗

PubMed1999

Activation of c-Jun NH2-terminal kinase and subsequent CPP32/Yama during topoisomerase inhibitor beta-lapachone-induced apoptosis through an oxidation-dependent pathway.

Beta-lapachone (beta-Lap) has been found to inhibit DNA topoisomerases (Topos) by a mechanism distinct from that of other commonly known Topo inhibitors. Here, we demonstrated a pronounced elevation of H2O2 and O2- in human leukemia HL-60 cells treated with beta-Lap. Treatment with other Topo poisons, such as camptothecin (CPT), Vbeta-16, and GL331, did not have the same effect. On the other hand, antioxidant vitamin C (Vit C) treatment effectively antagonized beta-Lap-induced apoptosis. This suggested that a reactive oxygen species (ROS)-related pathway was involved in beta-Lap-induced apoptosis program. We also found that c-Jun NH2-terminal kinase (JNK) but not p38 mitogen-activated protein kinase or extracellular signal-regulated kinase 1/2 was persistently activated in apoptosis induced by beta-Lap. Overexpression of a dominant-negative mutant mitogen-activated protein kinase kinase kinase 1 (MEKK1-DN) or treatment with JNK-specific antisense oligonucleotide or Vit C all prevented beta-Lap-induced JNK activation and the subsequent apoptosis. Only the expression of MEKK1-DN, not Vit C treatment, blocked the JNK activity induced by CPT, Vbeta-16, or GL331. These results confirm again that ROS acts as a mediator for JNK activation during beta-Lap-induced apoptosis. Furthermore, we found that beta-Lap can stimulate CPP32/Yama activity, which was, however, markedly inhibited by the MEKK1-DN expression or Vit C treatment. Again, CPT-induced CPP32/Yama activation can be abolished by MEKK1-DN but not by Vit C treatment. Taken together, these results indicate that beta-Lap but not other Topo inhibitors triggers apoptosis signaling, i.e., JNK and subsequent CPP32/Yama activation are mediated by the generation of ROS.

Apoptosis↗

PubMed1998

Bcl-2 prevents topoisomerase II inhibitor GL331-induced apoptosis is mediated by down-regulation of poly(ADP-ribose)polymerase activity.

Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme responsible for DNA strand breaks, has been recently suggested to be crucial for apoptosis induced by a number chemotherapeutic drugs. In this study, we demonstrated that the PARP activity could be evidently elevated with a peak at 6 h when HL-60 cells were treated with a new anticancer drug GL331. Coincident with the peak of PARP activity, an apparent DNA fragmentation and apoptotic morphology were observed in cells treated with GL331. The subsequent apoptotic DNA fragmentation induced by GL331 could be completely blocked by transfecting cells with anti-sense PARP retroviral vector or by treating cells with PARP inhibitor, 3-aminobenzamide (3-AB). This blocking effect thus suggests that activation of PARP was critically involved in GL331-induced apoptosis. The fact that Bcl-2 has been found to antagonize cell death induced by a wide variety of agents, accounts for why we examined whether if Bcl-2 could antagonize GL331 effects. Interestingly, ectopic overexpression of Bcl-2 in either HL-60 or U937 cells caused in resistance towards GL331-elicited DNA fragmentation and cytotoxic effect. Additionally, Bcl-2 also attenuated the poly(ADP-ribosyl)ation of PARP itself as well as Histone H1 at the early period of drug treatment. However, Bcl-2 did not influence the extent of DNA strand breaks induced by GL331 in either control or Bcl-2-overexpressing cells. In addition, analysis of basal PARP activity in control and several Bcl-2 overexpressing clones revealed that Bcl-2 down-regulated PARP activity under the condition without DNA damages. Above findings suggest that poly(ADP-ribosyl)ation of nuclear targets is important for apoptosis induced by DNA-reactive anticancer drugs.

Antineoplastic Agents↗

PubMed1998

Curcumin induces a p53-dependent apoptosis in human basal cell carcinoma cells.

Curcumin, a potent antioxidant and chemopreventive agent, has recently been found to be capable of inducing apoptosis in human hepatoma and leukemia cells by way of an elusive mechanism. Here, we demonstrate that curcumin also induces apoptosis in human basal cell carcinoma cells in a dose- and time-dependent manner, as evidenced by internucleosomal DNA fragmentation and morphologic change. In our study, consistent with the occurrence of DNA fragmentation, nuclear p53 protein initially increased at 12 h and peaked at 48 h after curcumin treatment. Prior treatment of cells with cycloheximide or actinomycin D abolished the p53 increase and apoptosis induced by curcumin, suggesting that either de novo p53 protein synthesis or some proteins synthesis for stabilization of p53 is required for apoptosis. In electrophoretic mobility gel-shift assays, nuclear extracts of cells treated with curcumin displayed distinct patterns of binding between p53 and its consensus binding site. Supportive of these findings, p53 downstream targets, including p21(CIP1/WAF1) and Gadd45, could be induced to localize on the nucleus by curcumin with similar p53 kinetics. Moreover, we immunoprecipitated extracts from basal cell carcinoma cells with different anti-p53 antibodies, which are known to be specific for wild-type or mutant p53 protein. The results reveal that basal cell carcinoma cells contain exclusively wild-type p53; however, curcumin treatment did not interfere with cell cycling. Similarly, the apoptosis suppressor Bcl-2 and promoter Bax were not changed with the curcumin treatment. Finally, treatment of cells with p53 antisense oligonucleotide could effectively prevent curcumin-induced intracellular p53 protein increase and apoptosis, but sense p53 oligonucleotide could not. Thus, our data suggest that the p53-associated signaling pathway is critically involved in curcumin-mediated apoptotic cell death. This evidence also suggests that curcumin may be a potent agent for skin cancer prevention or therapy.

Apoptosis↗

PubMed1998

Isolation and characterization of tilapia (Oreochromis mossambicus) insulin-like growth factors gene and proximal promoter region.

To understand the molecular mechanism which controls the transcription of the insulin-like growth factors (IGFs) gene, we have cloned and sequenced the cDNA for the proximal promoter region of the tilapia IGFs gene and have characterized its activity by chloramphenicol acetyltransferase (CAT) transient transfected expression assays. Tilapia (Oreochromis mossambicus) IGF-I cDNA (549 bp) was amplified by PCR from single-stranded cDNA of growth hormone (GH)-induced liver RNA using a pair of oligonucleotides specific for fish IGF-I as amplification primers. Tilapia IGF-I and IGF-II 5' termini were analyzed by rapid amplification of cDNA 5' ends (5'RACE). Analysis of the 5'RACE results revealed two transcription start sites in IGF-I and one transcription start site in IGF-II. Different fragments of the 5' flanking region were transfected into human lung adenocarcinoma cells. In the cell line, maximum promoter activity was located in the distal 657 basepairs of the IGF-I 5' flanking region and in the distal 450 basepairs of the IGF-II 5' flanking region. The in vivo actions of the IGFs promoter on developmental stage expression were investigated further in transgenic zebrafish in which an IGFs promoter-driven green fluorescent protein (GFP) encoding the cDNA transgene was microinjected into embryos. Morphologic and RT-PCR studies of the transgenic zebrafish indicated that IGF-I promoter-driven GFP transcripts appeared for the first time in the 1-K-cell stage and the IGF-II promoter-driven GFP transcripts appeared for the first time in the 32-cell stage. Fluorescent (GFP) distribution was apparent within 48 h in IGF-II-transgenic zebrafish embryos, especially in eye, muscle, corpuscle, floor plate, horizontal myoseptum, yolk sac extension, and yolk sac. These results indicate that the IGF-I and IGF-II promoters are active in tissue and in a development-specific manner. Our findings also indicate that the IGF-II promoter influences the growth of fish embryos earlier than does IGF-I, and IGF-II has higher levels of expression than does IGF-I. These results suggest that the IGF-II promoter plays a growth factor role in teleost embryo development.

Amino Acid Sequence↗

PubMed1998

Antiapoptotic but not antiviral function of human bcl-2 assists establishment of Japanese encephalitis virus persistence in cultured cells.

Upon infection of Japanese encephalitis virus (JEV), baby hamster kidney (BHK-21) and Chinese hamster ovary (CHO) cells were killed by a mechanism involved in apoptosis. While readily established in a variety of cell lines, JEV persistence has never been successfully instituted in BHK-21 and CHO cells. Since stable expression of human bcl-2 in BHK-21 cells has been shown to delay JEV-induced apoptosis, in this study we investigated whether JEV persistence could be established in such cells. When constitutively expressing bcl-2, but not its closest homolog, bcl-XL, following a primary lytic infection, approximately 5 to 10% of BHK-21 and CHO cells became persistently JEV infected during a long-term culture. From the persistent bulks, several independent clones were selected and expanded to form stable cell lines that continuously produced infectious virus without marked cytopathic effects (CPE). Among these stable cell lines, the truncated nonstructural protein 1 (NS1) was also detected and was indistinguishable from the NS1 truncations previously observed in JEV-persistent murine neuroblastoma N18 cells. However, the stable expression of NS1 alone, regardless of whether it was truncated or full length, failed to render the engineered cells persistently infected by JEV, implying that aberrant NS1 proteins were likely a consequence of, rather than a cause for, the viral persistence. Enforced bcl-2 expression, which did not affect virus replication and spread during the early phase of cytolytic infection, appeared to attain JEV persistence by restriction of virus-induced CPE. Our results suggest that it is the antiapoptotic, rather than the antiviral, effect of cellular bcl-2 which plays a role in the establishment of JEV persistence.

Amino Acid Sequence↗