Combined protective effects of icariin and selenomethionine on novel chronic tubulointerstitial nephropathy models in vivo and in vitro.

Chronic tubulointerstitial nephropathy (CTIN) is one of the most common kidney diseases. However, treatment for CTIN has multiple limits. Adjuvant therapy through nutritional regulation has become a hot research topic at present. Icariin (ICA), an extraction of Chinese herbal medicine epimedium, has many pharmacological functions including anti-inflammation and tonifying kidney. Selenomethionine (SeMet) possesses the effects of antioxidant and lightening nephrotoxicity. However, little is known about the combined nephroprotection of them. This study was investigated to evaluate the joint effects of ICA and SeMet on CTIN and explore the mechanism. Based on a novel CTIN model developed in our previous study, mice were randomly divided into five groups (a: control; b: model; c: model + ICA; d: model + SeMet; e: model + ICA + SeMet). Renal tubule epithelial cells were treated with cyclosporine A and ochratoxin A without/with ICA or/and SeMet. The results showed that ICA or/and SeMet ameliorated CTIN by inhibiting the uptrends of blood urine nitrogen, serum creatinine, urine protein, urine gravity, histopathological damage degree and collagen I deposition. ICA or/and SeMet also increased cell proliferation and decreased apoptosis and the expression of transforming growth factor-beta 1 and α-smooth muscle actin. Emphatically, ICA and SeMet joint had better nephroprotection than alone in most indexes including fibrosis. Furthermore, ICA and SeMet joint decreased the activation of toll-like receptor 4 (TLR4)/NFκB pathway induced by CTIN. TLR4 overexpression counteracted the joint protection of ICA and SeMet. Therefore, ICA and SeMet in combination could protect against CTIN through blocking TLR4/NFκB pathway. The study will provide novel insights to explore an adjuvant therapeutic orientation.

An ATF24 peptide-functionalized ß-elemene-nanostructured lipid carrier combined with cisplatin for bladder cancer treatment.

Objective: In this study, we aimed to develop an amino-terminal fragment (ATF) peptide-targeted liposome carrying ß-elemene (ATF24-PEG-Lipo-ß-E) for targeted delivery into urokinase plasminogen activator receptor-overexpressing bladder cancer cells combined with cisplatin (DDP) for bladder cancer treatment. Methods: The liposomes were prepared by ethanol injection and high-pressure microjet homogenization. The liposomes were characterized, and the drug content, entrapment efficiency, and in vitro release were studied. The targeting efficiency was investigated using confocal microscopy, ultra-fast liquid chromatography, and an orthotopic bladder cancer model. The effects of ATF24-PEG-Lipo-ß-E combined with DDP on cell viability and proliferation were evaluated by a Cell Counting Kit-8 (CCK-8) assay, a colony formation assay, and cell apoptosis and cell cycle analyses. The anticancer effects were evaluated in a KU-19-19 bladder cancer xenograft model. Results: ATF24-PEG-Lipo-ß-E had small and uniform sizes (˜79 nm), high drug loading capacity (˜5.24 mg/mL), high entrapment efficiency (98.37 ± 0.95%), and exhibited sustained drug release behavior. ATF24-PEG-Lipo-ß-E had better targeting efficiency and higher cytotoxicity than polyethylene glycol (PEG)ylated ß-elemene liposomes (PEG-Lipo-ß-E). DDP, combined with ATF24-PEG-Lipo-ß-E, exerted a synergistic effect on cellular apoptosis and cell arrest at the G2/M phase, and these effects were dependent on the caspase-dependent pathway and Cdc25C/Cdc2/cyclin B1 pathways. Furthermore, the in vivo antitumor activity showed that the targeted liposomes effectively inhibited the growth of tumors, using the combined strategy. Conclusions: The present study provided an effective strategy for the targeted delivery of ß-elemene (ß-E) to bladder cancer, and a combined strategy for bladder cancer treatment.

Erianin, a novel dibenzyl compound in Dendrobium extract, inhibits lung cancer cell growth and migration via calcium/calmodulin-dependent ferroptosis.

Ferroptosis, a novel form of programmed cell death, is characterized by iron-dependent lipid peroxidation and has been shown to be involved in multiple diseases, including cancer. Stimulating ferroptosis in cancer cells may be a potential strategy for cancer therapy. Therefore, ferroptosis-inducing drugs are attracting more attention for cancer treatment. Here, we showed that erianin, a natural product isolated from Dendrobium chrysotoxum Lindl, exerted its anticancer activity by inducing cell death and inhibiting cell migration in lung cancer cells. Subsequently, we demonstrated for the first time that erianin induced ferroptotic cell death in lung cancer cells, which was accompanied by ROS accumulation, lipid peroxidation, and GSH depletion. The ferroptosis inhibitors Fer-1 and Lip-1 but not Z-VAD-FMK, CQ, or necrostatin-1 rescued erianin-induced cell death, indicating that ferroptosis contributed to erianin-induced cell death. Furthermore, we demonstrated that Ca2+/CaM signaling was a critical mediator of erianin-induced ferroptosis and that blockade of this signaling significantly rescued cell death induced by erianin treatment by suppressing ferroptosis. Taken together, our data suggest that the natural product erianin exerts its anticancer effects by inducing Ca2+/CaM-dependent ferroptosis and inhibiting cell migration, and erianin will hopefully serve as a prospective compound for lung cancer treatment.

Combinative treatment of ß-elemene and cetuximab is sensitive to KRAS mutant colorectal cancer cells by inducing ferroptosis and inhibiting epithelial-mesenchymal transformation.

Background and Purpose: RAS mutations limit the effectiveness of anti-epidermal growth factor receptor (EGFR) monoclonal antibodies in combination with chemotherapy for metastatic colorectal cancer (mCRC) patients. Therefore, new cell death forms have focused on identifying indirect targets to inhibit Ras-induced oncogenesis. Recently, emerging evidence has shown the potential of triggering ferroptosis for cancer therapy, particularly for eradicating aggressive malignancies that are resistant to traditional therapies. Methods: KRAS mutant CRC cell HCT116 and Lovo were treated with cetuximab and ß-elemene, a bioactive compound isolated from Chinese herb Curcumae Rhizoma. Ferroptosis and epithelial-mesenchymal transformation (EMT) were detected in vitro and in vivo. Orthotopic CRC animal model were established and the tumor growth was monitored by IVIS bioluminescence imaging. Tumor tissues were collected to determine ferroptosis effect and the expression of EMT markers after the treatment. Results: CCK-8 assay showed that synergetic effect was obtained when 125 µg/ml ß-elemene was combined with 25 µg/ml cetuximab in KRAS mutant CRC cells. AV/PI staining suggested a non-apoptotic mode of cell death after the treatment with ß-elemene and cetuximab. In vitro, ß-elemene in combination with cetuximab was shown to induce iron-dependent reactive oxygen species (ROS) accumulation, glutathione (GSH) depletion, lipid peroxidation, upregulation of HO-1 and transferrin, and downregulation of negative regulatory proteins for ferroptosis (GPX4, SLC7A11, FTH1, glutaminase, and SLC40A1) in KRAS mutant CRC cells. Meanwhile, combinative treatment of ß-elemene and cetuximab inhibited cell migration and decreased the expression of mesenchymal markers (Vimentin, N-cadherin, Slug, Snail and MMP-9), but promoted the expression of epithelial marker E-cadherin. Moreover, ferroptosis inhibitors but not other cell death suppressors abrogated the effect of ß-elemene in combination with cetuximab on KRAS mutant CRC cells. In vivo, co-treatment with ß-elemene and cetuximab inhibited KRAS mutant tumor growth and lymph nodes metastases. Conclusions: Our data for the first time suggest that the natural product ß-elemene is a new ferroptosis inducer and combinative treatment of ß-elemene and cetuximab is sensitive to KRAS mutant CRC cells by inducing ferroptosis and inhibiting EMT, which will hopefully provide a prospective strategy for CRC patients with RAS mutations.

Efficacy and safety of elemene combined with chemotherapy in advanced gastric cancer: A Meta-analysis.

BACKGROUND: Elemene is a natural compound extracted from Zingiberaceae plants, and is used in various cancer. However, the efficacy and safety elemene combined with chemotherapy in advanced gastric cancer (GC) are lack of systematic assessment. METHODS: we searched the PubMed, EMBASE, Web of Science, Cochrane Library, China Academic Journals (CNKI), Chinese Science and Technology Journals (CQVIP) and Chinese Biomedical Literature databases. Randomized controlled trials (RCTs) comparing elemene plus chemotherapy with chemotherapy alone in participants with advanced GC and reporting at least one of the following outcomes were selected and assessed for inclusion. JADAD scale was used to assess the quality. Data was screened and extracted by two independent investigators. The primary clinical outcome was overall response rate (ORR); the secondary outcomes were quality of life (QOL) and adverse events (AEs). Analysis was performed using Review Manager 5.3. RESULTS: Sixteen RCTs matched the selection criteria, which reported on 969 subjects. Risk ratios (RR) and corresponding 95% confidence intervals (CIs) were pooled for ORR, life quality based on KPS, and risk of AEs. Compared to chemotherapy alone, elemene combined with chemotherapy in the treatment of GC may increase the efficiency of ORR(RR: 1.41; 95% CI: 1.23-1.60; P < .0001), improve their life quality based on KPS (RR: 1.84; 95% CI: 1.45-2.34; P < .00001), and reduce the adverse reactions, including leukopenia(RR: 0.73; 95% CI: 0.62-0.85; P < .00001), neutropenia (RR: 0.75; 95% CI: 0.60-0.95; P = .02), anemia (RR: 0.76; 95% CI: 0.60-0.95; P = .02), thrombocytopenia (RR: 0.56; 95% CI: 0.43-0.73; P < .00001). Nausea and vomiting (RR: 0.84; 95% CI: 0.84-1.07; P = .39), diarrhea (RR: 0.69; 95% CI: 0.41-1.15; P = .15), neurotoxicity (RR: 0.77; 95% CI: 0.59-1.00; P = .05) and hepatic dysfunction (RR: 0.95; 95% CI: 0.58-1.54; P = .83) were similar between two groups. CONCLUSIONS: Elemene may have the potential to improve the efficacy and reduce the AEs of chemotherapy for gastric cancer. However, the long-term, high-quality researches with a large sample size in different populations are required.

Differential effects of Chinese high-fat dietary habits on lipid metabolism: mechanisms and health implications.

BACKGROUND: The traditional Chinese diet blends lard with vegetable oil, keeping the fatty acid balance intake ratio of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids at nearly 1:1:1. However, the effects of a mixture of lard and vegetable oil on lipid metabolism have never been researched. In the present study, by simulating Chinese high-fat dietary habits, we explored the effects of a mixture of lard and vegetable oil on lipid metabolism. METHODS: We randomly assigned 50 male C57BL/6 J mice to 5 groups (10 in each group) and fed them lard, sunflower oil (SFO), soybean oil (SBO), lard blended with sunflower oil (L-SFO), or lard blended with soybean oil (L-SBO) for 12 weeks. RESULTS: We found that the final body weights of mice in the lard group were significantly higher than those of mice in the SFO and SBO groups. Body fat rate and volume of fat cell of the lard group were significantly higher than those of the SFO, SBO, and L-SBO groups. Liver triglyceride level of the lard group increased significantly compared to the other groups. Although body fat rate and liver triglyceride level in the SBO and SFO groups decreased compared to those in the other groups, the high-density lipoprotein cholesterol/low-density lipoprotein cholesterol ratio were also significantly decreased in the SBO and SFO groups. CONCLUSIONS: We found that a lard diet induced accumulation of body fat, liver and serum lipids, which can increase the risk of obesity, non-alcoholic fatty acid liver disease, and atherosclerosis. The vegetable oil diet resulted in cholesterol metabolism disorders even though it did not lead to obesity. The mixed oil diet induced body fat accumulation, but did not cause lipid accumulation in the liver and serum. Thus, differential oil/fat diets have an impact on differential aspects in mouse lipid metabolism.

Phenylethyl isothiocyanate induces oxidative damage of porcine kidney cells mediated by reactive oxygen species.

The aim of this study is to confirm the toxic effect of phenylethyl isothiocyanate (PEITC) on porcine kidney cells (PK-15) and explore the effect of oxidative damage mediated by reactive oxygen species (ROS) induced by PEITC in PK-15 cells. Porcine kidney cell line (PK-15) was treated with PEITC (2, 5, and 10 µM) for 24 hours, and the oxidative damage mediated by PEITC through ROS was investigated. The survival rate of PK-15 cells decreased in a dose-dependent manner after the treatment of PEITC in a dose-dependent manner. A high concentration of PEITC (10 µM) can change cell morphology, increase the content of malondialdehyde, ROS, and lactate dehydrogenase, and decrease the activity of SOD, CAT, GSH-PX, and GSH. PEITC has a toxic effect on PK-15 cells by inducing oxidative stress in PK-15 cells through the generation of ROS.

Molecular targets of ß-elemene, a herbal extract used in traditional Chinese medicine, and its potential role in cancer therapy: A review.

ß-Elemene is a sesquiterpene compound extracted from the herb Curcuma Rhizoma and is used in traditional Chinese medicine (TCM) to treat several types of cancer, with no reported severe adverse effects. Recent studies, using in vitro and in vivo studies combined with molecular methods, have shown that ß-elemene can inhibit cell proliferation, arrest the cell cycle, and induce cell apoptosis. Recent studies have identified the molecular targets of ß-elemene that may have a role in cancer therapy. This review aims to discuss the anticancer potential of ß-elemene through its actions on several molecular targets including kinase enzymes, transcription factors, growth factors and their receptors, and proteins. ß-Elemene also regulates the expression of several key molecules that are involved in tumor angiogenesis and metastasis including vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs), E-cadherin, N-cadherin, and vimentin. Also, ß-elemene has been shown to have regulatory effects on the immune response and increases the sensitivity of cancer cells to chemoradiotherapy and has shown effects on multidrug resistance (MDR) in malignancy. Recent studies have shown that ß-elemene can induce autophagy, which prevents cancer cells from undergoing apoptosis. Therefore, the molecular mechanisms for the treatment effects on cancer of the herbal extract, ß-elemene, which has been used for centuries in traditional Chinese medicine, are now being studied and identified.

Anti-obesity effect of a traditional Chinese dietary habit-blending lard with vegetable oil while cooking.

Obesity, which is associated with dietary habits, has become a global social problem and causes many metabolic diseases. In China, both percentages of adult obesity and overweight are far lower compared to western countries. It was designed to increase the two levels of daily intake in human, namely 3.8% and 6.5%, which are recommendatory intake (25 g/d) and Chinese citizens' practical intake (41.4 g/d), respectively. The mice were respectively fed with feeds added with soybean oil, lard or the oil blended by both for 12 weeks. In the mice fed with diet containing 3.8% of the three oils or 6.5% blended oil, their body weight, body fat rate, cross-sectional area of adipocytes, adipogenesis and lipogenesis in adipose were decreased, whereas hydrolysis of triglyserides in adipose was increased. This study demonstrated that the oil mixture containing lard and soybean oil had a remarkable anti-obesity effect. It suggests that the traditional Chinese dietary habits using oils blended with lard and soybean oil, might be one of the factors of lower percentages of overweight and obesity in China, and that the increasing of dietary oil intake and the changing of its component resulted in the increasing of obesity rate in China over the past decades.

Activation of HIV-1 expression in latently infected CD4+ T cells by the small molecule PKC412.

BACKGROUND: HIV-1 latency is a major obstacle for HIV-1 eradication. Extensive efforts are being directed toward the reactivation of latent HIV reservoirs with the aim of eliminating latently infected cells via the host immune system and/or virus-mediated cell lysis. RESULTS: We screened over 1,500 small molecules and kinase inhibitors and found that a small molecule, PKC412 (midostaurin, a broad-spectrum kinase inhibitor), can stimulate viral transcription and expression from the HIV-1 latently infected ACH2 cell line and primary resting CD4+ T cells. PKC412 reactivated HIV-1 expression in ACH2 cells in a dose- and time-dependent manner. Our results also suggest that the nuclear factor κB (NF-κB) signaling could be one of cellular pathways activated during PKC412-mediated activation of latent HIV-1 expression. Additionally, combining PKC412 with the HDAC inhibitor vorinostat (VOR) had an additive effect on HIV-1 reactivation in both ACH2 cells and infected resting CD4+ T cells. CONCLUSIONS: These studies provide evidence that PKC412 is a new compound with the potential for optimization as a latency-reactivator to eradicate HIV-1 infection.

Characterization of the inhibitory effect of an extract of Prunella vulgaris on Ebola virus glycoprotein (GP)-mediated virus entry and infection.

Currently, no approved antiviral therapeutic is available for treatment or prevention of Ebola virus (EBOV) infection. In this study, we characterized an EBOV-glycoprotein (GP) pseudotyped HIV-1-based vector system in different cell cultures, including human umbilical vein endothelial cells (HUVECs) and human macrophages, for the screening of anti-EBOV-GP agent(s). Based on this system, we demonstrated that an aqueous extract (CHPV) from the Chinese herb Prunella vulgaris displayed a potent inhibitory effect on EBOV-GP pseudotyped virus (EBOV-GP-V)-mediated infection in various cell lines, including HUVEC and macrophage. In addition, our results indicated that CHPV was able to block an eGFP-expressing Zaire ebola virus (eGFP-ZEBOV) infection in VeroE6 cells. The anti-EBOV activity of CHPV was exhibited in a dose-dependent manner. At a 12.5 µg/ml concentration, the CHPV showed a greater than 80% inhibition of EBOV-GP-V and eGFP-EBOV infections. Likewise, our studies suggested that the inhibitory effect of CHPV occurred by binding directly to EBOV-GP-Vs and blocking the early viral events. Interestingly, our results have shown that CHPV was able to enhance the anti-EBOV activity of the monoclonal antibody MAb 2G4 against EBOV-GP. Overall, this study provides evidence that CHPV has anti-EBOV activity and may be developed as a novel antiviral approach against EBOV infection.

Characterization of antiviral activity of benzamide derivative AH0109 against HIV-1 infection.

In the absence of an effective vaccine against HIV-1 infection, anti-HIV-1 strategies play a major role in disease control. However, the rapid emergence of drug resistance against all currently used anti-HIV-1 molecules necessitates the development of new antiviral molecules and/or strategies against HIV-1 infection. In this study, we have identified a benzamide derivative named AH0109 that exhibits potent anti-HIV-1 activity at an 50% effective concentration of 0.7 µM in HIV-1-susceptible CD4(+) C8166 T cells. Mechanistic analysis revealed that AH0109 significantly inhibits both HIV-1 reverse transcription and viral cDNA nuclear import. Furthermore, our infection experiments indicated that AH0109 is capable of disrupting the replication of HIV-1 strains that are resistant to the routinely used anti-HIV-1 drugs zidovudine, lamivudine, nevirapine, and raltegravir. Together, these findings provide evidence for a newly identified antiviral molecule that can potentially be developed as an anti-HIV-1 agent.

Relationships among genetic makeup, active ingredient content, and place of origin of the medicinal plant Gastrodia tuber.

Gastrodia tuber and its component gastrodin have many pharmacological effects. The chemical fingerprints and gastrodin contents of eight Gastrodia populations were determined, and the genomic DNA polymorphism of the populations was investigated. Genetic distance coefficients among the populations were calculated using the DNA polymorphism data. A dendrogram of the genetic similarities between the populations was constructed using the genetic distance coefficients. The results indicated that the genomic DNA of Gastrodia tubers was highly polymorphic; the eight populations clustered into three major groups, and the gastrodin content varied greatly among these groups. There were obvious correlations among genetic makeup, gastrodin content, and place of origin. The ecological environments in Guizhou and Shanxi may be conducive to evolution and to gastrodin biosynthesis, and more suitable for cultivation of Gastrodia tubers. These findings may provide a scientific basis for overall genetic resource management and for the selection of locations for cultivating Gastrodia tubers.

[Cloning of distinguishing DNA sequences of Gastrodia elata Blume and application of them in identifying gastrodia tuber].

Gastrodia elata Bl. is a famous and costful traditional Chinese medicine. Their genomic DNA fingerprints were investigated using a modified Randomly Amplified Polymorphic DNA method. DNA fragments common to all or to fine populations were identified and recovered. Five DNA fragments were proven not to be reported through DNA cloning, PCR identifying, nucleotide sequencing and bioinformatics analyses and were received in and recorded by NCBI GenBank. Gastrodine contents of the Gastrodia tuber samples were determined using high performance liquid chromatography technique. The distribution of the five DNA fragments in 9 Gastrodia elata Blue populations and the correlation with gastromedicine content were studied. The results show the distribution of these DNA sequences varied greatly among the populations whereby DNA Sequence 1 was the common and distinguishing molecular marker for all the populations studied and DNA Sequence 2 may relate to higher gastrodine content. In conclusion, these DNA marker sequences can be employed to identify genuine gastrodia tubers, better varieties and optimize their selection and cultivating.

Accumulation of mercury, selenium and their binding proteins in porcine kidney and liver from mercury-exposed areas with the investigation of their redox responses.

The subcellular localization of Se and Hg and their cytosolic binding proteins, including cellular oxidative status, in porcine liver and kidney have been studied by using samples from a chronic Hg-contaminated area and a non-Hg-contaminated area. Coaccumulation and redistribution of Se and Hg in subcellular fractions due to mercury exposure were found. The Hg and Se concentrations in tissues from Hg-exposed porcine were 80 fold and 5-20 fold higher than controls, respectively. Interestingly, the retention of both Se and Hg increased 10% in mitochondria, while decreased 10% in cytosol of Hg-exposed pig liver. Mercury was mainly in the form of MTs in the cytosol of the non-Hg-exposed porcine kidney. MT binds Hg in the cytosol with limited capacity, and the rest Hg was redistributed to the high molecular weight (MW) proteins (80-100 kDa) in the Hg-exposed group. The coaccumulation of Hg and Se was also found in high MW proteins, where their molar ratio tended to be 1:1. Moreover, the Se-containing polypeptides (3-6 kDa) increased significantly both in hepatic and renal cytosol of the Hg-exposed pigs. Se-dependent GSH-Px and SOD activity were increased to cope with Hg-induced oxidative stress. In previous studies, the roles of Se and MTs were generally taken into account separately; we discussed their combining roles in the case of high Hg exposure. The present results were beneficial to understand the existing states of Hg in vivo and evaluate the interaction of toxic and essential elements.

The roles of serum selenium and selenoproteins on mercury toxicity in environmental and occupational exposure.

Many studies have found that mercury (Hg) exposure is associated with selenium (Se) accumulation in vivo. However, human studies are limited. To study the interaction between Se and Hg, we investigated the total Se and Hg concentrations in body fluids and serum Se-containing proteins in individuals exposed to high concentrations of Hg. Our objective was to elucidate the possible roles of serum Se and selenoproteins in transporting and binding Hg in human populations. We collected data from 72 subjects: 35 had very low Hg exposure as evidenced by mean Hg concentrations of 0.91 and 1.25 ng/mL measured in serum and urine, respectively; 37 had high exposure (mean Hg concentrations of 38.5 and 86.8 ng/mL measured in serum and urine, respectively). An association between Se and Hg was found in urine (r = 0.625; p < 0.001) but not in serum. Hg exposure may affect Se concentrations and selenoprotein distribution in human serum. Expression of both selenoprotein P and glutathione peroxidase (GSH-Px) was greatly increased in Hg miners. These increases were accompanied by elevated Se concentrations in serum. In addition, selenoprotein P bound more Hg at higher Hg exposure concentrations. Biochemical observations revealed that both GSH-Px activity and malondialdehyde concentrations increased in serum of the Hg-exposed group. This study aids in the understanding of the interaction between Se and Hg. Selenoproteins play two important roles in protecting against Hg toxicity. First, they may bind more Hg through their highly reactive selenol group, and second, their antioxidative properties help eliminate the reactive oxygen species induced by Hg in vivo.

[Establishment and the significance of a cell model of secreted alkaline phosphatase co-controlled by HCV 5'NCR and NS3 serine protease].

OBJECTIVE: To establish a cell model of secreted alkaline phosphatase (SEAP) co-controlled by HCV 5'NCR and NS3 serine protease in an effort to develop new antiviral agents. METHODS: The fragments of HCV 5'NCR and NS3/4A-SEAP were amplified by PCR. They were fused into pBluescript SK+ to generate 5'NCR-NS3/4A-SEAP chimeric plasmid. The resulting chimeric gene was subcloned into HindIII/Bsu36 I site of pSEAP2-Control (a SEAP eukaryotic expression plasmid), to generate pNCR-NS3/4A-SEAP, in which the SEAP was fused in-frame to the downstream of NS4A/4B cleavage site. The SEAP activity in the culture media of transiently transfected cells was monitored quantitatively. The regulatory effect of HCV 5'NCR and NS3 serine protease on SEAP expression was measured by treatment of transfected cells with antisense oligodeoxynucleotide (ASODN) against HCV 5'NCR and TPCK, a irreversible serine protease inhibitor. RESULTS: The SEAP activity in the culture media reached 80801+/-4794 RLU, and was significantly inhibited by 5 micromol/L, 10 micromol/L of ASODN (t=4.315, p<0.01; t=6.985, p<0.001) and 100 micromol/L of TPCK (t=6.949, P<0.001). CONCLUSION: A cell model of SEAP co-controlled by HCV 5'NCR and NS3 serine protease has been successfully established. This might promote the screening of anti-viral drugs

[Screening and identification of novel genes involved in biosynthesis of ginsenoside in Panax ginseng plant].

The root of Panax ginseng plant undergoes a specific developmental process to become a biosynthesis and accumulation organ for ginsenosides. To identify and analyze genes involved in the biosynthesis of ginsenoside, suppression subtractive hybridization (SSH) between mRNAs of 4- and 1-year-old root tissues was performed, and a subtracted cDNA library specific to 4-year-old roots was constructed. Forty cDNA clones selected randomly from the subtracted cDNA library were sequenced. Sequence information of all clones was evaluated by Nucleotide Blast analysis in GenBank/DDBJ/EMBL. The results showed that six subtracted cDNA clones represented the novel genes (ESTs), because no sequence homology with any known sequences was found in the database. Expression in 4-year-old P. ginseng root tissues was verified by reverse Northern dot hybridization for the six clones. These six novel genes were named GBR1, GBR2, GBR3, GBR4, GBR5, and GBR6, and their Accession numbers of GenBank are AF485334, AF485335, AF485336, AF485337, AF485332, and AF485333, respectively. Finally, Northern blot analysis and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) confirmed that these six novel genes were differentially expressed in the defined development stage of P. ginseng plant roots. It is possible that their overexpression may play an important role in the ginsenoside biosynthesis. In addition, most of transcripts of all genes could also be detected in other P. ginseng plant tissues such as stem, leaf and seed. Our results provided a basis for obtaining the full-length cDNA sequences of such six novel genes, and for identifying their function involved in the biosynthesis of ginsenoside.