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Chikungunya virus (CHIKV), an alphavirus, has recently caused epidemic outbreaks and is therefore considered a re-emerging pathogen for which no effective treatment is available. In this study, a CHIKV replicon containing the virus replicase proteins together with puromycin acetyltransferase, EGFP and Renilla luciferase marker genes was constructed. The replicon was transfected into BHK cells to yield a stable cell line. A non-cytopathic phenotype was achieved by a Pro718 to Gly substitution and a five amino acid insertion within non-structural protein 2 (nsP2), obtained through selection for stable growth. Characterization of the replicon cell line by Northern blotting analysis revealed reduced levels of viral RNA synthesis. The CHIKV replicon cell line was validated for antiviral screening in 96-well format and used for a focused screen of 356 compounds (natural compounds and clinically approved drugs). The 5,7-dihydroxyflavones apigenin, chrysin, naringenin and silybin were found to suppress activities of EGFP and Rluc marker genes expressed by the CHIKV replicon. In a concomitant screen against Semliki Forest virus (SFV), their anti-alphaviral activity was confirmed and several additional inhibitors of SFV with IC50 values between 0.4 and 24 µM were identified. Chlorpromazine and five other compounds with a 10H-phenothiazinyl structure were shown to inhibit SFV entry using a novel entry assay based on a temperature-sensitive SFV mutant. These compounds also reduced SFV and Sindbis virus-induced cytopathic effect and inhibited SFV virion production in virus yield experiments. Finally, antiviral effects of selected compounds were confirmed using infectious CHIKV. In summary, the presented approach for discovering alphaviral inhibitors enabled us to identify potential lead structures for the development of alphavirus entry and replication phase inhibitors as well as demonstrated the usefulness of CHIKV replicon and SFV as biosafe surrogate models for anti-CHIKV screening. 相似文献
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Siltanen S Wahlfors T Schindler M Saramäki OR Mpindi JP Latonen L Vessella RL Tammela TL Kallioniemi O Visakorpi T Schleutker J 《PloS one》2011,6(10):e26595
ARLTS1 is a recently characterized tumor suppressor gene at 13q14.3, a region frequently deleted in both sporadic and hereditary prostate cancer (PCa). ARLTS1 variants, especially Cys148Arg (T442C), increase susceptibility to different cancers, including PCa. In this study the role of Cys148Arg substitution was investigated as a risk factor for PCa using both genetic and functional analysis. Cys148Arg genotypes and expression of the ARLTS1 were explored in a large set of familial and unselected PCa cases, clinical tumor samples, xenografts, prostate cancer cell lines and benign prostatic hyperplasia (BPH) samples. The frequency of the variant genotype CC was significantly higher in familial (OR = 1.67, 95% CI = 1.08–2.56, P = 0.019) and unselected patients (OR = 1.52, 95% CI = 1.18–1.97, P = 0.001) and the overall risk was increased (OR = 1.54, 95% CI = 1.20–1.98, P = 0.0007). Additional analysis with clinicopathological data revealed an association with an aggressive disease (OR = 1.28, 95% CI = 1.05-∞, P = 0.02). The CC genotype of the Cys148Arg variant was also contributing to the lowered ARLTS1 expression status in lymphoblastoid cells from familial patients. In addition significantly lowered ARLTS1 expression was observed in clinical tumor samples compared to BPH samples (P = 0.01). The ARLTS1 co-expression signature based on previously published microarray data was generated from 1587 cancer samples confirming the low expression of ARLTS1 in PCa and showed that ARLTS1 expression was strongly associated with immune processes. This study provides strong confirmation of the important role of ARLTS1 Cys148Arg variant as a contributor in PCa predisposition and a potential marker for aggressive disease outcome. 相似文献
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Moses K. Langat Neil R. Crouch Leena Pohjala Päivi Tammela Peter J. Smith Dulcie A. Mulholland 《Phytochemistry letters》2012,5(3):414-418
Two new ent-kauren-19-oic acid derivatives, ent-14S*-hydroxykaur-16-en-19-oic acid and ent-14S*,17-dihydroxykaur-15-en-19-oic acid together with eleven known compounds ent-kaur-16-en-19-oic acid, ent-kaur-16-en-19-al, ent-12β-hydroxykaur-16-en-19-oic acid, ent-12β-acetoxykaur-16-en-19-oic acid, 8R,13R-epoxylabd-14-ene, eudesm-4(15)-ene-1β,6α-diol, (?)-7-epivaleran-4-one, germacra-4(15), 5E,10(14)-trien-9β-ol, acetyl aleuritolic acid, β-amyrin, and stigmasterol were isolated from the stem bark of Croton pseudopulchellus (Euphorbiaceae). Structures were determined using spectroscopic techniques. Ent-14S*-hydroxykaur-16-en-19-oic acid, ent-kaur-16-en-19-oic acid, ent-12β-hydroxykaur-16-en-19-oic acid, ent-12β-acetoxykaur-16-en-19-oic acid and 8R,13R-epoxylabd-14-ene were tested for their effects on Semliki Forest virus replication and for cytotoxicity against human liver tumour cells (Huh-7 strain) but were found to be inactive. Ent-kaur-16-en-19-oic acid, the major constituent, showed weak activity against the Plasmodium falciparum (CQS) D10 strain. 相似文献
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Tammela P Hopia A Hiltunen R Vuorela H Nygren M 《Biochemical Society transactions》2000,28(6):878-879
Aging of Scots pine seeds (Pinus sylvestris L.) leads to changes in seed quality, such as loss of germinability, delayed growth and abnormality in developing seedlings. The knowledge of biochemical changes responsible for these aging processes is plentiful in some seeds, which are of world-wide interest, but for pine seeds these studies are rare. The aim of the present study, was to analyse pine seeds of varying ages in order to identify biochemical changes occurring in aged pine seeds, and to see if a correlation existed between these results and traditionally used seed-quality parameters, such as germinability and electrolyte leakage. 相似文献
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Voltage-operated calcium channels (VOCCs) play a significant role in the regulation of intracellular calcium concentrations in cardiovascular, neuronal and skeletal tissues. Therefore, physiologically relevant screening methods for calcium channel modulators are required. A 45Ca2+ uptake assay based on clonal rat pituitary cell line GH4C1, possessing L-type VOCCs, was miniaturised into a 96-well plate format. The assay was validated by known Ca2+ channel blockers, verapamil and nimodipine (IC50 values 3.4 and 0.007 microM, respectively) and by a set of natural compounds and their synthetic derivatives. The results were consistent with our previous data and demonstrated the reliability of the assay. The signal-to-background ratio was 3.9 +/- 0.4, signal-to-noise ratio 10.3 +/- 2.3, Z' factor 0.59 +/- 0.10, and day-to-day variability in positive control values 5%. Furthermore, experiments were also made on a Biomek FX workstation to evaluate the suitability of the assay for automation. With minor modifications the assay is applicable, e.g. for studying possible Ca2+ channel activators in detail. The established 96-well plate assay modification for screening of calcium channel modulators reduces considerably the time, labour and resources needed for cell culture and experiments, and has significant advantages in terms of automation suitability and overall cost-efficiency. 相似文献
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Androgen receptor CAG polymorphism and prostate cancer risk 总被引:4,自引:0,他引:4
Mononen N Ikonen T Autio V Rökman A Matikainen MP Tammela TL Kallioniemi OP Koivisto PA Schleutker J 《Human genetics》2002,111(2):166-171
Recent studies have suggested that polymorphisms of the androgen receptor gene ( AR) may influence the risk of prostate cancer (PC) development and progression. Here, we analyzed the length of the CAG repeat of the AR gene in 1363 individuals, including patients with PC, benign prostate hyperplasia (BPH), and population controls. There was a tendency for short CAG repeats to be associated with PC. The Odds Ratio (OR) for PC was 1.47 ( P=0.05) when individuals with short CAG repeats (=18) were compared with those having long repeats (>18). CAG repeat length was not significantly associated with family history, disease stage, grade, age at diagnosis, prostate-specific antigen (PSA) level at diagnosis, or prognosis of the patients. Unexpectedly, short CAG repeats were significantly less common in patients with BPH compared with controls (OR=0.47, P=0.03). Our results suggest that the CAG polymorphism of the AR gene is unlikely to have a major role in the development or progression of PC in the Finnish population. The association of CAG repeats with the risk of BPH warrants further study. 相似文献
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