Isolation, characterization, anti-MRSA evaluation, and in-silico multi-target anti-microbial validations of actinomycin X2 and actinomycin D produced by novel Streptomyces smyrnaeus UKAQ_23.

Streptomyces smyrnaeus UKAQ_23, isolated from the mangrove-sediment, collected from Jubail,Saudi Arabia, exhibited substantial antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), including non-MRSA Gram-positive test bacteria. The novel isolate, under laboratory-scale conditions, produced the highest yield (561.3 ± 0.3 mg/kg fermented agar) of antimicrobial compounds in modified ISP-4 agar at pH 6.5, temperature 35 °C, inoculum 5% v/w, agar 1.5% w/v, and an incubation period of 7 days. The two major compounds, K1 and K2, were isolated from fermented medium and identified as Actinomycin X2 and Actinomycin D, respectively, based on their structural analysis. The antimicrobial screening showed that Actinomycin X2 had the highest antimicrobial activity compared to Actinomycin D, and the actinomycins-mixture (X2:D, 1:1, w/w) against MRSA and non-MRSA Gram-positive test bacteria, at 5 µg/disc concentrations. The MIC of Actinomycin X2 ranged from 1.56-12.5 µg/ml for non-MRSA and 3.125-12.5 µg/ml for MRSA test bacteria. An in-silico molecular docking demonstrated isoleucyl tRNA synthetase as the most-favored antimicrobial protein target for both actinomycins, X2 and D, while the penicillin-binding protein-1a, was the least-favorable target-protein. In conclusion, Streptomyces smyrnaeus UKAQ_23 emerged as a promising source of Actinomycin X2 with the potential to be scaled up for industrial production, which could benefit the pharmaceutical industry.

Antitumor effects of curcumin in pediatric rhabdomyosarcoma in combination with chemotherapy and phototherapy in vitro.

Rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma, has an unfavorable outcome in advanced tumor stages with less than 30% failure­free survival. Curcumin (CUR) is a promising drug in complementary oncology with few side effects but proven efficacy in various adult oncological entities. The present study analyzed the effects of CUR on pediatric (RMS) cell lines in vitro. RMS cell lines (RD and RH30), and skeletal muscle cells (SKMC) were treated with different doses of CUR (1.5­30 µM) alone, with phototherapy (PDT, 488 nm) or in combination with vincristine (VCR) or dactinomycin (DAC). MTT assays were used for analysis of RMS tumor cell viability. Clonal cell growth was assessed via colony forming assays and migration of the cells was analyzed with scratch tests. Annexin V staining was used to determine apoptosis in flow cytometry. Possible RMS resistance towards CUR after long­term treatment was analyzed with MTT assays. CUR decreased cell viability in all assessed RMS cell lines in a concentration­dependent manner with IC50=14­20 µM. CUR enhanced the effects of the cytotoxic drugs VCR or DAC, and led to reduced migration and increased cell apoptosis. In combination with PDT, CUR decreased the cell viability in minute quantities with up to a 10­fold lower IC50 than without PDT. CUR effectively inhibited the malignant properties of pediatric RMS cells and should be focused on as a useful additional agent in standard chemotherapy of RMS in children.

Rapid and simple screening of the apoptotic compounds based on Hsp70 inhibition using luciferase as an intracellular reporter.

HSP70 is a powerful antiapoptotic protein that can block the extrinsic and intrinsic pathways of apoptosis. The present study describes a rapid, sensitive, and inexpensive system using luciferase as a reporter for the functional analysis of apoptotic compounds. For this approach, the co-transformation of Escherichia coli cells was performed with two expression vectors containing Hsp70 and firefly luciferase. It was found that the luciferase inactivated by heat treatment (40-46 °C for 10 min) was approximately reactivated at room temperature and regained 70% of its initial activity before heat inactivation after 60 min. The results show that the reactivation of thermally inactivated luciferase was inhibited in living cells by treatment with VER-155008 and pifitrin-µ as Hsp70 inhibitors, with half-maximal inhibitory concentration of 124 and 384 µM, respectively. The sensitivity of this method for detecting VER-155008 and pifitrin-µ was about 8 and 25 µM, respectively. Also, this reporter system showed no response to doxorubicin and dactinomycin, which bind to DNA, and we used these anticancer compounds as control compounds. Therefore, for the first time, a rapid and simple real-time system using luciferase as a reporter is introduced for the screening of apoptosis-inducing compounds based on suppression of Hsp70 in E. coli cells.

A new actinomycin Z analogue with an additional oxygen bridge between chromophore and ß-depsipentapeptide from Streptomyces sp. KIB-H714.

Actinomycin Z6 (1), a new member of the actinomycin family, along with three congeners of the Z-type (Z1, Z3, Z5) actinomycins, are produced from Streptomyces sp. KIB-H714. Their structures were authenticated by comprehensive spectroscopic data interpretation. Different from all the reported Z-type actinomycins, the ß-ring of the new compound actinomycin Z6 includes an additional ring linked between the actinoyl chromophore and ß-peptidolactone. In Z3 and Z5, the L-threonine in ß-depsipeptide is replaced by the unusual 4-chlorothreonine, an amino acid rarely found in actinomycin family. All isolates were evaluated for cytotoxicity against five human tumor cell lines and for inhibitory activity against Candida albicans and Staphylococcus aureus.

Cell growth potential drives ferroptosis susceptibility in rhabdomyosarcoma and myoblast cell lines.

PURPOSE: Ferroptosis is a programmed form of iron-dependent cell death caused by lipid hydroperoxide accumulation, which can be prevented by glutathione peroxidase 4 (GPx4) activity. Here we investigated the effects of ferroptosis inducers called erastin and RSL3, which act by glutathione depletion and GPx4 inactivation, respectively, on muscle-derived cell lines of embryonal and alveolar rhabdomyosarcoma (RMS), and mouse normal skeletal C2C12 myoblasts. METHODS: Myogenic lines were exposed to stepwise increasing concentrations of ferroptosis inducers either alone or in combination with iron supplementation, iron chelating agents (bathophenanthrolinedisulfonic acid, BPS), antioxidant molecules (glutathione, N-acetylcysteine), lipid peroxidation inhibitors (ferrostatin-1), and chemotherapeutic agents (doxorubicin and actinomycin D). Drug susceptibility was quantified by measuring cell viability, proliferation and differentiation via neutral red assay, crystal violet assay and Giemsa staining, respectively. The detection of lipid hydroperoxide and protein levels was performed by immunofluorescence and Western blot analysis, respectively. RESULTS: Erastin and RSL3 increased lipid hydroperoxide levels preferentially in the embryonal U57810 and myoblast C2C12 lines, leading to ferroptosis that was accentuated by iron supplementation or prevented by co-treatment with BPS, glutathione, N-acetylcysteine and ferrostatin-1. The inhibition of extracellular regulated kinases (ERK) pathway prevented ferroptosis in U57810 and C2C12 cells, whereas its increased activation in the embryonal RD cells mediated by caveolin-1 (Cav-1) overexpression led to augmented ferroptosis susceptibility. Finally, we observed the combination of erastin or RSL3 with chemotherapeutic doxorubicin and actinomycin D agents to be effective in increasing cell death in all RMS lines. CONCLUSIONS: Erastin and RSL3 trigger ferroptosis in highly proliferating myogenic lines through a ERK pathway-dependent fashion.

Antimicrobial investigation of selected soil actinomycetes isolated from unexplored regions of Kashmir Himalayas, India.

The aim of the present study was to isolate and evaluate the antimicrobial potential of soil actinomycetes of Kashmir Himalayas. The secondary metabolites of actinomycetes are the prominent source of antibiotics. A total of 121 morphologically different actinomycete strains were isolated and screened for antimicrobial activity against various human pathogens. The ethyl acetate extract of fermented broth an actinomycete strain, identified as Streptomyces pratensis exhibited significant antimicrobial activity against Staphylococcus aureus ATCC 29213 with MIC 0.25 µg/ml and Mycobacterium tuberculosis Strain H37Rv with MIC 0.062 µg/ml. The strain S. pratensis IIIM06 was grown on large scale and their broth was extracted with ethyl acetate. The extract was subjected to various chromatography techniques which led to the isolation of four compounds whose structures were established as actinomycin C1, actinomycin C2, actinomycin C3 and actiphenol on the basis of spectral data analysis. Actinomycin C1, C2 and C3 exhibited potent antimicrobial activity against S. aureus as well as M. tuberculosis. The isolated indigenous actinomycetes exhibited good antibacterial activity and the study reveals that IIIM06 is a promising strain and could be of great potential for industrial applications.

In vitro evaluation of combination of EGCG and Erlotinib with classical chemotherapeutics on JAR cells.

Gestational Trophoblastic Neoplasia (GTN) is a term used for a group of malignant gynecological tumors including choriocarcinoma. Low-risk neoplasias can be cured using single agents Methotrexate (MTX) and actinomycin-D (ACD), but in certain cases, decreased responsiveness and serious side effects occur. Therefore, researchers have been attempting to find new treatment modalities. One of the most popular way for increasing cancer patient survival rates is supporting treatment with adjuvant molecules or chemosensitizers. For this purpose, we investigated epigallocatechin-3-gallate (EGCG), a green tea cathecin, and Erlotinib, an EGFR tyrosine kinase inhibitor, as single agents and combined with MTX or ACD. In accordance with this, JAR (human placenta choriocarcinoma) cell line was used as an in vitro model and MTT, LDH, caspase-3 activation, RT-PCR, and Western Blot analyses were performed to investigate the effects of the test materials. Our studies demonstrate that combination of Erlotinib and EGCG with MTX and ACD decreases JAR cell proliferation and metastatic HER2 protein synthesis and increases caspase-3 activation compared to ACD or MTX alone. In addition, significant increase was observed in the apoptotic Bax gene, but no notable protein synthesis occurred in the Western Blot analysis, which suggests that combination of Erlotinib and EGCG with classical chemotherapeutics ACD or MTX may lead the JAR cells to apoptosis, but not by a mitochondrial pathway. All the results indicate that the synergetic effect of Erlotinib and EGCG with classical chemotherapeutics may help to increase patient survival rates of choriocarcinoma, but the detailed mechanism needs further investigation.

Developmental regulation and modulation of apoptotic genes expression in sheep oocytes and embryos cultured in vitro with L-carnitine.

The objective of this study was to find out the impact of L-carnitine (10 mM) on developmental regulation of preimplantation sheep embryos cultured in vitro when supplemented in maturation medium and post-fertilization medium separately. Subsequent objective was to observe the L-carnitine-mediated alteration in expression of apoptotic genes (Bcl2, Bax, Casp3 and PCNA) in sheep oocytes and developing embryos produced in vitro. Oocytes matured with L-carnitine showed significantly (p < .05) higher cleavage (67.23% vs 43.12%), morula (47.65% vs 28.58%) and blastocysts (32.12% vs 13.24%) percentage as compared to presumptive zygotes cultured with L-carnitine during post-fertilization period. So it is suggested to use L-carnitine during maturation than post-fertilization period. Antiapoptotic and proliferative effects of L-carnitine were confirmed by inducing culture medium with actinomycin D (apoptotic agent) and TNFα (antiproliferative agent), respectively, with and without L-carnitine. Oocytes and embryos cultured with actinomycin D and TNFα showed developmental arrest with significant (p < .05) decrease in morula and blastocysts percentage but supplementation of L-carnitine to actinomycin D and TNFα induced culture medium showed similar result as that of control. L-carnitine supplementation during IVM significantly (p < .05) upregulated the expression of Bcl2 and PCNA genes in majority of the developmental stages. Although L-carnitine upregulated the expression of Bax in initial developmental stages but downregulated at latter part, whereas the expression of Casp3 was upregulated upto 16-cell stage but after that there was no difference in expression. Expression of GAPDH gene was not affected by L-carnitine supplementation. In conclusion, L-carnitine acted as an antiapoptotic and proliferative compound during embryo development and supplementation of L-carnitine during IVM altered the expression of apoptotic genes in the developmental stages of embryos.

NGS Transcriptomes and Enzyme Inhibitors Unravel Complexity of Picrosides Biosynthesis in Picrorhiza kurroa Royle ex. Benth.

Picrorhiza kurroa is an important medicinal herb valued for iridoid glycosides, Picroside-I (P-I) and Picroside-II (P-II), which have several pharmacological activities. Genetic interventions for developing a picroside production platform would require knowledge on biosynthetic pathway and key control points, which does not exist as of today. The current study reports that geranyl pyrophosphate (GPP) moiety is mainly contributed by the non-mevalonate (MEP) route, which is further modified to P-I and P-II through phenylpropanoid and iridoid pathways, in total consisting of 41 and 35 enzymatic steps, respectively. The role of the MEP pathway was ascertained through enzyme inhibitors fosmidomycin and mevinolin along with importance of other integrating pathways using glyphosate, aminooxy acetic acid (AOA) and actinomycin D, which overall resulted in 17%-92% inhibition of P-I accumulation. Retrieval of gene sequences for enzymatic steps from NGS transcriptomes and their expression analysis vis-à-vis picrosides content in different tissues/organs showed elevated transcripts for twenty genes, which were further shortlisted to seven key genes, ISPD, DXPS, ISPE, PMK, 2HFD, EPSPS and SK, on the basis of expression analysis between high versus low picrosides content strains of P. kurroa so as to eliminate tissue type/ developmental variations in picrosides contents. The higher expression of the majority of the MEP pathway genes (ISPD, DXPS and ISPE), coupled with higher inhibition of DXPR enzyme by fosmidomycin, suggested that the MEP route contributed to the biosynthesis of P-I in P. kurroa. The outcome of the study is expected to be useful in designing a suitable genetic intervention strategy towards enhanced production of picrosides. Possible key genes contributing to picroside biosynthesis have been identified with potential implications in molecular breeding and metabolic engineering of P. kurroa.

Cortisol stimulates arginine vasotocin and isotocin release from the hypothalamo-pituitary complex of round goby (Neogobius melanostomus): Probable mechanisms of action.

There were two aims of this in vitro perfusion study. Firstly, to determine which class of receptors, glucocorticoid (GRs) or mineralocorticoid (MRs), are involved in cortisol regulation of arginine vasotocin (AVT) and isotocin (IT) release from the hypothalamo-pituitary (H-P) complex of round goby (Neogobius melanostomus). Secondly, to determine which pathways, genomic or non-genomic, are involved in the aformentioned process.The H-P explants were perfused with cortisol (1.4 × 10(-) (7) M, 2.8 × 10(-7) M, 0.4 × 10(-6) M); only the highest dose significantly increased a release of both nonapeptides. In the perfusion of H-P explants, we used cortisol (0.4 × 10(-6) M) in combination with GRs antagonist RU486 (0.3 × 10(-6) M) or MRs antagonist C03DA01 (0.36 × 10(-6) M) or transcription inhibitor Actinomycin D (1 × 10(-7) M). All inhibitors were also tested seperately. The contents of AVT and IT in the perfusion media was determined by high-performance liquid chromatography (HPLC) with UV detection. This study suggested that different mechanisms were involved in the regulation of AVT and IT release from H-P complex in round goby. Apparently it was GRs but not MRs that were involved in cortisol regulation of AVT and IT release. In the case of AVT, our data points to both genomic and non-genomic pathways mediating the effect of cortisol; in the case of IT, it is only the non-genomic pathway. This study presents the first feasible mechanisms of cortisol action on AVT and IT release from the H-P complex in round goby.

Prevention of capsule opacification after accommodating lens refilling: pilot study of strategies evaluated in a monkey model.

PURPOSE: To test 2 strategies to prevent capsule opacification after accommodating lens refilling in a rhesus monkey model. SETTING: Animal laboratory and laboratory of European university medical centers. DESIGN: Experimental study. METHODS: Six rhesus monkeys had refilling of the lens capsular bag. In the first strategy, before it was filled with a silicone polymer, the capsular bag was treated with noncommercial sodium hyaluronate 1.0% containing cytotoxic substances. In the second strategy, the capsular bag was filled with clinically used sodium hyaluronate 1.0% (Healon) after treatment with actinomycin-D. Slitlamp inspection was performed during a follow-up of 40 to 50 weeks. After enucleation, magnetic resonance images were obtained and confocal fluorescence imaging was performed. RESULTS: Using the first strategy, capsule opacification developed in all eyes. Using the second strategy, 1 monkey did not develop capsule opacification after a 9-month follow-up. In a second monkey, the lens capsule remained clear for 3 months, after which the hyaluronate refill material was exchanged with a silicone polymer and capsule opacification developed. Combining these results with those in a previous study, the difference in opacification between silicone and sodium hyaluronate as refilling materials was statistically significant (P<.01). CONCLUSIONS: That no capsular bag fibrosis occurred in the presence of hyaluronate suggests that the properties of hyaluronate are the reason that remaining lens epithelial cells do not develop into fibrotic cells. The choice of a suitable lens-refilling material prevents the development of capsule opacification. FINANCIAL DISCLOSURE: Mr. Terwee was an employee of Abbott Medical Optics B.V. during the study period. No other author has a financial or proprietary interest in any material or method mentioned.

Immunostimulating effect of a well-known Thai folkloric remedy in breast cancer patients.

The study aimed to evaluate immune-stimulating effects of a well-known Thai folkloric remedy when used for adjuvant therapy with conventional chemotherapeutics for treatment of breast cancer. Immunostimulating influence of the remedy (215 mg/kg body weight per day) on NK cell activity and TNF-α release from the monocytes/macrophages were investigated in a total of 15 healthy women and 13 female patients with breast cancer (Group 1). The effect of breast tumor surgery on NK cell activity was further investigated in 18 female patients with breast cancer (Group 2). NK cell cytotoxic activity was determined by chromium release cytotoxic assay using K562, an erythroleukemic cell line. TNF-α release from monocytes/macrophages separated from blood samples was determined through a biological assay using actinomycin D-treated L929 mouse fibroblast cells in the presence and absence of LPS. Baseline NK cell activity of the monocytes/macrophages separated from Group 2 patients expressed as %cytotoxicity was significantly lower than in the healthy subjects at E:T ratios of 100:1 and 25:1. In healthy subjects, there was no change in NK cell cytotoxic activity (%cytotoxicity or LU) following 1 and 2 weeks of treatment with the remedy compared with the baseline at various E:T ratios but the binding activity (%binding) was significantly increased after 2 weeks of treatment. The addition of one or two conventional chemotherapeutic regimens did not significantly reduce the NK cytotoxic activity but did affect release of TNF-α in both unstimulated and LPS-stimulated samples. Surgery produced a significant suppressive effect on NK cell activity. The use of the remedy as an adjunct therapy may improve therapeutic efficacy and safety profiles of conventional chemotherapeutic regimens through stimulation of the immune system in cancer patients.

Selected drugs with reported secondary cell-differentiating capacity prime latent HIV-1 infection for reactivation.

Reactivation of latent HIV-1 infection is considered our best therapeutic means to eliminate the latent HIV-1 reservoir. Past therapeutic attempts to systemically trigger HIV-1 reactivation using single drugs were unsuccessful. We thus sought to identify drug combinations consisting of one component that would lower the HIV-1 reactivation threshold and a synergistic activator. With aclacinomycin and dactinomycin, we initially identified two FDA-approved drugs that primed latent HIV-1 infection in T cell lines and in primary T cells for reactivation and facilitated complete reactivation at the population level. This effect was correlated not with the reported primary drug effects but with the cell-differentiating capacity of the drugs. We thus tested other cell-differentiating drugs/compounds such as cytarabine and aphidicolin and found that they also primed latent HIV-1 infection for reactivation. This finding extends the therapeutic promise of N'-N'-hexamethylene-bisacetamide (HMBA), another cell-differentiating agent that has been reported to trigger HIV-1 reactivation, into the group of FDA-approved drugs. To this end, it is also noteworthy that suberoylanilide hydroxamic acid (SAHA), a polar compound that was initially developed as a second-generation cell-differentiating agent using HMBA as a structural template and which is now marketed as the histone deacetylase (HDAC) inhibitor vorinostat, also has been reported to trigger HIV-1 reactivation. Our findings suggest that drugs with primary or secondary cell-differentiating capacity should be revisited as HIV-1-reactivating agents as some could potentially be repositioned as candidate drugs to be included in an induction therapy to trigger HIV-1 reactivation.

A micro-Raman spectroscopic investigation of leukemic U-937 cells treated with Crotalaria agatiflora Schweinf and the isolated compound madurensine.

In South Africa traditional medicine plays an important role in primary health care and therefore it is very important that the medicinal use of plants is scientifically tested for toxicity and effectiveness. It was established that the ethanolic extract of the leaves of Crotalaria agatiflora, as well as the isolated compound madurensine, is moderately toxic against leukemic U-937 cells. Light microscopic investigations indicated that symptoms of cell death are induced during treatments, but flow cytometry analysis of treated cells, using annexin-V and propidium iodide, showed that apoptosis and necrosis are insignificantly induced. The Raman results suggested that protein extraction and DNA melting occur in the cells during treatment with the ethanolic extracts (IC(50) value 73.9 µg/mL), drastically changing the molecular content of the cells. In contrast, treatment with madurensine (IC(50) value 136.5 µg/mL), an isolated pyrrolizidine alkaloid from the ethanolic extract of the leaves, did not have the same effect. The results are also compared to that of cells treated with actinomycin D, a compound known to induce apoptosis. The investigation showed that micro-Raman spectroscopy has great promise to be used for initial screening of samples to determine the effects of different treatments on cancerous cell lines together with conventional methods. The results highlight the fact that for many natural products used for medicinal purposes, the therapeutic effect of the crude plant extract tends to be significantly more effective than the particular action of its individual constituents.

Oridonin induces apoptosis in gastric cancer through Apaf-1, cytochrome c and caspase-3 signaling pathway.

AIM: To investigate the effect and mechanism of oridonin on the gastric cancer cell line HGC-27 in vitro. METHODS: The inhibitory effect of oridonin on HGC-27 cells was detected using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. After treatment with 10 µg/mL oridonin for 24 h and 48 h, the cells were stained with acridine orange/ethidium bromide. The morphologic changes were observed under an inverted fluorescence microscope. DNA fragmentation (a hallmark of apoptosis) and lactate dehydrogenase activity were examined using DNA ladder assay and lactate dehydrogenase-release assay. After treated with oridonin (0, 1.25, 2.5, 5 and 10 µg/mL), HGC-27 cells were collected for anexin V-phycoerythrin and 7-amino-actinomycin D double staining and tested by flow cytometric analysis, and oridonin- induced apoptosis in HGC-27 cells was detected. After treatment with oridonin for 24 h, the effects of oridonin on expression of Apaf-1, Bcl-2, Bax, caspase-3 and cytochrome c were also analyzed using reverse-transcript polymerase chain reaction (RT-PCR) and Western blotting. RESULTS: Oridonin significantly inhibited the proliferation of HGC-27 cells in a dose- and time-dependent manner. The inhibition rates of HGC-27 treated with four different concentrations of oridonin for 24 h (1.25, 2.5, 5 and 10 µg/mL) were 1.78% ± 0.36%, 4.96% ± 1.59%, 10.35% ± 2.76% and 41.6% ± 4.29%, respectively, which showed a significant difference (P < 0.05). The inhibition rates of HGC-27 treated with oridonin at the four concentrations for 48 h were 14.77% ± 4.21%, 21.57% ± 3.75%, 30.31% ± 4.91% and 61.19% ± 5.81%, with a significant difference (P < 0.05). The inhibition rates of HGC-27 treated with oridonin for 72 h at the four concentrations were 25.77% ± 4.85%, 31.86% ± 3.86%, 48.30% ± 4.16% and 81.80% ± 6.72%, with a significant difference (P < 0.05). Cells treated with oridonin showed typical apoptotic features with acridine orange/ethidium bromide staining. After treatment with oridonin, the cells became round, shrank, and developed small buds around the nuclear membrane while forming apoptotic bodies. Lactate dehydrogenase (LDH) release assay showed that after treated with 1.25 µg/mL and 20 µg/mL oridonin for 24 h, LDH release of HGC-27 caused by apoptosis increased from 22.94% ± 3.8% to 52.68% ± 2.4% (P < 0.001). However, the change in the release of LDH caused by necrosis was insignificant, suggesting that the major cause of oridonin-induced HGC-27 cell death was apoptosis. Flow cytometric analysis also revealed that oridonin induced significant apoptosis compared with the controls (P < 0.05). And the apoptosis rates of HGC-27 induced by the four different concentrations of oridonin were 5.3% ± 1.02%, 12.8% ± 2.53%, 28.5% ± 4.23% and 49.6% ± 3.76%, which were in a dose-dependent manner (P < 0.05). After treatment for 24 h, DNA ladder showed that oridonin induced a significant increase in DNA fragmentation in a dose-dependent manner. RT-PCR revealed that mRNA expression levels were up-regulated compared with the controls in caspase-3 (0.917 ± 0.103 vs 0.357 ± 0.019, P < 0.05), cytochrome c (1.429 ± 0.111 vs 1.002 ± 0.014, P < 0.05), Apaf-1 (0.688 ± 0.101 vs 0.242 ± 0.037, P < 0.05) and Bax (0.856 ± 0.101 vs 0.278 ± 0.027, P < 0.05) (P < 0.05), whereas down-regulated in Bcl-2 (0.085 ± 0.012 vs 0.175 ± 0.030, P < 0.05). Western blotting analysis also confirmed this result. CONCLUSION: Apoptosis of HGC-27 induced by oridonin may be associated with differential expression of Apaf-1, caspase-3 and cytochrome c, which are highly dependent upon the mitochondrial pathway.

Stabilization of small heterodimer partner mRNA by grape seed procyanidins extract in cultured hepatocytes.

SCOPE: Consumption of dietary grape seed procyanidins extract (GSPE) has improved the plasma lipid profile in humans and experimental animals. The effect of GSPE on the reduction of the postprandial plasma triglyceride (TG) levels has been attributed to the activation of the small heterodimer partner (SHP). GSPE increases SHP gene expression in rat liver and the TG-lowering effect of GSPE is abolished in SHP-deficient mice. However, the mechanism by which GSPE increases SHP mRNA levels remains unclear. This study addressed the effect of GSPE on SHP mRNA stability. METHODS AND RESULTS: The present study shows for the first time that SHP mRNA is rapidly degraded, as measured by actinomycin D-based mRNA chase experiments, and GSPE transiently stabilizes SHP mRNA in HepG2 cells. This degradative effect was completely abolished with 2 h of prolonged treatment with GSPE. However, treatment of fresh HepG2 cells with a pretreated GSPE-containing medium also stabilized SHP mRNA, indicating that GSPE inactivation is not responsible for the transient effects that GSPE has on SHP mRNA stability. CONCLUSION: SHP expression is intricately controlled by mRNA stabilization, which is transiently increased by GSPE, along with at the transcriptional and posttranslational levels.

Ginkgo biloba extract suppresses endotoxin-mediated monocyte activation by inhibiting nitric oxide- and tristetraprolin-mediated toll-like receptor 4 expression.

Monocytes expressing toll-like receptor 4 (TLR4) play a major role in regulating the innate immune response and are involved in systemic inflammation. Previous studies have shown that Ginkgo biloba extract (GBE) may act as a therapeutic agent for some cardiovascular and neurological disorders. The objective of this study was to determine whether GBE could modulate immunity in human cells. The monocytic cell line THP-1 was used. Enzyme-linked immunosorbent assay results showed that lipopolysaccharide (LPS) induces the expression of monocyte chemotactic protein-1 (MIP-1), tumor necrosis factor-α, stromal cell-derived factor-1, and MIP-1α, and this induction may be repressed by GBE treatment due to TLR4 blockade. The Griess reagent assay and western blot analysis showed that GBE-mediated inhibition of TLR4 expression was associated with the activation of mitogen-activated protein kinase and production of nitric oxide (NO). Actinomycin D chase experiments demonstrated that GBE decreased the TLR4 mRNA stability in cells. Confocal microscopy and real-time polymerase chain reaction showed that GBE induced the expression of intracellular tristetraprolin (TTP). Transfection with TTP siRNA reversed the effects of GBE in naïve or TLR4-overexpressing cells. Treatment with SNAP (an NO donor) may increase intracellular TTP expression in cells. Immunoprecipitation analysis showed that GBE mediates TTP activation and increases the interaction of TTP with the 3' untranslated region (UTR) of TLR4 mRNA by regulating NO production. Our findings indicate that GBE could decrease the sensitivity of monocytes to LPS. Utilizing TTP to control TLR4 expression may be a promising approach for controlling systemic inflammation, and GBE may have potential applications in the clinical treatment of immune diseases.

Involvement of the 3'-untranslated region of the brain-derived neurotrophic factor gene in activity-dependent mRNA stabilization.

Although gene transcription is controlled by neuronal activity, little is known about post-transcriptional regulation in neurons. Using cultured neurons, we found that the half-life of immediate-early gene transcripts is prolonged or shortened by membrane depolarization. Focusing on the activity-dependent stabilization of brain-derived neurotrophic factor (BDNF) mRNA, we constructed a series of plasmids, in which the short 3'-untranslated region (3'-UTR) of the BDNF gene was fused to the firefly luciferase gene, and found that the 3'-UTR prevented destabilization of luciferase mRNA through Ca(2+) signals evoked via depolarization. No such prevention was observed with the simian virus 40 late poly(A) site. The pre-mRNA covering the entire short 3'-UTR, where multiple poly(A) sites including novel ones are located, was stabilized. Deletion analyses of 3'-UTR revealed a core region (about 130 bases long) and a complementary region to be responsible for the prevention, well consistent with the formation of an extended stem-loop RNA structure and the production of poly(A) mRNAs. Thus, the mRNA stability is activity-dependently controlled in neurons and distinct regions of the 3'-UTR of BDNF mRNA are involved in stabilizing mRNA in response to Ca(2+) signals, suggesting a primary role of the RNA secondary structure affecting the availability of poly(A) sites in activity-dependent mRNA stabilization.

Bee venom protects hepatocytes from tumor necrosis factor-alpha and actinomycin D.

Honeybee (Apis mellifera) venom (BV) has a broad array of therapeutic applications in traditional medicine to treat variety of diseases. It is also known that BV possesses anti-inflammatory and anticancer effect and that it can inhibit proliferation and induces apoptosis in cancer cells, but there is no evidence of information regarding anti-apoptosis of BV on hepatocytes. In the present study, we investigated the anti-apoptotic effect of BV on tumor necrosis factor (TNF)-alpha with actinomycin (Act) D induces apoptosis in hepatocytes. TNF-alpha/Act D-treated hepatocytes were exposed to different low concentration (1, 10 and 100 ng/mL) of BV. Our results showed statistically significant inhibition in DNA damage caused by BV treatment compared to corresponding TNF-alpha/Act D-treated hepatocytes. BV suppressed TNF-alpha/Act Dtreated activation of bcl-2 family and caspase family, which resulted in inhibition of cytochrome c release and PARP cleavage. These results demonstrate that low concentration BV possess a potent suppressive effect on anti-apoptotic responses of TNF-alpha/Act D-treated hepatocytes and suggest that these compounds may contribute substantial therapeutic potential for the treatment of liver diseases.

Protein kinase C mediated intracellular signaling pathways are involved in the regulation of sodium-dependent glucose co-transporter SGLT1 activity.

The sodium-dependent glucose co-transporter (SGLT1) is regulated by protein kinases. The aim of the present study was to examine the role of protein kinase C (PKC) in the regulation of rabbit (rb) SGLT1 activity as determined by alpha-methyl-D-glucopyranoside (AMG) uptake and to identify the cellular mechanisms involved in this process. For this purpose Chinese hamster ovary cells expressing rbSGLT1 (CHO-G6D3) were treated with PKC activators and inhibitors. PKC activators did not exert any effect on AMG uptake, as corroborated by mutation of the putative phosphorylation sites of PKC. In contrast, the PKC inhibitor bisindolylmaleimide I (BIM) increased AMG uptake. This effect was associated with translocation of rbSGLT1 from the intracellular pool to the plasma membrane demonstrated by pre-treatment of G6D3 cells with cytochalasin D that abolished the effect of BIM. In addition, intracellular signaling pathways (p38/MAPK, ERK/MAPK, JNK/MAPK, and PI3K/Akt/mTOR) were associated with PKC-regulated AMG uptake. Moreover, rbSGLT1 mRNA level was higher in BIM-treated cells than in untreated, control cells. This effect was completely abolished by actinomycin D treatment. The present study demonstrates that PKC regulates rbSGLT1 activity via a complex intracellular mechanism that involves sorting and transcriptional regulation of rbSGLT1. The study findings suggest the involvement of two complementary opposite mechanism of action, in which the balance between two antagonistic effects, namely stimulation and inhibition of the transporter, regulates the activity of rbSGLT1 by PKC.