MicroRNA-196a promotes renal cancer cell migration and invasion by targeting BRAM1 to regulate SMAD and MAPK signaling pathways

Rationale: MicroRNAs (miRNAs) are endogenous ~22nt RNAs that play critical regulatory roles in various biological and pathological processes, including various cancers. Their function in renal cancer has not been fully elucidated. It has been reported that miR-196a can act as oncogenes or as tumor suppressors depending on their target genes. However, the molecular target for miR-196a and the underlying mechanism in miR-196a promoted cell migration and invasion in renal cancer is still not clear.Methods: The expression, survival and correlation between miR-196a and BRAM1 were investigated using TCGA analysis and validated by RT-PCR and western blot. To visualize the effect of Bram1 on tumor metastasis in vivo, NOD-SCID gamma (NSG) mice were intravenously injected with RCC4 cells (10⁶ cells/mouse) or RCC4 overexpressing Bram1. In addition, cell proliferation assays, migration and invasion assays were performed to examine the role of miR-196a in renal cells in vitro. Furthermore, immunoprecipitation was done to explore the binding targets of Bram1.Results: TCGA gene expression data from renal clear cell carcinoma patients showed a lower level of Bram1 expression in patients'' specimens compared to adjacent normal tissues. Moreover, Kaplan‑Meier survival data clearly show that high expression of Bram1correlates to poor prognosis in renal carcinoma patients. Our mouse metastasis model confirmed that Bram1 overexpression resulted in an inhibition in tumor metastasis. Target-prediction analysis and dual-luciferase reporter assay demonstrated that Bram1 is a direct target of miR-196a in renal cells. Further, our in vitro functional assays revealed that miR-196a promotes renal cell proliferation, migration, and invasion. Rescue of Bram1 expression reversed miR-196a-induced cell migration. MiR-196a promotes renal cancer cell migration by directly targeting Bram1 and inhibits Smad1/5/8 phosphorylation and MAPK pathways through BMPR1A and EGFR.Conclusions: Our findings thus provide a new mechanism on the oncogenic role of miR-196a and the tumor-suppressive role of Bram1 in renal cancer cells. Dysregulated miR-196a and Bram1 represent potential prognostic biomarkers and may have therapeutic applications in renal cancer.     

Isolation and purification of closely related citrus limonoid glucosides by flash chromatography

Several citrus limonoid glycosides have proved to be particularly difficult to purify using conventional techniques. A reversed-phase flash chromatographic technique has been developed for the separation and isolation of the closely related limonoid glucosides, nomilin 17-beta-D-glucopyranoside and nomilinic acid 17-beta-D-glucopyranoside, with confirmation of their identities by electrospray ionisation mass spectrometry. Furthermore, the semi-purification of the mixture of glucosides enriched with flavanone glucosides such as naringin, narirutin and other limonoid glucosides was obtained. The closely eluting glucosides were successfully separated to achieve a good yield and purity of 93%.     

A novel tunnel in mycobacterial type III polyketide synthase reveals the structural basis for generating diverse metabolites

The superfamily of plant and bacterial type III polyketide synthases (PKSs) produces diverse metabolites with distinct biological functions. PKS18, a type III PKS from Mycobacterium tuberculosis, displays an unusual broad specificity for aliphatic long-chain acyl-coenzyme A (acyl-CoA) starter units (C(6)-C(20)) to produce tri- and tetraketide pyrones. The crystal structure of PKS18 reveals a 20 A substrate binding tunnel, hitherto unidentified in this superfamily of enzymes. This remarkable tunnel extends from the active site to the surface of the protein and is primarily generated by subtle changes of backbone dihedral angles in the core of the protein. Mutagenic studies combined with structure determination provide molecular insights into the structural elements that contribute to the chain length specificity of the enzyme. This first bacterial type III PKS structure underlines a fascinating example of the way in which subtle changes in protein architecture can generate metabolite diversity in nature.     

Spermatotoxic effect of carbendazim

Carbendazim, suspended in sunflower oil, was administered to Wistar male rats through an oral intubation at a daily dose of 25 mg/kg body weight for 48 days, and the cauda epididymal sperm were analysed on day 49 for counts, motility and abnormalities. The study indicates that carbendazim affects the cauda epididymal sperm as seen in decreased sperm counts, inhibition of motility and increased incidence of abnormalities.     

Purification and characterization of an extracellular alpha-glucosidase protein from Trichoderma viride which degrades a phytotoxin associated with sheath blight disease in rice

AIMS: To purify and characterize an extracellular alpha-glucosidase from Trichoderma viride capable of inactivating a host-specific phytotoxin, designated RS toxin, produced by the rice sheath blight pathogen, Rhizoctonia solani Kühn. METHODS AND RESULTS: The host-specific RS toxin was purified from both culture filtrates (culture filtrate toxin, CFTox) and R. solani-inoculated rice sheaths (sheath blight toxin, SBTox). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analyses of extracellular proteins, purified from a biocontrol fungus T. viride (TvMNT7) grown on SBTox and CFTox separately, were carried out. The antifungal activity of the purified high molecular weight protein (110 kDa) was studied against RS toxin as well as on the sclerotial germination and mycelial growth of R. solani. Enzyme assay and Western blot analysis with the antirabbit TvMNT7 110-kDa protein indicated that the protein was an alpha-glucosidase. The 110-kDa protein was highly specific to RS toxin and its Michaelis-Menten constant value was 0.40 mmol l-1 when p-nitrophenyl alpha-D-glucopyranoside was used as the substrate. The isoelectric point of the protein was 5.2. N-terminal sequencing of the alpha-glucosidase protein showed that its amino acid sequence showed no homology with other known alpha-glucosidases. CONCLUSION: This appears to be the first report of the purification and characterization of an alpha-glucosidase capable of inactivating a host-specific toxin of fungal origin. The alpha-glucosidase is specific to RS toxin and is different from the known alpha-glucosidases. SIGNIFICANCE AND IMPACT OF THE STUDY: As RS toxin could be inactivated by the microbial alpha-glucosidase enzyme, isolation of the gene that codes for the enzyme from T. viride and transfer of the gene to rice plants would lead to enhanced resistance against sheath blight pathogen by inactivation of RS toxin.     

Inactivation of Rhizoctonia solani toxin by a putative α-glucosidase from coconut leaves for control of sheath blight disease in rice

Inactivation of a host-specific toxin, RS-toxin, induced by Rhizoctonia solani, the cause of rice sheath blight disease was investigated. A putative -glucosidase identified based on enzyme assay and Western blot analysis was purified from coconut (Cocos nucifera; the only known non-host of R. solani) leaves and tested for its efficacy in degrading RS-toxin. SDS–PAGE analysis showed the appearance of a 97 kDa protein, which appeared in proteins extracted from coconut leaf bits during 48 and 96 h after RS-toxin-treatment and the protein eventually disappeared. A comparison of the u.v. spectra read at 150–300 nm revealed conspicuous disturbances in the absorbance at 24 h of incubation of RS-toxin with the coconut leaf protein extracts as compared to that at 12 h, indicating the possible degradation of RS-toxin by coconut leaf -glucosidase during incubation. Incubation of rice leaf sheath bits with coconut leaf protein extracts significantly reduced electrolyte leakage due to RS-toxin 30 min after the toxin treatment. Simultaneously, there was a significant reduction in sheath blight symptoms when the incubation of rice leaf sheaths with the coconut leaf protein extracts was extended up to 96 or 120 h. This appears to be the first report of purification and characterization of a putative plant -glucosidase.     

Endonucleolytic cleavage of murine leukemia virus 35S RNA by microsome-associated nuclease.

Moloney murine leukemia virus 35S RNA (molecular weight 3 to 3.4 × 10⁶) is cleaved by nuclease activity present in microsomal fractions from MLV infected or uninfected mouse embryo cells to two RNA species of approximate molecular weights 1.8 × 10⁶ and 1.5 × 10⁶. Microsomal fractions from MLV infected and uninfected cells also contained nucleolytic activity that solubilized [³H]poly(A)·poly(U) but not [³H]poly(C) or [³H]poly(U); the cleavage of poly(A)·poly(U) was inhibited by ethidium bromide. The cleavage of MLV RNA was also inhibited by ethidium bromide, suggesting double stranded regions in 35S RNA as the site of cleavage.