The Thyroid Hormone Transporter Mct8 Restricts Cathepsin-Mediated Thyroglobulin Processing in Male Mice through Thyroid Auto-Regulatory Mechanisms That Encompass Autophagy.

The thyroid gland is both a thyroid hormone (TH) generating as well as a TH responsive organ. It is hence crucial that cathepsin-mediated proteolytic cleavage of the precursor thyroglobulin is regulated and integrated with the subsequent export of TH into the blood circulation, which is enabled by TH transporters such as monocarboxylate transporters Mct8 and Mct10. Previously, we showed that cathepsin K-deficient mice exhibit the phenomenon of functional compensation through cathepsin L upregulation, which is independent of the canonical hypothalamus-pituitary-thyroid axis, thus, due to auto-regulation. Since these animals also feature enhanced Mct8 expression, we aimed to understand if TH transporters are part of the thyroid auto-regulatory mechanisms. Therefore, we analyzed phenotypic differences in thyroid function arising from combined cathepsin K and TH transporter deficiencies, i.e., in Ctsk-/-/Mct10-/-, Ctsk-/-/Mct8-/y, and Ctsk-/-/Mct8-/y/Mct10-/-. Despite the impaired TH export, thyroglobulin degradation was enhanced in the mice lacking Mct8, particularly in the triple-deficient genotype, due to increased cathepsin amounts and enhanced cysteine peptidase activities, leading to ongoing thyroglobulin proteolysis for TH liberation, eventually causing self-thyrotoxic thyroid states. The increased cathepsin amounts were a consequence of autophagy-mediated lysosomal biogenesis that is possibly triggered due to the stress accompanying intrathyroidal TH accumulation, in particular in the Ctsk-/-/Mct8-/y/Mct10-/- animals. Collectively, our data points to the notion that the absence of cathepsin K and Mct8 leads to excessive thyroglobulin degradation and TH liberation in a non-classical pathway of thyroid auto-regulation.

Metabolome Comparison of Bioactive and Inactive Rhododendron Extracts and Identification of an Antibacterial Cannabinoid(s) from Rhododendron collettianum.

INTRODUCTION: The science of metabolomics offers the possibility to measure full secondary plant metabolomes with limited experimental effort to allow identification of metabolome differences using principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) of liquid chromatography mass spectrometry (LC-MS) data. OBJECTIVE: To demonstrate a bioinformatics driven hypothesis generator for identification of biologically active compounds in plant crude extracts, which is validated by activity guided fractionation. METHODOLOGY: Crude extracts of Rhododendron leaves were tested for their antibacterial activity using agar diffusion and minimum inhibitory concentration assays. Extracts were profiled by LC-MS. PCA and PLS-DA were used for differentiation of bioactive and inactive extracts and their metabolites. Preparative-high performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy were used for separation and structure elucidation of pure compound(s) respectively. RESULTS: An antibacterial Rhododendron collettianum was compared to a series of inactive extracts. Three metabolites were found to distinguish R. collettianum from other species indicating the ability of PCA and PLS-DA to suggest potential bioactive substances. An activity-guided fractionation of R. collettianum extracts was carried out and cannabiorcichromenic acid (CCA) was identified as antibacterial compound thereby validating the PCA and PLS-DA generated hypothesis. Four mammalian cell lines were used to estimate possible cytotoxicity of CCA. CONCLUSION: It was shown that bioinformatics tools facilitate early stage identification of a biologically active compound(s) using LC-MS data, which reduce complexity and number of separation steps in bioactive screening. Copyright © 2017 John Wiley & Sons, Ltd.

Assessment of cytotoxicity exerted by leaf extracts from plants of the genus Rhododendron towards epidermal keratinocytes and intestine epithelial cells.

BACKGROUND: Rhododendron leaf extracts were previously found to exert antimicrobial activities against a range of Gram-positive bacteria. In this study, we investigated which of the extracts with these antimicrobial properties would be best suited for further exploitation. Specifically, the project aims to identify biologically active compounds that affect bacterial but not mammalian cells when applied in medical treatments such as lotions for ectopic application onto skin, or as orally administered drugs. METHODS: Different concentrations of DMSO-dissolved remnants of crude methanol Rhododendron leaf extracts were incubated for 24 h with cultured epidermal keratinocytes (human HaCaT cell line) and epithelial cells of the intestinal mucosa (rat IEC6 cell line) and tested for their cytotoxic potential. In particular, the cytotoxic potencies of the compounds contained in antimicrobial Rhododendron leaf extracts were assessed by quantifying their effects on (i) plasma membrane integrity, (ii) cell viability and proliferation rates, (iii) cellular metabolism, (iv) cytoskeletal architecture, and (v) determining initiation of cell death pathways by morphological and biochemical means. RESULTS: Extracts of almost all Rhododendron species, when applied at 500 µg/mL, were potent in negatively affecting both keratinocytes and intestine epithelial cells, except material from R. hippophaeoides var. hippophaeoides. Extracts of R. minus and R. racemosum were non-toxic towards both mammalian cell types when used at 50 µg/mL, which was equivalent to their minimal inhibitory concentration against bacteria. At this concentration, leaf extracts from three other highly potent antimicrobial Rhododendron species proved non-cytotoxic against one or the other mammalian cell type: Extracts of R. ferrugineum were non-toxic towards IEC6 cells, and extracts of R. rubiginosum as well as R. concinnum did not affect HaCaT cells. In general, keratinocytes proved more resistant than intestine epithelial cells against the treatment with compounds contained in Rhododendron leaf extracts. CONCLUSIONS: We conclude that leaf extracts from highly potent antimicrobial R. minus and R. racemosum are safe to use at 50 µg/mL in 24-h incubations with HaCaT keratinocytes and IEC6 intestine epithelial cells in monolayer cultures. Extracts from R. rubiginosum as well as R. concinnum or R. ferrugineum are applicable to either keratinocytes or intestinal epithelial cells, respectively. Beyond the scope of the current study, further experiments are required to identify the specific compounds contained in those Rhododendron leaf extracts that exert antimicrobial activity while being non-cytotoxic when applied onto human skin or gastrointestinal tract mucosa. Thus, this study supports the notion that detailed phytochemical profiling and compound identification is needed for characterization of the leaf extracts from specific Rhododendron species in order to exploit their components as supplementary agents in antimicrobial phyto-medical treatments.

Phylogenetic spectrum and analysis of antibacterial activities of leaf extracts from plants of the genus Rhododendron.

BACKGROUND: Plants are traditionally used for medicinal treatment of numerous human disorders including infectious diseases caused by microorganisms. Due to the increasing resistance of many pathogens to commonly used antimicrobial agents, there is an urgent need for novel antimicrobial compounds. Plants of the genus Rhododendron belong to the woody representatives of the family Ericaceae, which are typically used in a range of ethno-medical applications. There are more than one thousand Rhododendron species worldwide. The Rhododendron-Park Bremen grows plants representing approximately 600 of the known Rhododendron species, and thus enables research involving almost two thirds of all known Rhododendron species. METHODS: Twenty-six bacterial species representing different taxonomic clades have been used to study the antimicrobial potential of Rhododendron leaf extracts. Agar diffusion assay were conducted using 80% methanol crude extracts derived from 120 Rhododendron species. Data were analyzed using principal component analysis and the plant-borne antibacterial activities grouped according the first and second principal components. RESULTS: The leaf extracts of 17 Rhododendron species exhibited significant growth-inhibiting activities against Gram-positive bacteria. In contrast, only very few of the leaf extracts affected the growth of Gram-negative bacteria. All leaf extracts with antimicrobial bioactivity were extracted from representatives of the subgenus Rhododendron, with 15 from the sub-section Rhododendron and two belonging to the section Pogonanthum. The use of bacterial multidrug efflux pump mutants revealed remarkable differences in the susceptibility towards Rhododendron leaf extract treatment. CONCLUSIONS: For the first time, our comprehensive study demonstrated that compounds with antimicrobial activities accumulate in the leaves of certain Rhododendron species, which mainly belong to a particular subgenus. The results suggested that common genetic traits are responsible for the production of bioactive secondary metabolite(s) which act primarily on Gram-positive organisms, and which may affect Gram-negative bacteria in dependence of the activity of multidrug efflux pumps in their cell envelope.

Toxicity of cucurbit[7]uril and cucurbit[8]uril: an exploratory in vitro and in vivo study.

Cucurbit[n]urils (CB[n]) are potential stabilizing, solubilizing, activating, and delivering agents for drugs. The toxicity of the macrocyclic host molecules cucurbit[7]uril (CB[7]), the most water-soluble homologue, as well as cucurbit[8]uril (CB[8]) has been evaluated. In vitro studies on cell cultures revealed an IC(50) value of 0.53 +/- 0.02 mM for CB[7], corresponding to around 620 mg of CB[7] per kg of cell material. Live-cell imaging studies performed on cells treated with subtoxic amounts of CB[7] showed no detrimental effects on the cellular integrity as assessed by mitochondrial activity. For CB[8], no significant cytotoxicity was observed within its solubility range. The bioadaptability of the compounds was further examined through in vivo studies on mice, where intravenous administration of CB[7] showed a maximum tolerated dosage of 250 mg kg(-1), while oral administration of a CB[7]/CB[8] mixture showed a tolerance of up to 600 mg kg(-1). The combined results indicate a sufficiently low toxicity to encourage further exploration of CB[n] as additives for medicinal and pharmaceutical use.