Regulation of the IGFBP-5 and MMP-13 genes by the microRNAs miR-140 and miR-27a in human osteoarthritic chondrocytes

<p>Abstract</p> <p>Background</p> <p>MMP-13 and IGFBP-5 are important factors involved in osteoarthritis (OA). We investigated whether two highly predicted microRNAs (miRNAs), miR-140 and miR-27a, regulate these two genes in human OA chondrocytes.</p> <p>Methods</p> <p>Gene expression was determined by real-time PCR. The effect of each miRNA on IGFBP-5 and MMP-13 expression/production was evaluated by transiently transfecting their precursors (pre-miRNAs) and inhibitors (anti-miRNAs) into human OA chondrocytes. Modulation of IGFBP-5, miR-140 and miR-27a expression was determined upon treatment of OA chondrocytes with cytokines and growth factors.</p> <p>Results</p> <p>IGFBP-5 was expressed in human chondrocytes with its level significantly lower (p < 0.04) in OA. Five computational algorithms identified miR-140 and miR-27a as possible regulators of MMP-13 and IGFBP-5 expression. Data showed that both miRNAs were expressed in chondrocytes. There was a significant reduction (77%, p < 0.01) in miR-140 expression in OA compared to the normal chondrocytes, whereas miR-27a expression was only slightly decreased (23%). Transfection with pre-miR-140 significantly decreased (p = 0.0002) and with anti-miR-140 significantly increased (p = 0.05) IGFBP-5 expression at 24 hours, while pre-miR-27a did not affect either MMP-13 or IGFBP-5. Treatment with anti-miR-27a, but not with anti-miR-140, significantly increased the expression of both MMP-13 (p < 0.05) and IGFBP-5 (p < 0.01) after 72 hours of incubation. MMP-13 and IGFBP-5 protein production followed the same pattern as their expression profile. These data suggest that IGFBP-5 is a direct target of miR-140, whereas miR-27a down-regulates, likely indirectly, both MMP-13 and IGFBP-5.</p> <p>Conclusion</p> <p>This study is the first to show the regulation of these miRNAs in human OA chondrocytes. Their effect on two genes involved in OA pathophysiology adds another level of complexity to gene regulation, which could open up novel avenues in OA therapeutic strategies.</p

Hydrocephalus induces dynamic spatiotemporal regulation of aquaporin-4 expression in the rat brain

<p>Abstract</p> <p>Background</p> <p>The water channel protein aquaporin-4 (AQP4) is reported to be of possible major importance for accessory cerebrospinal fluid (CSF) circulation pathways. We hypothesized that changes in AQP4 expression in specific brain regions correspond to the severity and duration of hydrocephalus.</p> <p>Methods</p> <p>Hydrocephalus was induced in adult rats (~8 weeks) by intracisternal kaolin injection and evaluated after two days, one week and two weeks. Using magnetic resonance imaging (MRI) we quantified lateral ventricular volume, water diffusion and blood-brain barrier properties in hydrocephalic and control animals. The brains were analysed for AQP4 density by western blotting and localisation by immunohistochemistry. Double fluorescence labelling was used to study cell specific origin of AQP4.</p> <p>Results</p> <p>Lateral ventricular volume was significantly increased over control at all time points after induction and the periventricular apparent diffusion coefficient (ADC) value significantly increased after one and two weeks of hydrocephalus. Relative AQP4 density was significantly decreased in both cortex and periventricular region after two days and normalized after one week. After two weeks, periventricular AQP4 expression was significantly increased. Relative periventricular AQP4 density was significantly correlated to lateral ventricular volume. AQP4 immunohistochemical analysis demonstrated the morphological expression pattern of AQP4 in hydrocephalus in astrocytes and ventricular ependyma. AQP4 co-localized with astrocytic glial fibrillary acidic protein (GFAP) in glia limitans. In vascular structures, AQP4 co-localized to astroglia but not to microglia or endothelial cells.</p> <p>Conclusions</p> <p>AQP4 levels are significantly altered in a time and region dependent manner in kaolin-induced hydrocephalus. The presented data suggest that AQP4 could play an important neurodefensive role, and may be a promising future pharmaceutical target in hydrocephalus and CSF disorders.</p

Ubiquitous molecular substrates for associative learning and activity-dependent neuronal facilitation.

Recent evidence suggests that many of the molecular cascades and substrates that contribute to learning-related forms of neuronal plasticity may be conserved across ostensibly disparate model systems. Notably, the facilitation of neuronal excitability and synaptic transmission that contribute to associative learning in Aplysia and Hermissenda, as well as associative LTP in hippocampal CA1 cells, all require (or are enhanced by) the convergence of a transient elevation in intracellular Ca2+ with transmitter binding to metabotropic cell-surface receptors. This temporal convergence of Ca2+ and G-protein-stimulated second-messenger cascades synergistically stimulates several classes of serine/threonine protein kinases, which in turn modulate receptor function or cell excitability through the phosphorylation of ion channels. We present a summary of the biophysical and molecular constituents of neuronal and synaptic facilitation in each of these three model systems. Although specific components of the underlying molecular cascades differ across these three systems, fundamental aspects of these cascades are widely conserved, leading to the conclusion that the conceptual semblance of these superficially disparate systems is far greater than is generally acknowledged. We suggest that the elucidation of mechanistic similarities between different systems will ultimately fulfill the goal of the model systems approach, that is, the description of critical and ubiquitous features of neuronal and synaptic events that contribute to memory induction

Circulating microRNAs as potential diagnostic biomarkers for osteoporosis

Osteoporosis is the most common age-related bone disease worldwide and is usually clinically asymptomatic until the first fracture happens. MicroRNAs are critical molecular regulators in bone remodelling processes and are stabilised in the blood. The aim of this project was to identify circulatory microRNAs associated with osteoporosis using advanced PCR arrays initially and the identified differentially-expressed microRNAs were validated in clinical samples using RT-qPCR. A total of 161participants were recruited and 139 participants were included in this study with local ethical approvals prior to recruitment. RNAs were extracted, purified, quantified and analysed from all serum and plasma samples. Differentially-expressed miRNAs were identified using miRNA PCR arrays initially and validated in 139 serum and 134 plasma clinical samples using RT-qPCR. Following validation of identified miRNAs in individual clinical samples using RT-qPCR, circulating miRNAs, hsa-miR-122-5p and hsa-miR-4516 were statistically significantly differentially-expressed between non-osteoporotic controls, osteopaenia and osteoporosis patients. Further analysis showed that the levels of these microRNAs were associated with fragility fracture and correlated with the low bone mineral density in osteoporosis patients. The results show that circulating hsa-miR-122-5p and hsa-miR-4516 could be potential diagnostic biomarkers for osteoporosis in the future

Monotropastrum humile var. humile is associated with diverse ectomycorrhizal Russulaceae fungi in Japanese forests

The original publication is available at www.springerlink.comArticleECOLOGICAL RESEARCH. 23(6):983-993(2008)journal articl

Mycorrhizal synthesis between Pisolithus arhizus and adult clones of Arbutus unedo in vitro and in nursery

Arbutoid mycorrhizae were synthesized between adult selected clones of Arbutus unedo L. and Pisolithus arhizus. Two micropropagated clones were tested: AL1, in vitro and C1 (acclimatized plants) in nursery and later in a field trial. In vitro, rooted shoots were transferred to test tubes containing the substrate previously inoculated with mycelium cultured on agar. In the nursery, two inoculation treatments were tested (vegetative inocula or dry sporocarps) and compared to control plants. In the field trial, plants from nursery inoculation treatments were compared and an additional control treatment using seedlings was implemented. Plant height was evaluated 4 months later in the nursery and 20 months later in the field trial. Roots were examined by morphological and histological studies: a) in vitro plantlets one month after inoculation and nine months after acclimatization; and b) 20 months after the field trial was established. Arbutoid mycorrhizae were observed in vitro one month after inoculation, indicating compatibility between A. unedo and P. arhizus. These showed the presence of a mantle, Hartig net, and intracellular hyphal complexes confined to the epidermal root cells. Arbutoid mycorrhizae were also observed nine months after acclimatiza- Fund project: This work was supported by a PhD fellowship (SFRH/BD/37170/2007) from the Portuguese Foundation for Science and Technology (FCT) The online version is available at http://link.springer.com Filomena Gomes ( ) • Esteban San Martin Filomena Gomes. CERNAS, Dep. Recursos Florestais, Escola Superior Agrária Coimbra, Bencanta, 3040-316, Coimbra, Portugal, Tel: 351 239 802940, Fax: 351 239 802979, Email: [email protected] Helena Machado INIAV, Instituto Nacional de Investigação Agrária e Veterinária, IP., Av. República, Quinta do Marquês 2780-159 Oeiras, Portugal A. Portugal • Jorge M. Canhoto Centre of Functional Ecology, Department of Life Sciences, University of Coimbra, Ap. 3046, 3001-401 Coimbra, Portugal. Corresponding editor: Chai Ruihai tion in inoculated and control plants. In order to confirm the identity of mycorrhizae, molecular techniques were used, in previously inoculated in vitro plants, 12 months after acclimatization. Thelephora and Hebeloma mycorrhizae, two types of highly competitive and widespread mycorrhizae on nurseries were identified. In the nursery, dry sporocarp treatment improved plant height after four months. In a field trial (20 months later), plants growth did not show significant differences. By this time, mycorrhized roots with Cenococcum geophilum and other types were identified. These results and their implications on A. unedo breeding program are discussed.F. Gomes was supported by a PhD fellowship (SFRH/BD/37170/2007) from the Portuguese Foundation for Science and Technology (FCT)