Calcium (Ca) homeostasis is essential for neuronal function and survival. Altered Ca homeostasis has been consistently observed in neurological diseases. How Ca homeostasis is achieved in various cellular compartments of disease-relevant cell types is not well understood. Here we show in Parkinson's disease (PD) models that Ca transport from the endoplasmic reticulum (ER) to mitochondria through the ER-mitochondria contact site (ERMCS) critically regulates mitochondrial Ca (mito-Ca) homeostasis in dopaminergic (DA) neurons, and that the PD-associated PINK1 protein modulates this process. In mutant DA neurons, the ERMCS is strengthened and mito-Ca level is elevated, resulting in mitochondrial enlargement and neuronal death. Miro, a well-characterized component of the mitochondrial trafficking machinery, mediates the effects of PINK1 on mito-Ca and mitochondrial morphology, apparently in a transport-independent manner. Miro overexpression mimics loss-of-function effect, whereas inhibition of Miro or components of the ERMCS, or pharmacological modulation of ERMCS function, rescued mutant phenotypes. Mito-Ca homeostasis is also altered in the LRRK2-G2019S model of PD and the PAR-1/MARK model of neurodegeneration, and genetic or pharmacological restoration of mito-Ca level is beneficial in these models. Our results highlight the importance of mito-Ca homeostasis maintained by Miro and the ERMCS to mitochondrial physiology and neuronal integrity. Targeting this mito-Ca homeostasis pathway holds promise for a therapeutic strategy for neurodegenerative diseases.

Background Cytoplasmic inclusions of transactive response DNA binding protein of 43 kDa (TDP-43) in neurons and astrocytes are a feature of some neurodegenerative diseases, such as frontotemporal lobar degeneration with TDP-43 (FTLD-TDP) and amyotrophic lateral sclerosis (ALS). However, the role of TDP-43 in astrocyte pathology remains largely unknown. Methods To investigate whether TDP-43 overexpression in primary astrocytes could induce inflammation, we transfected primary astrocytes with plasmids encoding Gfp or TDP-43-Gfp. The inflammatory response and upregulation of PTP1B in transfected cells were examined using quantitative RT-PCR and immunoblot analysis. Neurotoxicity was analysed in a transwell coculture system of primary cortical neurons with astrocytes and cultured neurons treated with astrocyte-conditioned medium (ACM). We also examined the lifespan, performed climbing assays and analysed immunohistochemical data in pan-glial TDP-43-expressing flies in the presence or absence of a Ptp61f RNAi transgene. Results PTP1B inhibition suppressed TDP-43-induced secretion of inflammatory cytokines (interleukin 1 beta (IL-1β), interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-α)) in primary astrocytes. Using a neuron-astrocyte coculture system and astrocyte-conditioned media treatment, we demonstrated that PTP1B inhibition attenuated neuronal death and mitochondrial dysfunction caused by overexpression of TDP-43 in astrocytes. In addition, neuromuscular junction (NMJ) defects, a shortened lifespan, inflammation and climbing defects caused by pan-glial overexpression of TDP-43 were significantly rescued by downregulation of ptp61f (the Drosophila homologue of PTP1B) in flies. Conclusions These results indicate that PTP1B inhibition mitigates the neuronal toxicity caused by TDP-43-induced inflammation in mammalian astrocytes and Drosophila glial cells.

We investigated potential protein markers of post-mortem interval (PMI) using rat kidney and psoas muscle. Tissue samples were taken at 12 h intervals for up to 96 h after death by suffocation. Expression levels of eight soluble proteins were analyzed by Western blotting. Degradation patterns of selected proteins were clearly divided into three groups: short-term, mid-term, and long-term PMI markers based on the half maximum intensity of intact protein expression. In kidney, glycogen synthase (GS) and glycogen synthase kinase-3β were degraded completely within 48 h making them short-term PMI markers. AMP-activated protein kinase α, caspase 3 and GS were short-term PMI markers in psoas muscle. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was a mid-term PMI marker in both tissues. Expression levels of the typical long-term PMI markers, p53 and β-catenin, were constant for at least 96 h post-mortem in both tissues. The degradation patterns of GS and caspase-3 were verified by immunohistochemistry in both tissues. GAPDH was chosen as a test PMI protein to perform a lateral flow assay (LFA). The presence of recombinant GAPDH was clearly detected in LFA and quantified in a concentration-dependent manner. These results suggest that LFA might be used to estimate PMI at a crime scene.

In this study, the antibacterial activity of essential oil from Chamaecyparis obtusa (Sieb. et Zucc) leaves and twigs was investigated. The test strains were Klebsiella pneumoniae, Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, Escherichia coli, Legionella pneumophila, and Methicilline-resistant Staphylococcus aureus. Antibacterial activity was estimated by measuring bacterial growth inhibition. Histopathological examination was also performed. C. obtusa oil distinctly inhibited the growth of all test strains and exhibited the strongest antibacterial activity against L. monocytogenes. It was chromatographically divided into several fractions. The fractions were further tested against antibacterial activity and their chemical compositions were analyzed. The fraction containing terpinen-4-ol (TA) showed high antibacterial activity toward all strains tested. Tests with authentic samples showed that TA played a major role in the antibacterial activity of C. obtusa oil, and in a mice test, the oil actively minimized inflammation by S. aureus.

No abstract

Key words: Amorpha fruticosa, isoflavone glucosides, 3′-hydroxy-4′-methoxyisoflavone-7-O-β-D-glucopyranoside,Amorpha fruticosa L. (Leguminosae) is a shrub that flowers in May to June and originates from North America. This plant has used as a chinese folk medicine for hypertension, hematomas, and contutions [1]. A. fruticosa was introduced to Korea through China in 1930s. Although numerous isoflavones [2], flavanones [3,4], and rotenoids [5-7] along with their biological activities have been reported from the fruit, leaf, and root of this plant, the chemical constituents in the bark of A. fruticosa have been less investigated.In this study, the methanol extract from the bark of A. fruticosa was separated by column chromatography and the four known isoflavone glucosides, 3′-hydroxy-4′-methoxyisoflavone-7-O-β-D-glucopyranoside (1), 4′,6-dimethoxyisoflavone-7-O-β-D-glucopyranoside (2), 4′-methoxyisoflavone-7-O-β-D-glucopyranoside (3), and 3′,5-dihydroxy-4′-methoxyisoflavone-7-O-β-Dglucopyranoside (4) were isolated for the first time. Their chemical structures were identified by instrumental analysis using UV, IR, MS, and NMR spectrometer. 1: R 1 = R 2 = H, R 3 = OH 2: R 1 = R 3 = H, R 2 = OMe 3: R 1 = R 2 = R 3 = H 4: R 1 =R 3 = OH, R 2 = H

The sap of Acer mono has been called 'bone-benefit-water' in Korea because of its mineral and sugar content. In particular, the calcium concentration of the sap of A. mono is 37·5 times higher than commercial spring water. In the current study, we examined whether A. mono sap could improve or prevent osteoporosis-like symptoms in a mouse model. Male mice (3 weeks old) were fed a low-calcium diet supplemented with 25, 50 or 100 % A. mono sap, commercial spring water or a high calcium-containing solution as a beverage for 7 weeks. There were no differences in weekly weight gain and food intake among all the groups. Mice that were given a low-calcium diet supplemented with commercial spring water developed osteoporosis-like symptoms. To assess the effect of sap on osteoporosis-like symptoms, we examined serum calcium concentration, and femur density and length, and carried out a histological examination. Serum calcium levels were significantly lower in mice that received a lowcalcium diet supplemented with commercial spring water (the negative control group), and in the 25 % sap group compared to mice fed a normal diet, but were normal in the 50 and 100 % sap and high-calcium solution groups. Femur density and length were significantly reduced in the negative control and 25 % sap groups. These results indicate that a 50 % sap solution can mitigate osteoporosis-like symptoms induced by a lowcalcium diet. We also examined the regulation of expression of calcium-processing genes in the duodenum and kidney. Duodenal TRPV6 and renal calbindin-D9k were up-regulated dose-dependently by sap, and the levels of these factors were higher than those attained in the spring water-treated control. The results demonstrate that the sap of A. mono ameliorates the low bone density induced by a low-calcium diet, most likely by increasing calcium ion absorption.

Resistance against anthracnose fungi was enhanced in transgenic pepper plants that accumulated high levels of a carboxylesterase, PepEST in anthracnose-susceptible fruits, with a concurrent induction of antioxidant enzymes and SA-dependent PR proteins. A pepper esterase gene (PepEST) is highly expressed during the incompatible interaction between ripe fruits of pepper (Capsicum annuum L.) and a hemibiotrophic anthracnose fungus (Colletotrichum gloeosporioides). In this study, we found that exogenous application of recombinant PepEST protein on the surface of the unripe pepper fruits led to a potentiated state for disease resistance in the fruits, including generation of hydrogen peroxide and expression of pathogenesis-related (PR) genes that encode mostly small proteins with antimicrobial activity. To elucidate the role of PepEST in plant defense, we further developed transgenic pepper plants overexpressing PepEST under the control of CaMV 35S promoter. Molecular analysis confirmed the establishment of three independent transgenic lines carrying single copy of transgenes. The level of PepEST protein was estimated to be approximately 0.002 % of total soluble protein in transgenic fruits. In response to the anthracnose fungus, the transgenic fruits displayed higher expression of PR genes, PR3, PR5, PR10, and PepThi, than non-transgenic control fruits did. Moreover, immunolocalization results showed concurrent localization of ascorbate peroxidase (APX) and PR3 proteins, along with the PepEST protein, in the infected region of transgenic fruits. Disease rate analysis revealed significantly low occurrence of anthracnose disease in the transgenic fruits, approximately 30 % of that in non-transgenic fruits. Furthermore, the transgenic plants also exhibited resistance against C. acutatum and C. coccodes. Collectively, our results suggest that overexpression of PepEST in pepper confers enhanced resistance against the anthracnose fungi by activating the defense signaling pathways.