Brain‐derived neurotrophic factor protects cementoblasts from serum starvation‐induced cell death

Our previous studies have shown that brain‐derived neurotrophic factor (BDNF) enhances bone/cementum‐related protein gene expression through the TrkB‐c‐Raf‐ERK1/2‐Elk‐1 signaling pathway in cementoblasts, which play a critical role in the establishment of a functional periodontal ligament. To clarify how BDNF regulates survival in cementoblasts, we examined its effects on cell death induced by serum starvation in immortalized human cementoblast‐like (HCEM) cells. BDNF inhibited the death of HCEM cells. Small‐interfering RNA (siRNA) for TRKB, a high affinity receptor for BDNF, and for Bcl‐2, countered the BDNF‐induced decrease in dead cell number. In addition, LY294002, a PI3‐kinase inhibitor; SH‐6, an Akt inhibitor; and PDTC, a nuclear factor kappa B (NF‐κB) inhibitor, but not PD98059, an ERK1/2 inhibitor, abolished the protective effect of BDNF against cell death. BDNF enhanced phosphorylated Akt levels, NF‐κB activity in the nucleus, Bcl‐2 mRNA levels, and mitochondrial membrane potential. The blocking of BDNF's actions by treatment with siRNA in all cases for TRKB and Bcl‐2, LY294002, SH‐6, and PDTC suppressed the enhancement. These findings provide the first evidence that a TrkB‐PI3‐kinase‐Akt‐NF‐κB‐Bcl‐2 signaling pathway triggered by BDNF and the subsequent protective effect of BDNF on mitochondrial membrane potential are required to rescue HCEM cells from serum starvation‐induced cell death. Furthermore, the survival and increased expression of bone/cementum‐related proteins induced by BDNF in HCEM cells occur through different signaling pathways. J. Cell. Physiol. 221: 696–706, 2009. © 2009 Wiley‐Liss, Inc.     

The SI strain of measles virus derived from a patient with subacute sclerosing panencephalitis possesses typical genome alterations and unique amino acid changes that modulate receptor specificity and reduce membrane fusion activity

Subacute sclerosing panencephalitis (SSPE) is a fatal sequela associated with measles and is caused by persistent infection of the brain with measles virus (MV). The SI strain was isolated in 1976 from a patient with SSPE and shows neurovirulence in animals. Genome nucleotide sequence analyses showed that the SI strain genome possesses typical genome alterations for SSPE-derived strains, namely, accumulated amino acid substitutions in the M protein and cytoplasmic tail truncation of the F protein. Through the establishment of an efficient reverse genetics system, a recombinant SI strain expressing a green fluorescent protein (rSI-AcGFP) was generated. The infection of various cell types with rSI-AcGFP was evaluated by fluorescence microscopy. rSI-AcGFP exhibited limited syncytium-forming activity and spread poorly in cells. Analyses using a recombinant MV possessing a chimeric genome between those of the SI strain and a wild-type MV strain indicated that the membrane-associated protein genes (M, F, and H) were responsible for the altered growth phenotype of the SI strain. Functional analyses of viral glycoproteins showed that the F protein of the SI strain exhibited reduced fusion activity because of an E300G substitution and that the H protein of the SI strain used CD46 efficiently but used the original MV receptors on immune and epithelial cells poorly because of L482F, S546G, and F555L substitutions. The data obtained in the present study provide a new platform for analyses of SSPE-derived strains as well as a clear example of an SSPE-derived strain that exhibits altered receptor specificity and limited fusion activity.     

ER-resident Gi2 protein controls sar1 translocation onto the ER during budding of transport vesicles

In our previous study, fluoride ([AlF(4) ](-) ) disturbed ER-to-Golgi transport through the activation of ER-resident heterotrimeric G protein (ER-G protein). Therefore, ER-G protein may be implicated in ER-to-Golgi transport at the early stage prior to coat protein assembly. Sar1 translocation onto the endoplasmic reticulum (ER) membrane is suppressed by non-selective protein kinase inhibitor H89, suggesting the participation of H89-sensitive kinase in this process. To investigate the involvement of ER-G protein in ER-to-Golgi transport, the effect of G(i) protein activator (mastoparan 7) was examined on Sar1 translocation onto the ER in a cell-free system consisting of microsome membrane and cytosol. Sar1 translocation onto the microsome membrane was induced by addition of GTPγS in the cell-free system. Translocation of Sar1 by GTPγS was suppressed significantly by both H89 and mastoparan 7. Mastoparan 7 suppressed the translocation of Sar1 onto the microsome membrane with dosage dependency, but mastoparan 17, the inactive analog of mastoparan 7, had no effect on Sar1 translocation. The suppressive effect of mastoparan 7 was recovered by treatment with pertussis toxin (IAP). Moreover, G(i2) protein was detected on the microsome membrane by western blotting for heterotrimeric G(i) proteins. These results indicate that ER-G(i2) protein modulated Sar1 translocation onto the ER, suggesting that ER-resident G(i2) protein is an important negative regulator of vesicular transport at the early stage of vesicle formation before coat protein assembly on the ER.     

Stable cesium uptake and accumulation capacities of five plant species as influenced by bacterial inoculation and cesium distribution in the soil

The effects of inoculation with Bacillus and Azospirillum strains on growth and cesium accumulation of five plant species, Komatsuna, Amaranth, sorghum, common millet and buckwheat, grown on cesium-spiked soil were assessed for potential use in cesium remediation. Pot experiments were performed using “artificially” Cs-contaminated soil. Three treatments were applied based on Cs location in the soil. For a soil height of 15 cm in the pots, Cs was added as follows: in the top five cm to imitate no ploughing condition; in the bottom five cm simulating inverted ploughing; and uniformly distributed Cs reproducing normal plowing. Generally, inoculation of Cs-exposed plants significantly enhanced growth and tolerance to this element. Transfer factor (ratio of Cs concentration in the plant tissues to that in surrounding soil) was strongly influenced by Cs distribution, with higher values in the top-Cs treatment. Within this treatment, inoculation of Komatsuna with Bacillus and Azospirillum strains resulted in the greatest transfer factors of 6.55 and 6.68, respectively. Cesium content in the shoots was high in the Azospirillum-inoculated Komatsuna, Amaranth, and buckwheat, i.e., 1,830, 1,220, and 1,030 µg per pot, respectively (five plants were grown in each pot). Therefore, inoculation of Komatsuna and Amaranth with the strains tested here could be effective in enhancing Cs accumulation. The decrease of Cs transfer under uniform- and bottom-Cs treatments would suggest that countermeasures aiming at decreasing the transfer of Cs could rely on ploughing practices.     

Changes in the potential habitats of 10 dominant evergreen broad-leaved tree species in the Taiwan-Japan archipelago

Ecosystem vulnerability to climate change remains elusive in the species-rich Taiwan-Japan archipelago. We predicted potential habitats (PHs) of ten dominant evergreen broad-leaved tree species by using the current and twenty potential climate change scenarios using generalised additive models. The presence/absence records of each species, extracted from vegetation database, were used as response variables. Four climatic and one spatial variables were used as explanatory variables. The results showed that the interaction terms of spatial variable, indicating historical range shifts or species interactions, restricted the distribution of all the target species as much as that by the each climatic variable. The PHs of all the target species were predicted to consistently increase, and in particular, to expand northward and upward to the cool temperate zone. However, the PHs were predicted to decrease within the range of 23.6–38.1 % in the Ryukyu Islands for Castanopsis sieboldii and Elaeocarpus japonica, respectively, and within the range of 32.4–42.3 % in Taiwan for Camellia japonica and Distylium racemosum, respectively. These findings suggest that the four species will be vulnerable at the southern range limits; however, the remaining six species will potentially increase within the PH areas in the future at all regions.     

Endogenous neurotoxic dopamine derivative covalently binds to Parkinson's disease‐associated ubiquitin C‐terminal hydrolase L1 and alters its structure and function

Parkinson's disease (PD) is a common neurodegenerative disease, but its pathogenesis remains elusive. A mutation in ubiquitin C‐terminal hydrolase L1 (UCH‐L1) is responsible for a form of genetic PD which strongly resembles the idiopathic PD. We previously showed that 1‐(3′,4′‐dihydroxybenzyl)‐1,2,3,4‐tetrahydroisoquinoline (3′,4′DHBnTIQ) is an endogenous parkinsonism‐inducing dopamine derivative. Here, we investigated the interaction between 3′,4′DHBnTIQ and UCH‐L1 and its possible role in the pathogenesis of idiopathic PD. Our results indicate that 3′,4′DHBnTIQ binds to UCH‐L1 specifically at Cys152 in vitro. In addition, 3′,4′DHBnTIQ treatment increased the amount of UCH‐L1 in the insoluble fraction of SH‐SY5Y cells and inhibited its hydrolase activity to 60%, reducing the level of ubiquitin in the soluble fraction of SH‐SY5Y cells. Catechol‐modified UCH‐L1 as well as insoluble UCH‐L1 were detected in the midbrain of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐treated PD model mice. Structurally as well as functionally altered UCH‐L1 have been detected in the brains of patients with idiopathic PD. We suggest that conjugation of UCH‐L1 by neurotoxic endogenous compounds such as 3′,4′DHBnTIQ might play a key role in onset and progression of idiopathic PD.

     

The inositol 5-phosphatase SHIP2 is an effector of RhoA and is involved in cell polarity and migration

Cell migration is essential for various physiological and pathological processes. Polarization in motile cells requires the coordination of several key signaling molecules, including RhoA small GTPases and phosphoinositides. Although RhoA participates in a front-rear polarization in migrating cells, little is known about the functional interaction between RhoA and lipid turnover. We find here that src-homology 2-containing inositol-5-phosphatase 2 (SHIP2) interacts with RhoA in a GTP-dependent manner. The association between SHIP2 and RhoA is observed in spreading and migrating U251 glioma cells. The depletion of SHIP2 attenuates cell polarization and migration, which is rescued by wild-type SHIP2 but not by a mutant defective in RhoA binding. In addition, the depletion of SHIP2 impairs the proper localization of phosphatidylinositol 3,4,5-trisphosphate, which is not restored by a mutant defective in RhoA binding. These results suggest that RhoA associates with SHIP2 to regulate cell polarization and migration.