Plant-growth inhibitory activity of heliannuol derivatives

In addition to (+)-, (-)- and (+/-)-heliannuol E, growth-inhibitory activities of five synthetic chromans and four tetrahydrobenzo[b]oxepins were examined against oat and cress. All heliannuol E isomers exhibited similar biological activities against cress, whereas when tested against oat roots, the unnatural optical isomer (+) showed no inhibitory activity. Four brominated chromans and two tetrahydrobenzo[b]oxepin derivatives also showed apparent inhibition against both cress and oat.     

Nerve growth factor-induced glutamate release is via p75 receptor,ceramide, and Ca(2+) from ryanodine receptor in developing cerebellar neurons

Very little is known about the contribution of a low affinity neurotrophin receptor, p75, to neurotransmitter release. Here we show that nerve growth factor (NGF) induced a rapid release of glutamate and an increase of Ca2+ in cerebellar neurons through a p75-dependent pathway. The NGF-induced release occurred even in the presence of the Trk inhibitor K252a. The release caused by NGF but not brain-derived neurotrophic factor was enhanced in neurons overexpressing p75. Further, after transfection of p75-small interfering RNA, which down-regulated the endogenous p75 expression, the NGF-induced release was inhibited, suggesting that the NGF-induced glutamate release was through p75. We found that the NGF-increased Ca2+ was derived from the ryanodine-sensitive Ca2+ receptor and that the NGF-increased Ca2+ was essential for the NGF-induced glutamate release. Furthermore, scyphostatin, a sphingomyelinase inhibitor, blocked the NGF-dependent Ca2+ increase and glutamate release, suggesting that a ceramide produced by sphingomyelinase was required for the NGF-stimulated Ca2+ increase and glutamate release. This action of NGF only occurred in developing neurons whereas the brain-derived neurotrophic factor-mediated Ca2+ increase and glutamate release was observed at the mature neuronal stage. Thus, we demonstrate that NGF-mediated neurotransmitter release via the p75-dependent pathway has an important role in developing neurons.     

Adenoviral transfection of hepatocytes with the thioredoxin gene confers protection against apoptosis and necrosis

A recombinant adenovirus vector containing the human thioredoxin (TRX) gene was constructed using the Cre-loxP recombination system and used to transfect rat hepatocytes with very high efficiency. The TRX gene was expressed in a dose-dependent manner and significantly modulated rat cellular functions. The TRX gene conferred resistance to oxidative stress, such as hydrogen peroxide treatment, on the host hepatocytes. FACS analysis of DNA fragmentation showed that the TRX gene suppressed hepatocyte apoptosis. It also significantly extended the life span of hepatocytes cultured conventionally on polystyrene plates. Liver-specific functions were maintained in the viability-modulated hepatocytes. Moreover, TRX expression did not affect hepatocyte spheroid formation and it extensively suppressed necrosis in the internal cells. Thus, the transfection of hepatocytes with the TRX gene successfully confers global maintenance of liver functions. These findings provide important information for the development of bioartificial liver support systems and gene therapy for liver diseases.     

Solution structure of the fibronectin type III domain from Bacillus circulans WL-12 chitinase A1

Growing evidence suggests that horizontal gene transfer plays an integral role in the evolution of bacterial genomes. One of the debated examples of horizontal gene transfer from animal to prokaryote is the fibronectin type III domain (FnIIID). Certain extracellular proteins of soil bacteria contain an unusual cluster of FnIIIDs, which show sequence similarity to those of animals and are likely to have been acquired horizontally from animals. Here we report the solution structure of the FnIIID of chitinase A1 from Bacillus circulans WL-12. To the best of our knowledge, this is the first tertiary structure to be reported for an FnIIID from a bacterial protein. The structure of the domain shows significant similarity to FnIIIDs from animal proteins. Sequence comparisons with FnIIIDs from other soil bacteria proteins show that the core-forming residues are highly conserved and, thus, are under strong evolutionary pressure. Striking similarities in the tertiary structures of bacterial FnIIIDs and their mammalian counterparts may support the hypothesis that the evolution of the FnIIID in bacterial carbohydrases occurred horizontally. The total lack of surface-exposed aromatic residues also suggests that the role of this FnIIID is different from those of other bacterial beta-sandwich domains, which function as carbohydrate-binding modules.     

Isolation and metabolic characteristics of previously uncultured members of the order aquificales in a subsurface gold mine

Culture-dependent and -independent techniques were combined to characterize the physiological properties and the ecological impacts of culture-resistant phylotypes of thermophiles within the order Aquificales from a subsurface hot aquifer of a Japanese gold mine. Thermophilic bacteria phylogenetically associated with previously uncultured phylotypes of Aquificales were successfully isolated. 16S ribosomal DNA clone analysis of the entire microbial DNA assemblage and fluorescence in situ whole-cell hybridization analysis indicated that the isolates dominated the microbial population in the subsurface aquifer. The isolates were facultatively anaerobic, hydrogen- or sulfur/thiosulfate-oxidizing, thermophilic chemolithoautotrophs utilizing molecular oxygen, nitrate, ferric iron, arsenate, selenate, and selenite as electron acceptors. Their versatile energy-generating systems may reflect the geochemical conditions of their habitat in the geothermally active subsurface gold mine.     

Regulatory expression of MDP77 protein in the skeletal and cardiac muscles

The mdp77 gene was first cloned from the cDNA library of denervated chick muscles, while its role(s) in vivo was unknown. In the present study, using specific polyclonal antibodies against MDP77, we show that MDP77 was expressed specifically in the skeletal and cardiac muscle, and confirm its presence in the cytoplasm of the extrafusal muscle fibers. In mature muscles, MDP77 immunoreactivity was observed in a repetitive manner along the sarcomere. The onset of MDP77 expression occurred just after myotube formation both in vivo and in vitro. Furthermore, MDP77 was enriched in the intrafusal muscle fibers. Our findings suggest that MDP77 plays an important role(s) in the differentiation, maturation and function of both the skeletal and cardiac muscles.     

Basic fibroblast growth factor evokes a rapid glutamate release through activation of the MAPK pathway in cultured cortical neurons

We examined the possibility that basic fibroblast growth factor (bFGF) is involved in synaptic transmissions. We found that bFGF rapidly induced the release of glutamate and an increase in the intracellular Ca2+ concentration through voltage-dependent Ca2+ channels in cultured cerebral cortical neurons. bFGF also evoked a significant influx of Na+. Tetanustoxin inhibited the bFGF-induced glutamate release, revealing that bFGF triggered exocytosis. The mitogen-activated protein kinase (MAPK) pathway was required for these acute effects of bFGF. We also found that pretreatment with bFGF significantly enhanced high K+-elicited glutamate release also in a MAPK activation-dependent manner. Therefore, we propose that bFGF exerts promoting effects on excitatory neuronal transmission via activation of the MAPK pathway.     

Cloning and characterization of a novel synaptosome-enriched mRNA that encodes 31 kDa protein

Although a subpopulation of mRNAs has been identified as translocated to the dendrites or the synaptic regions of neurons, the translocational mechanism has not been elucidated. To find mRNAs enriched in synapses, we compared the synaptosomal mRNAs with those from whole forebrain using differential display (DD). We cloned one of these mRNAs, which encoded a novel 31 kDa protein (PMES-2). PMES-2 mRNA was specifically transcribed in the brain and was present in the dendrites of the hippocampal neurons. PMES-2 protein was partly localized in the postsynaptic density. Although this protein is very similar to human NABC1 protein, its function is still unknown.     

TAK1-TAB1 fusion protein: a novel constitutively active mitogen-activated protein kinase kinase kinase that stimulates AP-1 and NF-kappaB signaling pathways

TAK1 mitogen-activated protein kinase kinase kinase (MAP3K) is activated by its specific activator, TAK1-binding protein 1 (TAB1). A constitutively active TAK1 mutant has not yet been generated due to the indispensable requirement of TAB1 for TAK1 kinase activity. In this study, we generated a novel constitutively active TAK1 by fusing its kinase domain to the minimal TAK1-activation domain of TAB1. Co-immunoprecipitation assay demonstrated that these domains interacted intra-molecularly. The TAK1-TAB1 fusion protein showed a significant MAP3K activity in vitro and activated c-Jun N-terminal kinase/p38 MAPKs and IkappaB kinase in vivo, which was followed by increased production of interleukin-6. These results indicate that the fusion protein is useful for characterizing the physiological roles of the TAK1-TAB1 complex.