Crystal structure of the rac activator, Asef, reveals its autoinhibitory mechanism

The Rac-specific guanine nucleotide exchange factor (GEF) Asef is activated by binding to the tumor suppressor adenomatous polyposis coli mutant, which is found in sporadic and familial colorectal tumors. This activated Asef is involved in the migration of colorectal tumor cells. The GEFs for Rho family GTPases contain the Dbl homology (DH) domain and the pleckstrin homology (PH) domain. When Asef is in the resting state, the GEF activity of the DH-PH module is intramolecularly inhibited by an unidentified mechanism. Asef has a Src homology 3 (SH3) domain in addition to the DH-PH module. In the present study, the three-dimensional structure of Asef was solved in its autoinhibited state. The crystal structure revealed that the SH3 domain binds intramolecularly to the DH domain, thus blocking the Rac-binding site. Furthermore, the RT-loop and the C-terminal region of the SH3 domain interact with the DH domain in a manner completely different from those for the canonical binding to a polyproline-peptide motif. These results demonstrate that the blocking of the Rac-binding site by the SH3 domain is essential for Asef autoinhibition. This may be a common mechanism in other proteins that possess an SH3 domain adjacent to a DH-PH module.     

A proposed role of oxalic acid in wood decay systems of wood-rotting basidiomycetes

Abstract: The possible roles of oxalic acid, veratryl alcohol, and manganese were investigated in relation to lignin biodegradation by white-rot basidiomycetes. Oxalate inhibited both lignin peroxidase (LiP) and manganese-peroxidase (MnP). and was decarboxylated by the mediation of veratryl alcohol and Mn. Oxalate was shown to regulate the mineralization of lignin in the in vivo system of Phanerochaete chrysosporium . In the brown-rot wood decay process, oxalic acid may serve as an acid catalyst as well as an electron donor for the Fenton reaction, to breakdown cellulose and hemicellulose. Oxaloacetase and glyoxylate oxidase may play a key role in production of oxalic acid by white-rot and brown-rot basidiomycetes such as Phanerochaete chrysosporium, Coriolus versicolor and Tyromyces palustris . A possible role of oxalate metabolism is discussed in relation to the physiology of wood-rotting fungi.     

Involvement of vesicle-cytoskeleton interaction in AQP5 trafficking in AQP5-gene-transfected HSG cells

We examined the regulatory role of a reduction/oxidation (redox) control protein, thioredoxin (TRX), in tumor necrosis factor-alpha (TNF-alpha)-induced p38 MAP kinase activation and p38 MAP kinase-mediated cytokine expression utilizing TRX-transfected murine L929 cells (TRX14). The results showed that TNF-alpha-induced p38 MAP kinase activation and interleukin-6 (IL-6) production by TRX 14 were less than those by the parental L cells and the control transfected L cells (Neo-1). SB 203580 as the specific inhibitor for p38 MAP kinase activity inhibited TNF-alpha-induced IL-6 production by the parental L cells, indicating that TNF-alpha-activated p38 MAP kinase regulates IL-6 production by the cell lines used in this study. These results showed that overexpression of TRX negatively regulates p38 MAP kinase activation and p38 MAP kinase-mediated IL-6 production by TNF-alpha-stimulated cells, indicating that TRX is critical for p38 MAP kinase activation which regulates cytokine expression.     

Histochemical,ultrastructural, and three-dimensional observation of smooth muscle cells in human gastric mucosa

The present study was undertaken to clarify the histochemical and ultrastructural properties and the three-dimensional distribution of the smooth muscle cells (SMCs) located in the lamina propria (LP) of the human gastric mucosa. Standard paraffin sections obtained from stomachs surgically resected for gastric cancer were immunostained for alpha-smooth muscle actin (alpha-SMA), vimentin, desmin, laminin, and type IV collagen. In addition, 100-m-thick sections were fluorostained for alpha-SMA and CD34, while three-dimensional images were prepared by confocal laser scanning microscope. Ultrastructural studies were carried out using normal gastric biopsy specimens. The results indicated that SMCs in the LP differed between the upper and lower regions, SMCs in the lower LP being fairly typical SMCs, whereas those in the upper LP had apparently lost reactivity for desmin but gained that for vimentin. The basal lamina became sparser, but a fibronexus was occasionally seen in SMCs in the upper LP. Three-dimensional images revealed bundles of SMCs in the upper LP encircling several foveolae to form acinus-like structures and, in the upper LP, SMCs branching into fine fibrils with a brush-like (corpus) or besom-like (i.e., a twiggy witchs broom) appearance (antrum).     

Qualitative and quantitative analyses of the decomposition products that arise from the exposure of thymine to monochromatic ultrasoft X rays and 60Co gamma rays in the solid state

HPLC analyses of condensed thymine irradiated with monochromatic synchrotron ultrasoft X rays in the energy region around nitrogen and oxygen K-shell edges were performed. Cobalt-60 gamma rays were used as a reference radiation. The radiation chemical dose response of each separated thymine decomposition product was also determined. Uracil (U), 5-(hydroxymethyl)uracil (HMU), 5,6-dihydrothymine (DHT), 5-formyluracil (foU) and four main unknown products were found in the HPLC chromatogram of the sample irradiated with ultrasoft X rays in vacuo. Similar spectra of the products were also found in the gamma-ray experiment; however, some unknown products that appeared after elution of the thymine peak were significantly larger than those in the ultrasoft X- ray experiment. This result indicates the difference in radiation quality. The G value of DHT produced by gamma radiation was 10 times larger than those produced by the ultrasoft X- ray photons with energies of 395 and 407 eV corresponding to below and on the nitrogen K-shell edge, respectively. This result suggests that the differences in the photon energy and/ or in the energy spectra of the secondary electron between ultrasoft X rays and gamma rays are causing differences in the process of the radiation chemistry. Moreover, the yields of all the thymine decomposition products induced by 538 eV photons (oxygen K-shell edge) were significantly smaller than those induced by photons around the nitrogen K-shell edge. The K-shell excitation of oxygen in thymine may efficiently promote the production of small thymine fragments susceptible to desorption from the sample.     

Salt-inducible multidrug efflux pump protein in the moderately halophilic bacterium Chromohalobacter sp

It has been known that halophilic bacteria often show natural resistance to antibiotics, dyes, and toxic metal ions, but the mechanism and regulation of this resistance have remained unexplained. We have addressed this question by identifying the gene responsible for multidrug resistance. A spontaneous ofloxacin-resistant mutant derived from the moderately halophilic bacterium Chromohalobacter sp. strain 160 showed a two- to fourfold increased resistance to structurally diverse compounds, such as tetracycline, cefsulodin, chloramphenicol, and ethidium bromide (EtBr), and tolerance to organic solvents, e.g., hexane and heptane. The mutant produced an elevated level of the 58-kDa outer membrane protein. This mutant (160R) accumulated about one-third the level of EtBr that the parent cells did. An uncoupler, carbonyl cyanide m-chlorophenylhydrazone, caused a severalfold increase in the intracellular accumulation of EtBr, with the wild-type and mutant cells accumulating nearly equal amounts. The hrdC gene encoding the 58-kDa outer membrane protein has been cloned. Disruption of this gene rendered the cells hypersusceptible to antibiotics and EtBr and led to a high level of accumulation of intracellular EtBr. The primary structure of HrdC has a weak similarity to that of Escherichia coli TolC. Interestingly, both drug resistance and the expression of HrdC were markedly increased in the presence of a high salt concentration in the growth medium, but this was not observed in hrdC-disrupted cells. These results indicate that HrdC is the outer membrane component of the putative efflux pump assembly and that it plays a major role in the observed induction of drug resistance by salt in this bacterium.     

Attenuation of a delayed increase in the extracellular glutamate level in the peri-infarct area following focal cerebral ischemia by a novel agent ONO-2506

A novel agent, ONO-2506 [(R)-(-)-2-propyloctanoic acid, ONO Pharmaceutical Co. Ltd.] was previously shown to mitigate delayed infarct expansion through inhibition of the enhanced production of S-100beta, while inducing a prompt symptomatic improvement that attained a significant level as early as 24h after drug administration. To elucidate the mechanism underlying the prompt symptomatic improvement, the present study aimed to examine whether ONO-2506 modulates the level of extracellular glutamate ([Glu]e) in the rat subjected to transient middle cerebral artery occlusion (tMCAO). In this model, it had been shown that ONO-2506 reduces the infarct volume, improves the neurological deficits, and enhances the mRNA expression of glial glutamate transporters (GLT-1 and GLAST). The [Glu]e levels in the ischemic cortices were continuously measured using intracerebral microdialysis. The alterations in the [Glu]e levels in the sham-operated and tMCAO-operated groups with or without drug administration were compared. In the tMCAO groups, the [Glu]e level increased during tMCAO to a similar extent, returned to normal on reperfusion, and increased again around 5h. In the saline-treated group, however, the [Glu]e level further increased from 15 h on to reach about 280% of the normal level at 24h. This secondary increase in the [Glu]e level in the late phase of reperfusion was prevented by ONO-2506. The intracerebral infusion of glutamate transporter inhibitor, l-trans-pyrrolidine-2,4-dicarboxylic acid, at 24h after tMCAO induced an increase in the [Glu]e level, which was marked in both the sham-operated and ONO-2506-treated groups, but much less pronounced in the saline-treated group. The above results suggest that functional modulation of activated astrocytes by pharmacological agents like ONO-2506 may inhibit the secondary rise of [Glu]e level in the late phase of reperfusion, leading to amelioration of delayed infarct expansion and neurological deficits.     

Changes in the fabric and compliance tensors of cancellous bone due to trabecular surface remodeling, predicted by a digital image-based model

Fabric and compliance tensors of a cube of cancellous bone with a complicated three-dimensional trabecular structure were obtained for trabecular surface remodeling by using a digital image-based model combined with a large-scale finite element method. Using mean intercept length and a homogenization method, the fabric and compliance tensors were determined for the trabecular structure obtained in the computer remodeling simulation. The tensorial quantities obtained indicated that anisotropic structural changes occur in cancellous bone adapting to the compressive loading condition. There were good correlations between the fabric tensor, bone volume fraction, and compliance tensor in the remodeling process. The result demonstrates that changes in the structural and mechanical properties of cancellous bone are essentially anisotropic and should be expressed by tensorial quantities.