Isolation and Crystallization of Extracellular 3β-Hydroxysteroid Oxidase of Brevibacterium sterolicum nov. sp.

Among about 500 strains tested, a newly isolated soil bacterium, Brevibacterium sterolicum nov. sp. KY 3463 (ATCC 21387) showed the highest potency in production of 3β-hydroxysteroid oxidase in the culture fluid.

The 3β-hydroxysteroid oxidase was purified from the culture filtrate by a procedure involving ammonium sulfate fractionation, DEAE-cellulose and hydroxyapatite column chromatographies and Sephadex G–75 gel filtration. Crystals of the enzyme were obtained from solutions of the purified preparation by the addition of ammonium sulfate. The crystals appeared as fine rods, with a bright yellow color.

The enzyme is homogeneous by disc gel electrophoresis and ultracentrifugation. Sedimentation velocity yields a value of . It exhibits a typical flavoprotein spectrum of absorption maxima at 280, 390, and 470 mμ.     

Expression of Cell Competition Markers at the Interface between p53 Signature and Normal Epithelium in the Human Fallopian Tube

There is a growing body of evidence regarding cell competition between normal and mutant mammalian cells, which suggest that it may play a defensive role in the early phase of carcinogenesis. In vitro study in the past has shown that overexpression of vimentin in normal epithelial cells at the contact surface with transformed cells is essential for the cell competition involved in epithelial defense against cancer. In this study, we attempted to examine cell competition in human tissue in vivo by investigating surgically resected human fallopian tubes that contain p53 signatures and serous tubal intraepithelial lesions (STILs), a linear expansion of p53-immunopositive/TP53 mutant tubal epithelial cells that are considered as precursors of pelvic high grade serous carcinoma. Immunofluorescence double staining for p53 and the cell competition marker vimentin was performed in 21 sections of human fallopian tube tissue containing 17 p53 signatures and 4 STILs. The intensities of vimentin expression at the interface between p53-positive cells at the end of the p53 signature/STIL and adjacent p53-negative normal tubal epithelial cells were compared with the background tubal epithelium. As a result, the average vimentin intensity at the interfaces relative to the background intensity was 1.076 (95% CI, 0.9412 – 1.211 for p53 signature and 0.9790 (95% CI, 0.7206 – 1.237) for STIL. Thus, it can be concluded that overexpression of the cell competition marker vimentin are not observed in human tissue with TP53 alterations.     

Stress distribution of three-unit fixed partial prostheses (conventional and pontic) supported by three or two implants: 3D finite element analysis of ductile materials

In implantology, when financial or biological feasibility limitations appear, it is necessary to use prostheses with geometries that deviate from the conventional, with a pontic in the absence of an intermediate implant. The aim of this study was analyze and understand the general differences in the stresses generated in implants, components and infrastructures according to the configuration of the prosthesis over three or two implants. Thus, this paper analyzes the von Mises equivalent stresses (VMES) of ductile materials on their external surfaces. The experimental groups: Regular Splinted Conventional Group (RCG), which had conventional infrastructures on 3 regular-length Morse taper implants (4x11?mm); Regular Splinted Pontic Group (RPG), which had infrastructures with intermediate pontics on 2 regular-length Morse taper implants (4x11?mm). The simulations of the groups were created with Ansys Workbench 10.0 software. The results revealed that the RPG presented greater areas of possible fragility due to higher stress concentrations, for example, in the cervical area of the union between the implant and component the top platform of the abutment, as well as greater coverage of the stress by the cervical implant threads. The RPG infrastructure was also more affected by stresses in the connection areas between the prostheses and on the occlusal surface. There is an advantage to using prostheses supported by a greater number of implants (RCG) because this decreases the stress in the analyzed structures and consequently improves stress dissipation to the supporting bone, which would preserve the system.     

Human Pluripotent Stem Cell-Derived Tumor Model Uncovers the Embryonic Stem Cell Signature as a Key Driver in Atypical Teratoid/Rhabdoid Tumor

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Redox-stratification controlled biofilm (ReSCoBi) for completely autotrophic nitrogen removal: the effect of co- versus counter-diffusion on reactor performance

A multi-population biofilm model for completely autotrophic nitrogen removal was developed and implemented in the simulation program AQUASIM to corroborate the concept of a redox-stratification controlled biofilm (ReSCoBi). The model considers both counter- and co-diffusion biofilm geometries. In the counter-diffusion biofilm, oxygen is supplied through a gas-permeable membrane that supports the biofilm while ammonia (NH(4)(+)) is supplied from the bulk liquid. On the contrary, in the co-diffusion biofilm, both oxygen and NH(4)(+) are supplied from the bulk liquid. Results of the model revealed a clear stratification of microbial activities in both of the biofilms, the resulting chemical profiles, and the obvious effect of the relative surface loadings of oxygen and NH(4)(+) (J(O(2))/J(NH(4)(+))) on the reactor performances. Steady-state biofilm thickness had a significant but different effect on T-N removal for co- and counter-diffusion biofilms: the removal efficiency in the counter-diffusion biofilm geometry was superior to that in the co-diffusion counterpart, within the range of 450-1,400 microm; however, the efficiency deteriorated with a further increase in biofilm thickness, probably because of diffusion limitation of NH(4)(+). Under conditions of oxygen excess (J(O(2))/J(NH(4)(+)) > 3.98), almost all NH(4)(+) was consumed by aerobic ammonia oxidation in the co-diffusion biofilm, leading to poor performance, while in the counter-diffusion biofilm, T-N removal efficiency was maintained because of the physical location of anaerobic ammonium oxidizers near the bulk liquid. These results clearly reveal that counter-diffusion biofilms have a wider application range for autotrophic T-N removal than co-diffusion biofilms.     

In vitro analysis of Bcl-2 proteins in mitochondria and endoplasmic reticulum: similarities in anti-apoptotic functions and differences in regulation

Anti-apoptotic Bcl-2 localizes in the membranes of mitochondria and endoplasmic reticulum (ER) and resists a broad range of apoptotic stimuli. However, the precise function of Bcl-2 in ER is still unclear. We herein examined the anti-apoptotic potencies of Bcl-2 in mitochondria and ER in vitro. The mitochondria isolated from HeLa cells, which have little or practically no Bcl-2, were apoptosis-competent. That is, membrane-bound Bax was activated and cytochrome c was released when the isolated mitochondria were incubated at 35 degrees C. Cytochrome c release from the apoptosis-competent mitochondria was suppressed by co-incubation with the mitochondria with overexpressed Bcl-2 (Bcl-2 mitochondria), suggesting that Bcl-2 anchored in one mitochondrion can suppress cytochrome c release from another mitochondrion. Similar results were obtained when microsomes with overexpressed Bcl-2 (Bcl-2 microsomes) were co-incubated with apoptosis-competent mitochondria. A quantitative titration analysis showed that Bcl-2 in the ER suppresses cytochrome c release as efficiently as that in the mitochondria. An immunoprecipitation assay showed that Bcl-2 in both mitochondria and ER binds to Bax at almost the same degree. However, in the presence of tBid, co-incubation of apoptosis-competent mitochondria with Bcl-2 microsomes, but not with Bcl-2 mitochondria, diminished the Bax-binding to Bcl-2 significantly, suggesting that Bcl-2 in ER is readily inactivated by tBid. Co-incubation assay further confirmed that Bcl-2 in the ER, but not Bcl-2 in the mitochondria, is potentially inactivated by tBid. Our quantitative in vitro studies indicate that Bcl-2 in mitochondria and ER are similarly potent in inhibiting Bax-associated apoptosis of other mitochondria, but are regulated by tBid differently.     

Specific role of the truncated betaIV-spectrin Sigma6 in sodium channel clustering at axon initial segments and nodes of ranvier

At axon initial segments and nodes of Ranvier in neurons, the spectrin membrane skeleton plays roles in physically stabilizing the plasma membrane integrity and in clustering voltage-gated sodium channels for proper conduction of the action potential. betaIV-Spectrin, an essential component of the membrane skeleton at these sites, has an N-terminal-truncated isoform, Sigma6, which is expressed at much higher levels than the full-length isoform Sigma1. To investigate the role of betaIV-spectrin Sigma6, we generated Sigma1-deficient mice with a normal level of Sigma6 expression (Sigma1(-/-) mice), and compared their phenotypes with those of previously generated mice lacking both Sigma1 and Sigma6(Sigma1Sigma6(-/-) mice). The gross neurological defects observed in Sigma1Sigma6(-/-) mice, such as hindleg contraction, were apparently ameliorated in Sigma1(-/-) mice. At cellular levels, Sigma1Sigma6(-/-) and Sigma1(-/-) neurons similarly exhibited waving and swelling of the plasma membrane at axon initial segments and nodes of Ranvier. By contrast, the levels of ankyrin G and voltage-gated sodium channels at these sites, which are significantly reduced in Sigma1Sigma6(-/-) mice, were substantially recovered in Sigma1(-/-) mice. We conclude that the truncated betaIV-spectrin isoform Sigma6 plays a specific role in clustering voltage-gated sodium channels, whereas it is dispensable for membrane stabilization at axon initial segments and nodes of Ranvier.     

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.     

Age-dependent changes in cell proliferation and cell death in the periodontal tissue and the submandibular gland in mice: a comparison with other tissues and organs

This study investigated the age-dependent changes in the number of BrdU- and TUNEL-positive cells in murine gingival tissue and submandibular gland, and compared the findings with those in other tissues and organs. The cell proliferative activity was decreased after 20 weeks of age in epithelial cells of the gingiva, tongue, buccal mucosa and skin. A decreased cell proliferative activity was also associated with aging in the liver and kidney parenchymal cells. Meanwhile, cell death showed peculiar changes in gingival subepithelial tissue, and mucous and serous acini of the submandibular gland. An increase of TUNEL-positive cells was demonstrated in gingival subepithelial tissue after 20-week-old of age. A significant increase of TUNEL-positive cells was also found in the mucous acinar cells in the 20-week-old mice and in the serous acini after 20 weeks. The fluctuation in the number of TUNEL-positive cells in the subepithelial tissue of the skin, and BrdU- and TUNEL-positive staining ratios in the liver was smaller than that in other tissue and organs throughout life. This study may provide useful information for better understanding the influence of aging on the functional alteration that occurs in the gingival tissue and submandibular gland of the elderly.     

Contrasting patterns of genetic structure and phylogeography in the marine agarophytes Gelidiophycus divaricatus and G. freshwateri (Gelidiales,Rhodophyta) from East Asia

The evolutionary and population demographic history of marine red algae in East Asia is poorly understood. Here, we reconstructed the phylogeographies of two upper intertidal species endemic to East Asia, Gelidiophycus divaricatus and G. freshwateri. Phylogenetic and phylogeographic inferences of 393 mitochondrial cox1, 128 plastid rbcL, and 342 nuclear ITS2 sequences were complemented with ecological niche models. Gelidiophycus divaricatus, a southern species adapted to warm water, is characterized by a high genetic diversity and a strong geographical population structure, characteristic of stable population sizes and sudden reduction to recent expansion. In contrast, G. freshwateri, a northern species adapted to cold temperate conditions, is genetically relatively homogeneous with a shallow population structure resulting from steady population growth and recent equilibrium. The overlap zone of the two species roughly matches summer and winter isotherms, indicating that surface seawater temperature is a key feature influencing species range. Unidirectional genetic introgression was detected at two sites on Jeju Island where G. divaricatus was rare while G. freshwateri was common, suggesting the occurrence of asymmetric natural hybrids, a rarely reported event for rhodophytes. Our results illustrate that Quaternary climate oscillations have left strong imprints on the current day genetic structure and highlight the importance of seawater temperature and sea level change in driving speciation in upper intertidal seaweed species.