Low pressure condition of a lipid core in an eccentrically developed carotid atheromatous plaque: a static finite element analysis

Plaque ruptures in atherosclerotic carotid arteries cause cerebral strokes. Accumulation of lipoproteins in the deep intimal layer forms a lipid core (LC), whose progression may be enhanced by mechanical conditions on the arterial wall. In this study, we investigated the pressure conditions of a liquid LC through numerical simulations of a sliced segment finite element (FE) model and a three-dimensional (3D) symmetric FE model. A model of an LC filled with nearly incompressible fluid was compared with incompressible and soft neo-Hookean LC models in a static FE analysis. Material constants for a nonlinear hyperelastic model of the arterial wall were identified based on an inflation test using a tube specimen. The results from the FE analysis of a sliced segment model show an LC fluid pressure as low as 1.9 kPa at a blood pressure of 16 kPa. A neo-Hookean LC model with a Young’s modulus of 0.06 kPa produced an almost uniform pressure in the LC within an error of 1.3 %. The 3D model predicted a similar level of LC pressure. Such low fluid pressure in the LC region may enhance the infiltration of lipoproteins and other substances from the lumen and facilitate transport through microvessels from the adventitia to the LC.     

Serological and Progression Differences of Joint Destruction in the Wrist and the Feet in Rheumatoid Arthritis - A Cross-Sectional Cohort Study

Objective

To investigate clinical and radiological differences between joint destruction in the wrist and the feet in patients with RA.

Methods

A cross-sectional clinical study was conducted in an RA cohort at a single institution. Clinical data included age, sex and duration of disease. Laboratory data included sero-positivity for anti-cyclic citrullinated peptide (CCP) antibody and RF. Radiological measurements included Larsen grades and the modified Sharp/van der Heijde method (SHS) for the hands/wrists and the feet. Statistical analyses were performed using the Kruskal—Wallis H-test, a dummy variable linear regression model and multivariate logistic regression analysis with 95% confidence interval and odds ratios.

Results

A total of 405 patients were enrolled, and 314 patients were analysed in this study. The duration of disease in the foot-dominant group was significantly less than that in the wrist-dominant group. When patients were subdivided by duration of disease, the Larsen grade of the feet was significantly higher than that of the wrist in the first quadrant subgroup, but this was reversed with increasing duration of disease. Anti-CCP status was a significant predictive factor for joint destruction in the wrist but not in the feet, while RF status was not predictive in either the wrist or the feet.

Conclusions

Joint destruction in the feet started earlier than in the wrist, but the latter progresses faster with increasing duration of disease. Anti-CCP status predicts joint destruction in the wrist better than in the feet.     

Purification, characterization, and molecular cloning of organic-solvent-tolerant cholesterol esterase from cyclohexane-tolerant Burkholderia cepacia strain ST-200

Extracellular cholesterol esterase of Burkholderia cepacia strain ST-200 was purified from the culture supernatant. Its molecular mass was 37 kDa. The enzyme was stable at pH 5.5–12 and active at pH 5.5–6, showing optimal activity at pH 7.0 at 45°C. Relative to the commercially available cholesterol esterases, the purified enzyme was highly stable in the presence of various water-miscible organic solvents. The enzyme preferentially hydrolyzed long-chain fatty acid esters of cholesterol, except for that of cholesteryl palmitate. The enzyme exhibited lipolytic activity toward various p-nitrophenyl esters. The hydrolysis rate of p-nitrophenyl caprylate was enhanced 3.5- to 7.2-fold in the presence of 5–20% (vol/vol) water-miscible organic solvents relative to that in the absence of organic solvents. The structural gene encoding the cholesterol esterase was cloned and sequenced. The primary translation product was predicted to be 365 amino acid residues. The mature product is composed of 325 amino acid residues. The amino acid sequence of the product showed the highest similarity to the lipase LipA (87%) from B. cepacia DSM3959.     

Characterization of a novel fungal protein, p15, which induces neuronal differentiation of PC12 cells

In our previous paper, we reported that a 15 kDa protein (p15) produced by a fungus, genus Helicosporium, enhanced NGF-induced neurite outgrowth from PC12 cells. Here we further characterized the actions of p15. The complete amino acid sequence of p15 was determined and it was shown to be a hydrophilic protein composed of 118 amino acid residues with two intramolecular disulfide bridges. p15-induced neurite outgrowth was blocked by the depletion of extracellular Ca(2+) in the culture medium and was significantly inhibited by L-type Ca(2+) channel inhibitor nicardipine. p15 stimulated Src kinase and MAPK activities, and neurite outgrowth was not observed in srcDN2, a dominant negative c-src(K295R)-expressing cell line, and was significantly reduced in RasN17-expressing cells. These results suggest that p15 stimulates neurite outgrowth through the potentiation of L-type Ca(2+) channels, thereby activating the Src-Ras-MAPK cascade.     

A method of tracking donor cells after simulated autologous transplantation: a study using synovial cells of transgenic rats

A transgenic rat was used as a transplantation donor to simulate autologous transplantation. The sex-matched transplantation between a female transgenic and a wild-type rat can theoretically be regarded as an autologous transplantation due to the genetic agreement of these rats except for the non-protein-producing transgenes. Transgene-containing synovial cells were tracked in the joint using this autologous transplantation model. The transgenes in the donor synovial cells were detected using in situ hybridization (ISH), while mitotic activities were simultaneously examined by immunodetection of 5-bromo-2'-deoxyuridine (BrdU). A defect was generated in the knee joint capsule of a Fischer 344 (wild-type) rat. The synovium of a transgenic rat was sutured to the defect of the wild-type rat in group 1 and was allowed to free float in the joint in group 2. A large number of BrdU-labeled, transgene-containing synovial cells were detected in both groups at 3 days. The number of these cells then decreased, but they could still be identified even at 4 weeks after autologous transplantation. These results indicated that transplanted synovial cells were viable in the joint for at least 4 weeks. Furthermore, the transgenic rat was shown to be an effective animal model for distinguishing the extrinsic from the intrinsic cells in the cellular intermixed tissues in vivo. The combined method of ISH for detecting transgene-containing cells and the immunohistochemistry of BrdU for detecting proliferating cells was also shown to be effective for tracking the viability of extrinsic cells after autologous transplantation.     

Rho guanine nucleotide exchange factor xNET1 implicated in gastrulation movements during Xenopus development

During Xenopus development, embryonic cells dramatically change their shape and position. Rho family small GTPases, such as RhoA, Rac, and Cdc42, play important roles in this process. These GTPases are generally activated by guanine nucleotide exchange factors (GEFs); however, the roles of RhoGEFs in Xenopus development have not yet been elucidated. We therefore searched for RhoGEF genes in our Xenopus EST database, and we identified several genes expressed during embryogenesis. Among them, we focused on one gene, designated xNET1. It is similar to mammalian NET1, a RhoA-specific GEF. An in vitro binding assay revealed that xNET1 bound to RhoA, but not to Rac or Cdc42. In addition, transient expression of xNET1 activated endogenous RhoA. These results indicated that xNET1 is a GEF for RhoA. Epitope-tagged xNET1 was localized mainly to the nucleus, and the localization was regulated by nuclear localization signals in the N-terminal region of xNET1. Overexpression of either wild-type or a mutant form of xNET1 severely inhibited gastrulation movements. We demonstrated that xNET1 was co-immunoprecipitated with the Dishevelled protein, which is an essential signaling component in the non-canonical Wnt pathway. This pathway has been shown to activate RhoA and regulate gastrulation movements. We propose that xNET1 or a similar RhoGEF may mediate Dishevelled signaling to RhoA in the Wnt pathway.     

Effects of protein transport inhibitors on the distribution and secretion of the fusion protein RntA-EGFP in Aspergillus oryzae

The distribution of the secreted protein ribonuclease T1 (RntA) fused with the enhanced green fluorescent protein (EGFP), RntA-EGFP, was visualized in hyphae of Aspergillus oryzae in the presence of a protein transport inhibitor, brefeldin A, cytochalasin A, or nocodazole. During treatment with the protein transport inhibitors, the distribution of RntA-EGFP changed and distinct patterns of fluorescence accumulation were observed. The addition of brefeldin A caused RntA-EGFP fluorescence to appear in reticular networks, and the disruption of the polymerization of actin filaments by cytochalasin A caused an increase in RntA-EGFP fluorescence intensity in the hyphae without accumulation in a specific cellular component. In contrast, RntA-EGFP fluorescence was distributed in different parts of a hypha during treatment with nocodazole, a compound that depolymerizes microtubules. In addition, quantitative analysis was performed using the RntA-EGFP visualization system to analyze the relative amount of RntA-EGFP secreted into the culture medium during treatment with the protein transport inhibitors.     

Molecular cloning and functional characterization of avaB, a gene encoding Vam6p/Vps39p-like protein in Aspergillus nidulans

It has been demonstrated that Saccharomyces cerevisiae Vam6p/Vps39p plays a critical role in the tethering steps of vacuolar membrane fusion by facilitating guanine nucleotide exchange on small guanosine triphosphatase (GTPase) Vam4p/Ypt7p. We report here the identification and characterization of a novel protein in Aspergillus nidulans, AvaB, that exhibits similarity to Vam6p/Vps39p and plays a critical role in vacuolar morphogenesis in A. nidulans. AvaB is comprised of 1058 amino acids with amino-terminal citron homology (CNH) and central clathrin homology (CLH) domains, as observed for other Vam6p/Vps39p family proteins. Disruption of avaB in A. nidulans resulted in the fragmentation of vacuoles and reduced growth rate under various growth conditions, implying its importance in maintaining vacuolar morphology and function. Yeast two-hybrid analysis demonstrated the interaction of AvaB with AvaA, a Vam4p/Ypt7p homolog in A. nidulans, as well as the homooligomer formation of AvaB, suggesting that AvaB performs its function through hetero- or homophilic protein-protein interactions.     

Plant-specific microtubule-associated protein SPIRAL2 is required for anisotropic growth in Arabidopsis

In diffusely growing plant cells, cortical microtubules play an important role in regulating the direction of cell expansion. Arabidopsis (Arabidopsis thaliana) spiral2 (spr2) mutant is defective in directional cell elongation and exhibits right-handed helical growth in longitudinally expanding organs such as root, hypocotyl, stem, petiole, and petal. The growth of spr2 roots is more sensitive to microtubule-interacting drugs than is wild-type root growth. The SPR2 gene encodes a plant-specific 94-kD protein containing HEAT-repeat motifs that are implicated in protein-protein interaction. When expressed constitutively, SPR2-green fluorescent protein fusion protein complemented the spr2 mutant phenotype and was localized to cortical microtubules as well as other mitotic microtubule arrays in transgenic plants. Recombinant SPR2 protein directly bound to taxol-stabilized microtubules in vitro. Furthermore, SPR2-specific antibody and mass spectrometry identified a tobacco (Nicotiana tabacum) SPR2 homolog in highly purified microtubule-associated protein fractions from tobacco BY-2 cell cultures. These results suggest that SPR2 is a novel microtubule-associated protein and is required for proper microtubule function involved in anisotropic growth.