Enhancement of Procollagen Biosynthesis by p180 through Augmented Ribosome Association on the Endoplasmic Reticulum in Response to Stimulated Secretion

A coiled-coil microtubule-bundling protein, p180, was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported a novel role for p180 in the trans-Golgi network (TGN) expansion following stimulated collagen secretion. Here, we show that p180 plays a key role in procollagen biosynthesis and secretion in diploid fibroblasts. Depletion of p180 caused marked reductions of secreted collagens without significant loss of the ER membrane or mRNA. Metabolic labeling experiments revealed that the procollagen biosynthetic activity was markedly affected following p180 depletion. Moreover, loss of p180 perturbs ascorbate-stimulated de novo biosynthesis mainly in the membrane fraction with a preferential secretion defect of large proteins. At the EM level, one of the most prominent morphological features of p180-depleted cells was insufficient ribosome association on the ER membranes. In contrast, the ER of control cells was studded with numerous ribosomes, which were further enhanced by ascorbate. Similarly biochemical analysis confirmed that levels of membrane-bound ribosomes were altered in a p180-dependent manner. Taken together, our data suggest that p180 plays crucial roles in enhancing collagen biosynthesis at the entry site of the secretory compartments by a novel mechanism that mainly involves facilitating ribosome association on the ER.     

Proteomic Analysis to Examine the Role of Matrix Proteins in a Gouty Tophus from a Patient with Recurrent Gout

To examine the role of matrix proteins in the formation of gouty tophus, we analyzed the crystalline components and matrix proteins in a gouty tophus from a patient with recurrent gout. Micro-area X-ray diffraction analysis and infrared spectroscopy indicated that the tophus was composed of monosodium urate monohydrate. Proteomic analysis identified 134 proteins from the tophus as matrix proteins. Many proteins relevant to inflammation and host defense were identified, and immunoglobulin was detected in all four extracted fractions (KCl, formic acid, guanidine-HCl, and ethylenediaminetetraacetic acid) and from many spots throughout a broad molecular weight range after electrophoresis. It is thought that the process of biological defense including the immunity has occurred in the gouty tophus.     

Intramembrane processing by signal peptide peptidase regulates the membrane localization of hepatitis C virus core protein and viral propagation

Hepatitis C virus (HCV) core protein has shown to be localized in the detergent-resistant membrane (DRM), which is distinct from the classical raft fraction including caveolin, although the biological significance of the DRM localization of the core protein has not been determined. The HCV core protein is cleaved off from a precursor polyprotein at the lumen side of Ala(191) by signal peptidase and is then further processed by signal peptide peptidase (SPP) within the transmembrane region. In this study, we examined the role of SPP in the localization of the HCV core protein in the DRM and in viral propagation. The C terminus of the HCV core protein cleaved by SPP in 293T cells was identified as Phe(177) by mass spectrometry. Mutations introduced into two residues (Ile(176) and Phe(177)) upstream of the cleavage site of the core protein abrogated processing by SPP and localization in the DRM fraction. Expression of a dominant-negative SPP or treatment with an SPP inhibitor, L685,458, resulted in reductions in the levels of processed core protein localized in the DRM fraction. The production of HCV RNA in cells persistently infected with strain JFH-1 was impaired by treatment with the SPP inhibitor. Furthermore, mutant JFH-1 viruses bearing SPP-resistant mutations in the core protein failed to propagate in a permissive cell line. These results suggest that intramembrane processing of HCV core protein by SPP is required for the localization of the HCV core protein in the DRM and for viral propagation.     

Do replicates of independent guppy lineages evolve similarly in a predator‐free laboratory environment?

The Trinidadian guppy is emblematic of parallel and convergent evolution, with repeated demonstrations that predation regime is a driver of adaptive trait evolution. A classic and foundational experiment in this system was conducted by John Endler 40 years ago, where male guppies placed into low‐predation environments in the laboratory evolved increased color in a few generations. However, Endler's experiment did not employ the now typical design for a parallel/convergent evolution study, which would employ replicates of different ancestral lineages. We therefore implemented an experiment that seeded replicate mesocosms with small founding populations of guppies originating from high‐predation populations of two very different lineages. The different mesocosms were maintained identically, and male guppy color was quantified every four months. After one year, we tested whether male color had increased, whether replicates within a lineage had parallel phenotypic trajectories, and whether the different lineages converged on a common phenotype. Results showed that male guppy color generally increased through time, primarily due to changes in melanic color, whereas the other colors showed inconsistent and highly variable trajectories. Most of the nonparallelism in phenotypic trajectories was among mesocosms containing different lineages. In addition to this mixture of parallelism and nonparallelism, convergence was not evident in that the variance in color among the mesocosms actually increased through time. We suggest that our results reflect the potential importance of high variation in female preference and stochastic processes such as drift and founder effects, both of which could be important in nature.     

IgM-type GM-CSF autoantibody is etiologically a bystander but associated with IgG-type autoantibody production in autoimmune pulmonary alveolar proteinosis

The granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibody (GMAb) is the causative agent underlying autoimmune pulmonary alveolar proteinosis (aPAP). It consists primarily of the IgG isotype. At present, information on other isotypes of the autoantibody is limited. We detected serum the IgM isotype of GMAb (IgM-GMAb) in more than 80% of patients with aPAP and 22% of healthy subjects, suggesting that a continuous antigen pressure may be present in most patients. Levels of the IgM isotype were weakly correlated with IgG-GMAb levels but not IgA-GMAb, suggesting that its production may be associated with that of IgG-GMAb. The mean binding avidity to GM-CSF of the IgM isotype was 100-fold lower than the IgG-GMAb isotype, whereas the IC(50) value for neutralizing capacity was 20,000-fold higher than that of IgG-GMAb, indicating that IgM-GMAb is only a very weak neutralizer of GM-CSF. In bronchoalveolar lavage fluid from nine patients, IgG-GMAb was consistently detected, but IgM-GMAb was under the detection limit in most patients, confirming that IgM-GMAb is functionally a bystander in the pathogenesis of aPAP. It rather may be involved in the mechanism for development of IgG-GMAb in vivo.     

Specific detection of buckwheat residues in processed foods by polymerase chain reaction

A specific and qualitative detection method for buckwheat in foods using the polymerase chain reaction (PCR) was developed. Trace amounts of buckwheat in commercial food products were qualitatively detected by this method. It should be reliable for detecting buckwheat residues in processed foods and practical for monitoring the labeling system for allergenic food materials.     

Cigarette smoking and serum soluble Fas levels: Findings from the JACC study

Cigarette smoking enhances low-grade systemic inflammation in the lung and other organs. Activated immune cells play an important role at early and late stages of inflammation, and in recent years, soluble Fas (sFas), an isoform of death molecule Fas, was found to interfere with the apoptotic pathways of these activated immune cells. The aim of this study was to confirm the association between cigarette smoking and sFas levels in healthy male subjects. We measured serum sFas levels of 4415 male subjects selected as controls for a nested case-control study within the large-scale cohort study conducted in Japan, called the JACC Study. Smoking status at baseline was evaluated by a self-administered questionnaire. Least square means of sFas according to smoking status and numbers of cigarettes smoked per day among smokers were calculated and adjusted for possible confounding factors. Mean sFas levels showed an increasing trend across never smokers, past smokers and current smokers, as 2.21 (95% CI: 2.14–2.27) ng/ml, 2.29 (2.22–2.36) ng/ml, and 2.36 (2.30–2.43) ng/ml, respectively. However, no dose-response relationship was observed between the number of cigarettes smoked per day and sFas levels among smokers.     

Differential interaction and aggregation of 3-repeat and 4-repeat tau isoforms with 14-3-3ζ protein

Tau isoforms, 3-repeat (3R) and 4-repeat tau (4R), are differentially involved in neuronal development and in several tauopathies. 14-3-3 protein binds to tau and 14-3-3/tau association has been found both in the development and in tauopathies. To understand the role of 14-3-3 in the differential regulation of tau isoforms, we have performed studies on the interaction and aggregation of 3R-tau and 4R-tau, either phosphorylated or unphosphorylated, with 14-3-3ζ. We show by surface plasmon resonance studies that the interaction between unphosphorylated 3R-tau and 14-3-3ζ is ∼3-folds higher than that between unphosphorylated 4R-tau and 14-3-3ζ. Phosphorylation of tau by protein kinase A (PKA) increases the affinity of both 3R- and 4R-tau for 14-3-3ζ to a similar level. An in vitro aggregation assay employing both transmission electron microscopy and fluorescence spectroscopy revealed the aggregation of unphosphorylated 4R-tau to be significantly higher than that of unphosphorylated 3R-tau following the induction of 14-3-3ζ. The filaments formed from 3R- and 4R-tau were almost similar in morphology. In contrast, the aggregation of both 3R- and 4R-tau was reduced to a similar low level after phosphorylation with PKA. Taken together, these results suggest that 14-3-3ζ exhibits a similar role for tau isoforms after PKA-phosphorylation, but a differential role for unphosphorylated tau. The significant aggregation of 4R-tau by 14-3-3ζ suggests that 14-3-3 may act as an inducer in the generation of 4R-tau-predominant neurofibrillary tangles in tauopathies.     

Changes in cell migration of mesenchymal cells during osteogenic differentiation

We showed that the migration, morphology and adhesiveness of undifferentiated mesenchymal cells dramatically changed during osteogenic differentiation. The migration of these cells was transiently upregulated early in osteogenic differentiation. At a later stage, migration was decreased but adhesiveness was increased. Furthermore, Cdc42 and Rac1 Rho-family small GTPases were activated at early stages of differentiation and the phosphorylation level of FAK decreased as differentiation progressed. We also showed cell migration was promoted by inhibition of the Rho-ROCK-myosin signaling. Finally, using a mouse model of ectopic bone formation, we confirmed that treatment with ROCK inhibitor, Y-27632 increased cell movement into bone formation sites, resulting in enhanced osteogenesis. These results provide a new insight into the link between cell migration and osteogenic differentiation.     

Virology of the family Togaviridae

Many pathogens important for medicine, veterinary medicine or public health belong to the genera alphavirus and rubivirus within the family Togaviridae. 29 species of alphaviruses have been reported, and most of them are arboviruses. Chikungnya virus re-emerged in Kenya in 2004 and the epidemics spread to the Indian Ocean islands and many countries in South Asia, South-East Asia and Europe. On the other hand, rubella virus, a sole member of the genus rubivirus, is the causative agent of rubella and congenital rubella syndrome (CRS). Because human is only a natural host of the virus and effective live attenuated vaccines are available, immunization activities are strengthened globally to eliminate rubella and CRS, together with measles.