Immunoregulatory role of ocular macrophages: the macrophages produce RANTES to suppress experimental autoimmune uveitis

Murine experimental autoimmune uveitis (EAU) is a model of human uveitis. Ocular-infiltrating macrophages play a crucial role in the generation of tissue damage in EAU. In fact, several chemokines are actually produced in the inflamed eye. The aim of this study was to elucidate the role of ocular macrophage-derived chemokines in EAU. C57BL/6 mice were immunized with human interphotoreceptor retinoid binding protein peptide 1-20, and the EAU severity was scored at multiple time points based on microscopic fundus observations (retinal vascular dilatation and exudates) and histological examinations. The peak inflammatory response was observed 1 wk (day 16) after the beginning of macrophage infiltration to the eye (day 9). Ocular-infiltrating cells were enriched or depleted of macrophages by magnetic beads and analyzed by real-time RT-PCR for chemokine mRNA production. We found that only the macrophage-enriched cells from the eye produced RANTES, and thus proposed that macrophage-derived RANTES facilitated the ocular inflammations. In contrast to our postulate, neutralization of RANTES by specific Ab in vivo on days 9 and 13 exacerbated EAU. We also found that the ratio of ocular CD4/CD8 T cells was markedly increased after treatment. As a result, RANTES neutralization might exacerbate EAU by modulating the type of T cell subsets recruited to the eye. In conclusion, our data provide insight into the immunoregulatory role of macrophages and RANTES in the pathogenesis of ocular inflammation. Not all macrophage-derived chemokines cause local inflammation, since RANTES produced by ocular macrophages appears to suppress EAU.     

V-1, a protein expressed transiently during murine cerebellar development,regulates actin polymerization via interaction with capping protein

V-1 is a 12-kDa protein consisting of three consecutive ANK repeats, which are believed to serve as the surface for protein-protein interactions. It is thought to have a role in neural development for its temporal profile of expression during murine cerebellar development, but its precise role remains unknown. Here we applied the proteomic approach to search for protein targets that interact with V-1. The V-1 cDNA attached with a tandem affinity purification tag was expressed in the cultured 293T cells, and the protein complex formed within the cells were captured and characterized by mass spectrometry. We detected two polypeptides specifically associated with V-1, which were identified as the alpha and beta subunits of the capping protein (CP, alternatively called CapZ or beta-actinin). CP regulates actin polymerization by capping the barbed end of the actin filament. The V-1.CP complex was detected not only in cultured cells transfected with the V-1 cDNA but also endogenously in cells as well as in murine cerebellar extracts. An analysis of the V-1/CP interaction by surface plasmon resonance spectroscopy showed that V-1 formed a stable complex with the CP heterodimer with a dissociation constant of 1.2 x 10(-7) m and a molecular stoichiometry of approximately 1:1. In addition, V-1 inhibited the CP-regulated actin polymerization in vitro in a dose-dependent manner. Thus, our results suggest that V-1 is a novel component that regulates the dynamics of actin polymerization by interacting with CP and thereby participates in a variety of cellular processes such as actin-driven cell movements and motility during neuronal development.     

Proteomic analysis of human Nop56p-associated pre-ribosomal ribonucleoprotein complexes. Possible link between Nop56p and the nucleolar protein treacle responsible for Treacher Collins syndrome

Nop56p is a component of the box C/D small nucleolar ribonucleoprotein complexes that direct 2'-O-methylation of pre-rRNA during its maturation. Genetic analyses in yeast have shown that Nop56p plays important roles in the early steps of pre-rRNA processing. However, its precise function remains elusive, especially in higher eukaryotes. Here we describe the proteomic characterization of human Nop56p (hNop56p)-associated pre-ribosomal ribonucleoprotein complexes. Mass spectrometric analysis of purified pre-ribosomal ribonucleoprotein complexes identified 61 ribosomal proteins, 16 trans-acting factors probably involved in ribosome biogenesis, and 29 proteins whose function in ribosome biogenesis is unknown. Identification of pre-rRNA species within hNop56p-associated pre-ribosomal ribonucleoprotein complexes, coupled with the known functions of yeast orthologs of the probable trans-acting factors identified in human, demonstrated that hNop56p functions in the early to middle stages of 60 S subunit synthesis in human cells. Interestingly, the nucleolar phosphoprotein treacle, which is responsible for the craniofacial disorder associated with Treacher Collins syndrome, was found to be a constituent of hNop56p-associated pre-rRNP complexes. The association of hNop56p and treacle within the complexes was independent of rRNA integrity, indicating a direct interaction. In addition, the protein compositions of the treacle-associated and hNop56p-associated pre-ribosomal ribonucleoprotein complexes were very similar, suggesting functional similarities between these two complexes with respect to ribosome biogenesis in human cells.     

A case of Turner syndrome with congenital hypothyroidism untreated until age 38 years

OBJECTIVE AND METHODS: The effect of thyroid hormone on human growth and maturation is considered 'permissive'. To evaluate the effect of a prolonged thyroid hormone defect, especially in the pubertal period, a woman with untreated congenital hypothyroidism underwent studies of thyroid function and bone maturation for the first time at age 38 years 10 months and received thyroid hormone replacement. RESULTS: The karyotype was 45,X/46,XX. Menstruation had occurred for 10 years, from menarche until she was about 31 years old. Epiphyseal closure of the left hand was incomplete. The serum thyroid hormone level was virtually undetectable, and her thyroid gland was not detectable in the normal position by ultrasonography. Her height increased by 3.5 cm in the first 9 months after starting thyroid hormone replacement; after 11 months, closure of the epiphysis was complete. CONCLUSION: Thyroid hormone is necessary to achieve bone maturation and epiphyseal closure, but its role is only permissive.     

Structure-activity relationship of 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone)

This paper describes the discovery of a novel free radical scavenger, 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone, 1), as a potent antioxidant agent against lipid peroxidation. The structure-activity relationship of edaravone indicated that lipophilic substituents were essential to show its lipid peroxidation-inhibitory activity. In vivo studies revealed that edaravone showed brain-protective activity in a transient ischemia model.     

Large-scale identification of Caenorhabditis elegans proteins by multidimensional liquid chromatography-tandem mass spectrometry

A proteome of a model organism, Caenorhabditis elegans, was analyzed by an integrated liquid chromatography (LC)-based protein identification system, which was constructed by microscale two-dimensional liquid chromatography (2DLC) coupled with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) on a high-resolution hybrid mass spectrometer with an automated data analysis system. Soluble and insoluble protein fractions were prepared from a mixed growth phase culture of the worm C. elegans, digested with trypsin, and fractionated separately on the 2DLC system. The separated peptides were directly analyzed by on-line ESI-MS/MS in a data-dependent mode, and the resultant spectral data were automatically processed to search a genome sequence database, wormpep 66, for protein identification. The total number of proteins of the composite proteome identified in this method was 1,616, including 110 secreted/targeted proteins and 242 transmembrane proteins. The codon adaptation indices of the identified proteins suggested that the system could identify proteins of relatively low abundance, which are difficult to identify by conventional 2D-gel electrophoresis (GE) followed by an offline mass spectrometric analysis such as peptide mass fingerprinting. Among the approximately 5,400 peptides assigned in this study, many peptides with post-translational modifications, such as N-terminal acetylation and phosphorylation, were detected. This expression profile of C. elegans, containing 571 hypothetical gene products, will serve as the basic data of a major proteome set expressed in the worm.     

Aggregate formation and degradation of overexpressed wild-type and mutant urate oxidase proteins. Quality control of organelle-destined proteins by the endoplasmic reticulum

To analyze the cellular response caused by the overexpression of proteins in subcellular compartments, we constructed four expression clones encoding wild-type peroxisomal urate oxidase (UO), truncated UO lacking the peroxisomal targeting signal (UOdC), and chimeric UOs with a mitochondrial targeting signal (MTS) at the N-terminus of UOdC (MUOdC) or UO (MUO). After transfection, we examined COS-1 and HEK293 cells by immunofluorescence and immunoelectron microscopy, transmission electron microscopy, and pulse-chase experiments. The overexpressed UO and UOdC formed large electron-dense aggregates with no limiting membrane in both the cytoplasm and the nucleus. The UO aggregates exhibited the crystalloid structure quite similar to that of rat liver peroxisomal cores, whereas the UOdC aggregates formed a loose mass consisting of small dense substructures. The overexpressed MUOdC and MUO, on the other hand, formed other types of aggregates which were distributed in the cytoplasm. They consisted of tubular and circular membrane structures, which were morphologically confirmed to be derived from the endoplasmic reticulum (ER). No immunolabeling signals for MUOdC and MUO were present free in the cytoplasm and most of them were associated with membrane structures, suggesting that overexpressed UO containing the MTS attached to the ER membranes soon after synthesis and segregated from the cytosolic compartment. All the UO aggregates were stained for ubiquitin antigen. Pulse-chase experiments in combination with proteasome inhibitors suggested that proteasomes did not contribute to the degradation of these products.