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Abstract Proteins harboring peroxisome-targeting signal type-2 (PTS2) are recognized in the cytosol by mobile PTS2 receptor Pex7p and associate with a longer isoform Pex5pL of the PTS1 receptor. Trimeric PTS2 protein-Pex7p-Pex5pL complexes are translocated to peroxisomes in mammalian cells. However, it remains unclear whether Pex5pL and Pex7p are sufficient cytosolic components in transporting of PTS2 proteins to peroxisomes. Here, we construct a semi-intact cell import system to define the cytosolic components required for the peroxisomal PTS2 protein import and show that the PTS2 pre-import complexes comprising Pex7p, Pex5p, and Hsc70 isolated from the cytosol of pex14 Chinese hamster ovary cell mutant ZP161 is import-competent. PTS2 reporter proteins are transported to peroxisomes by recombinant Pex7p and Pex5pL in semi-intact cells devoid of the cytosol. Furthermore, PTS2 proteins are translocated to peroxisomes in the presence of a non-hydrolyzable ATP analogue, adenylyl imidodiphosphate, and N-ethylmaleimide, suggesting that ATP-dependent chaperones including Hsc70 are dispensable for PTS2 protein import. Taken together, we suggest that Pex7p and Pex5pL are the minimal cytosolic factors in the transport of PTS2 proteins to peroxisomes. Highlights • Two targeting-signals for peroxisomal matrix proteins are identified: PTS1and PTS2. • PTS1 and PTS2 are recognized in the cytosol by Pex5p and Pex7p, respectively. • To delineate mechanisms of PTS2 protein import, we set up a PTS2 import assay system. • Pex7p and Pex5pL are found to be sufficient to transport PTS2-proteins to peroxisome. • PTS2 proteins are imported in a temperature-dependent and an ATP-independent manner. [ABSTRACT FROM AUTHOR]

Abstract: Simultaneous detection of multiple antigens by conventional immunological methods has been limited by the source of primary antibodies. Each antibody should be derived from a different host species (or subclass of immunoglobulin (Ig)) for suppressing the cross-reactions of secondary antibodies. Here we describe an innovative method for simultaneous, rapid, and sensitive detection of multiple antigens using ∼30-nm bio-nanocapsules (BNCs) displaying IgG Fc-binding Z domains derived from Staphylococcus aureus protein A (ZZ-BNC). When Cy2-labeled ZZ-BNC (Cy2-ZZ-BNC) was used instead of Cy2-labeled secondary antibody in western blot analysis, both sensitivity and signal intensity were significantly increased. The complex of Cy5-ZZ-BNC and mouse IgG2a (which shows moderate affinity to the Z domain) was not dissociated, even in the presence of 8-fold excess of free mouse IgG2a. In addition, crosslinking with BS3 (bis-sulfosuccinimidyl suberate) efficiently stabilized the interaction. The ZZ-BNCs labeled with various Cy dyes facilitated the simultaneous detection of multiple antigens using primary antibodies derived from the same host species, by western blot analysis, immunocytochemistry and flow cytometry, which could expand the possibility of bio-imaging probes in various immunofluorescence techniques. [Copyright &y& Elsevier]

We studied the hair-forming ability of epithelium and the relevant activity of dermal papilla (DP) in mouse vibrissal follicles during the hair cycle. Follicles were transversely cut into four pieces and each of them was associated with an isolated DP and grafted beneath the kidney capsule to induce hair formation. Various hair-cycle combinations of the fragments and DPs were examined. Hairs were generated not only in the follicle fragment containing the bulge (fragment III) but also in the fragment between the bulge and hair bulb (fragment II). The hair-forming frequencies were affected by the hair cycle stages of both the follicle fragments and DPs. Fragment III at late anagen (LA) and fragment II at catagen frequently generated hairs when associated with early anagen (EA)-DPs, but infrequently with mid-anagen (MA)-DPs. Oppositely, anagen fragment II produced hairs at a high frequency with MA-DPs and at a low frequency with EA-DPs. Hair generation in anagen fragment II is an unexpected finding because previous studies suggested that, during anagen, this region does not contain clonogenic epithelial cells that have been believed to be crucial for hair formation. Therefore, non-clonogenic epithelial cells would be able to generate hairs as well as clonogenic ones, and they should have a latent hair-forming ability that could be more effectively awakened by MA-DP than by EA-DP stimuli. Non-clonogenic epithelial cells might be a dormant phase of hair precursor cells. Proliferating follicular epithelial cells were detected in the middle and lower outer root sheath throughout the hair cycle but scarcely at LA. These findings suggest that the hair inductivity of DPs should be altered between EA and MA, and follicular epithelial cells would change their DP stimuli-directed hair-forming ability around LA, probably linked to the proliferative activity. [ABSTRACT FROM AUTHOR]

Dermal papilla cells in culture show a lower proliferative capacity compared with dermal fibroblasts, and lose their in situ potency to induce hair follicles in the epidermis at more than 10 passage numbers. This study overcomes these limitations of cultured papilla cells and for the first time demonstrates that papilla cells can be serially cultured for a long period without losing their hair‐inductive potency. Outgrowth and the ensuing proliferation of papilla cells were markedly stimulated when explants of rat vibrissa papillae were cultured with rat sole‐derived keratinocytes. Such feeder effects of the keratinocytes could be replaced to some extent with conditioned medium of the cells. Serial cultivation of papilla cells was established by maintaining them in the conditioned medium in which they were subcultured for more than 90 passages with an approximate population doubling time of 30 h, a value similar to that of rat dermal fibroblasts. During the subculture, they showed morphologic characteristics and phenotypic expressions of original papilla cells. Even after at least 70 passages, papilla cells sustained the innate hair follicle inductive ability at a level comparable with that of intact dermal papillae. The established cell lines did not show tumorigenicity when they were subcutaneously implanted into nude mice. The culture method developed in this study should facillitate the search for a biochemical entity of dermal papilla cells. [ABSTRACT FROM AUTHOR]

An α-selective glycosidation reaction of d -tagatofuranose was successfully achieved using 3,4- O -isopropylidene-protected d -tagatofuranose as a glycosyl donor. A variety of glycosyl acceptors, including primary, secondary, and β-amino alcohols, and carbohydrates, can be used for this d -tagatofuranosidation reaction with complete α-selectivities and good yields (57–83%). The stereochemistries at the anomeric positions were determined by nuclear Overhauser effect spectroscopic correlations, as well as comparison of the chemical shifts in the 13C NMR spectra. Image 1 [ABSTRACT FROM AUTHOR]

Abstract: Peroxisome is a single-membrane organelle in eukaryotes. The functional importance of peroxisomes in humans is highlighted by peroxisome-deficient peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome (ZS). Gene defects of peroxins required for both membrane assembly and matrix protein import are identified: ten mammalian pathogenic peroxins for ten complementation groups of PBDs, are required for matrix protein import; three, Pex3p, Pex16p and Pex19p, are shown to be essential for peroxisome membrane assembly and responsible for the most severe ZS in PBDs of three complementation groups 12, 9, and 14, respectively. Patients with severe ZS with defects of PEX3, PEX16, and PEX19 tend to carry severe mutation such as nonsense mutations, frameshifts and deletions. With respect to the function of these three peroxins in membrane biogenesis, two distinct pathways have been proposed for the import of peroxisomal membrane proteins in mammalian cells: a Pex19p- and Pex3p-dependent class I pathway and a Pex19p- and Pex16p-dependent class II pathway. In class II pathway, Pex19p also forms a soluble complex with newly synthesized Pex3p as the chaperone for Pex3p in the cytosol and directly translocates it to peroxisomes. Pex16p functions as the peroxisomal membrane receptor that is specific to the Pex3p-Pex19p complexes. A model for the import of peroxisomal membrane proteins is suggested, providing new insights into the molecular mechanisms underlying the biogenesis of peroxisomes and its regulation involving Pex3p, Pex19p, and Pex16p. Another model suggests that in Saccharomyces cerevisiae peroxisomes likely emerge from the endoplasmic reticulum. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of peroxisomes in Health and Disease. [Copyright &y& Elsevier]

Abstract: In contrast to the molecular mechanisms underlying import of peroxisomal matrix proteins, those involving the transport of membrane proteins remain rather elusive. At present, two targeting routes for peroxisomal membrane proteins (PMPs) have been depicted: class I PMPs are targeted from the cytoplasm directly to the peroxisome membrane, and class II PMPs are sorted indirectly to peroxisomes via the endoplasmic reticulum (ER). In addition, three peroxins – Pex3p, Pex16p, and Pex19p – have been identified as essential factors for PMP assembly in several species including humans: Pex19p is a predominantly cytoplasmic protein that shows a broad PMP-binding specificity; Pex3p serves as the membrane-anchoring site for Pex19p; and Pex16p – a protein absent in most yeasts – is thought to provide the initial scaffold for recruiting the protein import machinery required for peroxisome membrane biogenesis. Remarkably, the function of Pex16p does not appear to be conserved between different species. In addition, significant disagreement exists about whether Pex19p has a chaperone-like role in the cytosol or at the peroxisome membrane and/or functions as a cycling import receptor for newly synthesized PMPs. Here we review the recent progress made in our understanding of the role of two key players in PMP biogenesis, Pex3p and Pex19p. [Copyright &y& Elsevier]

Activin type II receptors (ActRIIs) are the primary receptors that transmit the activin signal to intracellular signaling pathways. Binding of activins to ActRIIs recruits the activin type I receptor and initiates downstream signaling. We have found that PDZ proteins, named activin receptor-interacting proteins (ARIPs), specifically associate with ActRIIs. We have studied the mechanism that ARIPs regulate cell surface expression and cellular localization of ActRIIs. ARIP2 interacts with both ActRIIs and RalBP1 (Ral binding protein 1) through different domains to dramatically change the localization of ActRIIs. Overexpression of ARIP2 enhances endocytosis of ActRIIs. These data indicate that ARIP2 is a novel factor regulating cell surface ActRII expression and activin function.A novel activin binding protein, follistatin-related gene (FLRG) was identified. FLRG protein binds activin and myostatin with a high affinity. The biological activity of FLRG is similar to those of follistatin, however, the regulation and expression patterns of follistatin and FLRG differ. Immunohistochemical analysis shows that FLRG is distributed in spermatogenic cells of the testis, renal tubules, epithelial cells of the lung, and myocardium. Thus, although structurally and functionally similar, follistatin and FLRG likely play distinct roles as activin/GDF binding proteins in vivo. [Copyright &y& Elsevier]

Follistatin (FS) is well characterized as an activin-binding protein. Recently, a novel follistatin-like protein called follistatin-related gene (FLRG) that has a similar domain organization to that of follistatin has been identified. Like follistatins, FLRG binds activins and bone morphogenetic proteins (BMPs). To study the regulation of FLRG expression, we have analyzed the genomic organization and promoter of the mouse FLRG gene. The mouse FLRG gene consists of five exons, and each encodes discrete functional regions. The overall genomic structure of FLRG is similar to that of FS except that the FLRG gene is missing one exon that codes a third FS domain found in FS. The promoter that covers 2.5 kbp and is linked to a luciferase reporter construct is active in human cervical carcinoma HeLa cells as well as in human embryonic kidney (HEK293) cells. Deletion analysis of the promoter regions indicates that a proximal 550 base pairs are enough for basal FLRG promoter activity in the cell lines. FLRG promoter activity is significantly augmented by phorbol 12-myristate 13-acetate (PMA) treatment, but not by cAMP stimulation. By contrast, FS promoter is activatable either by cAMP or PMA. Thus, although FS and FLRG are structurally and functionally related, their modes of regulation by external stimuli are different. [Copyright &y& Elsevier]

Background and purpose ST2 is one of the interleukin (IL)-1 receptor family members comprising of membrane-bound (ST2L) and soluble (sST2) isoforms. Clinical trials have revealed that serum sST2 levels predict outcome in patient with myocardial infarction or chronic heart failure (HF). Meanwhile, we and others have reported that ablation of ST2 caused exaggerated cardiac remodeling in both ischemic and non-ischemic HF. Here, we tested whether IL-33, the ligand for ST2, protects myocardium against HF induced by mechanical overload using ligand specific knockout (IL-33 −/− ) mice. Methods and results Transverse aortic constriction (TAC)/sham surgery were carried out in both IL-33 and WT-littermates. Echocardiographic measurements were performed at frequent interval during the study period. Heart was harvested for RNA and histological measurements. Following mechanical overload by TAC, myocardial mRNA expressions of Th1 cytokines, such as TNF-α were enhanced in IL-33 −/− mice than in WT mice. After 8-weeks, IL-33 −/− mice exhibited exacerbated left ventricular hypertrophy, increased chamber dilation, reduced fractional shortening, aggravated fibrosis, inflammation, and impaired survival compared with WT littermates. Accordingly, myocardial mRNA expressions of hypertrophic (c-Myc/BNP) molecular markers were also significantly enhanced in IL-33 −/− mice than those in WT mice. Conclusions We report for the first time that ablation of IL-33 directly and significantly leads to exacerbate cardiac remodeling with impaired cardiac function and survival upon mechanical stress. These data highlight the cardioprotective role of IL-33/ST2 system in the stressed myocardium and reveal a potential therapeutic role for IL-33 in non-ischemic HF. [ABSTRACT FROM AUTHOR]