Repulsive guidance molecule b inhibits neurite growth and is increased after spinal cord injury

Neuronal axons are guided by attractive and repulsive cues in their local environment. Since the identification of the repulsive guidance molecule (RGM) a (RGMa) as an axon repellent in the visual system, diverse functions, as part of the developing and adult central nervous system (CNS), have been ascribed to it. The binding of RGMa to its receptor neogenin has been shown to induce RhoA activation, leading to inhibitory/repulsive behavior and the collapse of the neuronal growth cone. In this paper, we provide evidence to suggest the involvement of RGMb, another member of the RGM family, in the rat CNS. RGMb inhibits neurite outgrowth in postnatal cerebellar granule neurons (CGNs) in vitro. RGMb is expressed by oligodendrocytes and neurons in the adult rat CNS, and the expression of this molecule is upregulated around the site of spinal cord injury. RGMb is present in myelin isolated from an adult rat brain. RGMb and neogenin are coexpressed in CGNs and entorhinal cortex neurons. These findings suggest that RGMb is a myelin-derived inhibitor of axon growth in the CNS. Inhibition of RGMb may provide an alternative approach for the treatment of spinal injuries.     

Proteomic analysis of membrane proteins expressed specifically in pluripotent murine embryonic stem cells

Embryonic stem cells (ESCs) are established from the inner cell mass of preimplantation embryos, are capable of self‐renewal, and exhibit pluripotency. Given these unique properties, ESCs are expected to have therapeutic potential in regenerative medicine and as a powerful tool for in vitro differentiation studies of stem cells. Various growth factors and extracellular matrix components regulate the pluripotency and differentiation of ESC progenies. Thus, the cell surface receptors that bind these regulatory factors are crucial for the precise regulation of stem cells. To identify membrane proteins that are involved in the regulation of pluripotent stem cells, the membrane proteins of murine ESCs cultured with or without leukemia inhibitory factor (LIF) were purified and analyzed by quantitative proteomics. 2‐D PAGE‐based analysis using fluorescently labeled proteins and shotgun‐based analysis with isotope‐labeled peptides identified 338 proteins, including transmembrane, membrane‐binding, and extracellular proteins, which were expressed specifically in pluripotent or differentiated murine ESCs. Functions of the identified proteins revealed cell adhesion molecules, channels, and receptors, which are expected to play important roles in the maintenance of murine ESC pluripotency. Membrane proteins that are expressed in pluripotent ESCs but not in differentiated cells such as Slc16a1 and Bsg could be useful for the selection of the stem cells in vitro.     

Thyroglobulin may affect telomerase activity in thyroid follicular cells

Telomerase (TA) activity is known to be present in malignant tumor cells, but not in most somatic differentiated cells. TA shows relatively high activity in thyroid cancer cells, but reports vary. This fact prompted us to elucidate whether cell component inhibitors of TA in the thyroid follicles can modulate its activity. The activity of TA extracted from Hela cells was inhibited by mixing with the supernatant fraction of human thyroid tissue extract. To examine the effect of iodine, thyroid hormones (l-T3 and l-T4) and human thyroglobulin (hTg) contained in the thyroid follicles, l-T3, l-T4 and hTg were added to the TRAP assay system in vitro, using TA from Hela cells. Iodine, l-T3 and l-T4 did not affect TA activity, but hTg inhibited the TA activity in a dose-dependent manner (IC(50) of hTg: ca 0.45 microM: inhibiting concentration of hTg was from 0.15 microM to 3.0 microM). The hTg inhibition was not evident in the RT-PCR system, suggesting no effect of hTg on Taq DNA polymerase activity. The hTg inhibition of TA activity was attenuated by dNTP but not significantly by TS primer. These data suggest that hTg contained in thyroid follicular cells of various thyroid diseases may affect the TA activity measured in biopsied thyroid specimens, and that the reduction of the TA activity by hTg may induce slow progression and growth, and low grade malignancy of thyroid cancer, particularly differentiated carcinoma.     

Focused differential glycan analysis with the platform antibody-assisted lectin profiling for glycan-related biomarker verification

Protein glycosylation is a critical subject attracting increasing attention in the field of proteomics as it is expected to play a key role in the investigation of histological and diagnostic biomarkers. In this context, an enormous number of glycoproteins have now been nominated as disease-related biomarkers. However, there is no appropriate strategy in the current proteome platform to qualify such marker candidate molecules, which relates their specific expression to particular diseases. Here, we present a new practical system for focused differential glycan analysis in terms of antibody-assisted lectin profiling (ALP). In the developed procedure, (i) a target protein is enriched from clinic samples (e.g. tissue extracts, cell supernatants, or sera) by immunoprecipitation with a specific antibody recognizing a core protein moiety; (ii) the target glycoprotein is quantified by immunoblotting using the same antibody used in (i); and (iii) glycosylation difference is analyzed by means of antibody-overlay lectin microarray, an application technique of an emerging glycan profiling microarray. As model glycoproteins having either N-linked or O-linked glycans, prostate-specific antigen or podoplanin, respectively, were subjected to systematic ALP analysis. As a result, specific signals corresponding to the target glycoprotein glycans were obtained at a sub-picomole level with the aid of specific antibodies, whereby disease-specific or tissue-specific glycosylation changes could be observed in a rapid, reproducible, and high-throughput manner. Thus, the established system should provide a powerful pipeline in support of on-going efforts in glyco-biomarker discovery.     

JNK and p38 MAPK are independently involved in tributyltin-mediated cell death in rainbow trout (Oncorhynchus mykiss) RTG-2 cells

Mitogen-activated protein kinases (MAPKs) are a family of Ser/Thr protein kinases that transmit various extracellular signals to the nucleus inducing gene expression, cell proliferation, and apoptosis. Recent studies have revealed that organotin compounds induce apoptosis and MAPK phosphorylation/activation in mammal cells. In this study, we elucidated the cytotoxic mechanism of tributyltin (TBT), a representative organotin compound, in rainbow trout (Oncorhynchus mykiss) RTG-2 cells. TBT treatment resulted in significant caspase activation, characteristic morphological changes, DNA fragmentation, and consequent apoptotic cell death in RTG-2 cells. TBT exposure induced the rapid and sustained accumulation of phosphorylated MAPKs, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAP kinase (p38 MAPK). Further analysis using pharmacological inhibitors against caspases and MAPKs showed that TBT also induced cell death in a caspase-independent manner and that p38 MAPK is involved in TBT-induced caspase-independent cell death, whereas JNK is involved in the caspase-dependent apoptotic pathway. Thus, TBT employs at least two independent signaling cascades to mediate cell death in RTG-2 cells. To our knowledge, this is the first study revealing the relationship between MAPK activation and TBT cytotoxicity in RTG-2 cells.     

Protective Effect of Geranylgeranylacetone via Enhancement of HSPB8 Induction in Desmin-Related Cardiomyopathy

Background

An arg120gly (R120G) missense mutation in HSPB5 (α-β-crystallin ), which belongs to the small heat shock protein (HSP) family, causes desmin-related cardiomyopathy (DRM), a muscle disease that is characterized by the formation of inclusion bodies, which can contain pre-amyloid oligomer intermediates (amyloid oligomer). While we have shown that small HSPs can directly interrupt amyloid oligomer formation, the in vivo protective effects of the small HSPs on the development of DRM is still uncertain.

Methodology/Principal Findings

In order to extend the previous in vitro findings to in vivo, we used geranylgeranylacetone (GGA), a potent HSP inducer. Oral administration of GGA resulted not only in up-regulation of the expression level of HSPB8 and HSPB1 in the heart of HSPB5 R120G transgenic (R120G TG) mice, but also reduced amyloid oligomer levels and aggregates. Furthermore, R120G TG mice treated with GGA exhibited decreased heart size and less interstitial fibrosis, as well as improved cardiac function and survival compared to untreated R120G TG mice. To address possible mechanism(s) for these beneficial effects, cardiac-specific transgenic mice expressing HSPB8 were generated. Overexpression of HSPB8 led to a reduction in amyloid oligomer and aggregate formation, resulting in improved cardiac function and survival. Treatment with GGA as well as the overexpression of HSPB8 also inhibited cytochrome c release from mitochondria, activation of caspase-3 and TUNEL-positive cardiomyocyte death in the R120G TG mice.

Conclusions/Significance

Expression of small HSPs such as HSPB8 and HSPB1 by GGA may be a new therapeutic strategy for patients with DRM.     

Saccadic Compression of Rectangle and Kanizsa Figures: Now You See It,Now You Don't

Background

Observers misperceive the location of points within a scene as compressed towards the goal of a saccade. However, recent studies suggest that saccadic compression does not occur for discrete elements such as dots when they are perceived as unified objects like a rectangle.

Methodology/Principal Findings

We investigated the magnitude of horizontal vs. vertical compression for Kanizsa figure (a collection of discrete elements unified into single perceptual objects by illusory contours) and control rectangle figures. Participants were presented with Kanizsa and control figures and had to decide whether the horizontal or vertical length of stimulus was longer using the two-alternative force choice method. Our findings show that large but not small Kanizsa figures are perceived as compressed, that such compression is large in the horizontal dimension and small or nil in the vertical dimension. In contrast to recent findings, we found no saccadic compression for control rectangles.

Conclusions

Our data suggest that compression of Kanizsa figure has been overestimated in previous research due to methodological artifacts, and highlight the importance of studying perceptual phenomena by multiple methods.