Effects of sodium chloride on growth of ectomycorrhizal fungal isolates in culture

We studied the tolerance of ectomycorrhizal (ECM) fungi to sodium chloride (NaCl) to find the best fungus to aid growth of Pinus thunbergii. Four ECM fungi, Cenococcum geophilum, Pisolithus tinctorius, Rhizopogon rubescens, and Suillus luteus, were grown in liquid MMN media with five different concentrations of NaCl for 30 days, and their mycelial weights were determined. Mycelial weights of P. tinctorius and R. rubescens were not significantly different between 0 mM and 200 mM, whereas those of C. geophilum and S. luteus decreased with increasing NaCl concentration, indicating that the former two species were more tolerant to higher NaCl concentrations than the latter species. We further studied the intraspecific differences in NaCl tolerance of nine P. tinctorius isolates. They were grown on MMN agar media with six different concentrations of NaCl for 21 days, and their radial growth was measured. In total, the hyphal growth at 25 mM NaCl was significantly higher than those at the other NaCl concentrations, and EC₅₀ values were confirmed at between 50 mM and 200 mM. Among the isolates, Pt03 and Pt21 showed measurable growth at 200 mM; the growth of Pt03 was not significantly different between 0 mM and 200 mM. The results indicate that there are intraspecific variations in NaCl tolerance of Pisolithus species.     

Generation of hybrid hepatocytes by cell fusion from monkey embryoid body cells in the injured mouse liver

Monkey embryonic stem (ES) cells have characteristics that are similar to human ES cells, and might be useful as a substitute model for preclinical research. When embryoid bodies (EBs) formed from monkey ES cells were cultured, expression of many hepatocyte-related genes including cytochrome P450 (Cyp) 3a and Cyp7a1 was observed. Hepatocytes were immunocytochemically observed using antibodies against albumin (ALB), cytokeratin-8/18, and α1-antitrypsin in the developing EBs. The in vitro differentiation potential of monkey ES cells into the hepatic lineage prompted us to examine the transplantability of monkey EB cells. As an initial approach to assess the repopulation potential, we transplanted EB cells into immunodeficient urokinase-type plasminogen activator transgenic mice that undergo liver failure. After transplantation, the hepatocyte colonies expressing monkey ALB were observed in the mouse liver. Fluorescence in-situ hybridization revealed that the repopulating hepatocytes arise from cell fusion between transplanted monkey EB cells and recipient mouse hepatocytes. In contrast, neither cell fusion nor repopulation of hepatocytes was observed in the recipient liver after undifferentiated ES cell transplantation. These results indicate that the differentiated cells in developing monkey EBs, but not contaminating ES cells, generate functional hepatocytes by cell fusion with recipient mouse hepatocytes, and repopulate injured mouse liver.     

CIN85 is localized at synapses and forms a complex with S-SCAM via dendrin

Membrane-associated guanylate kinase inverted (MAGI)-1 plays a role as a scaffold at cell junctions in non-neuronal cells, while S-SCAM, its neuronal isoform, is involved in the organization of synapses. A search for MAGI-1-interacting proteins by yeast two-hybrid screening of a kidney cDNA library yielded dendrin. As dendrin was originally reported as a brain-specific postsynaptic protein, we tested the interaction between dendrin and S-SCAM and revealed that dendrin binds to the WW domains of S-SCAM. Dendrin is known to be dendritically translated but its function is largely unknown. To gain insights into the physiological meaning of the interaction, we performed a second yeast two-hybrid screening using dendrin as a bait. We identified CIN85, an endocytic scaffold protein, as a putative dendrin-interactor. Immunocytochemistry and subcellular fractionation analysis supported the synaptic localization of CIN85. The first SH3 domain and the C-terminal region of CIN85 bind to the proline-rich region and the N-terminal region of dendrin, respectively. In vitro experiments suggest that dendrin forms a ternary complex with CIN85 and S-SCAM and that this complex formation facilitates the recruitment of dendrin and S-SCAM to vesicle-like structures where CIN85 is accumulated.     

Galectin-1 suppresses autoimmune retinal disease by promoting concomitant Th2- and T regulatory-mediated anti-inflammatory responses

Intraocular inflammatory diseases are a common cause of severe visual impairment and blindness. In this study, we investigated the immunoregulatory role of galectin-1 (Gal-1), an endogenous lectin found at sites of T cell activation and immune privilege, in experimental autoimmune uveitis (EAU), a Th1-mediated model of retinal disease. Treatment with rGal-1 either early or late during the course of interphotoreceptor retinoid-binding protein-induced EAU was sufficient to suppress ocular pathology, inhibit leukocyte infiltration, and counteract pathogenic Th1 cells. Administration of rGal-1 at the early or late phases of EAU ameliorated disease by skewing the uveitogenic response toward nonpathogenic Th2 or T regulatory-mediated anti-inflammatory responses. Consistently, adoptive transfer of CD4(+) regulatory T cells obtained from rGal-1-treated mice prevented the development of active EAU in syngeneic recipients. In addition, increased levels of apoptosis were detected in lymph nodes from mice treated with rGal-1 during the efferent phase of the disease. Our results underscore the ability of Gal-1 to counteract Th1-mediated responses through different, but potentially overlapping anti-inflammatory mechanisms and suggest a possible therapeutic use of this protein for the treatment of human uveitic diseases of autoimmune etiology.     

Embryonic stem cell-derived neuron models of Parkinson's disease exhibit delayed neuronal death

Establishment of a Parkinson's disease (PD) neuron model was attempted with mouse embryonic stem (ES) cells. ES cell lines over-expressing mouse nuclear receptor-related 1 (Nurr1), together with human wild-type and alanine 30 --> proline (A30P) and alanine 53 --> threonine (A53T) mutant alpha-synuclein were established and subjected to differentiation into dopaminergic neurons. The ES cell-derived dopaminergic neurons expressing wild-type or mutant alpha-synuclein exhibited the fundamental characteristics consistent with dopaminergic neurons in the substantia nigra. The ES cell-derived PD model neurons exhibited increased susceptibility to oxidative stress, proteasome inhibition, and mitochondrial inhibition. Cell viability of PD model neurons and the control neurons was similar until 28 days after differentiation. Nonetheless, after that time, PD model neurons gradually began to undergo neuronal death over the course of 1 month, showing cytoplasmic aggregate formation and an increase of insoluble alpha-synuclein protein. Such delayed neuronal death was observed in a mutant alpha-synuclein protein level-dependent manner, which was slightly inhibited by a c-jun N-terminal kinase inhibitor and a caspase inhibitor. Such cell death was not observed when the same ES cell lines were differentiated into oligodendrocytes. The ES cell-derived PD model neurons are considered as prospective candidates for a new prototype modelling PD that would allow better investigation of the underlying neurodegenerative pathophysiology.     

Expression of FGF23 is correlated with serum phosphate level in isolated fibrous dysplasia

Fibrous dysplasia (FD) patients sometimes suffer from concomitant hypophosphatemic rickets/osteomalacia, resulting from renal phosphate wasting. It was recently reported that FD tissue in the patients with McCune-Albright syndrome (MAS) expressed fibroblast growth factor-23 (FGF-23), which is now known to be as a pathogenic phosphaturic factor in patients with oncogenic osteomalacia and X-linked hypophosphatemic rickets. Since it remains controversial whether serum phosphate levels are influenced by FGF23 expressions in FD tissue, isolated FD patients without MAS syndrome were examined for the relationship between FGF23 expressions, circulating levels of FGF-23 and phosphate to negate the effects of MAS-associated endocrine abnormalities on serum phosphate. Eighteen paraffin embedded FD tissues and 2 frozen tissues were obtained for the study. Sixteen of 18 isolated FD tissues were successfully analyzed GNAS gene, which exhibited activated mutations observed in MAS. Eight of 16 FD tissues, which exhibited GNAS mutations, revealed positive staining for FGF-23. These evidence indicate that postzygotic activated mutations of GNAS is necessary for the FD tissue formation by mosaic distribution of mutated osteogenic cell lineage, but is not sufficient to elevate FGF23 expression causing generalized osteomalacia with severe renal phosphate wasting. The expression level of FGF23 in isolated FD tissue with hypophosphatemic osteomalacia determined by real-time PCR was abundant close to the levels in OOM tumors. Osteoblasts/osteocytes in woven bone were predominant source of circulating FGF-23 in FD tissues by immunohistochemistry. A negative correlation of the intensity of FGF-23 staining with serum inorganic phosphate levels indicated that the expression of FGF23 in focal FD tissues could be a prominent determinant of serum phosphate levels in isolated FD patient. These data provide novel insights into the regulatory mechanism of serum inorganic phosphate levels in isolated FD patients and extend the notion that FGF-23 originating from FD tissue may cause hypophosphatemia not only in isolated FD patients but also in the patients with MAS syndrome.     

3′-Untranslated region of doublecortin mRNA is a binding target of the Musashi1 RNA-binding protein

Musashi1 (Msi1) is an RNA-binding protein that is highly expressed in neural stem cells, and is considered to be a stemness factor. A known function of Msi1 is translational repression of specifically bound mRNAs. Although the basic mechanism and some target RNAs have been reported, further survey of interactors is necessary to understand the integrated function of Msi1. By screening using an mRNA display technique, we found that doublecortin (dcx) mRNA is a specific binding target of Msi1 in vitro. We confirmed that Msil repressed translation of a luciferase reporter gene linked to the selected 3′-untranslated region fragment of dcx in Neuro2A cells.     

Energy expenditure associated with softening and stiffening of echinoderm connective tissue

Catch connective tissue of echinoderms at rest (in the standard state) either stiffens or softens in response to different kinds of stimulation. The energy consumption associated with the changes was estimated by measurement of the oxygen consumption rate (VO(2)) in three types of connective tissues-echinoid catch apparatus (CA), holothuroid body-wall dermis (HD), and asteroid body-wall dermis (AD). Mechanical stimulation by repetitive compression (10%-15% strain), which increased viscosity measured by creep tests, was employed for inducing the stiff state. Noradrenaline (10(-3) mol l(-1)), which decreased viscosity of CA, and static 80% compressive strain, which decreased viscosity of HD, were used to induce the soft state in the respective tissues. The VO(2) (in μl/g/h) values of the standard state were 2.91 (CA), 1.41 (HD), and 0.56 (AD), which were less than 1/4 of the VO(2) of the resting body-wall muscle of the starfish. The VO(2) of the stiff state was about 1.5 times greater than that of the standard state in all types of connective tissues. The VO(2) of the soft state was 3.4 (CA)-9.1 (HD) times greater than that of the standard state. The economical nature of catch connective tissue in posture maintenance is discussed.     

Formation of tRNA granules in the nucleus of heat-induced human cells

The stress response, which can trigger various physiological phenomena, is important for living organisms. For instance, a number of stress-induced granules such as P-body and stress granule have been identified. These granules are formed in the cytoplasm under stress conditions and are associated with translational inhibition and mRNA decay. In the nucleus, there is a focus named nuclear stress body (nSB) that distinguishes these structures from cytoplasmic stress granules. Many splicing factors and long non-coding RNA species localize in nSBs as a result of stress. Indeed, tRNAs respond to several kinds of stress such as heat, oxidation or starvation. Although nuclear accumulation of tRNAs occurs in starved Saccharomyces cerevisiae, this phenomenon is not found in mammalian cells. We observed that initiator tRNA(Met) (Meti) is actively translocated into the nucleus of human cells under heat stress. During this study, we identified unique granules of Meti that overlapped with nSBs. Similarly, elongator tRNA(Met) was translocated into the nucleus and formed granules during heat stress. Formation of tRNA granules is closely related to the translocation ratio. Then, all tRNAs may form the specific granules.