Involvement of position-147 for HLA-E expression

HLA-E functions as an inhibitory signaling molecule of natural killer (NK) cell-mediated cytolysis. However, the cell surface expression of HLA-E molecules is quite restricted because of the limited repertoire of binding peptide sequences, such as signal peptides of other HLA molecules, especially on xenogeneic cells. In this study, we successfully determined that position-147 is an important amino acid position for cell surface expression by producing point substitutions. For further studies concerning transplantation therapy, the point substitution, Ser147Cys, that resulted in a single atom change, oxygen to sulfur, designated as HLA-Ev(147), led to a much higher expression on the human and pig cell surface and a greater inhibitory function against human NK cells than wild type HLA-E in an in vitro model system of pig to human xenotransplantation. Consequently, HLA-Ev(147) might be a promising alternative gene tool for future transplantation therapy such as xenotransplantation.     

Serum ethanolamine and hepatocyte proliferation in perinatal and partially hepatectomized rats

It has been shown that the administration of ethanolamine (Etn) to partially hepatectomized rats enhances stimulation of DNA synthesis in regenerating hepatocytes. The present study aimed to test the hypothesis that the level of serum Etn in vivo may be regulated to control the growth of hepatocytes. Concentrations of serum Etn were determined in rats 1) of varying ages (from embryonic-19 (E-19) to 7-week-old), and 2) during regeneration following two-thirds hepatectomy (PH), to investigate whether serum Etn concentration correlates with the rate of proliferation of hepatocytes in growing animals or during regeneration. Serum Etn levels were 3 fold higher in E-19 fetuses and newborns than in adults, and were increased 2 fold 4 h after PH and remained high for at least 24 h. Results in both systems indicated a significant positive correlation between the rate of hepatocyte proliferation and serum Etn levels. Furthermore, Etn supplementation of 0.1 to 1 mmol immediately after PH promoted a significant weight gain and stimulated phosphatidylethanolamine (PE) and phosphatidylcholine (PC) synthesis in the regenerating liver. We also observed that whenever serum Etn levels were elevated, the metabolism of PE and PC in the liver changed dynamically, first by elevating the net synthesis of PE. Taken together, these results suggested that the levels of serum Etn might be regulated based on the physiological state of an animal, which consequently regulates the proliferation of hepatocytes.     

An RMND1 Mutation Causes Encephalopathy Associated with Multiple Oxidative Phosphorylation Complex Deficiencies and a Mitochondrial Translation Defect

Mutations in the genes composing the mitochondrial translation apparatus are an important cause of a heterogeneous group of oxidative phosphorylation (OXPHOS) disorders. We studied the index case in a consanguineous family in which two children presented with severe encephalopathy, lactic acidosis, and intractable seizures leading to an early fatal outcome. Blue native polyacrylamide gel electrophoretic (BN-PAGE) analysis showed assembly defects in all of the OXPHOS complexes with mtDNA-encoded structural subunits, and these defects were associated with a severe deficiency in mitochondrial translation. Immunoblot analysis showed reductions in the steady-state levels of several structural subunits of the mitochondrial ribosome. Whole-exome sequencing identified a homozygous missense mutation (c.1250G>A) in an uncharacterized gene, RMND1 (required for meiotic nuclear division 1). RMND1 localizes to mitochondria and behaves as an integral membrane protein. Retroviral expression of the wild-type RMND1 cDNA rescued the biochemical phenotype in subject cells, and siRNA-mediated knockdown of the protein recapitulated the defect. BN-PAGE, gel filtration, and mass spectrometry analyses showed that RMND1 forms a high-molecular-weight and most likely homopolymeric complex (∼240 kDa) that does not assemble in subject fibroblasts but that is rescued by expression of RMND1 cDNA. The p.Arg417Gln substitution, predicted to be in a coiled-coil domain, which is juxtaposed to a transmembrane domain at the extreme C terminus of the protein, does not alter the steady-state level of RMND1 but might prevent protein-protein interactions in this complex. Our results demonstrate that the RMND1 complex is necessary for mitochondrial translation, possibly by coordinating the assembly or maintenance of the mitochondrial ribosome.     

The Mitochondrial RNA-Binding Protein GRSF1 Localizes to RNA Granules and Is Required for Posttranscriptional Mitochondrial Gene Expression

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Highlights? GRSF1 (G-rich sequence binding factor 1) is a mitochondrial RNA-binding protein ? GRSF1 localizes to granules containing newly synthesized RNA in close proximity to mitochondrial nucleoids ? GRSF1 specifically binds one mRNA and two long noncoding RNAs transcribed from contiguous genes on the light strand of mtDNA ? Loss of GRSF1 leads to misloading of RNAs onto the mitochondrial ribosome and a specific translation defect     

Furanoditerpenes from Arcangelisia flava (L.) Merr. and their antifungal activity

Two furanoditerpenes, 2α,3α-epoxy-2,3,7,8α-tetrahydropenianthic acid methyl ester (1) and 2α,3α-epoxy-2,3-dihydropenianthic acid methyl ester (2) were isolated and identified from the root of Arcangelisia flava (L.) Merr. The configuration of 1 was determined by X-ray crystallographic analysis and two-dimensional NMR. Fibraurin (3), fibleucin (4), 2β, 3α-dihydroxy-2,3,7,8α-tetrahydropenianthic acid-2,17-lactone (5), p-hydroxybenzaldehyde and vanillin were also isolated and identified by NMR and EI-MS or FAB-MS. The 2β, 3α-dihydroxy-2,3,7,8α-tetrahydropenianthic acid-2,17-lactone (5) showed the highest antifungal activity of the isolated five furanoditerpenes against a white-rot fungus (Trametes versicolor) and a brown-rot fungus (Fomitopsis palustris).     

Canonical Wnt signaling in the visceral muscle is required for left-right asymmetric development of the Drosophila midgut

Many animals develop left-right (LR) asymmetry in their internal organs. The mechanisms of LR asymmetric development are evolutionarily divergent, and are poorly understood in invertebrates. Therefore, we studied the genetic pathway of LR asymmetric development in Drosophila. Drosophila has several organs that show directional and stereotypic LR asymmetry, including the embryonic gut, which is the first organ to develop LR asymmetry during Drosophila development. In this study, we found that genes encoding components of the Wnt-signaling pathway are required for LR asymmetric development of the anterior part of the embryonic midgut (AMG). frizzled 2 (fz2) and Wnt4, which encode a receptor and ligand of Wnt signaling, respectively, were required for the LR asymmetric development of the AMG. arrow (arr), an ortholog of the mammalian gene encoding low-density lipoprotein receptor-related protein 5/6, which is a co-receptor of the Wnt-signaling pathway, was also essential for LR asymmetric development of the AMG. These results are the first demonstration that Wnt signaling contributes to LR asymmetric development in invertebrates, as it does in vertebrates. The AMG consists of visceral muscle and an epithelial tube. Our genetic analyses revealed that Wnt signaling in the visceral muscle but not the epithelium of the midgut is required for the AMG to develop its normal laterality. Furthermore, fz2 and Wnt4 were expressed in the visceral muscles of the midgut. Consistent with these results, we observed that the LR asymmetric rearrangement of the visceral muscle cells, the first visible asymmetry of the developing AMG, did not occur in embryos lacking Wnt4 expression. Our results also suggest that canonical Wnt/β-catenin signaling, but not non-canonical Wnt signaling, is responsible for the LR asymmetric development of the AMG. Canonical Wnt/β-catenin signaling is reported to have important roles in LR asymmetric development in zebrafish. Thus, the contribution of canonical Wnt/β-catenin signaling to LR asymmetric development may be an evolutionarily conserved feature between vertebrates and invertebrates.     

Evolution of metaplastic squamous cells of alveolar walls in pulmonary fibrosis produced by paraquat

Sequential histologic, ultrastructural, immunohistochemical and morphometric studies were made of the evolutional changes of metaplastic and regenerating alveolar epithelial cells in monkeys from 3 days to 8 weeks after paraquat administration. In the early proliferative phase, many alveoli were lined by single-layered and stratified squamous epithelium and bronchiolized epithelium (i.e., presumably derived from bronchi and bronchioles). The regenerating epithelial cells had well developed bundles of actin-like filaments, which were arranged parallel to the basal surfaces of the cells and were associated with zonulae adherentes; these cells also had intermediate filaments and some desmosomes, but lacked basement membranes, hemidesmosomes and anchoring fibrils. They covered either denuded, wavy and disrupted original epithelial basement membranes or areas of developing intraalveolar fibrosis. In zones of squamous epithelial cell metaplasia associated with intraalveolar fibrosis, fibronexus-like structures appeared to be responsible for the initial adhesion of the cells to the underlying connective tissue. In later phases, single-layered and stratified squamous epithelial cells disappeared, and only bronchiolized epithelial cells, with hemidesmosomes and anchoring fibrils on their basal surfaces, were found in fibrotic alveoli. Although bronchiolized and squamous metaplastic epithelial cells are generally thought to be formed as late events in pulmonary damage, such cells play an important role in early, temporary repair of damaged alveoli.     

Phosphatidylethanolamine deficiency in membrane lipids inhibits keratinocyte intercellular networks formation

Ethanolamine (Etn) is required for the growth of epithelial cells in culture. Without Etn, the amount of phosphatidylethanolamine (PE) in membrane lipids is reduced, and cell proliferation stops. When the membrane lipids are deficient of PE, some extracellular signaling processes become impaired. In this study, we examined the effect of Etn deprivation on the formation of intercellular networks in immortalized human oral keratinocytes. Keratinocytes proliferate with undifferentiated morphologies in a low-calcium medium, whereas they undergo differentiation to form intercellular networks in a high-calcium medium. The cells were first cultured with or without Etn supplement in a low-calcium (0.07 mM) medium, and then the calcium concentration was raised to 1.8 mM. The localization and organization of the following proteins were examined: (1) desmogleins and plakoglobin in desmosomes, (2) E-cadherin and beta-catenin in adherens junctions and (3) actin and keratin filaments in cytoskeletons. As expected, in the Etn-supplemented cells, the elevated level of calcium induced the junctional localization of the proteins associated with desmosomes and adherens junctions and also induced the formation of keratin and actin networks. On the contrary, in the Etn-deprived cells, the elevated level of calcium induced none of the above processes. The results suggest that having a sufficient amount of PE or proper phospholipid composition in the membranes is crucial for differentiation in epithelial cells.     

Immunohistochemical expression of G protein α-subunit isoforms in rat and monkey Merkel cell-neurite complexes

The true function of Merkel cells (MCs) is still enigmatic, though the localization of various kinds of neurotransmitter-like substances in MCs has been revealed by immunohistochemistry. Most of the neurotransmitters act on target cells via seven-transmembrane receptors coupled to heterotrimeric G proteins. The heterotrimeric G proteins include various subfamilies that contribute to different signal transduction pathways. Therefore investigation of specific types of G proteins in MCs and related axon terminals (MC-axon terminals) should contribute to the elucidation of the function of MCs. In this study, we investigated the expression patterns of alpha-subunit isoforms of G proteins in MC-neurite complexes of the rat and monkey by enzymatic and fluorescence immunohistochemistry. MC-axon terminals of the rat and monkey showed positive immunoreactions of Galphao and Galphai1. Those of the monkey also showed a weak immunoreaction of Galphas. On the other hand, MCs of both animals showed positive immunoreactions of Galphao, Galphai1, Galphaq, and Galphaz. In addition, MCs of the monkey showed weak immunoreactions of Galphas. Galphao- and Galphai1-like immunoreactions in the MC-axon terminals suggest that MCs suppressively regulate receptive functions of type I mechanosensory nerve terminals. On the other hand, the localization of Galpha-subunits in MCs suggests that these cells are regulated with hormones, neurotransmitter-like substances, or growth factors.     

Immunohistochemical expressions of mGluR5, P2Y2 receptor,PLC-β1, and IP3R-I and -II in Merkel cells in rat sinus hair follicles

We previously found that Merkel cells (MCs) of the rat and monkey show a strong immunoreaction of the -subunit of Gq protein. The Gq-subunit isoform activates isozymes of phospholipase C- (PLC-), which produces inositol-(1,4,5)-triphosphate (IP3) which mobilizes intracellular Ca⁺⁺ from calcium stores via IP3 receptors. Glutamate and adenosine triphosphate (ATP), which are candidates for neurotransmitters in Merkel endings, are known to couple to Gq. Although MCs showed positive immunoreactions of metabotropic glutamate receptor 5 (mGluR5) in our preliminary study, these cells were not reactive to all antibodies to PLC- isozymes. We, therefore, reinvestigated immunohistochemical affinities to MCs of antibodies to PLC- isozymes and mGluRs using frozen sections of rat sinus hair follicles that were briefly postfixed in formaldehyde. We also studied the immunohistochemical expressions of P2Y receptors for ATP and IP3 receptor subtypes using similar sections. Merkel cells showed positive immunoreactions of PLC-1 and mGluR5. It was also found that MCs show positive immunoreactions of P2Y2, IP3R-I, and IP3R-II receptors. These results suggest that the Gq isoform in MCs couples to both the P2Y2 receptor and mGluR5 and regulates the intracellular Ca⁺⁺ concentration via the PLC-–IP3 cascade.