Accumulation of noncrystalline cellulose in Physarum microplasmodia

Physarum plasmodium lives as a slimy mass of protoplast in the dark fragments into small multinucleated microplasmodia (mPL) in a liquid medium. When mPL are exposed to several unfavorable environments, they transform into “spherules” with a cell wall. Using a synchronous spherule-induction system for mPL, we examined the effect of 2,6-dichlorobenzonitrile on the synthesis of cellulose in mPL, by observing mPL under a fluorescence microscope, and isolated cellulose from mPL to identify them morphologically under scanning electron microscopy. Moreover, we examined in vivo labeling to determine when cellulose synthesis is activated in step 2. We found that the nourishment medium in step 2 was essential for mPL prior to spherulation and that the conversion starts at 48 h in step 2 of our system. From the experiments using Updegraff reagent for the sedimentation of cellulose in the cell wall fraction from mPL, we propose that cellulose produced in mPL is likely noncrystalline cellulose. We conclude that mPL of multinucleated protoplasts without the cell wall structure synthesize cellulose under constitutive condition and accumulate abundantly noncrystalline cellulose, in preparation for unfavorable environments that may occur in the future in which mPL must initiate the program to form the cell wall of spherules.     

Extracellular superoxide dismutase in tissues from obese (ob/ob) mice

We have examined the protein content and gene expression of three superoxide dismutase (SOD) isoenzymes in eight tissues from obese ob/ob mice, particularly placing the focus on extracellular-SOD (EC-SOD) in the white adipose tissue (WAT). Obesity significantly increased EC-SOD level in liver, kidney, testis, gastrocnemius muscle, WAT, brown adipose tissue (BAT), and plasma, but significantly decreased the isoenzyme level in lung. Tumor necrosis factor-α and interleukin-1β contents in WAT were significantly higher in obese mice than in lean control mice. Immunohistochemically, both WAT and BAT from obese mice could be stained deeply with anti-mouse EC-SOD antibody compared with those from lean mice. Each primary culture per se almost time-dependently enhanced EC-SOD production, and overtly expressed its mRNA. The loss of heparin-binding affinity of EC-SOD type C with high affinity for heparin occurred in kidney of obese mice. These results suggest that the physiological importance of this SOD isoenzyme in WAT may be a compensatory adaptation to oxidative stress.     

Hepatitis E virus infection in two different regions of Indonesia with identification of swine HEV genotype 3

Hepatitis E is an emerging disease with a high incidence globally. Few data are available on hepatitis E virus (HEV) infection in Indonesia. To obtain molecular information on HEV infection in two regions of Indonesia with different customs and swine breeding conditions, serum samples from 137 swine farm workers, 100 blood donors and 100 swine (27 fecal samples also obtained) in Yogyakarta (Central Java) and from 12 and 64 swine farm workers, 42 and 135 local residents and 89 and 119 swine in Tulungagung (East Java) and Mengwi (Bali), respectively, from our previous study, were compared. Serological tests for anti‐HEV antibodies by ELISA, HEV‐RNA detection by RT‐PCR and phylogenetic analysis were performed. The total prevalence of anti‐HEV antibodies in humans was higher in Bali (11.6%) than in Java (5.1%; P = 0.015). No significant differences in anti‐HEV prevalence among swine farm workers and local residents in Java were found. The finding of swine HEV genotype 3 in specimens from Yogyakarta and genotype 4 from Tulungagung and Bali is somewhat different from other reports. We suggest other factors in addition to close contact with swine might play an important role in HEV transmission of non‐endemic/related custom groups. To the best of our knowledge, this is the first report on swine HEV genotype 3 in Indonesia.     

Influence of Dietary Protein,Carbohydrate, and Fat on Pyruvate Kinase Activity in Rat Liver and Kidney

The effect of individual nutrients on pyruvate kinase activity was studied in the rat liver and kidney, and experimental results are summarized as follows: The level of the enzyme in the liver increased with the feeding of carbohydrate, and unaffected by the feeding of protein or fat. On the other hand, the level of renal enzyme was influenced by the amount of protein ingested. The feeding of protein led to increased enzyme activity in this organ. The results presented show that there is a clear difference in the response of pyruvate kinase level in the liver and kidneys to changes in the diet.     

Moesin Controls Clathrin-Mediated S1PR1 Internalization in T Cells

The lipid mediator sphingosine 1-phosphate (S1P) regulates a wide range of cellular activities, including vascular maturation, angiogenesis, and immune-cell trafficking. Among the five known receptors for S1P (S1PR1-S1PR5), S1PR1 is a critical regulator of lymphocyte trafficking: its signaling is required for lymphocyte egress from lymphoid organs, while its down-modulation by agonist-induced internalization is a prerequisite for lymphocyte entry into lymphoid organs from the bloodstream. Despite the importance of S1PR1 down-regulation in determining lymphocyte behavior, the molecular mechanism of its internalization in lymphocytes has not been defined. Here we show that agonist-induced S1PR1 internalization in T cells occurs via clathrin-mediated endocytosis and is regulated by moesin, an ezrin-radixin-moesin (ERM) family member. In S1P-stimulated T cells, S1PR1 relocalized within clathrin-coated vesicles (CCVs) and early endosomes, and S1PR1 internalization was blocked when clathrin was pharmacologically inhibited. Stimulating moesin-deficient T cells with S1P failed to induce S1PR1 internalization and CCV formation. Furthermore, treating moesin-deficient mice with FTY720, an S1P receptor agonist known to internalize S1PR1, caused delayed lymphopenia, and lymphocytes isolated from FTY720-treated moesin-deficient mice still responded to S1P ex vivo in chemotaxis assays. These results reveal a novel role for moesin in regulating clathrin-dependent S1PR1 internalization through CCV formation.     

Plasmablasts as Migratory IgG-Producing Cells in the Pathogenesis of Neuromyelitis Optica

Neuromyelitis optica (NMO) is an inflammatory disease characterized by recurrent attacks of optic neuritis and myelitis. It is generally accepted that autoantibodies against aquaporin 4 water channel protein play a pathogenic role in neuromyelitis optica. We have recently reported that plasmablasts are increased in the peripheral blood of this autoimmune disease, and are capable of producing autoantibodies against aquaporin 4. Here, we demonstrate that CD138⁺HLA-DR⁺ plasmablasts, a subset of IgG-producing cells, are increased in the peripheral blood and are enriched among the cerebrospinal fluid (CSF) lymphocytes during the relapse of neuromyelitis optica. Notably, these CD138⁺HLA-DR⁺ plasmablasts overexpress CXCR3, whose ligands are present in the cerebrospinal fluid during the relapse of neuromyelitis optica. These results led us to speculate that plasmablasts producing anti-aquaporin 4 autoantibodies might traffic toward the central nervous system (CNS). Furthermore, we performed single-cell sorting of plasmablasts from peripheral blood and CSF samples from NMO and sequenced the complementarity-determining regions (CDRs) of the IgG heavy chain expressed by the sorted plasmablast clones. There were high frequencies of mutations in the CDRs compared with framework regions, indicating that these plasmablast clones would represent a post-germinal center B-cell lineage. Consistent with the preceding results, the plasmablast clones from the peripheral blood shared the same CDR sequences with the clones from the CSF. These results indicate that IgG-producing plasmablasts, which are guided by helper T-cells, may migrate from the peripheral blood preferentially to the CSF. Since migratory plasmablasts could be involved in the inflammatory pathology of NMO, the B-cell subset and their migration might be an attractive therapeutic target.     

Interleukin-6 Mediates Epithelial–Stromal Interactions and Promotes Gastric Tumorigenesis

Interleukin-6 (IL-6) is a pleiotropic cytokine that affects various functions, including tumor development. Although the importance of IL-6 in gastric cancer has been documented in experimental and clinical studies, the mechanism by which IL-6 promotes gastric cancer remains unclear. In this study, we investigated the role of IL-6 in the epithelial–stromal interaction in gastric tumorigenesis. Immunohistochemical analysis of human gastritis, gastric adenoma, and gastric cancer tissues revealed that IL-6 was frequently detected in the stroma. IL-6–positive cells in the stroma showed positive staining for the fibroblast marker α-smooth muscle actin, suggesting that stromal fibroblasts produce IL-6. We compared IL-6 knockout (IL-6^−/−) mice with wild-type (WT) mice in a model of gastric tumorigenesis induced by the chemical carcinogen N-methyl-N-nitrosourea. The stromal fibroblasts expressed IL-6 in tumors from WT mice. Gastric tumorigenesis was attenuated in IL-6^−/− mice, compared with WT mice. Impaired tumor development in IL-6^−/− mice was correlated with the decreased activation of STAT3, a factor associated with gastric cancer cell proliferation. In vitro, when gastric cancer cell line was co-cultured with primary human gastric fibroblast, STAT3–related genes including COX-2 and iNOS were induced in gastric cancer cells and this response was attenuated with neutralizing anti-IL-6 receptor antibody. IL-6 production from fibroblasts was increased when fibroblasts were cultured in the presence of gastric cancer cell–conditioned media. IL-6 production from fibroblasts was suppressed by an interleukin-1 (IL-1) receptor antagonist and siRNA inhibition of IL-1α in the fibroblasts. IL-1α mRNA and protein were increased in fibroblast lysate, suggesting that cell-associated IL-1α in fibroblasts may be involved. Our results suggest the importance of IL-6 mediated stromal-epithelial cell interaction in gastric tumorigenesis.     

Plasma lysophosphatidic acid level and serum autotaxin activity are increased in liver injury in rats in relation to its severity

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological actions. We have reported that LPA stimulates hepatic stellate cell proliferation and inhibits DNA synthesis in hepatocytes, suggesting that LPA might play some role in the liver. We have found that plasma LPA level and serum autotaxin (ATX) activity were increased in patients with chronic hepatitis C. However, the clinical significance of LPA and its synthetic enzyme, autotaxin (ATX), is still unclear. To determine whether the increase of plasma LPA level and serum ATX activity might be found generally in liver injury, we examined the possible modulation of them in the blood in rats with various liver injuries. Plasma LPA level and serum ATX activity were increased in carbon tetrachloride-induced liver fibrosis correlatively with fibrosis grade, in dimethylnitrosamine-induced acute liver injury correlatively with serum alanine aminotransferase level or in 70% hepatectomy as early as 3 h after the operation. Plasma LPA level was correlated with serum ATX activity in rats with chronic and acute liver injury. ATX mRNA in the liver was not altered in carbon tetrachloride-induced liver fibrosis. Plasma LPA level and serum ATX activity are increased in various liver injuries in relation to their severity. Whether increased ATX and LPA in the blood in liver injury is simply a result or also a cause of the injury should be further clarified.     

Stimulation by glutamine and proline of HGF production in hepatic stellate cells

Amino acids regulate cellular functions in a variety of cell types. Most notably, leucine stimulates protein production through the mammalian target of rapamycin (mTOR)-dependent signaling pathway. We investigated the effect of amino acids on hepatocyte growth factor (HGF) production. Treatment with glutamine and proline, as well as leucine, increased HGF levels in the culture medium of a rat hepatic stellate cell clone in a dose-dependent manner. Up-regulation of phosphorylation of 70 kDa ribosomal protein S6 kinase and eukaryotic initiation factor 4E-binding protein 1 was not apparent in the cells after treatment with glutamine or proline. When rats received injections of glutamine or proline, hepatic and circulating HGF levels increased and peaked around 12 h after treatment. Glutamine and proline may have the potential to stimulate HGF production but the mechanism underlying this stimulation seems not to be through the mTOR-dependent signaling pathway.