Hypomorphic phenotype of <Emphasis Type="Italic">Foxn1</Emphasis> gene-modified rats by CRISPR/Cas9 system

The Foxn1 gene is known as a critical factor for the differentiation of thymic and skin epithelial cells. This study was designed to examine the phenotype of Foxn1-modified rats generated by the CRISPR/Cas9 system. Guide-RNA designed for first exon of the Foxn1 and mRNA of Cas9 were co-injected into the pronucleus of Crlj:WI zygotes. Transfer of 158 injected zygotes resulted in the birth of 50 offspring (32 %), and PCR identified five (10 %) as Foxn1-edited. Genomic sequencing revealed the deletion of 44 or 60 bp from and/or insertion of 4 bp into the Foxn1 gene in a single allele. The number of T-cells in the peripheral blood lymphocytes of mutant rats decreased markedly. While homozygous deleted mutant rats had no thymus, the mutant rats were not completely hairless and showed normal performance in delivery and nursing. Splicing variants of the indel-mutation in the Foxn1 gene may cause hypomorphic allele, resulting in the phenotype of thymus deficiency and incomplete hairless. In conclusion, the mutant rats in Foxn1 gene edited by the CRISPR/Cas9 system showed the phenotype of thymus deficiency and incomplete hairless which was characterized by splicing variants.     

Characterization of three capsular polysacharides produced by Burkholderia pseudomallei

Three kinds of capsular polysaccharide (CP) were found to be produced by Burkholderia pseudomallei. When the bacterium was grown with the medium without glycerol, CP-1a and CP-1b were produced. CP-1a was mainly 1.4-linked glucan and CP-1b was identified as a polymer composed of galactose and 3-deoxy-D-manno-octulosonic acid, whose chemical structure was recently reported by other laboratories. When the bacterium was grown with the medium containing 5" glycerol. CP-2 was synthesized. CP-2 contained galactose, rhamnose, mannose, glucose and a uronic acid in a ratio of approximately 3:1:0.3:1:1. Methylation analysis of the purified polysaccharides demonstrated that the two acidic polysaccharides. CP-1b and CP-2 shared no common structure, indicating that CP-2 was an acidic capsular polysaccharide whose chemical characters were not reported previously.     

Inhibitory effect of hot-water extract of quince (<Emphasis Type="Italic">Cydonia oblonga</Emphasis>) on immunoglobulin E-dependent late-phase immune reactions of mast cells

We evaluated the effect of a crude hot-water extract (HW) of quince (Cydonia oblonga Miller) fruit on immunoglobulin E (IgE)-dependent late-phase immune reactions of mast cells using in vitro system. Mast cell-like RBL-2H3 cells were treated with quince HW and late-phase reaction was then induced by stimulation with IgE + Antigen. Quince HW reduced the elevation of interleukin-13 and tumor necrosis factor-α expression level. Furthermore, quince HW suppressed these cytokine expressions of mouse bone marrow-derived mast cells (BMMCs), a normal mast cell model. Leukotriene C₄ and prostaglandin D₂ production in BMMCs after 1 and 6 h of stimulation, respectively, were also reduced by treating the cells with quince HW. We found that the induction of intracellular cyclooxygenase (COX)-2 expression but not COX-1 expression in BMMCs was reduced by quince HW. These results suggest that quince HW has an inhibitory effect on broad range of the late-phase immune reactions of mast cells.     

Sofalcone, a gastric mucosa protective agent, increases vascular endothelial growth factor via the Nrf2-heme-oxygenase-1 dependent pathway in gastric epithelial cells

Sofalcone, 2′-carboxymethoxy-4,4-bis(3-methyl-2-butenyloxy)chalcone, is an anti-ulcer agent that is classified as a gastric mucosa protective agent. Recent studies indicate heat shock proteins such as HSP32, also known as heme-oxygenase-1(HO-1), play important roles in protecting gastrointestinal tissues from several stresses. We have previously reported that sofalcone increases the expression of HO-1 in adipocytes and pre-adipocytes, although the effect of sofalcone on HO-1 induction in gastrointestinal tissues is not clear. In the current study, we investigated the effects of sofalcone on the expression of HO-1 and its functional role in rat gastric epithelial (RGM-1) cells. We found that sofalcone increased HO-1 expression in RGM-1 cells in both time- and concentration-dependent manners. The HO-1 induction was associated with the nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in RGM-1 cells. We also observed that sofalcone increased vascular endothelial growth factor (VEGF) production in the culture medium. Treatment of RGM-1 cells with an HO-1 inhibitor (tin-protoporphyrin), or HO-1 siRNA inhibited sofalcone-induced VEGF production, suggesting that the effect of sofalcone on VEGF expression is mediated by the HO-1 pathway. These results suggest that the gastroprotective effects of sofalcone are partly exerted via Nrf2-HO-1 activation followed by VEGF production.     

Diagnostic evaluation of 2', 5'-oligoadenylate synthetase activities and antibodies against Epstein-Barr virus and Coxiella burnetii in patients with chronic fatigue syndrome in Japan

To investigate the association of viral infections with chronic fatigue syndrome (CFS), we assayed 2', 5'-oligoadenylate synthetase (2-5AS) activities in peripheral blood mononuclear cells from CFS patients in Japan. These patients were diagnosed in two hospitals, H1 and H2, located in different areas of the country. The activities were detected in 19 (86%) and 7 (32%) of each of the 22 patients in H1 and H2, respectively, while they were detected in only four (11%) out of the 38 healthy controls. IFN-alpha was similarly detected in a few CFS patients and healthy controls. We also assayed the antibody titers against Epstein-Barr virus (EBV) and Coxiella burnetii in these patients. The EBV anti-EA-IgG antibodies were detected in two (9%) and seven (32%) of each of the 22 patients in H1 and H2, respectively. Anti-C. burnetii IgG antibodies were detected in six (27%) out of 22 patients in H1 but not in 22 patients in H2, while they were detected in one (11%) of the nine healthy controls. Some CFS patients may be associated with EBV or C. burnetii infection. There were some statistical correlations between the 2-5AS activities and antibody titers of EA-IgG (P < 0.05, Student's t-test) but not to the antibody titers of C. burnetii. The up-regulation of 2-5AS activities suggests immunological dysfunctions with some virus infections in the CFS patients. Our results indicate that 2-5AS activities are useful for a diagnostic marker of CFS and for exploring the complicated pathogenesis of CFS.     

Acoustic emissions of Sorex unguiculatus (Mammalia: Soricidae): Assessing the echo‐based orientation hypothesis

Shrew species have been proposed to utilize an echo‐based orientation system to obtain additional acoustic information while surveying their environments. This system has been supported by changes in vocal emission rates when shrews encounter different habitats of varying complexity, although detailed acoustic features in this system have not been reported. In this study, behavioral experiments were conducted using the long‐clawed shrew (Sorex unguiculatus) to assess this orientation system. Three experimental conditions were set, two of which contained obstacles. Short‐click, noisy, and different types of tonal calls in the audible‐to‐ultrasonic frequency range were recorded under all experimental conditions. The results indicated that shrews emit calls more frequently when they are facing obstacles or exploring the experimental environment. Shrews emitted clicks and several different types of tonal calls while exploring, and modified the use of different types of calls for varying behavior. Furthermore, shrews modified the dominant frequency and duration of squeak calls for different types of obstacles, that is, plants and acrylic barriers. The vocalizations emitted at short inter‐pulse intervals could not be observed when shrews approached these obstacles. These results are consistent with the echo‐based orientation hypothesis according to which shrews use a simple echo‐orientation system to obtain information from their surrounding environments, although further studies are needed to confirm this hypothesis.     

Isolation of a new potent cytotoxic pigment along with indigotin from the pathogenic basidiomycetous fungus Schizophyllum commune

An indole derivative, schizocommunin, was isolated along with indigotin (indigo), indirubin, isatin, and tryptanthrin, from the liquid culture medium in which a culture of Schizophyllum commune, isolated from the bronchus of a human patient with allergic bronchopulmonary mycosis, had been grown. The structure of schizocommunin was established by spectroscopic investigation. Schizocommunin showed the strong cytotoxicity against murine lymphoma cells. The assignments of the ¹H- and ¹³C-NMR signals of indigotin were also listed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Intracellularly inducible, ubiquitin hydrolase-insensitive tandem ubiquitins inhibit the 26S proteasome activity and cell division

We constructed polyubiquitin derivatives that contain a tandem repeat of ubiquitins and were insensitive to ubiquitin hydrolases. They were designated tandem ubiquitin (tUb) with the number of repeats, such as tUb2. When tUbs were expressed under the control of the GAL1 promoter in the wild-type yeast strain, growth was strongly inhibited. Under these conditions, the degradation of N-end rule substrates, a UFD substrate and Gcn4 was inhibited, indicating that the tUb inhibits 26S proteasome activity. Consistent with this, tUb binds to the 26S proteasome. We showed that tUb inhibited the in vitro degradation of polyubiquitinylated Sic1 by the 26S proteasome. When tUB6 messenger RNA was injected into Xenopus embryos, cell division was inhibited, suggesting that tUb can be used as a versatile inhibitor of the 26S proteasome.     

Synthesis and pharmacological properties of benzamide derivatives as selective serotonin 4 receptor agonists

A series of 4-amino-5-chloro-2-methoxy-N-(piperidin-4-ylmethyl)benzamides with a polar substituent group at the 1-position of the piperidine ring was synthesized and evaluated for its effect on gastrointestinal motility. The benzoyl, phenylsulfonyl, and benzylsulfonyl derivatives accelerated gastric emptying and increased the frequency of defecation. One of them, 4-amino-N-[1-[3-(benzylsulfonyl)propyl]piperidin-4-ylmethyl]-5-chloro-2-methoxybenzamide (13a, Y-36912), was a selective 5-HT4 receptor agonist offering potential as a novel prokinetic with reduced side effects derived from 5-HT3- and dopamine D2 receptor-binding affinity. In the oral route of administration, this compound enhanced gastric emptying and defecation in mice, and has a possibility as a prokinetic agent, which is effective on both the upper and the lower gastrointestinal tract.     

Stimulatory effect of a dietary casein phosphopeptide preparation on the mucosal IgA response of mice to orally ingested lipopolysaccharide from Salmonella typhimurium

The effect on immunoglobulin production of a commercially available casein phosphopeptide preparation (CPP-III) consisting mainly of bovine alpha s2-casein (1-32) and beta-casein (1-28) in mice that had orally ingested lipopolysaccharide (LPS) from Salmonella typhimurium was investigated. No significant difference in body weight gain was observed between the mice fed on the CPP-III-added diet and those fed on the control diet. The mice fed on the CPP-III-added diet exhibited similar serum and intestinal IgG, IgM, and IgE responses towards LPS to those fed on the control diet. In contrast, fecal and intestinal anti-LPS IgA and total IgA in mice fed on the CPP-III-added diet were significantly higher than in those fed on the control diet. Spleen cells from mice fed on the CPP-III-added diet produced larger amounts of IgA, IL-5, and IL-6 than cells from mice fed on the control diet. These results suggest that dietary casein phosphopeptide may protect a host from invasion of the intestinal mucosa by food-born pathogenic microorganisms.