FCGR3A-158 polymorphism influences the biological response to infliximab in Crohn’s disease through affecting the ADCC activity

An association between FCGR3A-158 V/F polymorphism and biological responses to infliximab has been reported in Crohn’s disease (CD) in Western countries. However, little is known about the mechanism by which gene polymorphism affects the responses to infliximab. The aims of this study were to confirm the association in Japanese CD patients and to reveal the effect of gene polymorphism on biological responses to infliximab. Japanese CD patients were examined retrospectively at weeks 8 and 30. Clinical and biological responses were assessed by the Crohn’s disease activity index and C-reactive protein levels, respectively. The infliximab-binding affinity of natural killer (NK) cells from FCGR3A-158 V/V, V/F and F/F donors was examined. Infliximab-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) activities were also determined using transmembrane TNF-α-expressing Jurkat T cells as target cells and peripheral blood mononuclear cells (PBMCs) from V/V, V/F and F/F donors as effector cells. Biological responses at week 8 were statistically higher in V/V patients, whereas no significant differences were observed in either clinical responses at weeks 8 and 30 or biological responses at week 30 among the three genotypes. NK cells and PBMCs from V/V patients also showed higher infliximab-binding affinity and infliximab-mediated ADCC activity, respectively. Our results suggest that FCGR3A-158 polymorphism is a predicting factor of biological responses to infliximab in the early phases. FCGR3A-158 polymorphism was also found to affect the infliximab-binding affinity of NK cells and infliximab-mediated ADCC activity in vitro, suggesting that an effect on ADCC activity influences biological responses to infliximab in CD patients.     

Purification and properties of human serum dopamine-β -hydroxylase

1. Two different molecular forms of dopamine-beta-hydroxylase were isolated from human serum; a major component (Peak I enzyme) with a molecular weight of 368000 and with a higher specific activity and a minor component (Peak II enzyme) with a molecular weight of 188000 and with a lower specific activity. 2. Both forms require ascorbic acid for the activity, and are stimulated by fumarate. Addition of N-ethylmaleimide or copper also increased the activity. The optimal pH of both forms in the presence of 20mM tyramine as substrate is 5.0. 3. Km values toward tyramine of Peak I enzyme and Peak II enzyme were 1.67 mM and 14.2 mM respectively. 4. Both Peak I enzyme and Peak II enzyme are glycoprotein.     

Clearance rates of biotinylated ferritins in canine: A possible physiological role of canine serum ferritin as an iron transporter

To elucidate the physiological role of canine serum ferritin, we measured clearance rates of biotinylated ferritins in beagle. Biotinylated canine tissue ferritins were cleared rapidly from circulation. The clearance time (T_1/2) of liver ferritin (H/L subunit ratio=0.43) was 6.8 to 11.8 min, and that of heart ferritin (H/L=3.69) was 9.3 to 25.0 min. T_1/2 of biotinylated canine liver ferritin was independent of iron content, whereas canine heart apoferritin (T_1/2=31.2 and 32.7 min) was more slowly removed from circulation than the holoferritin. On the other hand, biotinylated recombinant bovine H-chain ferritin homopolymer show a much slower rate of removal (T_1/2=153.8 and 155.0 min) compared with the L-chain ferritin homopolymer (T_1/2=26.4 and 31.3 min). The rapid clearance of canine tissue ferritin suggests that serum ferritin is an iron transporter in canines.     

Effect of cold stimulation during slow-wave sleep on sleep cycle

To clarify the effect of cold stimulation during slow-wave sleep (SWS) on the sleep cycle, we conducted a sleep experiment. Five healthy males slept on a bedding system we developed to make the inside of bedding cooler. When the subject was sleeping deeply in the second and fourth SWS, the system cooled their bedding. When the subject's sleep condition shifted toward arousal, the cold air was stopped. As a result, all subjects’ sleep stage shifted to light sleep and reached arousal. After stopping stimulation, they immediately returned to the SWS at the first stimulation. But at the second stimulation, the sleep state did not return to the SWS.     

A cytoplasmic RNA vector derived from nontransmissible Sendai virus with efficient gene transfer and expression

We have recovered a virion from defective cDNA of Sendai virus (SeV) that is capable of self-replication but incapable of transmissible-virion production. This virion delivers and expresses foreign genes in infected cells, and this is the first report of a gene expression vector derived from a defective viral genome of the Paramyxoviridae. First, functional ribonucleoprotein complexes (RNPs) were recovered from SeV cloned cDNA defective in the F (envelope fusion protein) gene, in the presence of plasmids expressing nucleocapsid protein and viral RNA polymerase. Then the RNPs were transfected to the cells inducibly expressing F protein. Virion-like particles thus obtained had a titer of 0.5 x 10(8) to 1. 0 x 10(8) cell infectious units/ml and contained F-defective RNA genome. This defective vector amplified specifically in an F-expressing packaging cell line in a trypsin-dependent manner but did not spread to F-nonexpressing cells. This vector infected and expressed an enhanced green fluorescent protein reporter gene in various types of animal and human cells, including nondividing cells, with high efficiency. These results suggest that this vector has great potential for use in human gene therapy and vaccine delivery systems.     

PERK eIF2 alpha kinase is required to regulate the viability of the exocrine pancreas in mice

Background

The lymphatic system complements the blood circulatory system in absorption and transport of nutrients, and in the maintenance of homeostasis. Angiopoietins 1 and 2 (Ang1 and Ang2) are regulators of both angiogenesis and lymphangiogenesis through the Tek/Tie-2 receptor tyrosine kinase. The response of endothelial cells to stimulation with either Ang1 or Ang2 is thought to be dependent upon the origin of the endothelial cells. In this study, we examined the effects of the angiopoietins on lymphatic, venous and arterial primary endothelial cells (bmLEC, bmVEC and bmAEC, respectively), which were isolated and cultured from bovine mesenteric vessels.

Results

BmLEC, bmVEC and bmAEC cell populations all express Tie-2 and were shown to express the appropriate cellular markers Prox-1, VEGFR3, and Neuropilin-1 that define the particular origin of each preparation. We showed that while bmLECs responded slightly more readily to angiopoietin-2 (Ang2) stimulation, bmVECs and bmAECs were more sensitive to Ang1 stimulation. Furthermore, exposure of bmLECs to Ang2 induced marginally higher levels of proliferation and survival than did exposure to Ang1. However, exposure to Ang1 resulted in higher levels of migration in bmLECs than did to Ang2.

Conclusion

Our results suggest that although both Ang1 and Ang2 can activate the Tie-2 receptor in bmLECs, Ang1 and Ang2 may have distinct roles in mesenteric lymphatic endothelial cells.     

Partial requirement of endothelin receptor B in spiral ganglion neurons for postnatal development of hearing

Impairments of endothelin receptor B (Ednrb/EDNRB) cause the development of Waardenburg-Shah syndrome with congenital hearing loss, hypopigmentation, and megacolon disease in mice and humans. Hearing loss in Waardenburg-Shah syndrome has been thought to be caused by an Ednrb-mediated congenital defect of melanocytes in the stria vascularis (SV) of inner ears. Here we show that Ednrb expressed in spiral ganglion neurons (SGNs) in inner ears is required for postnatal development of hearing in mice. Ednrb protein was expressed in SGNs from WT mice on postnatal day 19 (P19), whereas it was undetectable in SGNs from WT mice on P3. Correspondingly, Ednrb homozygously deleted mice (Ednrb(-/-) mice) with congenital hearing loss showed degeneration of SGNs on P19 but not on P3. The congenital hearing loss involving neurodegeneration of SGNs as well as megacolon disease in Ednrb(-/-) mice were markedly improved by introducing an Ednrb transgene under control of the dopamine β-hydroxylase promoter (Ednrb(-/-);DBH-Ednrb mice) on P19. Neither defects of melanocytes nor hypopigmentation in the SV and skin in Ednrb(-/-) mice was rescued in the Ednrb(-/-);DBH-Ednrb mice. Thus, the results of this study indicate a novel role of Ednrb expressed in SGNs distinct from that in melanocytes in the SV contributing partially to postnatal hearing development.     

A simple method for enrichment of uninucleate conidia of Aspergillus oryzae

Aspergillus oryzae produces multinucleate conidia, which makes the obtaining of homokaryons labor-intensive. Analysis of conidia by flow cytometry clarified the relationship that conidia of lower nuclear number were smaller in size. Based on this, we have developed a simple way to enrich uninucleate conidia with a membrane filter. Our results also suggest that the method is useful for elimination of heterokaryons.     

Enhancement of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production in the transgenic Arabidopsis thaliana by the in vitro evolved highly active mutants of polyhydroxyalkanoate (PHA) synthase from Aeromonas caviae

In this study, the enhancement of photosynthetic PHA production was achieved using the highly active mutants of PHA synthase created by the in vitro evolutionally techniques. The wild-type and mutated PHA synthase genes from Aeromonas caviae were introduced into Arabidopsis thaliana together with the NADPH-dependent acetoacetyl-CoA reductase gene from Ralstonia eutropha. Expression of the highly active mutated PHA synthase genes, N149S and D171G, led to an 8-10-fold increase in PHA content in the T1 transgenic Arabidopsis, compared to plants harboring the wild-type PHA synthase gene. In homozygous T2 progenies, PHA content was further increased up to 6.1 mg/g cell dry weight. GC/MS analysis of the purified PHA from the transformants revealed that these PHAs were poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymers consisting of 0.2-0.8 mol % 3HV. The monomer composition of the P(3HB-co-3HV) copolymers synthesized by the wild-type and mutated PHA synthases reflected the substrate specificities observed in Escherichia coli. These results indicate that in vitro evolved PHA synthases can enhance the productivity of PHA and regulate the monomer composition in transgenic plants.     

Histochemical studies of mucosubstances in metaplastic epithelium of the stomach,with special reference to the development of intestinal metaplasia

Summary Processes in the development of intestinal metaplasia of the stomach were investigated from the morphological and histochemical approaches using light and electron microscopic techniques. The specimens taken from 38 gastric carcinomas and 15 gastric and/or duodenal ulcers were subjected to this study. Morphological appearances of the intestinal metaplasia observed in routine examination with hematoxylin and eosin staining was able to be divided into complete and incomplete metaplasia by the light and electron microscopic histochemical stainings of the mucosubstances. The columnar cells at the area of the incomplete metaplasia had both the properties of the intestinal epithelia and the gastric foveolar epithelia. The incomplete as well as the complete metaplasia arose from the generative cells at the isthmus of the gland. The generative cells, however, sometimes gradually transformed to produce the complete metaplastic cells. The two processes of the development of the intestinal metaplasia were proposed and discussed.