Detection of vitellogenin incorporation into zebrafish oocytes by FITC fluorescence

<p>Abstract</p> <p>Background</p> <p>Large volumes of lymph can be collected from the eye-sacs of bubble-eye goldfish. We attempted to induce vitellogenin (Vtg) in the eye-sac lymph of bubble-eye goldfish and develop a method for visualizing Vtg incorporation by zebrafish oocytes using FITC-labeling.</p> <p>Methods</p> <p>Estrogen efficiently induced Vtg in the eye-sac lymph of goldfish. After FITC-labeled Vtg was prepared, it was injected into mature female zebrafish.</p> <p>Results</p> <p>Incorporation of FITC-labeled Vtg by zebrafish oocytes was detected in <it>in vivo </it>and <it>in vitro </it>experiments. The embryos obtained from zebrafish females injected with FITC-labeled Vtg emitted FITC fluorescence from the yolk sac and developed normally.</p> <p>Conclusion</p> <p>This method for achieving Vtg incorporation by zebrafish oocytes could be useful in experiments related to the development and endocrinology of zebrafish oocytes.</p

Archaeal Phospholipid Biosynthetic Pathway Reconstructed in Escherichia coli

A part of the biosynthetic pathway of archaeal membrane lipids, comprised of 4 archaeal enzymes, was reconstructed in the cells of Escherichia coli. The genes of the enzymes were cloned from a mesophilic methanogen, Methanosarcina acetivorans, and the activity of each enzyme was confirmed using recombinant proteins. In vitro radioassay showed that the 4 enzymes are sufficient to synthesize an intermediate of archaeal membrane lipid biosynthesis, that is, 2,3-di-O-geranylgeranyl-sn-glycerol-1-phosphate, from precursors that can be produced endogenously in E. coli. Introduction of the 4 genes into E. coli resulted in the production of archaeal-type lipids. Detailed liquid chromatography/electron spray ionization-mass spectrometry analyses showed that they are metabolites from the expected intermediate, that is, 2,3-di-O-geranylgeranyl-sn-glycerol and 2,3-di-O-geranylgeranyl-sn-glycerol-1-phosphoglycerol. The metabolic processes, that is, dephosphorylation and glycerol modification, are likely catalyzed by endogenous enzymes of E. coli

Mother-to-embryo vitellogenin transport in a viviparous teleost Xenotoca eiseni

魚類がお腹の子供に与える栄養素を解明 --哺乳類が失った遺伝子を利用して胎生機構を獲得--. 京都大学プレスリリース. 2019-10-09.Vitellogenin (Vtg), a yolk nutrient protein that is synthesized in the livers of female animals, and subsequently carried into the ovary, contributes to vitellogenesis in oviparous animals. Thus, Vtg levels are elevated during oogenesis. In contrast, Vtg proteins have been genetically lost in viviparous mammals, thus the yolk protein is not involved in their oogenesis and embryonic development. In this study, we identified Vtg protein in the livers of females during the gestation of the viviparous teleost, Xenotoca eiseni. Although vitellogenesis is arrested during gestation, biochemical assays revealed that Vtg protein was present in ovarian tissues and lumen fluid. The Vtg protein was also detected in the trophotaeniae of the intraovarian embryo. Immunoelectron microscopy revealed that Vtg protein is absorbed into intracellular vesicles in the epithelial cells of the trophotaeniae. Furthermore, extraneous Vtg protein injected into the abdominal cavity of a pregnant female was subsequently detected in the trophotaeniae of the intraovarian embryo. Our data suggest that the yolk protein is one of the matrotrophic factors supplied from the mother to the intraovarian embryo during gestation in X. eiseni

Role of interleukin 6 for differential responsiveness of naive and memory CD4+ T cells in CD2-mediated activation

The present study was undertaken to elucidate different requirements for CD2-mediated activation of naive (CD45RO-) and memory (CD45RO+) CD4+ T cells. A mitogenic combination of anti-CD2 (anti-T112 and anti-T113) mAbs could effectively induce the proliferation of memory CD4+ T cells even in the absence of monocytes. In marked contrast, naive CD4+ T cells did not disclose any proliferative responses to antiCD2 mAbs, when monocytes were absent in culture. This differential responsiveness of naive and memory CD4+ T cells appeared to be related largely to a difference in IL-6 - producing ability between both populations. IL-6 among monocyte-derived cytokines could correct unresponsiveness of naive CD4+ T cells to anti-CD2 stimulation. Unlike naive CD4+ T cells, memory CD4+ T cells produced IL-6 by themselves, with its mRNA being expressed on anti-CD2 stimulation. Anti-IL-6R mAb significantly inhibited proliferation of memory CD4+ T cells seen in the anti-CD2-stimulated cultures without monocytes, indicating the involvement of their own production of IL-6 in CD2-mediated activation. The results suggest an essential role of IL-6 for triggering of CD4+ T cells via the CD2 molecule

焼結ルチル二酸化チタン上の骨芽細胞様細胞の増殖およびアルカリホスファターゼ活性

The purpose of this study is creation of biomaterials from titanium dioxide (TiO2). This TiO2 has known for photocatalysis and osteogenesis. For the purpose of applying this function to orthodontic brackets and coating materials for implant, the relationship between surface of sintered and cell proliferation were examined. In addition, crystal structure and the surface property of sintering TiO2 were investigated. TiO2 were sintered at 1300°C for use as samples. We examined surface roughness, x-ray diffraction and scanning electron microscopy to make observations of the surface properties and texture. Moreover, mouse osteoblast-like cell line, MC3T3-E1 was cultured on sintered TiO2 in order to evaluate the cell proliferation and ALP. For the samples sintered at 1300°C, the crystalline phase of rutile-type TiO2 was confirmed.5000-fold magnified SEMimages of the surface of the unsintered samples, needle-like TiO2 crystals were pressure welded and showed mutual overlap, with pores occurring among the crystals. Sintering at 1300°C produced numerous small pores. Rutile TiO2 as a starting material was sintered at 1300°C and subjected to a cell culture experiment in which MC3T3-E1 cells were cultured on the sample, followed by viable cell counting and cell morphology observationon days 7, 14, 21, and 28 of culture. In the test of cell proliferation, sintered at 1300°C samples was found to remarkable cell proliferation even after time had passed. ALP activity of cells on 1300°C TiO2 sample, the values were 110% and 126% on days 14 and 28 of culture, respectively. These changes were calculated using polystyrene dish as the reference condition. Thus, TiO2 sintered at 1300°C showed good compatibility and increase in the ALP activity in MC3T3-E1 cells

A bone substitute with high affinity for vitamin D-binding protein―relationship with niche of osteoclasts

The biological activity of osteoblasts and osteoclasts is regulated not only by hormones but also by local growth factors, which are expressed in neighbouring cells or included in bone matrix. Previously, we developed hydroxyapatite (HA) composed of rod-shaped particles using applied hydrothermal methods (HHA), and it revealed mild biodegradability and potent osteoclast homing activity. Here, we compared serum proteins adsorbed to HHA with those adsorbed to conventional HA composed of globular-shaped particles (CHA). The two ceramics adsorbed serum albumin and γ-globulin to similar extents, but affinity for γ-globulin was much greater than that to serum albumin. The chemotactic activity for macrophages of serum proteins adsorbed to HHA was significantly higher than that of serum proteins adsorbed to CHA. Quantitative proteomic analysis of adsorbed serum proteins revealed preferential binding of vitamin D-binding protein (DBP) and complements C3 and C4B with HHA. When implanted with the femur of 8-week-old rats, HHA contained significantly larger amount of DBP than CHA. The biological activity of DBP was analysed and it was found that the chemotactic activity for macrophages was weak. However, DBP-macrophage activating factor, which is generated by the digestion of sugar chains of DBP, stimulated osteoclastogenesis. These results confirm that the microstructure of hydroxyapatite largely affects the affinity for serum proteins, and suggest that DBP preferentially adsorbed to HA composed of rod-shaped particles influences its potent osteoclast homing activity and local bone metabolism

Establishment of Functioning Human Corneal Endothelial Cell Line with High Growth Potential

Hexagonal-shaped human corneal endothelial cells (HCEC) form a monolayer by adhering tightly through their intercellular adhesion molecules. Located at the posterior corneal surface, they maintain corneal translucency by dehydrating the corneal stroma, mainly through the Na+- and K+-dependent ATPase (Na+/K+-ATPase). Because HCEC proliferative activity is low in vivo, once HCEC are damaged and their numbers decrease, the cornea begins to show opacity due to overhydration, resulting in loss of vision. HCEC cell cycle arrest occurs at the G1 phase and is partly regulated by cyclin-dependent kinase inhibitors (CKIs) in the Rb pathway (p16-CDK4/CyclinD1-pRb). In this study, we tried to activate proliferation of HCEC by inhibiting CKIs. Retroviral transduction was used to generate two new HCEC lines: transduced human corneal endothelial cell by human papillomavirus type E6/E7 (THCEC (E6/E7)) and transduced human corneal endothelial cell by Cdk4R24C/CyclinD1 (THCEH (Cyclin)). Reverse transcriptase polymerase chain reaction analysis of gene expression revealed little difference between THCEC (E6/E7), THCEH (Cyclin) and non-transduced HCEC, but cell cycle-related genes were up-regulated in THCEC (E6/E7) and THCEH (Cyclin). THCEH (Cyclin) expressed intercellular molecules including ZO-1 and N-cadherin and showed similar Na+/K+-ATPase pump function to HCEC, which was not demonstrated in THCEC (E6/E7). This study shows that HCEC cell cycle activation can be achieved by inhibiting CKIs even while maintaining critical pump function and morphology

A Case of Wilsonism with Torsion Spasm-like Symptoms

In view of the scarcity in number of the reports on clinical cases of torsion spasm, the authors believe that the case presented here will be of some interest. This patient was a 15-year old boy who had had, since early part of his twelfth year, the episodes of the torsion on the anterior backbone accompanied by that peculiar twisting and turning along with cramping of muscles, which alternately increased or decreased, facial expression blank and entire movement simple and sluggish, without any apparent causative factors. He had been clinically diagnosed as having torsion spasm reactions, but he soon died of somatic weakening. The autopsy findings of this case were the destruction of the lense nucleus, the caudate nucleus, the thalamus, the dentate nucleus, and a marked proliferation of neuroglia, especially, the appearance of giant glia as well as liver cirrhosis; and it was diagnosed as Wilson-pseudosclerosis. Thereupon, the authors have attempted to present their opinions on this particular case here