Improvement of porcine oocytes with low developmental ability after fusion of cytoplasmic fragments prepared by serial centrifugation

We have shown in pigs that oocytes denuded of cumulus cells at 24 h of in vitro maturation culture and subsequently matured for a total of 46 h (DO24 oocytes) have lower cytoplasmic maturity than those matured with cumulus cells for 46 h and then denuded (DO46 oocytes). In the present study, DO24 zona-free oocytes were fused with one (1C) or two (2C) cytoplasmic fragments produced by serial centrifugation ("centri-fusion") of DO46 oocytes (DO24+1C and DO24+2C oocytes, respectively). Groups of (1) DO46 (a control), (2) DO24, (3) DO24+1C and (4) DO24+2C oocytes were partheno-activated by an electrical pulse or fertilized in vitro and subsequently cultured for 6 days. In the fused groups, female pronucleus (FPN) formation rates were higher than that in the DO24 group after parthenogenetic activation (PA); however, the blastocyst rates were intermediate between those of the control and DO24 groups. After in vitro fertilization, the male pronucleus (MPN) formation rates in the fused groups were similar to that in the control group and higher than that in the DO24 group; the normal fertilization rate in the DO24+2C group was higher than that in the DO24 group and similar to that in the control group, resulting in significantly higher blastocyst rates in the DO24+2C and control groups than that in the DO24 group. These results suggest that centri-fusion using ooplasm from fully matured DO46 oocytes can offer a potentially novel approach for restoration of cytoplasmic maturity to oocytes with low developmental ability and subsequent improvement of fertilization and developmental competence.     

Susceptibility to N-methyl-N-nitrosourea-induced retinal degeneration in different rat strains

To evaluate the potential role of genetic background in the susceptibility to retinal degeneration induced by N-methyl-N-nitrosourea (MNU), female rats of the Sprague-Dawley (SD), Long-Evans (LE) and Copenhagen (CH) strains were administered 50 mg/kg MNU or saline at 7 weeks of age. Retina morphology and morphometric analysis of all rats was performed 7 days after MNU administration. Atrophy of both the peripheral and central outer retina occurred in all rat strains exposed to MNU. Decreased photoreceptor cell ratio and increased retinal damage ratio were observed. The severities of the retinal atrophy were similar among all three rat strains. In conclusion, MNU-induced photoreceptor degeneration developed consistently in all three strains regardless of the absence (SD rats) or presence (LE and CH rats) of melanin in the retina, suggesting that genetic and melanin factors did not affect photoreceptor cell death after MNU.     

Maintenance of mouse trophoblast stem cells in KSR-based medium allows conventional 3D culture

Mouse trophoblast stem cells (TSCs) can differentiate into trophoblast cells, which constitute the placenta. Under conventional culture conditions, in a medium supplemented with 20% fetal bovine serum (FBS), fibroblast growth factor 4 (FGF4), and heparin and in the presence of mouse embryonic fibroblast cells (MEFs) as feeder cells, TSCs maintain their undifferentiated, proliferative status. MEFs can be replaced by a 70% MEF-conditioned medium (MEF-CM) or by TGF-ß/activin A. To find out if KnockOut^TM Serum Replacement (KSR) can replace FBS for TSC maintenance, we cultured mouse TSCs in KSR-based, FBS-free medium and investigated their proliferation capacity, stemness, and differentiation potential. The results indicated that fibronectin, vitronectin, or laminin coating was necessary for adhesion of TSCs under KSR-based conditions but not for their survival or proliferation. While the presence of FGF4, heparin, and activin A was not sufficient to support the proliferation of TSCs, the addition of a pan-retinoic acid receptor inverse agonist and a ROCK-inhibitor yielded a proliferation rate comparable to that obtained under the conventional FBS-based conditions. TSCs cultured under the KSR-based conditions had a gene expression and DNA methylation profile characteristic of TSCs and exhibited a differentiation potential. Moreover, under KSR-based conditions, we could obtain a suspension culture of TSCs using extracellular matrix (ECM) coating-free dishes. Thus, we have established here, KSR-based culture conditions for the maintenance of TSCs, which should be useful for future studies.     

Identification of an Avirulence Gene in the Fungus Magnaporthe grisea Corresponding to a Resistance Gene at the Pik Locus

ABSTRACT A rice isolate of Magnaporthe grisea collected from China was avirulent on rice cvs. Hattan 3 and 13 other Japanese rice cultivars. The rice cv. Hattan 3 is susceptible to almost all Japanese blast fungus isolates from rice. The genetic basis of avirulence in the Chinese isolate on Japanese rice cultivars was studied using a cross between the Chinese isolate and a laboratory isolate. The segregation of avirulence or virulence was studied in 185 progeny from the cross, and monogenic control was demonstrated for avirulence to the 14 rice cultivars. The resistance gene that corresponds to the avirulence gene (Avr-Hattan 3) is thought to be located at the Pik locus. Resistance and susceptibility in response to the Chinese isolate in F(3) lines of a cross of resistant and susceptible rice cultivars were very similar to the Pik tester isolate, Ken54-20. Random amplified polymorphic DNA markers and restriction fragment length polymorphism markers from genetic maps of the fungus were used to construct a partial genetic map of Avr-Hattan 3. We obtained several flanking markers and one co-segregated marker of Avr-Hattan 3 in the 144 mapping population.     

Characteristic cellular composition of germinal centers

We established the method of isolating individually encapsulated germinal centers (GCs) from immunized spleen and analyzed single cell suspension of GCs by flowcytometry. In GCs, the high frequency of sIgG+ cells (29%) and sIgA+ cells (5%) was detected. Two-color flowcytometry analysis showed that GCs contained 27% of sIgM-IgG+ cells, in which isotype switch from IgM to IgG had occurred, and 5% of Bu1-IgG+ cells, which were differentiating into plasma cells. On the other hand, sIgM-IgG+ and Bu1-IgG+ cells were not detected in the bursa, which contained 95% of B cells and only 1% of T cells. CD4+ but not CD8+ T cells were detected in the light zone of GCs and these CD4+ T cells are supposed to play a key role in isotype switching and differentiation into plasma cells in GCs. These results clearly demonstrate that GCs provide a site for isotype switching and differentiation into plasma cells.     

Preparation of anion-exchange resins from pine sulfuric acid lignin,one of the acid hydrolysis lignins

To utilize acid hydrolysis lignin effectively, chemical conversion to anion-exchange resin was investigated by two methods. Sulfuric acid lignin (SAL) was selected as a typical acid hydrolysis lignin in this experiment. Because it is less reactive, SAL was phenolated with sulfuric acid catalyst to yield reactive phenolized SAL (P-SAL) with p-hydroxyphenyl nuclei. One method was the restricted resinification of P-SAL followed by the Mannich reaction with formaldehyde and dimethylamine to yield a weakly basic anion-exchange resin with an ion-exchange capacity of 2.4mEq/g. Another method was to react resinified P-SAL with glycidyltrimethylammonium chloride to yield a strongly basic anion-exchange resin with an ion-exchange capacity of 2.0mEq/g. The reaction of a simple P-SAL model compound with an epoxide suggested that the phenolic hydroxyl group of the p-hydroxyphenyl nucleus had slightly higher reactivity than that of the guaiacyl nucleus.Part of this report was presented at the 47th Lignin Symposium, Fukuoka, October 2002     

Use of trioxane for improvement of hygroscopic and acoustic properties of wood for musical instruments

Summary As a reagent source for the formalization of wood, trioxane was used instead of conventional paraformaldehyde. From preliminary experiments it became apparent that trioxane depolymerizes to formaldehyde by heating in the presence of some metallic salts. Formalization of wood with trioxane was carried out under the catalysis of sulfur dioxide (SO₂) along with these salts. The hygroscopic and acoustic properties were considerably improved by the treatment. Especially when the reaction was catalyzed by a combination of SO₂ and Fe₂(SO₄)₃, the antiswelling efficiency (ASE) reached nearly 70%, the specific dynamic Young's modulus (E/) in the radial direction increased about 20%, and the loss tangent (tan ) decreased about 30% and 50% in the longitudinal and radial directions, respectively. In this case, the modulus of rupture did not appreciably decrease in the radial direction. This is comparable to the most prominent results obtained so far by SO₂ catalyzed formalization with tetraoxane. It can be concluded that trioxane is very effective as a reagent source for formalization. The increase in acoustic converting efficiency, , of about twofold in the radial direction and decrease in hygroscopicity of more than 60% should not only enrich the sound volume of musical instruments but also stabilize their tone quality against humidity changes.     

Formation and chemical structures of acid-soluble lignin II: reaction of aromatic nuclei model compounds with xylan in the presence of a counterpart for condensation,and behavior of lignin model compounds with guaiacyl and syringyl nuclei in 72% sulfuric acid

To elucidate the formation and chemical structures of water-soluble material in acid-soluble lignin (ASL), lignin aromatic nuclei model compounds of creosol (I) and 5-methoxycreosol (II) were reacted with xylose or xylan in the presence of apocynol as a counterpart for condensation in 72% sulfuric acid (SA). The reaction of I gave mainly condensation product. However, the condensation reaction of II with apocynol was suppressed because of steric hindrance from the methoxyl group, and II yielded a C-xyloside after refluxing in 3% SA together with condensation products. To obtain information on CHCl₃-soluble material in ASL, model compounds of arylglycerol--aryl ethers with guaiacyl (VIII) and syringyl (X) nuclei were treated by the Klason procedure. VIII gave only insoluble polymerized product, while X gave insoluble polymerized product and CHCl₃-soluble low molecular weight products, which were dissolved in 3% SA. These results prove earlier views that water-soluble material in ASL consists of condensation products formed from syringyl lignin and monosaccharide units in hemicellulose. In addition, the CHCl₃-soluble material in ASL appears to be composed of low molecular weight degradation products from SA treatment of Klason lignin with the syringyl nucleus.Part of this report was presented at the 51st Annual Meeting of the Japan Wood Research Society, Tokyo, April 2001 and at the 47th Lignin Symposium, Fukuoka, October 2002, and was reviewed in Mokuzai Gakkaishi (2002) 48:55–62     

Determination of the distribution and reaction of polysaccharides in wood cell walls by the isotope tracer technique VII: Double radiolabeling of xylan and pectin in magnolia (Magnolia kobus DC) and comparison of their behaviors during kraft pulping by radiotracer technique

myo-Inositol-[2-³H] and d-glucuronic acid-[6-¹⁴C] were administered simultaneously to a growing stem of magnolia (Magnolia kobus DC) to label xylan and pectin, respectively, in the cell wall. Determination of the radioactivity of nitrobenzene oxidation products and sulfuric acid hydrolysates of the newly formed xylem indicated that xylan and pectin were labeled with ³H and ¹⁴C, respectively. The doubly labeled wood tissue was treated to kraft pulping, and the radioactivity of the pulping black liquor and treated wood tissue were determined at various stages of the pulping to compare the dissolving behavior between pectin and xylan during the pulping. The results showed that pectin was not dissolved as easily as xylan and was not redeposited on pulp fiber at the late stage of the pulping.Part of this report was presented at the 40th Lignin Symposium at Tsukuba, October 1995