Characteristics of cytotoxic cells induced by Toxoplasma lysate antigen in mouse spleen.

Spleen cells from Toxoplasma lysate antigen (TLA)-sensitized BALB/c mice showed the strong cytotoxic activity against both natural killer (NK)-sensitive cells (YAC-1 and RL male-1) and NK-insensitive cells (P-815), when incubated with TLA or recombinant human IL-2 (rhIL-2). The increment of TLA concentration in culture medium increased the cytotoxic activity. Treatment of effector cells; spleen cells from TLA-sensitized mice incubated with TLA, with anti-asialo GM1 or anti-Thy-1 plus complement inhibited the cytotoxic activity of effector cells, whereas treatment with anti-mouse Lyt-2.2 serum plus complement had no effect on the cytotoxic activity. Treatment of spleen cells from TLA-sensitized mice with anti-asialo GM1 and/or anti-Thy-1 plus complement inhibited cytotoxic activities of effector cells. These results suggested that spleen cells sensitized with TLA both in vivo and in vitro were asialo GM1 positive and Thy-1 positive, and the majority of cytotoxic cells induced by TLA were similar to lymphokine-activated killer (LAK) cells induced by IL-2.     

Distributions of extracellular matrix and carbonic anhydrase-III during bovine palatine ridge development.

The present study describes histological alterations and immunohistochemical distributions of extracellular matrices (ECMs) and the carbonic anhydrase isozyme-III (CA-III) during the period of bovine palatine ridge formation. Morphogenesis of bovine palatine ridges was preceded by epidermal placodes and the mesenchymal condensation (MC). During the early stages of less than 44 cm crown rump length (CRL), fibronectin (FN) was distributed densely in the MC. Strong reactions against type I collagen (C-1) were detected outer to the FN positive site. In the stages of more than 44 cm CRL, FN and C-1 were distributed diffusely in subepithelial mesenchyme. Laminin (LN) and type IV collagen were distributed in the epithelial and endothelial basement membranes (BMs) in all of the stages examined, except in the stage of 7 cm CRL, where LN was not detected only in the BM just beneath the epidermal placode. CA-III was detected in basal epithelial cells except for palatine ridge rudiments in the stages of more than 21 cm CRL. It is suggested that the expressions of LN and CA-III might play a role in the spatial determination of rudiments of bovine fetal palatine ridges.     

Impaired female fertility in tubulointerstitial antigen-like 1-deficient mice

Tubulointerstitial nephritis antigen-like 1 (Tinagl1, also known as adrenocortical zonation factor 1 [AZ-1] or lipocalin 7) is a matricellular protein. Previously, we demonstrated that Tinagl1 expression was restricted to extraembryonic regions during the postimplantation period and detected marked expression in mouse Reichert’s membranes. In uteri, Tinagl1 is markedly expressed in the decidual endometrium during the postimplantation period, suggesting that it plays a physical and physiological role in embryo development and/or decidualization of the uterine endometrium during pregnancy. In the present study, in order to determine the role of Tinagl1 during embryonic development and pregnancy, we generated Tinagl1-deficient mice. Although Tinagl1^–/– embryos were not lethal during development to term, homologous matings of Tinagl1^–/– females and Tinagl1^–/– males showed impaired fertility during pregnancy, including failure to carry pregnancy to term and perinatal lethality. To examine ovarian function, ovulation was induced with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG); the number of ovulated oocytes did not differ between Tinagl1^–/– and Tinagl1^flox/flox. In vitro fertilization followed by embryo culture also demonstrated the normal developmental potential of Tinagl1-null embryos during the preimplantation period. Our results demonstrate that Tinagl1 deficiency affects female mice and results in subfertility phenotypes, and they suggest that although the potential of Tinagl1^–/– oocytes is normal, Tinagl1 is related to fertility in adult females but is not essential for either fertilization or preimplantation development in vitro.     

Influence of volcanic ash sediments on spore adhesion of the red alga Gelidium elegans from the coastal seabed around Miyakejima Island, Japan

The populations of red alga Gelidium elegans along the coast of Miyakejima Island were severely damaged by a volcanic eruption in 2000. The effect of this volcanic eruption has been long lasting, and populations of this red alga still have not recovered. We investigated the effect of seabed sediment particles derived from volcanic ash on the substrate adhesion of G.?elegans spores. The analysis provides evidence that increasing amounts of sediment particles result in lower adhesion rates of G.?elegans spores, and that smaller sediment particles have a greater influence on adhesion. The amount of seabed sediment particles around Miyakejima Island was 9.3?C1815.4?mg/cm². This amount has changed greatly from year to year. The adhesion rate of G.?elegans spores in water around Miyakejima Island was 0?% at all points in 2008 and 2010, but it was estimated as 6.3?C38.6?% in 2009. These results suggest that there is significant inhibition of algal spore adhesion by seabed sediment particles derived from volcanic ash around Miyakejima Island.     

Effect of amending materials on growth of radish plant in salinized soil

The critical Na levels in soil which restricted seed germination and growth of radish, and effect of the amending materials on reducing the salinity hazard were determined by performing petri dish and pot experiments. The plants were grown in amended salinized soil in a phytotron for 21 days. Excess accumulation of Na in cells suppressed seed germination and plant growth. The critical Na level in control soil which restricted germination and growth was 12.5mg Na/g soil. At the same salinity level, amending materials promoted seed germination and plant growth mainly by inhibiting the excess flow of Na into the cell through ion holding mechanism. Among the materials used, the commercial manure of 4 months old held greater amount of Na(3.7mg/g) and thus more efficiently reduced the ion damage. Plants grown under such treatment had the highest dry matter yield (28.Gmg/plant), water content was normal(84%), and the ratio for Na in root to Na in plant's shoot was 2.4. As for the case of plant grown in control soil the respective data were 8. 5mg, 54%, and 4.8.     

Isolation and culture of rabbit primordial germ cells

Primordial germ cells (PGCs) are embryonic precursors of the gametes of adult animals and are considered stem cells of the germline. Since their proliferation in vitro correlates well with the schedule of developmental changes in vivo, they might be interesting research tools for genomic imprinting, germ-cell tumors and fertility. Furthermore, once primordial germ cells are separated and placed on a feeder layer with cytokines, they become cultured pluripotent cell lines called embryonic germ (EG) cells. EG cells share several important characteristics with embryonic stem (ES) cells as they can also contribute to the germ line of chimeras. To investigate the characteristics of PGCs and establish rabbit EG (rEG) cells, we cultured rabbit PGCs (rPGCs) in vitro with various combinations of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and forskolin on inactivated mouse embryonic fibroblast (MEF) feeder layers. The present study found PGC proliferation in early cultures and induction of rEG-like colonies. These cells expressed pluripotent markers, such as alkaline phosphatase activity, OCT-4, Sox-2 and SSEA-1, in the undifferentiated state; however, the cells did not develop into a teratoma when injected into the kidney capsules of SCID mice, although the restricted differentiation potentials to neural cells were determined via embryoid body formation. From these characteristics and further characterization of the germ stem cell markers Vasa, SCP-1 and SCP-3, we suggested that these were hybrid cells with characteristics somewhere between PGC and EG cells.     

Down regulation of a novel protein,PSP, in rat hepatoma cdRLh 84-beared tumor

Perchloric acid-soluble protein (PSP) is a members of a new hypothetical family (YER057c/YJGF family) of small proteins with presently unknown function. The high degree of evolutionary conservation of these proteins reflect an involvement in basic cellular regulation. The expression of PSP was examined in rat hepatoma cell dRLh 84-beared rats. The tumor weight increased to 4.24 g at 3 weeks after the transplantation of hepatoma cells and hepatoma which has less differentiated characteristics were observed in rat liver. The expression of PSP in rat hepatoma was down regulated as compared with intact tissue. Thus the expression of PSP seems to be associated with the differentiation process in these transformed cells. On the other hand, some positive cells against the PSP-antibody were observed in the central region of tumor tissue by immunohistochemistry. These cells were shown to be the lymphocytes and the macrophages. The involvement of PSP to cellular growth and differentiation is discussed.     

Bovine herpesvirus-1 (BHV-1) recombinant expressing pseudorabies virus (PrV) glycoproteins B and C induces type 1 immune response in BALB/c mice

Bovine herpesvirus 1 (BHV-1) attached poorly and penetrated into a mouse cell line, BALB 3T3/A31, but a recombinant BHV-1/TF7-6, which expresses pseudorabies virus (PrV) gB and gC genes, did attach and penetrated into cells more efficiently. In this study the gene green fluorescent protein (GFP) has been integrated into genome of BHV-1/TF7-6 and its parental line of BHV-1. When the mouse mesenteries were incubated in vitro and infected with BHV-1/TF7-6/GFP, strong fluorescence was observed while BHV-1/GFP infection hardly demonstrated fluorescence, suggesting that BHV-1 recombinant expressing PrV gB and gC can infect mouse tissue cells more efficiently than the parental BHV-1 does. When BALB/c mice were inoculated with purified BHV-1/TF7-6 or its parental BHV-1, the former induced lower level of anti-BHV-1 immunoglobulin G (IgG) than the latter did. When sub-classes of anti-BHV-1 IgG were analyzed, it was found that mice immunized with BHV-1/TF7-6 or the parental BHV-1 demonstrated the same level of IgG2a. Since anti-BHV-1 IgG1 level was lower in mice inoculated with BHV-1/TF7-6, the IgG2a:IgG1 ratio was higher in BHV-1/TF7-6 inoculated mice than in the parental BHV-1 inoculated ones. These results indicate that BHV-1/TF7-6 induces type 1 predominant immune to BALB/c mice.     

Horizontal transmission and host-virulence attenuation of totivirus in violet root rot fungus Helicobasidium mompa

Monokaryotic strains of Helicobasidium mompa were used as vectors of a mycovirus between various H. mompa isolates to examine the transmissibility of one of the mycoviruses, totivirus (HmTV1–17 virus) in the hypovirulent isolate V17 of H. mompa. The isolates that acquired HmTV1–17 virus were also examined for any alteration in the virulence. Twelve dikaryotic isolates of H. mompa, belonging to 11 mycelial compatibility groups (MCGs) and being mycelially incompatible with isolate V17, were used as recipients of HmTV1–17 virus. Two monokaryotic isolates that were mycelially incompatible with isolate V17 and all of the recipients were also used as vectors of HmTV1-17 virus between isolate V17 and the recipients. When isolate V17 and recipients were directly paired on plate media, HmTV1-17 virus was transmitted from isolate V17 into 2 of the 12 recipients (i.e., 2 of the 11 MCGs). Two monokaryotic strains were paired with isolate V17, and the monokaryotic strains that acquired HmTV1-17 virus were then used as new virus donors. When the monokaryotic strains containing HmTV1-17 virus were paired with the 12 recipients, HmTV1-17 virus was transmitted into 7 of the 12 recipients from the monokaryotic strains (i.e., 7 of 11 MCGs). Based on these results, we concluded that monokaryotic strains could act as vectors to transmit HmTV1-17 virus into H. mompa isolates. When four of the H. mompa isolates that acquired HmTV1-17 virus were used to inoculate apple rootstock Malus prunifolia, the virulence of all of the isolates was attenuated from that of their parental isolates. Moreover, because the DNA fingerprints of the fungal isolates that acquired HmTV1-17 virus were the same as those of their parental isolates, the infection with HmTV1-17 virus is considered the cause of virulence attenuation of H. mompa.