Destructive effect of heating against toxoplasma oocysts.

Heating was examined for destructive effect against Toxoplasma oocysts, both unsporulated and sporulated, of the O-1 and O-3 strains. Sporulation-inhibition rates were measured by counting sporulated and unsporulated oocysts in each examination. As a result, the sporulation of Toxoplasma oocysts was completely inhibited by exposure to 60 or 70 degrees C for 10 seconds, 55 degrees C for 30 seconds, 50 degrees C for 2.5 minutes. 45 degrees C for 1 hour, or 37 degrees C for 48 hours. When examined by the mouse inoculation method, the infectivity of sporulated oocysts became extinct after heating at 90 degrees C for 30 seconds, 80 degrees C for 1 minute, 70 degrees C for 2 minutes, 55 or 60 degrees C for 15 minutes, or 50 degrees C for 30 minutes. It was confirmed that heating was effective for the sterilization of Toxoplasma oocysts.     

Industrial production of disodium 5′-guanylate

A process for manufacturing disodium 5′-guanylate was devised. 5′-Amino 4-imidazole carboxamide riboside (AICA-R) was accumulated with an amount over 100 times those reported in the literature by fermentation of D -glucose with a non-exacting purineless mutant derived from Bacillus megaterium JAM 1245) by x-ray irradiation. The influence of RNA, amino acids, and salts on AICA-R accumulation was clarified. Appropriate aeration and agitation was found necessary. The (60-hr, cultivation of the medium containing 8% of D -glucose gave AICA-R in the concentration above, 11 g/l. AICA-R thus accumulated was separated from the fermentation broth by ion-exchange technique and subjected to synthetic processes to yield disodium 5′-guanylatc with the yield over 40%, based on AICA-R.     

Effect of anti-pig zona pellucida serum on fertilization in several mammals

The effect of goat antiserum against isolated pig zonae pellucidae on fertilization in vivo was examined in the pig, cow, sheep, rabbit, rat, and mouse. As shown by indirect immunofluorescence, anti-pig zona serum reacted strongly with the zonae of pig, cow, sheep, and rabbit, but the reaction with the zonae of mouse and rat was weak. Passive immunization with anti-pig zona serum significantly, or completely, inhibited fertilization in all species. However, inhibition of fertilization was more pronounced in the pig, cow, sheep, rabbit, and mouse than in the rat. Inhibition of fertilization in the rabbit was also observed after passive immunization with antiserum absorbed with rabbit liver and kidney. All of the zonae recovered from the pig, cow, sheep, rat, and mouse after passive immunization with anti-pig zona serum exhibited strong fluorescence, regardless of the incidence of fertilization. It was concluded that the pig and other mammalian zonae pellucidae tested have tissue-specific antigens.     

A comparative investigation on the potency of cells from the inner cell mass and trophectoderm of mouse blastocysts to produce chimeras

The ability of trophectoderm (TE) cells to produce chimeric mice (pluripotency) was compared with that of inner cell mass (ICM) cells. TE and ICM cells of blastocysts and hatching or hatched blastocysts derived from albino mice (CD-1, Gpi-1a/a) were aggregated with zona cut 8- to 16-cell stage embryos or injected into the blastocoele from non-albino mice (C57BL/6 x C3H/He, Gpi-1b/b). After transfer to pseudopregnant female mice, the contribution of the donor cells was examined by glucose phosphate isomerase (GPI) analysis of embryos, membrane and placenta at mid-gestation (Day 10.5 and 12.5) or by the coat color of newborn mice. In contrast to ICM cells, there was no contribution of TE cells in the conceptuses and no coat color chimeric young were obtained. After pre-labeling of TE cells with fluorescent latex microparticles, they were aggregated with embryos and the allocation of TE cells at the compacted morula and blastocyst stages was observed under a fluorescent microscope. Although the TE cells were observed attached onto the surface of the embryos at morula and blastocyst stages, unlike the ICM cells, they were not positively incorporated into the embryos. Thus, the pluripotency of TE cells from mouse blastocysts was not induced by the aggregation and injection methods.     

Transforming growth factor-beta inhibits phosphorylation of the retinoblastoma susceptibility gene product in human monocytic leukemia cell line JOSK-I.

Proliferation of the human monocytic leukemia cell line JOSK-I is inhibited by transforming growth factor-beta (TGF-beta). Growth inhibition by TGF-beta was not due to either a toxic effect or to induction of differentiation. TGF-beta induced a cell cycle arrest at late G1 phase and was not found to be inhibitory to JOSK-I cells in S phase or G2/M. This G1 cell cycle arrest was associated with an accumulation of the unphosphorylated form of the retinoblastoma susceptibility gene product (Rb) in good correlation with inhibition of DNA synthesis. In contrast to the effects of TGF-beta, two other agents which induced a G1 arrest of JOSK-I cells had a different effect on Rb. Aphidicolin blocked cells at G1/S but could not reduce Rb phosphorylation as great as that seen with TGF-beta. 12-O-Tetradecanoylphorbol-13-acetate, an inducer of differentiation, did reduce Rb phosphorylation, but not until 72 h, when differentiation had already occurred. The identities of the Rb kinases are unknown, but recent evidence suggests that the cdc2 gene product could participate in Rb phosphorylation. Although cdc2 mRNA and total protein levels were not affected, TGF-beta inhibited the rate of translation and kinase activity of cdc2 in JOSK-I cells. These results suggest that growth inhibition of hematopoietic cells by TGF-beta is linked to suppression of Rb phosphorylation to retain Rb in an unphosphorylated, growth-inhibitory state. The suppression of Rb phosphorylation is suggested to be mediated through inhibition of cdc2 kinase activity by TGF-beta.     

A short-term hypotonic treatment for chromosome preparation of intact and zona-penetrated mouse embryos

An air-drying method for chromosome preparation was improved for use in zona-penetrated mouse embryos. This method consisted of a sequence of short-term (1 to 3 min) hypotonic treatments, a two-step fixation process and slow air-drying under heavy moisture. The method is also applicable to intact embryos at the one-cell, morula, and blastocyst stages for chromosome analysis of embryos at each of these stages.     

Production of identical twin and triplet mice by nuclear transplantation

Transplantation of a single nucleus from two- or four-cell embryos into one of the enucleated blastomeres of a two-cell embryo resulted in successful production of identical triplet and twin mice. The proportion of reconstituted embryos that developed in blastocysts was 71% (84/118) when four-cell embryos were used as donors of nuclei; 10 sets of quadruplet and nine sets each of triplet and twin blastocysts were obtained by this technique. After transfer to recipients, 30% (18/61) developed to term, and one set of identical triplet and four sets of identical twin mice were obtained. When two-cell embryos were used as donors of nuclei, 79 (95%) sets of twin embryos developed to blastocysts. Of 38 twin blastocysts transferred to recipients, 21 sets (55%) developed to term as identical twin mice. These results demonstrate that the enucleated two-cell embryo develops in vitro after transfer of a nucleus from a two- or four-cell embryo and the resultant blastocyst has high potential for development to term after transfer to a recipient.     

The effect of radiation therapy combined with natural killer cells against spontaneous murine fibrosarcoma

The effect of radiation therapy combined with lymphoid cells against spontaneous murine fibrosarcoma (FSa-II) was investigated bothin vivo andin vitro. In thein vivo experiment, syngeneic C3H mice were divided into 3 groups. Animals in the first group were injected with 1 x 10⁵ tumor cells into the right hind leg. Animals in the second and third groups were injected with 1 x 10⁵ tumor cells mixed with 1 x 10⁷ normal lymphoid cells (NLC) or effector lymphoid cells (ELC), respectively. ELC were obtained from spleen and lymph nodes of FSa-II-bearing mice and incubatedin vitro for 40 hr to eliminate suppressor T cell function. NLC were obtained from normal mice and incubated in the same way. Irradiation was given using¹³⁷Cs unit 3 days after cell inoculation. 12 out of 14 mice (85.7%) inoculated with tumor cells mixed with NLC did not show any tumor growth at 60 Gy local irradiation. 12 out of 21 mice (57.1 %) inoculated with tumor cells alone and 6 out of 10 (60%) with tumor cells mixed with ELC rejected tumors at the same radiation dose. This synergistic effect with NLC was not observed when NLC was inoculated after irradiation, indicating that lymphoid cells should be in contact with tumor cells before irradiation. In the⁵¹Cr release assay, lymphoid cells obtained from whole body irradiated (WBI) mice showed 17.8% lysis without irradiation and 28.8% lysis at 5 Gy irradiation. Untreated NLC showed almost no cytotoxic effect at the same radiation dose. This synergistic effect disappeared when WBI lymphoid cells were treated with anti asialo GM1 and complement. These results suggested that NK cells might be important in this synergistic effect with irradiation. To obtain a sufficient level of synergistic effect by in vitro combined treatment of mixed tumor cell - NLC culture and irradiation - incubation for more than 12 hrs and 8 hrs appeared to be necessary before and after irradiation, respectively.     

Heterogenous distribution of free calcium and propagation of calcium transient in gastric parietal cells revealed by digital imaging microscopy

Spatial and temporal changes of cytoplasmic free calcium concentration ([Ca2+]i) in single parietal cells of guinea pig were investigated with a digital imaging microscope equipped with a microspectrofluorometer, using a Ca2+-sensitive dye, fura-2. Intracellular distribution of [Ca2+]i was not homogeneous, but there were two kinds of [Ca2+]i gradient in the resting parietal cells, one a continuous gradient increasing towards the plasma membrane and a second discontinuous gradient (Ca2+ plateau) in some restricted regions of the cytoplasm. When treated with gastrin, only about 40% of parietal cells in the gastric gland responded with an almost twofold increase in the average resting [Ca2+]i of 52.4 +/- 7.1 nM. In the responding cells, the discontinuous plateaus transiently enlarged to the entire cytoplasm. In marked contrast, all of these cells responded to Ca2+ ionophore ionomycin. We also found that when provoked by gastrin Ca2+ transient in the parietal cells in the gastric gland often propagated to some adjacent cells, and occasionally spontaneous Ca2+ transient and oscillation were observed even in the resting state.     

Apolipoprotein E Polymorphism in Sheep

Using polyacrylamide gel isoelectric focusing followed by immunoblotting with anti-human apolipoprotein E (APO E) antibody, the genetic polymorphism of APO E was determined from desialylated plasma of 554 unrelated adults of four European sheep (Suffolk, Corriedale, Cheviot, and Finnish Landrace) and five Asian local sheep (Bhyanglung, Baruwal, Kagi, Lampuchhre, and Vietnamese). Twenty phenotypes consisting of the homozygous and heterozygous combinations of two APO E variants within the seven variants (E1-E7) detected were identified. Family and population data supported the hypothesis that the phenotypes are controlled by seven codominant alleles, designated APOE1 to APOE7, at a single autosomal locus. The common alleles, APOE4, APOE5, and APOE7 were observed at mean frequencies of 0.5763, 0.1471, and 0.1921 in the European sheep group and 0.4920, 0.1123, and 0.2995 in the Asian local sheep group, respectively.