Involvement of interleukin-1 in lipopolysaccaride-induced microglial activation and learning and memory deficits.

We have developed an animal model of learning and memory impairment associated with activation of microglia in the mouse brain. Injection of lipopolysaccharide into the CA1 region of the mouse hippocampus resulted in an increased production of inflammatory cytokines, such as interleukin-1ß. Immunostaining for interleukin-1ß revealed an increase in the signal at 6 hr after lipopolysaccharide injection. Immunopositive cells for interleukin-1ß were colocalized with those immunopositive for CD11b. When subacute lipopolysaccharide treatment (20 µg/2 µl/injection, bilaterally for 5 consecutive days) was performed, long-term activation of microglia and learning and memory deficits as evaluated using a step-through passive avoidance test were observed in the wild-type mice. Gene expression of the N-methyl-D-aspartate receptor NR1 and NR2A subunits was also decreased by the lipopolysaccharide treatment. In contrast, activation of microglia and the associated behavioral deficits were not observed in mice lacking interleukin-1α and -1ß following the subacute lipopolysaccharide treatment, together with little change in the gene expression of NR1 and NR2A subunits. However, the subacute lipopolysaccharide treatment produced almost similar changes in those parameters in the tumor necrosis factor-α knockout mice as in the wild-type animals. The injection of interleukin-1ß neutralizing antibody with lipopolysaccharide for 5 consecutive days resulted in the improvement of lipopolysaccharide-induced learning and memory deficits. These findings suggest that the expression of interleukin-1 plays an important role in lipopolysaccharide-induced activation of microglia and the associated functional deficits in learning and memory.

Increased expression of UDP-galactose transporter messenger RNA in human colon cancer tissues and its implication in synthesis of Thomsen-Friedenreich antigen and sialyl Lewis A/X determinants.

A series of human nucleotide sugar transporters of the Golgi apparatus was recently cloned, including the transporters for UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP-GlcNAc) and CMP-sialic acid (CMP-SA). We have examined the mRNA expression of these three transporters in human colon cancer tissues by reverse transcription-PCR analysis and compared it with that in nonmalignant colonic mucosa prepared from the same patients. The amount of mRNA for UDP-Gal transporter was significantly increased in colon cancer tissues compared with nonmalignant mucosa tissues (P = 0.035; n = 20). The increase was more prominent in patients with advanced colorectal cancer of Dukes' stages C and D, in which the amount of UDP-Gal transporter mRNA in cancer tissues showed on average about a 3.6-fold increase over the paired nonmalignant mucosa (statistically significant at P = 0.004; n = 14). The mRNA content of the other two transporters showed no significant difference between the paired cancer and normal tissues. When UDP-Gal transporter cDNA was stably transfected to cultured human colon cancer cells, the expression of Thomsen-Friedenreich (TF) antigen and of sialyl Lewis A (NeuAcalpha2-->3Galbeta1-->3[Fucalpha1-->4]GlcNAcbeta1-->R) and sialyl Lewis X (NeuAcalpha2-->3Galbeta1-->4[Fucalpha1-->3]GlcNAcbeta1-->R) determinants was significantly induced on transfectant cells, which resulted in markedly enhanced cell adhesion to vascular E-selectin. These findings suggest that the increase of UDP-Gal transporter mRNA is involved in the enhanced expression of cancer-associated carbohydrate determinants such as TF and sialyl Lewis A/X antigens in colon cancers.

Quantitative trait loci that regulate plasma lipid concentration in hereditary obese KK and KK-Ay mice.

To identify quantitative trait loci (QTLs) responsible for regulating plasma lipid concentration associated with obesity, linkage analysis was carried out on the 190 F2 progeny of a cross between C57BL/6J female and KK-Ay (Ay allele at the agouti locus congenic) male. In F2 a/a (agouti locus genotype) mice, two QTLs were identified on chromosome 1 and a QTL on chromosome 3 for total-cholesterol. A QTL for HDL-cholesterol was identified on chromosome 1 and a QTL for NEFA on chromosome 9. In F2 Ay/a mice, two QTLs for HDL-cholesterol were found on chromosome 1. Loci for other lipids with suggestive linkage were also identified. In both F2 mice, one QTL on chromosome 1 for total- and HDL-cholesterol was mapped near D1Mit150, in the vicinity of the apolipoprotein A-II (Apoa2) locus. Seven nucleotide substitutions out of 309 nucleotide apolipoprotein A-II cDNA sequences were identified between KK and C57BL/6J. The Ay allele may be an indication of the plasma lipid levels, but its influence was less apparent than in the case of weight control. The loci for lipids were not on identical chromosomes with those previously identified for obesity, suggesting that hyperlipidemia in KK does not coincidentally occur with obesity.

Tumor necrosis factor-alpha (TNF-alpha) plays a protective role in acute viralmyocarditis in mice: A study using mice lacking TNF-alpha.

BACKGROUND: It has been reported that tumor necrosis factor-alpha (TNF-alpha) is expressed in the heart with viral myocarditis and that its expression aggravates the condition. The pathophysiological effects of TNF-alpha on viral myocarditis, however, have not been fully elucidated. METHODS AND RESULTS: To investigate the role of TNF-alpha in the progression of viral myocarditis, we used TNF-alpha gene-deficient mice (TNF-alpha(-/-)) and induced acute myocarditis by infection with encephalomyocarditis virus (EMCV). The survival rate of TNF-alpha(-/-) mice after EMCV infection was significantly lower than that of TNF-alpha(+/+) mice (0% versus 67% on day 14). Injection of recombinant human TNF-alpha (0.2 to 4.0 microg/mouse IV) improved the survival of TNF-alpha(-/-) mice in a dose-dependent manner, indicating that TNF-alpha is essential for protection against viral myocarditis. The levels of viral titer and viral genomic RNA of EMCV in the myocardium were significantly higher in TNF-alpha(-/-) than in TNF-alpha(+/+) mice. Histopathological examination showed that the inflammatory changes of the myocardium were less marked in TNF-alpha(-/-) than in TNF-alpha(+/+) mice. Immunohistochemical analysis revealed that the levels of immunoreactivity of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in the myocardium were decreased in TNF-alpha(-/-) mice compared with TNF-alpha(+/+) mice. CONCLUSIONS: These observations suggested that TNF-alpha is necessary for adhesion molecule expression and to recruit leukocytes to inflammatory sites, and thus, the lack of this cytokine resulted in failure of elimination of infectious agents. We concluded that TNF-alpha plays a protective role in the acute stage of viral myocarditis.

Lipopolysaccharide-induced HIV-1 expression in transgenic mice is mediated by tumor necrosis factor-alpha and interleukin-1, but not by interferon-gamma nor interleukin-6.

BACKGROUND: As serum HIV-1 load correlates well with the prognosis of the disease, it is suggested that the viral load is one of the major determinants of the disease progression of AIDS. Accordingly, HIV-1 activation mechanisms were extensively studied in vitro, and involvement of cytokines including tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, IL-6 and interferon (IFN)-gamma has been suggested in this process. However, so far the roles of these cytokines in the HIV-1 expression in vivo have not been well elucidated because of the lack of appropriate animal disease models. OBJECTIVE: To elucidate the roles of cytokines in HIV-1 activation in vivo. DESIGN AND METHODS: Transgenic mice carrying a defective HIV-1 genome were used as a model for HIV-1 carriers. In order to examine the possible involvement of cytokines in HIV-1 expression, TNF-alpha-, IL-1-, IL-6- and IFN-gamma-deficient HIV-1 transgenic mice, were produced and HIV-1 expression was analyzed after activation with bacterial lipopolysaccharides (LPS). RESULTS: HIV-1 expression in the transgenic mouse spleen was activated 10- to 20-fold by LPS, and the serum p24 Gag protein levels reached 400 pg/ml, which is nearly equal to the levels that occur in AIDS patients. However, this augmentation was suppressed by 60% in TNF-alpha-deficient mice and by 40% in IL-1alpha/beta-deficient mice. In contrast, no suppression was observed in either IL-6-, IFN-gamma-, IL-1alpha, or IL-1beta-deficient mice. CONCLUSIONS: Results suggest that TNF-alpha and IL-1 play important roles in HIV-1 gene activation and selective suppression of these cytokines could improve clinical prognosis and potentially slow progression of the disease.

The roles of Nramp1 and Tnfa genes in nitric oxide production and their effect on the growth of Salmonella typhimurium in macrophages from Nramp1 congenic and tumor necrosis factor-alpha-/- mice.

The macrophages from Nramp1 congenic mice and tumor necrosis factor (TNF)-alpha(-/-) mice were used to examine the functions of Nramp1 and Tnfa genes in nitric oxide (NO) production and Salmonella typhimurium infection. It was confirmed that the level of inducible NO synthase (iNOS)-mediated NO production in Nramp1(r) peritoneal macrophages was generally higher than that of Nramp1(s) macrophages after stimulation by interferon-gamma (IFN-gamma), lipopolysaccharide (LPS), and tumor necrosis factor-alpha (TNF-alpha) alone or in combination. Nramp1 mRNA expression in both Nramp1 congenic macrophages was constitutive notwithstanding cytokine stimulation. During infection with S. typhimurium strain 6203, Nramp1(r) macrophages produced a lower amount of NO because of an initial strong reaction and unsustained iNOS gene expression as compared with Nramp1(s) macrophages. An inhibitory effect of the Nramp1(r) gene on bacterial replication was also observed during the early stage of S. typhimurium infection, whereas the effect of TNF-alpha occurred later. NO production and iNOS expression in TNF-alpha(-/-) macrophages were not detected from the start of the bacterial infection or at 24 h after infection. We also observed that S. typhimurium strain 6203 grew more profoundly without TNF-alpha, especially in Nramp1(s) macrophages. These data, therefore, demonstrate that there is cooperation of the Nramp1 and Tnfa genes in NO production and a growth inhibitory effect in response to S. typhimurium infection.

Neurobehavioral alterations in mice with a targeted deletion of the tumor necrosis factor-alpha gene: implications for emotional behavior.

Tumor necrosis factor-alpha (TNF-alpha) is emerging as an important modulator of the function of the central nervous system. In the present study, we investigated a role of endogenous TNF-alpha in cognitive and emotional function using mice with targeted deletions of the TNF-alpha gene. TNF-alpha-(-/-) mice showed normal diurnal rhythms of spontaneous locomotor activity and cognitive functions. Emotional behavior in the mutant mice, however, was significantly altered, which manifested in the performance in the open-field, elevated plus maze, and forced swimming tests. The altered performance in the elevated plus maze test was significantly alleviated by treatment with diazepam. Postmortem brain analysis of TNF-alpha-(-/-) mice revealed a significant increase in serotonin metabolism in the brain. These findings suggest a role for endogenous TNF-alpha in emotional behavior, which may possibly be related to alterations of serotonine metabolism.

Establishment of bovine leukemia virus-producing and -nonproducing B-lymphoid cell lines and their proviral genomes.

We have established four cell lines in vitro from peripheral leukemic cells of four independent enzootic bovine leukosis (EBL) cattle. All cell lines exhibited differentiated B-cell characters. Two of them produced infectious bovine leukemia virus (BLV), but the others did not. Nonproducer cell lines contained single copies of defective BLV proviral genomes with the same integration profiles as the uncultured cells. On the other hand, numerous proviral copies were detected in producer cell lines. One of the producer cell lines, BL407, whose original uncultured cell contained complete and defective proviral genomes retained the original two copies and had increased only complete genomes in different integration sites after long term culture. These findings suggest that the monoclonal leukemic cells from EBL cases are preferentially established in vitro irrespective of their proviral structures, and the producer B-lymphoid cells amplify their proviral copies by reinfection with viruses re-expressed from the cells during in vitro cultivation.

Genetic analysis of non-insulin-dependent diabetes mellitus in KK and KK-Ay mice.

The KK mouse is considered suitable as a polygenic model for human non-insulin-dependent diabetes mellitus. To identify the quantitative trait loci (QTLs) responsible for hyperglycemia and impaired glucose tolerance in KK mice, linkage analysis using 97 microsatellite markers was carried out in a 192 F2 progeny, comprising 93 mice with the a/a genotype at the agouti locus (chromosome 2) and 99 mice with the Ay/a genotype, produced by a cross between a C57BL/6J female and a KK-Ay (Ay congenic) male. In F2 a/a progenies, we identified a QTL for fasting glucose levels on chromosome 6 (LOD score 6.0) and three loci with suggestive linkage on chromosomes 3, 5 and 14, but could not identify loci accounting for glucose tolerance and plasma insulin levels. In F2 Ay/a progenies, there were no loci with statistically significant linkage, but three suggestive loci were identified: a locus for fasting glucose on chromosome 9, and two loci for glucose tolerance on chromosomes 1 and 8. It would thus appear that. although the fasting glucose level is controlled by QTLs in KK mice, these QTLs may be masked by the strong hyperglycemic influence of the Ay allele. Suggestive loci accounting for glucose tolerance may interact with the Ay allele, since these loci were identified only in F2 Ay/a progeny. This is consistent with the finding that the impaired glucose tolerance in KK mice is moderate and becomes overt when associated with the Ay allele.

Effect of a matrix metalloproteinase inhibitor on host resistance against Listeria monocytogenes infection.

Hydroxy acid-based matrix metalloproteinase (MMP) inhibitors have been shown to inhibit tumor infiltration and growth, endotoxin shock, and acute graft-versus-host disease. Blockade of the release of soluble tumor necrosis factor-alpha (TNF-alpha) and CD95 ligand (CD95L; FasL) from cell-associated forms is reportedly involved in the mechanism of the drug effect. We investigated the effect of a MMP inhibitor, KB-R7785, on host resistance against Listeria monocytogenes infection, in which TNF-alpha is essentially required for the defense, in mice. The administration of KB-R7785 exacerbated listeriosis, while the drug prevented lethal shock induced by lipopolysaccharide and D-galactosamine. KB-R7785 inhibited soluble TNF-alpha production in spleen cell cultures stimulated by heat-killed L. monocytogenes and the drug treatment reduced serum TNF-alpha levels in infected mice, whereas the compound was ineffective on the modulation of interferon-gamma and interleukin-10 production. The effect of KB-R7785 was considered to be dependent on TNF-alpha because the drug failed to affect L. monocytogenes infection in anti-TNF-alpha monoclonal antibody-treated mice and TNF-alpha knockout mice. Anti-CD95L monoclonal antibody was also ineffective on the infection. These results suggest that induction of infectious diseases, to which TNF-alpha is critical in host resistance, should be considered in MMP inhibitor-treated hosts.

Roles of endogenous cytokines in liver apoptosis of mice in lethal Listeria monocytogenes infection.

Various bacterial pathogens have been identified as mediators of apoptosis. Apoptosis reportedly shows both detrimental and beneficial effects on biological functions. We studied the role of liver apoptosis in lethal Listeria monocytogenes infection and the regulation of apoptosis by endogenous cytokines during infection. Apoptosis was observed in the spleen but not in the liver of infected mice, whereas the induction of liver necrosis was evident by rising levels of serum aminotransferases in these animals. Apoptosis was detected in the liver of L. monocytogenes-infected mice which had been treated with monoclonal antibody (mAb) against tumor necrosis factor-alpha (TNF-alpha) or interleukin-6 (IL-6), or in TNF-alpha(-/-) mice, but not in gamma- interferon (IFN-gamma)(-/-) mice or mice which had been treated with mAb against IL-4 or IL-10. Augmentation of liver apoptosis in mice treated with mAb against TNF-alpha or IL-6 or in TNF-alpha(-/-) mice correlated with the increase in bacterial numbers in the organ, while no augmentation of apoptosis was observed in the liver of IFN-gamma(-/-) mice irrespective of the marked increase in bacterial numbers in the organs, indicating that augmentation of liver apoptosis may not be merely due to the increase in bacterial growth in the organs. These results suggest that TNF-alpha and IL-6 may play an important role in protecting the liver from apoptosis in lethal L. monocytogenes infection.

Inhibition in a microgravity environment of the recovery of Escherichia coli cells damaged by heavy ion beams during the NASDA ISS phase I program of NASA Shuttle/Mir mission no. 6.

We participated in a space experiment, part of the National Space Development Agency of Japan (NASDA) Phase I Space Radiation Environment Measurement Program, conducted during the National Aeronautics and Space Administration (NASA) Shuttle/Mir Mission No. 6 (S/MM-6) project. The aim of our study was to investigate the effects of microgravity on the DNA repair processes of living organisms in the in orbit. Heavy ion beam radiation- or ç-irradiation-damaged biological samples of Escherichia coli and the radioresistant bacterium Deinococcus radiodurans were prepared and placed in a biospecimen box, which was loaded into the RRMD III sensor unit of the Space Shuttle. Two identical sets of samples were left in the Spacehab's Payload Processing Facility (SPPF) in Florida, USA, as a control. (flight No. STS-84) was launched from NASA John F. Kennedy Space Center (KSC) in Florida, USA, on May 15, 1997. The mission duration was 9.22 days. An astronaut activated the biological samples in the biospecimen box in the Spacehab during orbit in order to start repair of the DNA damaged by heavy ion beams or ç-irradiation and the samples were incubated for 19 h 35 min at about 22ûC, the cabin temperature. The control specimens in the SPPF were subjected to the same treatment under terrestrial gravity. After returned to earth, we investigated cell recovery by comparing the repair of the radiation-damaged DNA of E. coli and D. radiodurans in the microgravity environment in space with that on Earth. The results indicated that the DNA repair process of E. coli, but not of D. radiodurans, cells was inhibited in a microgravity environment.

The effects of microgravity on ligase activity in the repair of DNA double-strand breaks.

PURPOSE: In recent years, contradictory data have been reported about the effects of microgravity on radiation-induced biological responses in space experiments. The aim of the present study was to clarify whether enzymatic repair of DNA double-strand breaks is affected by microgravity using an in vitro enzymatic reaction system. MATERIALS AND METHODS: The DNA repair activity of T4 DNA ligase (EC 6.5.1.1) was measured in vitro for a DNA substrate damaged by restriction enzyme digestion during a US Space Shuttle mission (Discovery; STS-91). After the flight, the amount of ligated DNA molecules was measured using an electrophoresis method. RESULTS: Ligated products (closed circular DNA, open circular DNA and multimeric ligated products) were produced by T4 DNA ligase treatment of linear DNA containing double-strand breaks, and they increased with increasing T4 DNA ligase concentration (0-3 units per microg of plasmid DNA). Almost no difference in T4 DNA ligase activity was detected between the space experiments and the control ground experiments. CONCLUSIONS: No significant effect of microgravity on ligation of damaged DNA was found during space flight. Therefore, other mechanisms must account for the synergism between radiation and microgravity, if it exists.

Differential expression of activin subunits, activin receptors and follistatin genes in bovine oocytes and preimplantation embryos.

The presence of inhibin subunits (alpha, betaA and betaB), activin binding protein follistatin and activin receptors (ActR-I, ActR-IB, ActR-II and ActR-IIB) mRNA transcripts in bovine oocytes and preimplantation embryos was examined by reverse transcription-polymerase chain reaction. Immature and matured oocytes, zygotes, 2-cell, 3-4-cell, 5-8-cell, 9-16-cell embryos, morulae, blastocysts and hatched blastocysts were produced by in vitro maturation, fertilization and culture for this study. Activin betaA subunit and follistatin transcripts were detected in all of the stages tested. However, alpha subunit transcripts were detected only in oocytes (immature and matured), blastocysts and hatched blastocysts, and no betaB subunit transcripts were found in any of the stages. Whereas ActR-I transcripts were detectable in all stages, ActR-IB transcripts were not detectable in oocytes or embryos except in matured oocytes. Activin type II receptors (ActR-II and ActR-IIB) transcripts were detectable in oocytes and embryos except for ActR-IIB, which was not found in the 5-8-cell stage embryos. The results suggest that bovine embryos may produce activin A (homodimer of the betaA subunits) at least from zygotes to morulae. Furthermore, bovine oocytes and embryos express the activin receptors mainly by the combination of ActR-I and either ActR-II or ActR-IIB.

Mutational analysis of TGF-beta type II receptor, Smad2, Smad3, Smad4, Smad6 and Smad7 genes in colorectal cancer.

Transforming growth factor-beta(TGF-beta) is known to play an important role in controlling embryonal development, cell proliferation and homeostasis. The purpose of this study is to elucidate the involvement of the TGF-beta pathway in colorectal carcinogenesis. DNA was extracted from 100 patients with colorectal cancer. Then, all coding regions of the TGF-beta type II receptor (TRII) and the genes for Smad2, Smad3, Smad4, Smad6, and Smad7 were analyzed by PCR-SSCP and direct sequencing. Also, a LOH analysis of 18q21, where the Smad2 and Smad4 genes are located, was performed. We detected 11 cases of frameshift mutation in the TRII gene (11%) and 5 cases of point mutations in the Smad4 gene (5.0%); LOH at 18q21 was detected with 33% frequency. No abnormalities were found in the genes for Smad2, Smad3, Smad6, and Smad7. These results suggest that the abnormalities of TRII and Smad4 play an important role inhibiting TGF-beta signaling in colorectal carcinogenesis.

Differential patterns of blastulation in bovine morulae cultured in synthetic oviduct fluid medium containing FCS or BSA.

This study examined the effects of fetal calf serum (FCS) supplementation of culture medium on blastulation and hatching of bovine morulae cultured in vitro. The presumptive zygotes derived from in vitro maturation and fertilization (IVM/IVF) were cultured in the modified synthetic oviduct fluid medium containing 3 mg/ml BSA (mSOF-BSA). At 120 h post insemination, morulae were randomly assigned to culture with mSOF-BSA (control) or mSOF containing 5% FCS (mSOF-FCS) instead of BSA. The replacement of BSA with FCS in mSOF significantly increased the percentage of blastocyst formation from Day 6 to Day 10 (Day 0 = the day of in vitro insemination) and the hatching rate of embryos on Days 8 and 9. The total number of cells in morulae and blastocysts on Day 6, in blastocysts on Day 7, and in blastocysts and hatched blastocysts on Day 8 were similar among the treatments. However, the replacement of BSA with FCS in mSOF significantly increased the total number of cells in hatched blastocysts on Day 10. Although the time of blastulation of embryos was significantly accelerated by the replacement of BSA with FCS in mSOF, the total number of cells in embryos at blastulation was lowered. The total number of cells in embryos at blastulation showed a time-dependent decrease when the embryos were cultured in mSOF-BSA. In contrast, the total number of cells in embryos that were cultured in mSOF-FCS depended little on the time after in vitro insemination. The results indicate that FCS supplementation of culture medium increased the percentage of embryos developing to the blastocyst stage without an increase in the total number of cells. However, an acceleration in the hatching rate and an increase in the total number of cells in hatched blastocysts were observed, compared with that in BSA-supplemented medium. It is suggested that FCS in the culture medium initiates earlier blastulation with fewer total numbers of cells in the morulae than BSA during in vitro culture of bovine embryos.

Molecular cloning of bovine CD14 gene.

Genomic DNA encoding bovine CD14 was isolated from a bovine (Holstein) genomic library. Utilizing PCR fragment of mouse CD14 gene as a probe, we screened 9 x 10(5) plaques and obtained 3 clones containing the bovine CD14 gene. DNA sequencing showed that bovine CD14 gene encodes 373 amino acids, and the coding sequence was separated by a 90 nt intron. The identity of the deduced amino acid sequence of bovine CD14 was 61-73% to those of mouse, rabbit and human. Northern blot analysis revealed that CD14 mRNA (1.5 kb) was expressed in the lung. Expression of CD14 mRNA was stimulated about 2-fold in bovine peripheral blood macrophage activated with LPS in vitro.

Molecular cloning of bovine thyrotropin-releasing hormone receptor gene.

The genomic DNA encoding bovine Thyrotropin-Releasing Hormone Receptor (TRHR) was isolated from a bovine (Holstein) genomic library. Using PCR fragments of bovine candidate TRHR Transmembrane domain (TMD-III-IV) and C-terminus domain of mouse TRHR cDNA as probes, 9 x 10(5) plaques were screened to obtain several clones each containing the N-terminus or C-terminus domain. The bovine TRHR gene encoded 398 amino acids and has a long intron. The identity of the deduced amino acid sequence of bovine TRHR exceeded 88% that of mouse, rat or human. RT-PCR analysis indicated TRHR mRNA to be expressed in the pituitary and brain.

Differentiation of Dictyostelium discoideum vegetative cells into spores during Earth orbit in space.

We reported previously that emerged amoebae of Dictyostelium (D.) discoideum grew, aggregated and differentiated to fruiting bodies with normal morphology in space. Here, we investigated the effects of space radiation and/or microgravity on the number, viability, kinetics of germination, growth rate and mutation frequency of spores formed in space in a radiation-sensitive strain, gamma s13, and the parental strain, NC4. In gamma s13, there were hardly spores in the fruiting bodies formed in space. In NC4, we found a decrease in the number of spores, a delay in germination of the spores and delayed start of cell growth of the spores formed in space when compared to the ground control. However, the mutation frequency of the NC4 spores formed in space was similar to that of the ground control. We conclude that the depression of spore formation might be induced by microgravity and/or space radiation through the depression of some stage(s) of DNA repair during cell differentiation in the slime mold.

The effects of microgravity on induced mutation in Escherichia coli and Saccharomyces cerevisiae.

We examined whether microgravity influences the induced-mutation frequencies through in vivo experiments during space flight aboard the space shuttle Discovery (STS-91). We prepared dried samples of repair-deficient strains and parental strains of Escherichia (E.) coli and Saccharomyces (S.) cerevisiae given DNA damage treatment. After culture in space, we measured the induced-mutation frequencies and SOS-responses under microgravity. The experimental findings indicate that almost the same induced-mutation frequencies and SOS-responses of space samples were observed in both strains compared with the ground control samples. It is suggested that microgravity might not influence induced-mutation frequencies and SOS-responses at the stages of DNA replication and/or DNA repair. In addition, we developed a new experimental apparatus for space experiments to culture and freeze stocks of E. coli and S. cerevisiae cells.