Effects of different Bacillus licheniformis and Bacillus subtilis ratios on nutrient digestibility, fecal microflora, and gas emissions of growing pigs.

The objective of this study was to evaluate the effects of different mixing ratios of Bacillus licheniformis and Bacillus subtilis in diets on nutrient digestibility, fecal microflora, and odor gas emissions of growing pigs. A total of four crossbred ([Landrace × Yorkshire] × Duroc) barrows with average body weight (BW) of 41.2 ± 0.7 kg were randomly allotted four diets over four periods in a 4 × 4 Latin square design. Treatments were as follows: Control (CON, basal diet), CON + 0.2% probiotic complex (L4S6, B. licheniformis and B. subtilis at a 4:6 ratio), CON + 0.2% probiotic complex (L5S5, B. licheniformis and B. subtilis at a 5:5 ratio), CON + 0.2% probiotic complex (L6S4, B. licheniformis and B. subtilis at a 6:4 ratio). Dietary probiotic supplementation showed higher crude protein (CP) digestibility values and lower Escherichia coli counts in fecal samples than the CON group (p < 0.05). There was no significant difference in NH3 or H2S emission until day 3. The positive effect of H2S and NH3 emissions was detected earlier with the L4S6 and L5S5 compared to the L6S4, which had a lower ratio of B. subtilis. Both the L4S6 and L5S5 probiotic complexes significantly decreased the fecal H2S and NH3 emission in days 4 and 6 (p < 0.05). On day 7, all probiotic complexes decreased (p < 0.05) H2S and NH3 emissions than the CON group. Our results agreed that the dietary supplementation of Bacillus licheniformis and Bacillus subtilis complexes in growing pigs can significantly improve CP digestibility and reduce fecal E. coli counts, NH3 and H2S emissions. Notably, the higher mixing ratio of Bacillus subtilis in probiotic supplementation is more effective in reducing the odor of manure.

Induction of APOBEC3B cytidine deaminase in HTLV-1-infected humanized mice.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma (ATL). Following viral infection with HTLV-1, certain infected cells exhibit clonal proliferation. Additional genetic and epigenetic changes in these clonally proliferating cells provide them with the selective advantage of growth, which eventually results in ATL. The precise mechanism, however, has yet to be completely elucidated. It has previously been established that APOBEC3 enzymes are potent host-antiviral restriction factors. Conversely, previous studies have reported that the A3B level is increased in tumor virus infections, such as those caused by HBV and HPV, suggesting that A3B exerts a function as a mutagen. Therefore, the present study analyzed the expression of APOBEC3 family members in various HTLV-1 infection states. No significant differences were observed in the expression between healthy donors and patients with HTLV-1-associated myelopathy. Although no significant changes in the expressions of A3C, A3D, A3F and A3G between uninfected and HTLV-1-infected mice were observed, an increased A3B expression was observed in a short-term humanized mouse model following HTLV-1 infection. In a long-term humanized mouse model following HTLV-1 infection, the gene expression array data exhibited an apparent increase in A3B and CADM1, which are indicators of ATL. Collectively, the results of the present study suggest that A3B is likely involved in the development of ATL in HTLV-1-infected humanized mice.

Evaluation of the secretion and release of vascular endothelial growth factor from two-dimensional culture and three-dimensional cell spheroids formed with stem cells and osteoprecursor cells.

BACKGROUND: Co-culture has been applied in cell therapy, including stem cells, and has been reported to give enhanced functionality. OBJECTIVES: In this study, stem-cell spheroids were formed in concave micromolds at different ratios of stem cells to osteoprecursor cells, and the amount of secretion of vascular endothelial growth factor (VEGF) was evaluated. MATERIAL AND METHODS: Gingiva-derived stem cells and osteoprecursor cells in the amount of 6 × 105 were seeded on a 24-well culture plate or concave micromolds. The ratios of stem cells to osteoprecursor cells included: 0:4 (group 1), 1:3 (group 2), 2:2 (group 3), 3:1 (group 4), and 4:0 (group 5). RESULTS: The morphology of cells in a 2-dimensional culture (groups 1-5) showed a fibroblast-like appearance. The secretion of VEGF increased with the increase in stem cells, and a statistically significant increase was noted in groups 3, 4 and 5 when compared with the media-only group (p < 0.05). Osteoprecursor cells formed spheroids in concave microwells, and no noticeable change in the morphology was noted with the increase in stem cells. Spheroids containing stem cells were positive for the stem-cell markers SSEA-4. The secretion of VEGF from cell spheroids increased with the increase in stem cells. CONCLUSIONS: This study showed that cell spheroids formed with stem cells and osteoprecursor cells with different ratios, using microwells, had paracrine effects on the stem cells. The secretion of VEGF increased with the increase in stem cells. This stem-cell spheroid may be applied for tissue-engineering purposes.

Enhanced Osteogenic Differentiation Potential of Stem-Cell Spheroids Created From a Coculture of Stem Cells and Endothelial Cells.

PURPOSE: This study was performed to fabricate stem-cell spheroids formed with human gingiva-derived stem cells and endothelial cells and to evaluate their viability and osteogenic differentiation potential. MATERIALS AND METHODS: Gingiva-derived stem cells were isolated, and stem cells and endothelial cells with a total of 6 × 10 cells were seeded into concave micromolds with different ratios of 6:0 (group 1), 4:2 (group 2), 3:3 (group 3), and 2:4 (group 4). RESULTS: Gingiva-derived stem cells and/or endothelia cells formed spheroids in concave microwells. There was a decreasing trend in the diameter of spheroids with increasing amounts of endothelial cells, but there were no statistically significant differences between the groups. The secretion of vascular endothelial growth factor from the spheroids was noted. The results of the alkaline phosphatase activity assays showed significantly higher values for groups 2, 3, and 4 when compared with the value of group 1. CONCLUSIONS: Conclusively, stem-cell spheroids formed with human gingiva-derived stem cells and endothelial cells using concave microwells enhanced osteogenic differentiation potential, and multicell spheroid-based cell delivery could be a simple and effective strategy for improving stem-cell therapy.

Isolation and characterization of novel Ty1-copia-like retrotransposons from lily.

Species of the genus Lilium are well known for their large genomes. Although expansion of noncoding repeated DNA is believed to account for this genome size, retroelement del Ty3-gypsy is the only one described so far in the genus Lilium. We isolated Ty1-copia elements from Lilium longiflorum and named them LIREs (lily retrotransposons). The long terminal repeats, primer binding site, and polypurine tract sequences are highly similar among the LIRE elements, indicating that they are in the same lineage. Although the protein-coding regions were highly decayed, the sequence motifs of the integrase, reverse transcriptase, and RNase H domains were identifiable as belonging to the order of Ty1-copia elements. Phylogenetic analysis and primer binding site sequences revealed that these elements belonged to the Ale lineage among the six lineages of plant Ty1-copia elements. Base substitutions in the long terminal repeats estimated that the integration times of the LIRE Ty1-copia elements were between 0.7 and 5.5 mya. In situ hybridization showed that the LIRE elements were present in all the chromosomes of L. longiflorum and L. lancifolium, but absent in centromeres, telomeres, and 45S rRNA sites in both species. The LIRE elements were present very abundantly in species of the genus Lilium, but absent in other genera of the family Liliaceae, implying that the LIRE elements might have contributed to the expansion of the genome in the genus Lilium.

Construction of artificial promoters sensitively responsive to sonication in vitro.

PURPOSE: To develop artificial promoters that are activated in response to sonication and to determine these properties in vitro. METHODS: The binding sites of four transcription factors (nuclear factor-kappa B, activating protein-1, nuclear factor-Y, and CArG element binding factor A) that are activated by oxidative stress were randomly ligated and linked to a TATA-box sequence to control the luciferase gene located downstream. Transiently transfected HeLa cells from human cervical cancer with a plasmid vector containing such a gene cassette were exposed to sonication, and enhancement of luciferase expression was assessed by dual luciferase assay. RESULTS: Of 62 promoters constructed, two promoters, designated clone 31 and clone 62 promoters, showed a more than tenfold enhancement 6 h after sonication with 1-MHz ultrasound at 1.0 W/cm(2) for 60 s. These promoters were activated in a dose-dependent manner with the intensity and duration of sonication. The activation was attenuated by addition of dimethyl sulfoxide, an antioxidant, suggesting that oxidative stress was involved. The clone 31 promoter responded to each of two serial sonications. When sonicated 24 h after the first sonication, the peak of promoter enhancement was higher than that after the first sonication. CONCLUSIONS: A promoter sensitively responsive to sonication was constructed using the above method, possibly leading to the construction of a promoter of interest that could be applied for clinical use.

Enhancement of artificial promoter activity by ultrasound-induced oxidative stress.

We previously developed artificial promoters that were activated in response to X-ray irradiation. Sonication with 1.0MHz ultrasound that causes intracellular oxidative stress was found to activate some of these promoters though to lesser degrees. The most sensitive one among these promoters showed intensity- and duration-dependent activations by sonication. In addition, its activation by sonication was attenuated when N-acetyl cysteine was present, suggesting the involvement of intracellular oxidative stress in the activation mechanism. Improved promoters for sensitivity to X-ray irradiation were also found more sensitive to sonication. The most improved one showed 6.0 fold enhancement after sonication with 1.0MHz ultrasound at 1.0W/cm2 for 60s. This enhancement was also attenuated with the presence of N-acetyl cysteine. When stably transfected HeLa cells with the most sensitive promoter were transplanted on to mice and sonicated, luciferase activity by the promoter increased to 1.35 fold in average though it was not statistically significant compared to control. Although gene regulation in vivo by sonication was not clear, this is the first report on artificially constructed promoters responsive to ultrasound.

Construction of X-ray-inducible promoters through cis-acting element elongation and error-prone polymerase chain reaction.

BACKGROUND: A promoter that is activated by ionizing radiation may be a useful tool for cancer therapy since, with such a promoter, the therapeutic gene can be expressed only in cancer tissues by irradiation. An artificially constructed promoter is advantageous as natural promoters may have physiological limitations. However, reasonably designing a promoter is hampered by shortage of information about the relationship between the structure and properties of a promoter DNA. MATERIALS AND METHODS: Binding sites of four transcription factors that were activated by radiation were randomly ligated and linked to a TATA-box sequence to control the luciferase gene located downstream. Transiently transfected cancer cells with such a vector were exposed to X-ray irradiation and enhancement of luciferase expression was assessed. To improve promoter sensitivity, mutations were randomly introduced into a constructed promoter by error-prone polymerase chain reaction (epPCR). RESULTS: Of the 11 promoters constructed, the clone 11 promoter (clone 11 + TATA-box) showed a 5-fold enhancement 6 h after the 10 Gy X-ray irradiation in HeLa cells. A mutant designated the clone 11-9-37 promoter generated through two steps of epPCR showed a sensitivity 4.8 times higher than the clone 11 promoter to the 10 Gy X-rays, showing 21.6-fold enhancement of luciferase expression. Clone 11 was composed of 16 cis-acting elements, and the clone 11-9-37 promoter carried six point mutations. CONCLUSION: A sensitively responsive promoter to radiation could be constructed using this method, possibly leading to the construction of a promoter of interest that could be applied for clinical use.

A highly E-stereoselective approach to beta-iodo Morita-Baylis-Hillman esters: synthesis of secokotomolide A.

A geometric-selective synthesis of (E)-beta-iodo Morita-Baylis-Hillman esters has been developed through a three-component aldol-type reaction using BF3.Et2O and TMS-I. The synthetic utility of the (E)-beta-iodo Morita-Baylis-Hillman esters was demonstrated in the first reported short synthesis of secokotomolide A.

Development of a nested polymerase chain reaction method to detect oncogenic Marek's disease virus from feather tips.

For the easy survey of Marek's disease virus (MDV), feather tip-derived DNA from MDV-infected chickens can be used because feather tips are easy to collect and feather follicle epithelium is known to be the only site of productive replication of cell-free MDV. To develop a diagnostic method to differentiate highly virulent strains of MDV from the attenuated MDV vaccine strain, CVI988, which is widely used, nested polymerase chain reaction (PCR) was performed to detect a segment of the meq gene in feather tip samples of chickens experimentally infected with MDV. In chickens infected with Md5, a strain of oncogenic MDV, the meq gene was consistently detected, whereas the L-meq gene, in which a 180-base pair (180-bp) sequence is inserted into the meq gene, was detected in CVI988-infected chickens. Moreover, the meq gene was mainly detected even in chickens co-infected with both Md5 and CVI988. These results suggest that this method is appropriate for the surveillance of the highly virulent MDV infection in the field.

Detection of the meq gene in the T cell subsets from chickens infected with Marek's disease virus serotype 1.

The meq gene was thought to be only detected in Marek's disease virus serotype 1 (MDV 1) including a very virulent strain, Md5, while L-meq, in which a 180-bp sequence is inserted into the meq open reading frame, is found in other strains of MDV 1, such as CVI 988/R6. However, both meq and L-meq were previously detected by PCR in chickens infected with MDV 1, suggesting that MDV 1 may consists of at least two subpopulations, one with meq, the other with L-meq. To further analyze these subpopulations, we analyzed the time course changes in distribution of these subpopulations among T cell subsets from chickens infected with MDV 1. Both meq and L-meq were detected in CD4+ and CD8+ T cells infected with strain Md5 or CVI 988/R6. The shift in MDV subpopulations from one displaying meq to the other displaying L-meq and/or the conversion from meq to L-meq occurred mainly in the CD8+ T cell subset from Md5-infected chickens. PCR products corresponding to L-meq rather than meq were frequently amplified from the CD8+ T cell subset from CVI 988/R 6 -infected chickens. These results suggest that a dominant subpopulation of MDV 1 changes depending on the T cell subsets, and that L-meq is dominantly present in the CD8+ T cells which play a role in the clearance of pathogenic agents.

Phenotypic alteration of eukaryotic cells using randomized libraries of artificial transcription factors.

We have developed a method in which randomized libraries of zinc finger-containing artificial transcription factors are used to induce phenotypic variations in yeast and mammalian cells. By linking multiple zinc-finger domains together, we constructed more than 100,000 zinc-finger proteins with diverse DNA-binding specificities and fused each of them to either a transcription activation or repression domain. The resulting transcriptional regulatory proteins were expressed individually in cells, and the transfected cells were screened for various phenotypic changes, such as drug resistance, thermotolerance or osmotolerance in yeast, and differentiation in mammalian cells. Genes associated with the selected phenotypes were also identified. Our results show that randomized libraries of artificial transcription factors are useful tools for functional genomics and phenotypic engineering.

The detection of the meq gene in chicken infected with Marek's disease virus serotype 1.

In the genome of strains of very virulent Marek's disease virus serotype 1(vvMDV1), such as Md5 and RB1B, the meq open reading frame (ORF) encoding a 339-amino-acid bZIP protein, is present, while a slightly longer meq ORF, termed as L-meq, in which a 180-bp sequence is inserted into the meq ORF is found in other strains of MDV1, such as CV1988/R6 and attenuated JM. When chickens were infected with vvMDV1 strains and the meq gene was amplified by nested polymerase chain reaction (PCR), the meq gene was detected throughout the experimental period for 7 weeks post inoculation (pi). However, the L-meq gene was also detected at 3 to 5 weeks and 3 to 4 weeks pi. in Md5-infected and RB1B-infected chickens, respectively. In the case of chickens infected with an attenuated MDV1, the JM strain, the L-meq gene was detected at 2 to 7 weeks pi., and the meq gene was also detected at 2 to 6 weeks pi. Both L-meq and meq genes were detected in chickens infected with an attenuated nononcogenic vaccine strain of MDV1 (CVI988/R6), throughout the experimental period. Though quantitative PCR was not performed, a larger amount of the PCR products corresponding to the L-meq than the meq gene was amplified from chickens infected with JM or CVI988/R6. These results suggest that a dynamic population shift between the MDV subpopulations displaying meq and L-meq genes occurs in chickens during the course of MDV infection. Since the MDV subpopulation that displays the L-meq gene only displays it during the latent phase, the L-meq and its gene product, if any, might contribute to the maintenance of the MDV latency.