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This experiment was carried out to develop a new technique by identifying XX-bearing embryos prior to implantation by immunological method. H-Y antiserum was prepared in inbred Wistar female rats by repeated immunization with spleen cells from males of the same strain. The reactivity of H-Y antibody was confirmed by culturing mouse embryos in the medium containing H-Y antiserum and complement obtained from the guinea pig. The optimal condition for the activity of H-Y antibody was also investigated by culturing embryos under the various conditions of equilibration times, complement concentrations and various media. The results obtained in this experiment are as follows: When the embryos were cultured in the medium of H-Y antiserum and complement which was given the equilibration time of less than 30 minutes in CO₂ incubator, the lysis-rate of embryo was 89.3%. The embryo lysis-rates in the equilibration time of 1-1.5, 3-3.5, or 24-26 hours were 48.1, 47.7 and 48.2%, respectively. When the concentration rate of complement to H-Y antiserum varied from 0.25 - 4.0, the lysis-rate of embryo was 43.2 to 52.7%. The concentration rate of complement did not influence the lysis-rate of embryos. The meda of D-PBS + 0.3% BSA, D-PBS + 20% FCS, Ham's F-10 + 0.3% BSA and Ham's F-10 + 20% FCS showed the embryo lysis-rate of 46.4, 57.4, 49.3 and 49.1%, respectively. The culture media used in this experiment did not show any significant difference in the embryo lysis-rate. After the embryos were cultured to the late blastocyst in the media of D-PBS + NGPS + H-Y antiserum or D-PBS + NGPS + normal female rat serum the normally developed embryos were selected and transferred to the pseudo pregnant recipients. The percentages of their female offspring were 82.3%(14/17) in H-Y antiserum treatment and 53.6%(15/28) in normal serum treatment and showed a significant difference between the two treatments(p $lt;0.001).
This experiment was carried out to develop a new technique by immunological method. H-Y antiserum was prepared in inbred Wistar female rats by repeated immunization with new-born testis supernatant and spleen cells from males of the same strain. The activity of H-Y antibody in antiserum was tested by cytotoxicity and biological tests. The results obtained in this experiment. are as follows: In the sperm cytotoxicity test it was showed about 70% of the sperm were dead in the 1/2 to 1/8 dilution of H-Y antiserum immunized with spleen cells, new-born testis or the compound of both antigens. As the dilution increased, the death rate of sperm decreased markedly. H-Y antibody absorbed with female-rat spleen cells showed higher death-rate of sperm than that with male-rat spleen cells. The normal female-rat serum showed the sperm death-rate of 14.6 to 27.9% irrespective of the dilution rate. The difference between the two sperm death-rates was significant. The embryos cultured in the medium of complement and H-Y antiserum immunized with male-rat spleen, new-born testis or the compound of both antigens showed the lysis-rates of 48.9, 50.0 and 46.3% respectively. There was no significant difference among each lysis-rate. But the lysis-rate of the embryos cultured in the medium of complement and normal female rat serum was 5.1% and it was markedly different from the above lysis rates(p $lt;0.001).
To understand the effect of Y<SUB>2</SUB>BaCuO<SUB>5</SUB> (Y211)/YBa<SUB>2</SUB>Cu<SUB>3</SUB>O<SUB>7-y</SUB> (Y123) interfaces on the oxygen diffusion in single grain YBa<SUB>2</SUB>Cu<SUB>3</SUB>O<SUB>7-y</SUB> superconductors, single grain Y123 superconductors with 0.05 and 0.3moles of Y<SUB>2</SUB>O<SUB>3</SUB> additions were fabricated by a top-seeded melt growth (TSMG) process. Y123 compacts with Y<SUB>2</SUB>O<SUB>3</SUB> additions were subjected to melt growth heating cycles with a cooling rate of 1<SUP>o</SUP>C/h through a peritectic temperature (1015<SUP>o</SUP>C) and then annealed at 450<SUP>o</SUP>C for 200h in flowing oxygen. The superconducting temperature (T<SUB>c</SUB>) and critical current density (J<SUB>c</SUB>) were estimated for the three different regions (top surface (s), intermediate (i) and center (c)) of samples. The amount of Y211/Y123 interface area in single grain Y123 superconductors was successfully controlled by Y<SUB>2</SUB>O<SUB>3</SUB> additions. The T<SUB>c</SUB> values of s regions were higher than those of i and c regions, which indicates the presence of more oxygen at the sample surfaces. In addition, the T<SUB>c</SUB> values of i and c regions of the Y123 sample with 0.3mole Y<SUB>2</SUB>O<SUB>3</SUB> addition were higher than those of the same regions of the Y123 sample with 0.05mole Y<SUB>2</SUB>O<SUB>3</SUB> addition due to the promoted oxygen diffusion through Y211/Y123 interfaces and other related defects. In spite of the promoted oxygen diffusion by Y<SUB>2</SUB>O<SUB>3</SUB> addition, the large T<SUB>c</SUB> difference among the regions still existed, which suggests sluggish oxygen diffusion into single Y123 grains.
The purpose of this study was to detect association between genetic variation and economic trait in the porcine heart type fatty acid-binding protein gene as a candidate gene for the traits related with growth and meat quality in pigs. The H-FABP is a 15-kDa protein expressed in several tissues with high demand for fat metabolism such as cardiac and skeletal muscle and lactating mammary gland. H-FABP is small intracellular protein involved in fatty acid transport from the plasma membrane to the site of β-oxidation and/or triacylglycerol or phospholipid synthesis. In this study, H-FABP PCR-RFLP was performed in F_(2) population composed of 214 individuals form an intercross between Korean Native Boars and Landrace sows. PCR products form tow primer sets within H-FABP gene were amplified in 850bp and 700bp. Digestion of PCR products with the restriction digestion enzymes HaeⅢ and Hinf Ⅰ, revealed fragment length polymorphisms(RFL. Ps). The genotype frequencies from H-FABP/HaeⅢ was .29 for genotype DD, .53 for genotype Dd, and .15 for genotype dd, respectively. The genotype frequencies of HH, Hh, and hh from H-FABP(hinf Ⅰ was .38, .41, and .20, respectively, in the population.Relationships between their genotypes and economic traits were estimated. In H-FABP/HaeⅢ locus, there were specific genotypes(Dd and dd) associated with economic traits such as body weight. In H-FABP/Hinf Ⅰ Iocus, Genotypes of HH and Hh associated with growth traits such as body weights at 5, 12, and 30 week of age (p<.05 or p<.001) and back fat thickness, body fat including abdominal and trimmed fat (p<.001) and intramuscular fat(p<.05). The 'H'allele was positivecly associated with gaining of body weight and fatness deposition. In conclusion, a significant association of the H-FABP gene from its genetic variation was found on body weight, intramuscular fat and backfat thickness.
The objective of this study was to investigate the anti-oxidative effects of taurine on sperm characteristics for in vitro storage of boar semen. Semen was randomly divided into 10 groups in conical tubes and treated with different concentrations of taurine (25-100 mM) with or without 250 ???H2O2. The percentage of motile spermatozoa in taurine groups after 6 and 9 h were significantly higher at >94% and 87%, respectively, compared to the control group (85.1??.5 and 72.4??.3, p<0.05). The sperm motility in taurine with H2O2 after 6 h incubation was slightly decreased compared to the taurine alone treatment, but after 9 and 12 h incubation % sperm motility dropped sharply in taurine with H2O2 (75.3??.3 and 69.6??.9, p<0.05). For 3, 9 and 12 h incubation, sperm viability in the control was lower than in taurine groups, irrespective of taurine concentration. In eosin Y and nigrosin staining (ENS), the sperm survival rates (%) for 6 h incubation were significantly higher in 25 mM (76.0??.6) and 50 mM taurine groups (78.0??.7), respectively. Sperm survival rates for 9 and 12 h incubation were higher in taurine groups (??8% in 9 h and ??2% in 12 h) compared to controls (43.0??.1 and 31.0??.6, respectively). In the hyoosmotic swelling test (HOST), sperm membrane integrity was similar to the results of sperm survival. These experiments indicate that supplementation of taurine to the semen extender can increase the sperm characteristics (motility, viability, survival and membrane integrity).
We analyzed haplotypes for 22 Y chromosomal STRs (Y-STRs), including 17 Yfiler loci (DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DY438, DYS439, DYS448, DYS456, DYS458, DYS635 and Y-GATA-H4) and five additional STRs (DYS388, DYS446, DYS447, DYS449 and DYS464), and Y chromosomal haplogroup distribution in 270 unrelated individuals from the Pathans residing in the Federally Administered Tribal Areas and the North-West Frontier Province of Pakistan using in-house multiplex PCR systems. Each Y-STR showed diversities ranging from 0.2506 to 0.8538, and the discriminatory capacity (DC) was 73.7% with 199 observed haplotypes using 17 Yfiler loci. By the addition of 5 Y-STRs to the Yfiler system, the DC was increased to 85.2% while showing 230 observed haplotypes. Among the additional 5 Y-STRs, DYS446, DYS447 and DYS449 were major contributors to enhancing discrimination. In the analysis of molecular variance, the Pathans of this study showed significant differences from other Pathan populations as well as neighboring population sets. In Y-SNP analysis, a total of 12 Y chromosomal haplogroups were observed and the most frequent haplogroup was R1a1a with 49.3% frequency. To obtain insights on the origin of Pathans, the network analysis was performed for the haplogroups G and Q observed from the Pathans and the Jewish population groups including Ashkenazim and Sephardim, but little support for a Jewish origin could be found. In the present study, we report Y-STR population data in Pathans of Pakistan, and we emphasize the need for adding additional markers to the commonly used 17 Yfiler loci to achieve more improved discriminatory capacity in a population with low genetic diversity.
Testis supernatant, a source of H-Y, obtained from BALB/c mice was used to immunize females of same strain. B lymphocytes of mouse producing antibodies to H-Y were fused with SP2/0-Ag 14 myeloma cells and distributed to 384 wells of 96-well microtiter plates. Eighty hybridoma colonies were formed, resulting in 20.8 percent of fusion efficiency. Three strong positive wells from hybridoma colonies were selected for cloning by ELISA and two of them were also found to be positive by indirect immunofluorescence test. Twelve wells of ELISA-positive were selected after cloning and 2D45D4 clones from them were confirmed to produce monoclonal antibodies to H-Y by indirect immunofluorescence test.
Next-generation sequencing (NGS) can produce massively parallel sequencing (MPS) data for many targeted regions with a high depth of coverage, suggesting its successful application to the amplicons of forensic genetic markers. In the present study, we evaluated the practical utility of MPS in Y-chromosome short tandem repeat (Y-STR) analysis using a multiplex polymerase chain reaction (PCR) system. The multiplex PCR system simultaneously amplified 24 Y-chromosomal markers, including the PowerPlex<SUP>®</SUP> Y23 loci (DYS19, DYS385ab, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS481, DYS533, DYS549, DYS570, DYS576, DYS635, DYS643, and YGATAH4) and the M175 marker with the small-sized amplicons ranging from 85 to 253bp. The barcoded libraries for the amplicons of the 24 Y-chromosomal markers were produced using a simplified PCR-based library preparation method and successfully sequenced using MPS on a MiSeq<SUP>®</SUP> System with samples from 250 unrelated Korean males. The genotyping concordance between MPS and the capillary electrophoresis (CE) method, as well as the sequence structure of the 23 Y-STRs, were investigated. Three samples exhibited discordance between the MPS and CE results at DYS385, DYS439, and DYS576. There were 12 Y-STR loci that showed sequence variations in the alleles by a fragment size determination, and the most varied alleles occurred in DYS389II with a different sequence structure in the repeat region. The largest increase in gene diversity between the CE and MPS results was in DYS437 at +34.41%. Single nucleotide polymorphisms (SNPs), insertions, and deletions (indels) were observed in the flanking regions of DYS481, DYS576, and DYS385, respectively. Stutter and noise ratios of the 23 Y-STRs using the developed MPS system were also investigated. Based on these results, the MPS analysis system used in this study could facilitate the investigation into the sequences of the 23 Y-STRs in forensic genetics laboratories.
Y chromosome single nucleotide polymorphisms (Y-SNPs) are useful markers for reconstructing male lineages through hierarchically arranged allelic sets known as haplogroups, and are thereby widely used in the fields such as human evolution, anthropology and forensic genetics. The Y haplogroup tree was recently revised with newly suggested Y-SNP markers for designation of several subgroups of haplogroups C2, O2b and O3a, which are predominant in Koreans. Therefore, herein we analyzed these newly suggested Y-SNPs in 545 unrelated Korean males who belong to the haplogroups C2, O2b or O3a, and investigated the reconstructed topology of the Y haplogroup tree. We were able to confirm that markers L1373, Z1338/JST002613-27, Z1300, CTS2657, Z8440 and F845 define the C2 subhaplogroups, C2b, C2e, C2e1, C2e1a, C2e1b and C2e2, respectively, and that markers F3356, L682, F11, F238/F449 and F444 define the O subhaplogroups O2b1, O2b1b, O3a1c1, O3a1c2 and O3a2c1c, respectively. Among six C2 subhaplogroups (C2b, C2e, C2e1*, C2e1a, C2e1b and C2e2), the C2e haplogroup and its subhaplogroups were found to be predominant, and among the four O2b subhaplogroups (O2b*, O2b1*, O2b1a and O2b1b), O2b1b was most frequently observed. Among the O3a subhaplogroups, O3a2c1 was predominant and it was further divided into the subhaplogroups O3a2c1a and O3a2c1c with a newly suggested marker. However, the JST002613-27 marker, which had been known to define the haplogroup C2f, was found to be an ancestral marker of the C2e haplogroup, as is the Z1338 marker. Also, the M312 marker for the O2b1 haplogroup designation was replaced by F3356, because all of the O2b1 haplotypes showed a nucleotide change at F3356, but not at M312. In addition, the F238 marker was always observed to be phylogenetically equivalent to F449, while both of the markers were assigned to the O3a1c2 haplogroup. The confirmed phylogenetic tree of this study with the newly suggested Y-SNPs could be valuable for anthropological and forensic investigations of East Asians including Koreans.
In this study, 363 Korean father-son haplotype transfers in 351 families were analyzed using an in-house multiplex PCR system for 14 Y-STRs (DYS385a/b, DYF387S1, DYS391, DYS449, DYS460, DYS481, DYS518, DYS533, DYS549, DYS570, DYS576, DYS627 and DYS643), that included 11 loci newly added to the PowerPlex Y23 system or the Yfiler Plus system. The Y-STRs showed gene diversity values ranging from 0.2499 to 0.9612; the multicopy Y-STR loci DYS385 and DYF387S1 had high gene diversity of 0.9612 and 0.9457, respectively. In addition, DYF387S1, which has two copies, showed three alleles in seven individuals, and micro-variant alleles were observed in 14 individuals at four loci (DYS448, DYS518, DYS570 and DYS627). Among 351 haplotypes for the 11 newly added Y-STRs, 350 different haplotypes were observed, with an overall haplotype diversity of 0.9999 and discrimination capacity of 99.72%. In 363 haplotype transfers from 351 pedigrees, 29 single-step mutations were observed at 11 Y-STRs. Locus-specific mutation rate estimates varied from 0.0 to 1.93x10<SUP>-2</SUP>, with an average estimated mutation rate of 6.66x10<SUP>-3</SUP>. Two father-son pairs had mutations at two different loci in 11 Y-STRs. The number of pairs with mutations at multiple loci increased to five when the mutation event was investigated for haplotype transfer at 28 Y-STRs including 17 Yfiler loci and 11 Y-STRs examined in this study: four father-son pairs had mutations at two loci, and one pair had mutations at three loci. Overall, mutations were frequently observed at DYS449, DYS576 and DYS627 loci, which are known to be rapidly mutating Y-STRs. Mutation rate estimates at most loci were not significantly different from rates in other populations, but estimates for DYF387S1, DYS518 and DYS570 were considerably lower in the Korean population than in other populations.