• Asian-Australasian Journal of Animal Sciences
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• 제29권3호
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• pp.315-320
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• 2016

Porcine beta-defensin-1 (PBD-1) gene plays an important role in the innate immunity of pigs. The peptide encoded by this gene is an antimicrobial peptide that has direct activity against a wide range of microbes. This peptide is involved in the co-creation of an antimicrobial barrier in the oral cavity of pigs. The objective of the present study was to detect polymorphisms, if any, in exon-1 and exon-2 regions of PBD-1 gene in Large White Yorkshire (LWY) and native Ankamali pigs of Kerala, India. Blood samples were collected from 100 pigs and genomic DNA was isolated using phenol chloroform method. The quantity of DNA was assessed in a spectrophotometer and quality by gel electrophoresis. Exon-1 and exon-2 regions of PBD-1 gene were amplified by polymerase chain reaction (PCR) and the products were subjected to single strand conformation polymorphism (SSCP) analysis. Subsequent silver staining of the polyacrylamide gels revealed three unique SSCP banding patterns in each of the two exons. The presence of single nucleotide polymorphisms (SNPs) was confirmed by nucleotide sequencing of the PCR products. A novel SNP was found in the 5'-UTR region of exon-1 and a SNP was detected in the mature peptide coding region of exon-2. In exon-1, the pooled population frequencies of GG, GT, and TT genotypes were 0.67, 0.30, and 0.03, respectively. GG genotype was predominant in both the breeds whereas TT genotype was not detected in LWY breed. Similarly, in exon-2, the pooled population frequencies of AA, AG, and GG genotypes were 0.50, 0.27, and 0.23, respectively. AA genotype was predominant in LWY pigs whereas GG genotype was predominant in native pigs. These results suggest that there exists a considerable genetic variation at PBD-1 locus and further association studies may help in development of a PCR based genotyping test to select pigs with better immunity.

• 한국식품영양과학회지
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• 제33권3호
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• pp.560-565
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• 2004

배추김치로부터 식품유해균인 Listeria monocytogenes를 저해하는 박테리오신을 생산하는 유산균, Lactobacillus sakei P3-1이 분리되었다. 형태학적, 생화학적 특성조사와 최종적으로 PCR로 증폭하여 얻은 16S rDNA 염기서열 결정을 통해서 L. sakei로 동정되었다. L. sakei P3-1이 분비하는 박테리오신은 여러 그람 양성 및 음성균들 중에서 단지 L. monocytogenes만을 저해하는 그래서 저해범위가 매우 좁은 박테리오신으로 확인되었다. 이온교환 크로마토그래피에 의해서 박테리오신은 부분 정제되었으며 박테리오신의 열처리 안정성을 조사한 결과 121$^{\circ}C$에서 15분간 그리고10$0^{\circ}C$에서 10분간 열처리 후에도 각각 12.5%와 50%의 역가가 잔존하여 상당한 열안정성을 지니고 있음을 알 수 있었다. MRS배지에서 배양중 배양온도가 박테리오신 역가에 미치는 영향을 조사한 결과 3$0^{\circ}C$에서 배양할 때 그리고 18시간 이상 배양에서 가장 높은 1,000 AU/mL 역가를 보였다. 한편 SDS-PAGE 및 activity staining에 의해 측정된 박테리오신의 분자량은 4,000이었다. L. monocytogenes 생육 억제능, 작은 분자량 및 높은 열안정성 등의 성질들을 종합적으로 고려할 때 L. sakei P3-1이 생산하는 박테리오신은 박테리오신들 중에서 class II-a에 속하는 것으로 추정된다.

• Tuberculosis and Respiratory Diseases
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• 제72권2호
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• pp.124-131
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• 2012

Background: DNA damage-inducible 1 (Ddi1), one of the ubiquitin-like and ubiquitin-associated family of proteins, may function in the regulation of the ubiquitin-proteasome pathway, which has been validated as a target for antineoplastic therapy. We investigated Ddi1 expression in human lung cancer tissues and evaluated the relationship of this expression pattern with clinicopathological factors in patients with non-small-cell lung cancer (NSCLC). Methods: Ddi1 expression was examined by immunohistochemistry in tumor tissues from 97 patients with stage I NSCLC, who had undergone curative surgical resection at two tertiary referral hospitals from 1993~2004. None of the patients received preoperative chemotherapy and/or radiation therapy. Results: Thirty-nine (40.2%) of the 97 cases were positive for Ddi1. Ddi1 expression was dominantly seen in cytoplasm rather than in the nuclei of cancer cells in all histological types, whereas adjacent nontumoral lung tissue showed negative Ddi1 staining in most cases. Ddi1 expression tended to increase in well-differentiated tumors but without statistical significance. Positive Ddi1 expression was associated with a tendency for better disease-free survival and disease-specific survival, although the difference was not significant. Conclusion: Ddi1 expression is a property of NSCLC. Because Ddi1 could be a potential target for cancer therapy, more research is needed to evaluate its role in NSCLC.

• BMB Reports
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• 제40권2호
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• pp.270-276
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• 2007

We have cloned a novel mouse zinc finger protein gene Znf313 by rapid amplification of cDNA ends (RACE) according to the homologue of human ZNF313 gene. The cDNA is 2,163 base pairs (bp) in length and encodes a 229 amino acids (aa) protein with a $C_3HC_4$ ring finger domain and three $C_2H_2$ domains. 89% and 93% nucleotide (nt) and aa sequence identity is observed with its human homologue. Revealed by Northern blot and RT-PCR, full mRNA consists of 2.16 kb and widely expresses in tissues as a single transcript, most abundantly in heart, liver, kidney and testis. The expression of Znf313 in testis is detected in all development stages. Western blot analysis also reveals that Znf313 is expressed in the tissues. Immunohistochemical staining and subcellular localization demonstrate that Znf313 is expressed both in the cytoplasm and nucleus whereas predominantly localized in the nucleus. Present data suggests that Znf313 gene might play a fundamental role in gene transcription and regulation in organism and relates to spermatogenesis.

• 미생물학회지
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• 제28권2호
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• pp.120-127
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• 1990

전분 분해능이 있는 담자균성 효모, Filohasidium capsuligenum의 종내 및 Saccharomyces cerevisiae와의 속간 원형질체융합을 시도하였다. F capsuligenum CBS 6122-2의 종내 융합율은 각각 $2.9\times10^{-3}$ (ade+mel trp), $8.5\times10^{-3}$(ade+met)이고, F. capsuligemum CBS 4381 (cys his)과 S.cerevisiae 262-12-1 (hom3 thr1), X2180-1A (ade thr)와의 속간 융합율은 각각 $8.8\times10^{-6}$, $9.5\times10^{-4}$이었다. DNA 정량, 핵 염색, 자외선 조사에 따른 생존력 비교 그리고 형질 분리 분석 결과 등을 통해 종내와 속간에서 핵 융합이 일어난 것을 알 수 있었다. 또한, 종내 융합체의 경우 세포당 $\alpha$-amylase와 glucoamylase 생성능이 양천형에 비해 1.6-2.1배의 증진 효과가 있었다.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
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• pp.261-261
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• 1994

Bovine milk xanthine oxidase (E.C.1.1.3.22, XO) purchased from Sigma Chemical Co. had the three protein fragments below 150 kDa on 7.5％ SDS-PAGE, which did not show enzyme activity. To remove these fragments, the enzyme preparation was further purified through Sephadex G-200 column chromatography. Two peaks exhibiting enzymatic activity were separated very closely to the void volume, which were revealed as two different enzyme forms, dimeric and monomeric, confirmed by activity staining on native PAGE. Anti sera-against each of the two enzyme forms were raised by subcutaneous injection at multiple sites on the back of rabbits during 4 weeks. On the immunodiffusion test, it was found that both of the antisera of the two forms could react with each other, which implied that their epitopes were identical In the Western blot analysis of mouse liver cytosol fraction, it was found that rabbit anti-XO antibody bound well with the protein band of monomeric mouse liver XO of about 150kDa. Based on this result, mouse liver cDNA 1 ibrary was screened by in situ hybridizat ion wi th rabbi t anti -XO antibody as probe. Through the immunological screening, recombinant phages giving positive signal by the production of XO were selected and further purified. To validate these clones, purified phages were lysogenized in E. coli Y1089 and their lysates were analysed for enzyme activity and immunoreactivity, It was verified that lysates of the purified recombinant phage lysogens exhibited the enzymatic activity as well as bound wi th XO antibody, when induced by IPTG. The above results assert that selected recombinant phage carries mouse liver XO gene.

• Natural Product Sciences
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• 제26권3호
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• pp.191-199
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• 2020

Fucoidan, a natural component of brown seaweed, has various biological activities such as anti-cancer activity, anti-oxidant, and anti-inflammatory against various cancer cells. However, the fucoidan has been implicated in melanoma cells via apoptosis signaling pathway. Therefore, we investigated apoptosis with fucoidan in A2058 human melanoma cells with dose- and time-dependent manners. In our results, A2058 cells viability decreased at relatively short-time and low-concentration through fucoidan. This effects of fucoidan on A2058 cells appeared to be mediated by the induction of apoptosis, as manifested by morphological changes through DNA-binding dye Hoechst 33342 staining. When a dose of 80 ㎍/mL fucoidan was treated, the cells were observed: crescent or ring-like structure, chromatin condensation, and nuclear fragmentation. With the increase at 100 ㎍/mL fucoidan, the cell membrane is intact throughout the total process, including membrane blebbing and loss of membrane integrity as well as increase of sub-G1 DNA. Furthermore, to understand the exact mechanism of fucoidan-treated in A2058 cells, western blotting was performed to detect apoptosis-related protein expression. In this study, Bcl-2 family proteins can be regulated by fucoidan, suggesting that fucoidan-induced apoptosis is modulated by intrinsic pathway. Therefore, expression of Bcl-2 and Bax may result in altered permeability, activating caspase-3 and caspase-9. And the cleaved form of poly ADP-ribose polymerase was detected in fucoidan-treated A2058 cells. These results suggest that A2058 cells are highly sensitive to growth inhibition by fucoidan via apoptosis, as evidenced by activation of extracellular signal-regulated kinases/p38/Bcl-2 family signaling, as well as alteration in caspase-9 and caspase-3.

• 한국발생생물학회지:발생과생식
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• 제1권1호
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• pp.79-89
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• 1997

Recent studies have demonstrated that apoptotic cell death plays an important role in the mechanism underlying follicular atresia and luteolysis. However, the mechanisms responsible for initiating these processes have not been elucidated. In in vitro fertilization (IVF) programs, it is highly possible that continuous and repeated administration of FSH/hMG and GnRH agonists for the usage of ovarian hyperstimulation may induce apoptotic death of granulosa cells leading to atresia in the human ovarian follicles. The present study was performed to investigate whether FSH/hMG and GnRh agonists used for a longer period in controlled ovarian hyperstimulation has any effect on the apoptosis of granulosa-luteal (GL) cells obtained from hyperstimulated ovaries. To examine apoptotic cell death in the GL cells, cells were stained with acridie orange followed by observed in some of GL cells. Similar but distinct staining of apoptotic GL cells was observed when the cells were examined by using in situ TUNEL method. The healthy-looking cells with normal nuclear morphology were not stained, whereas cells with pyknotic nuclei or with apoptotic nuclei were intensively stained. After examining the ultrastructural features of GL cells by TEM, it was confirmed that the majority of cells seemed to have normal nuclei while GL cells undergoing apoptotic cel death were rarely found. The DNA extracted from GL cells showed a typical pattern of fragmentation following DNA electrophoretic analysis. We have confirmed that the apoptosis occurs in granulosa-luteal cells obtained from hyperstimulated ovaries. Technically, in situ apoptosis detection method is simple and reproducible and is well suited to identify the quality of oocytes retrieved from hyperstimulated ovaries.

• Journal of Microbiology and Biotechnology
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• 제15권4호
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• pp.734-739
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• 2005

Myxobacterium sp. HK1, isolated from Korean soil, degrades cellulose, differentiates to fruiting body, and its 16s rDNA has $95\%$ similarity to Polyangium sp. An anticancer molecule, CDMHK, was identified from culture broth of Myxobacterium sp. HK1, and purified by Diaion HP20, Silica gel, Sephadex LH-20 chromatography, and preparative HPLC using an YMC OSD-A C18 column. The molecular structure and formula were determined to be $C_{l2}H_{l9}N_3O_2$ (M.W 237) by MS spectrometry, 300 MHz $^{1}H\;and\;^{13}C$ NMR. The CDMHK was not active against Escherichia coli, Staphylococcus aureus, and Candida albicans. However, this molecule inhibited the growth of various cancer cell lines. The $ED_{50}$ values of CDMHK were determined to be 0.147, 0.086, 0.18, 0.166, and 0.142 $\mu$g/ml against A549, SK-OV-3, SK-MEL-2, VF498, and HCTl5 cancer cell lines, respectively. In addition, the CDMHK was able to induce apoptosis of the CCRF-CEM cancer cell line, evidenced by DNA fragmentation assay and DAPI staining.

• Asian-Australasian Journal of Animal Sciences
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• 제19권5호
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• pp.622-626
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• 2006

Molecular characterization of Hallikar, the native cattle breed of Karnataka, was undertaken using 19 cattle specific, highly polymorphic microsatellite markers recommended by FAO. The genomic DNA was subjected to PCR amplification and alleles were resolved through six per cent denaturing PAGE with a 10 bp DNA ladder followed by silver staining. Genotyping of animals was done based on allele size. The number of alleles ranged from three to nine with allele sizes ranging from 102 bp to 294 bp. These alleles were distributed in the frequency range between 0.0306 and 0.8673 in the population. The mean observed number of alleles was $6.368{\pm}1.4225$. The mean observed and expected heterozygosities were $0.7515{\pm}0.1734$ and $0.7850{\pm}0.1381$, respectively. The high heterozygosity observed implies presence of higher genetic variability within Hallikar breed. The PIC (Polymorphism Information Content) values ranged from 0.2322 (ETH152) to 0.8654 (ETH225). The percentage of polymorphic loci obtained was 100 as all the 19 microsatellite markers were found to be polymorphic. Except for ETH152, all the other loci had high PIC values, indicating that these markers are highly informative for characterization of Hallikar breed. The population was tested for Hardy-Weinberg equilibrium at 19 microsatellite loci, and at 74 per cent of the loci the population was found to be in disequilibrium.