• Journal of Microbiology and Biotechnology
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• v.10 no.3
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• pp.312-320
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• 2000

Abstract The full sequence of the plasmid pKJ36, which was derived from Bifidobacterium longum KJ, was determined and analyzed to construct shuttle vectors between E. coli and Bifidobacterium. The plasmid pKJ36 was composed of 3,625 base pairs with a 65.1% G+C content. The structural organization of pKJ36 was highly similar to that of pKJ50, and the three major ORFs on pKJ36 showed high amino acid sequence homologies with those of pKJ50. The putative proteins coded by these three ORFs were designated as RepB (32.0 kDa, pI=9.25), MembB (29.0 kDa, pI=12.25), and MobB (39.0 kDa, pI=IO.66), respectively. The amino acid sequence of RepB showed a 57% identity and 70% similarity with that of the RepA protein of pKJ50. Upstream of the repB gene, the so-called iteron sequence was directly repeated four-and-ahalf times and a conserved dnaA box was identified. An amino acid sequence comparison between the MobB and MobA of pKJ50 revealed a 48% identity and 61 % similarity. A conserved oriT sequence with an inverted repeat identical to that of pKJ50 was also found upstream of the mobB gene. A hydropathy analysis of MembB revealed four possible transmembrane regions. The expressions of the repB and membB genes were confirmed by RT-PCR. The in vitro translation reaction of pKJ36 showed protein bands with anticipated sizes with respect to each putative gene product. S 1 endonuclease treatment and Southern hybridization suggested that pKJ36 replicates by a rolling circle mechanism via a single-stranded DNA (ssDNA) intermediate. A shuttle vector between E. coli and Bifidobacterium sp. was constructed using the pKJ36, pBR322, and staphylococcal chloramphenicol acetyl transferase (CAT) gene. The successful transformation of the Bifidobacterium strains was shown by Southern hybridization and PCR. The transformation efficiency differed from strain to strain and, depending on the electroporation conditions, with a range between $1.2{\times}10^1-2.6{\times}10^2{\;}cfu/\mu\textrm{g}$ DNA.X> DNA.

• Pediatric Infection and Vaccine
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• v.11 no.2
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• pp.153-157
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• 2004

Purpose : Ttransfusion transmitted virus(TTV) is a circular DNA and consists of diverse genotypes and variants. The pathogenecity of TTV is still unclear. Recently another circular single stranded DNA virus, distantly related to TTV was isolated from the sera of blood donors, designated as Transfusion transmitted virus like minivirus(TLMV). TTV and TLMV show greater sequence divergence from each other than between genotypes of TTV. We planned to know the prevalence of TLMV in children. Methods : TLMV DNA was detected by PCR primers from noncoding region of the genome in 88 children without hepatitis, aged 0~15 years. PCR products derived from 10 children were directly sequenced and phylogenetic analysis was undertaken. Results : TLMV DNA was detected in 49% of 88 children without hepatitis. The prevalence of TLMV varied with age : <1 y, 16%(4/25); 1~3 y, 62%(18/29); 4~6 y, 43%(7/16); 7~9 y. 16%(1/6); 10~15 y, 66%(8/12). Mixed infection with TTV was confirmed in 22% of 88 children. Pyhlogenetic analysis of 10 TLMV sequences showed much heterogeneity compared to sequences of GenBank. Conclusion : TLMV prevalence in children was 49% in Korean children. Our TLMV sequence did not cluster in any sequence of TLMV in the GenBank.

• Journal of Microbiology and Biotechnology
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• v.17 no.2
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• pp.262-270
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• 2007

Of the twelve lytic bacteriophages recovered from five different fermenting cucumber tanks that were inoculated with Pediococcus sp. LA0281, a lytic phage, ${\phi}ps05$, was characterized in the present study. The plaques were mostly clear and round-shaped on the lawn of starter strain, indicating lytic phage. Overall appearance indicated that it belongs to the Siphoviridae family or Bradley's group B1, with a small isometric head and a flexible noncontractile tail with swollen base plate. The average size was found to be 51.2 nm in head diameter and 11.6 nm wide ${\times}$ 129.6 nm long for the tail. The single-step growth kinetics curve showed that the eclipse and the latent period were 29 min and 34 min, respectively, and an average burst size was calculated to be 12 particles per infective center. The optimum proliferating temperature ($35^{\circ}C$) was slightly lower than that of cell growth ($35\;to\;40^{\circ}C$). The structural proteins revealed by SDS-PAGE consisted of one main protein of 33 kDa and three minor proteins of 85, 58, and 52 kDa. The phage genome was a linear double-stranded DNA without cohesive ends. Based on the single and double digestion patterns obtained by EcoRI, HindIII, and SalI, the physical map was constructed. The overall size of the phage genome was estimated to be 24.1 kb. The present report describes the presence of a lytic phage active against a commercial starter culture Pediococcus sp. LA0281 in cucumber fermentation, and a preliminary study characterizes the phage on bacterial successions in the process of starter-added cucumber fermentation.

• Journal of the Korea Organic Resources Recycling Association
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• v.23 no.1
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• pp.70-81
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• 2015

Helicases are known to be a proteins that use the chemical energy of NTP binding and hydrolyze to separate the complementary strands of double-stranded nucleic acids to single-stranded nucleic acids. They participate in various cellular metabolism in many organisms. DEAD-box proteins are ATP-dependent RNA helicase that participate in all biochemical steps involving RNA. DEAD-box3 (DDX3) gene is belonging to the DEAD-box family and plays an important role in germ cell development in many organisms including not only vertebrate, but also invertebrate during asexual and sexual reproduction and participates in stem cell differentiation during regeneration. In this study, in order to identify and characterize DDX3 gene in the earthworm, Perionyx excavatus having a powerful regeneration capacity, total RNA was isolated from adult head containing clitellum. Full length of DDX3 gene from P. excavatus, Pe-DDX3, was identified by RT-PCR using the total RNA from head as a template. Pe-DDX3 encoded a putative protein of 607 amino acids and it also has the nine conserved motifs of DEAD-box family, which is characteristic of DEAD-box protein family. It was confirmed that Pe-DDX3 has the nine conserved motifs by the comparison of entire amino acids sequence of Pe-DDX3 with other species of different taxa. Phylogenetic analysis revealed that Pe-DDX3 belongs to a DDX3 (PL10) subgroup of DEAD-box protein family. And it displayed a high homology with PL10a, b from P. dumerilii.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.18
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• pp.8503-8512
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• 2016

The purpose of this study was to provide a detailed explanation of the mechanism of bisanthracycline, WP 631 in comparison to doxorubicin (DOX), a first generation anthracycline, currently the most widely used pharmaceutical in clinical oncology. Experiments were performed in SKOV-3 ovarian cancer cells which are otherwise resistant to standard drugs such as cis-platinum and adriamycin. As attention was focused on the ability of WP 631 to induce apoptosis, this was examined using a double staining method with Annexin V and propidium iodide probes, with measurement of the level of intracellular calcium ions and cytosolic cytochrome c. The western blotting technique was performed to confirm PARP cleavage. We also investigated the involvement of caspase activation and DNA degradation (comet assay and immunocytochemical detection of phosphorylated H2AX histones) in the development of apoptotic events. WP 631 demonstrated significantly higher effectiveness as a pro-apoptotic drug than DOX. This was evident in the higher levels of markers of apoptosis, such as the externalization of phosphatidylserine and the elevated level of cytochrome c. An extension of incubation time led to an increase in intracellular calcium levels after treatment with DOX. Lower changes in the calcium content were associated with the influence of WP 631. DOX led to the activation of all tested caspases, 8, 9 and 3, whereas WP 631 only induced an increase in caspase 8 activity after 24h of treatment and consequently led to the cleavage of PARP. The lack of active caspase 3 had no outcome on the single and double-stranded DNA breaks. The obtained results show that WP 631 was considerably more genotoxic towards the investigated cell line than DOX. This effect was especially visible after longer times of incubation. The above detailed studies indicate that WP 631 generates early apoptosis and cell death independent of caspase-3, detected at relatively late time points. The observed differences in the mechanisms of the action of WP631 and DOX suggest that this bisanthracycline can be an effective alternative in ovarian cancer treatment.

• Molecules and Cells
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• v.26 no.2
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• pp.131-139
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• 2008

Expression of the seven open reading frames (ORFs) of single-stranded DNA Curtoviruses such as Beet curly top virus (BCTV) and Beet severe curly top virus (BSCTV) is driven by a bi-directional promoter. To investigate this bidirectional promoter activity with respect to viral late gene expression, transgenic Arabidopsis plants expressing a GUS reporter gene under the control of either the BCTV or BSCTV bi-directional promoter were constructed. Transgenic plants harboring constructs showed higher expression levels when the promoter of the less virulent BCTV was used than when the promoter of the more virulent BSCTV was used. In transgenic seedlings, the reporter gene constructs were expressed primarily in actively dividing tissues such as root tips and apical meristems. As the transgenic plants matured, reporter gene expression diminished but viral infection of mature transgenic plants restored reporter gene expression, particularly in transgenic plants containing BCTV virion-sense gene promoter constructs. A 30 base pair conserved late element (CLE) motif was identified that was present three times in tandem in the BCTV promoter and once in that of BSCTV. Progressive deletion of these repeats from the BCTV promoter resulted in decreased reporter gene expression, but BSCTV promoters in which one or two extra copies of this motif were inserted did not exhibit increased late gene promoter activity. These results demonstrate that Curtovirus late gene expression by virion-sense promoters depends on the developmental stage of the host plant as well as on the number of CLE motifs present in the promoter.

• International Journal of Industrial Entomology and Biomaterials
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• v.24 no.1
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• pp.23-27
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• 2012

Porcine circovirus type 2 (PCV2) is a single-stranded circular DNA virus associated with Postweaning multisystemic wasting syndrome (PMWS), which is considered to be an important infectious swine viral disease. PCV2 capsid protein encoded by ORF2 is a structural protein and expected as the high immunogenicity protein. In this study, we generated recombinant baculovirus containing ORF2 of PCV2 and analyzed the optimal conditions for the production of capsid protein in insect cell. Production and status of recombinant capsid protein in insect cell were confirmed by SDS-PAGE and Western blot analysis using His tag antibody and anti-PCV2 serum. The yield of recombinant capsid protein was high like as shown visible on SDS-PAGE. Optimal multiplicity of infection (MOI) and infection time of recombinant virus were determined as 5 MOI and 4 days, respectively. ORF2 is known to have N-linked glycosylation site, but we couldn't detect the glycosylation of recombinant protein in insect cells.

• Childhood Kidney Diseases
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• v.9 no.2
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• pp.275-281
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• 2005

Parvovirus B19(PV B19) is a nonenveloped single-stranded DNA virus that causes a wide variety of diseases ranging from benign childhood infection such as slapped-cheek rash(fifth disease) to life threatening diseases such as hydrous fetalis in fetuses or aplastic anemic crises in patients with hemolytic anemia. In immunocompromised hosts including organ transplant recipients, this infection can cause chronic anemia. Recently, the reports of cases of PV B19 infection have been increasing in transplant recipients and most reported cases of PV B19 infection-associated anemia in renal transplant recipients were successfully treated with intravenous immunoglobulin infusion. We experienced two cases of aplastic anemia caused by PV B19 infection in pediatric renal transplant recipients. The patients were an 8 year-old boy and 12-year-old boy who received allograft kidneys from their mothers. Anemia developed 2 weeks after transplantation and their serum was positive for PV B19 PCR. They were treated with 400 mg/kg of intravenous immunoglobulin(IVIG) for 5 consecutive days. In one of the case, anemia was corrected promptly after the first 5-day course of IVIG therapy but in the other, anemia persisted but responded to the second course of IVIG therapy. One year later, the patients have normal hematocrit levels and stable renal function These are the first cases of PV B19 infection treated successfully with IVIG in pediatric renal transplant recipients in Korea. (J Korean Soc Pediatr Nephrol 2005;9:275-281)

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.5
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• pp.3309-3314
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• 2013

Spleen tyrosine kinase (SYK) is a non-receptor type cytoplasmic protein and a known tumor suppressor gene in breast cancer. Polymorphisms in SYK have been reported to be associated with cell invasion/cell morality and an increased risk of cancer development. In this case control study, all exons of the SYK gene and its exon/ intron boundaries were amplified in 200 breast cancer cases and 100 matched controls and then analyzed by single stranded conformational polymorphism. Amplified products showing altered mobility patterns were sequenced and analyzed. Twelve variations were identified in exonic and intronic regions of DNA encoding SH2 domain and kinase domain of the SYK gene. All of these mutations are novel. Among them, 5 missense mutations were observed in exon 15 while one missense mutation was found in exon 8. In addition to these mutations, six mutations were also identified in intronic regions. We found a significant association between SYK mutations and breast cancer and observed that Glu241Arg, a missense mutation is associated with an increase risk of ~7 fold (OR=6.7, 95% CI=1.54-28.8), Thr581Pro (missense mutation) is associated with increased risk of ~16 fold (OR=15.5, 95%CI=2.07-115.45) and 63367 T>G (missense mutation) is associated with increased risk of ~13 fold (OR=12.8, 95%CI=1.71-96.71) for breast cancer. Significant associations were observed for each of these variations with both late menopause (p<0.01) and early menarche (p<0.005) cases when compared to controls. Our findings suggest that the polymorphic gene SYK may contribute to the development of breast cancer in at least the Pakistani population. This study provides an insight view of SYK which may provide a significant finding for the pharmaceutical and biotechnology industry.

• BMB Reports
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• v.39 no.6
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• pp.686-695
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• 2006

Interleukin-2 enhancer binding factor 2 (ILF2) was reported to regulate transcription of interleukin-2 (IL-2), a central cytokine in the regulation of T-cell responses. This property of ILF2 was well characterized in human and mammals, but little is known in bony fish. In this paper, an ILF2 homologue was cloned and well characterized from Tetraodon nigrovirid is for the further investigation of the function of ILF2 in bony fish. The full-length Tetraodon ILF2 cDNA was 1380 bp in size and contained an open reading frame (ORF) of 1164 bp that translates into a 387 amino-acid peptide with a molecular weight of 42.9 kDa, a 5' untranslated region (UTR) of 57 bp, and a 3' UTR of 159 bp containing a poly A tail. The deduced peptide of Tetraodon ILF2 shared an overall identity of 58%~93% with other known ILF2 sequences, and contained two N-glycosylation sites, two N-myristoylation sites, one RGD cell attachment sequence, six protein kinase C phosphorylation sites, one amino-terminal RGG-rich single-stranded RNA-binding domain, and a DZF zinc-finger nucleic acid binding domain, most of which were highly conserved through species compared. Constitutive expression of Tetraodon ILF2 was observed in all tissues examined, including gill, gut, head kidney, spleen, liver, brain and heart. The highest expression was detected in heart, followed by liver, head kidney and brain. Stimulation with LPS did not significantly alter the expression of Tetraodon ILF2. Gene organization analysis showed that the Tetraodon ILF2 gene have fifteen exons, one more than other known ILF2 genes in human and mouse. Genes up- and down-stream from the Tetraodon ILF2 were Rpa12, Peroxin-11b, Smad4, Snapap and Txnip homologue, which were different from that in human and mouse.