• Journal of Microbiology
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• v.38 no.2
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• pp.80-87
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• 2000

The nucleotide sequence of the luxC gene coding for lux-specific fatty acyl-CoA reductase and the upstream DNA (325bp)of the structural gene from bioluminescent bacterium, Photobacterium phosphoreum, has been deternubed. An open reading frame extending for more than 20 codons in 325 bp DNA upstream of luxC was not present in both directions. The lux gene can be translated into a polypeptide of 54 kDa and the amino acid sequences of lux specific reductases of P. phosphoreum shares 80, 65, 58, and 62% identity with those of the Photobacterium leiognathi, Vibrio fischeri, Vibrio harveyi, and Xehnorhabdus luminescenens reductases, respectively. Analyses of codon usage, showing that a high frequency (2.3%) of the isoleucine codon, AUA, in the luxC gene compared to that found in Escherichia coli genes (0.2%) and its absence in the luxA and B genes, suggested that the AUA codon may play a modulator role in the expression of lux gene in E. coli. The structural genes (luxC, D, A, B, E) of the P. phosphoreum coding for luciferase (${\alpha}$,${\beta}$) and fatty acid reductase (r, s, t) polypeptides can be expressed exclusively in E. coli under the T7 phage RNA polymerase/promoter system and identificationof the [35S]methionine labelled polypeptide products. The degree of expression of lux genes in analyses of codon usage. High expression of the luxC gene could only be accomplished in a mutant E. coli 43R. Even in crude extracts, the acylated acyl-CoA reductase intermediate as well as acyl-CoA reductrase activities could be readily detected.

• Journal of Veterinary Science
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• v.24 no.3
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• pp.38.1-38.12
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• 2023

Background: Poor disease management and irregular vector control could predispose sheltered animals to disease such as feline Bartonella infection, a vector-borne zoonotic disease primarily caused by Bartonella henselae. Objectives: This study investigated the status of Bartonella infection in cats from eight (n = 8) shelters by molecular and serological approaches, profiling the CD4:CD8 ratio and the risk factors associated with Bartonella infection in shelter cats. Methods: Bartonella deoxyribonucleic acid (DNA) was detected through polymerase chain reaction (PCR) targeting 16S-23S rRNA internal transcribed spacer gene, followed by DNA sequencing. Bartonella IgM and IgG antibody titre, CD4 and CD8 profiles were detected using indirect immunofluorescence assay and flow cytometric analysis, respectively. Results: B. henselae was detected through PCR and sequencing in 1.0% (1/101) oral swab and 2.0% (1/50) cat fleas, while another 3/50 cat fleas carried B. clarridgeiae. Only 18/101 cats were seronegative against B. henselae, whereas 30.7% (31/101) cats were positive for both IgM and IgG, 8% (18/101) cats had IgM, and 33.7% (34/101) cats had IgG antibody only. None of the eight shelters sampled had Bartonella antibody-free cats. Although abnormal CD4:CD8 ratio was observed in 48/83 seropositive cats, flea infestation was the only significant risk factor observed in this study. Conclusions: The present study provides the first comparison on the Bartonella spp. antigen, antibody status and CD4:CD8 ratio among shelter cats. The high B. henselae seropositivity among shelter cats presumably due to significant flea infestation triggers an alarm of whether the infection could go undetectable and its potential transmission to humans.

• Journal of Periodontal and Implant Science
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• v.41 no.5
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• pp.227-233
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• 2011

Purpose: The aim of this study was to compare osteoblast behavior on zirconia and titanium under conditions cultured with bone morphogenetic protein-2. Methods: MC3T3-E1 cells were cultured on sandblasted zirconia and sandblasted/etched titanium discs. At 24 hours after seeding MC3T3-E1, the demineralized bone matrix (DBM) gel alone and the DBM gel with bone morphogenetic protein-2 (BMP-2) were added to the culture medium. The surface topography was examined by confocal laser scanning microscopy. Cellular proliferation was measured at 1, 4, and 7 days after gel loading. Alkaline phosphatase activity was measured at 7 days after gel loading. The mRNA expression of ALPase, bone sialoprotein, type I collagen, runt-related transcription factor 2 (Runx-2), osteocalcin, and osterix were evaluated by real-time polymerase chain reaction at 4 days and 7 days. Results: At 1, 4, and 7 days after loading the DBM gel alone and the DBM gel with BMP-2, cellular proliferation on the zirconia and titanium discs was similar and that of the groups cultured with the DBM gel alone and the DBM gel with BMP-2 was not significantly different, except for titanium with BMP-2 gel. ALPase activity was higher in the cells cultured with BMP-2 than in the other groups, but there was no difference between the zirconia and titanium. In ALPase, bone sialoprotein, osteocalcin, Runx-2 and osterix gene expression, that of cells on zirconia or titanium with BMP-2 gel was much more highly increased than titanium without gel at day 7. The gene expression level of cells cultured on zirconia with BMP-2 was higher than that on titanium with BMP-2 at day 7. Conclusions: The data in this study demonstrate that the osteoblastic cell attachment and proliferation of zirconia were comparable to those of titanium. With the stimulation of BMP-2, zirconia has a more pronounced effect on the proliferation and differentiation of the osteoblastic cells compared with titanium.

• Journal of Korean Medical Science
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• v.33 no.47
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• pp.303.1-303.10
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• 2018

Background: Cell division cycle 6 (CDC6) is an essential regulator of DNA replication and plays important roles in the activation and maintenance of the checkpoint mechanisms in the cell cycle. CDC6 has been associated with oncogenic activities in human cancers; however, the clinical significance of CDC6 in prostate cancer (PCa) remains unclear. Therefore, we investigated whether the CDC6 mRNA expression level is a diagnostic and prognostic marker in PCa. Methods: The study subjects included 121 PCa patients and 66 age-matched benign prostatic hyperplasia (BPH) patients. CDC6 expression was evaluated using real-time polymerase chain reaction and immunohistochemical (IH) staining, and then compared according to the clinicopathological characteristics of PCa. Results: CDC6 mRNA expression was significantly higher in PCa tissues than in BPH control tissues (P = 0.005). In addition, CDC6 expression was significantly higher in patients with elevated prostate-specific antigen (PSA) levels (> 20 ng/mL), a high Gleason score, and advanced stage than in those with low PSA levels, a low Gleason score, and earlier stage, respectively. Multivariate logistic regression analysis showed that high expression of CDC6 was significantly associated with advanced stage (${\geq}T3b$) (odds ratio [OR], 3.005; confidence interval [CI], 1.212-7.450; P = 0.018) and metastasis (OR, 4.192; CI, 1.079-16.286; P = 0.038). Intense IH staining for CDC6 was significantly associated with a high Gleason score and advanced tumor stage including lymph node metastasis stage (linear-by-linear association, P = 0.044 and P = 0.003, respectively). Conclusion: CDC6 expression is associated with aggressive clinicopathological characteristics in PCa. CDC6 may be a potential diagnostic and prognostic marker in PCa patients.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• v.4 no.2
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• pp.213-217
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• 2001

Purpose: Glycogen storage disease type Ia (GSD Ia) is an autosomal recessive disorder of glycogen metabolism caused by glucose-6-phosphatase (G6Pase) deficiency. The clinical manifestations of G6Pase deficiency include growth retardation, hepatomegaly, hypoglycemia, lactic acidemia, hyperlipidemia and hyperuricemia. Many mutations of this gene have been found worldwide in various ethnic groups, establishing the molecular basis of GSD Ia. To elucidate a spectrum of the G6Pase gene mutations in Korean, we analyzed mutations in Korean patients with GSD Ia. Methods: Both alleles of 9 unrelated GSD 1a patients were studied by PCR and direct DNA sequencing methods. In all patients, GSD 1a was diagnosed by the enzyme assay for the liver biopsy specimen. Results: In Korean, the most prevalent mutation was g727t substitution in exon 5, which has been reported to cause abnormal mRNA splicing: Sixteen out of 18 alleles were found to have this mutation. In addition, we identified one novel mutation, a c611g, converting a proline to an alanine at codon 178. Conclusion: Our findings suggest that a screening for the g727t mutation by noninvasive molecular method can detect most cases of GSD Ia in Korean patients.

• Journal of Korean Medicine for Obesity Research
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• v.19 no.2
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• pp.106-112
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• 2019

Objectives: The aim of this study was to observe the reduction of lipid accumulation by treatment with Akkermansia muciniphila extract on 3T3-L1 adipocytes. Methods: After treating pasteurized Akk. muciniphila strains in HT-29 colorectal cancer cell, the relative expression of interleukin (IL)-8, tumor necrosis factor-α, IL-6, and IL-1β mRNA was analyzed by real time polymerase chain reaction, respectively. 27 strains of Akk. muciniphila which have anti-inflammatory effects were selected. 3T3-L1 pre-adipocytes were treated with Akk. muciniphila for 24 hr and then measured the toxicity using water soluble tetrazolium salt assay. The cells were incubated for 4 days and then differentiated into adipocytes using the medium including adipogenic reagents for 10 days. The Akk. muciniphila was treated when the medium was exchanged for differentiation medium at 4^th day and insulin medium at 6^th day. To observe the lipid accumulation, the cells were stained with Oil red O dye and were measured using a spectrophotometer. Results: In the cytotoxicity test, the cell viability of 3T3-L1 pre-adipocytes was significantly increased compared to the control group which untreated with Akk. muciniphila, and there was no cytotoxicity of Akk. muciniphila at 1×10⁷ CFU/mL. The results on Oil red O staining and absorbance measurements were showed a significant decrease in lipid accumulation in the group which was treated with Akk. muciniphila compared to the control group. Conclusions: In our results, Akk. muciniphila has the inhibitory effect of lipid accumulation in 3T3-L1 adipocytes. This suggests that Akk. muciniphila could be help to improve obesity.

• The Journal of Korean Society of Virology
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• v.30 no.1
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• pp.71-81
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• 2000

We have determined and analyzed the full-length cDNA sequence of a coxsackievirus B3 (CVB3) Korean isolate (CVB3-Korea/97) which has been known as a general human pathogen. The whole genome contains 7,400 nucleotides and has a single large open reading frame with 6,555 nucleotides that encodes a potential polyprotein precursor of 2,185 amino acids. The genome also contains a 5' non-coding region (NCR) of 741 bases and a 3' NCR of 104 bases followed by poly(A) tail. Sequence homologies of nucleotides and deduced amino acids between the CVB3-Korea/97 strain and the prototype (Nancy strain) were 81.7% and 91.5%, respectively. The genes encoding the functional proteins including viral protease and RNA dependent RNA polymerase showed higher homology than those encoding the structural proteins. We have further analyzed the sequences of 5' NCR, VP1 and VP2 of CVB3-Korea/97, which are known as cardiovirulent determining factors at the nucleotide and amino acid levels. Although the CVB 3-Korea/97 strain was isolated from an aseptic meningitis patient without cardiomyopathy, its 234th nucleotide and 165th amino acid were uracil and Asn as same as those of other cardiovirulent strains one. However, the 155th amino acid of VP1, which closely associated with cardiovirulence, was replaced with $Arg^{155}$ by single nucleotide substitution from $A^{2916}$ to $T^{2916}$. Moreover, additional amino acid substitutions were observed in the flanking region of $Asp^{155}$. Taken together, amino acid(s) substitution in VP1 may playa critical role in determining cardiovirulence of the CVB3-Korea/97 strain rather than individual nucleotide replacements in the 5' NCR and/or an amino acid substitution in VP2.

• Journal of Veterinary Clinics
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• v.32 no.2
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• pp.191-195
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• 2015

A 7-year-old castrated male Korean Shorthair cat was referred with lethargy and anorexia. Laboratory examination revealed moderate degenerative changes of peripheral neutrophils on blood smear examination and decreased levels of free and total thyroxine ($T_4$) as well as bacterial growth on blood culture. Molecular analyses of the 16S ribosomal RNA gene and heat shock protein 60 gene confirmed the bacterium as Enterobacter cloacae. A minimal inhibitory concentration test showed multidrug resistance of the bacterium against 16 antibiotics. Polymerase chain reaction (PCR) and subsequent sequencing specifically for $bla_{TEM}$, $bla_{SHV}$, $bla_{CTX-M}$, and plasmid-mediated ampC genes revealed positive results to $bla_{TEM-1}$, $bla_{CTX-M-15}$, and plasmid-mediated $bla_{ACT-1}$ genes, indicating that the isolated bacterium contains plasmids containing genes encoding extended-spectrum beta-lactamase and plasmid-mediated ampC beta-lactamase. After 1 month of treatment with antibiotics and levothyroxine, the cat's condition improved; both the thyroid function test and the blood culture showed no abnormalities. This is the first report of community-acquired multidrug-resistant E. cloacae-induced euthyroid sick syndrome in a cat. By the prompt diagnostic procedures and properly selected antibiotic therapy, the cat was recovered from the multidrug-resistant bacterium-induced septicaemia.

• Korean Journal of Microbiology
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• v.38 no.2
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• pp.86-95
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• 2002

As a result of genome projects, the research to elucidate the function of a protein of interest has recently been well-recognized. In order to facilitate functional genomics, a useful mammalian gene expression vector is required. Using an infectious CDNA clone of BVDV pNADLclns-, we have developed a mammalian gene expression vector. In this study, a replication-competent full-length infectious CDNA clone containing puremycin acetyltransferase (pac) gene (pNADLclns-/pac) was successfully generated. The viral RNA replication and viral protein NS3 synthesis were examined by detecting metabollically $^{32}P$-labelled genomic viral RNA and immunoblotting with a mouse anti-NS3 antibody. To generate viral replicon as an expression vector, we examine if the viral structural genes (C, E0, El, E2) are required for viral replication by deletion analysis. As a result, all of the structural proteins are dispensable for viral replication per se, but essential for infectious viral particle formation. Based on our deletion analysis, we have generated a replication-competent BVDV viral replicon (pNADLclns-/pac/${\Delta}S$), whose structural genes are all deleted. In addition to NADLclns- /pac/${\Delta}S$, NADLclns-/ luc/${\Delta}S$ viral replicon containing luciferase gene as a reporter was constructed and fecund to be replication-compotent in HeLa and BHK cells as well as MDBK cells. Therefore, BVDV viral replicon developed in our study will be a useful tool to express a protein of interest in various mammalian cells.

• Tuberculosis and Respiratory Diseases
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• v.52 no.2
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• pp.107-116
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• 2002

Background: Matrix metalloproteinases(MMP) are essential enzymes for tumor invasion and metastasis. Among the MMP family, elevated MMP-9 and stromelysin-3(STR-3) expression have been reported to be poor prognostic factors in lung cancer patients. To evaluate the possibility of a molecular diagnosis of lung cancer using peripheral blood, the mRNA expression level of MMP-9 and STR-3 was measured using a reverse transcriptase-polymerase chain reaction (RT-PCR) in patients with lung cancer. Methods : Ninety six patients(44 patients with lung cancer, 19 pulmonary infection, and 33 control) were included. To detect MMP-9 and STR-3 mRNA expression, RT-PCR was performed in peripheral blood mononuclear cells. ELISA was also used to measure the serum level of MMP-9. Results : MMP-9 was expressed more frequently in patients with a pulmonary infection(18/19, 94.7%) compared to lung cancer patients(26/44, 59.1%) or the controls (23/33, 69.7%) (p=0.018). On the other hand, STR-3 expression was observed more frequently in patients with lung cancer(37/44, 84.1%) compared to the lung infection patients(8/19, 42.1%) or control(20/33, 60.6%) (p=0.003). Among the lung cancer patients, MMP-9 was expressed more frequently when a tumor invaded the lymph nodes(17/24, 70.8%) compared to when a tumor did not(3/13, 23.1%) (p=0.005). The MMP-9 and STR-3 expression levels had no relationship with age, sex, tumor size, distant metastasis, or tumor histology. The serum MMP-9 concentration was not higher in lung cancer patients compared to patients with a pulmonary infection or the control subjects. Conclusion : STR-3 may be used as a diagnostic marker in the peripheral blood of lung cancer patients using RT-PCR. Further studies to evaluate the clinical significance of elevated STR-3 expression in lung cancer patients is recommended.