• Journal of Food Hygiene and Safety
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• v.22 no.4
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• pp.248-256
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• 2007

In this study, the competitive polymerase chain reaction (cPCR) was used to develop a direct enumeration method of Salmonella spp. in pork meat. After comparing three DNA extraction methods, the modified guanidine thiocyanate-phenol-chloroform method was chosen for Salmonella DNA extraction in artificially inoculated pork meat. The previously reported 284-bp invA gene (Rahn et al. Mol. Cell. Probes 1992) was tested for specificity, and 57 Salmonella strains and 24 non-Salmonella strains were evaluated. All Salmonella strains tested were invA positive, and all non-Salmonella strains produced no false positive amplification products. The detection limit achieved was as low as 1,460 colony-forming units (cfu) per 0.1g of pork meat. For cPCR, the invA gene, which features a 82 bp-deletion, was cloned in the pGEM-4Z vector. A known amount of competitor DNA, which has the same primer binding sites, was co-amplified with Salmonella chromosomal DNA from the artificially inoculated pork meat. The cell-number determined by cPCR was approximately equal to the cfu from the most probable number (MPN) method. Finally, the whole procedure took only 5 hr.

• Journal of Microbiology and Biotechnology
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• v.30 no.8
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• pp.1261-1271
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• 2020

Cytochrome c_L (Cytc_L) is an essential protein in the process of methanol oxidation in methylotrophs. It receives an electron from the pyrroloquinoline quinone (PQQ) cofactor of methanol dehydrogenase (MDH) to produce formaldehyde. The direct electron transfer mechanism between Cytc_L and MDH remains unknown due to the lack of structural information. To help gain a better understanding of the mechanism, we determined the first crystal structure of heme c containing Cytc_L from the aquatic methylotrophic bacterium Methylophaga aminisulfidivorans MP^T at 2.13 Å resolution. The crystal structure of Ma-Cytc_L revealed its unique features compared to those of the terrestrial homologues. Apart from Fe in heme, three additional metal ion binding sites for Na⁺, Ca⁺, and Fe²⁺ were found, wherein the ions mostly formed coordination bonds with the amino acid residues on the loop (G93-Y111) that interacts with heme. Therefore, these ions seemed to enhance the stability of heme insertion by increasing the loop's steadiness. The basic N-terminal end, together with helix α4 and loop (G126 to Y136), contributed positive charge to the region. In contrast, the acidic C-terminal end provided a negatively charged surface, yielding several electrostatic contact points with partner proteins for electron transfer. These exceptional features of Ma-Cytc_L, along with the structural information of MDH, led us to hypothesize the need for an adapter protein bridging MDH to Cytc_L within appropriate proximity for electron transfer. With this knowledge in mind, the methanol oxidation complex reconstitution in vitro could be utilized to produce metabolic intermediates at the industry level.

• Fisheries and Aquatic Sciences
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• v.14 no.1
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• pp.43-54
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• 2011

Gene structure of copper/zinc-superoxide dismutase (Cu/Zn-SOD; sod1) was characterized in Hemibarbus mylodon (Teleostei; Cypriniformes), an endangered freshwater fish species in Korean peninsula. Full-length cDNA of H. mylodon SOD1 consisted of a 796-bp open reading frame sequence encoding 154 amino acids, and the deduced polypeptide sequence shared high sequence homology with other orthologs, particularly with regard to metal-coordinating ligands. Genomic structure of the H. mylodon sod1 gene (hmsod1; 1,911 bp from the ATG start codon to the stop codon) was typical quinquepartite (i.e., five exons interrupted by four introns); the lengths of the exons were similar among species belonging to various taxonomic positions. The molecular phylogeny inferred from sod1 genes in the teleost lineage was in accordance with the conventional taxonomic assumptions. 5'-flanking upstream region of hmsod1, obtained using the genome walking method, contained typical TATA and CAAT boxes. It also showed various transcription factor binding motifs that may be potentially involved in stress/immune response (e.g., sites for activating proteins or nuclear factor kappa B) or metabolism of xenobiotic compounds (e.g., xenobiotic response element; XRE). The hmsod1 transcripts were ubiquitously detected among tissues, with the liver and spleen showing the highest and lowest expression, respectively. An experimental challenge with Edwardsiella tarda revealed significant upregulation of the hmsod1 in kidney (4.3-fold) and spleen (3.1-fold), based on a real-time RT-PCR assay. Information on the molecular characteristics of this key antioxidant enzyme gene could be a useful basis for a biomarker-based assay to understand cellular stresses in this endangered fish species.

• Journal of Korean Neurosurgical Society
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• v.49 no.2
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• pp.83-91
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• 2011

Objective : Minocycline, a second-generation tetracycline-class antibiotic, has been well established to exert a neuroprotective effect in animal models and neurodegenerative disease through the inhibition of microglia. Here, we investigated the effects of minocycline on motor recovery and neuropathic pain in a rat model of spinal cord injury. Methods : To simulate spinal cord injury, the rats' spinal cords were hemisected at the 10th thoracic level (T10). Minocycline was injected intraperitoneally, and was administered 30 minutes prior surgery and every second postoperative day until sacrifice 28 days after surgery. Motor recovery was assessed via the Basso-Beattie-Bresnahan test Mechanical hyperalgesia was measured throughout the 28-day post -operative course via the von Frey test Microglial and astrocyte activation was assessed by immunohistochemical staining for ionized calcium binding adaptor molecule 1 (lba1) and glial fibrillary acidic protein (GFAP) at two sites: at the level of hemisection and at the 5th lumbar level (L5). Results : In rats, spinal cord hemisection reduced locomotor function and induced a mechanical hyperalgesia of the ipsilateral hind limb. The expression of lba1 and GFAP was also increased in the dorsal and ventral horns of the spinal cord at the site of hemisection and at the L5 level. Intraperitoneal injection of minocycline facilitated overall motor recovery and attenuated mechanical hyperalgesia. The expression of lba1 and GFAP in the spinal cord was also reduced in rats treated with minocycline. Conclusion : By inhibiting microglia and astrocyte activation, minocycline may facilitate motor recovery and attenuate mechanical hyperalgesia in individuals with spinal cord injuries.

• International Journal of Oral Biology
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• v.30 no.1
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• pp.17-22
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• 2005

In the previous studies of Saccharomyces cerevisiae, Abp140p (actin binding protein 140) fused to GFP has been only a protein that can label actin cables of yeast cells so far. However, the role of Abp140p in actin dynamics was remained elusive. In this study, the function of Abp140p was investigated with a deletion mutant and overexpression of GFP fused Abp140p. The deletion mutant was slightly more susceptible to Latrunculin-A (Lat-A), an actin-monomer sequestering agent, than wild type, although no significant deformation of actin structures was caused by ABP 140 deletion. Overexpression of Abp140p-GFP retarded cell growth, and produced thick and robust actin cables. Lat-A was not able to destabilize the thick actin cables, which suggests that actin dynamics was compromised in the cells with surplus of Abp140p. Therefore, Abp140p seems to stabilize actin cables together with other bundling proteins. Recently, actin cable dynamics of budding yeast was found to have a resemblance to that of filopodial tip of cultured mammalian cells. Retrograde movement of actin cables from buds to mother cells indicated local generation of the cable at bud sites. By using Abp140p-GFP, we traced the steps in the generation of a new actin cable after elimination of old cables by sodium azide. Before the appearance of a new actin cable, Abp140p-GFP concentrated in buds and disappeared, as mother cells became abundant in actin cables. Our observations provide a direct evidence of actin cable formation at buds of budding cells.

• BMB Reports
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• v.45 no.5
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• pp.275-280
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• 2012

Nicotinic acetylcholine receptors (nAChRs) are a diverse family of homo- or heteropentameric ligand-gated ion channels. Understanding the physiological role of each nAChR subtype and the key residues responsible for normal and pathological states is important. ${\alpha}$-Conotoxin neuropeptides are highly selective probes capable of discriminating different subtypes of nAChRs. In this study, we performed homology modeling to generate the neuronal ${\alpha}3$, ${\beta}2$ and ${\beta}4$ subunits using the x-ray structure of the ${\alpha}1$ subunit as a template. The structures of the extracellular domains containing ligand binding sites in the ${\alpha}3{\beta}2$ and ${\alpha}3{\beta}4$ nAChR subtypes were constructed using MD simulations and ligand docking processes in their free and ligand-bound states using ${\alpha}$-conotoxin GIC, which exhibited the highest ${\alpha}3{\beta}2$ vs. ${\alpha}3{\beta}4$ discrimination ratio. The results provide a reasonable structural basis for such a discriminatory ability, supporting the idea that the present strategy can be used for future investigations on nAChR-ligand complexes.

• BMB Reports
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• v.41 no.1
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• pp.29-34
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• 2008

The aim of this study was to determine whether single nucleotide polymorphisms (SNP) in the beef cattle adipocyte fatty-acid binding protein 3 and 4 (FABP3 and FABP4) genes are associated with carcass weight (CW) and back fat thickness (BF) of beef cattle. By direct DNA sequencing in 24 unrelated Korean native cattle, we identified 20 SNPs in FABP3 and FABP4. Among them, 10 polymorphic sites were selected for genotyping in our beef cattle. We performed SNP, haplotype and linkage disequilibrium studies on 419 Korean native cattle with the 10 SNPs in the FABP genes. Statistical analysis revealed that 220A>G (I74V) and 348+303T>C polymorphisms in FABP4 showed putative associations with BF traits (P=0.02 and 0.01, respectively). Our findings suggest that the polymorphisms in FABP4 may play a role in determining one of the important genetic factors that influence BF in beef cattle.

• Proceedings of the Korean Society of Applied Pharmacology
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• 2001.11a
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• pp.94-94
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• 2001

Prostaglandin endoperoxide H synthase (PGHS) catalyzes the committed step in prostaglandins and thromboxane A$_2$-- oxygenation of arachidonic acid to the hydroperoxy endoperoxide PGG$_2$, followed by reduction PGG$_2$to the alcohol PGH$_2$. The two reactions by PGHS -- cyclooxygenase and peroxidase -- occur at distinct but structurally and functionally interconnected sites. The peroxidase reaction occurs at a heme-containing active site located near the protein surface. The cyclooxygenase reaction occurs in a hydrophobic channel in the core of the enzyme. Initially a peroxide reacts with the heme group, yielding Compound I and an alcohol derived from the oxidizing peroxide. Compound I next undergoes an intramolecular reduction by a single electron traveling from Tyr385 along the peptide chain to the proximal heme ligand, His388, and finally to the heme group. Following the binding of arachidonic acid, Tyr385 tyrosyl radical initiates the cyclooxygenase reaction by abstracting the 13-pro(5) hydrogen atom to give an arachidonyl radical, which sequentially reacts with two molecules of oxygen to yield PGG$_2$. In order to characterize PGHS peroxidase active site, we examined various lipid peroxides with purified recombinant ovine PGHS proteins and determined the rate constants. The results have shown that twenty-carbon unsaturated fatty acid hydroperoxides have similar efficiency in peroxidation by PGHS, irrespective of either the location of hydroperoxy group or the number of double bonds. It was also confirmed by the subsequent study with PGHS peroxidase active site mutants.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.10
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• pp.1399-1403
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• 2006

A total of 80 cows, including 40 top mastitis resistant and 40 top mastitis susceptible animals as Group I and Group II, were selected from a population of 520 cows based on clinical mastitis occurrence. PCR-SSCP analysis on four fragments within the 5'region and two fragments of Exons 4,15 of bovine lactoferrin (bLF) revealed that four fragments-P1,P4,E4,E15-had polymorphisms which totally included six base mutations, and only two of them had significant differences in allele frequencies between resistant and susceptible groups, P1 (53.7% vs. 70.0%, p<0.05) and P4 (55.0% vs. 68.8%, p<0.05). Further study on these two promising markers combined with the milk performance traits of cows demonstrated that their selection would result in higher fat percentage (p<0.05), lower Somatic Cell Score (SCS) (p<0.05) and Clinical Mastitis Residuals (CMR) (p<0.01) indicating higher mastitis resistance and lower milk yield (p<0.05). The putative transcription factor binding sites in the 5'region were also studied by using MatInspector 7.2.2 software, and two signal pathways regulating the expression of bLF including the NF-${\kappa}B$ pathway and nuclear hormone receptor pathway were predicted.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.4
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• pp.1783-1789
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• 2014

Background: The EMSY gene encodes a BRCA2-binding partner protein that represses the DNA repair function of BRCA2 in non-hereditary breast cancer. Although amplification of EMSY gene has been proposed to have prognostic value in breast cancer, no data have been available concerning EMSY tissue expression patterns and its associations with clinicopathological features. Materials and Methods: In the current study, we examined the expression and localization pattern of EMSY protein by immunohistochemistry and assessed its prognostic value in a well-characterized series of 116 unselected breast carcinomas with a mean follow up of 47 months using tissue microarray technique. Results: Immunohistochemical expression of EMSY protein was detected in 76% of primary breast tumors, localized in nuclear (18%), cytoplasmic (35%) or both cytoplasmic and nuclear sites (23%). Univariate analysis revealed a significant positive association between EMSY expression and lymph node metastasis (p value=0.045) and larger tumor size (p value=0.027), as well as a non-significant relation with increased risk of recurrence (p value=0.088), whereas no association with patients' survival (log rank test, p value=0.482), tumor grade or type was observed. Conclusions: Herein, we demonstrated for the first time the immunostaining pattern of EMSY protein in breast tumors. Our data imply that EMSY protein may have impact on clinicipathological parameters and could be considered as a potential target for breast cancer treatment.