• Korean Journal of Medicinal Crop Science
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• v.22 no.5
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• pp.398-404
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• 2014

Salt stress is one of the major abiotic stresses affecting the yield of ginseng (Panax ginseng C. A. Meyer). The objective of this study was to identify bio-marker, which is early responsive in salt stress in ginseng, using proteomics approach. Ginseng plants were exposed to 5 ds/m salt concentration and samples were harvested at 0, 6, 12 and 18 hours after exposure. Total proteins were extracted from ginseng leaves treated with salt stress using Mg/NP-40 buffer and were separated on high resolution 2-DE. Approximately $1003{\pm}240$ (0 h), $992{\pm}166$ (6 h), $1051{\pm}51$ (12 h) and $990{\pm}160$ (18 h) spots were detected in colloidal CBB stained 2D maps. Among these, 8 spots were differentially expressed and were identified by using MALDI-TOF/TOF MS or/and LC-MS/MS. Ethylene response sensor-1 (spot GL 1), nucleotide binding protein (spot GL 2), carbonic anhydrase-1 (spot GL 3), thylakoid lumenal 17.9 kDa protein (spot GL 4) and Chlorophyll a/b binding protein (spot GL 5, GL 6) were up-regulated at the 12 and 18 hour, while RuBisCO activase B (spot GL 7) and DNA helicase (spot GL 8) were down-regulated. Thus, we suggest that these proteins might participate in the early response to salt stress in ginseng leaves.

• Journal of Microbiology and Biotechnology
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• v.24 no.7
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• pp.998-1003
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• 2014

Aspartate aminotransferase (AST; E.C. 2.6.1.1), a vitamin B6-dependent enzyme, preferentially promotes the mutual transformation of aspartate and ${\alpha}$-ketoglutarate to oxaloacetate and glutamate. It plays a key role in amino acid metabolism and has been widely recommended as a biomarker of liver and heart damage. Our study aimed to evaluate the extensive preparation of AST and its application in quality control in clinical laboratories. We describe a scheme to express and purify the 6His-AST fusion protein. An optimized sequence coding AST was synthesized and transformed into Escherichia coli BL21 (DE3) strain for protein expression. Ideally, the fusion protein has a volumetric productivity achieving 900 mg/l cultures. After affinity chromatography, the enzyme activity of purified AST reached 150,000 U/L. Commutability assessment between the engineered AST and standard AST from Roche suggested that the engineered AST was the better candidate for the reference material. Moreover, the AST showed high stability during long-term storage at $-20^{\circ}C$. In conclusion, the highly soluble 6His-tagged AST can become a convenient tool for supplying a much better and cheaper standard or reference material for the clinical laboratory.

• Journal of dental hygiene science
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• v.21 no.1
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• pp.45-51
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• 2021

Background: Periodontal disease, also known as gum disease, is a major dental inflammatory disease with a very high prevalence; it is the main cause of tooth loss. Therefore, diagnostic biomarkers that can monitor gum inflammation are important for oral healthcare. Since the gingival crevicular fluid (GCF) adequately reflects changes in the periodontal environment, they have become a target for the development of effective diagnostic biomarkers for periodontitis. In the present study, the level of the target molecules suggested as diagnostic biomarkers for periodontitis were analyzed in GCF samples collected from healthy individuals and periodontitis patients. In addition, useful targets for the diagnosis of periodontitis were evaluated. Methods: GCF samples were collected from healthy individuals and periodontitis patients using absorbent paper points. SDS-PAGE and Coomassie staining were performed for protein analysis. The protein concentrations of GCF specimens were determined using the Bradford method. The levels of the target molecules appropriate for diagnosing periodontal disease were measured by ELISA, according to the manufacturer's protocol. Results: The protein concentration of GCF collected from periodontitis patients was 3.72 fold higher than that in an equal volume of GCF collected from healthy individuals. ELISA analysis showed that the level of interukin-6 (IL-6), IL-8, metalloproteinases 2 (MMP-2), MMP-9, tumor necrosis factor-alpha (TNF-α), azurocidin, and odontogenic ameloblast-associated protein (ODAM) were higher in the GCF samples from the periodontitis patients than in those from the healthy individuals. However, the level of IL-6 and TNF-α were relatively low (> 5 pg/ml). The prostaglandin E2 (PGE2) levels were not significantly different between the two GCF samples. Conclusion: These results indicate that IL-8, MMP-2, MMP-9, azurocidin, and ODAM are potentially useful diagnostic biomarkers for periodontitis; combining multiple biomarkers will improve the diagnostic accuracy of periodontitis.

• Clinical and Experimental Reproductive Medicine
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• v.50 no.3
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• pp.185-191
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• 2023

Objective: Although intracytoplasmic sperm injection (ICSI) is a way to deal with in vitro fertilization failure, 3% of couples still experience repeated fertilization failure after attempted ICSI, despite having sperm within normal parameters. These patients are a challenging group whose sperm cannot fertilize the egg during ICSI. Unfortunately, no test can predict the risk of fertilization failure. Phospholipase C zeta (PLCζ) and transition nuclear proteins (TNPs) are essential factors for chromatin packaging during sperm maturation. This study aimed to assess PLCζ1 and TNP1 expression in the sperm of patients with fertilization failure and the correlations among the DNA fragmentation index, PLCζ1 and TNP1 gene and protein expression, and the risk of fertilization failure. Methods: In this study, 12 infertile couples with low fertilization rates (<25%) and complete failure of fertilization in their prior ICSI cycles despite normal sperm parameters were chosen as the case group. Fifteen individuals who underwent ICSI for the first time served as the control group. After sperm analysis and DNA fragmentation assays, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blot analyses were performed to compare the gene and protein expression of PLCζ and TNP1 in both groups. Results: DNA fragmentation was significantly higher in the fertilization failure group. The qRT-PCR and Western blot results demonstrated significantly lower PLCζ and TNP1 gene and protein expression in these patients than in controls. Conclusion: The present study showed that fertilization failure in normozoospermic men was probably due to deficient DNA packaging and expression of TNP1.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.9
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• pp.4541-4544
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• 2012

Background: Breast cancer is the most common malignancy with the highest incidence rates among women worldwide. Triple-negative breast cancer (TNBC) disease is diagnosed more frequently in younger women, and is associated with a poor prognosis. Elevated levels of serum haptoglobin protein (Hp) are observed in many malignant diseases including breast cancer. We evaluated the expression and prognostic value of Hp among patients with TNBC. Materials and Methods: Serum Hp levels were determined by Elisa in 41 patients with TNBC and 10 normal individuals. Hp status was correlated with other clinico-pathological parameters including patient survival. Results: Of the 41 patients with TNBC, Hp over expression was detected in 24 (59%) by Elisa. Hp up-regulation was confirmed by Elisa based quantification in the serum of 41 TNBC patients against lower grades and 10 normal individuals. Survival analysis revealed that Hp ($p=2.016{\times}10^{-5}$), stage ($p=2.166{\times}10^{-5}$), distant metastasis ($p=2.217{\times}10^{-5}$), tumor size ($p=1.053{\times}10^{-5}$), and tumor grade (p=0.001), correlated with patient survival on univariate analysis. Multivariate analysis revealed that Hp (p=0.001), and grade of the disease (p=0.008) were independent predictors of survival. Conclusion: Our results indicate that serum levels of Hp may play a role as a potential serum biomarker and prognostic indicator among TNBC patients. Thus, Hp may present a new promising prognostic biomarker in TNBC patients, but independent validations are now necessary for confirmation.

• Tuberculosis and Respiratory Diseases
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• v.75 no.3
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• pp.104-110
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• 2013

Background: Osteopontin (OPN) and carbonic anhydrase IX (CAIX), which are expressed on the surface of tumor cells, are associated with hypoxia during tumor development and progression. However, the roles of these proteins in the plasma of patients with non-small cell lung cancer (NSCLC) are poorly understood. Herein, we hypothesized that plasma OPN and CAIX levels could be used as diagnostic and prognostic tumor markers in patients with NSCLC. Methods: Fifty-three patients with NSCLC and 50 healthy control subjects were enrolled. We selected controls without malignancy and matched them with NSCLC patient cases according to age and gender. Blood samples were collected at the time of diagnosis; the plasma levels of OPN and CAIX were measured by enzyme-linked immunosorbent assays. Results: The plasma levels of OPN in the patients with NSCLC were significantly elevated as compared to those in the controls (p=0.016). However, there was no difference in the plasma level of CAIX between the NSCLC patients and controls. NSCLC patients with a distant metastasis had a remarkable increase in plasma OPN compared with patients without metastasis (p=0.026), but no such correlation was found for CAIX. There was no difference in overall survival rates according to the plasma level of OPN between the two groups (by Kaplan-Meier survival analysis). Conclusion: Plasma OPN levels were elevated in patients with NSCLC as compared with the controls, with greater elevation of OPN levels in the advanced stages of disease. Therefore, plasma OPN may have utility as a diagnostic, but not prognostic, biomarker of advanced NSCLC.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.2
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• pp.513-517
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• 2015

Background: Growing evidence suggests that the members of the ubiquitin-proteasome system (UPS) are important for tumorigenesis. HERC4, one component, is a recently identified ubiqutin ligase. However, the expression level and function role of HERC4 in lung cancer remain unknown. Our objective was to investigate any correlation between HERC4 and development of lung cancer and its clinical significance. Materials and Methods: To determine HERC4 expression in lung cancer, an immunohistochemistry analysis of a tissue microarray containing samples of 10 lung normal tissues, 15 pulmonary neuroendocrine carcinomas, 45 squamous epithelial cancers and 50 adenocarcinomas was conducted. Receiver operating characteristic (ROC) curve analysis was applied to obtain a cut-off point of 52.5%, above which the expression of HERC4 was regarded as "positive". Results: On the basis of ROC curve analysis, positive expression of HERC4 was detected in 0/10 (0.0%) of lung normal tissues, in 4/15 (26.7%) of pulmonary neuroendocrine carcinomas, in 13/45 (28.9%) of squamous epithelial cancers and in 19/50 (38.0%) of adenocarcinomas. It showed that lung tumors expressed more HERC4 protein than adjacent normal tissues (${\chi}^2$=4.675, p=0.031). Furthermore, HERC4 positive expression had positive correlation with pT status (${\chi}^2$=44.894, p=0.000), pN status (${\chi}^2$=43.628, p=0.000), histological grade (${\chi}^2$=7.083, p=0.029) and clinical stage (${\chi}^2$=72.484, p=0.000), but not age (${\chi}^2$=0.910, p=0.340). Conclusions: Our analysis suggested that HERC4 is likely to be a diagnostic biomarker for lung cancer.

• Molecules and Cells
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• v.41 no.3
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• pp.179-187
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• 2018

Proteomic analysis of extracellular vesicles (EVs) from biological fluid is a powerful approach to discover potential biomarkers for human diseases including cancers, as EV secreted to biological fluids are originated from the affected tissue. In order to investigate significant molecules related to the pathogenesis of bladder cancer, EVs were isolated from patient urine which was analyzed by mass spectrometry based proteomics. Comparison of the EV proteome to the whole urine proteome demonstrated an increased number of protein identification in EV. Comparative MS analyses of urinary EV from control subjects and bladder cancer patients identified a total of 1,222 proteins. Statistical analyses provided 56 proteins significantly increased in bladder cancer urine, including proteins for which expression levels varied by cancer stage (P-value < 0.05). While urine represents a valuable, non-invasive specimen for biomarker discovery in urologic cancers, there is a high degree of intra- and inter-individual variability in urine samples. The enrichment of urinary EV demonstrated its capability and applicability of providing a focused identification of biologically relevant proteins in urological diseases.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.5
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• pp.2133-2137
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• 2012

Objective: To investigate the prognostic role of antigen KI-67 (Ki-67) and G1/S-specific cyclin-D1 (cyclin-D1) in patients with laryngeal squamous cell carcinoma (LSCC). Methods: Immunohistochemical staining (IHS) was used to determine the protein expression of Ki-67 and cyclin-D1 in LSCC tissues. Kaplan-Meier survival curves was calculated with reference to Ki-67 and cyclin-D1 levels. Results: Cyclin-D1 and Ki67 were expressed in the nuclei of cancer cells. Among the total of 92 cancer tissues examined by immunohistochemistry, 60 (65.22%) had cyclin-D1 overexpression and 56 (60.87%) had Ki67 overexpression. Cyclin-D1 overexpression is associated with the advanced stage of the cancer (P=0.029), but not with gender, age, stage of cancer, histological differentiation, anatomical site, smoking history and alcohol consumption history. Ki67 overexpression is not associated with the advanced stage, gender, age, histological differentiation, anatomical site, smoking history and alcohol consumption history. A statistically significant correlation was found between lymph node status and the expression of Ki67 (p = 0.025). Overexpression of cyclin-D1 was correlated to shorter relapse-free survival period (P<0.001). Conclusions: Overexpression of cyclin-D1 can be used as a marker to predict relapse in patients with LSCC after primary curative resection.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.2
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• pp.829-834
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• 2013

Background: MicroRNAs are noncoding RNA molecules that posttranscriptionally regulate gene expression. The aim of this study was to determine the role of microRNA-21 in cholangiocarcinomas and its relationship to cholangiocarcinoma RBE cell capacity for invasion and metastasis. Methods: MicroRNA-21 expression was investigated in 41 cases of cholangiocarcinoma samples by in situ hybridization and real-time PCR. Influence on cholangiocarcinoma cell line invasion and metastasis was analyzed with microRNA-21 transfected cells. In addition, regulation of reversion-inducing-cysteine-rich protein with kazal motifs (RECK) by microRNA-21 was elucidated to identify mechanisms. Results: In situ hybridization and real-time quantitative PCR results for patients with lymph node metastasis or perineural invasion showed significantly high expression of microRNA-21 (P<0.05). There was a dramatic decrease in cholangiocarcinoma cell line invasion and metastasis ability after microRNA-21 knockdown (P<0.05). However, overexpression significantly increased invasion and metastasis (P<0.05). Real-time PCR and Western-blot analysis showed that microRNA-21 could potentially inhibit RECK expression in RBE cells. Survival analysis showed that patients with higher expression levels of microRNA-21 more often had a poor prognosis (P<0.05). Conclusions: MicroRNA-21 may play an important role in cholangiocarcinoma invasion and metastasis, suggesting that MicroRNA-21 should be further evaluated as a biomarker for predicting cholangiocarcinoma prognosis.