Biomedical subjects

Chan-Wha Kim

Publications and source records attributed to Chan-Wha Kim.

At least 19 recordsLinked to original sources

PubMed2006

Human blood plasma preparation for two-dimensional gel electrophoresis.

Human plasma consists of mainly large proteins, which vary in terms of both composition and concentration with the physiological state of the individual. Alterations in protein concentrations reflect the current state of the individual's health and thus may be utilized as valuable biomarkers for a specific biological process or disease. Two-dimensional gel electrophoresis (2-DE) has proven to be a valuable method for the separation and comparison of complex protein mixtures, for example, from disease and healthy states, as this method provides information regarding the variation, relative quantities, and structures of the intact proteins. The procedures utilized for the preparation of samples for 2-DE are critical to the acquisition of high-quality results for the discovery of biomarkers. The objective of this study was to review the preparation methods of plasma for 2-DE, particularly those designed to improve the detection of proteins in low abundance in plasma on 2-DE. The use of anticoagulants and protease inhibitors during the collection of blood, the removal of abundant proteins using multicomponent immunodepletion system, and desalting procedure allow us to compile profiles of proteins occurring in low concentrations in the plasma and to improve the pattern generated during 2-DE.

Biomarkers↗

PubMed2006

Proteome analysis of Halobacterium salinarum and characterization of proteins related to the degradation of isopropyl alcohol.

We reported in a previous study that proteomic approach, coupled with genomic techniques, could be used to screen and develop multiple candidates for halophilic enzymes from Halobacterium salinarum. In order to evaluate the biodegradation of isopropyl alcohol (IPA) by H. salinarum, the amounts of residual IPA and acetone generated in the growth media were determined using a gas chromatography-flame ionization detector (GC-FID). The protein expression profiles of cells which had been cultured with IPA were obtained with the two-dimensional gel electrophoresis. Proteins evidencing different expression levels in the presence of 0.5% IPA were identified by electrospray ionization-quadruple-time of flight (ESI-Q-TOF) mass spectrometry. We found 12 proteins which were down-regulated, and another 12 proteins which were up-regulated, in the presence of 0.5% IPA and we further identified 17 proteins among them using ESI-TOF MS/MS. Among these identified proteins, we selected glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for further characterization as a halophilic enzyme. We have demonstrated for the first time that H. salinarum possesses the ability to degrade IPA and GAPDH was both stable and active at high salt concentrations, with maximum activity occurring at 1 M NaCl, although the optimal salt concentration with regard to the growth of H. salinarum is 4.3 M.

2-Propanol↗

PubMed2006

The proteomic analysis of an adipocyte differentiated from human mesenchymal stem cells using two-dimensional gel electrophoresis.

Adipose tissues play a crucial endocrine role in the control of whole body glucose homeostasis and insulin sensitivity. Considering the current substantial rise in obesity and obesity-related diseases, including diabetes, it is important to understand the molecular basis of adipocyte differentiation and its control. In this study, we have analyzed the protein expression inherent to adipogenic differentiation, by 2-DE, MALDI-TOF, and RT-PCR. This study focused on proteins that were differentially expressed by the differentiation of human mesenchymal stem cells (hMSCs) to adipocytes. We conducted 2-DE for each set of proteins in the cytosol of adipocytes that had differentiated from hMSC, in a pH range from 3-10. Thirty-two protein spots were shown to have different expression levels. Among these, eight up-regulated proteins were identified by MALDI-TOF/MS, as the following: syntaxin binding protein 3, OSBP-related protein 3, phosphodiesterase, glycophorin, immunoglobulin kappa chain variable region, peroxisome proliferative activated receptor gamma (PPAR-gamma), bA528A10.3.1 (novel protein similar to KIAA01616, isoform 1), and T cell receptor V-beta 4. Four proteins: syntaxin-3, OSBP-related protein 3, PPAR-gamma and glycophorin were associated with adipogenesis.

Adipocytes↗

PubMed2006

Halophile aldehyde dehydrogenase from Halobacterium salinarum.

Halobacterium salinarum is a member of the halophilic archaea. In the present study, H. salinarum was cultured at various NaCl concentrations (3.5, 4.3, and 6.0 M NaCl), and its proteome was determined and identificated via proteomics technique. We detected 14 proteins which were significantly down-regulated in 3.5 M and/or 6 M NaCl. Among the identified protein spots, aldehyde dehydrogenase (ALDH) was selected for evaluation with regard to its potential applications in industry. The most effective metabolism function exhibited by ALDH is the oxidation of aldehydes to carboxylic acids. The ALDH gene from H. salinarum (1.5 kb fragment) was amplified by PCR and cloned into the E. coli strain, BL21 (DE3), with the pGEX-KG vector. We subsequently analyzed the enzyme activity of the recombinant ALDH (54 kDa) at a variety of salt concentrations. The purified recombinant ALDH from H. salinarum exhibited the most pronounced activity at 1 M NaCl. Therefore, the ALDH from H.salinarum is a halophilic enzyme, and may prove useful for applications in hypersaline environments.

Aldehyde Dehydrogenase↗

PubMed2006

Enhanced hypoglycemic activity following intratracheal administration of insulin microcrystal suspension with injection adjuvant.

The pulmonary route appears to be the most attractive alternative for non-invasive systemic delivery of insulin. We have shown the feasibility of insulin microcrystals as a long-acting formulation for pulmonary delivery. In this study, we examined the effects of adjuvant for pulmonary formulations of insulin, such as protamine, zinc, and glycerol. In an in vivo experiment with rats, only zinc enhanced the hypoglycemic effect of insulin microcrystals, with 17% of minimum reductions in blood glucose (%MRBG) and a 44% decrement in the blood glucose level (D%9h).

Adjuvants, Pharmaceutic↗

PubMed2006

Crystallization of antiangiogenic Kringle V derived from human apolipoprotein A: crystallization applied to purification and formulation.

In this study, the Kringle V domain (Glu4225-Ser4310) of human apolipoprotein A, an antiangiogenic polypeptide, was expressed as a secreted form in Pichia pastoris, and was purified via a process consisting of three chromatographic steps. The chromatographically purified kringle V domain contained a C-terminal serine-deleted form and several high-molecular-weight forms, which were suspected to represent glycosylated derivatives. In order to remove these derivatives, we employed a crystallization process. The crystallization of kringle V resulted in an 85% recovery yield, and also resulted in the complete removal of the aforementioned high-molecular-weight forms. However, we were still able to detect a trace of the C-terminal serine-deleted form. The prepared Kringle V crystals were stable within a pH range of 7.0 to 8.0, and were completely dissolved by dilution, which is a crucial factor in the preparation of a highly concentrated formulation. The chromatogram of the crystallized kringle V on reversed-phase HPLC analysis was identical to that observed without crystallization. Also, we noted that the original anti-wound migration activities of the molecule toward human umbilical vein endothelial cells were completely retained.

Angiogenesis Inhibitors↗

PubMed2005

Purification and characterization of a recombinant anti-angiogenic kringle fragment expressed in Escherichia coli: Purification and characterization of a tri-kringle fragment from human apolipoprotein (a) (kringle IV (9)-kringle IV (10)-kringle V).

A kringle fragment (type IV (9)-IV (10)-V) from human apolipoprotein (a) (called LK68) was expressed in an inclusion body in Escherichia coli. The LK68 in this inclusion body was rendered soluble with urea, and efficiently refolded via oxidation in the presence of re-dox couple. The refolded LK68 was then purified via two steps of ion exchange chromatography, concentrated via preparative reversed-phase chromatography, and freeze-dried, at a final yield of approximately 30%. The purified LK68 exhibited profound affinity for lysine and fibrinogen, which suggests the proper folding of the kringle fragment, and also indicates that the native characteristics of apolipoprotein (a) were preserved. The purified LK68 was determined to be highly homogeneous upon reversed-phase HPLC analysis and size-exclusion HPLC analysis, in the presence of 20% (v/v) acetonitrile. However, on size-exclusion HPLC analysis without acetonitrile, it was determined to be somewhat heterogeneous, and this was corroborated by native analyses, including native PAGE and IEF.

Angiogenesis Inhibitors↗

PubMed2005

Proteome analysis of rat pancreas induced by pancreatectomy.

The previous study demonstrated that the streptozotocin (STZ)-induced diabetic mice can be cured by injecting the regenerating pancreatic extract (RPE) of the partially pancreatectomized Wistar-Kyoto rats. In this study, to characterize the complex pattern of protein expression in RPE, the proteins of altered expression level after the pancreatectomy were identified by 2-dimensional electrophoresis (2-DE) and mass spectrometry. Of 76 significantly up- or down-regulated protein spots, 61 were identified by MALDI-TOF/MS. Moreover, the whole RPE was fractionated into 4 groups using an anion-exchange chromatography and each fraction's cell proliferating activity was measured by MTT assay. Compared to the normal pancreatic extract, fraction 3 and 4 of RPE showed the maximal cell proliferating activity. On 2-DE of 3 and 4 fractions, a total of 10 spots, which are differentially expressed after the pancreatectomy, were identified by MS/MS. Of these identified proteins, Reg III which might be functionally associated with well known regenerating factor (Reg I) was found. Taken together, our results demonstrated that the differential protein expression associated with pancreas regeneration could be sought by 2-DE and mass spectroscopy and suggested that the pre-fractionation method combined with in vitro cell proliferation assay is effectively used to pinpoint the active components for pancreas regeneration.

Animals↗

PubMed2005

In vitro trans-differentiation of rat mesenchymal cells into insulin-producing cells by rat pancreatic extract.

Recent reports have suggested that mesenchymal cells derived from bone marrow may differentiate into not only mesenchymal lineage cells but also other lineage cells. There is possibility for insulin-producing cells (IPCs) to be differentiated from mesenchymal cells. We used self-functional repair stimuli of stem cells by partial injury. Rat pancreatic extract (RPE) from the regenerating pancreas (2 days after 60% pancreatectomy) was treated to rat mesenchymal cells. After the treatment of RPE, they made clusters like islet of Langerhans within a week and expressed four pancreatic endocrine hormones; insulin, glucagon, pancreatic polypeptide, and somatostatin. Moreover, IPCs released insulin in response to normal glucose challenge. Here we demonstrate that the treatment of RPE can differentiate rat mesenchymal cells into IPCs which can be a potential source for the therapy of diabetes.

Animals↗

PubMed2005

Proteomics of Halophilic archaea.

Halophilic archaea is a member of the Halobacteriacea family, the only family in the Halobacteriales order. Most Halophilic archaea require 1.5M NaCl both to grow and retain the structural integrity of the cells. The proteins of these organisms have thus been adapted to be active and stable in the hypersaline condition. Consequently, the unique properties of these biocatalysts have resulted in several novel applications in industrial processes. Halophilic archaea are also to be useful for bioremediation of hypersaline environment. Proteome data have expended enormously with the significant advance recently achieved in two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). The whole genome sequencing of Halobacterium species NRC-1 was completed and this would also provide tremendous help to analyze the protein mass data from the similar strain Halobacterium salinarum. Proteomics coupled with genomic databases now has become a basic tool to understand or identify the function of genes and proteins. In addition, the bioinformatics approach will facilitate to predict the function of novel proteins of Halophilic archaea. This review will discuss current proteome study of Halophilic archaea and introduce the efficient procedures for screening, predicting, and confirming the function of novel halophilic enzymes.

Amino Acid Sequence↗

PubMed2005

An efficient proteomics based strategy for the functional characterization of a novel halophilic enzyme from Halobacterium salinarum.

The extremely halophilic archaeon, Halobacterium salinarum grows in environments containing over 25% NaCl. The enzymes of this organism have thus been adapted to be active and stable in hypersaline conditions, which makes them strong candidates as robust industrial enzymes. In this study, the proteomics approach was applied to screen novel halophilic enzymes. We focused initially on proteins that are differentially expressed under different salt concentrations in culture media. After two-dimensional gel electrophoresis over a pH 3.5-4.5 range, 29 differentially expressed protein spots were identified by tandem mass spectrometry and six of these had no similarity to preexisting genes of known function. To predict the function of them, we used various bioinformatic methods. Among other proteins, we selected Vng0487h, which showed a high similarity to acetyltransferases. As a step toward assaying the enzymatic activity of this protein, we cloned the Vng0487h gene of H. salinarum and expressed and purified the recombinant protein with a glutathione-S-transferase (GST) tag in Escherichia coli. Using a GST-pulldown assay, a protein fragment derived from E. coli could interact with recombinant Vng0487h, and was identified to be the ribosomal protein L3. This protein showed high sequence homology with ribosomal protein L7/12 from E. coli and ribosomal protein L13p from H. salinarum. This suggests that Vng0487h acetylates a subunit of ribosomal protein, possibly L13p, in H. salinarum. During the present study, an efficient procedure was established to screen novel halophilic enzymes, and to predict and assess their functions.

Acetyltransferases↗

PubMed2004

Enhancement of radiosensitivity by combined ceramide and dimethylsphingosine treatment in lung cancer cells.

Ceramide generated from sphingomyelin in response to ionizing radiation has been implicated as a second messenger to induce cellular proapoptotic signals. Both ceramide and its metabolic inhibitor, N, N-dimethyl-D-erythro-sphingosine (DMS), might lead to sustained ceramide accumulation in cells more efficiently, thereby sensitizing them to gamma-radiation-induced cell death. To delineate this problem, the clonogenic survival of Lewis lung carcinoma (LLC) cells was evaluated following exposure to radiation together with or without C2-ceramide, DMS, or both. The treatment of ceramide/DMS synergistically decreased the survival of the irradiated cells compared with treatment with ceramide or DMS alone. Ceramide/DMS-treated cells displayed several apoptotic features after gamma-irradiation, including increased sub G(1) population, TUNEL-positive fraction, and poly-(ADP-ribose) polymerase (PARP) cleavage. We also observed ceramide/ DMS induced disruption of mitochondrial membrane potential (MMP) and activation of caspase- 9 and -3 in a radiation-dose-dependent manner. Furthermore, pretreatment of LLC cells with ceramide/DMS not only increased the protein expression level of Bax, but also decreased Bcl-2 after gamma-irradiation. Taken together, the present study indicates that the radiosensitizing activity of ceramide/DMS on LLC cells most likely reflects the dominance of pro-apoptotic signals related to the mitochondria-dependent pathway.

Animals↗

PubMed2004

Human beta-defensin 2 is induced by interleukin-1beta in the corneal epithelial cells.

Mammalian epithelia produce the various antimicrobial peptides against the bacterial or viral infection, thereby acting as the active immune modulators in the innate immunity. In this study, we examined the effects of the various proinflammatory cytokines or LPS on cell viability and antimicrobial beta-defensin gene expressions in human corneal epithelial cells. Results showed that the cytokines or LPS did not exert severe cytotoxic effects on the cells, and that beta-defensin 1 was constitutively expressed, while beta-defensin 2 was specifically induced by IL-1beta, supporting the idea that these cytokines or LPS involve the defense mechanism in the cornea. Furthermore, the reporter and gel shift assay to define the induction mechanism of beta-defensin 2 by IL-1beta demonstrated that the most proximal NF-kappaB site on the promoter region of beta-defensin 2 was not critical for the process. Data obtained from the normal or patients with the varying ocular diseases showed that our in vitro results were relevant in the clinical settings. Our results clearly demonstrated that beta-defensin 1 and 2 are important antimicrobial peptides in the corneal tissues, and that the mechanistic induction process of beta-defensin 2 by IL-1beta is not solely dependent on proximal NF-kappaB site activation, thus suggesting that the long distal portion of the promoter is needed for the full responsiveness toward IL-1beta.

Binding, Competitive↗

PubMed2004

Proteomic analysis of plasma proteins of workers exposed to benzene.

In this study, we analyzed the proteins in plasma of workers exposed to benzene by two-dimensional gel electrophoresis, in the hope of finding a specific protein suitable for the biomonitoring of benzene exposure. Comet assays were also carried out to evaluate lymphocytes DNA damage. Fifty workers from a printing company and 38 matched unexposed healthy subjects were enrolled in the study. DNA damage was found to be significantly higher in the exposed workers than in the controls. The tail moments of the two groups were 2.07 +/- 0.35 and 1.48 +/- 0.41, respectively (P < 0.0001). The mean values of trans, trans-muconic acid (t,t-MA) in workers exposed to benzene and in unexposed subjects were 1.011 +/- 0.249 and 0.026 +/- 0.028 mg/g creatinine, respectively. Protein profiles were significantly different (P < 0.05) in the two groups, as identified by matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) mass spectrometry and confirmed by Western blot. T cell receptor beta chain (TCR beta), FK506-binding protein (FKBP51) and matrix metalloproteinase-13 (MMP13) were found to be up-regulated in the benzene-exposed workers. In addition, the correlation between TCR beta and the tail moments of lymphocytes was statistically significant (r-value, 0.428). We conclude that TCR beta in plasma could be used for the early detection of exposure to benzene.

Adult↗

PubMed2004

Isolation of ethanol-induced genes in pancreatic beta-cells by representational difference analysis (RDA).

Recent epidemiological studies suggest that alcohol consumption is one of the risk factors leading to type 2 diabetes, but the direct effect of ethanol on beta-cell gene expression is not known. Here, using cDNA RDA method, we isolated 43 ethanol-induced genes in pancreatic beta-cells, and confirmed their differential expression by Northern blot or semi-quantitative RT-PCR. These genes were further categorized by the functional criteria based on the published data; Translation, Transcription, Metabolism, Signal transduction, Transport, Structure, Cytoskeleton, Regulation, or Putative/Unknown genes. The effects of each gene on beta-cell function need to be further investigated, however, the present data strongly suggest that these genes might be related to the metabolic alterations caused by ethanol as indicated in earlier study. In particular, RPS3 gene expression was increased by ethanol, glucosamine, and cytokines, implying that ethanol might decrease the metabolic activity by oxidative stress in beta-cells. Therefore, cloning of these genes in full-length and the detailed studies of each gene on beta-cell functions might provide clues on the pathophysiology of type 2 diabetes caused by alcohol.

Alcohol Drinking↗

PubMed2004

Quantitative profiling of plasma peptides in asthmatic mice using liquid chromatography and mass spectrometry.

Asthma is increasing in prevalence worldwide as a result of factors associated with a Western lifestyle. However, simple and reliable diagnostic and prognostic markers are yet to be found. In an attempt to identify protein biomarker profiles among small molecular weight ranges, we employed an approach combining liquid chromatography with mass spectrometry, instead of two-dimensional gel electrophoresis (2-DE), which has previously been used to analyze protein expression patterns. Here we described its application to compare plasma peptides from control and chronic asthma mice. Peptides were quantitatively profiled as a multidimensional peptide mass fingerprint by a combination of reverse-phase high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. They were identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry. In this study, we quantitatively identified the fragment f of complement 3 (C3f), which is important in inflammation. C3f was significantly higher in controls than chronic asthma mice. Our strategy allowed the detection and identification of different plasma peptides between control and chronic asthma mice on a proteomic scale. Therefore, these results suggest that native small peptides detected by non-2-DE techniques may be useful and specific biomarkers of disease.

Animals↗

PubMed2004

Identification and characterization of inosine monophosphate dehydrogenase from Halobacterium salinarum.

Extremely halophilic Archaea, Halobacterium salinarum live in hypersaline habitats and maintain an osmotic balance of their cytoplasm by accumulating high concentrations of salt (mainly KCl). Therefore, their enzymes adapted to high NaCl concentrations offer a multitude of acutal or potential applications such as biocatalysts in the presence of high salt concentrations. In this study, the protein expression profile of H. salinarum cultured under different NaCl concentrations (3.5 M, 4.3 M, and 6.0 M) was investigated using two-dimensional gel electrophoresis (2-DE). As a result of 2-DE, the protein spots concentrated in acidic range at pH 3-10 were separated effectively using pH 3.5-4.5 ultrazoom IPG DryStrips. The proteins which proved to be upregulated or downregulated in 2-DE gel were digested with trypsin and identified with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray ionization quadrupole (ESI-Q) TOF-mass spectrometry. Most proteins were identified as known annotated proteins based on sequence homology and few as unknown hypothetical proteins. Among proteins identified, an enzyme named inosine monophosphate dehydrogenase (IMPDH) was selected based on the possibility of its industrial application. IMPDH gene (1.6 kb fragment) expected to exist in H. salinarum was amplified by polymerase chain reaction (PCR) and expressed in Escherichia coli strain, BL21 (DE3) using a pGEX-KG vector. Recombinant IMPDH purified from H. salinarum has a higher activity in the presence of salt than in the absence of salt.

Base Sequence↗

PubMed2004

Identification of a novel protein D3UPCA from Halobacterium salinarum and prediction of its function.

Halobacterium salinarum is an extremely halophilic archaea, which is able to live in highly saline environments. In a recent study, several halophilic archaea were found to have the ability to biodegrade organic hydrocarbon pollutants, but protein information regarding hydrocarbon degradation and tolerance in halophilic archaea has been relatively rare. In this study, the protein expression profile of H. salinarum cultured under different diesel concentrations (0, 2 and 4%) was investigated by two-dimensional gel electrophoresis. Proteins which increased their expression levels in diesel media were identified using matrix-assisted laser desorption/ionization-time of flight and electrospray ionization-tandem mass spectrometry analysis. Among these, a protein spot (named D3UPCA) which was up-regulated about nine-fold and found to have COG3388, an uncharacterized protein conserved in archaea, was selected in order to further characterize its functions. The D3UPCA coding gene (named d3upca) was cloned and expressed in Escherichia coli, and purified by the glutathione-S-transferase-fusion method. The function of the protein was estimated using various bioinformatics tools and was predicted to be related to the regulation of transcription and/or translation of genes needed to tolerate stresses associated within the presence of diesel oil.

Amino Acid Sequence↗