• Journal of Microbiology and Biotechnology
• /
• v.22 no.5
• /
• pp.654-658
• /
• 2012

Osteoarthritis (OA) is the most common rheumatic pathology. One of the major objectives of OA research is the development of early diagnostic strategies such as those using proteomic technology. Synovial fluid (SF) in OA patients is a potential source of biomarkers for OA. The efficient and reliable preparation of SF proteomes is a critical step towards biomarker discovery. In this study, we have optimized a pretreatment method for twodimensional gel electrophoresis (2DE) separation of the SF proteome, by enriching low-abundance proteins and simultaneously removing hyaluronic acid, albumin, and IgG. SF samples pretreated using this optimized method were then evaluated by 1DE and 2DE separation followed by immunodetection of cartilage oligomeric matrix protein (COMP), a known OA biomarker, and by the identification of 3 proteins (apolipoprotein, haptoglobin precursor, and fibrinogen D fragment) that are related to joint diseases.

• Bulletin of the Korean Chemical Society
• /
• v.33 no.11
• /
• pp.3681-3685
• /
• 2012

We developed a simple and effective method for the SERS-based detection of protein-small molecule complexes and label-free proteins using avidin-induced silver aggregation. Upon excitation with light of the appropriate wavelength (633 and 532 nm), the aggregated silver nanoparticles generate a strong electric field that couples with the resonance of the molecules (atto610 and cytochrome c), increasing the characteristic signals of these molecules and resulting in sensitive detection. The detection limit of biotin with the proposed method is as low as 48 ng/mL. The most important aspect of this method is the induction of silver aggregation by a protein (avidin), which makes the silver more biocompatible. This technique is very useful for the detection of protein-small molecule complexes.

• BMB Reports
• /
• v.41 no.3
• /
• pp.194-203
• /
• 2008

Nitrosative modifications regulate cellular signal transduction and pathogenesis of inflammatory responses and neuro-degenerative diseases. Protein tyrosine nitration is a biomarker of oxidative stress and also influences protein structure and function. Recent advances in mass spectrometry have made it possible to identify modified proteins and specific modified amino acid residues. For analysis of nitrated peptides with low yields or only a subset of peptides, affinity 'tags' can be bait for 'fishing out' target analytes from complex mixtures. These tagged peptides are then extracted to a solid phase, followed by mass analysis. In this review, we focus on protein tyrosine modifications caused by nitrosative stresses and proteomic methods for selective enrichment and identification of nitrosative protein modifications.

• Journal of Plant Biotechnology
• /
• v.7 no.2
• /
• pp.97-103
• /
• 2005

This study was carried out to obtain basic information for gene manipulation in potent edible tobacco (Nicotiana tabacum cv. TI 516). N. tabacum cv. TI 516 is a plant for a possible candidate to use as an edible vaccine, since it contains a low level of nicotine. The effective plant regeneration system through leaf disc culture was achieved using a MS basal medium supplemented with 0.1 mg $1^{-1}$ NAA and 0.5 mg $1^{-1}$ BA. In order to transform the N. tabacum cv. TI 516 with the green fluorescent protein (GFP) gene, Agrobacterium tumefaciens LBA 4404 containing the GFP gene was used. Genomic PCR confirmed the integration of the GFP gene into nuclear genome of transgenic plants. Expression of the GFP gene was identified in callus, apical meristem and root tissue of transgenic N. tabacum cv. TI 516 plants using fluorescence microscopy. Western blot analysis revealed the expression of GFP protein in the transgenic edible tobacco plants. The amount of GFP protein detected in the transgenic tobacco plants was approximately 0.16% of the total soluble plant protein (TSP), which was determined by ELISA.

• Animal Bioscience
• /
• v.36 no.3
• /
• pp.451-460
• /
• 2023

Objective: This program aimed to reveal the association of feed intrinsic molecular structure with nutrient supply to animals from canola feedstocks and co-products from bio-oil processing. The special objective of this study was to quantify the relationship between molecular spectral feature and nutrient availability and develop nutrient prediction equation with vibrational molecular structure spectral profiles. Methods: The samples of feedstock (canola oil seeds) and co-products (meals and pellets) from different bio-oil processing plants in Canada (CA) and China (CH) were submitted to this molecular spectroscopic technique and their protein and carbohydrate related molecular spectral features were associated with the nutritional results obtained through the conventional methods of analyses for chemical and nutrient profiles, rumen degradable and intestinal digestible parameters. Results: The results showed that the spectral structural carbohydrates spectral peak area (ca. 1,487.8 to 1,190.8 cm^-1) was the carbohydrate structure that was most significant when related to various carbohydrate parameters of canola meals (p<0.05, r>0.50). And spectral total carbohydrate area (ca. 1,198.5 to 934.3 cm^-1) was most significant when studying the various carbohydrate parameters of canola seeds (p<0.05, r>0.50). The spectral amide structures (ca. 1,721.2 to 1,480.1 cm^-1) were related to a few chemical and nutrient profiles, Cornell Net Carbohydrate and Protein System (CNCPS) fractions, truly absorbable nutrient supply based on the Dutch protein system (DVE/OEB), and NRC systems, and intestinal in vitro protein-related parameters in co-products (canola meals). Besides the spectral amide structures, α-helix height (ca. 1,650.8 to 1,643.1 cm^-1) and β-sheet height (ca. 1,633.4 to 1,625.7 cm^-1), and the ratio between them have shown to be related to many protein-related parameters in feedstock (canola oil seeds). Multi-regression analysis resulted in moderate to high R² values for some protein related equations for feedstock (canola seeds). Protein related equations for canola meals and carbohydrate related equations for canola meals and seeds resulted in weak R² and low p values (p<0.05). Conclusion: In conclusion, the attenuated total reflectance Fourier transform infrared spectroscopy vibrational molecular spectroscopy can be a useful resource to predict carbohydrate and protein-relates nutritional aspects of canola seeds and meals.

• Molecules and Cells
• /
• v.44 no.10
• /
• pp.710-722
• /
• 2021

Hypoxia, or low oxygen tension, is a hallmark of the tumor microenvironment. The hypoxia-inducible factor-1α (HIF-1α) subunit plays a critical role in the adaptive cellular response of hypoxic tumor cells to low oxygen tension by activating gene-expression programs that control cancer cell metabolism, angiogenesis, and therapy resistance. Phosphorylation is involved in the stabilization and regulation of HIF-1α transcriptional activity. HIF-1α is activated by several factors, including the mitogen-activated protein kinase (MAPK) superfamily. MAPK phosphatase 3 (MKP-3) is a cytoplasmic dual-specificity phosphatase specific for extracellular signal-regulated kinase 1/2 (Erk1/2). Recent evidence indicates that hypoxia increases the endogenous levels of both MKP-3 mRNA and protein. However, its role in the response of cells to hypoxia is poorly understood. Herein, we demonstrated that small-interfering RNA (siRNA)-mediated knockdown of MKP-3 enhanced HIF-1α (not HIF-2α) levels. Conversely, MKP-3 overexpression suppressed HIF-1α (not HIF-2α) levels, as well as the expression levels of hypoxia-responsive genes (LDHA, CA9, GLUT-1, and VEGF), in hypoxic colon cancer cells. These findings indicated that MKP-3, induced by HIF-1α in hypoxia, negatively regulates HIF-1α protein levels and hypoxia-responsive genes. However, we also found that long-term hypoxia (>12 h) induced proteasomal degradation of MKP-3 in a lactic acid-dependent manner. Taken together, MKP-3 expression is modulated by the hypoxic conditions prevailing in colon cancer, and plays a role in cellular adaptation to tumor hypoxia and tumor progression. Thus, MKP-3 may serve as a potential therapeutic target for colon cancer treatment.

• International Journal of Industrial Entomology and Biomaterials
• /
• v.48 no.2
• /
• pp.86-97
• /
• 2024

Alkaline- or salt-assisted extractions have been widely used to extract edible insect proteins, however, there is a need for extraction techniques that balance cost-efficient production as well as preserving the protein properties. Mealworm proteins (Tenebrio molitor larvae) were extracted using three different extraction methods-alkali (AMP), salt (SMP), and water (WMP)-and then physicochemical and techno-functional properties were examined. AMP had high yield, protein, and amino acid contents, whereas WMP had high moisture, ash, and fat contents. SDS-PAGE showed a wide range of molecular weights in WMP whereas mostly low molecular weights were observed in AMP and SMP. AMP had poor protein solubilities compared to SMP and WMP across all pHs. AMP had enhanced water-holding capacity and emulsion stability, whereas WMP had improved oil-holding capacity and foaming properties. WMP formed a gel with and without the transglutaminase. The physicochemical and techno-functional properties demonstrated that water-soluble mealworm protein was superior to alkali-and salt-soluble mealworm proteins. Considering the cost efficiency and minimal impact on the environment as well, a cold press juicer could be utilized for mass production of mealworm protein compared to the conventional methods of protein extraction using alkali and salt.

• Preventive Nutrition and Food Science
• /
• v.2 no.3
• /
• pp.219-224
• /
• 1997

The pancreas is an important organ in the maintenance of zine homeostasis. The pancreatic tissue used in this study was obtained from rats fed varying levels of dietary Ca nd phytate followed by intraperitoneal {TEX}${65}^Zn${/TEX} injection. THe objective of this study was to determine the molecular size and distribution of compounds that may represent zinc-binding complexes in pancreatic tissue homogenates. The supernatant of the homogenized pancreatic tissue was separated using a Sephadex G-75 column with Tris buffer at pH 8.1. All subfractions were assayed for zinc, protein and {TEX}${65}^Zn${/TEX} activity. The elution of subfractions from pancreatic tissue homogenates showed a prominent peak corresponding to the high molecular weight protein standard (>66kd). A sall molecular weigth protein (<6.5kd), that was absorbed at 280nm, was also present: prominently in low Ca group, however not much as in high Ca group. These small compounds may combine weakly with zinc in pancreatic tissue an serve as zinc-binding ligands in pancreatic/biliary fluid. In the duodenum, these ligands dissociate zinc into an ionic form which becomes vulnerable to phytate complexation.

• BMB Reports
• /
• v.29 no.5
• /
• pp.436-441
• /
• 1996

The palmitoyl acyl carrier protein (ACP) specific thioesterase (EC 3.1.2.14) from Iris pseudoacorus was purified and characterized. The thioesterase which was very unstable in relatively high salt concentrations was eluted using a co-gradient of Triton X-100 and low concentration of KCl or Na-phosphate from Q-Sepharose, DEAE-Sepharose, and hydroxyapatite chromatography. SDS-PAGE analysis showed a single band with a molecular weight of 35,000. The native molecular weight of approximately 37,000 was estimated by Sephacryl S-200 chromatography, indicating that the enzyme is a monomer. The thioesterase activity was inhibited about 75% and 50% by N-ethylmaleimide (2 mM) and phenylmethylsulfonyl fluoride (2 mM). respectively. The N-ethylmaleimide-inactivation was protected by sodium palmitate but the inactivation with phenylmethylsulfonyl fluoride was not protected. Oxidation of thiols by 2 mM 5.5'-dithio-bis-(2-nitrobenzoic acid) resulted in 65% inactivation of the enzyme. These results suggest that a cysteinyl residue is essential to the catalytic reaction of the enzyme. The enzyme activity was increased by sodium citrate and also by $Cu^{2+}$

• Journal of Photoscience
• /
• v.4 no.3
• /
• pp.133-140
• /
• 1997

A system for studying oxidative hemolysis has been used by controling UV-irradiation and concentration of a novel molecular probe, N,N'-bis(2-hydroperoxy-2-methoxyethyl)-1,4,5,8-naphthalenetetra-carboxylic diimide (NP-III), which generates hydroxyl radical upon longer wavelength photoirradiation (> 350 nm). NP-III induces 25~30% of hemolysis at low concentration (50 $\mu$M) for 3h-irradiation of UVA. The simultaneous treatment of N-ethylmaleimide (NEM) with NP-IH completely hemotyzed erythrocytes under the same conditions as NP-III alone by both decreasing thiol group and increasing lipid peroxidation in erythrocyte membrane. However. thiol-reducing agents prevented the protein-crosslinking and lipid peroxidation on the NEM-synergistic hemolysis by partially scavenging hydroxyl radical and maintaining the thiol group of erythrocyte membrane in the reduced state. In addition, erythrocytes pretreated with 2,2,5,7,8-pentamethyl-6-hydroxychromane (PMC), vitamin E homologue was able to delay and decrease the lipid peroxidation when compared to cells pretreated with both NEM and PMC. We suggest that the presence of reduced thiols in inner membrane protein by GSH can prevent the protein-crosslinking and the lipid peroxidation, and eventually prevent the oxidative hemolysis of erythrocyte.