Identification and validation of LGR5-binding peptide for molecular imaging of gastric cancer.

Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is a stem cell marker in gastric cancer. In this study, we aimed to produce the LGR5-targeting peptide probe for the use of molecular imaging for gastric cancer. We used phage display libraries to produce a LGR5-specific peptide probe. This peptide was validated for targeting gastric cancer with in vitro and in vivo studies. This peptide was tagged with fluorescein isothiocyanate (FITC) and cyanine 5.5 (Cy5.5). We used two normal and three gastric cancer cell lines. Immunocytochemistry (ICC) and fluorescence-activated cell sorting (FACS) analysis were used to validate the target specificity of the peptide. After three rounds of bio-panning, we found a novel 7-mer peptides, IPQILSI (IPQ∗). FITC-conjugated IPQ∗ showed 2 to 10 times higher fluorescence in gastric cancer cells vs. control cells in ICC. This discrimination was consistently observed using Cy5.5-conjugated IPQ∗ in ICC. FACS analysis showed right shift of peak point in gastric cancers compared to the control cells. In the peritoneal metastasis animal model, we could find Cy5.5-conjugated IPQ∗ accumulated specifically to gastric tumors. In conclusion, IPQ∗ peptide showed a specific probe for gastric cancer diagnosis. This probe can be applied to theragnosis for gastric cancer diagnosis including peritoneal metastasis.

Gelatin-chlorin e6 conjugate for in vivo photodynamic therapy.

BACKGROUND: Improving the water solubility of hydrophobic photosensitizer and increasing its accumulation in tumor tissue are essential for in vivo photodynamic therapy (PDT). Considering commercialization or clinical application in future, it will be promising to achieve these purposes by developing new agents with simple and non-toxic structure. RESULTS: We conjugated multiple chlorin e6 (Ce6) molecules to gelatin polymer, synthesizing two types of gelatin-Ce6 conjugates with different amounts of Ce6: gelatin-Ce6-2 and gelatin-Ce6-8. The resulting conjugates remained soluble in aqueous solutions for a longer time than hydrophobic Ce6. The conjugates could generate singlet oxygen and kill tumor cells upon laser irradiation. After intravenous injection into SCC-7 tumor-bearing mice, gelatin-Ce6-2 showed prolonged blood circulation and highly increased accumulation in tumor tissue as observed in real-time imaging in vivo. After laser irradiation, gelatin-Ce6-2 suppressed tumor growth completely and enabled improved PDT compared to free Ce6 and gelatin-Ce6-8. CONCLUSIONS: This work demonstrates that a simple structure based on photosensitizer and gelatin can highly improve water solubility and stability. Superior tumor tissue accumulation and increased therapeutic efficacy of gelatin-Ce6 during in vivo PDT showed its high potential for clinical application.

Neuroprotective effects of fermented soybean products (Cheonggukjang) manufactured by mixed culture of Bacillus subtilis MC31 and Lactobacillus sakei 383 on trimethyltin-induced cognitive defects mice.

OBJECTIVES: This study aimed to investigate the beneficial effects of Cheonggukjang (CGK) manufactured by mixed culture of Bacillus subtilis MC31 and Lactobacillus sakei 383 on neurotoxic damages. METHODS: The specific aspects of brain functions were measured in Institute for Cancer Research (ICR) mice that had been pretreated for 4 weeks with three difference doses of CGK before trimethyltin (TMT) treatment. RESULTS: The short- and long-term memory loss induced by TMT treatment was significantly improved in the CGK-pretreated group in a dose-dependent manner. The number of dead cells in the granule cell layer of the dentate gyrus was decreased in the TMT/CGK-cotreated group relative to the TMT/vehicle-treated group, whereas significant suppression of acetylcholinesterase (AChE) activity was observed in the same group. Additionally, a dose-dependent increase in nerve growth factor (NGF) concentration, activation of the NGF receptor signaling pathway including the TrkA high affinity receptor and p75(NTR) low affinity receptor, and decline in Bax/Bcl-2 level was measured in all TMT/CGK-treated groups, although a decrease in the active form of caspase-3 was observed in the TMT/H-CGK-treated group. Furthermore, superoxide dismutase (SOD) activity was enhanced in the TMT/CGK-treated group, whereas the level of malondialdehyde (MDA), a marker of lipid peroxidation, was 43-58% lower in the TMT/CGK-treated group than the TMT/vehicle-treated group. DISCUSSION: These results demonstrate that CGK fermented by mixed culture of B. subtilis and L. sakei could exert a wide range of beneficial activities for neurodegenerative diseases, including Alzheimer, Parkinson, and Huntington disease.

Cellulose film regenerated from Styela clava tunics have biodegradability, toxicity and biocompatibility in the skin of SD rats.

Cellulose is one of the most widespread biomolecules in nature and has been exploited in various applications including scaffolding, tissue engineering, and tissue formation. To evaluate the biocompatibility of cellulose film manufactured from Styela clava tunics (SCT-CF), these films were implanted in Sprague-Dawley (SD) rats for various lengths of time, after which they were subjected to mechanical and biological analyses. The cellulose powders (12-268 m) obtained from SCT was converted into films via casting methods without adding any additives. SCT-CF contained about 98 % α-cellulose and very low concentrations of ßß-cellulose. Additionally, the crystallinity index (CrI) of SCT-CF was lower (10.71 %) than that of wood pulp-cellulose films (WP-CF) (33.78 %). After implantation for 90 days, the weight loss and formation of surface corrugations were greater in SCT-CF than that of WP-CF, while the surface roughness was significantly higher in WP-CF than SCT-CF. However, there were no differences in the number of white blood cells between SCT-CF implanted rats and vehicle implanted rats. The level of metabolic enzymes representing liver and kidney toxicity in the serum of SCT-CF implanted rats was maintained at levels consistent with vehicle implanted rats. Moreover, no significant alteration of the epidermal hyperplasia, inflammatory cell infiltration, redness, and edema were observed in SD rats implanted with SCT-CF. Taken together, these results indicate that SCT-CF showed good degradability and non-toxicity without inducing an immune response in SD rats. Further, the data presented here constitute strong evidence that SCT-CF has the potential for use as a powerful biomaterial for medical applications including stitching fiber, wound dressing, scaffolding, absorbable hemostats and hemodialysis membrane.

Gastric cancer specific drug delivery with hydrophilic peptide probe conjugation.

Cancer-specific diagnosis is challenging. Phage display is an approach that could contribute to finding new specific biomarkers. In this study, we developed a new peptide probe specific for gastric cancer and validated it for gastric cancer-specific theranostics. We isolated linear peptides by screening a combinatorial phage library for a cancer stem cell marker, LGR5 protein. Among these, peptides with high selectivity against gastric cancer cells were selected and examined for therapeutic poteintial in vitro as well as in vivo. Through leucine-rich G protein-coupled receptor 5 (LGR5) protein-based phage display, we obtained a hydrophilic 7-mer peptide sequence (STCTRSR, named STC). Both the STC-peptide-conjugated fluorescent dye and chlorin e6 (Ce6) displayed a significantly higher intensity in gastric cancer cells compared to that in healthy cells. In mice with gastric cancer, the fluorescence in the tumors was 3.4× more intense when treated with the Ce6-STC conjugate compared to that with free Ce6 and conferred higher phototoxicity after single laser irradiation. Repeated photodynamic therapy could further reduce the tumor volume after treating these mice with the Ce6-STC conjugate. The treatment with the Ce6-STC conjugate exhibited a significantly lower fluorescence in the liver than that with free Ce6. In conclusion, we confirmed that the STC peptide is a gastric cancer-specific probe that could be useful in gastric cancer theranostics. In conclusion, considering its targeting ability and hydrophilicity, various hydrophobic chemotherapeutic agents could be revisited for gastric cancer treatment in combination with the probe described in this study.

Proteomic analysis of kidneys from selenoprotein M transgenic rats in response to increased bioability of selenium.

BACKGROUND: To characterize changes in global protein expression in kidneys of transgenic rats overexpressing human selenoprotein M (SelM) in response to increased bioabivility of selenium (Sel), total proteins extracted from kidneys of 10-week-old CMV/hSelM Tg and wild-type rats were separated by 2-dimensional gel electrophoresis and measured for changes in expression. RESULTS: Ten and three proteins showing high antioxidant enzymatic activity were up- and down-regulated, respectively, in SelM-overexpressing CMV/hSelM Tg rats compared to controls based on an arbitrary 2-fold difference. Up-regulated proteins included LAP3, BAIAP2L1, CRP2, CD73 antigen, PDGF D, KIAA143 homolog, PRPPS-AP2, ZFP313, HSP-60, and N-WASP, whereas down-regulated proteins included ALKDH3, rMCP-3, and STC-1. After Sel treatment, five of the up-regulated proteins were significantly increased in expression in wild-type rats, whereas there were no changes in CMV/hSelM Tg rats. Only two of the down-regulated proteins showed reduced expression in wild-type and Tg rats after Sel treatment. CONCLUSIONS: These results show the primary novel biological evidences that new functional protein groups and individual proteins in kidneys of Tg rats relate to Sel biology including the response to Sel treatment and SelM expression.

Aqueous extracts of Liriope platyphylla induced significant laxative effects on loperamide-induced constipation of SD rats.

BACKGROUND: Liriope platyphylla has long been reported as a therapeutic drug for treatment of various human chronic diseases including inflammation, diabetes, neurodegenerative disorders, obesity, and atopic dermatitis. To investigate the laxative effects of L. platyphylla, alterations in excretion parameters, histological structure, mucin secretion, and related protein levels were investigated in rats with loperamide (Lop)-induced constipation after treatment with aqueous extract of L. platyphylla (AEtLP). METHODS: Alterations on constipation phenotypes were measured in rats with Lop-induced constipation after treatment with AEtLP using excretion parameter analysis, histological analysis, RT-PCR, western blot and transmission electron microscope (TEM) analysis. RESULTS: The amounts of stool and urine excretion were significantly higher in the Lop + AEtLP-treated group than in the Lop + vehicle-treated group, whereas food intake and water consumption were maintained at constant levels. AEtLP treatment also induced an increase in villus length, crypt layer, and muscle thickness in the constipation model. Total mucin secretion was higher in the Lop + AEtLP-treated group than in the Lop + vehicle-treated group, although mucin secretion per crypt was very similar among all groups. Furthermore, RT-PCR and western blot revealed a dramatic reduction of key factors level on the muscarinic acetylcholine receptors (mAChRs) signaling pathway in the Lop + AEtLP-treated group relative to the Lop + vehicle-treated group. Especially, the accumulation of lipid droplets in enterocytes of crypts following Lop treatment was improved to the level of the No-treated group in response to AEtLP treatment. CONCLUSION: These results suggest that AEtLP improves constipation induced by Lop treatment through an increase in crypt layer and stimulation of lipid droplet secretions. These data are the first to show that the laxative effects of AEtLP are closely related to the down-regulation of mAchRs and their downstream signals.

Duodenal mucosal resurfacing with photodynamic therapy using methylene blue in a mouse model.

BACKGROUND: The duodenum has emerged as a key player in metabolic diseases. The objective was to evaluate the safety and efficacy of intra-duodenal PDT using methylene blue in managing glycemic control and weight reduction. METHODS: Optimal concentration of methylene blue and conditions for intra-duodenal PDT were determined through in vitro experiments. After injecting methylene blue into the duodenum, we performed intra-duodenal PDT. High-fat diet rats were used to assess the efficacy of intra-duodenal PDT through measures of oral glucose tolerance, insulin sensitivity, and weight change. Immunohistochemical staining was also conducted to examine GLP-1 and GIP-producing cells in the ileum and duodenum, respectively. RESULTS: Introduodenal PDT reduced villous height of duodenum at 48 h, which was fully recovered at 30 days without complications. Rats treated with PDT showed significantly lower blood glucose levels with glucose loading and improved insulin sensitivity than rats in the sham-treatment group. The PDT group also had a significant reduction in body weight compared to the sham-treatment group at 30 days after intervention, although food intake was not significantly different between the two groups. Numbers of GLP-1 and GIP producing cells in the ileum and irradiated area were significantly higher in the PDT group than in the sham-treatment group. CONCLUSIONS: Intra-duodenal PDT using methylene blue showed a feasible therapeutic modality in improving metabolic parameters. However, large animal experiments and mechanism studies are needed to determine the clinical relevance. The possibility of repeating this treatment every 30 days and its accompanying complications should be further studied.

A Dodecapeptide Selected by Phage Display as a Potential Theranostic Probe for Colon Cancers.

AIM: Colon cancer is one of the leading causes of cancer-related mortality. However, specific biomarkers for its diagnosis or treatment are not established well. METHODS: We developed a colon-cancer specific peptide probe using phage display libraries. We validated the specificity of this probe to colon cancer cells with immunohistochemical staining and FACS analysis using one normal cell and five colon cancer cell lines. RESULTS: This peptide probe maintained binding affinity even after serum incubation. For therapeutic applications, this peptide probe was conjugated to hematoporphyrin, a photosensitizer, which showed a significantly enhanced cellular uptake and high photodynamic effect to kill tumor cells. As another application, we made a nanoparticle modified from the peptide probe. It efficiently delivered SN-38, an anticancer drug, into tumor cells, and its tumor-targeting ability was observed in vivo after intravenous injection to the same xenograft model. CONCLUSION: The noble dodecapeptide probe can be a promising candidate for both colon tumor diagnosis and targeted drug delivery.

Hydrogen Peroxide Enhances the Antibacterial Effect of Methylene Blue-based Photodynamic Therapy on Biofilm-forming Bacteria.

Recently, increased attention has been focused on endoscopic disinfection after outbreaks of drug-resistant infections associated with gastrointestinal endoscopy. The aims of this study were to investigate the bactericidal efficacy of methylene blue (MB)-based photodynamic therapy (PDT) on Pseudomonas aeruginosa (P. aeruginosa), which is the major cause of drug-resistant postendoscopy outbreak, and to assess the synergistic effects of hydrogen peroxide addition to MB-based PDT on biofilms. In planktonic state of P. aeruginosa, the maximum decrease was 3 log10 and 5.5 log10 at 20 and 30 J cm-2 , respectively, following MB-based PDT. However, the maximum reduction of colony forming unit (CFU) was decreased by 2.5 log10 and 3 log10 irradiation on biofilms. The biofilm formation was significantly inhibited upon irradiation with MB-based PDT. When the biofilm state of P. aeruginosa was treated with MB-based PDT with hydrogen peroxide, the CFU was significantly decreased by 6 log10 after 20 J cm-2 , by 7 log10 after 30 J cm-2 irradiation, suggesting significantly higher efficacy than MB-based PDT alone. The implementation of the combination of hydrogen peroxide with MB-based PDT through working channels might be appropriate for preventing early colonization and biofilm formation in the endoscope and postendoscopy outbreak.

[Development of a Pancreatic Cancer Specific Binding Peptide Using Phage Display].

BACKGROUND/AIMS: Pancreatic cancer has a very poor prognosis, and early diagnosis is a way to increase the survival rate of patients. The purpose of this study was to develop pancreatic cancer-specific peptides for imaging studies. METHODS: Three pancreatic cancer cell lines, MIA PaCa-2, UACC-462, and BxPC-3, and a control cell line, CCD841, were used. Biopannings were performed on MIA PaCa-2 using a phage display library. After this, the peptides were synthesized and labeled with fluorescein isothiocyanate (FITC). Immunocytochemistry (ICC), enzyme-linked immunosorbent assay (ELISA), and fluorescence- activated cell sorter (FACS) were performed to examine the specific binding. To examine its therapeutic applications, a photosensitizer, chlorin e6 (Ce6), was conjugated on the peptide and photodynamic therapy was performed. Cell survival was investigated using a [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay. RESULTS: After three biopannings, the phages were amplified from 1.4×104 to 3.2×105 plaque-forming units. The most strongly binding phage was selected from the ELISA and ICC results. FITC-labeled peptide, M5, in the three pancreatic cancer cell lines showed significantly higher immunofluorescence in the ICC experiments than that of CCD841. The higher binding ability to MIA PaCa-2 cells was confirmed from FACS analysis, which showed a right shift compared to CCD841. M5 bound to Ce6 showed a significantly lower cell survival rate than that of Ce6 alone in photodynamic therapy, which was observed consistently as a change in the tumor size and fluorescence intensity in MIA PaCa-2 cell-implanted animal models. CONCLUSIONS: This study showed that the noble peptide, M5, binds specifically to the pancreatic cancer cell line, MIA PaCa-2. The M5 peptide has potential use in future optical diagnostic and therapeutic purposes.

Accelerated healing of cutaneous wounds using phytochemically stabilized gold nanoparticle deposited hydrocolloid membranes.

Rapid healing of dermatological wounds is of vital importance in preventing infection and reducing post-treatment side-effects. Here we report the therapeutic effects of phytochemically stabilized gold nanoparticles (pAuNPs) coated on a hydrocolloid membrane (HCM) for curing cutaneous wounds. Furthermore, the remedial effects of pAuNPs on skin regeneration and angiogenesis were examined using Sprague Dawley® (SD) rats with skin injuries after a pAuNP-deposited hydrocolloid membrane (pAuNP-HCM) had been applied for 15 days. The rate of wound closure was 4 times faster in the pAuNP-HCM-treated group than in the gauze (GZ)- or HCM-treated groups in the first 5 days. Moreover, wound widths in the pAuNP-HCM-treated group were significantly reduced after 5-15 days of treatment following the injury, compared with the other groups. In addition, a significant increase in collagen expression and a decrease in matrix metalloproteinase (MMP)-1 expression and transforming growth factor (TGF-ß1) concentration were observed in the pAuNP-HCM-treated group on day 5. Wound tissue applied with the pAuNP-HCM showed enhancement of vascular endothelial growth factor (VEGF), angiopoietin 1 (Ang-1), and angiopoietin 2 (Ang-2) expression. Furthermore, the activity of superoxide dismutases (SODs) was significantly increased in the skin tissue of the pAuNP-HCM-treated group, compared with the GZ- or HCM-treated groups. It is probable that the accelerated process of wound healing in the injured skin of SD rats via pAuNP-HCM results from the synergistic regulation of angiogenesis and connective tissue formation, as well as the stimulation of antioxidant effects.

Characterization of Changes in Global Genes Expression in the Distal Colon of Loperamide-Induced Constipation SD Rats in Response to the Laxative Effects of Liriope platyphylla.

To characterize the changes in global gene expression in the distal colon of constipated SD rats in response to the laxative effects of aqueous extracts of Liriope platyphylla (AEtLP), including isoflavone, saponin, oligosaccharide, succinic acid and hydroxyproline, the total RNA extracted from the distal colon of AEtLP-treated constipation rats was hybridized to oligonucleotide microarrays. The AEtLP treated rats showed an increase in the number of stools, mucosa thickness, flat luminal surface thickness, mucin secretion, and crypt number. Overall, compared to the controls, 581 genes were up-regulated and 216 genes were down-regulated by the constipation induced by loperamide in the constipated rats. After the AEtLP treatment, 67 genes were up-regulated and 421 genes were down-regulated. Among the transcripts up-regulated by constipation, 89 were significantly down-regulated and 22 were recovered to the normal levels by the AEtLP treatment. The major genes in the down-regulated categories included Slc9a5, klk10, Fgf15, and Alpi, whereas the major genes in the recovered categories were Cyp2b2, Ace, G6pc, and Setbp1. On the other hand, after the AEtLP treatment, ten of these genes down-regulated by constipation were up-regulated significantly and five were recovered to the normal levels. The major genes in the up-regulated categories included Serpina3n, Lcn2 and Slc5a8, whereas the major genes in the recovered categories were Tmem45a, Rerg and Rgc32. These results indicate that several gene functional groups and individual genes as constipation biomarkers respond to an AEtLP treatment in constipated model rats.

Bacterial cellulose membrane produced by Acetobacter sp. A10 for burn wound dressing applications.

Bacteria cellulose membranes (BCM) are used for wound dressings, bone grafts, tissue engineering, artificial vessels, and dental implants because of their high tensile strength, crystallinity and water holding ability. In this study, the effects of BCM application for 15 days on healing of burn wounds were investigated based on evaluation of skin regeneration and angiogenesis in burn injury skin of Sprague-Dawley (SD) rats. BCM showed a randomly organized fibrils network, 12.13 MPa tensile strength, 12.53% strain, 17.63% crystallinity, 90.2% gel fraction and 112.14 g × m(2)/h highest water vapor transmission rate (WVTR) although their swelling ratio was enhanced to 350% within 24h. In SD rats with burned skin, the skin severity score was lower in the BCM treated group than the gauze (GZ) group at all time points, while the epidermis and dermis thickness and number of blood vessels was greater in the BCM treated group. Furthermore, a significant decrease in the number of infiltrated mast cells and in vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) expression was observed in the BCM treated group at day 10 and 15. Moreover, a significant high level in collagen expression was observed in the BCM treated group at day 5 compared with GZ treated group, while low level was detected in the same group at day 10 and 15. However, the level of metabolic enzymes representing liver and kidney toxicity in the serum of BCM treated rats was maintained at levels consistent with GZ treated rats. Overall, BCM may accelerate the process of wound healing in burn injury skin of SD rats through regulation of angiogenesis and connective tissue formation as well as not induce any specific toxicity against the liver and kidney.

Quantitative evaluation of the therapeutic effect of fermented soybean products containing a high concentration of GABA on phthalic anhydride-induced atopic dermatitis in IL-4/Luc/CNS-1 Tg mice.

Cheonggukjang (CKJ) is a fermented soybean product that exhibits diverse biological and pharmacological activities, including anti-obesity, anti-diabetic, and anti-inflammatory effects on human chronic diseases. In this study, the effects of the aqueous extract of CKJ containing a high concentration of GABA on atopic dermatitis (AD) were quantified using the luciferase reporter system in IL-4/Luc/CNS-1 transgenic (Tg) mice. Alterations of the luciferase signal and phenotypes of AD were quantified in the IL-4/Luc/CNS-1 Tg mice co-treated with phthalic anhydride (PA) and CKJ for 4 weeks using the IVIS imaging system. A strong luciferase signal was detected in the abdominal region of IL-4/Luc/CNS-1 Tg mice treated with PA alone. However, this signal was significantly reduced in IL-4/Luc/CNS-1 Tg mice co-treated with PA and CKJ. The thymus showed the greatest decrease in luciferase following CKJ treatment, but the level increased after PA treatment. Furthermore, the CKJ-treated group showed improvement of common allergic responses including decreased ear thickness, dermis thickness, auricular lymph node (ALN) weight and infiltrating mast cells. However, IgE concentration and epidermis thickness were maintained a constant level. These results indicated that the luciferase signal may successfully reflect the therapeutic effects of CKJ in IL-4/Luc/CNS-1 Tg mice. The results also suggested that CKJ may be considered an effective substance for the treatment of AD.

Fermented soybean product (Cheonggukjang) improved some attributes of protein and growth hormone measurements in Sprague-Dawley rats.

We hypothesized that the administration of Cheonggukjang (CKJ) would exert positive effects on factors implicated with growth in Sprague-Dawley (SD) rats. To test this hypothesis, we measured specific aspects of bone and organ growth in male SD rats that were treated for 6 weeks with 3 concentrations of CKJ. Although the CKJ extract contained high concentrations of flavonoids and phenolic compounds, no significant differences in body length, organ weights, or femur weight were detected between the CKJ- and vehicle-treated groups. However, thicknesses of the epiphyseal growth plate in the proximal femoral epiphysis and the compact bone in the linea aspera were broadest in the femur of the 2 CKJ-treated groups when compared with the vehicle-treated groups. Furthermore, the levels of growth hormone (GH) and calcium ion were higher in the sera of the high-concentration CKJ-treated groups, whereas the expression level of GH receptor was higher in muscle tissue of all CKJ-treated groups and in the liver tissue of the high-concentration CKJ-treated group. In the GH receptor downstream signaling pathway, the phosphorylation levels of Akt and Erk were expressed differently between liver and muscle tissues upon CKJ treatment. However, the phosphorylation level of STAT5 was very similar to the expression level of the GH receptor in all CKJ-treated groups. These results indicate that CKJ extract may increase the thickness of the epiphyseal growth plate and the compact bone of the femur, elevate GH secretion, and stimulate regulation of the GH receptor downstream signaling pathway in the liver and muscle tissues of SD rats.

Identification of the responsible proteins for increased selenium bioavailability in the brain of transgenic rats overexpressing selenoprotein M.

The present study was conducted to investigate whether the high antioxidant activity induced by selenium (Sel) treatment and selenoprotein M (SelM) overexpression affected the protein profile of the brain cortex. To accomplish this, the changes in global protein expression were measured in transgenic (Tg) rats expressing human SelM (CMV/hSelM) and non­Tg rats using two-dimensional electrophoresis (2-DE). The results revealed that: ⅰ) CMV/hSelM Tg rats showed a high level of enzyme activity for antioxidant protein in the brain cortex compared to non-Tg rats; ⅱ) the high activity of these enzymes induced a decrease in total antioxidant concentration and γ-secretase activity in CMV/hSelM Tg rats; ⅲ) five proteins were upregulated and three were downregulated by SelM overexpression; ⅳ) among the five upregulated proteins, two associated with creatine kinase B-type (B-CK) and E3 ubiquitin-protein ligase RING1 (RING finger protein 1) were further increased in the two groups following Sel treatment, whereas synaptotagmin-15 (SytXV), eukaryotic translation initiation factor 4H (eIF-4H) and lactate dehydrogenase B (LDH-B) were increased or decreased under the same conditions; ⅴ) the three downregulated proteins did not induce a significant change in expression following Sel treatment; and ⅵ) the protein expression level alterations of the two selected spots (B-CK and SytXV) identified by 2-DE were extremely similar to the results from western blot analysis. Overall, the results of the present study provide primary novel biological evidence that new functional protein groups and individual proteins in the brain cortex of CMV/hSelM Tg rats are associated with Sel biology, including the response to Sel treatment and SelM overexpression.

Selenium treatment significantly inhibits tumor necrosis factor-α-induced cell death and tau hyperphosphorylation in neuroblastoma cells.

The hyperphosphorylation of the protein tau disrupts its normal function on regulating axonal transport and leads to the accumulation of neurofibrillary tangles (NFT), which are involved in the pathogenesis of Alzheimer's disease (AD). This study was performed to investigate whether sodium selenite may inhibit the hyperphosphorylation of tau induced by treatment with tumor necrosis factor­α (TNF­α). For this purpose, we studied the changes in cell viability, tau phosphorylation and activity of tau kinases in TNF­α+selenite-treated neuroblastoma cells. Cell viability was significantly recovered in the group cotreated with TNF­α and 5 µM selenite for 24 h, but not in the groups treated with TNF­α and lower concentrations of selenite. Tau phosphorylation was significantly higher in the group treated with TNF­α+vehicle (instead of selenite) compared to the non­treated group. However, in the TNF­α+selenite­treated group, the total phosphorylation level of tau protein at the Ser404 site was significantly reduced compared to the TNF­α+vehicle group, although western blot analysis revealed one band of increased intensity in the p­tau sample, corresponding to a phosphorylated tau isoform of 65­70 kDa. In addition, sodium selenite treatment led to a significant recovery in the immunofluorescence intensity of the p­tau protein in the cytoplasm and nucleus and in the apoptotic rate of neuroblastoma cells stained with the p­tau antibody and 4',6­diamidino­2­phenylindole (DAPI). The phosphorylation of two protein kinases responsible for phosphorylation of tau, glycogen synthase kinase 3ß (GSK­3ß) and Akt, also known as protein kinase B, was markedly decreased in the TNF­α+selenite­treated group relative to the TNF­α+vehicle­treated group. Overall, these results provide strong evidence that sodium selenite (selenium) can inhibit cell death and tau phosphorylation induced by TNF­α in neuroblastoma cells, through the inhibition GSK­3ß and Akt phosphorylation.

Toxicity of fermented soybean product (cheonggukjang) manufactured by mixed culture of Bacillus subtilis MC31 and Lactobacillus sakei 383 on liver and kidney of ICR mice.

To investigate the toxic effects of cheonggukjang (CKJ) manufactured using mixed cultures of Bacillus subtilis MC31 and Lactobacillus sakei 383 on the liver and kidney of ICR mice, an alteration on the related markers including body weight, organ weight, urine composition, liver pathology and kidney pathology were analyzed after oral administration at dosage of 25, 50 and 100 mg/kg body weight/day of CKJ for 14 days. Any significant toxicity was not observed on the body and organ weight, clinical phenotypes, urine parameters and mortality in the CKJ-treated group compared with the vehicle-treated group. Also, liver toxicity analysis revealed no significant increase in alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) or lactate dehydrogenase (LDH) in response to CKJ. Additionally, the specific pathological features induced by most toxic compounds were not observed upon liver histological analysis. Furthermore, kidney toxicological analysis revealed that blood urea nitrogen (BUN) and the serum creatinine (Cr) levels and pathological features on histological sections did not differ significantly between the vehicle- and CKJ-treated groups. Overall, these results suggest that CKJ does not induce any specific toxicity in liver and kidney organs of ICR at dose of 100 mg/kg body weight/day as no observed adverse effect level (NOAEL).

Metabolomics approach to serum biomarker for loperamide-induced constipation in SD rats.

Loperamide has long been known as an opioid-receptor agonist useful as a drug for treatment of diarrhea resulting from gastroenteritis or inflammatory bowel disease as well as to induce constipation. To determine and characterize putative biomarkers that can predict constipation induced by loperamide treatment, alteration of endogenous metabolites was measured in the serum of Sprague Dawley (SD) rats treated with loperamide for 3 days using (1)H nuclear magnetic resonance ((1)H NMR) spectral data. The amounts and weights of stool and urine excretion were significantly lower in the loperamide-treated group than the No-treated group, while the thickness of the villus, crypt layer, and muscle layer was decreased in the transverse colon of the same group. The concentrations of aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and creatinine (Cr) were also slightly changed in the loperamide-treated group, although most of the serum components were maintained at a constant level. Furthermore, pattern recognition of endogenous metabolites showed completely separate clustering of the serum analysis parameters between the No-treated group and loperamide-treated group. Among 35 endogenous metabolites, four amino acids (alanine, glutamate, glutamine and glycine) and six endogenous metabolites (acetate, glucose, glycerol, lactate, succinate and taurine) were dramatically decreased in loperamide-treated SD rats. These results provide the first data pertaining to metabolic changes in SD rats with loperamide-induced constipation. Additionally, these findings correlate the changes in 10 metabolites with constipation.