Chicken PRDX3 is required for proliferation of chicken embryo fibroblast cells.

1. This experiment investigated the influence of chicken PRDX3 on cell proliferation in chick embryo fibroblast cells using PRDX3 knockdown technology.2. A methyl thiazolyl tetrazolium (MTT) assay was performed to assess the effect of chPRDX3 knockdown on fibroblast proliferation. The antioxidant effect was investigated to determine if it directly mediated fibroblast cell proliferation.3. To determine the role of chPRDX3 on cell proliferation, an siRNA mediated knockdown was performed in chick fibroblast cells using an in vitro assay. The proliferation of fibroblast cells transfected with siPRDX3 #3 and siPRDX3 Mix was significantly decreased after 48 h (P < 0.01). In addition, the knockdown of chicken PRDX3 suppressed cell proliferation through an increase in oxidative stress.4. The results demonstrated that chPRDX3 is required for cell proliferation in chicken fibroblast cells. Such findings have important implications for the maintenance of chicken fibroblast cells.

GRP78 is required for cell proliferation and protection from apoptosis in chicken embryo fibroblast cells.

Chicken serum has been suggested as a supplement to promote chicken cell proliferation and development. However, the molecular mechanisms by which chicken serum stimulates chicken cell proliferation remain unknown. Here, we evaluated the effects of chicken serum supplementation on chicken embryo fibroblast (CEF) and DF-1 cell proliferation. We also sought to elucidate the molecular pathways involved in mediating the effects of chicken serum on fibroblasts and DF-1 cells by overexpression of chicken 78 kDa glucose-regulated protein (chGRP78), which is important for cell growth and the prevention of apoptosis. Our data demonstrated that the addition of 5% chicken serum significantly enhanced fibroblast proliferation. Moreover, knockdown of chGRP78 using siRNA decreased fibroblast proliferation and increased apoptosis. Based on these results, we suggest that the chGRP78-mediated signaling pathway plays a critical role in chicken serum-stimulated fibroblast survival and anti-apoptosis. Therefore, our findings have important implications for the maintenance of chicken fibroblast cells through the inhibition of apoptosis and may lead to the development of new treatments for avian disease.

Rescue endoscopic bleeding control for nonvariceal upper gastrointestinal hemorrhage using clipping and detachable snaring.

Nonvariceal upper gastrointestinal (UGI) bleeding recurs after appropriate endoscopic therapy in 10 % - 15 % of cases. The mortality rate can be as high as 25 % when bleeding recurs, but there is no consensus about the best modality for endoscopic re-treatment. The aim of this study was to evaluate clipping and detachable snaring (CDS) for rescue endoscopic control of nonvariceal UGI hemorrhage. We report a case series of seven patients from a Korean tertiary center who underwent endoscopic hemostasis using the combined method of detachable snares with hemoclips. The success rate of endoscopic hemostasis with CDS was 86 %: six of the seven patients who had experienced primary endoscopic treatment failure or recurrent bleeding after endoscopic hemostasis were treated successfully. In conclusion, rescue endoscopic bleeding control by means of CDS is an option for controlling nonvariceal UGI bleeding when no other method of endoscopic treatment for recurrent bleeding and primary hemostatic failure is possible.

A comparison of outcomes between concurrent chemoradiotherapy and radiotherapy alone in cancer of the uterine cervix: a single institutional experience.

PURPOSE: To compare failure patterns and evaluate prognostic factors related to survival rates after concurrent chemoradiotherapy (CCRT) or radiotherapy (RT) alone in cervical cancer. MATERIALS AND METHODS: From January 1996 to December 2006, 218 patients with cervical cancer (FIGO Stage IB2 - III) treated with CCRT or RT alone as primary treatments were included, retrospectively. One-hundred eight patients were treated with CCRT and 110 with RT alone. RESULTS: There was no significant difference in failure patterns between the treatment groups, but distant metastasis was the predominant pattern in both groups. The frequent metastatic sites were supraclavicular lymph node, lung, and brain. Treatment group, diabetes, and FIGO Stage were found to be significant for overall survival (OS) and disease-free survival (DFS), and initial hemoglobin level for DFS. CONCLUSION: Distant metastasis is the predominant failure pattern and diabetes is one of the independent prognostic factors to survival rates in this study.

The Association with rhegmatogenous retinal detachment and paediatric atopic dermatitis: a 12-year Nationwide Cohort Study.

PURPOSE: Historically, atopic dermatitis (AD) is associated with an increased risk of rhegmatogenous retinal detachment (RRD). However, uncertainty remained regarding the effect of AD itself and comorbidities (e.g., allergic diseases, cataract surgery) on RRD occurrence in a large, population-based paediatric population. PATIENTS AND METHODS: We analysed the 12-year National Health Insurance Service database (2002-2013) covering the entire Korean population to estimate the association between AD and RRD in people aged under 20 years. RESULTS: We identified 3142 RRD patients, and matched 18,852 controls (six controls to each RRD patient); therefore, we included 21,994 peoples under aged 20 years in the analyses. AD was more prevalent in the RRD group (329 patients, 10.47%) than the control group (1043 patients, 5.53%; P < 0.001), and so were severe AD (153 patients [4.87%] and 223 patients [1.18%], respectively; P < 0.001). In conditional logistic regression analysis, AD was associated with RRD (OR, 1.61; 95% CI, 1.93-1.87) even after adjusting for allergic conditions, connective tissue disease, uveitis, and cataract surgery. In addition, severity of AD was associated with an increased risk of RRD (OR for non-severe AD and severe AD, 1.26 [95% CI, 1.05-1.51] and 2.88 [95% CI, 2.25-3.68]). CONCLUSION: This study suggests that AD itself is a risk factor of RRD in children by showing the association between AD and RRD occurrence and the biologic gradient even after adjustment for known confounders including allergic conditions, uveitis, and cataract surgery.

Effects of dietary recombinant chlorella supplementation on growth performance, meat quality, blood characteristics, excreta microflora, and nutrient digestibility in broilers.

The use of chlorella as an immune stimulant to enhance nonspecific host defense mechanisms or as an antimicrobial to inhibit bacterial growth has been reported. Thus, the aim of the present study was to clarify the effect of recombinant chlorella supplementation on growth performance, meat quality, and the blood profile, excreta microflora, and nutrient digestibility in broilers. A total of 375 one-day-old ROSS 308 broilers (male and female) were allotted to 5 dietary treatments using 5 cages with 15 chicks per cage. Treatments were: 1) NC, basal diet supplemented with 1.0% E. coli fermented liquor (EFL); 2) PC1, 0.2% EFL with chlorella; 3) PC2, 1.0% EFL with chlorella; 4) T1, 0.2% EFL with chlorella (anti-viral); and 5) T2, 1.0% EFL with chlorella (anti-viral). The broilers in the T2 treatment groups showed higher body weight gain (BGW) by 2.55% (P < 0.01) and lower feed conversion ratio (FCR) by 2.75% (P < 0.05) compared with those fed the control NC treatment group. Moreover, the blood contents of blood urea nitrogen (BUN), creatinine, and IgA in the broilers of the T2 treatment group were significantly increased by 28.12, 23.07, and 29.72%, respectively -more than those found in the broilers of the NC treatment group (P < 0.01). In contrast, the LDL/C in the blood from the animals in the T2 treatment group was significantly decreased by 23.23% - more than that in the blood from the NC broilers (P < 0.05). Based on these results, we suggest that the dietary supplementation of broilers with recombinant chlorella could improve their growth performance, increase the concentration of IgA and apparently metabolizable nitrogen in the blood, and decrease ammonia emissions. Therefore, our findings have important implications for the effect of recombinant chlorella supplementation through increasing the concentration of IgA and the level of metabolizable nitrogen.

In vivo effects of s-pantoprazole, polaprenzinc, and probiotic blend on chronic small intestinal injury induced by indomethacin.

Treatment and prevention methods for non-steroidal anti-inflammatory drug-induced enteropathy have not yet been established. We tested the preventive effects of s-pantoprazole sodium trihydrate (PAN), polaprezinc (PZ), and probiotics on an indomethacin (Indo)-induced small intestinal injury in a rat model. Rats were randomised into 6 groups to receive: normal saline (control), Indo (6 mg/kg), PZ plus Indo, PAN plus Indo, or probiotics plus Indo (at 108 and 109 cfu/head) for 2 weeks. We measured body weight, food intake, severity of small intestinal damage, haemoglobin (Hb) levels in the small intestinal fluid, intestinal inflammatory cytokines, and a few groups of faecal bacteria. The experimental groups were found to have the following survival rates: 0% for the Indo, PZ, and PAN groups; 50% for both probiotic groups; and 100% for control. Treatment with probiotics of different concentrations reduced small intestinal lesion scores and intestinal fluid Hb as compared with the Indo group, while these parameters did not reduce in the PZ and PAN groups. The anti-inflammatory marker interleukin 10 increased in both probiotic groups. Analysis of a few groups of faecal bacteria revealed that Indo-induced a significant increase in Gram-negative bacteria and decreases in Bifidobacterium and Lactobacillus. Similar changes were also observed in the PZ and PAN groups. However, opposite effects were found in both probiotic groups. The use of probiotics appeared to be beneficial in preventing Indo-induced chronic small intestinal injury.

Identification of cDNA encoding a serine protease homologous to human complement C1r precursor from grafted mouse skin.

We isolated a cDNA clone from grafted mouse skin that encodes a serine protease homologous to human C1r. The C1r protease is involved in the activation of the first component of the classical pathway in the complement system. In order to identify novel transcripts whose expression is regulated in grafted mouse skin, we first performed differential display reverse transcription polymerase chain reaction analysis and obtained 18 partial cDNA clones whose protein products are likely to play an important role in allograft rejection. One of these showed significant sequence homology with human complement C1r precursor. The other clones displayed no homology to any known sequences, however. Northern blot analysis demonstrated that the level of this transcript was upregulated in day 8 postgrafted skin. The full-length cDNA 2121 nucleotides in length obtained from screening a mouse skin cDNA library contained a single open reading frame encoding 707 amino acid residues with a calculated molecular weight of 80,732 Da. Its deduced amino acid sequence revealed an 81% identity and 89% similarity to the human C1r counterpart. In particular, mouse C1r contained His501, Asp559, and Ser656, which were conserved among this group of serine proteases. This protein was thus designated as mouse C1r. We have expressed a truncated fragment of C1r protein without the N-terminal hydrophobic sequence in Escherichia coli and generated a polyclonal antibody against it. Subsequent immunohistochemical analysis confirmed that mouse C1r was significantly expressed 8 d after the skin graft in both allografted and autografted skins, compared with normal skins. These collective data suggest that a component of the complement system, C1r, might contribute to the graft versus host immune responses in mice.

Differentially expressed genes in cultured aortic smooth muscle cells by cholesterol-loading.

Differentially expressed genes generated by cholesterol-loading in the culture medium of aortic smooth muscle cells (SMC) were screened using the DDRT-PCR technique in order to identify the genes that are possibly involved in the pathogenesis of atherosclerosis in the artery. Twenty-eight genes were initially isolated and three differentially expressed cDNAs were finally selected by Northern blot analysis. All three cDNAs were up-regulated (designated CRGSM-1 through -3) by the cholesterol-loading. Upon nucleotide sequencing and homology search in the databases, the first cDNA (CRGSM-1) had a high homology (97%) with the corresponding segment of the acyl-CoA synthetase II gene from rat brain, which participates in fatty acid synthesis. The second one (CRGSM-2) had a high homology (91%) with a part of Mus musculus (mouse) LIM protein 1, and with human skeletal muscle LIM-protein 1 genes (80%) and the third gene (CRGSM-3) had no significant homology match in the database. A full size cDNA isolated from the cDNA library of rat aortic smooth muscle cell using the CRGSM-2 as a probe was identified to have a high homology with muscle LIM protein (MLP). The isolated cDNA contained a segment of DNA that encodes for a zinc-finger motif and two LIM domains. Proteins bearing the LIM domain, defined as a unique double zinc-finger structure associated with a subclass of proteins involved in the determination of cell identity, cell differentiation and control of cell growth, have previously been suggested to play an important role in the pathogenesis of atherosclerosis by others.

Cytoprotective effect of ß-lapachone by inducing heme oxygenase-1 expression and AMP-activated protein kinase activation in human endothelial cells.

OBJECTIVES: AMP-activated protein kinase (AMPK) is suggested to exert cytoprotective and anti-inflammatory effects in endothelial cells, but the precise mechanisms are not fully understood. It has been reported that pharmacological activation of AMPK induces endothelial heme oxygenase-1 (HO-1) expression. ß-Lapachone (BL), a well-known substrate of NAD(P)H: quinone oxidoreductase (NQO1), stimulates AMPK activation via NQO1 activation. Here we examined whether AMPK activation by BL would be linked to HO-1 expression in ECV304 endothelial cells and whether HO-1 expression could mediate the cytoprotective effect of BL. MATERIALS AND METHODS: Endothelial cells were pre-incubated for 6 h with BL or 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) in the absence or presence of dicoumarol (DC), compound C (CC), or tin protoporphyrin-IX (SnPP), and then challenged with tumor necrosis factor-α (TNF-α) for 24 h. Cell viability was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. AMPK phosphorylation and HO-1 expression were detected by Western blot analysis. RESULTS: At non-cytotoxic concentrations, BL induced AMPK phosphorylation and HO-1 expression. AICAR, an AMPK activator, also induced HO-1 expression. In contrast, CC, an inhibitor of AMPK activation, and DC, an inhibitor of NQO1, prevented the increase in BL-induced HO-1 expression. Pretreatment with BL or AICAR reduced TNF-α-induced endothelial cell death. Cytoprotection by BL was almost completely abolished by CC and DC and partly by SnPP, a competitive inhibitor of HO-1. CONCLUSIONS: Our results suggest that BL induces cytoprotective HO-1 expression in endothelial cells via AMPK activation, providing one of possible mechanisms by which BL can exert beneficial effects.

Differentiation between malignant and benign pathologic fractures with F-18-fluoro-2-deoxy-D-glucose positron emission tomography/computed tomography.

OBJECTIVE: To evaluate the efficacy of F-18-fluoro-2-deoxy-D: -glucose positron emission tomography/computed tomography (FDG PET/CT) in differentiating malignant from benign pathologic fractures. MATERIALS AND METHODS: F-18 FDG PET/CT was performed on 34 patients with pathologic fractures between May 2004 and June 2007. Fractures were located in tubular bones (26), in the pelvis (six), in the spine (one) and in a rib (one). The FDG uptake pattern at the fracture site was described, whether FDG uptake occurred in the marrow or cortex and soft tissue. Maximum standardized uptake values (SUVmax, the largest value at the region of interest) were measured at the fracture site, including cortical bone, bone marrow and soft tissue. As a reference standard, biopsy was used for 12 patients and clinical follow-up for 22 patients. Sensitivity, specificity and diagnostic accuracy of PET/CT were calculated. RESULTS: There were 19 malignant and 15 benign fractures. In the malignant fractures, PET/CT demonstrated high (mean SUVmax 12.0, range 4.3 to 45.7) F-18 FDG uptake in bone marrow in most cases (17 of 19). In benign fractures, there was low FDG uptake (mean SUVmax 2.9, range 0.6 to 5.5) within cortical bone or adjacent soft tissue around the fracture, rarely in the marrow. There were significant differences in the pattern of intramedullary FDG uptake (P < 0.001) and in the mean SUVmax (P < 0.01) between malignant and benign fractures. The sensitivity, specificity and diagnostic accuracy of F-18 FDG PET/CT were 89.5%, 86.7% and 88.2%, respectively, with a cut-off SUVmax set at 4.7. The time interval between fracture and PET/CT did not significantly influence FDG uptake at the fracture site. CONCLUSION: F-18 FDG PET/CT reliably differentiated between malignant and benign fractures based on the SUVmax and based on medullary uptake, which was characteristic for malignant fractures.

Extracellular superoxide dismutase tissue distribution and the patterns of superoxide dismutase mRNA expression following ultraviolet irradiation on mouse skin.

Superoxide dismutases (SODs) are believed to play a crucial role in protecting cells against oxygen toxicity. There are three forms of SOD: cytosolic Cu-Zn SOD, mitochondrial Mn SOD, and extracellular SOD (EC SOD). Extracellular SOD is primarily a tissue enzyme, but the role of EC SOD in skin is unclear. Therefore, this study investigated the distribution of EC SOD in the skin using immunohistochemistry and examining the patterns of EC SOD gene expression following ultraviolet (UV) irradiation in comparison with those of Cu-Zn SOD and Mn SOD in mouse dorsal skin using Northern blot analysis. Immunohistochemical analysis showed that EC SOD was abundantly located in the epidermis as well as in the dermis, but the gene expression of EC SOD mRNA was more abundant in the dermis than in the epidermis. The gene expression levels of all three types of SODs after UV irradiation were induced differently according to the type and UV irradiation dose. The EC SOD mRNA expression level was increased relatively later than that of Cu-Zn SOD and Mn SOD. The EC SOD mRNA level was significantly higher at 6 h and 48 h after UVA irradiation and psoralen plus ultraviolet-A treatment, respectively. Ultraviolet-B irradiation increased the EC SOD mRNA expression level, with maximum at 48 h. These suggest that EC SOD participates in the majority of antioxidant systems in the skin, and it may have different defensive roles from Cu-Zn SOD and Mn SOD against UV-induced injury of the skin.

Supplementation of Areca catechu L. extract alters triglyceride absorption and cholesterol metabolism in rats.

Areca extracts have already been found to exhibit a strong inhibitory activity on cholesterol absorption in high-cholesterol-fed rats. Accordingly, this study was performed to determine whether Areca extracts also exert an inhibitory activity on triglyceride absorption in triglyceride-fed rats. Male rats were fed a diet containing corn oil (10%, w/w) with or without an Areca nut extract supplement (0.5%, w/w). The supplementation of the Areca extract significantly lowered the absorption of triglyceride and the plasma lipid concentration. The absorbed triglyceride that appeared in the blood after an oral dose of [9,10(n)-(3)H] triglyceride was significantly lower in the rats supplemented with the Areca nut extract, compared with the control group. The supplementation also significantly lowered the small intestinal pCEase (pancreatic cholesterol esterase) activity by 22.5% compared to the control group. The hepatic and intestinal ACAT (acyl-CoA:cholesterol acyltransferase) activities were significantly decreased in the Areca group compared with the control group. Hence, further studies are needed to elucidate the structure and chemical properties of the active compound in the water-soluble Areca extract that lowers cholesterol absorption.

Lower absorption of cholesteryl oleate in rats supplemented with Areca catechu L. extract.

Areca catechu L. extracts I and II, prepared using two different solvent systems, exhibited strong inhibitory activities against pancreatic cholesterol esterase (pCEase) in vitro. To determine their cholesterol-lowering effects, these two extracts were investigated by analyzing plasma lipid levels, intestinal enzyme activities, and the absorption of cholesteryl oleate. For 6 days, male rats were fed a diet containing cholesteryl oleate (0.5 g/100 g of body weight) either with or without the Areca nut extract supplements. The supplementation of the two Areca nut extracts significantly lowered the concentrations of plasma cholesterol by 13. 4 and 11.7% and plasma triglycerides by 35.0 and 36.9%, respectively, compared with the pre-experimental values. However, when the cholesteryl oleate diet was fed without any Areca nut extract in high-cholesterol control, the plasma cholesterol and triglyceride concentrations significantly increased by 13.6 and 15.9%, respectively, compared with the pre-experimental values. After 6 days of treatment, the intestinal pCEase activities were significantly lower in the groups supplemented with the Areca nut extracts (37.8 and 26.5%) than in the group with no extract supplement (83.2%). The supplements also significantly elevated the excretion of [1,2(n)-(3)H]cholesteryl oleate administered orally, when determined by the large intestinal contents, 930.5 Bq/day (Areca I) and 1,766.3 Bq/day (Areca II) vs. 98.1 Bq/day (high-cholesteryl oleate (CO) control). The inhibition of pCEase activity with the supplementation of the Areca nut extracts could account for the decrease in [1,2(n)-(3)H]cholesteryl oleate absorption that resulted in decreased radioactivity in blood.

Expression of recombinant human granulocyte macrophage-colony stimulating factor (hGM-CSF) in mouse urine.

We have generated transgenic mice expressing human granulocyte macrophage-colony stimulating factor (hGM-CSF) in urine. In particular, the expression plasmid DNA containing mouse uroplakin II promoter was used to direct uroepithelium-specific transcription of transgene. In this study, hGM-CSF transcript was detected only in bladder uroepithelium as determined by northern blot analysis. Furthermore, hGM-CSF protein was detected in the suprabasal layer of the uroepithelium and ureter by immunohistochemistry. The hGM-CSF was secreted into urine at high level (up to 180 ng/ml), and enhanced proliferation of hGM-CSF-dependent human acute monocyte leukemic cells, suggesting that transgenic urine-derived hGM-CSF was bioactive. This is the first case of demonstrating biological activity of a cytokine produced in the urine of a transgenic animal. Our results demonstrate that bladder can be used as a bioreactor to produce biologically important substances. In addition, it suggests a potential application of bladder expression system to livestock for high-yield production of pharmaceuticals.