scholarly journals Expression of Thrombin-Cleaved Osteopontin and Integrin α9 and β1 Signaling Pathway Molecules in Chronic Subdural Hematomas

2021 ◽  
Author(s):  
Koji Osuka ◽  
Yusuke Ohmichi ◽  
Mika Ohmichi ◽  
Chiharu Suzuki ◽  
Masahiro Aoyama ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Western Blot ◽  
Outer Membrane ◽  
Signaling Pathway ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Matrix Protein ◽  
Chronic Subdural Hematoma ◽  
Extracellular Matrix Protein

Abstract Chronic subdural hematoma (CSDH) is considered to be an inflammatory and angiogenic disease. Osteopontin is an extracellular matrix protein. Osteopontin is cleaved by thrombin, resulting in N-half osteopontin, which is more prominent in integrin signal transduction. We examined the expression of N-half osteopontin in the CSDH fluid and the expression of integrin α9 and β1 and the downstream components of the angiogenic signaling pathways in the outer membrane of CSDHs. Twenty samples of CSDH fluid and 8 samples of CSDH outer membrane were included. The concentrations of N-half osteopontin in the CSDH fluid were measured using ELISA kits. The expression of integrin α9 and β1, vinculin, talin-1, focal adhesion kinase (FAK), paxillin, α-actin, Src and β-actin was examined by western blot analysis. The expression of integrin α9 and β1, FAK and paxillin was also examined by immunohistochemistry. We investigated whether CSDH fluid could activate FAK in cultured endothelial cells in vitro. The concentration of N-half osteopontin in CSDH fluid was significantly higher than that in the serum. Western blot analysis revealed above-mentioned molecules. In addition, integrin α9 and β1, FAK and paxillin were localized in the endothelial cells of vessels within the CSDH outer membrane. FAK was significantly phosphorylated immediately after treatment with CSDH fluid. Our data suggest that N-half osteopontin in CSDH fluid promotes neovascularization in endothelial cells through integrins α9 and β1. The N-half osteopontin and integrin signaling pathway might be a useful therapeutic target for treating the growth of refractory CSDH.

scholarly journals Exosomes derived from platelet-rich plasma present a novel potential in alleviating knee osteoarthritis by promoting proliferation and inhibiting apoptosis of chondrocyte via Wnt/β-catenin signaling pathway

2019 ◽  
Vol 14 (1) ◽  
Author(s):  
Xuchang Liu ◽  
Lubo Wang ◽  
Chengshan Ma ◽  
Guozong Wang ◽  
Yuanji Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Platelet Rich Plasma ◽  
Therapeutic Effects ◽  
Significant Difference ◽  
Better Than

Abstract Background Platelet-rich plasma (PRP) provides a nonsurgical approach for treating osteoarthritis (OA). Exosomes that play vital roles in intercellular communication have been studied extensively. Here, we investigated the therapeutic potential and molecular mechanism of exosomes derived from PRP (PRP-Exos) in alleviating OA. Methods Exosomes derived from PRP(PRP-Exos) were isolated and purified using the exoEasy Maxi Kit and then identified and analyzed. Primary rabbit chondrocytes were isolated and treated with interleukin 1 beta (IL-1β) to establish the OA model in vitro. Proliferation, migration, and apoptosis assays were measured and compared between PRP-Exos and activated PRP (PRP-As) to evaluate the therapeutic effects on OA. The mechanism involving the Wnt/β-catenin signaling pathway was investigated by Western blot analysis. In vivo, we established animal knee OA model by surgery to compare the therapeutic effect of PRP-Exos and PRP-As. Results We successfully isolated and purified exosomes from PRP using the exoEasy Maxi Kit. We also isolated and identified chondrocytes from the New Zealand white rabbit and established the IL-1β-induced OA model; meanwhile, PRP-Exos and PRP-As both inhibited the release of tumor necrosis factor-α(TNF-α) and there was no statistically significant difference between the two. In proliferation, migration, scratch assay, the promoting effect of PRP-Exos was significantly more better than PRP-As. Furthermore, PRP-Exos could significantly decreased apoptotic rate of OA chondrocyte compared with PRP-As. In Western blot analysis, the expression of β-catenin, and RUNX2, Wnt5a were increased in IL-1β-treated chondrocytes, but PRP-Exos and PRP-As could both reverse these changes, and the reversal effect of the former was better than the latter. In vivo, we found that both PRP-Exos and PRP-As displayed the progression of OA, and the effect of PRP-Exos was obviously better than PRP-As by chondrocyte count and Osteoarthritis Research Society International (OARSI) scoring system. Conclusion The therapeutic effects of PRP-Exos on OA were similar or better compared with those of PRP-As in vitro or in vivo. PRP-Exos acting as carriers containing growth factors derived from PRP present a novel therapy for OA by activating the Wnt/β-catenin signaling pathway.

Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

2010 ◽  
Vol 113 (Special_Supplement) ◽  
pp. 228-235 ◽  
Author(s):  
Qiang Jia ◽  
Yanhe Li ◽  
Desheng Xu ◽  
Zhenjiang Li ◽  
Zhiyuan Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Gamma Knife ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

scholarly journals Punicalagin Prevents Inflammation in LPS-Induced RAW264.7 Macrophages by Inhibiting FoxO3a/Autophagy Signaling Pathway

Nutrients ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 2794 ◽  
Author(s):  
Cao ◽  
Chen ◽  
Ren ◽  
Zhang ◽  
Tan ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Inflammatory Cytokines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Inflammatory Responses ◽  
Raw264.7 Macrophages ◽  
Tnf Α ◽  
Autophagy Inhibition ◽  
Pro Inflammatory Cytokines

Punicalagin, a hydrolysable tannin of pomegranate juice, exhibits multiple biological effects, including inhibiting production of pro-inflammatory cytokines in macrophages. Autophagy, an intracellular self-digestion process, has been recently shown to regulate inflammatory responses. In this study, we investigated the anti-inflammatory potential of punicalagin in lipopolysaccharide (LPS) induced RAW264.7 macrophages and uncovered the underlying mechanisms. Punicalagin significantly attenuated, in a concentration-dependent manner, LPS-induced release of NO and decreased pro-inflammatory cytokines TNF-α and IL-6 release at the highest concentration. We found that punicalagin inhibited NF-κB and MAPK activation in LPS-induced RAW264.7 macrophages. Western blot analysis revealed that punicalagin pre-treatment enhanced LC3II, p62 expression, and decreased Beclin1 expression in LPS-induced macrophages. MDC assays were used to determine the autophagic process and the results worked in concert with Western blot analysis. In addition, our observations indicated that LPS-induced releases of NO, TNF-α, and IL-6 were attenuated by treatment with autophagy inhibitor chloroquine, suggesting that autophagy inhibition participated in anti-inflammatory effect. We also found that punicalagin downregulated FoxO3a expression, resulting in autophagy inhibition. Overall these results suggested that punicalagin played an important role in the attenuation of LPS-induced inflammatory responses in RAW264.7 macrophages and that the mechanisms involved downregulation of the FoxO3a/autophagy signaling pathway.

scholarly journals A Dot Immunobinding Assay (Dot-ELISA) for the Rapid Serodiagnosis of Salmonella Enteritidis Infection in Chickens

1996 ◽  
Vol 8 (3) ◽  
pp. 310-314 ◽  
Author(s):  
Samuel D. Charles ◽  
Srinand Sreevatsan ◽  
Russell F. Bey ◽  
V. Sivanandan ◽  
D. A. Halvorson ◽  
...  
Keyword(s):  
Western Blot ◽  
Outer Membrane ◽  
Blot Analysis ◽  
Screening Test ◽  
Western Blot Analysis ◽  
Salmonella Enteritidis ◽  
Outer Membrane Proteins ◽  
Sensitive Test ◽  
Molecular Masses ◽  
Dot Elisa

A dot immunobinding assay (DIA) was developed for the detection of antibodies to Salmonella enteritidis. Western blot analysis of outer membrane proteins from SE identified 2 polypeptides of molecular masses 43 and 46 kD that were specific for S. enteritidis. These 2 polypeptides were utilized as antigens in the DIA. The DIA was tested on sera from chickens experimentally infected with S. enteritidis. Results of the DIA were compared with that of conventional microagglutination and serum plate tests. The DIA was a highly specific and sensitive test that can be useful for screening birds to determine if they are infected with S. enteritidis. Its simplicity, reliability, reproducibility, and speed in interpreting the assay results makes it a useful screening test for flock monitoring.

Regulation of Rhcg, an ammonia transporter, by aldosterone in the kidney

2021 ◽  
Author(s):  
Koji Eguchi ◽  
Yuichiro Izumi ◽  
Yukiko Yasuoka ◽  
Terumasa Nakagawa ◽  
Makoto Ono ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Membrane Fraction ◽  
Potassium Level ◽  
Collecting Duct ◽  
Subcutaneous Administration ◽  
Extracellular Potassium ◽  
Membrane Expression

Rh C glycoprotein (Rhcg), an ammonia transporter, is a key molecule in urinary acid excretion and is expressed mainly in the intercalated cells (ICs) of the renal collecting duct. In the present study we investigated the role of aldosterone in the regulation of Rhcg expression. In in vivo experiments using C57BL/6J mice, Western blot analysis showed that continuous subcutaneous administration of aldosterone increased the expression of Rhcg in membrane fraction of the kidney. Supplementation of potassium inhibited the effect of aldosterone on the Rhcg. Next, mice were subjected to adrenalectomy with or without administration of aldosterone, and then ad libitum 0.14M NH4Cl containing water was given. NH4Cl load increased the expression of Rhcg in membrane fraction. Adrenalectomy decreased NH4Cl-induced Rhcg expression, which was restored by administration of aldosterone. Immunohistochemical studies revealed that NH4Cl load induced the localization of Rhcg at the apical membrane of ICs in the outer medullary collecting duct. Adrenalectomy decreased NH4Cl-induced membrane localization of Rhcg, which was restored by administration of aldosterone. For in vitro experiments, IN-IC cells, an immortalized cell line stably expressing Flag-tagged Rhcg (Rhcg-Flag), were used. Western blot analysis showed that aldosterone increased the expression of Rhcg-Flag in membrane fraction, while the increase in extracellular potassium level inhibited the effect of aldosterone. Both spironolactone and Gӧ6983, a PKC inhibitor, inhibited the expression of Rhcg-Flag in the membrane fraction. These results suggest that aldosterone regulates the membrane expression of Rhcg through the mineralocorticoid receptor and PKC pathways, which is modulated by extracellular potassium level.

Interfering With Hyaluronic Acid Metabolism Suppresses Glioma Cell Proliferation by Regulating Autophagy

2020 ◽  
Author(s):  
Tao Yan ◽  
Xin Chen ◽  
Hua Zhan ◽  
Penglei Yao ◽  
Ning Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Hyaluronic Acid ◽  
Tumor Microenvironment ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cell ◽  
Glioma Cells

Abstract BackgroundThe tumor microenvironment plays an important role in tumor progression. Hyaluronic acid (HA), an important component of the extracellular matrix in the tumor microenvironment, abnormally accumulates in a variety of tumors. Whereas the role of abnormal HA metabolism in glioma remains unclear. MethodsThe expression level of hyaluronic acid (HA) was analyzed by ELISA assay and proteins such as HAS3, CD44, P62, LC3, CCND1 and CCNB1 were measured with Western blot analysis. The cell viability and proliferation were measured by MTT and KI67 immunofluorescence staining respectively. Autophagic vesicles and autophagosomes were quantified by transmission electron microscopy (TEM) and GFP-RFP-LC3 fluorescence analysis respectively. Cell cycle was analyzed by flowcytometry and Western blot analysis. Immunohistochemical (IHC) staining was used to detect expression levels of HA, Ki67, HAS3 and CD44 in human and mouse tumor tissues. Lentivirus constructed HAS3 and CD44 knockout stable glioma cells were transplanted to BALB/C nude mice for in vivo experiments. 4-Methylumbelliferone (4MU) was also used to treat glioma bearing mice for verifing its anti-tumor ability. The expression curve of HAS3, CD44 and the disease-free survival (DFS) curves for HAS3, CD44 in patients with LGG and GBM was performed based on TCGA database. ResultsAs shown in the present study, HA, hyaluronic acid synthase 3 (HAS3) and a receptor of HA named CD44 are expressed at high levels in human glioma tissues and negatively correlated with the prognosis of patients with glioma. Silencing HAS3 or blocking CD44 inhibited the proliferation of glioma cells in vitro and in vivo. The underlying mechanism was attributed to the inhibition of autophagy flux and further maintaining glioma cell cycle arrest in G1 phase. More importantly, 4-Methylumbelliferone (4-MU), a small competitive inhibitor of UDP with the ability to penetrate the blood-brain barrier (BBB), also inhibited the proliferation of glioma cells in vitro and in vivo. ConclusionApproaches that interfere with HA metabolism by altering the expression of HAS3 and CD44 and the administration of 4-MU potentially represent effective strategies for glioma treatment.

scholarly journals A39 NOVEL GAIN OF FUNCTION NON-RECEPTOR TYROSINE KINASE MUTATIONS ARE LINKED TO THE PATHOGENESIS OF VEOIBD

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. 46-48
Author(s):  
M Mehta ◽  
L Wang ◽  
C Guo ◽  
N Warner ◽  
Q Li ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Western Blot ◽  
Receptor Tyrosine Kinase ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Intestinal Inflammation ◽  
De Novo ◽  
Single Gene ◽  
Gain Of Function

Abstract Background Very early-onset inflammatory bowel disease (VEOIBD) is an emerging global disease, that results in inflammation of the digestive tract. Severe forms of VEOIBD can be caused by mutations in a single gene (monogenic variants) and, can result in death. A candidate gene which codes for a non-receptor tyrosine kinase (nRTK) has recently been implicated as a monogenic cause of IBD (unpublished). Whole exome sequencing was performed in two unrelated children who presented with symptoms of IBD identifying two distinct de novo gain of function mutations (S550Y and P342T). Both mutations are located in the highly conserved region of the nRTK, and were predicted to have similar downstream effects. Furthermore, four other patients with a variety of adult-onset immune disorders have recently been identified with rare variants in the same gene (M450I, R42P, A353T, V433M, S550F) but, their potential gain of function status remains to be determined. Studies show that this nRTK is an essential mediator in inflammation. It is expressed in both intestinal epithelial and immune cells however, its role in infantile IBD is unclear. This protein is first activated by phosphorylation and is linked to activating downstream transcription factors such as ERK and JNK. All these target proteins play a meaningful role in intestinal inflammation in patients with IBD. Aims Since we identified P342T and S550Y to be gain of function, we wanted to determine if the new variants exhibit a similar downstream impact on target protein expression levels when compared with S550Y and P342T. We also wanted to identify if all variants can be rescued with a known nRTK inhibitor. It is hypothesized that the new variants are gain of function and that all variants can be rescued with the inhibitor. Methods Using western blot analysis, the activation of ERK, JNK and nRTK was compared between wildtype (WT) and mutants. This in vitro method helped identify the degree of activation. For the second part of the study, HEK293T cells were treated with inhibitor to test for a rescue of phenotypes via western blot analysis. Results Results show an increased activation of nRTK, ERK and JNK in all variants with S550Y and S550F having the highest activation. Furthermore, pharmacological inhibition using small molecular kinase inhibitors resulted in decreased activation of nRTK, ERK and JNK suggesting a rescue of phenotypes. Conclusions Characterizing the downstream functional impact of these nRTK variants is an important first step to determine if gain of function nRTK mutations drive IBD. With a rising prevalence of IBD worldwide, these findings may lead to the development of pharmacological nRTK inhibitors as a novel personalized therapeutic approach for these patients and possibly for the broader IBD population. Funding Agencies CIHR

scholarly journals In Vitro and In Vivo Effects of Fermented Oyster-Derived Lactate on Exercise Endurance Indicators in Mice

2020 ◽  
Vol 17 (23) ◽  
pp. 8811
Author(s):  
Storm N. S. Reid ◽  
Joung-Hyun Park ◽  
Yunsook Kim ◽  
Yi Sub Kwak ◽  
Byeong Hwan Jeon
Keyword(s):  
Lactate Dehydrogenase ◽  
Protein Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Biochemical Analysis ◽  
Positive Control ◽  
Exercise Endurance

Exogenous lactate administration has more recently been investigated for its various prophylactic effects. Lactate derived from potential functional foods, such as fermented oyster extract (FO), may emerge as a practical and effective method of consuming exogenous lactate. The current study endeavored to ascertain whether the lactate derived from FO may act on muscle cell biology, and to what extent this may translate into physical fitness improvements. We examined the effects of FO in vitro and in vivo, on mouse C2C12 cells and exercise performance indicators in mice, respectively. In vitro, biochemical analysis was carried out to determine the effects of FO on lactate content and muscle cell energy metabolism, including adenosine triphosphate (ATP) activity. Western blot analysis was also utilized to measure the protein expression of total adenosine monophosphate-activated protein kinase (AMPK), p-AMPK (Thr172), lactate dehydrogenase (LDH), succinate dehydrogenase (SDHA) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in response to FO administration. Three experimental groups were formed: a positive control (PC) treated with 1% horse serum, FO10 treated with 10 μg/mL and FO50 treated with 50 μg/mL. In vivo, the effects of FO supplementation on exercise endurance were measured using the Rota-rod test, and Western blot analysis measured myosin heavy-chain 2 (MYH2) to assess skeletal muscle growth, alongside p-AMPK, total-AMPK, PGC-1α, cytochrome C and UCP3 protein expression. Biochemical analysis was also performed on muscle tissue to measure the changes in concentration of liver lactate, lactate dehydrogenase (LDH), glycogen and citrate. Five groups (n = 10/per group) consisted of a control group (CON), exercise group (Ex), positive control treated with Ex and 500 mg/kg Taurine (Ex-Tau), Ex and 100 mg/kg FO supplementation (Ex-FO100) and Ex and 200 mg/kg FO supplementation (Ex-FO200) orally administered over the 4-week experimental period.FO50 significantly increased PGC-1α expression (p < 0.001), whereas both FO10 and FO50 increased the expression of p-AMPK (p < 0.001), in C2C12 muscle cells, showing increased signaling important for mitochondrial metabolism and biogenesis. Muscle lactate levels were also significantly increased following FO10 (p < 0.05) and FO50 (p < 0.001). In vivo, muscle protein expression of p-AMPK (p < 0.05) and PGC-1α were increased, corroborating our in vitro results. Cytochrome C also significantly increased following FO200 intake. These results suggest that the effects of FO supplementation may manifest in a dose-response manner. FO administration, in vitro, and supplementation, in vivo, both demonstrate a potential for improvements in mitochondrial metabolism and biogenesis, and even for potentiating the adaptive effects of endurance exercise. Mechanistically, lactate may be an important molecule in explaining the aforementioned positive effects of FO.

The N-Terminally Truncated p53 Isoform Δ40p53 Influences Prognosis in Mucinous Ovarian Cancer

2012 ◽  
Vol 22 (3) ◽  
pp. 372-379 ◽  
Author(s):  
Gerda Hofstetter ◽  
Astrid Berger ◽  
Regina Berger ◽  
Arijana Zorić ◽  
Elena I. Braicu ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Confidence Interval ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Quantitative Polymerase Chain Reaction ◽  
Hazard Ratio ◽  
Histological Subtypes ◽  
Mutational Status

ObjectiveThe tumor suppressor p53 generates the N-terminally truncated isoforms Δ40p53 and Δ133p53 that possess the ability to modulate p53 function in vitro. The aim of the present study was to evaluate the clinical relevance of p53 isoforms in the main histological subtypes of ovarian cancer.MethodsΔ40p53, Δ133p53, and full-length p53 (FLp53) expression was determined in 45 mucinous, 30 endometrioid, and 91 serous ovarian cancer specimens as well as 42 normal ovarian tissues using reverse transcriptase–quantitative polymerase chain reaction. In a subgroup of mucinous ovarian cancer cases, Δ40p53 expression was examined using Western blot analysis. A functional yeast-based assay and subsequent sequencing were performed to analyze the p53 mutational status.ResultsIn endometrioid cancer specimens, Δ133p53 expression was significantly lower than in mucinous and serous cases (P = 0.016) or in normal tissues (P = 0.004). Mucinous cancer samples showed elevated Δ40p53 expression as compared with normal ovarian tissues (P = 0.003). In addition, high Δ40p53 expression constituted an independent prognostic marker for recurrence-free but not for overall survival in patients with mucinous ovarian cancer (hazard ratio, 0.267; 95% confidence interval, 0.094–0.756 [P = 0.013]; hazard ratio, 0.453, 95% confidence interval, 0.193–1.064 [P = 0.069]). Western blot analysis confirmed the presence of p53β and Δ40p53α in a subset of patients with mucinous ovarian cancer. Expression of p53 isoforms was not associated with p53 mutational status or clinicopathologic parameters.ConclusionsWe show that expression of p53 isoforms differs in histological subtypes, thus supporting the hypothesis that histological subtypes represent distinct disease entities. In addition, we provide first evidence for a favorable role of Δ40p53 in patients with mucinous ovarian cancer.

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