scholarly journals The diurnal pattern of salivary IL-1β in healthy young adults

2017 ◽  
Vol 31 (5) ◽  
Author(s):  
Nur Basirah Ghazali ◽  
Michael Steele ◽  
David Koh ◽  
Adi Idris
Keyword(s):  
Young Adults ◽  
Circadian Rhythm ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Inflammatory Cytokine ◽  
Enzyme Linked Immunosorbent Assay ◽  
Diurnal Pattern ◽  
Healthy Individuals

Abstract Disruption in circadian rhythm affects the production of inflammatory cytokines. Understanding how it behaves in diseased conditions is essential. Despite the role of the interleukin-1β (IL-1β), a potent inflammatory cytokine, in human diseases, little is known about the steady-state circadian rhythm of IL-1β in healthy individuals. This short study investigates the diurnal pattern of salivary IL-1β throughout the day in healthy young adults. Twelve participants provided saliva samples at various times throughout the day. Salivary IL-1β were assessed using enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Salivary IL-1β levels were highest at 0430 h and lowest at 0000 h and shared a similar diurnal pattern to that of salivary IL-6. Western blot analysis showed that these levels correspond to the mature form of IL-1β. Our findings are important as it established the diurnal pattern of salivary IL-1β is fluctuating normally throughout the day. The findings also open an incredible opportunity for developing research conducted in the field with saliva as the diagnostic tool.

Abstract 255: Cytoprotective Effects of Erythropoietin and Erythropoietin-derivatives in Ischaemic Human Myotubes and the Role of Pi3k/akt Signalling Pathway

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Rebekah Sian Hwee Yu ◽  
Daryll Baker ◽  
David Abraham ◽  
Janice Tsui
Keyword(s):  
Skeletal Muscle ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
Signalling Pathway ◽  
Critical Limb Ischaemia ◽  
Human Myotubes ◽  
Muscle Biopsies

Objectives Erythropoietin (Epo) has tissue-protective effects in response to injury, acting through the EpoR-βcR heteroreceptor. We have previously demonstrated the presence and interaction of the EpoR and βcR in human skeletal muscle. Here we aim to investigate the potential cytoprotective effects of Epo and an Epo-derivative (ARA-290) in a human in vitro model of skeletal muscle and establish a potential downstream signalling pathway utilised in protecting cells from apoptosis (including Jak-2, PI3k/Akt, NFkB). Methods Gastrocnemius muscle biopsies were obtained from patients with critical limb ischaemia and control samples were obtained from non-ischaemic patients. Human myoblasts were isolated from muscle biopsies, cultured, and allowed to differentiate into myotubes in order to investigate the cytoprotective effects of Epo and ARA-290 on myotubes subjected to simulated ischaemia. The PI3k inhibitors, LY294002 and wortmannin, were then used to determine the role of PI3k/Akt pathway in mediating cytoprotection. Following this, inhibitors against the upstreatm (Jak-2) and downstream (NFkB) molecules were also investigated. Western blot analysis, using the pro-apoptotic marker cleaved caspase-3 was performed and compared with levels of Akt and phosphorylated-Akt, using western blot analysis. Results Exogenous administration of Epo and ARA-290 were able to ameliorate the ischaemia-induced apoptosis on isolated human myotubes as shown by a significant reduction in cleaved caspase-3 expression. Addition of all inhibitors, to ARA-290 or Epo pre-treated cells, abolished the reduction in apoptosis. Conclusion The ability of ARA-290 to attenuate apoptosis in human myotubes undergoing ischaemic insult suggests a potential role in tissue protection in skeletal muscle injury. We propose that the PI3k/Akt signalling pathway is involved in mediating this cytoprotection.

scholarly journals Investigations on ornithobacterium rhinotracheale in broiler flocks in elazig province located in the east of turkey

2012 ◽  
Vol 49 (No. 8) ◽  
pp. 305-311 ◽  
Author(s):  
G. Ozbey ◽  
H. Ongor ◽  
D. T Balik ◽  
V. Celik ◽  
A. Kilic ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rrna Gene ◽  
Major Band ◽  
Cell Extracts ◽  
Sds Page ◽  
Ornithobacterium Rhinotracheale

In the present study, lung, trachea and serum samples from broiler flocks slaughtered at an abattoir in Elazig province located in the East of Turkey were examined for the presence of Ornithobacterium rhinotracheale using culture and enzyme-linked immunosorbent assay (ELISA). The identity was latter proved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot analysis, and polymerase chain reaction (PCR) assays. A total of 324 serum and 250 lung and trachea samples were collected from 10 commercially reared chicken flocks showing respiratory manifestations. The samples were obtained from different flocks. The causative agent (ORT) was isolated from trachea (1.5%) of five chickens and from both lung and trachea (0.4%) of only one chicken in the bacteriological examination of tissues. The presence of antibodies against ORT was detected in 33 (10.2%) of the 324 sera by ELISA. A 784 bp fragment of the 16S rRNA gene was amplified using specific primers in the PCR. All ORT isolates that were positive by culture were also detected to be positive by the PCR. SDS-PAGE protein profiles of whole cell extracts showed a high similarity for all the isolates with a major band of the molecular weight of 33 kDa (kiloDalton). Results of Western blot analysis indicate four antigenic fractions predominantly with molecular weights of 33, 42, 52 and 66 kDa.

TIPE1 suppresses the invasion and migration of breast cancer cells and inhibits epithelial-to-mesenchymal transition primarily via the ERK signaling pathway

2019 ◽  
Vol 51 (10) ◽  
pp. 1008-1015 ◽  
Author(s):  
Shusheng Qiu ◽  
Wei Hu ◽  
Qiuhong Ma ◽  
Yi Zhao ◽  
Liang Li ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Western Blot ◽  
Cancer Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Breast Cancer Cells ◽  
Mesenchymal Transition ◽  
Invasion And Migration ◽  
And Migration

Abstract Tumor necrosis factor α-induced protein 8-like-1 (TIPE1) functions as an activator or a repressor in a tumor cell type-specific manner. However, the role of TIPE1 in breast cancer, especially regarding metastasis, is unknown. In this study, we aimed to investigate the TIPE1 expression in breast cancer tissues, the biological functions, and the underlying mechanisms of TIPE1 regarding the metastatic properties of breast cancer cells. The results of immunohistochemical staining and western blot analysis indicated that TIPE1 expression was associated with tumor size and lymph node metastasis, and the expression of TIPE1 was downregulated in the tissues of patients with lymph node metastasis. Transwell and wound healing assay results showed that TIPE1 inhibited the invasive and migratory capacities of breast cancer cells. Moreover, the epithelial-mesenchymal transition (EMT) was suppressed in TIPE1-overexpressing cells, as demonstrated by western blot analysis. In addition, western blot analysis also showed that TIPE1 reduced the expression levels of MMP2 and MMP9 and decreased the phosphorylation level of ERK. These results suggested that TIPE1 might suppress the invasion and migration of breast cancer cells and inhibit EMT primarily via the ERK signaling pathway. Our findings revealed the anti-tumor metastasis role of TIPE1 in breast cancer and TIPE1 might be a new candidate prognostic indicator and a potential molecular target for the treatment of breast cancer.

scholarly journals Primary cilia respond to intermittent low-magnitude, high-frequency vibration and mediate vibration-induced effects in osteoblasts

2020 ◽  
Vol 318 (1) ◽  
pp. C73-C82 ◽  
Author(s):  
Yan-Hui Li ◽  
Dong Zhu ◽  
Zongbing Cao ◽  
Yanwei Liu ◽  
Jian Sun ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
High Frequency ◽  
Western Blot Analysis ◽  
Primary Cilia ◽  
Critical Role ◽  
Rt Pcr ◽  
High Frequency Vibration ◽  
Low Magnitude

Our objective was to investigate the role of primary cilia in low-magnitude, high-frequency vibration (LMHFV) treatment of MC3T3-E1 osteoblasts (OBs). We used chloral hydrate (CH), which has a well-characterized function in chemically removing primary cilia, to elucidate the role of primary cilia in LMHFV-induced OB osteogenic responses through cell viability assay, Western blot analysis, real-time quantitative RT-PCR, and histochemical staining methods. We observed a significant, 30% decrease in the number of MC3T3-E1 OBs with primary cilia (reduced from 64.3 ± 5%) and an approximately 50% reduction in length of primary cilia (reduced from 3 ± 0.8 μm) after LMHFV stimulation. LMHFV stimulation upregulated protein expression of the bone matrix markers collagen 1 (COL-1), osteopontin (OPN), and osteoclacin(OCN) in MC3T3-E1 OBs, indicating that LMHFV induces osteogenesis. High-concentration or long-duration CH exposure resulted in inhibition of MC3T3-E1 OB survival. In addition, Western blot analysis and RT-PCR revealed that CH treatment prevented LMHFV-induced osteogenesis. Furthermore, decreased alkaline phosphate activity, reduced OB differentiation, mineralization, and maturation were observed in CH-pretreated and LMHFV-treated OBs. We showed that LMHFV induces morphological changes in primary cilia that may fine-tune their mechanosensitivity. In addition, we demonstrated the significant inhibition by CH of LMHFV-induced OB mineralization, maturation, and differentiation, which might reveal the critical role of primary cilia in the process.

FGF-7 facilitates the process of psoriasis by inducing TNF-α expression in HaCaT cells

2019 ◽  
Vol 51 (10) ◽  
pp. 1056-1063 ◽  
Author(s):  
Jiaojiao Pu ◽  
Rui Wang ◽  
Guanglin Zhang ◽  
Ju Wang
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Specific Antibody ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Quantitative Polymerase Chain Reaction ◽  
Hacat Cells ◽  
Tnf Α

Abstract The purpose of this study was to uncover the mechanism of tumor necrosis factor (TNF)-α induction by fibroblast growth factor-7 (FGF-7) in human HaCaT cells and the potential role of FGF-7-specific antibody F-9 in psoriatic therapy. TNF-α expression in HaCaT cells induced by FGF-7 was analyzed by quantitative polymerase chain reaction, western blot analysis, and enzyme-linked immunosorbent assays. In vivo, the BALB/c mouse psoriasis model established by topical application of imiquimod (IMQ) was used to determine the role of FGF-7-specific antibody (F-9) in skin inflammation. We found that induction of TNF-α expression by FGF-7 in HaCaT cells was suppressed by FGF-7-specific antibody F-9. Western blot analysis results showed that FGF-7 induced TNF-α expression in HaCaT cells via the FGF receptor 2 (FGFR2)/AKT/NF-κB signaling pathway. In vivo, F-9 could significantly ameliorate the inflammations in a mouse psoriatic model evaluated by Psoriasis Area and Severity Index scores and ear thickness, which was consistent with the results of hematoxylin–eosin staining, immunohistochemistry assay, and western blot analysis. These results indicate that FGF-7 induces TNF-α expression in HaCaT cells and FGF-7 antibody F-9 alleviates IMQ-induced psoriasiform in mice. Therefore, FGF-7/FGFR2 signaling pathway is a potential target for psoriasis treatment.

Em18 and Em16, new serologic marker epitopes for alveolar echinococcosis in Western blot analysis, are the only two epitopes recognized by commercially available weak positive (cut off) sera for Em2plus-ELISA

1995 ◽  
Vol 69 (4) ◽  
pp. 369-371 ◽  
Author(s):  
A. Ito ◽  
Y. Osawa ◽  
M. Nakao ◽  
T. Horii ◽  
M. Okamoto ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Alveolar Echinococcosis ◽  
Specific Antigen ◽  
Antibody Responses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Western Blot Assay ◽  
Molecular Weights ◽  
Serologic Marker

AbstractThe assay system for antibody responses against Em2, the most specific antigen for serodiagnosis of alveolar echinococcosis (AE), has been established by enzyme-linked immunosorbent assay (ELISA) but not by Western blot assay, since Em2 antigen is not protein but carbohydrate in nature. Recently we reported that previously undescribed protein epitopes, designated Em18 and Em16 due to their molecular weights, were good serologic markers for AE by Western blot analysis. It has been shown that Em18 and Em16 are the only two epitopes recognized by commercially available weak positive (cut off) sera for the Em2plus-ELISA.

Galectin-1 induces chemokine production and proliferation in pancreatic stellate cells

2006 ◽  
Vol 290 (4) ◽  
pp. G729-G736 ◽  
Author(s):  
Atsushi Masamune ◽  
Masahiro Satoh ◽  
Jun Hirabayashi ◽  
Kenichi Kasai ◽  
Kennichi Satoh ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Stellate Cells ◽  
Pancreatic Tissue ◽  
Immunofluorescent Staining ◽  
Pancreatic Stellate Cells ◽  
Chemokine Production ◽  
Cell Functions

Galectin-1 is a β-galactoside-binding lectin. Previous studies have shown that galectin-1 was expressed in fibroblasts of chronic pancreatitis and of desmoplastic reaction associated with pancreatic cancer. These fibroblasts are now recognized as activated pancreatic stellate cells (PSCs). Here, we examined the role of galectin-1 in cell functions of PSCs. PSCs were isolated from rat pancreatic tissue and used in their culture-activated phenotype unless otherwise stated. Expression of galectin-1 was assessed by Western blot analysis, RT-PCR, and immunofluorescent staining. The effects of recombinant galectin-1 on chemokine production and proliferation were evaluated. Activation of transcription factors was assessed by EMSA. Activation of MAPKs was examined by Western blot analysis using anti-phosphospecific antibodies. Galectin-1 was strongly expressed in culture-activated but not freshly isolated PSCs. Recombinant galectin-1 increased proliferation and production of monocyte chemoattractant protein-1 and cytokine-induced neutrophil chemoattractant-1. Galectin-1 activated ERK, JNK, activator protein-1, and NF-κB, but not p38 MAPK or Akt. Galectin-1 induced proliferation through ERK and chemokine production mainly through the activation of NF-κB and in part by JNK and ERK pathways. These effects of galectin-1 were abolished in the presence of thiodigalactosie, an inhibitor of β-galactoside binding. In conclusion, our results suggest a role of galectin-1 in chemokine production and proliferation through its β-galactoside binding activity in activated PSCs.

scholarly journals The role of activating transcription factor 6 in hydroxycamptothecin-induced fibroblast autophagy and apoptosis

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Jin Tao ◽  
Hui Chen ◽  
Xiaolei Li ◽  
Jingcheng Wang
Keyword(s):  
Transcription Factor ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Counting ◽  
Immunofluorescent Staining ◽  
Fibroblast Proliferation ◽  
Activating Transcription Factor ◽  
Related Proteins

Abstract Background The over-proliferation of fibroblasts is considered to be the main cause of scar adhesion after joint surgery. Hydroxycamptothecin (HCPT), though as a potent antineoplastic drug, shows preventive effects on scar adhesion. This study aimed to investigate the role of activating transcription factor 6 (ATF-6) in the HCPT-induced inhibition of fibroblast viability. Methods The cell counting kit-8 (CCK-8) assay, western blot analysis, lentivirus-mediated gene silencing, transmission electron microscopy (TEM) analysis, immunofluorescent staining for autophagy-related protein light chain 3 (LC3) were used to explore the effect of HCPT on triggering fibroblast apoptosis and inhibiting fibroblast proliferation, and the involvement of possible signaling pathways. Results It was found that HCPT exacerbated fibroblast apoptosis and repressed its proliferation. Subsequently, endoplasmic reticulum stress (ERS)-related proteins were determined by western blot prior to ATF6 p50 was screened out and reexamined after it was silenced. As a result, ATF6-mediated ERS played a role in HCPT-induced fibroblast apoptosis. Autophagy-related proteins and autophagosomes were detected after the HCPT administration using western blot and TEM analyses, respectively. Autophagy was activated after the HCPT treatment. With the co-treatment of autophagy inhibitor 3-methyladenine (3-MA), both the western blot analysis and the CCK-8 assay showed inhibited autophagy, which indicated that the effect of HCPT on fibroblast proliferation was partially reversed. Besides, the LC3 immunofluorescence staining revealed suppressed autophagy after silencing ATF6 p50. Conclusion Our results demonstrate that HCPT acts as a facilitator of fibroblast apoptosis and inhibitor of fibroblast proliferation for curbing the postoperative scar adhesion, in which the ATF6-mediated ERS pathway and autophagy are involved.

scholarly journals Post-translational regulation of the RpoS and PsrA genes in pseudomonas putida WCS358: The role of ClpXP protease

2008 ◽  
Vol 60 (1) ◽  
pp. 1-4
Author(s):  
B. Jovcic ◽  
Jelena Begovic ◽  
Jelena Lozo ◽  
Lj. Topisirovic ◽  
M. Kojic
Keyword(s):  
Western Blot ◽  
Pseudomonas Putida ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Translational Regulation ◽  
Stationary Phases ◽  
Transcriptional Regulators ◽  
Exponential Phase ◽  
Early Exponential Phase

The RpoS and PsrA proteins are key transcriptional regulators that are activated in response to the stationary phase of growth in pseudomonads. This study was designed to establish whether ClpXP (ATP-dependent serine protease) regulates levels of RpoS and PsrA in Pseudomonas putida WCS358. Western blot analysis of P. putida WCS358 protein extracts from the early exponentianl, late exponential, and stationary phases of growth with antibodies against RpoS and PsrA revealed that these proteins are degraded by ClpXP in the early exponential phase of growth. The obtained results demonstrate a role for ClpXP protease in post-translational regulation of proteins encoded by the rpoS and psrA genes in Pseudomonas spp.

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