scholarly journals Role of Annexin A2 isoform 2 on the aggregative growth of dermal papillae cells

2018 ◽  
Vol 38 (6) ◽  
Author(s):  
Jing Gu ◽  
Yinni Ma ◽  
Lijia Yang ◽  
Feng Wang ◽  
Cao Lei ◽  
...  
Keyword(s):  
Western Blot ◽  
Mrna Expression ◽  
Expression Vector ◽  
Dermal Papilla ◽  
Annexin A2 ◽  
Expression Level ◽  
Rt Pcr ◽  
Aggregative Growth ◽  
Dermal Papillae

The dermal papilla is a major component of hair, which signals the follicular epithelial cells to prolong the hair growth process. Human Annexin A2 was preliminarily identified by two-dimensional gel electrophoresis (2-DE), MALDI-TOF-MS and database searching. The aim of the present study was to explore the role of Annexin A2 in the aggregative growth of dermal papillae cells (DPC). Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were adopted to detect the expression of Annexin A2. And siRNA technique was used to suppress the expression of Annexin A2. Construction of over-expression vector was used to up-regulate the expression of Annexin A2. Cell Counting Kit 8 (CCK-8) and proliferating cell nuclear antigen (PCNA) were taken to detect the proliferation of DPC. The expression of Annexin A2 mRNA was up-regulated in passage 3 DPC compared with passage 10 DPC by RT-PCR. In line with the results at the mRNA level, Western blot analysis revealed that Annexin A2 isoform 2 was up-regulated significantly in passage 3 DPC compared with passage 10 DPC. The Annexin A2 isoform 2 siRNA was synthesized and transfected into passage 3 DPC. RT-PCR data showed the mRNA expression of Annexin A2 isoform 2 was suppressed in passage 3 DPC. Western blot results showed the expression level of Annexin A2 isoform 2 and PCNA were suppressed in passage 3 DPC. CCK-8 results showed that the proliferation of passage 3 DPC was suppressed (P < 0.05). Recombinant plasmid PLJM-Annexin A2 isoform 2-expression vector were constructed and were transfected into passage 10 DPC. RT-PCR data showed the mRNA expression of Annexin A2 isoform 2 was up-regulated in passage 10 DPC. Western blot results showed the expression level of annexin A2 isoform 2 and PCNA were up-regulated in passage 10 DPC. CCK-8 assay showed the proliferation of DPC was stimulated compared with control group (*P < 0.05). Our study proved that Annexin A2 isoform 2 may participate in regulating the proliferation of DPC and may be related to aggregative growth of dermal papilla cells. Therefore, our study suggests that Annexin A2 may be linked to hair follicle growth cycle.

Abstract 3474: Cardiac -Specific SOCS3 Dificient Mice Shows Resistance to Lipopolysaccharide-Induced Cardiac Dysfunction

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Futamata Nobuyoshi ◽  
Hldeo Yasukawa ◽  
Toyoharu Ohba ◽  
Kazutoshi Mawatari ◽  
Daisuke Fukui ◽  
...  
Keyword(s):  
Western Blot ◽  
Left Ventricular ◽  
Negative Regulator ◽  
Left Ventricular Ejection ◽  
Cytokine Signaling ◽  
Rt Pcr ◽  
Lv Dysfunction ◽  
Stat3 Signaling ◽  
Cardiomyocyte Survival

Background : Lypopolysaccharide (LPS)-induced left ventricular (LV) dysfunction is a well-established model for sepsis-induced acute heart failure. STAT3 signaling in the heart has been shown to promote cardiomyocyte survival during LPS-induced LV dysfunction. Little is known, however, about the role of negative regulation of STAT3 signaling during LPS-induced LV dysfunction. Suppressor of cytokine signaling 3 (SOCS3) is an intrinsic negative regulator of gp130 cytokine-induced STAT3 signaling that plays an important role in cardiomyocyte survival. In this study, we determined whether STAT3 signaling and its negative regulator SOCS3 would play a role in LPS-induced LV dysfunction. Methods and Results : We examined the activation of STAT3 and inductions of gp130 cytokines and SOCS3 in the wild-type (WT) mice hearts after LPS injection by western blot and real-time PCR (RT-PCR). RT-PCR revealed that gp130 cytokines were markedly increased after AMI. Western blot revealed that STAT3 was markedly phosphorylated and SOCS3 was induced in WT mice hearts after LPS injection. To investigate the role of STAT3 signaling and SOCS3 in LPS-induced LV dysfunction, we generated cardiac-specific SOCS3 knockout mice (SOCS3-CKO). Left ventricular ejection fraction (LVEF) of SOCS3-CKO mice was similar to that of WT mice at baseline (64.2 ± 6.1 vs. 62.4 ± 4.4%). LPS (30mg/kg) elicited a significant and robust reduction of LVEF in both SOCS3-CKO mice and WT mice 3 hr after LPS injection (18 ± 4.5 vs. 16 ± 5.2%, p <0.01). LVEF in WT mice was further reduced 6 hr after LPS injection. On the other hand, interestingly, LVEF was restored to the baseline in SOCS3-CKO mice 6 hr after LPS injection (10.4 ± 3.9 vs. 62.2 ± 8.1%, p <0.01). Also the duration and intensity of STAT3 phosphorylation after LPS injection was greater in SOCS3-CKO mice than WT mice. Furthermore, SOCS3-CKO mice showed greater survival rate than WT mice after LPS injection ( p <0.01). Conclusion : Our data show that the deletion of SOCS3 in cardiomyocytes prevents the LPS-induced LV dysfunction in mice, possibly by augmenting the STAT3-mediated gp130 cytokine signaling.

Abstract 18391: Accumulation of Adiponectin in Association With Up-regulated T-cadherin in the Aortic Wall of Acute and Chronic Aortic Dissection

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Yoshiki Watanabe ◽  
Shigeru Miyagawa ◽  
Satsuki Fukushima ◽  
Norikazu Maeda ◽  
Atsuhiro Saito ◽  
...  
Keyword(s):  
Aortic Dissection ◽  
Western Blot ◽  
Blood Concentration ◽  
Aortic Wall ◽  
Acute Type ◽  
Type B ◽  
Rt Pcr ◽  
Multiple Organs ◽  
Aortic Media

Introduction: Adiponectin (APN) is a major adipokine, which has been reported to accumulate into the damaged tissues in multiple organs. However, the role of APN and its receptor, T-cadherin (T-cad), in the pathology of the human aortic wall (AW) is poorly understood. In this study, we examined the distributions of APN and T-cad in the dissected AW and measured the serum APN concentration in patients with aortic dissection (AD). Methods: Diseased AW tissues were collected from patients with acute or chronic AD at the time of surgery, and a healthy aorta was used as a control (n = 6 per group). Blood was serially sampled to measure the APN concentrations in non-surgically-treated patients with acute type B AD (n = 10). Results: Immunohistochemically, normal aortic walls weakly expressed APN or T-cad on the intimal surface, whereas the AWs of acute AD patients showed marked expression of both APN and T-cad on the surface of the dissected aortic media. In chronic AD, APN and T-cad were diffusely expressed on the medial layers of the thickened AW (Fig. A-C). Western blot analysis showed that APN expression in the AW was significantly greater in acute and chronic AD than in the normal aorta (151 ± 2% and 220 ± 27% vs. normal, respectively) (Fig. D). RT-PCR revealed no expression of APN mRNA in any AW, and stronger expression of T-cad mRNA in acute and chronic AD than in the normal aorta (108 ± 9% and 194 ± 101% vs. normal, respectively). The blood concentration of APN in type B AD patients decreased by 13.3 ± 3.8% in 24-78 hours, and by 22.7 ± 7.2% in over 78 hours, as compared to those monitored within 24 hours after the onset, indicating that APN was supplied to the affected aortic wall from the blood. Conclusions: APN markedly accumulated into the dissected aortic media in acute and chronic AD, and T-cad was upregulated in the corresponding area. In contrast, the blood concentration of APN reciprocally decreased after AD onset, indicating a possibility of APN contribution to protect a diseased aortic wall of AD.

Regulation of Human Thrombin-Activable Fibrinolysis Inhibitor Gene Expression in Megakaryocyte-Like (Dami) and Monocyte/Macrphage- Like (THP-1) Cell Lines

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3078-3078
Author(s):  
Joellen H. H. Lin ◽  
Michael B. Boffa ◽  
Marlys L. Koschinsky
Keyword(s):  
Gene Expression ◽  
Western Blot ◽  
Mrna Expression ◽  
Fibrin Clot ◽  
Mrna Abundance ◽  
Luciferase Reporter ◽  
Rt Pcr ◽  
Monocytes And Macrophages ◽  
Fibrinolysis Inhibitor ◽  
Flanking Region

Abstract Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase zymogen defining a pathway that functions as a molecular link between coagulation and fibrinolysis. Activation by thrombin, the thrombin-thrombomodulin complex, or plasmin, the resultant enzyme (TAFIa) affects the balance between these two cascades by attenuating positive feedback in the fibrinolytic cascade, thereby inhibiting fibrin clot lysis. Plasma TAFI antigen levels vary significantly between individuals, which has implicated TAFI as a risk factor for thrombotic diseases. TAFIa can also inactivate pro-inflammatory peptides such as the anaphylatoxins and bradykinin, suggesting a role for the TAFI pathway as a link between coagulation and inflammation. TAFI expression in cultured hepatic cells is decreased by interleukins −1 and −6, and plasma TAFI levels in human are decreased in experimental endotoxemia. Although the liver is the main source of plasma TAFI, TAFI has also been identified in platelets, and TAFI mRNA has been detected in the Dami (megakaryoblastic) cell line (but not the MEG-01 cell line). TAFI mRNA has also been detected in adipocytes of patients with type 2 diabetes; however, TAFI mRNA expression in human umbilical vein endothelial cells is still a point of controversy. It has been hypothesized that platelet TAFI arises from TAFI gene expression in megakaryocytes (MK). Using RT-PCR and real-time RT-PCR, we not only confirmed the presence of TAFI mRNA in Dami cells, but also found that TAFI mRNA abundance was increased throughout Dami cell differentiation along the megakaryocytes/platelet lineage (up to 8 fold increase after 48 hours) stimulated by phorbol myristate acetate (PMA) treatment. The quantitative real-time RT-PCR experiments revealed that TAFI mRNA is present in differentiated Dami cells at a level that is only one-hundredth of that observed in HepG2 (hepatoma) cells. Using transfection experiments with luciferase reporter plasmids containing progressive deletions of the human TAFI 5′-flanking region, we identified the sequence between −438 and −257 (relative to the initiator methionine codon) to be responsible for the enhanced TAFI gene transcription as Dami cells differentiate into more mature MK-like cells. Moreover, using western blot analysis, we detected TAFI protein expression in the medium of differentiated Dami cells, but not untreated Dami cells. Together, these data provide further evidence supporting the idea that platelet TAFI is generated from TAFI gene expression in megakaryocytes rather than by uptake from the plasma. To study TAFI gene regulation in monocytes and macrophages, RT-PCR and realtime RT-PCR were used to detected and quantify, respectively, TAFI mRNA expression in both THP-1 and THP-1 cells that have been differentiated into macrophage-like cells (THP-1ma) by PMA treatment. TAFI mRNA abundance was similar in THP-1 cells as what was observed in differentiated Dami cells. In addition, we found a progressive decrease in TAFI mRNA abundance throughout the THP-1 differentiation with an 85% decrease after 24 hours of PMA treatment. Transfection experiments using luciferase reporter plasmids representing progressive deletions of the human TAFI 5′-flanking region identified sequences between −151 and −121 as harboring key promoter elements for the differentiation-associated decrease in TAFI gene expression as THP-1 differentiate into macrophage-like cells. However, no TAFI protein was detected in either THP-1 or THP-1ma conditioned medium using western blot analyses. Nonetheless, extra-hepatic expression of TAFI, such as platelet, monocytes and macrophages, suggests novel roles for TAFI pathway beyond regulation of fibrin clot breakdown.

Aldosterone Stimulates a Degranulation Response in Human Neutrophils: Role of Protein Disulfide Isomerase

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1034-1034
Author(s):  
Daphne Diaz ◽  
Gregory N. Prado ◽  
Patricia Neuman ◽  
Adriana Nieva ◽  
Manuel Torres-Grajales ◽  
...  
Keyword(s):  
Western Blot ◽  
Protein Disulfide Isomerase ◽  
Mineralocorticoid Receptor ◽  
Inflammatory Responses ◽  
Rt Pcr ◽  
Disulfide Isomerase ◽  
Net Production ◽  
Protein Disulfide ◽  
Dose Dependent

Abstract Abstract 1034 There is growing evidence for an important role of aldosterone (ALDO) in inflammatory responses in addition to its well-described effects on sodium homeostasis via activation of the mineralocorticoid receptor (MR). We studied the effects of ALDO on activation of ex vivo human polymorphonuclear leukocytes (PMNC). We isolated untouched circulating human PMNC by immunomagnetic isolation following density gradient sedimentation with PolymorphPrep from otherwise healthy subjects. Flow cytometric analyses showed greater than 97% of PMNC were positive for myeloid-neutrophil markers, CD45, CD16 and CD66b. We show that PMNC express MR by western blot and RT-PCR analyses and when incubated with ALDO (10−9 −10−7 M) showed a dose-dependent rise in cytosolic Ca2+ that peaked within 2 min using FURA-2AM fluorescence. We then studied the effect of ALDO on PMNC degranulation following incubations with ALDO (10−9 −10−7 M) for 30 min and observed a significant increase in β–glucuronidase release (P<0.001, n=3) by established fluorescent detection methods, an event that was blocked by pre-incubation of cells with 1μM canrenoic acid (CA), an MR antagonist (P<0.04, n=3). PMA and N-Formyl-Methionyl-Leucyl-Phenylalanine (fMLP) were used as positive controls for PMNC activation. We then studied the effects of ALDO on HL-60, a human promyelocytic cell line, induced to differentiate into neutrophil-like cells by incubation for 5 days with 1.3% DMSO. We detected the presence of the mineralocorticoid receptor (MR), the receptor for ALDO, by western blot analyses and MR transcripts by quantitative RT-PCR using TaqMan detection probes in these cells and as reported in kidney and endothelial cells. Cells incubated with ALDO (10−8-10−7 M) showed a dose-dependent rise in cytosolic Ca2+ that peaked within 3 min using FURA-2AM fluorescence. To assess the degranulation response of these cells we quantified the in vitro release of myeloperoxidase (MPO) and observed that 10−8M ALDO was likewise associated with increased degranulation when compared to vehicle treated cells (AUC: 590±14 to 185±11, P<0.01, n=6). To characterize the mechanisms by which ALDO regulates the degranulation responses of these cells we studied the effects of Protein Disulfide Isomerase (PDI) on ALDO-stimulated cells. PDI catalyzes the oxidation or reduction of thiol/disulfide groups and modulates leukocyte function. Our results show that blockade of PDI, by bacitracin, led to a blunted ALDO-stimulated degranulation response in both cell types. Consistent with these observations, we show that in differentiated HL-60 cells, siRNA against PDI likewise led to reduced MPO responses (AUC: 590±14 to 290±13, P<0.01, n=6) that were associated with significantly reduced PDI mRNA levels but not with scrambled siRNA as determined by quantitative RT-PCR with ABI TaqMan detection probes and GAPDH and β2 microglobulin as endogenous controls (0.55 ± 0.02, ΔΔCT of PDI siRNA relative to scrambled transfected cells, P<0.01, n=6). These results suggest that ALDO stimulates MPO release. MPO has been shown to be one of the predominant granule proteins associated with Neutrophil Extracelullar Traps (NETs), extracellular structures that contain chromatin (DNA and histones) that can also trap microorganisms. We studied the effects of ALDO following digestion of the NETs by DNAse, and observed that 30–35% of the total cellular MPO was NET-associated. We also observed that incubation with 10−8 M ALDO led to increases in the oxidative-respiratory burst [superoxide production] (P<0.01, n=3), a responses that was blocked by pre-incubation of cells with 1 uM CA (P<0.03, n=3). Consistent with these results, we observed that ALDO likewise led to significant increases in the oxidative-respiratory burst in human PMNC (P<0.01, n=3). Thus our results suggest that activation of MR by ALDO leads to degranulation and NET production in neutrophils that may contribute to the inflammatory responses associated with MR activation in vivo. Furthermore, the association between degranulation and NET release implicates PDI as a novel regulator of MPO generated NET production. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Lycopene Treatment Ameliorates Amyloid Plaque Deposition and Memory Impairment in the APP/PS1 Mice

2021 ◽  
Author(s):  
Shanshan Ou ◽  
Yinchao Fang ◽  
Tong Wu ◽  
Jie Xu ◽  
Kaihua Guo
Keyword(s):  
Oxidative Stress ◽  
Oxidative Damage ◽  
Western Blot ◽  
Chronic Inflammation ◽  
Signal Pathway ◽  
Qualitative Assessment ◽  
Cell Cytotoxicity ◽  
Rt Pcr

Abstract Alzheimer’s disease (AD) is a neurodegenerative condition associated with oxidative stress and neuroinflammation. Lycopene has previously been shown to ameliorate neuroinflammation and exert protection against oxidative damage in neuroblastoma cells. The role of this compound in reversing cognitive dysfunction in AD has yet to be determined. The present study investigates the role of lycopene in AD with an in vitro Aβ1-42-induced cell cytotoxicity model as well as the in vivo APP/PS1 mouse model. The activation of Nrf2 signal pathway was assessed using western blot and RT-PCR. MDA, 8-OHdG, ROS, SOD, GHS and GSSG measurements were carried out using the specialized assay kits. The Morris water maze was used to examine qualitative assessment of memory and spatial learning. Immunofluorescence was used to visualize astrocytes and microglia activation as well as brain β-amyloid (Aβ) deposition. The NeuN positive cells were detected by immunofluorescence and western blot. Levels of cerebral cytokines were quantified using RT-PCR. Lycopene ameliorates oxidative damage in the Aβ1-42-triggered cell cytotoxicity model via Nrf2-ARE signal pathway activation, which is regulated by AKT-GSK3β pathway. In addition, lycopene improves the cognitive impairment and reduces the Aβ deposition. Mechanistically, lycopene attenuates neuron loss, decreases chronic inflammation and activates cerebral Nrf2-ARE signaling pathway in APP/PS1 mice. The results suggest that lycopene alleviates oxidative stress via AKT- Nrf2-ARE pathway. And early administration of lycopene improves cognitive deficits by reducing Aβ deposition, neuronal loss and decreasing the degree of chronic inflammation.

scholarly journals Markedly Reduced Expression of Annexin A2 Is Associated with Hypofibrinolysis and Provoked and Unprovoked Venous Thromboembolism

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 443-443
Author(s):  
Hannah Fassel ◽  
Huigen Chen ◽  
Mary Ruisi ◽  
Maria Teresa De Sancho ◽  
Katherine A. Hajjar
Keyword(s):  
Cell Surface ◽  
Protein Expression ◽  
Annexin A2 ◽  
Clot Lysis ◽  
Healthy Controls ◽  
Rt Pcr ◽  
Generating Capacity ◽  
Dependent Cell ◽  
The Mean

Introduction: Recent evidence indicates that plasma hypofibrinolysis, as measured by clot lysis times (CLT) at or above the 90th percentile, can double the risk of venous thromboembolism (VTE). To our knowledge, there are no studies on the role of cell surface fibrinolysis in thrombosis. Annexin A2 (A2) is a calcium dependent, phospholipid binding protein that forms a heterotetramer with S100A10 (A2-S100A10)2 on the surface of endothelial cells (ECs). This complex serves as a co-receptor for plasminogen and tissue plasminogen activator (tPA), and increases the catalytic efficiency of tPA-dependent cell surface plasmin generation by 60-fold. In mouse studies, global knockout of the annexin A2 gene (Anxa2) is associated with fibrin accumulation and impaired clearance of arterial thrombi. In addition, there are several examples of the regulatory role of A2 in fibrinolysis in human diseases such as antiphospholipid antibody syndrome, cerebral venous thrombosis, and sickle cell disease. In the current study, we aimed to explore the potential role of the A2 system and cell surface based fibrinolysis in the development of VTE. Methods: Study subjects included patients 18-65 years old with history of VTE and healthy controls. Subjects were classified as having provoked or unprovoked VTE based on the presence or absence of identifiable environmental risk factors. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood collected in EDTA tubes, and used as a surrogate for ECs. Assays performed on PBMCs included determination of the rate of tPA-dependent cell surface plasmin generation using a fluorogenic substrate and analysis of CLT in the presence of phospholipid vesicles. Because A2 accounts for approximately 50% of cell surface based plasmin generation on both ECs and PBMCs, we analyzed total A2 protein expression relative to GAPDH in whole cell lysates of PBMCs using semi-quantitative western blotting. Additional assays included quantitative RT-PCR and ANXA2 gene sequencing. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparisons test. Results: Overall, 116 subjects with VTE (76 with provoked and 40 with unprovoked VTE) and 87 healthy controls were studied between September 2010 and May 2019. Plasma based clot lysis assays revealed that the mean CLT for subjects with VTE was significantly longer than that for healthy controls (37.5 versus 30.7 minutes, p=0.0001). Additionally, the mean rate of cell surface plasmin generation was significantly reduced in subjects with VTE as compared with healthy controls (0.33 RFU/min2 versus 0.49 RFU/min2, p=0.0021). Moreover, none of the 60 healthy controls (0%), but 18 of the 107 subjects with thrombosis (17%) had cell surface plasmin generating capacity in the lowest 10th percentile. In protein expression studies, we observed that none of the 21 healthy controls (0%), but 5 of 41 subjects with thrombosis (12%) had A2 expression in the lowest 10th percentile for the group; 4 of 18 (22%) of those with unprovoked VTE and 1 of 23 (4%) of those with provoked VTE fell into the lowest decile for protein expression. For plasmin generating capacity, 8 of 36 (22%) of subjects with unprovoked VTE and 10 of 71 (14%) of those with provoked VTE occupied the lowest decile. Probing of western blots of samples obtained on two separate occasions several months apart with A2 epitope-specific antibodies revealed abnormalities within either the tPA binding N-terminal tail or C-terminal core domain in proximity to the plasminogen binding site. Neither quantitative RT-PCR nor ANXA2 gene sequencing of selected samples revealed abnormalities in either mRNA or genomic DNA that could explain the reduced A2 expression. Conclusion: These data confirm findings previously reported by Lisman that plasma hypofibrinolysis is associated with VTE and may represent an independent risk factor for VTE. Additionally, we demonstrate for the first time that impaired cell surface based fibrinolysis and aberrations in A2 protein expression are associated with both provoked and unprovoked VTE, and may represent a novel risk factor for thrombosis. Possible explanations for reduced A2 expression include dysfunctional translation of mRNA into protein or post-translational proteolysis of the translated protein. Compromise of the A2-based fibrinolytic system may represent a previously unrecognized contributor to thrombophilia in VTE. Disclosures Ruisi: BMY: Equity Ownership; EMD, subsidiary of Merck KGaA: Employment. De Sancho:Apellis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

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.

Novel alternative splice variant of IGF-1R and its mRNA expression patterns in BaMa and Landrace pigs

2019 ◽  
Author(s):  
Rui Yang ◽  
Lijie Dong ◽  
Songcai Liu ◽  
Yunyun Cheng ◽  
Wenzhen Wei ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Splice Variant ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Expression Level ◽  
Alternative Splice Variant ◽  
Nucleotide Polymorphism ◽  
Rt Pcr ◽  
Single Nucleotide ◽  
Muscle Tissues

The transcript variants of Insulin-like growth factor 1 receptor (IGF-1R) and their expression profiles had never been illuminated in pigs until now. Herein, we identified IGF-1R AS02 as a novel splice variant of IGF-1R gene by RT-PCR and analyzed its mRNA expression level by qRT-PCR in liver, cartilage and muscle tissues, while also detecting the single-nucleotide polymorphism (SNP) site near the splice site of the IGF-1R gene (in intron 19) of BaMa and Landrace pigs. Results demonstrated that the IGF-1R AS02 variant showed a significantly (P less than 0.05) higher expression level in cartilage than in muscle and liver across two pig breeds respectively. The expression level of the normal transcript (IGF-1R ISO01) of IGF-1R in cartilage was markedly lower than that in the other two tissues (P less than 0.05). In cartilage, IGF-1R ISO01 expression was higher in BaMa than in Landrace (P less than 0.05), while the expression level of IGF-1R AS02 was lower in BaMa than in Landrace (P less than 0.05). The SNP was detected in intron 19 of the IGF-1R gene of BaMa and Landrace pigs. These results contributed to facilitating a better understanding of IGF-1R gene in pigs.

255 GENE EXPRESSION OF Cox 5a, 5b, AND 6b1 AND THEIR ROLES IN PRE-IMPLANTATION OF MOUSE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 235
Author(s):  
S.-E. Lee ◽  
X.-Y. Li ◽  
X.-S. Cui ◽  
N.-H. Kim
Keyword(s):  
Rna Interference ◽  
Mrna Expression ◽  
Real Time ◽  
Oocyte Maturation ◽  
Blastocyst Stage ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Target Mrna ◽  
Protein Levels

Despite clear evidence of regulation of mitochondrial respiration by nuclear encoded genes, cytochrome oxidase (Cox), little information is available on their expression and functional roles during early embryonic development. To examine the role of Cox in oocyte maturation and embryogenesis, we first characterized mRNA and protein levels of nuclear encoded genes, Cox 5a, 5b, and 6b1, in mouse oocytes and during early embryogenesis, using real-time RT-PCR and immunocytochemistry. We then examined the possible role of these genes in oocyte maturation and pre-implantation development using RNA interference analysis. The relative abundances of Cox 5a, 5b, and 6b1 transcripts was measured by real time RT-PCR. After normalization by comparison to histone H2a mRNA levels, the mRNA expression of Cox 5a, 5b, and 6b1 were found to be considerable in mature oocytes and zygotes, but reduced slightly in 2-cell embryos. From the 2-cell to the blastocyst stage, mRNA expression is dependent on the number of blastomeres, as expression increases only gradually with development. Immunocytochemical studies revealed that Cox 5a, 5b, and 6b1 proteins were expressed in all blastomeres of the blastocyst. Injection of Cox 5a, 5b, or 6b1 siRNA into GV stage oocytes decreased expression of the target mRNA specifically, while not affecting the expression of mRNAs for the other subunits in mature oocytes. Similarly, each siRNA injection into zygotes specifically reduced target mRNA expression at the 2-cell, morula and blastocyst stages (P < 0.05). Silencing of mRNA expression by RNA interference (siRNA) did not inhibit oocyte maturation or developmental events up to the morula and blastocyst stages. The expression level of mtDNA9, as well as overall levels of mitochondrial mRNAs, was not different following injection of siRNA for Cox 5a, 5b, or 6b1. However, it is evident that the number of mitochondria in siRNA treated blastocysts was greatly reduced, and they appeared to be morphologically abnormal. Significantly higher apoptosis and lower cell numbers were observed in siRNA treated blastocysts. Real time RT PCR revealed that silencing of Cox 5a, 5b, and 6b1 decreased mRNA and protein levels of E-cadherin. These results suggest that the Cox subunits, Cox 5a, 5b, and 6b1, play an important role in mitochondrial function during pre-implantation development. This work was funded by a grant from the National Research Laboratory Program in Korea.

scholarly journals Ontogeny of sulfonylurea-binding regulatory subunits of KATP channels in the pregnant rat myometrium

Reproduction ◽  
2011 ◽  
Vol 142 (1) ◽  
pp. 175-181 ◽  
Author(s):  
N Lovasz ◽  
E Ducza ◽  
R Gaspar ◽  
G Falkay
Keyword(s):  
Potassium Channels ◽  
Western Blot ◽  
Channel Blocker ◽  
Katp Channels ◽  
Rt Pcr ◽  
Pregnant Rat ◽  
Regulatory Subunits ◽  
Tocolytic Agents ◽  
Late Stages

ATP-sensitive potassium channels (KATPchannels) are composed of sulfonylurea receptors (SURs) and potassium inward rectifiers (Kir6.x) that assemble to form a large octameric channel. This study was designed to examine the expression and role of sulfonylurea-binding regulatory subunits 1 (SUR1 (ABCC8)) and 2 (SUR2 (ABCC9)) of the KATPchannels in the pregnant rat myometrium with particular regard to the contractility. RT-PCR and western blot analyses were performed to detect the presence of SUR1 and SUR2. The SUR1 levels were markedly increased in the early stages of pregnancy. The highest level was detected on day 6 of pregnancy, whereas in the late stages, the levels of SUR1 were significantly decreased. The SUR2 level remained unchanged throughout pregnancy. The SUR non-selective diazoxide and the SUR2-selective pinacidil inhibited oxytocin-induced contractions. Glibenclamide, a KATPchannel blocker, antagonized both pinacidil- and diazoxide-induced relaxations. It was established that SURs are responsible for pharmacological reactivity of KATPchannel openers. We conclude that both SURs are involved in the KATPchannel in the pregnant rat myometrium. It may further be concluded that ‘pinacidil-like’ KATPchannel openers may be of therapeutic relevance as tocolytic agents in the future.

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