scholarly journals Expression and activity of Apaf1 and caspase-9 in granulosa cells during follicular atresia in pig ovaries

Reproduction ◽  
2003 ◽  
pp. 113-120 ◽  
Author(s):  
T Matsui ◽  
N Manabe ◽  
Y Goto ◽  
N Inoue ◽  
S Nishihara ◽  
...  
Keyword(s):  
Western Blot ◽  
Granulosa Cells ◽  
Blot Analysis ◽  
Crucial Role ◽  
Mitochondrial Pathway ◽  
Signalling Pathway ◽  
Follicular Atresia ◽  
Rt Pcr ◽  
Caspase 9 ◽  
Expression And Activity

Apoptosis in granulosa cells plays a crucial role in ovarian follicular atresia, but the intracellular regulating mechanism, especially the mitochondrion-dependent apoptosis signalling pathway, is still largely unknown. This study examined whether the mitochondrial pathway is associated with granulosa cell apoptosis during atresia in pig ovaries. Both mRNAs of caspase-9 and apoptotic protease-activating factor 1 (Apaf1), which are major signal transducing components in the mitochondrial pathway, were detected in granulosa cells in healthy, early atretic and progressed atretic follicles by RT-PCR. No changes in the expression of Apaf1 mRNA were seen during follicular atresia, but the expression of caspase-9 mRNA increased during atresia. Apaf1 protein was steadily detected in granulosa cells prepared from healthy, early atretic and progressed atretic follicles by western blot analysis, but high expression of the precursor of caspase-9 (procaspase-9) was detected only in granulosa cells of healthy follicles. Decreased procaspase-9 protein was demonstrated during follicular atresia. Proteolytic activity of caspase-9 increased during atresia, in agreement with the diminution of procaspase-9 protein. Intensive expression of caspase-9 mRNA was demonstrated in the granulosa cells of early atretic and progressed atretic follicles but not in those of healthy follicles. These results indicate that the mitochondrial signalling pathway, which is mediated by Apaf1 and caspase-9, plays a crucial role in determining the fate of granulosa cells during atresia in pig ovaries.

scholarly journals Increased MMPs expression and decreased contraction in the rat myometrium during pregnancy and in response to prolonged stretch and sex hormones

2012 ◽  
Vol 303 (1) ◽  
pp. E55-E70 ◽  
Author(s):  
Zongzhi Yin ◽  
Alaa A. Sada ◽  
Ossama M. Reslan ◽  
Neha Narula ◽  
Raouf A. Khalil
Keyword(s):  
Western Blot ◽  
Sex Hormones ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Gelatin Zymography ◽  
Normal Pregnancy ◽  
Rt Pcr ◽  
Pregnant Rats ◽  
Uterine Relaxation ◽  
Expression And Activity

Normal pregnancy is associated with uterine relaxation to accommodate the stretch imposed by the growing fetus; however, the mechanisms underlying the relationship between pregnancy-associated uterine stretch and uterine relaxation are unclear. We hypothesized that increased uterine stretch during pregnancy is associated with upregulation of matrix metalloproteinases (MMPs), which in turn cause inhibition of myometrium contraction and promote uterine relaxation. Uteri from virgin, midpregnant ( day 12), and late-pregnant rats ( day 19) were isolated, and myometrium strips were prepared for measurement of isometric contraction and MMP expression and activity using RT-PCR, Western blot analysis, and gelatin zymography. Oxytocin caused concentration-dependent contraction of myometrium strips that was reduced in mid- and late-pregnant rats compared with virgin rats. Pretreatment with the MMP inhibitors SB-3CT (MMP-2/MMP-9 Inhibitor IV), BB-94 (batimastat), or Ro-28–2653 (cipemastat) enhanced contraction in myometrium of pregnant rats. RT-PCR, Western blot analysis, and gelatin zymography demonstrated increased mRNA expression, protein amount, and activity of MMP-2 and MMP-9 in myometrium of late-pregnant>midpregnant>virgin rats. Prolonged stretch of myometrium strips of virgin rats under 8 g basal tension for 18 h was associated with reduced contraction and enhanced expression and activity of MMP-2 and MMP-9, which were reversed by MMP inhibitors. Concomitant treatment of stretched myometrium of virgin rats with 17β-estradiol (E2), progesterone (P4), or E2+P4 was associated with further reduction in contraction and increased MMP expression and activity. MMP-2 and MMP-9 caused significant reduction of oxytocin-induced contraction of myometrium of virgin rat. Thus, normal pregnancy is associated with reduced myometrium contraction and increased MMPs expression and activity. The results are consistent with the possibility that myometrium stretch and concomitant increase in sex hormones during pregnancy are associated with increased expression/activity of specific MMPs, which in turn inhibit uterine contraction and promote uterine relaxation.

scholarly journals NHE1, NHE2, and NHE3 contribute to regulation of intracellular pH in murine duodenal epithelial cells

2000 ◽  
Vol 278 (2) ◽  
pp. G197-G206 ◽  
Author(s):  
J. Praetorius ◽  
D. Andreasen ◽  
B. L. Jensen ◽  
M. A. Ainsworth ◽  
U. G. Friis ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Western Blot ◽  
Intracellular Ph ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mrna Level ◽  
Rt Pcr ◽  
Acid Extrusion ◽  
Molecular Expression ◽  
Nhe Isoforms

Na+/H+-exchangers (NHE) mediate acid extrusion from duodenal epithelial cells, but the isoforms involved have not previously been determined. Thus we investigated 1) the contribution of Na+-dependent processes to acid extrusion, 2) sensitivity to Na+/H+ exchange inhibitors, and 3) molecular expression of NHE isoforms. By fluorescence spectroscopy the recovery of intracellular pH (pHi) was measured on suspensions of isolated acidified murine duodenal epithelial cells loaded with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Expression of NHE isoforms was studied by RT-PCR and Western blot analysis. Reduction of extracellular Na+ concentration ([Na+]o) during pHirecovery decreased H+ efflux to minimally 12.5% of control with a relatively high apparent Michaelis constant for extracellular Na+. The Na+/H+exchange inhibitors ethylisopropylamiloride and amiloride inhibited H+ efflux maximally by 57 and 80%, respectively. NHE1, NHE2, and NHE3 were expressed at the mRNA level (RT-PCR) as well as at the protein level (Western blot analysis). On the basis of the effects of low [Na+]o and inhibitors we propose that acid extrusion in duodenal epithelial cells involves Na+/H+ exchange by isoforms NHE1, NHE2, and NHE3.

scholarly journals FSH Promotes Rat Follicle Development Through Inhibition of the Hippo Signalling Pathway

2021 ◽  
Author(s):  
Tairen Chen ◽  
Mengjing Wu ◽  
Yuting Dong ◽  
Bin Kong ◽  
Yufang Cai ◽  
...  
Keyword(s):  
Western Blot ◽  
In Vitro Culture ◽  
Granulosa Cells ◽  
Signalling Pathway ◽  
Control Group ◽  
Follicle Development ◽  
Follicle Growth ◽  
Expression Levels ◽  
Hippo Signalling

Abstract Purpose: Whether FSH promotes follicle growth by inhibiting the Hippo signalling pathway.METHODS: Ovaries were cultured in vitro into a control group (no intervention), an FSH group (0.3 IU/mL FSH), and a VP group (10 µg/mL vetiporfin). HE staining and follicle counts were performed at each stage after 3 hours of in vitro culture. Immunohistochemistry was performed to study the expression levels of LATS2, YAP, PLATS2, and PYAP, and their expression levels in each group were also analysed by Western blot.The number of secondary follicles was significantly increased in the FSH group, the arrangement of granulosa cells was neater, the nuclear fixation was reduced, and the number of atretic follicles was decreased in the VP group. The number of secondary follicles was significantly increased, the number of atretic follicles was reduced, and granulosa cell nuclear consolidation was reduced in the VP+FSH group. Immunohistochemistry showed that LATS2 and YAP expression levels were significantly increased and PLATS2 and PYAP expression levels were relatively decreased in the FSH group, PYAP and PLATS2 expression levels were significantly increased and YAP expression was significantly decreased in the VP group, and YAP and LATS2 expression levels were significantly increased and PYAP and PLATS2 expression levels were significantly decreased in the VP+FSH group. By Western blot, LATS2 and YAP were elevated and PYAP and PLAT2 were decreased in the FSH group, LATS2 and YAP were decreased and PYAP and PLATS were significantly elevated in the VP group, and LATS2 and YAP were elevated and PYAP and PLATS2 were decreased in the VP+FSH group.CONCLUSION: FSH promotes follicle development by inhibiting the Hippo signalling pathway.

scholarly journals Activation of CD147 with Cyclophilin A Induces the Expression of IFITM1 through ERK and PI3K in THP-1 Cells

2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Ju-Young Kim ◽  
Ho Kim ◽  
Kyoungho Suk ◽  
Won-Ha Lee
Keyword(s):  
Monoclonal Antibody ◽  
Western Blot ◽  
Blot Analysis ◽  
Cyclophilin A ◽  
Cross Linking ◽  
Specific Monoclonal Antibody ◽  
Rt Pcr ◽  
Transmembrane Glycoprotein ◽  
Proinflammatory Mediators ◽  
Inflammatory Activation

CD147, as a receptor for Cyclophilins, is a multifunctional transmembrane glycoprotein. In order to identify genes that are induced by activation of CD147, THP-1 cells were stimulated with Cyclophilin A and differentially expressed genes were detected using PCR-based analysis. Interferon-induced transmembrane 1 (IFITM1) was detected to be induced and it was confirmed by RT-PCR and Western blot analysis. CD147-induced expression of IFITM1 was blocked by inhibitors of ERK, PI3K, or NF-κB, but not by inhibitors of p38, JNK, or PKC. IFITM1 appears to mediate inflammatory activation of THP-1 cells since cross-linking of IFITM1 with specific monoclonal antibody against it induced the expression of proinflammatory mediators such as IL-8 and MMP-9. These data indicate that IFITM1 is one of the pro-inflammatory mediators that are induced by signaling initiated by the activation of CD147 in macrophages and activation of ERK, PI3K, and NF-κB is required for the expression of IFITM1.

scholarly journals Integrin β3 Mediates the Endothelial-to-Mesenchymal Transition via the Notch Pathway

2018 ◽  
Vol 49 (3) ◽  
pp. 985-997 ◽  
Author(s):  
Weisen Wang ◽  
Zhi Wang ◽  
Dingyuan Tian ◽  
Xi Zeng ◽  
Yangdong Liu ◽  
...  
Keyword(s):  
Western Blot ◽  
Notch Signaling ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Notch Signaling Pathway ◽  
Rt Pcr ◽  
Mesenchymal Transition ◽  
Integrin Β3 ◽  
Endothelial To Mesenchymal Transition ◽  
Tgf Β1

Background/Aims: Neointimal hyperplasia is responsible for stenosis, which requires corrective vascular surgery, and is also a major morphological feature of many cardiovascular diseases. This hyperplasia involves the endothelial-to-mesenchymal transition (EndMT). We investigated whether integrin β3 can modulate the EndMT, as well as its underlying mechanism. Methods: Integrin β3 was overexpressed or knocked down in human umbilical vein endothelial cells (HUVECs). The expression of endothelial markers and mesenchymal markers was determined by real-time reverse transcription PCR (RT-PCR), immunofluorescence staining, and western blot analysis. Notch signaling pathway components were detected by real-time RT-PCR and western blot analysis. Cell mobility was evaluated by wound-healing, Transwell, and spreading assays. Fibroblast-specific protein 1 (FSP-1) promoter activity was determined by luciferase assay. Results: Transforming growth factor (TGF)-β1 treatment or integrin β3 overexpression significantly promoted the EndMT by downregulating VE-cadherin and CD31 and upregulating smooth muscle actin α and FSP-1 in HUVECs, and by enhancing cell migration. Knockdown of integrin β3 reversed these effects. Notch signaling was activated after TGF-β1 treatment of HUVECs. Knockdown of integrin β3 suppressed TGF-β1-induced Notch activation and expression of the Notch downstream target FSP-1. Conclusion: Integrin β3 may promote the EndMT in HUVECs through activation of the Notch signaling pathway.

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 Investigation into imbalance of Th1/Th2 cells in cirrhotic, hypersplenic rats

2019 ◽  
Vol 48 (3) ◽  
pp. 030006051988944 ◽  
Author(s):  
Yunfu Lv ◽  
Yejuan Li ◽  
Ning Liu ◽  
Yonghong Dong ◽  
Jie Deng
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Chemokine Receptors ◽  
Immunohistochemical Staining ◽  
Th2 Cells ◽  
Intragastric Administration ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Chemokine Receptor Ccr3

Objectives To evaluate the Th1/Th2 cell profile in spleens of cirrhotic and hypersplenic rats by investigating the expression of Th1-associated chemokine receptors CXCR3, CCR5 and Th2-associated chemokine receptor CCR3. Methods Experimental liver cirrhosis and hypersplenism were induced in rats by the intragastric administration of carbon tetrachloride (CCl4; 40% solution [0.3 ml/100g, twice/week for 8 weeks]) and confirmed by pathology and hemogram. Presence of the three chemokine receptors was investigated by real-time polymerase chain reaction (RT-PCR), immunohistochemical staining, and western blot analysis. Results By comparison with control animals (n=10), RT-PCR demonstrated that CXCR3 and CCR5-mRNA levels were significantly elevated in the hypersplenic rats (n=26) and CCR3-mRNA levels were lower. Immunohistochemical staining showed that by comparison with controls, the mean density of the Th1-associated CXCR3 and CCR5 receptors was significantly increased but there was no difference between groups in Th2-associated CCR3 receptors. Western blot analysis showed that by comparison with controls, hypersplenic rats had higher levels of CXCR3 and CCR5 protein but lower levels of CCR3 protein. Conclusions The abnormal expression of Th1-associated chemokine receptors in spleens of rats with cirrhosis and hypersplenism induced by CCL4 suggests that a functional imbalance between Th1/Th2 cells may play a role in the pathogenesis of hypersplenism.

scholarly journals Melatonin Reduces Inflammatory Injury through Inhibiting NF-κB Activation in Rats with Colitis

2005 ◽  
Vol 2005 (4) ◽  
pp. 185-193 ◽  
Author(s):  
Jun-Hua Li ◽  
Jie-Ping Yu ◽  
Hong-Gang Yu ◽  
Xi-Ming Xu ◽  
Liang-Liang Yu ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Rt Pcr ◽  
Colon Tissue ◽  
Inflammatory Injury ◽  
Proinflammatory Mediators ◽  
Proinflammatory Molecule ◽  
Colitis Model

Proinflammatory mediators are important in the pathogenesis of IBD, which are regulated by activation of NF-κB. The aim of this study was to investigate whether melatonin reduces inflammatory injury and inhibits proinflammatory molecule and NF-κB in rats with colitis. Rat colitis model was established by TNBS enema. NF-κB p65, TNF-α, ICAM-1, and IκBα in colon tissue were examined by immunohistochemistry, EMSA, RT-PCR, and Western blot analysis. Expression of proinflammatory molecule and activation of NF-κB were upregulated and IκB level decreased in rats with colitis. Melatonin reduces colonic inflammatory injury through downregulating proinflammatory molecule mediated by NF-κB inhibition and blockade of IκBα degradation.

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