scholarly journals The role of the Siglec-G ITIM domain during bacterial infection

2022 ◽  
Vol 67 (4) ◽  
pp. 163-169
Author(s):  
Yin Wu ◽  
Darong Yang ◽  
Guo-Yun Chen
Keyword(s):  
Western Blot ◽  
Bacterial Infection ◽  
Expression Vectors ◽  
Immunoglobulin Superfamily ◽  
Western Blot Assay ◽  
Downstream Signaling ◽  
Sialic Acid Binding ◽  
Associated Proteins ◽  
Membrane Bound

Siglecs, membrane-bound lectins of the sialic acid-binding immunoglobulin superfamily, inhibit immune responses by recruiting tyrosine phosphatases (e.g., SHP-1 and SHP-2) through their cytoplasmic immunoreceptor tyrosine-based inhibition motif (ITIM) domain. The role of Siglecs in infection has been extensively studied, but downstream signaling through the ITIM domain remains unclear. Here, we used a GST pull-down assay to identify additional proteins associated with the ITIM domain during bacterial infection. Gdi2 bound to ITIM under normal homeostasis, but Rab1a was recruited to ITIM during bacterial infection. Western blot analysis confirmed the presence of SHP-1 and SHP-2 in eluted ITIM-associated proteins under normal homeostasis. We confirmed the association of ITIM with Gdi2 or Rab1a by transfection of corresponding expression vectors in 293T cells followed by immunoprecipitation-western blot assay. Thus, ITIM’s role in the inhibition of the immune response during bacterial infection may be regulated by interaction with Gdi2 and Rab1a in addition to SHP-1 and SHP-2.

Role of western blot assay for the diagnosis of histoplasmosis inAIDSpatients from a National Institute of Infectious Diseases in Rio de Janeiro, Brazil

Mycoses ◽  
2019 ◽  
Vol 62 (3) ◽  
pp. 261-267 ◽  
Author(s):  
Marcos Abreu Almeida ◽  
Lisandra Serra Damasceno ◽  
Cláudia Vera Pizzini ◽  
Mauro de Medeiros Muniz ◽  
Rodrigo Almeida‐Paes ◽  
...  
Keyword(s):  
Infectious Diseases ◽  
Western Blot ◽  
Rio De Janeiro ◽  
Western Blot Assay

Vectorial transport of proteins by membrane-bound ribosomes of nongrowing and growing Jerusalem artichoke tuber cells

1978 ◽  
Vol 56 (3) ◽  
pp. 167-173
Author(s):  
Joachim Sparkuhl ◽  
George Setterfield
Keyword(s):  
Reductase Activity ◽  
Jerusalem Artichoke ◽  
Jerusalem Artichoke Tuber ◽  
Protease Digestion ◽  
Associated Proteins ◽  
Membrane Bound ◽  
Nadh Cytochrome ◽  
Vectorial Transport

Both nongrowing (water-incubated) and growing (hormonally stimulated) Jerusalem artichoke tuber cells contain membrane-bound (mb) ribosomes. Using a rapid flotation procedure, a membrane fraction was prepared from both types of cells. This fraction was enriched in mb ribosomes, contained NADH cytochrome c reductase activity, had RNA:phospholipid and RNA:protein ratios similar to those reported for rough microsomes from animal tissues, and supported synthesis of preinitiated proteins in vitro. Using puromycin and detergent release, vectorial transport of labelled polypeptides was measured in the in vitro system. Of proteins made by mb ribosomes from nongrowing cells, only 12% remained associated with microsome membranes following chain termination. The comparable figure for proteins from mb ribosomes of growing tissue was 42%. The membrane-associated proteins were preferentially protected from protease digestion. Some possible reasons are suggested for the correlation between cell growth and the association of newly synthesized proteins with microsomes. The role of proteins synthesized by mb ribosomes but not vectorially transported, in both growing and nongrowing cells, is unknown.

scholarly journals miR-27a-3p attenuates induce autophagy-related cell death by suppressing Inhibitor of growth family 5 in cholangiocarcinoma

2020 ◽  
Author(s):  
Ming WAN ◽  
Fu-min Zhang ◽  
Peng-cheng Kang ◽  
Xing-ming Jiang ◽  
yunfu cui
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Luciferase Reporter ◽  
Immunofluorescence Analysis ◽  
Ki 67 ◽  
Western Blot Assay ◽  
Qrt Pcr ◽  
Related Cell ◽  
The Relationship

Abstract Background MicroRNAs (miRNAs) are abnormally expressed in human tumors, including cholangiocarcinoma (CCA). miR-27a-3p was observed up-regulated in CCA, but its functions in CCA are largely unknown.Methods CCK8 assay, Colony formation assays and Ki-67 staining was employed to detect the cell growth. The autophagy and proliferation relative-protein analyzed by western blot. The immunofluorescence staining was applied to analyze the expression level of LC3 I/II. Tumor xenografts was used to test the role of miR-27a-3p. Luciferase reporter assay, western bolt and qRT-PCR showed the relationship between miR-27a-3p and ING5.Results miR-27a-3p expression was increased in human CCA tissues. Inhibition of miR-27a-3p suppressed the proliferative capacity of CCA cells, silencing of miR-27a-3p dramatically induced cell death and suppressed tumor growth in vivo. The proteins, such as Beclin-1, p62, p21, p-p53, CDK4 and CDK6, were decreased upon miR-27a-3p inhibitor transfection. Western blot assay and immunofluorescence analysis were showed the induced-autophagy after transfecting with miR-27a-3p or inhibitor of growth family 5 (ING5) in RBE. ING5 as a direct miR-27a-3p target in CCA. Co-transfect of miR-27a-3p and ING5 can reverse CCA cell death which induced by miR-27a-3p inhibitor alone.Conclusions miR-27a-3p promotes oncogenesis of CCA by triggering autophagy-related cell death by interacting with ING5 directly.

scholarly journals Malate-Aspartate Shuttle Plays an Important Role in LPS-Induced Neuroinflammation of Mice Due to its Effect on STAT3 Phosphorylation

2021 ◽  
Vol 8 ◽  
Author(s):  
Cuiyan Zhou ◽  
Wangsong Shang ◽  
Shan-Kai Yin ◽  
Haibo Shi ◽  
Weihai Ying
Keyword(s):  
Western Blot ◽  
Microglial Activation ◽  
Critical Role ◽  
Mrna Levels ◽  
Western Blot Assay ◽  
Stat3 Phosphorylation ◽  
Nadh Ratio ◽  
Malate Aspartate Shuttle

Neuroinflammation is a key pathological factor in numerous neurological disorders. Cumulating evidence has indicated critical roles of NAD+/NADH metabolism in multiple major diseases, while the role of malate-aspartate shuttle (MAS) - a major NADH shuttle - in inflammation has remained unclear. In this study we investigated the roles of MAS in LPS-induced neuroinflammation both in vivo and in vitro. Immunofluorescence staining, Western blot assay and Real-time PCR assays were conducted to determine the activation of Iba-1, the protein levels of iNOS and COX2 and the mRNA levels of IL-1β, IL-6, and TNF-α in vivo, showing that both pre-treatment and post-treatment of aminooxyacetic acid (AOAA) - an MAS inhibitor - profoundly decreased the LPS-induced neuroinflammation in mice. BV2 microglia was also used as a cellular model to investigate the mechanisms of this finding, in which such assays as Western blot assay and nitrite assay. Our study further indicated that AOAA produced its effects on LPS-induced microglial activation by its effects on MAS: Pyruvate treatment reversed the effects of AOAA on the cytosolic NAD+/NADH ratio, which also restored the LPS-induced activation of the AOAA-treated microglia. Moreover, the lactate dehydrogenase (LDH) inhibitor GSK2837808A blocked the effects of pyruvate on the AOAA-produced decreases in both the cytosolic NAD+/NADH ratio and LPS-induced microglial activation. Our study has further suggested that AOAA produced inhibition of LPS-induced microglial activation at least partially by decreasing STAT3 phosphorylation. Collectively, our findings have indicated AOAA as a new and effective drug for inhibiting LPS-induced neuroinflammation. Our study has also indicated that MAS is a novel mediator of LPS-induced neuroinflammation due to its capacity to modulate LPS-induced STAT3 phosphorylation, which has further highlighted a critical role of NAD+/NADH metabolism in inflammation.

scholarly journals Extracellular Cleavage of Cadherin-11 by ADAM Metalloproteases Is Essential for Xenopus Cranial Neural Crest Cell Migration

2009 ◽  
Vol 20 (1) ◽  
pp. 78-89 ◽  
Author(s):  
Catherine McCusker ◽  
Hélène Cousin ◽  
Russell Neuner ◽  
Dominique Alfandari
Keyword(s):  
Cell Adhesion ◽  
Neural Crest ◽  
Neural Crest Cell ◽  
Cranial Neural Crest ◽  
Downstream Signaling ◽  
Cranial Neural Crest Cell ◽  
Neural Crest Cell Migration ◽  
Membrane Bound

Cell adhesion molecules such as cadherins alternate their expression throughout cranial neural crest (CNC) development, yet our understanding of the role of these molecules during CNC migration remains incomplete. The “mesenchymal” cadherin-11 is expressed in the CNC during migration yet prevents migration when overexpressed in the embryo, suggesting that a defined level of cadherin-11–mediated cell adhesion is required for migration. Here we show that members of the meltrin subfamily of ADAM metalloproteases cleave the extracellular domain of cadherin-11 during CNC migration. We show that a fragment corresponding to the putative shed form of cadherin-11 retains biological activity by promoting CNC migration in vivo, in a non-cell–autonomous manner. Additionally, cleavage of cadherin-11 does not affect binding to β-catenin and downstream signaling events. We propose that ADAM cleavage of cadherin-11 promotes migration by modifying its ability to support cell–cell adhesion while maintaining the membrane-bound pool of β-catenin associated with the cadherin-11 cytoplasmic domain.

scholarly journals TLR4-NLRP3-GSDMD-Mediated Pyroptosis Plays an Important Role in Aggravated Liver Injury of CD38-/- Sepsis Mice

2021 ◽  
Vol 2021 ◽  
pp. 1-15
Author(s):  
Huiqing Zhang ◽  
Yuna Du ◽  
Yujie Guo ◽  
Zeyu Wang ◽  
Hua Li ◽  
...  
Keyword(s):  
Liver Injury ◽  
Western Blot ◽  
Bacterial Infection ◽  
Hepatic Dysfunction ◽  
Bacterial Culture ◽  
Multiple Organ ◽  
Hematoxylin And Eosin ◽  
Bacterial Stimulation ◽  
Deficient Mice

Clinically, severe bacterial infection can cause septicemia and multiple organ dysfunction syndrome, especially liver injury. CD38 is closely related to many inflammatory pathways, but its role in liver injury caused by bacterial infection remains unclear. The purpose of this study is to discuss the specific role of CD38 in bacterial liver injury. Eight-week-old male C57BL/6 mice (WT, CD38-/- and CD38-/-TLR4mut) were used and stimulated with Escherichia coli (ATCC25922) or PBS, intraperitoneally. After 3 hours of bacterial stimulation, serum was collected to detect ALT and AST concentration, and liver tissue was harvested for hematoxylin and eosin staining and bacterial culture. The mRNA expressions of TLR4, NLRP3, IL-1β, IL-18, and GSDMD were quantitatively determined by RT-qPCR. The expressions of TLR4, MyD88, TRIF, NF-κB p65, NLRP3, GSDMD, and cytokines were detected by Western blot. The expression and localization of ERK1/2 were detected by immunohistochemistry and Western blot. The results showed that bacterial stimulation could upregulate the expression of inflammatory cytokines, leading to hepatic dysfunction. Moreover, bacterial stimulation of CD38-deficient mice can aggravate the inflammatory response, the expressions of TLR4, NF-κB, and ERK1/2 were significantly increased, and the biomarkers related to pyroptosis also manifested more obvious pyroptosis. However, TLR4 mutation significantly alleviated inflammation and pyroptosis in the liver caused by bacteria, on the basis of CD38 deficiency. Overall, CD38 knockout exacerbates bacteria-induced liver damage through TLR4-NLRP3-GSDMD-mediated pyroptosis.

scholarly journals LncRNA HOXA-AS2 promotes glioblastoma carcinogenesis by targeting miR-885-5p/RBBP4 axis

2020 ◽  
Author(s):  
Jixin Shou ◽  
Haidong Gao ◽  
Sen Cheng ◽  
Bingbing Wang ◽  
Haibo Guan
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Regulatory Mechanism ◽  
Luciferase Assay ◽  
Pcr Analysis ◽  
Western Blot Assay ◽  
Qrt Pcr ◽  
Brdu Assay ◽  
Rip Assay

Abstract Background: LncRNA HOXA-AS2 has been found in the literature to deteriorate glioblastoma. However, its regulatory mechanism is yet to be fully investigated. Our study focused chiefly on the interaction and role of the HOXA-AS2/miR-885-5p/RBBP4 axis in the development of glioblastoma. Methods: qRT-PCR analysis was performed to detect the expression of lncRNA, miRNA and mRNA in glioblastoma tissues and cells. Dual-luciferase assay, RIP assay and RNA pull-down assay were later carried out to reveal the interactions among HOXA-AS2, miR-885-5p and RBBP4. After that, CCK-8 assay, BrdU assay, nude mice xenografting assay, western blot assay, and flow cytometry were carried out to analyze the effect of the HOXA-AS2/miR-885-5p/RBBP4 axis on glioblastoma samples. Results: HOXA-AS2 and RBBP4 were found to be overexpressed in glioblastoma. Experimental results showed that HOXA-AS2 and RBBP4 contributed to the tumorigenesis of glioblastoma cells. However, miR-885-5p was observed to be downregulated in glioblastoma. Findings also indicated that HOXA-AS2 could negatively regulate miR-885-5p, thereby enhancing RBBP4 expression. Conclusion: Overall, HOXA-AS2 promoted the tumorigenesis of glioblastoma by targeting and regulating miR-885-5p to induce the expression of RBBP4.

scholarly journals Prodigiosin inhibits cholangiocarcinoma cell proliferation and induces apoptosis via suppressing SNAREs-dependent autophagy

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Dijie Zheng ◽  
Shiyu Chen ◽  
Kun Cai ◽  
Linhan Lei ◽  
Chunchen Wu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Western Blot ◽  
Cancer Biology ◽  
Bacterial Species ◽  
Inhibitory Effect ◽  
Cell Counting ◽  
Dependent Manner ◽  
Western Blot Assay

Abstract Background Prodigiosin (PG), a natural red pigment produced by numerous bacterial species, has been a eye-catching research point in recent years for its anticancer activity. However, the role of PG in the cancer biology of cholangiocarcinoma (CCA) remains vague. Methods The proliferation of CCA cells was detected by Cell Counting Kit-8(CCK-8), Colony formation assay and 5-ethynyl-2′-deoxyuridine (EdU) assay. Cell apoptosis was evaluated by flow cytometry assay and western blot assay. The effects of PG or SNAREs on cell autophagy were measured by autophagy flux assay and western blot assay. Xenograft mouse models were used to assess the role of PG in CCA cells in vivo. Results PG could inhibit the proliferation and viability of CCA cells in a concentration- and time-dependent manner via suppressing the late stage of autophagy. Mechanistically, PG inhibits the fusion of autophagosomes and lysosomes by blocking STX17 and SNAP29, components of soluble N-ethyl-maleimide-sensitive factor attachment protein receptors (SNAREs)complex. When STX17 and SNAP29 were overexpressed, the inhibitory effect of PG on CCA cells autophagy was relieved. In addition, PG showed obvious inhibitory effects on cancer cell viability but no toxic effects on organs in xenotransplantation models. Conclusion Taken together, our results demonstrated that PG inhibits CCA cell proliferation via suppressing SNAREs-dependent autophagy, implying that PG could be a potential chemotherapy drug for advanced CCA.

scholarly journals LncRNA HOXA-AS2 promotes glioblastoma carcinogenesis by targeting miR-885-5p/RBBP4 axis

2020 ◽  
Author(s):  
Jixin Shou ◽  
Haidong Gao ◽  
Sen Cheng ◽  
Bingbing Wang ◽  
Haibo Guan
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Regulatory Mechanism ◽  
Luciferase Assay ◽  
Pcr Analysis ◽  
Western Blot Assay ◽  
Qrt Pcr ◽  
Brdu Assay ◽  
Rip Assay

Abstract Background: LncRNA HOXA-AS2 has been found in the literature to deteriorate glioblastoma. However, its regulatory mechanism is yet to be fully investigated. Our study focused chiefly on the interaction and role of the HOXA-AS2/miR-885-5p/RBBP4 axis in the development of glioblastoma. Methods: qRT-PCR analysis was performed to detect the expression of lncRNA, miRNA and mRNA in glioblastoma tissues and cells. Dual-luciferase assay, RIP assay and RNA pull-down assay were later carried out to reveal the interactions among HOXA-AS2, miR-885-5p and RBBP4. After that, CCK-8 assay, BrdU assay, nude mice xenografting assay, western blot assay, and flow cytometry were carried out to analyze the effect of the HOXA-AS2/miR-885-5p/RBBP4 axis on glioblastoma samples. Results: HOXA-AS2 and RBBP4 were found to be overexpressed in glioblastoma. Experimental results showed that HOXA-AS2 and RBBP4 contributed to the tumorigenesis of glioblastoma cells. However, miR-885-5p was observed to be downregulated in glioblastoma. Findings also indicated that HOXA-AS2 could negatively regulate miR-885-5p, thereby enhancing RBBP4 expression. Conclusion: Overall, HOXA-AS2 promoted the tumorigenesis of glioblastoma by targeting and regulating miR-885-5p to induce the expression of RBBP4.

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