scholarly journals Concanavalin A receptor ‘tipping’ in Tetrahymena and its relationship to cell adhesion during conjugation

Development ◽  
1988 ◽  
Vol 102 (4) ◽  
pp. 699-708 ◽  
Author(s):  
J. Wolfe ◽  
S. Feng
Keyword(s):  
Cell Adhesion ◽  
Western Blot ◽  
Kinetic Analysis ◽  
Concanavalin A ◽  
Banding Pattern ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Binding Proteins ◽  
Mating Types ◽  
Receptor Localization

Shortly after mixing cells of complementary mating types of Tetrahymena, the cells develop the ability to pair, a process inhibited by ConA, and the region joining the cells becomes ringed with ConA receptors. This study examines the arrival of ConA receptors at the conjugation junction by looking at cells in the period between mixing and pairing. By brief incubations with F-ConA at intervals after mixing, it was ascertained that some cells had fluorescent tips as early as 15min. A kinetic analysis revealed that ‘tipping’ occurs in a manner that appears to be related to subsequent cell pairing. Cytoskeletal frameworks (CFs) were isolated under conditions in which ConA receptors remain attached. Western blot analysis of these structures revealed four major and several minor ConA-binding proteins. However, between mixing and the establishment of over 80% paired cells, changes occurring in the banding pattern were slight. This indicates that new populations of ConA receptors are not produced to any great extent after mixing. Head- on examination of CFs showed that it was possible to monitor simultaneously the process of tip transformation (widening of the nonciliated area of the tip) and ConA-receptor localization. ConA receptors originate posterior to the tip, begin to occupy the surface of the tip in clusters as the tip widens and eventually coat the transformed tip. Finally, as cells join pairs, the receptors relocate to a ring around the conjugation junction. These data suggest that ConA receptors accumulate at the anterior tips and then concentrate at the edge of the junction. This could provide a mechanism for controlling cell-cell adhesion.

Overexpression of the EphB4 Receptor Contributes to Imatinib Resistance in Chronic Myeloid Leukemia Through Regulating Mlcp and VAV1

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4421-4421
Author(s):  
Liu Xiaoli ◽  
Jinfang Zhang ◽  
Qingfeng Du ◽  
Na Xu ◽  
Lulu Xu ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Chronic Myeloid Leukemia ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Myeloid Leukemia ◽  
K562 Cells ◽  
Differential Expression Genes

Abstract Abstract 4421 Objective: To study the role of EphB4 in imatinib (IM) resistant chronic myeloid leukemia (CML) and investigate the mechanism. Methods: We derived IM-resistant cells, K562-R cells, from wild K562 cells under gradually increasing IM concentrations. We analysed expression level of EphB4 in CML patients, wild K562 and K562-R cell lines by real-time reverse transcription PCR and Western blot analysis. Then we established stable under-expressing EphB4 cell (K562-R-EphB4-sh) lines. We analysed the sensitive for IM of K562, K562-R, K562-R-EphB4-sh cell lines by CCK8 assay. Microarray analysis was used to screen differential expression genes between K562-R and K562-R-EphB4-sh cell lines. Results: The mRNA and protein of EphB4 were significantly increased in IM resistant CML patients compared to IM sensitive CML patients (p<0.05). The Similar results were observed in K562-R and K562 cells (p<0.01). To analyze the role of EphB4 in IM resistance, EphB4 was knocked down with shRNA expressed by pLL3.7 lentivirus vector. We established stable under-expressing EphB4 cell line K562-R-EphB4-sh. RT-PCR and western blot analysis showed that mRNA and protein expression of EphB4 in K562-R-EphB4-sh cells were reduced (p<0.05). CCK8 assay found K562 cells (IC50 0.1207±0.0234μM), K562-R-EphB4-sh cells (IC50 0.7228±0.04752μM) were sensitive to IM but K562-R (IC50 2.8101±0.04674μM) still showed IM resistance (p<0.05). Those suggested K562-R-EphB4-sh cells resensitize to IM when the expression of EphB4 was down regulated. However, these cells were still less sensitive than K562 cells. Microarray analysis between K562-R and K562-R-EphB4-sh cell lines found 641 differential expression genes, most of them were related to cell adhesion and cell cytoskeleton. We confirmed MLCP and VAV1 were down regulated in K562-R-EphB4-sh cells compared to K562-R cell lines by western blot analysis. Conclusion: Our study suggest EphB4 receptor contributes to IM-resistant in CML through regulating cell adhesion molecular MLCP and VAV1, which may provide new biomarkers and contribute to] developping new drugs for the disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Structural and Antigenic Characteristics of Streptococcus sobrinus Glucan Binding Proteins

1998 ◽  
Vol 66 (11) ◽  
pp. 5565-5569 ◽  
Author(s):  
Daniel J. Smith ◽  
William F. King ◽  
Christine D. Wu ◽  
Bella I Shen ◽  
Martin A. Taubman
Keyword(s):  
Western Blot ◽  
Streptococcus Mutans ◽  
Mass Spectroscopy ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Binding Proteins ◽  
Antigenic Relationship ◽  
Streptococcus Sobrinus ◽  
Proteolytic Fragment

ABSTRACT Three purified glucan binding proteins (GBP-2, GBP-3, and GBP-5) from Streptococcus sobrinus 6715 were compared structurally by mass spectroscopy of tryptic fragments and antigenically by Western blot analysis with rat antisera to each GBP or to peptides containing putative glucan binding epitopes of mutans streptococcal glucosyltransferases. Structural and antigenic analyses indicated that GBP-3 and GBP-5 are very similar but that both are essentially unrelated to GBP-2. None of theseS. sobrinus GBPs appeared to have a strong antigenic relationship with GBPs from Streptococcus mutans. Thus,S. sobrinus GBP-2 and GBP-3 appear to be distinct proteins with potentially different functions. S. sobrinusGBP-5 may be a proteolytic fragment of GBP-3, or, alternatively, the genes coding for these proteins may be closely related.

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.

Synthesis and Characterization of Quinoline-3-Carboxamide Derivatives as Inhibitors of the ATM Kinase

2020 ◽  
Vol 20 (23) ◽  
pp. 2070-2079
Author(s):  
Srimadhavi Ravi ◽  
Sugata Barui ◽  
Sivapriya Kirubakaran ◽  
Parul Duhan ◽  
Kaushik Bhowmik
Keyword(s):  
Western Blot ◽  
Cancer Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cancer Cell Lines ◽  
Atm Kinase ◽  
Cytotoxicity Studies

Background: The importance of inhibiting the kinases of the DDR pathway for radiosensitizing cancer cells is well established. Cancer cells exploit these kinases for their survival, which leads to the development of resistance towards DNA damaging therapeutics. Objective: In this article, the focus is on targeting the key mediator of the DDR pathway, the ATM kinase. A new set of quinoline-3-carboxamides, as potential inhibitors of ATM, is reported. Methods: Quinoline-3-carboxamide derivatives were synthesized and cytotoxicity assay was performed to analyze the effect of molecules on different cancer cell lines like HCT116, MDA-MB-468, and MDA-MB-231. Results: Three of the synthesized compounds showed promising cytotoxicity towards a selected set of cancer cell lines. Western Blot analysis was also performed by pre-treating the cells with quercetin, a known ATM upregulator, by causing DNA double-strand breaks. SAR studies suggested the importance of the electron-donating nature of the R group for the molecule to be toxic. Finally, Western-Blot analysis confirmed the down-regulation of ATM in the cells. Additionally, the PTEN negative cell line, MDA-MB-468, was more sensitive towards the compounds in comparison with the PTEN positive cell line, MDA-MB-231. Cytotoxicity studies against 293T cells showed that the compounds were at least three times less toxic when compared with HCT116. Conclusion: In conclusion, these experiments will lay the groundwork for the evolution of potent and selective ATM inhibitors for the radio- and chemo-sensitization of cancer cells.

The Antitumor Effects of Britanin on Hepatocellular Carcinoma Cells and its Real-Time Evaluation by In Vivo Bioluminescence Imaging

2020 ◽  
Vol 20 (9) ◽  
pp. 1147-1156
Author(s):  
Hanrui Li ◽  
GeTao Du ◽  
Lu Yang ◽  
Liaojun Pang ◽  
Yonghua Zhan
Keyword(s):  
Hepatocellular Carcinoma ◽  
Western Blot ◽  
Blot Analysis ◽  
Tumor Size ◽  
Hepg2 Cells ◽  
Western Blot Analysis ◽  
Bioluminescence Imaging ◽  
Antitumor Effects ◽  
In Vivo Bioluminescence Imaging

Background: Hepatocellular carcinoma is cancer with many new cases and the highest mortality rate. Chemotherapy is the most commonly used method for the clinical treatment of hepatocellular carcinoma. Natural products have become clinically important chemotherapeutic drugs due to their great potential for pharmacological development. Many sesquiterpene lactone compounds have been proven to have antitumor effects on hepatocellular carcinoma. Objective: Britanin is a sesquiterpene lactone compound that can be considered for the treatment of hepatocellular carcinoma. The present study aimed to investigate the antitumor effect of britanin. Methods: BEL 7402 and HepG2 cells were used to study the cytotoxicity and antitumor effects of britanin. Preliminary studies on the nuclear factor kappa B pathway were conducted by western blot analysis. A BEL 7402-luc subcutaneous tumor model was established for the in vivo antitumor studies of britanin. In vivo bioluminescence imaging was conducted to monitor changes in tumor size. Results: The results of the cytotoxicity analysis showed that the IC50 values for britanin in BEL 7402 and HepG2 cells were 2.702μM and 6.006μM, respectively. The results of the colony formation demonstrated that the number of cells in a colony was reduced significantly after britanin treatment. And the results of transwell migration assays showed that the migration ability of tumor cells was significantly weakened after treatment with britanin. Tumor size measurements and staining results showed that tumor size was inhibited after britanin treatment. The western blot analysis results showed the inhibition of p65 protein expression and reduced the ratio of Bcl-2/Bax after treatment. Conclusion: A series of in vitro and in vivo experiments demonstrated that britanin had good antitumor effects and provided an option for hepatocellular carcinoma treatment.

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 Protocol for determining protein cysteine thiol redox status using western blot analysis

2021 ◽  
Vol 2 (2) ◽  
pp. 100566
Author(s):  
Bikram Datt Pant ◽  
Sunhee Oh ◽  
Kirankumar S. Mysore
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Redox Status ◽  
Thiol Redox

Visfatin facilitates gastric cancer malignancy by targeting snai1 via the NF-κB signaling

2021 ◽  
pp. 096032712110061
Author(s):  
D Cao ◽  
L Chu ◽  
Z Xu ◽  
J Gong ◽  
R Deng ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Migration ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Epithelial Mesenchymal Transition ◽  
Migration And Invasion ◽  
Mesenchymal Transition ◽  
Cell Migration And Invasion ◽  
Protein Levels

Background: Visfatin acts as an oncogenic factor in numerous tumors through a variety of cellular processes. Visfatin has been revealed to promote cell migration and invasion in gastric cancer (GC). Snai1 is a well-known regulator of EMT process in cancers. However, the relationship between visfatin and snai1 in GC remains unclear. The current study aimed to explore the role of visfatin in GC. Methods: The RT-qPCR and western blot analysis were used to measure RNA and protein levels, respectively. The cell migration and invasion were tested by Trans-well assays and western blot analysis. Results: Visfatin showed upregulation in GC cells. Additionally, Visfatin with increasing concentration facilitated epithelial-mesenchymal transition (EMT) process by increasing E-cadherin and reducing N-cadherin and Vimentin protein levels in GC cells. Moreover, endogenous overexpression and knockdown of visfatin promoted and inhibited migratory and invasive abilities of GC cells, respectively. Then, we found that snai1 protein level was positively regulated by visfatin in GC cells. In addition, visfatin activated the NF-κB signaling to modulate snai1 protein expression. Furthermore, the silencing of snai1 counteracted the promotive impact of visfatin on cell migration, invasion and EMT process in GC. Conclusion: Visfatin facilitates cell migration, invasion and EMT process by targeting snai1 via the NF-κB signaling, which provides a potential insight for the treatment of GC.

scholarly journals OC125, M11 and OV197 Epitopes Are Not Uniformly Distributed in the Tandem-Repeat Region of CA125 and Require the Entire SEA Domain

2013 ◽  
Vol 34 (4) ◽  
pp. 257-267 ◽  
Author(s):  
Alessandro Bressan ◽  
Francesca Bozzo ◽  
Carlo Alberto Maggi ◽  
Monica Binaschi
Keyword(s):  
Ovarian Cancer ◽  
Monoclonal Antibodies ◽  
Western Blot ◽  
Tandem Repeat ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Human Cancer ◽  
Ovarian Cancer Cells ◽  
Repeat Region ◽  
Tandem Repeat Region

The human cancer antigen 125 (CA125) is over-expressed in epithelial ovarian cancer cells and it plays a role in the pathogenesis of ovarian cancer. This protein presents a repeat region containing up to sixty tandem repeat units. The anti-CA125 monoclonal antibodies have been previously classified into three groups: two major families, the OC125-like antibodies and M11-like antibodies, and a third group, the OV197-like antibodies. A model in which a single repeat unit contains all the epitopes for these antibodies has been also proposed, even if their exact position is still undetermined. In the present work, the affinities of the monoclonal antibodies, representative of the three families, have been investigated for different CA125-recombinant repeats through Western blot analysis. Different patterns of antibody recognition for the recombinant repeats show that CA125 epitopes are not uniformly distributed in the tandem repeat region of the protein. The minimal region for the recognition of these antibodies has been also individuated in the SEA domain through the subcloning of deleted sequences of the highly recognized repeat-25 (R-25), their expression as recombinant fragments inE. coliand Western blot analysis. Obtained data have been further confirmed by ELISA using the entire R-25 as coating antigen.

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