scholarly journals Identification of a Human Lactoferrin-Binding Protein in Gardnerella vaginalis

2000 ◽  
Vol 68 (6) ◽  
pp. 3443-3447 ◽  
Author(s):  
Gregory P. Jarosik ◽  
Carol Beth Land
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Binding Protein ◽  
Sole Source ◽  
Binding Activity ◽  
Human Lactoferrin ◽  
Gardnerella Vaginalis ◽  
Dot Blot ◽  
Heat Stable ◽  
Dot Blot Assay

ABSTRACT Previous studies have shown that Gardnerella vaginaliscan utilize iron-loaded human lactoferrin as a sole source of iron. In this study, G. vaginalis cells were shown to bind digoxigenin (DIG)-labeled human lactoferrin in a dot blot assay. Using the DIG-labeled human lactoferrin, a 120-kDa human lactoferrin-binding protein was detected by Western blot analysis of G. vaginalis proteins. The lactoferrin-binding activity of this protein was found to be heat stable. Competition studies indicated that this binding activity was specific for human lactoferrin. Treatment ofG. vaginalis cells with proteases suggested that this protein was surface exposed. An increase in lactoferrin binding by the 120-kDa protein was observed in G. vaginalis cells grown under iron-restrictive conditions, suggesting that this activity may be iron regulated.

Use of monoclonal and polyclonal antibodies to detect prostatic acid phosphatase by immunoblotting.

1986 ◽  
Vol 32 (10) ◽  
pp. 1832-1835 ◽  
Author(s):  
P C Patel ◽  
L Aubin ◽  
J Côte
Keyword(s):  
Monoclonal Antibodies ◽  
Acid Phosphatase ◽  
Western Blot ◽  
Polyclonal Antibodies ◽  
Prostatic Acid Phosphatase ◽  
The Other ◽  
Dot Blot ◽  
Western Blots ◽  
Dot Blot Assay ◽  
Polyclonal Antisera

Abstract We investigated two techniques of immunoblotting--the Western blot and the dot blot--for use in detecting prostatic acid phosphatase (PAP, EC 3.1.3.2). We used polyclonal antisera to human PAP, produced in rabbits by hyperimmunization with purified PAP, and PAP-specific monoclonal antibodies in the immunoenzymatic protocols. We conclude that PAP can be readily detected by Western blots with use of polyclonal antisera, but not with monoclonal antibodies. On the other hand, using a dot blot assay, we could easily detect PAP with both polyclonal and monoclonal antibodies.

scholarly journals The ErbB2/Neu/HER2 receptor is a new calmodulin-binding protein

2004 ◽  
Vol 381 (1) ◽  
pp. 257-266 ◽  
Author(s):  
Hongbing LI ◽  
Juan SÁNCHEZ-TORRES ◽  
Alan del CARPIO ◽  
Valentina SALAS ◽  
Antonio VILLALOBO
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Tyrosine Kinases ◽  
Western Blot Analysis ◽  
Binding Protein ◽  
Signalling Pathways ◽  
Breast Adenocarcinoma ◽  
Dependent Manner ◽  
Her2 Receptor

We have demonstrated previously that the EGFR (epidermal growth factor receptor) is a calmodulin (CaM)-binding protein. To establish whether or not the related receptor ErbB2/Neu/HER2 also binds CaM, we used human breast adenocarcinoma SK-BR-3 cells, because these cells overexpress this receptor thus facilitating the detection of this interaction. In the present paper, we show that ErbB2 could be pulled-down using CaM–agarose beads in a Ca2+-dependent manner, as detected by Western blot analysis using an anti-ErbB2 antibody. ErbB2 was also isolated by Ca2+-dependent CaM-affinity chromatography. We also demonstrate using an overlay technique with biotinylated CaM that CaM binds directly to the immunoprecipitated ErbB2. The binding of biotinylated CaM to ErbB2 depends strictly on the presence of Ca2+, since it was prevented by the presence of EGTA. Moreover, the addition of an excess of free CaM prevents the binding of its biotinylated form, demonstrating that this was a specific process. We excluded any interference with the EGFR, as SK-BR-3 cells express considerably lower levels of this receptor, and no detectable EGFR signal was observed by Western blot analysis in the immunoprecipitated ErbB2 preparations used to perform the overlay assays with biotinylated CaM. We also demonstrate that treating living cells with W7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide], a cell-permeant CaM antagonist, down-regulates ErbB2 phosphorylation, and show that W7 does not interfere non-specifically with the activity of ErbB tyrosine kinases. We also show that W7 inhibits the phosphorylation (activation) of both ERK1/2 (extracellular-signal-regulated kinases 1 and 2) and Akt/PKB (protein kinase B), in accordance with the inhibition observed in ErbB2 phosphorylation. In contrast, W7 treatment increased the phosphorylation (activation) of CREB (cAMP-response-element-binding protein) and ATF1 (activating transcription factor-1), two Ca2+-sensitive transcription factors that operate downstream of these ErbB2 signalling pathways, most likely because of the absence of calcineurin activity. We conclude that ErbB2 is a new CaM-binding protein, and that CaM plays a role in the regulation of this receptor and its downstream signalling pathways in vivo.

scholarly journals Effect of Immunization with Recombinant OspA on Serologic Tests for Lyme Borreliosis

2001 ◽  
Vol 8 (1) ◽  
pp. 79-84 ◽  
Author(s):  
Paul T. Fawcett ◽  
Carlos D. Rose ◽  
Sandra M. Budd ◽  
Kathleen M. Gibney
Keyword(s):  
Western Blot ◽  
Lyme Borreliosis ◽  
Vaccine Recipient ◽  
Enzyme Linked Immunosorbent Assay ◽  
Test Results ◽  
Dot Blot ◽  
Western Blot Assay ◽  
Serologic Tests ◽  
Human Volunteers ◽  
Dot Blot Assay

ABSTRACT This study evaluated the effects of vaccination with OspA on the use of serologic tests as aids in the diagnosis of Lyme borreliosis. Sera from control and OspA-immunized mice and from OspA-immunized human volunteers were tested for serologic reactivity to Borrelia burgdorferi. Testing was performed with samples obtained prior to administration of vaccine and at 30 days following administration of an initial and a second dose of OspA vaccine. The assays used to assess serologic reactivity included an in-house-developed enzyme-linked immunosorbent assay (ELISA), an in-house-developed Western blot assay, two commercial Western blot tests, and a commercially available dot blot assay. Data obtained from this study demonstrate that immunization with the OspA vaccine will cause ELISA to yield positive results (as reported previously) for the majority of vaccine recipients. Results obtained from Western blot analysis indicate that vaccination with recombinant OspA induces production of antibodies which bind to several different borrelial proteins. The degree of reactivity detected by Western blotting varied greatly between the three assays used. The in-house assay showed the least reactivity, while one commercial Western blot test actually yielded positive test results for infection with B. burgdorferi. The usefulness of all three Western blot assays for the diagnosis of potential infection in a vaccine recipient is severely limited by the extensive reactivity caused by vaccination alone. Antibodies produced in response to OspA vaccination did not significantly affect the performance of the dot blot test; thus, it could provide a reliable means to test for infection withB. burgdorferi in OspA vaccine recipients.

Abstract 9781: Deletion of Prolyl Hydroxylase Domain Protein 2 in Myeloid Lineage suppressed Experimental Abdominal Aortic Aneurysm via NF-κB Inactivation

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Tomotake Tokunou ◽  
Chikahiro Sankoda ◽  
Aya Watanabe ◽  
Yusuke Takahara ◽  
Hiroshi Kojima ◽  
...  
Keyword(s):  
Abdominal Aortic Aneurysm ◽  
Aortic Aneurysm ◽  
Protein Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Peritoneal Macrophages ◽  
Binding Activity ◽  
Abdominal Aortic ◽  
Domain Protein

Background: Macrophage migration to the vessels is important for vascular inflammation and induces vascular degenerative diseases. Macrophages secrete matrix metalloprotainases (MMPs), which activate many cytokines and digest extracellular matrix of aorta. MMPs play an important role in the progression of aortic aneurysm. We previously reported that non-specific prolyl hydroxylase domain protein (PHD) inhibitor, Cobalt chloride (CoCl2), suppressed MMP-2 and -9 expression and attenuated experimental abdominal aortic aneurysm (AAA) formation in mice. In this report we investigated the myeloid specific PHD2 knockout effect on MMPs expression and aneurysm formation. Methods: Myeloid specific PHD2 conditional knockout (MyPHD2KO) mice were generated. Experimental AAA was induced by periaortic application of Calcium chloride (CaCl2) for 6 weeks. In vitro Lipopolysaccharide (LPS, 100ng/ml) was used to induce MMP-2 and MMP-9 expression with peritoneal macrophages. MMPs mRNA, protein expression and protein activity were analyzed by qRT-PCR, Western blot analysis and Zymography, respectively. ELISA-based NF-κB p65 Transcription Factor Assay was used to examine NF-κB p65 binding activity with consensus DNA binding site. Results: CaCl2-induced AAA was suppressed with MyPHD2KO mice (max diameter of aneurysm: 1.03mm±0.14mm in MyPHD2KO group, 1.63mm±0.34mm in Control AAA group, p<0.01). In peritoneal macrophages CoCl2 reduced LPS-induced MMP-2 and MMP-9 mRNA and protein expression. PHD2 deletion in peritoneal macrophages from MyPHD2KO mice also suppressed LPS-induced MMP-2 and MMP-9 mRNA, protein expression and activity. PHD2 deletion suppressed LPS-induced NF-κB p65 phosphorylation via IκBα stabilization by Western blot analysis. NF-κB p65 binding activity was suppressed in MyPHD2KO macrophages (p<0.01 vs control). Conclusion: Deletion of PHD2 in myeloid lineage attenuated MMPs expression by NF-κB inactivation and suppressed AAA formation. PHD2 in macrophage may be a novel target for cardiovascular disease treatment.

scholarly journals Differential interactions of human kallikrein-binding protein and α1-antitrypsin with human tissue kallikrein

1990 ◽  
Vol 267 (1) ◽  
pp. 79-84 ◽  
Author(s):  
L M Chen ◽  
L Chao ◽  
R K Mayfield ◽  
J Chao
Keyword(s):  
Complex Formation ◽  
Human Serum ◽  
Human Tissue ◽  
Binding Protein ◽  
Binding Activity ◽  
Stable Complex ◽  
Tissue Kallikrein ◽  
Heat Stable ◽  
Sds Page ◽  
Alpha 1 Antitrypsin

The characteristics of a new kallikrein-binding protein in human serum and its activities were studied. Both the kallikrein-binding protein and alpha 1-antitrypsin form 92 kDa SDS-stable and heat-stable complexes with human tissue kallikrein. In non-SDS/PAGE, the mobility of these complexes differ. Complex-formation between kallikrein and the binding protein is inhibited by heparin, whereas that between kallikrein and alpha 1-antitrypsin is heparin-resistant. In normal or alpha 1-antitrypsin-deficient-serum, the amount of 92 kDa SDS-stable complex formed upon addition of kallikrein is not related to serum alpha 1-antitrypsin levels. The rate of complex-formation between kallikrein and the binding protein is 12 times higher than that between kallikrein and alpha 1-antitrypsin. Purified alpha 1-antitrypsin, which exhibits normal elastase binding, has a kallikrein-binding activity less than 5% of that of serum. Binding of tissue kallikrein in serum is not inhibited by increasing elastase concentrations, and elastase binding in serum is not inhibited by excess tissue kallikrein. A specific monoclonal antibody to human alpha 1-antitrypsin does not bind to either 92 kDa endogenous or exogenous kallikrein complexes isolated from human serum. The studies demonstrate a new tissue kallikrein-binding protein, distinct from alpha 1-antitrypsin, is present in human serum.

Adult and fetal human mesangial cells interact with specific laminin domains

1991 ◽  
Vol 261 (4) ◽  
pp. F688-F695
Author(s):  
B. S. Weeks ◽  
J. B. Kopp ◽  
S. Horikoshi ◽  
F. B. Cannon ◽  
M. Garrett ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mesangial Cell ◽  
Mesangial Cells ◽  
Cell Attachment ◽  
Binding Protein ◽  
Laminin Binding Protein ◽  
Laminin Binding

Mesangial cells are centrally located pericytes in the renal glomerulus. They are surrounded by an extracellular matrix and directly contact the glomerular basement membrane in vivo. Because these interactions are critical for renal development and function, we have studied human mesangial cell interactions with laminin, a major adhesive component of basement membranes present in the extracellular matrix of the mesangium. Human fetal and adult mesangial cell attachment was stimulated by both laminin and the laminin-derived synthetic peptides YIGSR-NH2, CQAGTFALRGDNPQG-NH2, and CIKVAVS-NH2. Furthermore, mesangial cells spread on laminin as well as on both the RGD-containing and CIKVAVS peptides. When added in solution, all three peptides inhibited mesangial cell attachment to laminin, and the latter two peptides inhibited mesangial cell spreading on laminin. Laminin affinity column chromatography demonstrated several low-molecular-mass laminin-binding proteins ranging from between 35 and 42 kDa, which predominated in fetal mesangial cells, whereas a higher molecular mass laminin-binding protein of 65 kDa was predominant in adult mesangial cells. Western blot analysis with an anti-32-kDa laminin-binding protein antibody showed increased expression of both 31- and 42-kDa proteins in fetal mesangial cells when compared with the adult. The antisera to the 32-kDa laminin-binding protein also inhibited fetal mesangial spreading on the CIKVAVS peptide. Western blot analysis with an anti-67-kDa laminin-binding protein antibody revealed a 110-kDa protein in adult mesangial cells that was not present in fetal mesangial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Active domains of salivary statherin on apatitic surfaces for binding to Fusobacterium nucleatum cells

Microbiology ◽  
2004 ◽  
Vol 150 (7) ◽  
pp. 2373-2379 ◽  
Author(s):  
Shinichi Sekine ◽  
Kosuke Kataoka ◽  
Muneo Tanaka ◽  
Hideki Nagata ◽  
Toru Kawakami ◽  
...  
Keyword(s):  
Inhibitory Activity ◽  
Fusobacterium Nucleatum ◽  
Inhibitory Effect ◽  
Salivary Protein ◽  
Binding Activity ◽  
Peptide Fragments ◽  
Dot Blot ◽  
Tooth Surfaces ◽  
Dot Blot Assay ◽  
Active Segment

Fusobacterium nucleatum can bind to saliva-coated tooth surfaces. However, the nature of the domains of salivary protein that interact with F. nucleatum remains unclear. The ability of individual proteins in human submandibular-sublingual saliva (HSMSL) to bind F. nucleatum cells was examined by dot blot assay; statherin displayed the strongest binding activity. Statherin binding sites were determined based on binding of 125I-labelled F. nucleatum to statherin-coated hydroxyapatite (sHAP) beads via inhibition assays using synthetic analogous peptide fragments of whole statherin. Analogous peptides corresponding to residues 19–26 and 32–39 of statherin inhibited binding by 77 % and 68 %, respectively. Synthetic peptides were also prepared by serial deletions of individual residues from N- and C-termini of the peptides GPYQPVPE (aa 19–26) and QPYQPQYQ (aa 32–39). The inhibitory effects of peptides YQPVPE (aa 21–26) and PYQPQYQ (aa 33–39) were very similar to those of GPYQPVPE and QPYQPQYQ, respectively. However, additional deletion of residues resulted in significant reduction of the inhibitory effect. Alanine-scan analysis of YQPVPE revealed that all tested peptides retained inhibitory activity; only YAPVPE exhibited significantly decreased inhibitory activity. These findings suggest that YQPVPE and PYQPQYQ may represent the minimal active segments of statherin for binding to F. nucleatum; moreover, Gln may be a key amino acid in the active segment.

scholarly journals Ribonucleic acid-binding protein CPSF6 promotes glycolysis and suppresses apoptosis in hepatocellular carcinoma cells by inhibiting the BTG2 expression

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Yang Liu ◽  
Hongbo Zou ◽  
Qichao Xie ◽  
Lan Zou ◽  
Rui Kong ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Rna Binding ◽  
Binding Protein ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Glucose Assay ◽  
Normal Hepatocyte ◽  
Hcc Cells

AbstractHepatocellular carcinoma (HCC) is currently the sixth most common malignancy and the second major cause of tumor-related deaths in the world. This study aimed to investigate the role of cleavage and polyadenylation factor-6 (CPSF6) and B-cell translocation gene 2 (BTG2) in regulating the glycolysis and apoptosis in HCC cells. The RNA and protein expression of CPSF6 and BTG2 in normal hepatocyte and HCC were, respectively, detected by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis and Western blot analysis. The viability and apoptosis of transfected Huh-7 cells were, respectively, analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay. The expression of apoptosis-related proteins and HK-2 in transfected Huh-7 cells was also detected by Western blot analysis. The levels of glucose and lactate in the culture supernatant of transfected Huh-7 cells were, respectively, detected with the glucose assay kit and lactate assay kit. The interaction of CPSF6 and BTG2 was confirmed by RNA binding protein immunoprecipitation (RIP) assay. As a result, CPSF6 expression was increased while BTG2 expression was decreased in Huh-7 cells. Interference with CPSF6 suppressed the viability and glycolysis, and promoted the apoptosis of Huh-7 cells. Furthermore, CPSF6 interacted with BTG2 and interference with CPSF6 upregulated the BTG2 expression and inhibited the protein kinase B (AKT)/extracellular signal-regulated kinase (ERK)/nuclear factor (NF)-κB pathway. Interference with BTG2 could partially reverse the above cell changes caused by interference with CPSF6. In conclusion, CPSF6 inhibited the BTG2 expression to promote glycolysis and suppress apoptosis in HCC cells by activating AKT/ERK/NF-κB pathway.

scholarly journals Universal Immunoprobe for (Per)Chlorate-Reducing Bacteria

2002 ◽  
Vol 68 (6) ◽  
pp. 3108-3113 ◽  
Author(s):  
Susan M. O'Connor ◽  
John D. Coates
Keyword(s):  
Blot Analysis ◽  
Polyclonal Antibodies ◽  
Binding Activity ◽  
Dot Blot ◽  
Chlorite Dismutase ◽  
Couple Growth ◽  
Cell Lysates ◽  
Phylogenetic Affiliation ◽  
Dot Blot Analysis ◽  
Reducing Bacteria

ABSTRACT Recent studies in our lab have demonstrated the ubiquity and diversity of microorganisms which couple growth to the reduction of chlorate or perchlorate [(per)chlorate] under anaerobic conditions. We identified two taxonomic groups, the Dechloromonas and the Dechlorosoma groups, which represent the dominant (per)chlorate-reducing bacteria (ClRB) in the environment. As part of these studies we demonstrated that chlorite dismutation is a central step in the reductive pathway of (per)chlorate that is common to all ClRB and which is mediated by the enzyme chlorite dismutase (CD). Initial studies on CD suggested that this enzyme is highly conserved among the ClRB, regardless of their phylogenetic affiliation. As such, this enzyme makes an ideal target for a probe specific for these organisms. Polyclonal antibodies were commercially raised against the purified CD from the ClRB Dechloromonas agitata strain CKB. The obtained antiserum was deproteinated by ammonium sulfate precipitation, and the antigen binding activity was assessed using dot blot analysis of a serial dilution of the antiserum. The titers obtained with purified CD indicated that the antiserum had a high affinity for the CD enzyme, and activity was observed in dilutions as low as 10−6 of the original antiserum. The antiserum was active against both cell lysates and whole cells of D. agitata, but only if the cells were grown anaerobically with (per)chlorate. No response was obtained with aerobically grown cultures. In addition to D. agitata, dot blot analysis employed with both whole-cell suspensions and cell lysates of several diverse ClRB representing the alpha, beta, and gamma subclasses of Proteobacteria tested positive regardless of phylogenetic affiliation. Interestingly, the dot blot response obtained for each of the ClRB cell lysates was different, suggesting that there may be some differences in the antigenic sites of the CD protein produced in these organisms. In general, no reactions were observed with cells or cell lysates of the organisms closely related to the ClRB which could not grow by (per)chlorate reduction. These studies have resulted in the development of a highly specific and sensitive immunoprobe based on the commonality of the CD enzyme in ClRB which can be used to assess dissimilatory (per)chlorate-reducing populations in environmental samples regardless of their phylogenetic affiliations.

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