LL-37 Recruits Immunosuppressive Regulatory T Cells to Ovarian Tumors

DTIC Science & Technology

2009-11-01

receptor. Western blot analysis of MSC lysates showed that ERK-1 and -2 are robustly phosphorylated beginning 10 minutes after LL-37 treatment and...Carretero, Escamez et al. 2008; von Haussen, Koczulla et al. 2008). Western blot analysis of LL-37-treated SK-OV-3 cell lysates showed the robust...mesenchymal stem cells in the treatment of gliomas ." Cancer Res 65(8): 3307-18. Studeny, M., F. C. Marini, et al. (2004). "Mesenchymal stem cells: potential

A Proteomics Approach to the Protein Normalization Problem: Selection of Unvarying Proteins for MS-Based Proteomics and Western Blotting.

PubMed

Wiśniewski, Jacek R; Mann, Matthias

2016-07-01

Proteomics and other protein-based analysis methods such as Western blotting all face the challenge of discriminating changes in the levels of proteins of interest from inadvertent changes in the amount loaded for analysis. Mass-spectrometry-based proteomics can now estimate the relative and absolute amounts of thousands of proteins across diverse biological systems. We reasoned that this new technology could prove useful for selection of very stably expressed proteins that could serve as better loading controls than those traditionally employed. Large-scale proteomic analyses of SDS lysates of cultured cells and tissues revealed deglycase DJ-1 as the protein with the lowest variability in abundance among different cell types in human, mouse, and amphibian cells. The protein constitutes 0.069 ± 0.017% of total cellular protein and occurs at a specific concentration of 34.6 ± 8.7 pmol/mg of total protein. Since DJ-1 is ubiquitous and therefore easily detectable with several peptides, it can be helpful in normalization of proteomic data sets. In addition, DJ-1 appears to be an advantageous loading control for Western blot that is superior to those used commonly used, allowing comparisons between tissues and cells originating from evolutionarily distant vertebrate species. Notably, this is not possible by the detection and quantitation of housekeeping proteins, which are often used in the Western blot technique. The approach introduced here can be applied to select the most appropriate loading controls for MS-based proteomics or Western blotting in any biological system.

A research design for the quantification of the neuropeptides substance p and calcitonin gene-related Peptide in rat skin using Western blot analysis.

PubMed

Lapin, Guilherme Abbud Franco; Hochman, Bernardo; Nishioka, Michele Akemi; Maximino, Jessica Ruivo; Chadi, Gerson; Ferreira, Lydia Masako

2015-06-01

To describe and standardize a protocol that overcomes the technical limitations of Western blot (WB) analysis in the quantification of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) following nociceptive stimuli in rat skin. Male Wistar rats (Rattus norvegicus albinus) weighing 250 to 350 g were used in this study. Elements of WB analysis were adapted by using specific manipulation of samples, repeated cycles of freezing and thawing, more thorough maceration, and a more potent homogenizer; increasing lytic reagents; promoting greater inhibition of protease activity; and using polyvinylidene fluoride membranes as transfer means for skin-specific protein. Other changes were also made to adapt the WB analysis to a rat model. University research center. Western blot analysis adapted to a rat model. This research design has proven effective in collecting and preparing skin samples to quantify SP and CGRP using WB analysis in rat skin. This study described a research design that uses WB analysis as a reproducible, technically accessible, and cost-effective method for the quantification of SP and CGRP in rat skin that overcomes technical biases.

Anti-RAINBOW dye-specific antibodies as universal tools for the visualization of prestained protein molecular weight markers in Western blot analysis.

PubMed

Schüchner, Stefan; Andorfer, Peter; Mudrak, Ingrid; Ogris, Egon

2016-08-17

Western blotting is one of the most widely used techniques in molecular biology and biochemistry. Prestained proteins are used as molecular weight standards in protein electrophoresis. In the chemiluminescent Western blot analysis, however, these colored protein markers are invisible leaving researchers with the unsatisfying situation that the signal for the protein of interest and the signal for the markers are not captured simultaneously and have to be merged in an error-prone step. To allow the simultaneous detection of marker proteins we generated monoclonal antibodies specific for the protein dyes. To elicit a dye rather than protein specific immune response we immunized mice sequentially with dye-carrier protein complexes, in which a new carrier protein was used for each subsequent immunization. Moreover, by sequentially immunizing with dye-carrier protein complexes, in which different but structurally related dyes were used, we could also generate an antibody, termed anti-RAINBOW, that cross-reacted even with structurally related dyes not used in the immunizations. Our novel antibodies represent convenient tools for the simultaneous Western blot detection of commercially available prestained marker proteins in combination with the detection of any specific protein of interest. These antibodies will render obsolete the anachronistic tradition of manually charting marker bands on film.

Anti-RAINBOW dye-specific antibodies as universal tools for the visualization of prestained protein molecular weight markers in Western blot analysis

PubMed Central

Schüchner, Stefan; Andorfer, Peter; Mudrak, Ingrid; Ogris, Egon

2016-01-01

Western blotting is one of the most widely used techniques in molecular biology and biochemistry. Prestained proteins are used as molecular weight standards in protein electrophoresis. In the chemiluminescent Western blot analysis, however, these colored protein markers are invisible leaving researchers with the unsatisfying situation that the signal for the protein of interest and the signal for the markers are not captured simultaneously and have to be merged in an error-prone step. To allow the simultaneous detection of marker proteins we generated monoclonal antibodies specific for the protein dyes. To elicit a dye rather than protein specific immune response we immunized mice sequentially with dye-carrier protein complexes, in which a new carrier protein was used for each subsequent immunization. Moreover, by sequentially immunizing with dye-carrier protein complexes, in which different but structurally related dyes were used, we could also generate an antibody, termed anti-RAINBOW, that cross-reacted even with structurally related dyes not used in the immunizations. Our novel antibodies represent convenient tools for the simultaneous Western blot detection of commercially available prestained marker proteins in combination with the detection of any specific protein of interest. These antibodies will render obsolete the anachronistic tradition of manually charting marker bands on film. PMID:27531616

Deregulation of miRNAs Contributes to Development and Progression of Prostate Cancer

DTIC Science & Technology

2011-09-01

blocking with 5% non- fat dry milk in Tris-buffered saline/0.05% Tween 20 (TBST), the membrane was incubated with a specific primary antibody...androgen R1881, and p53 protein was detected by Western blot analysis. Consistent with our previous observation (2), untreated LNCaP-R273H cells expressed...Cell Biochem 2009; 106(3): 363–371. 7 Appendices Figure 1. Western blot analysis of p53 protein in 5.0 nM R1881-treated LNCaP cells and LNCaP-R273H

Isolation and purification assay of ex vivo photosystem II D1 protein toward integrated biointeraction analysis.

PubMed

Muktiono, B; Schulten, C; Heemken, O; Gandrass, J; Prange, A; Schnabl, H; Cerboncini, C

2008-02-01

Protein extracts of photosystem II were prepared from leaf chloroplasts of different plant species by fast and nondenaturing methods. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot analysis of the proteins obtained showed that the extracts were enriched by D1 proteins, which appeared putatively in association with the 33-kDa oxygen-evolving-complex subunits. In further isolation steps D1 proteins were purified using salt-gradient chromatography (fast protein liquid chromatography) and characterized by western blot and mass spectrometry.

Identification of Causes and Treatments for Chronic Pain in a Model of Gulf War Illness

DTIC Science & Technology

2017-10-01

saline delivered. Euthanasia and necropsy at days 5 and 20 post acidic/normal saline, and tissue analysis performed ( ELISA , Western Blot, LC-MS...performed ( ELISA , Western Blot, LC-MS). Milestone 3: the association between GWI-induced musculoskeletal pain and neuroinflammation, as well as the...thresholds. Serum samples analysed by ELISA . Major Task 7: Acidic/normal saline administered 180 days after final DFP injection; pharmacological reversal

Use of a sensitive EnVision +-based detection system for Western blotting: avoidance of streptavidin binding to endogenous biotin and biotin-containing proteins in kidney and other tissues.

PubMed

Banks, Rosamonde E; Craven, Rachel A; Harnden, Patricia A; Selby, Peter J

2003-04-01

Western blotting remains a central technique in confirming identities of proteins, their quantitation and analysis of various isoforms. The biotin-avidin/streptavidin system is often used as an amplification step to increase sensitivity but in some tissues such as kidney, "nonspecific" interactions may be a problem due to high levels of endogenous biotin-containing proteins. The EnVision system, developed for immunohistochemical applications, relies on binding of a polymeric conjugate consisting of up to 100 peroxidase molecules and 20 secondary antibody molecules linked directly to an activated dextran backbone, to the primary antibody. This study demonstrates that it is also a viable and sensitive alternative detection system in Western blotting applications.

Evaluation of two commercial systems for automated processing, reading, and interpretation of Lyme borreliosis Western blots.

PubMed

Binnicker, M J; Jespersen, D J; Harring, J A; Rollins, L O; Bryant, S C; Beito, E M

2008-07-01

The diagnosis of Lyme borreliosis (LB) is commonly made by serologic testing with Western blot (WB) analysis serving as an important supplemental assay. Although specific, the interpretation of WBs for diagnosis of LB (i.e., Lyme WBs) is subjective, with considerable variability in results. In addition, the processing, reading, and interpretation of Lyme WBs are laborious and time-consuming procedures. With the need for rapid processing and more objective interpretation of Lyme WBs, we evaluated the performances of two automated interpretive systems, TrinBlot/BLOTrix (Trinity Biotech, Carlsbad, CA) and BeeBlot/ViraScan (Viramed Biotech AG, Munich, Germany), using 518 serum specimens submitted to our laboratory for Lyme WB analysis. The results of routine testing with visual interpretation were compared to those obtained by BLOTrix analysis of MarBlot immunoglobulin M (IgM) and IgG and by ViraScan analysis of ViraBlot and ViraStripe IgM and IgG assays. BLOTrix analysis demonstrated an agreement of 84.7% for IgM and 87.3% for IgG compared to visual reading and interpretation. ViraScan analysis of the ViraBlot assays demonstrated agreements of 85.7% for IgM and 94.2% for IgG, while ViraScan analysis of the ViraStripe IgM and IgG assays showed agreements of 87.1 and 93.1%, respectively. Testing by the automated systems yielded an average time savings of 64 min/run compared to processing, reading, and interpretation by our current procedure. Our findings demonstrated that automated processing and interpretive systems yield results comparable to those of visual interpretation, while reducing the subjectivity and time required for Lyme WB analysis.

Cross-reactivity of anti-chicken IgY antibody with immunoglobulins of exotic avian species.

PubMed

Cray, Carolyn; Villar, David

2008-09-01

A major challenge in the serologic diagnosis of infectious diseases in exotic birds is the limited availability of species-specific antibodies. The purpose of the current study was to determine if there is cross reactivity between commercially available anti-chicken IgY antibodies and immunoglobulins of several avian species, with particular emphasis on psittacines. To quantitate the reactivity with anti-chicken IgY, Western blot analysis was performed using plasma samples from many different avian species. Results were compared with gamma globulin fraction quantitation obtained by protein electrophoresis. By Western blot, 2 protein bands corresponding to the heavy and light chains of chicken IgY were identified in species from 21 avian orders using 1 of 2 rabbit anti-chicken IgY antibodies. Densitometric analysis showed that the amount of immunoglobulin estimated from Western blots correlated strongly with data from protein electrophoresis assays. The results demonstrate that some commercially available anti-chicken IgY antibodies exhibit good cross-reactivity with most avian species.

[Purification of human goose-type lysozyme 2 (HLysG2) from human seminal plasma and analysis of its enzymatic properties].

PubMed

Huang, Peng; Yang, Zhifang; Bao, Jianying; Zhang, Ning; Li, Wenshu

2017-03-01

Objective To purify human goose-type lysozyme 2 (HLysG2) from human seminal plasma by chromatography and analyze its enzymatic properties. Methods The distribution of HLysG2 in semen was analyzed by Western blot analysis. Seminal plasma was subjected to the separation of target protein using cation-exchange chromatography, chitin affinity chromatography and size-exclusion chromatography. The purified product was identified by Western blot analysis and mass spectrometry (MS).The purity was analyzed by high performance liquid chromatography (HPLC). Then, the optimum pH, ion concentration and temperature of HLysG2 and its standard activity were determined by the turbidimetric assay. The bactericidal activity of HLysG2 was assessed by the colony-forming assay. Results The existence of HLysG2 in seminal plasma was confirmed by Western blot analysis. A protein of about 21.5 kDa was purified from seminal plasma by the three kinds of chromatography and identified as HLysG2 by Western blot analysis and MS. The final purity of the purified product was above 99.0% and the peak enzymatic activity reached 13 800 U/mg under the condition of pH 6.4, 0.09 mol/L Na + , 30DegreesCelsius. In vitro assay indicated that HLysG2 had a significant killing effect on Micrococcus lysodeikticus, Bacillus subtilis and Staphylococcus aureus, but not on Pseudomonas aeruginosa and Escherichia coli. Conclusion Native HLysG2 can be obtained from seminal plasma by chromatography. It has in vitro bactericidal activity against Gram-positive bacteria, suggesting that it might play a role in innate immunity of the male reproductive system.

[Detection of stable expression of human interlukin-2 gene in transfected keratinocytes].

PubMed

Liao, W; Liu, Y; Ye, L

1999-09-01

To investigate the stable expression and secretion of human interlukin-2 gene in transfected keratinocytes. Keratinocytes were transfected with lipofectamine and selected by G418. Then the samples were analyzed with the techniques of DNA dot blot, RNA dot blot, hybridization in situ, immunohistochemistry, Western blot and MTT. The positive signals were observed in transfected keratinocytes by DNA dot blot, RNA dot blot, hybridization in situ and immunohistochemistry. With Western blot analysis, a specific band exhibiting a molecular weight of 15,000 was detected in transfected keratinocytes, which was in acordance with that of IL-2. The expression of IL-2 can maintain for up to 1 month. The amounts of IL-2 in the supernatants of two and four passages transfected keratinocytes were 27.7 U/ml and 15.0 U/ml, respectively. Keratinocytes have the potential for stable gene expression and secretion of active transgene products. Thus, it is possible to use keratinocytes as a target cell for gene transfection, gene expression and even gene therapy.

Automated capillary Western dot blot method for the identity of a 15-valent pneumococcal conjugate vaccine.

PubMed

Hamm, Melissa; Ha, Sha; Rustandi, Richard R

2015-06-01

Simple Western is a new technology that allows for the separation, blotting, and detection of proteins similar to a traditional Western except in a capillary format. Traditionally, identity assays for biological products are performed using either an enzyme-linked immunosorbent assay (ELISA) or a manual dot blot Western. Both techniques are usually very tedious, labor-intensive, and complicated for multivalent vaccines, and they can be difficult to transfer to other laboratories. An advantage this capillary Western technique has over the traditional manual dot blot Western method is the speed and the automation of electrophoresis separation, blotting, and detection steps performed in 96 capillaries. This article describes details of the development of an automated identity assay for a 15-valent pneumococcal conjugate vaccine, PCV15-CRM197, using capillary Western technology. Copyright © 2015 Elsevier Inc. All rights reserved.

Single-Cell Western Blotting after Whole-Cell Imaging to Assess Cancer Chemotherapeutic Response

PubMed Central

2015-01-01

Intratumor heterogeneity remains a major obstacle to effective cancer therapy and personalized medicine. Current understanding points to differential therapeutic response among subpopulations of tumor cells as a key challenge to successful treatment. To advance our understanding of how this heterogeneity is reflected in cell-to-cell variations in chemosensitivity and expression of drug-resistance proteins, we optimize and apply a new targeted proteomics modality, single-cell western blotting (scWestern), to a human glioblastoma cell line. To acquire both phenotypic and proteomic data on the same, single glioblastoma cells, we integrate high-content imaging prior to the scWestern assays. The scWestern technique supports thousands of concurrent single-cell western blots, with each assay comprised of chemical lysis of single cells seated in microwells, protein electrophoresis from those microwells into a supporting polyacrylamide (PA) gel layer, and in-gel antibody probing. We systematically optimize chemical lysis and subsequent polyacrylamide gel electrophoresis (PAGE) of the single-cell lysate. The scWestern slides are stored for months then reprobed, thus allowing archiving and later analysis as relevant to sparingly limited, longitudinal cell specimens. Imaging and scWestern analysis of single glioblastoma cells dosed with the chemotherapeutic daunomycin showed both apoptotic (cleaved caspase 8- and annexin V-positive) and living cells. Intriguingly, living glioblastoma subpopulations show up-regulation of a multidrug resistant protein, P-glycoprotein (P-gp), suggesting an active drug efflux pump as a potential mechanism of drug resistance. Accordingly, linking of phenotype with targeted protein analysis with single-cell resolution may advance our understanding of drug response in inherently heterogeneous cell populations, such as those anticipated in tumors. PMID:25226230

Western Blot of Stained Proteins from Dried Polyacrylamide Gels

NASA Technical Reports Server (NTRS)

Gruber, Claudia; Stan-Lotter, Helga

1996-01-01

Western blotting of proteins is customarily performed following their separation on polyacrylamide gels, either prior to staining (1) or, as recently reported, following staining (2). We describe here Western blotting with stained gels, which had been dried and some of which had been stored for years. This procedure permits immunological analysis of proteins, to which antisera may have become available only later, or where the application of newly developed sensitive detection methods is desired. Once rehydration of the gels is achieved, proteins can be-transferred to blotting membranes by any appropriate protocol. Proteins stained with Coomassie Blue have to be detected with a non-chromogenic method, such as the film-based enhanced chemiluminescence (ECL)2) procedure (3). Silver stained proteins, which transfer in the colorless form, may be visualized by any detection method, although, because of the usually very low amounts of proteins, detection by ECL is preferable. Blotting of stained proteins from rehydrated gels is as rapid and as quantitative as from freshly prepared gels, in contrast to blotting from wet stained gels, which requires extensive washing and results in low transfer efficiency (2). Together with a photographic record of the gel pattern, unambiguous identification of immunoreactive proteins from complex mixtures is possible. Some further applications of this work are discussed.

Development of EMab-51, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in Flow Cytometry, Western Blot, and Immunohistochemistry.

PubMed

Itai, Shunsuke; Kaneko, Mika K; Fujii, Yuki; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Handa, Saori; Chang, Yao-Wen; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

2017-10-01

The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and is involved in cell growth and differentiation. EGFR homodimers or heterodimers with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many cancers. In this study, we developed novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. First, we expressed the full-length or ectodomain of EGFR in LN229 glioblastoma cells and then immunized mice with LN229/EGFR or ectodomain of EGFR, and performed the first screening using enzyme-linked immunosorbent assays. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical (fourth screening) analyses. Among 100 mAbs, only one clone EMab-51 (IgG 1 , kappa) reacted with EGFR in Western blot analysis. Finally, immunohistochemical analyses with EMab-51 showed sensitive and specific reactions against oral cancer cells, warranting the use of EMab-51 to detect EGFR in pathological analyses of EGFR-expressing cancers.

Apatinib promotes apoptosis of the SMMC-7721 hepatocellular carcinoma cell line via the PI3K/Akt pathway.

PubMed

Zhang, Hua; Cao, Yumei; Chen, Yuru; Li, Guangxi; Yu, Hanshu

2018-04-01

The present study investigated the inhibitory effects of apatinib on the proliferation of the SMMC-7721 hepatocellular carcinoma cell line to explore the possible mechanism. The MTT assay was used to detect the inhibitory effects of the different concentrations of apatinib on the proliferation of SMMC-7721 cells. Annexin V/PI double staining was performed to investigate the effects of apatinib on the apoptosis of SMMC-7721 cells. Expression of the apoptosis-related genes Bcl-2, Bax and caspase-9 after apatinib treatment was detected by reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis. Expression of the PI3K, p-PI3K, Akt and p-Akt proteins after apatinib treatment was detected using western blot analysis. The MTT results showed that apatinib inhibited the in vitro proliferation of SMMC-7721 cells. Annexin V/PI double staining showed that apatinib induced the apoptosis of SMMC-7721 cells in a concentration-dependent manner. Results of RT-qPCR and western blot analysis showed that apatinib was able to induce the expression of pro-apoptotic genes Bax and caspase-9 and inhibited the expression of anti-apoptotic gene Bcl-2 . In addition, the western blot analysis revealed that p-PI3K and p-Akt was significantly decreased following apatinib treatment, while no significant differences were found in the total protein levels of PI3K and Akt. The results of the present show that apatinib is capable of promoting the apoptosis of SMMC-7721 cells by inhibiting the PI3K/Akt signal transduction pathway, upregulating the expression of pro-apoptotic genes Bax and caspase-9 , and downregulating the expression level of the anti-apoptotic gene Bcl-2 .

Neurotrophins and Neurotrophin Receptors in Proliferative Diabetic Retinopathy

PubMed Central

Abu El-Asrar, Ahmed M.; Mohammad, Ghulam; De Hertogh, Gert; Nawaz, Mohd Imtiaz; Van Den Eynde, Kathleen; Siddiquei, Mohammad Mairaj; Struyf, Sofie; Opdenakker, Ghislain; Geboes, Karel

2013-01-01

Neurotrophins (NTs) are emerging as important mediators of angiogenesis and fibrosis. We investigated the expression of the NTs nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) and their receptors TrkA, TrkB, and TrkC in proliferative diabetic retinopathy (PDR). As a comparison, we examined the expression of NTs and their receptors in the retinas of diabetic rats. Vitreous samples from 16 PDR and 15 nondiabetic patients were studied by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). Epiretinal membranes from 17 patients with PDR were studied by immunohistochemistry. Rats were made diabetic with a single high dose of streptozotocin and retinas of rats were examined by Western blot analysis. Western blot analysis revealed a significant increase in the expression of NT-3 and NT-4 and the shedding of receptors TrkA and TrkB in vitreous samples from PDR patients compared to nondiabetic controls, whereas NGF and BDNF and the receptor TrkC were not detected with the use of Western blot analysis and ELISA. In epiretinal membranes, vascular endothelial cells and myofibroblasts expressed NT-3 and the receptors TrkA, TrkB and TrkC in situ, whereas NT-4 was not detected. The expression levels of NT-3 and NT-4 and the receptors TrkA and TrkB, both in intact and solubilized forms, were upregulated in the retinas of diabetic rats, whereas the receptor TrkC was not detected. Co-immunoprecipitation studies revealed binding between NT-3 and the receptors TrkA and TrkB in the retinas of diabetic rats. Our findings in diabetic eyes from humans and rats suggest that the increased expression levels within the NT-3 and NT-4/Trk axis are associated with the progression of PDR. PMID:23762379

Evaluation of Two Commercial Systems for Automated Processing, Reading, and Interpretation of Lyme Borreliosis Western Blots▿

PubMed Central

Binnicker, M. J.; Jespersen, D. J.; Harring, J. A.; Rollins, L. O.; Bryant, S. C.; Beito, E. M.

2008-01-01

The diagnosis of Lyme borreliosis (LB) is commonly made by serologic testing with Western blot (WB) analysis serving as an important supplemental assay. Although specific, the interpretation of WBs for diagnosis of LB (i.e., Lyme WBs) is subjective, with considerable variability in results. In addition, the processing, reading, and interpretation of Lyme WBs are laborious and time-consuming procedures. With the need for rapid processing and more objective interpretation of Lyme WBs, we evaluated the performances of two automated interpretive systems, TrinBlot/BLOTrix (Trinity Biotech, Carlsbad, CA) and BeeBlot/ViraScan (Viramed Biotech AG, Munich, Germany), using 518 serum specimens submitted to our laboratory for Lyme WB analysis. The results of routine testing with visual interpretation were compared to those obtained by BLOTrix analysis of MarBlot immunoglobulin M (IgM) and IgG and by ViraScan analysis of ViraBlot and ViraStripe IgM and IgG assays. BLOTrix analysis demonstrated an agreement of 84.7% for IgM and 87.3% for IgG compared to visual reading and interpretation. ViraScan analysis of the ViraBlot assays demonstrated agreements of 85.7% for IgM and 94.2% for IgG, while ViraScan analysis of the ViraStripe IgM and IgG assays showed agreements of 87.1 and 93.1%, respectively. Testing by the automated systems yielded an average time savings of 64 min/run compared to processing, reading, and interpretation by our current procedure. Our findings demonstrated that automated processing and interpretive systems yield results comparable to those of visual interpretation, while reducing the subjectivity and time required for Lyme WB analysis. PMID:18463211

Serological investigation of enterococcal infections using western blot.

PubMed

Sulaiman, A; Rakita, R M; Arduino, R C; Patterson, J E; Steckelberg, J M; Singh, K V; Murray, B E

1996-10-01

To assess the usefulness of Western blot in the diagnosis of enterococcal infections, a pilot study was conducted with a newly developed Western blot using sera from patients with confirmed enterococcal infections. Sera from 17 of 19 patients with enterococcal endocarditis reacted strongly to enterococcal antigens on the Western blot, and most produced specific bands at molecular weights 98 kDa and 54 kDa. Sera from patients with bacteremic cholangitis and pyelonephritis reacted frequently as well, but the pattern of bands was different from that observed with endocarditis. Eighty-five percent of 26 sera tested from patients with bacteremia and associated deep-seated infections (endocarditis, cholangitis, and pyelonephritis) were positive on Western blot, compared to 30% of sera from bacteremic patients with no clinically determined deep focus of infection (p < 0.001).

Avoiding pitfalls of internal controls: validation of reference genes for analysis by qRT-PCR and Western blot throughout rat retinal development.

PubMed

Rocha-Martins, Maurício; Njaine, Brian; Silveira, Mariana S

2012-01-01

Housekeeping genes have been commonly used as reference to normalize gene expression and protein content data because of its presumed constitutive expression. In this paper, we challenge the consensual idea that housekeeping genes are reliable controls for expression studies in the retina through the investigation of a panel of reference genes potentially suitable for analysis of different stages of retinal development. We applied statistical tools on combinations of retinal developmental stages to assess the most stable internal controls for quantitative RT-PCR (qRT-PCR). The stability of expression of seven putative reference genes (Actb, B2m, Gapdh, Hprt1, Mapk1, Ppia and Rn18s) was analyzed using geNorm, BestKeeper and Normfinder software. In addition, several housekeeping genes were tested as loading controls for Western blot in the same sample panel, using Image J. Overall, for qRT-PCR the combination of Gapdh and Mapk1 showed the highest stability for most experimental sets. Actb was downregulated in more mature stages, while Rn18s and Hprt1 showed the highest variability. We normalized the expression of cyclin D1 using various reference genes and demonstrated that spurious results may result from blind selection of internal controls. For Western blot significant variation could be seen among four putative internal controls (β-actin, cyclophilin b, α-tubulin and lamin A/C), while MAPK1 was stably expressed. Putative housekeeping genes exhibit significant variation in both mRNA and protein content during retinal development. Our results showed that distinct combinations of internal controls fit for each experimental set in the case of qRT-PCR and that MAPK1 is a reliable loading control for Western blot. The results indicate that biased study outcomes may follow the use of reference genes without prior validation for qRT-PCR and Western blot.

SDS-Polyacrylamide Electrophoresis and Western Blotting Applied to the Study of Asthma.

PubMed

García-Solaesa, Virginia; Abad, Sara Ciria

2016-01-01

Western blotting is used to analyze proteins after being separated by electrophoresis and subsequently electro-transferred to a membrane. Once immobilized, a specific protein can be identified through its reaction with a labeled antibody or antigen. It is a methodology commonly used in biomedical research such as asthma studies, to assess the pathways of inflammatory mediators involved in the disease.Here, we describe an example of western blotting to determine the factors involved in asthma. In this chapter, the methodology of western blotting is reviewed, paying attention on potential problems and giving interesting recommendations.

Deregulation of miRNAs Contributes to Development and Progression of Prostate Cancer

DTIC Science & Technology

2012-09-01

p14ARF gene were co-transfected with miR-125b into LNCaP cells. Cotransfection resulted in approximately 50% reduction of the enzyme activity (Fig...Figure3. Downregulation of miR-125b activity induces apoptosis in p53-null CaP cells. A) Western blot analysis of p14ARF and...miR-124-mediated downregulation of the AR affects the AR activity , both AR- positive LNCaP and C4-2B were treated with miR-124 mimic. Western blot

Serologic detection of antibodies against Fasciola hepatica in sheep in the middle Black Sea region of Turkey.

PubMed

Acici, Mustafa; Buyuktanir, Ozlem; Bolukbas, Cenk Soner; Pekmezci, Gokmen Zafer; Gurler, Ali Tumay; Umur, Sinasi

2017-06-01

The aim of the present study was to estimate the prevalence of Fasciola hepatica infection in sheep in the Black Sea region of Turkey. Samples from 213 sheep were collected randomly in Samsun, Tokat, and Sinop from September 2005 to January 2007 and tested by indirect enzyme-linked immunosorbent assay (ELISA) and Western blot analysis using F. hepatica excretory-secretory (E/S) antigens. The distribution of ELISA-positive samples for F. hepatica infections out of a total of 213 sheep serum samples was 23/71 (32.4%), 15/59 (25.4%), and 29/83 (34.9%) in Samsun, Sinop, and Tokat, respectively. The immunodominant proteins were determined by Western blot analysis using molecular weight markers of 14 kDa, 20 kDa, 24 kDa, 27 kDa, 33 kDa, 45 kDa, and 66 kDa and extracted from sera of sheep that were positive for Fasciola spp. eggs and also hyperimmune sera from rabbits immunized with E/S antigens. The ELISA-positive results were confirmed by Western blot analysis. As a result, seroprevalence of F. hepatica infection was found in 31.4% of sheep from the Karayaka breed in the Middle Black sea region of Turkey. Copyright © 2015. Published by Elsevier B.V.

Expression and purification of the antimicrobial peptide GSL1 in bacteria for raising antibodies.

PubMed

Meiyalaghan, Sathiyamoorthy; Latimer, Julie M; Kralicek, Andrew V; Shaw, Martin L; Lewis, John G; Conner, Anthony J; Barrell, Philippa J

2014-11-04

The Gibberellin Stimulated-Like (GSL) or Snakin peptides from higher plants are cysteine-rich, with broad spectrum activity against a range of bacterial and fungal pathogens. To detect GSL peptides in applications such as western blot analysis and enzyme-linked immunosorbent assays (ELISA), specific antibodies that recognise GSL peptides are required. However, the intrinsic antimicrobial activity of these peptides is likely to prevent their expression alone in bacterial or yeast expression systems for subsequent antibody production in animal hosts. To overcome this issue we developed an Escherichia coli expression strategy based on the expression of the GSL1 peptide as a His-tagged thioredoxin fusion protein. The DNA sequence for the mature GSL1 peptide from potato (Solanum tuberosum L.) was cloned into the pET-32a expression vector to produce a construct encoding N-terminally tagged his6-thioredoxin-GSL1. The fusion protein was overexpressed in E. coli to produce soluble non-toxic protein. The GSL1 fusion protein could be easily purified by using affinity chromatography to yield ~1.3 mg of his6-thioredoxin-GSL1 per L of culture. The fusion protein was then injected into rabbits for antibody production. Western blot analysis showed that the antibodies obtained from rabbit sera specifically recognised the GSL1 peptide that had been expressed in a wheat germ cell-free expression system. We present here the first report of a GSL1 peptide expressed as a fusion protein with thioredoxin that has resulted in milligram quantities of soluble protein to be produced. We have also demonstrated that a wheat germ system can be used to successfully express small quantities of GSL1 peptide useful as positive control in western blot analysis. To our knowledge this is the first report of antibodies being produced against GSL1 peptide. The antibodies will be useful for analysis of GSL1peptides in western blot, localization by immunohistochemistry (IHC) and quantitation by ELISA.

Immunohistochemical localization of galectin-3 in the pig retina during postnatal development

PubMed Central

Kim, Jihoon; Moon, Changjong; Ahn, Meejung; Joo, Hong-Gu; Jin, Jae-Kwang

2009-01-01

Purpose The differential level and localization of galectin-3 protein were examined in the retinas of two-day-old pigs and six-month-old pigs. Methods The retinas sampled from two-day-old and six-month-old pigs were analyzed by western blot and immunohistochemistry. Results western blot analysis detected galectin-3 in both age groups, although the levels were significantly higher in six-month-old pigs. Immunohistochemical staining showed that galectin-3 was localized in the retinas of both two-day-old pigs and six-month-old pigs; the galectin-3 immunostaining was more intense in the six-month-old pig retina, as shown in the western blot analysis. Galectin-3 was expressed in glial cells, particularly in glutamine synthetase-positive Müller cells and their processes, across all retina layers in both age groups; however, it was not found in ganglion cells of the ganglion cell layer or neuronal cells of the inner and outer nuclear cell layers in either age group. Conclusions This is the first demonstration that galectin-3 is detected in the retinas of two-day-old pigs and that the expression in Müller cells increases with postnatal development. PMID:19816601

Multistrip western blotting to increase quantitative data output.

PubMed

Kiyatkin, Anatoly; Aksamitiene, Edita

2009-01-01

The qualitative and quantitative measurements of protein abundance and modification states are essential in understanding their functions in diverse cellular processes. Typical western blotting, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. Multistrip western blotting is a modified immunoblotting procedure based on simultaneous electrophoretic transfer of proteins from multiple strips of polyacrylamide gels to a single membrane sheet. In comparison with the conventional technique, Multistrip western blotting increases the data output per single blotting cycle up to tenfold, allows concurrent monitoring of up to nine different proteins from the same loading of the sample, and substantially improves the data accuracy by reducing immunoblotting-derived signal errors. This approach enables statistically reliable comparison of different or repeated sets of data, and therefore is beneficial to apply in biomedical diagnostics, systems biology, and cell signaling research.

Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots.

PubMed

Abeyrathne, Priyanka D; Lam, Joseph S

2007-04-01

A major hurdle in characterizing bacterial membrane proteins by Western blotting is the ineffectiveness of transferring these proteins from sodium dodecyl sulfate -- polyacrylamide gel electrophoresis (SDS-PAGE) gel onto nitrocellulose membrane, using standard Western blot buffers and electrophoretic conditions. In this study, we compared a number of modified Western blotting buffers and arrived at a composition designated as the SDS-PAGE-Urea Lysis buffer. The use of this buffer and specific conditions allowed the reproducible transfer of highly hydrophobic bacterial membrane proteins with 2-12 transmembrane-spanning segments as well as soluble proteins onto nitrocellulose membranes. This method should be broadly applicable for immunochemical studies of other membrane proteins.

Western Blotting of the Endocannabinoid System.

PubMed

Wager-Miller, Jim; Mackie, Ken

2016-01-01

Measuring expression levels of G protein-coupled receptors (GPCRs) is an important step for understanding the distribution, function, and regulation of these receptors. A common approach for detecting proteins from complex biological systems is Western blotting. In this chapter, we describe a general approach to Western blotting protein components of the endocannabinoid system using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nitrocellulose membranes, with a focus on detecting type 1 cannabinoid (CB1) receptors. When this technique is carefully used, specifically with validation of the primary antibodies, it can provide quantitative information on protein expression levels. Additional information can also be inferred from Western blotting such as potential posttranslational modifications that can be further evaluated by specific analytical techniques.

Alterations in the lenticular protein profile in experimental selenite-induced cataractogenesis and prevention by ellagic acid.

PubMed

Sakthivel, Muniyan; Geraldine, Pitchairaj; Thomas, Philip A

2011-08-01

Accumulating evidence suggests that oxidative stress underlies age-related formation of cataract, and that antioxidants retard cataractogenesis. This study aimed to evaluate whether ellagic acid, a natural polyphenol with antioxidant properties, prevents alterations in the lenticular protein profile in an experimental model of selenite cataract. Alterations in lenticular protein were determined by two-dimensional electrophoresis (2DE) and image analysis. Eluted αA-crystallin spots were analyzed by mass spectrometry. Western blot analysis was also performed to confirm the differential expression of certain crystallins and cytoskeletal proteins. In cataractous lenses, 2DE and image analysis revealed approximately 45 and 60 prominent spots in soluble and insoluble protein fractions respectively. Analysis of the pI and molecular weight of protein spots revealed differences in the expression of crystallin proteins in soluble and insoluble fractions. Western blot analysis confirmed changes in the expression of αA- and βB1- crystallins in both soluble and insoluble protein fractions, while mass spectrometry confirmed the degradation of αA-crystallin in selenite cataractous lenses. Western blot analysis also confirmed the occurrence of altered expression of certain cytoskeletal proteins in insoluble fractions. However, the lenticular protein profile in lenses from selenite-challenged, ellagic acid-treated rats was essentially similar to that noted in lenses from normal rats. The present study confirms the importance of structural and cytoskeletal proteins in the maintenance of lenticular transparency; the results also suggest that ellagic acid prevents lenticular protein alterations induced by selenite in an experimental setting.

Other notable protein blotting methods: a brief review.

PubMed

Kurien, Biji T; Scofield, R Hal

2015-01-01

Proteins have been transferred from the gel to the membrane by a variety of methods. These include vacuum blotting, centrifuge blotting, electroblotting of proteins to Teflon tape and membranes for N- and C-terminal sequence analysis, multiple tissue blotting, a two-step transfer of low- and high-molecular-weight proteins, acid electroblotting onto activated glass, membrane-array method for the detection of human intestinal bacteria in fecal samples, protein microarray using a new black cellulose nitrate support, electrotransfer using square wave alternating voltage for enhanced protein recovery, polyethylene glycol-mediated significant enhancement of the immunoblotting transfer, parallel protein chemical processing before and during western blot and the molecular scanner concept, electronic western blot of matrix-assisted laser desorption/ionization mass spectrometric-identified polypeptides from parallel processed gel-separated proteins, semidry electroblotting of peptides and proteins from acid-urea polyacrylamide gels, transfer of silver-stained proteins from polyacrylamide gels to polyvinylidene difluoride (PVDF) membranes, and the display of K(+) channel proteins on a solid nitrocellulose support for assaying toxin binding. The quantification of proteins bound to PVDF membranes by elution of CBB, clarification of immunoblots on PVDF for transmission densitometry, gold coating of nonconductive membranes before matrix-assisted laser desorption/ionization tandem mass spectrometric analysis to prevent charging effect for analysis of peptides from PVDF membranes, and a simple method for coating native polysaccharides onto nitrocellulose are some of the methods involving either the manipulation of membranes with transferred proteins or just a passive transfer of antigens to membranes. All these methods are briefly reviewed in this chapter.

A brief review of other notable protein blotting methods.

PubMed

Kurien, Biji T; Scofield, R Hal

2009-01-01

A plethora of methods have been used for transferring proteins from the gel to the membrane. These include centrifuge blotting, electroblotting of proteins to Teflon tape and membranes for N- and C-terminal sequence analysis, multiple tissue blotting, a two-step transfer of low and high molecular weight proteins, blotting of Coomassie Brilliant Blue (CBB)-stained proteins from polyacrylamide gels to transparencies, acid electroblotting onto activated glass, membrane-array method for the detection of human intestinal bacteria in fecal samples, protein microarray using a new black cellulose nitrate support, electrotransfer using square wave alternating voltage for enhanced protein recovery, polyethylene glycol-mediated significant enhancement of the immunoblotting transfer, parallel protein chemical processing before and during western blot and the molecular scanner concept, electronic western blot of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-identified polypeptides from parallel processed gel-separated proteins, semidry electroblotting of peptides and proteins from acid-urea polyacrylamide gels, transfer of silver-stained proteins from polyacrylamide gels to polyvinylidene difluoride (PVDF) membranes, and the display of K(+) channel proteins on a solid nitrocellulose support for assaying toxin binding. The quantification of proteins bound to PVDF membranes by elution of CBB, clarification of immunoblots on PVDF for transmission densitometry, gold coating of nonconductive membranes before MALDI tandem mass spectrometric analysis to prevent charging effect for analysis of peptides from PVDF membranes, and a simple method for coating native polysaccharides onto nitrocellulose are some of the methods involving either the manipulation of membranes with transferred proteins or just a passive transfer of antigens to membranes. All these methods are briefly reviewed in this chapter.

Prolonged incubation and stacked film exposure improve sensitivity in western blotting.

PubMed

Luo, Haitao; Rankin, Gary O; Straley, Shannon; Chen, Yi Charlie

2011-01-01

Western blotting is a basic technique for protein detection. For proteins of less abundance or antibodies of poorer quality, an increased sensitivity is often desired. Although it is commonly known that higher concentrations of antibodies and prolonged film exposure times will help improve sensitivity in western blots, both measures come with their own risks, and it is often unclear to which extent these measures should be applied. We conducted time-course studies to investigate protein-antibody interactions and primary antibody-secondary antibody interactions in western blotting. We also propose a protocol of stacked film exposure and have tested it in standard curves and cancer cell samples. Our study found that protein-primary antibody interactions and primary antibody-secondary antibody interactions could take a longer time than commonly used "one hour" or "overnight", and in some cases longer than 48h, to reach its maximum binding. We also show that the modified protocol of stacked film exposure works well for both standard curves and biological samples, reaching a maximum sensitivity in western blots without blurring target signals or increasing backgrounds. In addition to regular optimization of antibody concentrations and film exposure time, a prolonged incubation with antibodies and stacked film exposure will also help improve sensitivity and reduce background in western blotting. Copyright © 2011 Elsevier Inc. All rights reserved.

Protein blotting protocol for beginners.

PubMed

Petrasovits, Lars A

2014-01-01

The transfer and immobilization of biological macromolecules onto solid nitrocellulose or nylon (polyvinylidene difluoride (PVDF)) membranes subsequently followed by specific detection is referred to as blotting. DNA blots are called Southerns after the inventor of the technique, Edwin Southern. By analogy, RNA blots are referred to as northerns and protein blots as westerns (Burnette, Anal Biochem 112:195-203, 1981). With few exceptions, western blotting involves five steps, namely, sample collection, preparation, separation, immobilization, and detection. In this chapter, protocols for the entire process from sample collection to detection are described.

[Wood smoke condensate induced epithelial-mesenchymal transition in human airway epithelial cells].

PubMed

Li, Wenxi; Zou, Weifeng; Li, Bing; Ran, Pixin

2014-01-01

To observe the detrimental effects of wood smoke condensate (WSC) exposure on human bronchial epithelial cells (HBEC), and to explore the expression of epithelial-mesenchymal transition (EMT) markers in HBEC exposed to WSC. HBEC were exposed respectively to 5, 10, 20, 40 and 50 mg/L of WSC /CSC for 7 days, with control groups only in cell culture medium at the same time, then the total cytoactivity was detected by cell counting kit-8. After observing the cellular morphology of WSC-stimulated HBEC. Western blot and immunofluorescence method were used to evaluate the expression levels of type I collagen, vimentin, E-cad and MMP-9 in HBEC exposed to WSC (10 mg/L) and cigarette smoke condensate (CSC) (10 mg/L) for 7 days. Statistical evaluation of the continuous data was performed by ANOVA. Independent-Samples t-test for between-group comparisons. After 7 days of exposure to WSC, HBEC manifested a morphological characteristic of loss of cell-cell contact and elongated shape. The level of E-cad was decreased in WSC exposure groups (Western blot: 0.30 ± 0.05, F = 22.07, P < 0.05) compared with the groups without WSC exposure (Western blot: 0.59 ± 0.08, F = 22.07, P < 0.05). In contrast, an upregulation in expression of type I collagen (Western blot: 0.58 ± 0.04 vs 0.26 ± 0.02, F = 119.72, P < 0.05) and MMP-9 (0.56 ± 0.08 vs 0.19 ± 0.03, F = 21.79, P < 0.05) was observed in the presence of WSC, compared with the control groups. Immunofluorescence analysis showed that after a 7-day exposure to WSC in these cells, the E-cad protein was lost whereas type I collagen, vimentin and MMP-9 were acquired. Both Western blot and immunofluorescence analysis showed no difference in expression levels of E-cad, type I collagen, vimentin and MMP-9 between WSC and CSC exposure groups. WSC exposure could induce EMT-like process in human airway epithelial cells.

Direct analysis of the secretions of the potato cyst nematode Globodera rostochiensis.

PubMed

Robertson, L; Robertson, W M; Jones, J T

1999-08-01

Secretions were induced from second (invasive) stage juveniles (J2s) of the potato cyst nematode Globodera rostochiensis by exposing them to 5-methoxy-N,N-dimethyl tryptamine oxalate (DMT). Secretions were collected from J2s in sufficient quantity to allow direct analysis. Gel electrophoresis followed by monochromatic silver staining demonstrated the presence of at least 10 proteins. The presence of several enzymes, including superoxide dismutase and proteases, was demonstrated using Western blots and activity assays. Antisera raised against the secretions recognized bands on Western blots consistent in molecular mass with those identified on silver stained gels. The antisera recognized structures implicated in the production of secretions including the subventral gland cells and surface of J2s.

Fluorescent Labeling of Proteins and Its Application to SDS-PAGE and Western Blotting.

PubMed

Alba, F Javier; Bartolomé, Salvador; Bermúdez, Antonio; Daban, Joan-Ramon

2015-01-01

This chapter describes very simple fluorescent methods developed in our laboratory allowing the rapid monitoring of total protein patterns on both sodium dodecyl sulfate (SDS) polyacrylamide gels and western blots. The noncovalent dye Nile red (9-diethylamino-5H-benzo[α]phenoxazine-5-one) is used for the sensitive staining of proteins in SDS gels. This method is compatible with the electroblotting of protein bands and with the staining of the resulting blot with the covalent dye MDPF (2-methoxy-2,4-diphenyl-3(2H)-furanone). These staining procedures are applied sequentially; there is no need to run a duplicate unstained gel for protein blotting. Furthermore, since only the adduct formed by the reaction of MDPF with proteins is fluorescent, there is no need to destain the membrane after protein labeling. In addition, MDPF staining is compatible with further immunodetection of specific bands with polyclonal antibodies. Finally, using the adequate conditions described below, MDPF staining does not preclude the N-terminal sequence analysis of proteins in selected bands.

Analysis of amyloid fibrils in the cheetah (Acinonyx jubatus).

PubMed

Bergström, Joakim; Ueda, Mitsuharu; Une, Yumi; Sun, Xuguo; Misumi, Shogo; Shoji, Shozo; Ando, Yukio

2006-06-01

Recently, a high prevalence of amyloid A (AA) amyloidosis has been documented among captive cheetahs worldwide. Biochemical analysis of amyloid fibrils extracted from the liver of a Japanese captive cheetah unequivocally showed that protein AA was the main fibril constituent. Further characterization of the AA fibril components by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis revealed three main protein AA bands with approximate molecular weights of 8, 10 and 12 kDa. Mass spectrometry analysis of the 12-kDa component observed in SDS-PAGE and Western blotting confirmed the molecular weight of a 12,381-Da peak. Our finding of a 12-kDa protein AA component provides evidence that the cheetah SAA sequence is longer than the previously reported 90 amino acid residues (approximately 10 kDa), and hence SAA is part of the amyloid fibril.

Regulation of human corneal epithelial mucins by rebamipide.

PubMed

Itoh, Shinsaku; Itoh, Kuni; Shinohara, Hisashi

2014-02-01

Membrane-associated mucins (MAMs) play important roles in barrier function and tear stability, and their expression on the ocular surface is altered in dry eye disease. Rebamipide is a mucin secretagogue that promotes the production of mucin-like glycoproteins in human corneal epithelial (HCE) cells. However, the expression of MAMs on the corneal epithelia (MUC1, MUC4, MUC16), which is induced by rebamipide, is poorly understood. In this study, we investigated the effect of rebamipide on the regulation of MAM expression in HCE cells. MUC16, Ki67 and PCNA expression levels in HCE cells isolated at confluence and at 24 hours after confluence were examined by Western blotting to assess cell proliferation. HCE cells isolated at 24 hours after confluence were cultured in medium supplemented with 1-10 µM rebamipide or 0.3-30 nM of epidermal growth factor (EGF). Real-time PCR (RT-PCR) and Western blot analysis of MAMs were performed to evaluate the effect of rebamipide. Western blot analysis of cells treated with an EGF receptor inhibitor (AG1478) or MEK1/2 inhibitor (U0126) was performed to reveal the relationship between EGF receptor activation and rebamipide-induced MAM expression. HCE cells isolated at 24 hours after confluence had lower cell proliferation activity and increased MUC16 expression compared with cells isolated at confluence. RT-PCR and Western blot analysis revealed that rebamipide increased MAM gene expression for 2 hours and protein expression for 24 hours in HCE cells. EGF inhibitor treatment led to reduced levels of all three MAMs that are normally induced by rebamipide, whereas EGF induced the expression of all three MAMs. We suggested that rebamipide increased MUC1, MUC4 and MUC16 expression levels through signals involved in EGF receptor activation in the human corneal epithelia. These data suggest that rebamipide may improve subjective symptoms of dry eye disease by upregulating MAM expression.

Dual effects of ouabain on the regulation of proliferation and apoptosis in human umbilical vein endothelial cells: involvement of Na(+)-K(+)-ATPase α-subunits and NF-κB.

PubMed

Ren, Yan-Ping; Zhang, Ming-Juan; Zhang, Ting; Huang, Ruo-Wen

2014-01-01

To elucidate the effect of ouabain on the regulation of proliferation and apoptosis of HUVECs and involvement of different Na(+)-K(+)-ATPase α-subunits and NF-κB. HUVECs were isolated by collagenase perfusion, and MTT assays and cell cycle analysis were performed to study proliferation. NF-κB expression and function were examined by immunohistochemical staining and western blotting. Na(+)-K(+)-ATPase activity was determined by measuring released ouabain inhibitable inorganic phosphate (Pi). The expression of different α-subunits was investigated by real RT-PCR, western blotting and cell immunofluorescence. 0.3 nM ouabain treatment for 0.5 h triggered the proliferation of HUVECs, peaking at 1-2 h. At 1.8 nM for 0.5 h, ouabain induced an increase of cell proliferation for a short time, and then triggered a decrease after 1 h. Cell cycle analysis show that 37% of HUVECs were in G2/M phase of the cell cycle following incubation with 1.8 nM ouabain, compared with 18% with 0.3 nM ouabain. NF-κB activity was assessed by western blot analysis of IκB expression, which was significantly reduced with 0.3 nM ouabain treatment; there was no different between 1.8 nM ouabain treatment and untreated cells. Na(+)-K(+)-ATPase activity in HUVECs was markedly reduced after treatment with 0.3 nM and 1.8 nM ouabain. Real RT-PCR and western blotting indicated that Na(+)-K(+)-ATPase α1-subunit mRNA expression levels increased after 0.3 nM ouabain treatment and decreased after 1.8 nM ouabain treatment. However, α2- and α3-subunit mRNA decreased after 0.3 nM ouabain treatment and increased after 1.8 nM ouabain treatment. Ouabain at different concentrations caused dual effects on proliferation and apoptosis in HUVECs.

Quantum dot-based western blot for sensitive detection of pig serum antibody to actinobacillus pleuropneumoniae

NASA Astrophysics Data System (ADS)

Cişmileanu, Ana; Sima, Cornelia; Grigoriu, Constantin

2007-08-01

A quantum dot - immunoglobulin conjugate specific for pig IgG, was obtained by carbodiimide chemistry. We used a Western blot technique for detecting specific antibodies against Actinobacillus pleuropneumoniae (A. pp), which cause porcine pleuropneumonia. The antigen used in this technique was Apx haemolysin which is an important virulence factor of A. pp and it induces protective immunity in vaccined pigs. The detection on Western blot membrane was possible at 1/50 dilution of quantum dot conjugate at a dilution of pig serum till 1/6400. The results for pig serum demonstrated a higher sensitivity of QD-based Western blot technique for the presence of antibodies specific for Apx haemolysin in comparison with similar classical techniques (with coloured substrate for enzyme present in secondary antibody conjugate).

TGF-Beta Antibody for Prostate Cancer: Role of ERK

DTIC Science & Technology

2011-07-01

St. Louis, MO). rotein concentration was assayed and adjusted to 1 mg/mL ith the lysis/wash buffer. An aliquot of 600 L of cell lysates as precleared...kit. Precleared lysate was immunoprecip- ated by the crosslinked antibody and agarose mixture for over- ight on 4°C. Control agarose resin in the kit...was used as a egative control when western-blot analysis was conducted. estern Blot Analysis ell lysates were prepared by using cell lysis buffer

[Comparison between old and new methods for detection of allergenic substances (egg and milk)].

PubMed

Watanabe, Hiroko; Akaboshi, Chie; Saita, Kiyotaka; Sekido, Haruko; Hashiguchi, Shigeki; Watabe, Kenjiro; Tanaka, Kouki

2011-01-01

The old ELISA method for detection of allergenic substances (egg and milk) in Kanagawa prefecture from 2003 to 2007, employed before improvement of the food allergen labeling system, yielded detection rates of 20% for egg and 30% for milk. In 2005, after improvement of the labeling system, the detection rate using the new ELISA in solutions containing 1% SDS and 7% 2-mercaptoethanol increased by about 10% for egg, but decreased by half for milk. There were 4 positive samples (over 10 µg/g) for both egg and milk proteins, on account of contamination by ingredients at the manufacturing line and the lack of proper food labeling. In 2009, the contamination levels of egg and milk proteins in labeled commercial foods were low. In a comparison between the new and old methods with the same samples, both the new ELISA and Western-blot analyses showed an increase in the detection rate of egg protein. In relation to milk protein, the detection rates were decreased with the new ELISA, although the ELISA detection rate and consistency rates with Western-blot analysis were increased. On the other hand, in the case of a protein content below 5 µg/g, it was impossible to determine ovomucoid and casein by Western-blot analysis.

Establishment of H2Mab-119, an Anti-Human Epidermal Growth Factor Receptor 2 Monoclonal Antibody, Against Pancreatic Cancer.

PubMed

Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Chang, Yao-Wen; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

2017-12-01

Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer and is associated with poor clinical outcomes. In addition, HER2 expression has been reported in other cancers, such as gastric, colorectal, lung, and pancreatic cancers. An anti-HER2 humanized antibody, trastuzumab, leads to significant survival benefits in patients with HER2-overexpressing breast cancers and gastric cancers. Herein, we established a novel anti-HER2 monoclonal antibody (mAb), H 2 Mab-119 (IgG 1 , kappa), and characterized its efficacy against pancreatic cancers using flow cytometry, Western blot, and immunohistochemical analyses. H 2 Mab-119 reacted with pancreatic cancer cell lines, such as KLM-1, Capan-2, and MIA PaCa-2, but did not react with PANC-1 in flow cytometry analysis. Western blot analysis also revealed a moderate signal for KLM-1 and a weak signal for MIA PaCa-2, although H 2 Mab-119 reacted strongly with LN229/HER2 cells. Finally, immunohistochemical analyses with H 2 Mab-119 revealed sensitive and specific reactions against breast and colon cancers but did not react with pancreatic cancers, indicating that H 2 Mab-119 is useful for detecting HER2 overexpression in pancreatic cancers using flow cytometry and Western blot analyses.

A Novel Quantitative Method for Diabetic Cardiac Autonomic Neuropathy Assessment in Type 1 Diabetic Mice

PubMed Central

Yang, Bufan; Posada-Quintero, Hugo F.; Siu, Kin L.; Rolle, Marsha; Brink, Peter; Birzgalis, Aija; Moore, Leon C.

2014-01-01

In this work, we used a sensitive and noninvasive computational method to assess diabetic cardiovascular autonomic neuropathy (DCAN) from pulse oximeter (photoplethysmographic; PPG) recordings from mice. The method, which could be easily applied to humans, is based on principal dynamic mode (PDM) analysis of heart rate variability (HRV). Unlike the power spectral density, PDM has been shown to be able to separately identify the activities of the parasympathetic and sympathetic nervous systems without pharmacological intervention. HRV parameters were measured by processing PPG signals from conscious 1.5- to 5-month-old C57/BL6 control mice and in Akita mice, a model of insulin-dependent type 1 diabetes, and compared with the gold-standard Western blot and immunohistochemical analyses. The PDM results indicate significant cardiac autonomic impairment in the diabetic mice in comparison to the controls. When tail-cuff PPG recordings were collected and analyzed starting from 1.5 months of age in both C57/Bl6 controls and Akita mice, onset of DCAN was seen at 3 months in the Akita mice, which persisted up to the termination of the recording at 5 months. Western blot and immunohistochemical analyses also showed a reduction in nerve density in Akita mice at 3 and 4 months as compared to the control mice, thus, corroborating our PDM data analysis of HRV records. Western blot analysis of autonomic nerve proteins corroborated the PPG-based HRV analysis via the PDM approach. In contrast, traditional HRV analysis (based on either the power spectral density or time-domain measures) failed to detect the nerve rarefaction. PMID:25097056

Development of transgenic finger millet (Eleusine coracana (L.) Gaertn.) resistant to leaf blast disease.

PubMed

Ignacimuthu, S; Ceasar, S Antony

2012-03-01

Finger millet plants conferring resistance to leaf blast disease have been developed by inserting a rice chitinase (chi11) gene through Agrobacterium-mediated transformation. Plasmid pHyg-Chi.11 harbouring the rice chitinase gene under the control of maize ubiquitin promoter was introduced into finger millet using Agrobacterium strain LBA4404 (pSB1). Transformed plants were selected and regenerated on hygromycin-supplemented medium. Transient expression of transgene was confirmed by GUS histochemical staining. The incorporation of rice chitinase gene in R0 and R1 progenies was confirmed by PCR and Southern blot analyses. Expression of chitinase gene in finger millet was confirmed by Western blot analysis with a barley chitinase antibody. A leaf blast assay was also performed by challenging the transgenic plants with spores of Pyricularia grisea. The frequency of transient expression was 16.3% to 19.3%. Stable frequency was 3.5% to 3.9%. Southern blot analysis confirmed the integration of 3.1 kb chitinase gene. Western blot analysis detected the presence of 35 kDa chitinase enzyme. Chitinase activity ranged from 19.4 to 24.8. In segregation analysis, the transgenic R1 lines produced three resistant and one sensitive for hygromycin, confirming the normal Mendelian pattern of transgene segregation. Transgenic plants showed high level of resistance to leaf blast disease compared to control plants. This is the first study reporting the introduction of rice chitinase gene into finger millet for leaf blast resistance.

BMP4 Signaling Is Able to Induce an Epithelial-Mesenchymal Transition-Like Phenotype in Barrett’s Esophagus and Esophageal Adenocarcinoma through Induction of SNAIL2

PubMed Central

Kestens, Christine; Siersema, Peter D.; Offerhaus, G. Johan A.; van Baal, Jantine W. P. M.

2016-01-01

Background Bone morphogenetic protein 4 (BMP4) signaling is involved in the development of Barrett’s esophagus (BE), a precursor of esophageal adenocarcinoma (EAC). In various cancers, BMP4 has been found to induce epithelial-mesenchymal transition (EMT) but its function in the development of EAC is currently unclear. Aim To investigate the expression of BMP4 and several members of the BMP4 pathway in EAC. Additionally, to determine the effect of BMP4 signaling in a human Barrett’s esophagus (BAR-T) and adenocarcinoma (OE33) cell line. Methods Expression of BMP4, its downstream target ID2 and members of the BMP4 pathway were determined by Q-RT-PCR, immunohistochemistry and Western blot analysis using biopsy samples from EAC patients. BAR-T and OE33 cells were incubated with BMP4 or the BMP4 antagonist, Noggin, and cell viability and migration assays were performed. In addition, expression of factors associated with EMT (SNAIL2, CDH1, CDH2 and Vimentin) was evaluated by Q-RT-PCR and Western blot analysis. Results Compared to squamous epithelium (SQ), BMP4 expression was significantly upregulated in EAC and BE. In addition, the expression of ID2 was significantly upregulated in EAC and BE compared to SQ. Western blot analysis confirmed our results, showing an upregulated expression of BMP4 and ID2 in both BE and EAC. In addition, more phosphorylation of SMAD1/5/8 was observed. BMP4 incubation inhibited cell viability, but induced cell migration in both BAR-T and OE33 cells. Upon BMP4 incubation, SNAIL2 expression was significantly upregulated in BAR-T and OE33 cells while CDH1 expression was significantly downregulated. These results were confirmed by Western blot analysis. Conclusion Our results indicate active BMP4 signaling in BE and EAC and suggest that this results in an invasive phenotype by inducing an EMT-like response through upregulation of SNAIL2 and subsequent downregulation of CDH1. PMID:27191723

Development of Monoclonal Antibodies Recognizing Linear Epitope: Illustration by Three Bacillus thuringiensis Crystal Proteins of Genetically Modified Cotton, Maize, and Tobacco.

PubMed

Cao, Zhen; Zhang, Wei; Ning, Xiangxue; Wang, Baomin; Liu, Yunjun; Li, Qing X

2017-11-22

Bacillus thuringiensis Cry1Ac, Cry1Ia1, and Cry1Ie are δ-endotoxin insecticidal proteins widely implemented in genetically modified organisms (GMO), such as cotton, maize, and potato. Western blot assay integrates electrophoresis separation power and antibody high specificity for monitoring specific exogenous proteins expressed in GMO. Procedures for evoking monoclonal antibody (mAb) for Western blot were poorly documented. In the present study, Cry1Ac partially denatured at 100 °C for 5 min was used as an immunogen to develop mAbs selectively recognizing a linear epitope of Cry1Ac for Western blot. mAb 5E9C6 and 3E6E2 selected with sandwich ELISA strongly recognized the heat semidenatured Cry1Ac. Particularly, 3E6E2 recognized both E. coli and cotton seed expressed Cry1Ac in Western blot. Such strategy of using partially denatured proteins as immunogens and using sandwich ELISA for mAb screening was also successfully demonstrated with production of mAbs against Cry1Ie for Western blot assay in maize.

SEROEPIDEMIOLOGY

EPA Science Inventory

ELIZA and Western blot assays have been used to detect serological responses to Cryptosporidium antigens. 1-16. A Western blot assay htat uses a miniblot was developed by the Loveland Clinic Foundation (LCF) with funding from the U.S. Environmental Protection Agency (EPA). This...

Improved high-throughput quantification of luminescent microplate assays using a common Western-blot imaging system.

PubMed

Hawkins, Liam J; Storey, Kenneth B

2017-01-01

Common Western-blot imaging systems have previously been adapted to measure signals from luminescent microplate assays. This can be a cost saving measure as Western-blot imaging systems are common laboratory equipment and could substitute a dedicated luminometer if one is not otherwise available. One previously unrecognized limitation is that the signals captured by the cameras in these systems are not equal for all wells. Signals are dependent on the angle of incidence to the camera, and thus the location of the well on the microplate. Here we show that: •The position of a well on a microplate significantly affects the signal captured by a common Western-blot imaging system from a luminescent assay.•The effect of well position can easily be corrected for.•This method can be applied to commercially available luminescent assays, allowing for high-throughput quantification of a wide range of biological processes and biochemical reactions.

Expression of chloroperoxidase from Pseudomonas pyrrocinia in tobacco plastids for fungal resistance.

PubMed

Ruhlman, Tracey A; Rajasekaran, Kanniah; Cary, Jeffrey W

2014-11-01

The chloroperoxidase (cpo) gene from Pseudomonas pyrrocinia was transformed into the plastid genome (plastome) of Nicotiana tabacum var. Petit Havana and transplastomic lines were compared with a nuclear transformant for the same gene. Southern analysis confirmed integration in the plastome and western blotting confirmed the presence of the chloroperoxidase protein (CPO) in higher abundance in transplastomic plants than in cpo nuclear transformants. Northern analysis of primary plastome transformants for cpo showed 15-fold higher transcript abundance than in the nuclear transformant, yet this extent of enhancement was not observed in western blot, enzyme or bioassay, indicating a bottleneck at the post-transcriptional level. Representative plants from the two transplastomic lines showed resistance to fungal pathogens in vitro (Aspergillus flavus, Fusarium verticillioides, and Verticillium dahliae) and in planta (Alternaria alternata). Published by Elsevier Ireland Ltd.

Characterization of a simian T-lymphotropic virus from a wild-caught orang-utan (Pongo pygmaeus) from Kalimantan, Indonesia.

PubMed

Verschoor, E J; Warren, K S; Niphuis, H; Heriyanto; Swan, R A; Heeney, J L

1998-01-01

In a recent serological survey among 143 ex-captive orang-utans two individuals were found that reacted positive in an ELISA detecting antibodies which cross-react with human T-lymphotropic virus type I (HTLV-I) antigens. Infection of both animals with an HTLV-I or simian T-lymphotropic virus (STLV)-like virus was confirmed by Western blot analysis. A third wild-caught animal, which was not part of the original serological survey, was also found to be infected with an HTLV-related virus in a diagnostic PCR assay and Western blot assay. Nucleotide sequence analysis of the 709 bp PCR fragment from the tax/rex region of the HTLV/STLV genome confirmed infection of orang-utans with an STLV similar to but clearly distinct from other Asian STLVs.

The Role of IQGAP1 in Breast Carcinoma

DTIC Science & Technology

2011-10-01

study! of! the! pathogenesis! of! breast! cancer.! These! include! analysis ! of! intracellular! signaling!by!Western!blotting,! determination!of! cell...proliferation!by! sulforhodamine!B! staining,! fluorescence: activated!cell!sorting!(FACS)! analysis ,!stable!cell!line!generation,!production!of!and...transduction!using!retroviral! and!lentiviral!supernatants,! immunocytochemistry!and!confocal! laser!microscopy,! immunohistochemistry,!and! analysis

Western blotting: an introduction.

PubMed

Kurien, Biji T; Scofield, R Hal

2015-01-01

Western blotting is an important procedure for the immunodetection of proteins, particularly proteins that are of low abundance. This process involves the transfer of protein patterns from gel to microporous membrane. Electrophoretic as well as non-electrophoretic transfer of proteins to membranes was first described in 1979. Protein blotting has evolved greatly since the inception of this protocol, allowing protein transfer to be accomplished in a variety of ways.

Modifying a standard method allows simultaneous extraction of RNA and protein, enabling detection of enzymes in the rat retina with low expressions and protein levels.

PubMed

Agardh, Elisabet; Gustavsson, Carin; Hagert, Per; Nilsson, Marie; Agardh, Carl-David

2006-02-01

The aim of the study was to evaluate messenger RNA and protein expression in limited amounts of tissue with low protein content. The Chomczynski method was used for simultaneous extraction of RNA, and protein was modified in the protein isolation step. Template mass and cycling time for the complementary DNA synthesis step of real-time reverse transcription-polymerase chain reaction (RT-PCR) for analysis of catalase, copper/zinc superoxide dismutase, manganese superoxide dismutase, the catalytic subunit of glutamylcysteine ligase, glutathione peroxidase 1, and the endogenous control cyclophilin B (CypB) were optimized before PCR. Polymerase chain reaction accuracy and efficacy were demonstrated by calculating the regression (R2) values of the separate amplification curves. Appropriate antibodies, blocking buffers, and running conditions were established for Western blot, and protein detection and multiplex assays with CypB were performed for each target. During the extraction procedure, the protein phase was dissolved in a modified washing buffer containing 0.1% sodium dodecyl sulfate, followed by ultrafiltration. Enzyme expression on real-time RT-PCR was accomplished with high reliability and reproducibility (R2, 0.990-0.999), and all enzymes except for glutathione peroxidase 1 were detectable in individual retinas on Western blot. Western blot multiplexing with CypB was possible for all targets. In conclusion, connecting gene expression directly to protein levels in the individual rat retina was possible by simultaneous extraction of RNA and protein. Real-time RT-PCR and Western blot allowed accurate detection of retinal protein expressions and levels.

A novel mAb against a human CD34 peptide reacts with the native protein on CD34+ cells.

PubMed

Shams, Mahmood; Jeddi-Tehrani, Mahmood; Notash Haghighat, Farzaneh; Bayat, Ali Ahmad; Mahmoudian, Jafar; Rezvani, Mohammad Reza

2013-12-01

‎Human CD34 is a transmembrane glycoprotein which is expressed in human hematopoietic stem ‎cells (HSCs) and the small-‎vessel endothelial cells of a variety of tissues. CD34 plays a critical role as a ‎marker for diagnosis ‎and classification of leukemia. Anti CD34 antibodies are used for isolation and ‎purification ‎of HSCs from bone marrow, peripheral blood and cord blood. To characterize a newly produced monoclonal antibody against a human CD34 peptide. Anti CD34 monoclonal antibody (Clone 2C10-D3) was purified from mouse ascitic fluid and hybridoma cell culture supernatants by affinity chromatography and its immune reactivity was examined by ELISA. The purified antibody was further characterized using Western blot and flow cytometry on TF1 (Human Erythroblast) cell line. ‎ELISA experiment revealed that the antibody recognized CD34 peptide. Western ‎blot analysis on TF1 ‎cell lysate confirmed the reactivity of the antibody with a 42 KDa protein. Blocking the antibody with a saturating concentration of specific CD34 peptide resulted in loss of its activity with TF1 lysate in Western blot. The 2C10-D3 antibody reacted with TF1 cells in flow cytometry in a similar manner to a commercial anti CD34 monoclonal antibody.‎ ‎Our data suggest that the anti CD34 monoclonal antibody (Clone 2C10-D3) is an appropriate antibody to study the CD34+ cells by flow cytometry and Western blot.

Detection of serum antibodies to hepatitis E virus in domestic pigs in Italy using a recombinant swine HEV capsid protein.

PubMed

Ponterio, Eleonora; Di Bartolo, Ilaria; Orrù, Ginevra; Liciardi, Manuel; Ostanello, Fabio; Ruggeri, Franco Maria

2014-06-16

The hepatitis E virus (HEV) has been detected in both humans and animals, particularly pigs, worldwide. Several evidences, including human infection following consumption of raw contaminated meat, suggest a zoonotic transmission of HEV. In Italy, large circulation of genotype 3 HEV has been reported in swine, and recent studies have confirmed the involvement of this genotype in autochthonous human cases. In this study 111 sera collected from healthy pigs in two Italian regions were tested for anti-HEV IgG antibodies. For specific HEV antibody detection in swine, we developed ELISA and Western blotting methods, using a truncated capsid (ORF2) protein lacking the first 111 amino acids of a swine HEV genotype 3 strain. The ORF2-based ELISA revealed anti-HEV antibodies in 104 out of 111 pigs compared with 102 detected with a commercial ELISA kit. A lower number of sera reacted with the recombinant ORF2 protein in a Western blotting format (81/111). Using a Latent class analysis (LCA), the estimated sensitivities for ELISA-ORF2 and ELISA-kit tests were 0.961 and 0.936, respectively, whereas specificities were 0.599 and 0.475. The estimated sensitivity of Western blotting was 0.775, and the specificity was 0.944. The overall results confirm the high prevalence of HEV seropositive healthy pigs in Italy. Through comparisons with a commercial ELISA test, the swine genotype 3 HEV antigen produced in this study was proven suitable to detect anti-HEV antibodies in pig sera by both ELISA and Western Blotting.

Western blotting.

PubMed

Kurien, Biji T; Scofield, R Hal

2006-04-01

Western blotting (protein blotting or immunoblotting) is a powerful and important procedure for the immunodetection of proteins post-electrophoresis, particularly proteins that are of low abundance. Since the inception of the protocol for protein transfer from an electrophoresed gel to a membrane in 1979, protein blotting has evolved greatly. The scientific community is now confronted with a variety of ways and means to carry out this transfer. This review describes the various procedures that have been used to transfer proteins from a gel to a membrane based on the principles of simple diffusion, vacuum-assisted solvent flow and electrophoretic elution. Finally, a brief description of methods generally used to detect antigens on blots is also described.

Identification of recombinant human EPO variants in greyhound plasma and urine by ELISA, LC-MS/MS and western blotting: a comparative study.

PubMed

Timms, Mark; Steel, Rohan; Vine, John

2016-02-01

The recombinant human erythropoietins epoetin alfa (Eprex®), darbepoetin (Aranesp®) and methoxy polyethylene glycol-epoetin beta (Mircera®) were administered to greyhounds for 7, 10 and 14 days respectively. Blood and urine samples were collected and analysed for erythropoietin by ELISA, LC-MS/MS and western blotting. Limits of confirmation in plasma for western blotting and LC-MS/MS methods ranged from a low of 2.5mIU/mL, and closely matched the sensitivity of ELISA screening. Copyright © 2015 John Wiley & Sons, Ltd.

Analysis of immunostaining and western blotting of endothelin 1 and its receptors in mitral stenosis

PubMed Central

Leão, Sydney Correia; Dashwood, Michael R.; de Andrade, Mateus Santana; Santos, Nicolas Nascimento; Teles, Olivia Regina Lins Leal; de Souza, Williasmin Batista; Rodrigues, Tania Maria de Andrade

2015-01-01

Introduction Rheumatic Fever represents a serious public health problem in developing countries, with thousands of new cases each year. It is an autoimmune disease, which occurs in response to infection by streptococcus A. Objective The aim of this study was to evaluate the immunolabeling and protein expression for endothelin-1 and 3 (ET-1, ET-3) and its receptors (ETA, ETB) in rheumatic mitral valves. Methods Immunohistochemistry was used to identify ET-1/ET-3 and ETA/ETB receptors in rheumatic and control mitral valves. Quantitative analysis of immunostaining for ET-1/ET-3 and ETA/ETB receptors was performed. In addition, western blot analysis was carried out to assess protein levels in tissue samples. Results ET-1 and ETA receptor immunostaining predominated in stenotic valves, mainly associated with fibrotic regions, inflammatory areas and neovascularization. Quantitative analysis showed that the average area with positive expression of ET-1 was 18.21±14.96%. For ETA and ETB, the mean expressed areas were respectively 15.06±13.13% and 9.20±11.09%. ET-3 did not have a significant expression. The correlation between the expression of both endothelin receptors were strongly positive (R=0.74, P=0.02), but the correlation between ET-1 and its receptor were negative for both ETA (R=-0.37, P=0.25), and ETB (R=-0.14, P=0.39). This data was supported by western blot analysis. Conclusion The strong correlation between ET-1 and its receptors suggests that both play a role in the pathophysiology of rheumatic mitral valve stenosis and may potentially act as biomarkers of this disease. PMID:26107453

Identification of proteins in a human pleural exudate using two-dimensional preparative liquid-phase electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry.

PubMed

Nilsson, C L; Puchades, M; Westman, A; Blennow, K; Davidsson, P

1999-01-01

Pleural effusion may occur in patients suffering from physical trauma or systemic disorders such as infection, inflammation, or cancer. In order to investigate proteins in a pleural exudate from a patient with severe pneumonia, we used a strategy that combined preparative two-dimensional liquid-phase electrophoresis (2-D LPE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and Western blotting. Preparative 2-D LPE is based on the same principles as analytical 2-D gel electrophoresis, except that the proteins remain in liquid phase during the entire procedure. In the first dimension, liquid-phase isoelectric focusing allows for the enrichment of proteins in liquid fractions. In the Rotofor cell, large volumes (up to 55 mL) and protein amounts (up to 1-2 g) can be loaded. Several low abundance proteins, cystatin C, haptoglobin, transthyretin, beta2-microglobulin, and transferrin, were detected after liquid-phase isoelectric focusing, through Western blotting analysis, in a pleural exudate (by definition, >25 g/L total protein). Direct MALDI-TOF-MS analysis of proteins in a Rotofor fraction is demonstrated as well. MALDI-TOF-MS analysis of a tryptic digest of a continuous elution sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fraction confirmed the presence of cystatin C. By applying 2-D LPE, MALDI-TOF-MS, and Western blotting to the analysis of this pleural exudate, we were able to confirm the identity of proteins of potential diagnostic value. Our findings serve to illustrate the usefulness of this combination of methods in the analysis of pathological fluids.

Confirmation of the immunoreactivity of monoclonal anti-human C-terminal EGFR antibodies in bronze Corydoras Corydoras aeneus (Callichthyidae Teleostei) by Western Blot method.

PubMed

Mytych, Jennifer; Satora, Leszek; Kozioł, Katarzyna

2018-02-01

Bronze corydoras (Corydoras aeneus) uses the distal part of the intestine as accessory respiratory organ. Our previous study showed the presence of epidermal growth factor receptor (EGFR) cytoplasmic domain in the digestive tract of the bronze corydoras. In this study, using Western Blot method, we validated the results presented in the previous research. In detail, results of Western Blot analysis on digestive and respiratory part of bronze corydoras intestine homogenates confirmed the immunoreactivity of anti-cytoplasmic domain (C-terminal) human EGFR antibodies with protein band of approximately 180kDa (EGFR molecular weight). This indicates a high homology of EGFR domain between these species and the possibility of such antibody use in bronze corydoras. Statistically significantly higher EGFR expression was observed in the respiratory part of intestine when compared to the digestive part. This implies higher proliferation activity and angiogenesis of epithelium in this part of intestine, creating conditions for air respiration. Therefore, Corydoras aeneus may be considered as a model organism for the molecular studies of the mechanisms of epithelial proliferation initiation and inhibition depending on hypoxia and normoxia. Copyright © 2017. Published by Elsevier GmbH.

Pirfenidone Inhibits Proliferation and Promotes Apoptosis of Hepatocellular Carcinoma Cells by Inhibiting the Wnt/β-Catenin Signaling Pathway.

PubMed

Zou, Wei-Jie; Huang, Zhi; Jiang, Tian-Peng; Shen, Ya-Ping; Zhao, An-Su; Zhou, Shi; Zhang, Shuai

2017-12-25

BACKGROUND Hepatocellular carcinoma (HCC) is the most important cause of cancer-related deaths worldwide. Pirfenidone is an orally available small molecule with therapeutic potential for fibrotic diseases. MATERIAL AND METHODS In this study, we analyzed the effects of different pirfenidone concentrations on the proliferation of HepG2 HCC cells using Cell Counting Kit-8 (CCK-8) and colony formation assays. Flow cytometry was performed to measure the apoptotic effects of pirfenidone on HepG2 cells. Western blot analysis was performed to detect the expression of β-catenin and p-β-catenin. RESULTS Pirfenidone inhibited proliferation and promoted HepG2 cell apoptosis. In addition, Western blot results indicated that pirfenidone suppressed b-catenin expression in HepG2 cells. To assess the mechanism, we treated HepG2 cells with pirfenidone, and pirfenidone plus the β-catenin activator, SB-216763. The results revealed that SB-216763 accelerated proliferation and inhibited apoptosis in HepG2 cells treated with pirfenidone. Western blot results showed that SB-216763 upregulated β-catenin expression in HepG2 cells treated with pirfenidone. CONCLUSIONS In conclusions, pirfenidone may be a potential drug for HCC treatment.

Transfer in SDS of biotinylated proteins from acrylamide gels to an avidin-coated membrane filter.

PubMed

Karlin, Arthur; Wang, Chaojian; Li, Jing; Xu, Qiang

2004-06-01

Avidin was covalently linked to aldehyde-derivatized polyethersulfone membrane filters. These filters were used in Western blot analysis of proteins reacted with biotinylation reagents and electrophoresed in sodium dodecyl sulfate (SDS) on polyacrylamide gels. Electrophoretic transfer from the gels to these filters was in 0.1% SDS, in which the covalently bound avidin retained its biotin-binding capacity. We compared Western blots on avidin-coated membrane filters of biotinylated and nonbiotinylated forms of mouse immunoglobulin G (IgG), mouse IgG heavy chain, muscle-type acetylcholine receptor alpha subunit, and fused alpha and beta subunits of receptor. Biotinylated proteins were captured with high specificity compared to their nonbiotinylated counterparts and sensitively detected on the avidin-coated membranes.

Rapid Preparation of a Plasma Membrane Fraction: Western Blot Detection of Translocated Glucose Transporter 4 from Plasma Membrane of Muscle and Adipose Cells and Tissues.

PubMed

Yamamoto, Norio; Yamashita, Yoko; Yoshioka, Yasukiyo; Nishiumi, Shin; Ashida, Hitoshi

2016-08-01

Membrane proteins account for 70% to 80% of all pharmaceutical targets, indicating their clinical relevance and underscoring the importance of identifying differentially expressed membrane proteins that reflect distinct disease properties. The translocation of proteins from the bulk of the cytosol to the plasma membrane is a critical step in the transfer of information from membrane-embedded receptors or transporters to the cell interior. To understand how membrane proteins work, it is important to separate the membrane fraction of cells. This unit provides a protocol for rapidly obtaining plasma membrane fractions for western blot analysis. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Determining Zebrafish Epitope Reactivity to Commercially Available Antibodies.

PubMed

Villarreal, Michael A; Biediger, Nicole M; Bonner, Natalie A; Miller, Jennifer N; Zepeda, Samantha K; Ricard, Benjamin J; García, Dana M; Lewis, Karen A

2017-08-01

Antibodies raised against mammalian proteins may exhibit cross-reactivity with zebrafish proteins, making these antibodies useful for fish studies. However, zebrafish may express multiple paralogues of similar sequence and size, making them difficult to distinguish by traditional Western blot analysis. To identify the zebrafish proteins that are recognized by an antimammalian antibody, we developed a system to screen putative epitopes by cloning the sequences between the yeast SUMO protein and a C-terminal 6xHis tag. The recombinant fusion protein was expressed in Escherichia coli and analyzed by Western blot to conclusively identify epitopes that exhibit cross-reactivity with the antibodies of interest. This approach can be used to determine the species cross-reactivity and epitope specificity of a wide variety of peptide antigen-derived antibodies.

BEVACIZUMAB LEVELS IN BREAST MILK AFTER LONG-TERM INTRAVITREAL INJECTIONS.

PubMed

McFarland, Trevor J; Rhoads, Andrew D; Hartzell, Matthew; Emerson, Geoffrey G; Bhavsar, Abdhish R; Stout, J Timothy

2015-08-01

The purpose of this study is to determine whether bevacizumab is detectable in the breast milk of nursing mothers. Breast milk samples were collected from 2 patients receiving monthly intravitreal bevacizumab injections for choroidal neovascularization over the course of 16 months. Enzyme-linked immunosorbent assay and Western blot analysis was used to determine the levels of bevacizumab in the milk samples. An enzyme-linked immunosorbent assay was developed using antibodies specific to bevacizumab in which the sensitivity threshold was 3 ng/mL. All breast milk samples assayed from the two patients actively undergoing treatment did not have detectable levels of bevacizumab. Samples collected 1.5 hours and 7 hours after an injection and 2 randomly chosen samples were negative by Western blot analysis. A sensitive assay to detect bevacizumab in breast milk samples assayed suggests that intravitreal injections do not result in detectable bevacizumab in breast milk.

Human thyrotropin receptor subunits characterized by thyrotropin affinity purification and western blotting.

PubMed

Leedman, P J; Newman, J D; Harrison, L C

1989-07-01

We studied the subunit structure of the human TSH receptor in thyroid tissue from patients with Graves' disease and multinodular goiter by TSH affinity chromatography, immunoprecipitation with Graves' immunoglobulins (Igs), and a modified technique of Western blotting. Human TSH receptor-binding activity was purified about 1,270-fold by sequential affinity chromatography on wheat germ lectin-agarose and TSH-agarose. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of nonreduced affinity-purified receptors eluted in sodium dodecyl sulfate sample buffer revealed three noncovalently linked subunits of 70,000, 50,000, and 35,000 mol wt. When reduced, a major subunit of 25,000 mol wt was identified. When 3 mol/L NaCl was used to elute affinity-purified receptors only the 50,000 mol wt nonreduced subunit was detected. This subunit bound [125I]bovine TSH and was precipitated by Graves' Igs. Modifications to the conventional Western blotting technique enabled thyroglobulin components (approximately 220,000 mol wt), thyroid microsomal antigen (a doublet of approximately 110,000 mol wt), and putative TSH receptor subunits of 70,000 and 50,000 mol wt to be identified in thyroid particulate membranes by Graves' Igs. Blotting of affinity-purified receptors eluted in sodium dodecyl sulfate sample buffer revealed subunits of either 70,000 or 50,000 mol wt, with a minority of Graves' serum samples. We conclude that the nonreduced human TSH receptor is an oligomeric complex comprising three different subunits of 70,000, 50,000, and 35,000 mol wt. The reduced receptor exists as a single subunit of 25,000 mol wt, which may be disulfide linked to form the higher mol wt forms. The 70,000 and 50,000 mol wt subunits contain epitopes that bind Graves' Igs in modified Western blots, thus directly confirming that the human TSH receptor is a target for Graves' Igs.

Protein Stains to Detect Antigen on Membranes.

PubMed

Dsouza, Anil; Scofield, R Hal

2015-01-01

Western blotting (protein blotting/electroblotting) is the gold standard in the analysis of complex protein mixtures. Electroblotting drives protein molecules from a polyacrylamide (or less commonly, of an agarose) gel to the surface of a binding membrane, thereby facilitating an increased availability of the sites with affinity for both general and specific protein reagents. The analysis of these complex protein mixtures is achieved by the detection of specific protein bands on a membrane, which in turn is made possible by the visualization of protein bands either by chemical staining or by reaction with an antibody of a conjugated ligand. Chemical methods employ staining with organic dyes, metal chelates, autoradiography, fluorescent dyes, complexing with silver, or prelabeling with fluorophores. All of these methods have differing sensitivities and quantitative determinations vary significantly. This review will describe the various protein staining methods applied to membranes after western blotting. "Detection" precedes and is a prerequisite to obtaining qualitative and quantitative data on the proteins in a sample, as much as to comparing the protein composition of different samples. "Detection" is often synonymous to staining, i.e., the reversible or irreversible binding by the proteins of a colored organic or inorganic chemical.

The Association of Palmitoylethanolamide with Luteolin Decreases Neuroinflammation and Stimulates Autophagy in Parkinson's Disease Model.

PubMed

Siracusa, Rosalba; Paterniti, Irene; Impellizzeri, Daniela; Cordaro, Marika; Crupi, Rosalia; Navarra, Michele; Cuzzocrea, Salvatore; Esposito, Emanuela

2015-01-01

Parkinson's disease (PD) is a disorder resulted by degeneration of dopaminergic neurons. To counteract the neuroinflammation and oxidative stress of PD, we decided to test a new composite constituted by palmitoylethanolamide (PEA) and luteolin (Lut), in a mass ratio of 10:1, respectively (co-ultraPEALut). In this study the neuroprotective property of the new compound was investigated. For the in vivo model of PD, mice received four injections of the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP). Starting 24 h after the first administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we treated animals with co-ultraPEALut daily until 7 days. On day 8, brains were processed for Western blotting and immunohistochemical analysis. Treatment with co-ultraPEALut reduced the specific markers of PD (tyrosine hydroxylase immunopositive), and the increased levels of activated astrocytes and pro-inflammatory cytokines as well as inducible nitric oxide synthase. Further, the possible association of autophagy with the beneficial effects of coultraPEALut. Western blot analysis and immunofluorescence staining showed that co-ultraPEALut administration increased autophagy process. These data were confirmed by an in vitro model, using SH-SY5Y neuroblastoma cells. Western blot analysis showed that co-ultraPEALut pre-treatment maintained high Beclin-1 and p62 expression, while continued to inhibit the p70S6K expression. Altogether, these results put forward that treatment with co-ultraPEALut is able to modulate both the neuroinflammatory process and the autophagic pathway involved in PD, actions which may underlie its neuroprotective effect.

TAZ promotes epithelial to mesenchymal transition via the upregulation of connective tissue growth factor expression in neuroblastoma cells.

PubMed

Wang, Qiang; Xu, Zhilin; An, Qun; Jiang, Dapeng; Wang, Long; Liang, Bingxue; Li, Zhaozhu

2015-02-01

Neuroblastoma (NB) is a neuroendocrine cancer that occurs most commonly in infants and young children. The Hippo signaling pathway regulates cell proliferation and apoptosis, and its primary downstream effectors are TAZ and yes‑associated protein 1 (YAP). The effect of TAZ on the metastatic progression of neuroblastoma and the underlying mechanisms involved remain elusive. In the current study, it was determined by western blot analysis that the migratory and invasive properties of SK‑N‑BE(2) human neuroblastoma cells are associated with high expression levels of TAZ. Repressed expression of TAZ in SK‑N‑BE(2) cells was shown to result in a reduction in aggressiveness of the cell line, by Transwell migration and invasion assay. In contrast, overexpression of TAZ in SK‑N‑SH human neuroblastoma cells was shown by Transwell migration and invasion assays, and western blot analysis, to result in epithelial‑mesenchymal transition (EMT) and increased invasiveness. Mechanistically, the overexpression of TAZ was demonstrated to upregulate the expression levels of connective tissue growth factor (CTGF), by western blot analysis and chromatin immunoprecipitation assay, while the knockdown of TAZ downregulated it. Furthermore, TAZ was shown by luciferase assay to induce CTGF expression by modulating the activation of the TGF‑β/Smad3 signaling pathway. In conclusion, the present study is, to the best of our knowledge, the first to demonstrate that the overexpression of TAZ induces EMT, increasing the invasive abilities of neuroblastoma cells. This suggests that TAZ may serve as a potential target in the development of novel therapies for the treatment of neuroblastoma.

A hybrid of coumarin and phenylsulfonylfuroxan induces caspase-dependent apoptosis and cytoprotective autophagy in lung adenocarcinoma cells.

PubMed

Wang, Qian; Guo, Yalan; Jiang, Shanshan; Dong, Mengxue; Kuerban, Kudelaidi; Li, Jiyang; Feng, Meiqing; Chen, Ying; Ye, Li

2018-01-15

Lung adenocarcinoma is the most primary histologic subtype of non-small cell lung cancer (NSCLC). Compound 8b, a novel coumarin derivative with phenylsulfonylfuroxan group, shows significant antiproliferation activity against lung adenocarcinoma cell with low toxicity. This study aims to uncover the potential of compound 8b in relation to apoptosis as well as autophagy induction in lung adenocarcinoma cells. The cytotoxicity and apoptosis of A549 and H1299 cells induced by compound 8b were detected by MTT, microscope and western blot analysis. Autophagy was determined by TEM, confocal microscopy and western blot analysis. Akt/mTOR and Erk signaling pathway were also examined by western blot analysis. First, significant growth inhibition and caspase-dependent apoptosis were observed in compound 8b-treated A549 and H1299 cells. Then, we confirmed compound 8b-induced autophagy by autophagosomes formation, upregulated expression of autophagy-related protein LC3-II and autophagic flux. Importantly, abolishing autophagy using inhibitors and ATG5 siRNA enhanced the cytotoxicity of compound 8b, indicating the cytoprotective role of autophagy in lung adenocarcinoma. Further mechanistic investigations suggested that Akt/mTOR and Erk signaling pathways contributed to autophagy induction by compound 8b. This results demonstrate that compound 8b induces caspase-dependent apoptosis as well as cytoprotective autophagy in lung adenocarcinoma cells, which may provide scientific evidence for developing this furoxan-based NO-releasing coumarin derivative as a potential anti-lung adenocarcinoma therapeutic agents. Copyright © 2017 Elsevier GmbH. All rights reserved.

The function of oxytocin: a potential biomarker for prostate cancer diagnosis and promoter of prostate cancer.

PubMed

Xu, Huan; Fu, Shi; Chen, Qi; Gu, Meng; Zhou, Juan; Liu, Chong; Chen, Yanbo; Wang, Zhong

2017-05-09

To measure the level of oxytocin in serum and prostate cancer (PCa) tissue and study its effect on the proliferation of PCa cells. Oxytocin level in serum was significantly increased in PCa patients compared with the no-carcinoma individuals. Additionally, the levels of oxytocin and its receptor were also elevated in the PCa tissue. However, no significant difference existed among the PCa of various Gleason grades. Western blot analysis confirmed the previous results and revealed an increased expression level of APPL1. The level of oxytocin in serum was measured by ELISA analysis. The expression of oxytocin and its receptor in prostate was analyzed by immunohistochemistry. The proliferation and apoptosis of PCa cells were assessed by the Cell Counting Kit 8 (CCK8) assay, cell cycle analysis and caspase3 activity analysis, respectively. Western blot analysis was used for the detection of PCNA, Caspase3 and APPL1 protein levels. Serum and prostatic oxytocin levels are increased in the PCa subjects. Serum oxytocin level may be a biomarker for PCa in the future. Oxytocin increases PCa growth and APPL1 expression.

Western blotting using chemiluminescent substrates.

PubMed

Alegria-Schaffer, Alice

2014-01-01

Western blotting is a powerful and commonly used tool to identify and quantify a specific protein in a complex mixture (Towbin et al., 1979). The technique enables indirect detection of protein samples immobilized on a nitrocellulose or polyvinylidene fluoride (PVDF) membrane. Copyright © 2014 Elsevier Inc. All rights reserved.

Ferulic acid enhances IgE binding to peanut allergens in western blots.

USDA-ARS?s Scientific Manuscript database

Phenolic compounds at high concentrations are known to form insoluble complexes with proteins. We hypothesized that this complex formation could interfere with Western blot and ELISA assays for peanut allergens. To verify this, three simple phenolic compounds (ferulic, caffeic, and chlorogenic acids...

COMPARISONS OF ELISA AND WESTERN BLOT ASSAYS FOR DETECTION OF CRYPTOSPORIDIUM ANTIBODY

EPA Science Inventory

A seroprevalence survey was conducted using ELISA and Western blot (WB) assays for antibody to three Cryptosporidium antigens on 380 blood donors in Jackson County, Oregon. The purpose was to determine if either assay could detect serological evidence of an outbreak which occurre...

The early days of blotting.

PubMed

Southern, Edwin

2015-01-01

The history of the development of DNA blotting is described in this chapter. DNA blotting, involving the transfer of electrophoretically separated DNA fragments to a membrane support through capillary action, is also known as Southern blotting. This procedure enables the detection of a specific DNA sequence by hybridization with probes. The term Southern blotting led to a "geographic" naming tradition, with RNA blotting bearing the name Northern blotting and protein transfer to membranes becoming known as Western blotting.

Molecular evidence and functional expression of multidrug resistance associated protein (MRP) in rabbit corneal epithelial cells.

PubMed

Karla, Pradeep K; Pal, Dananjay; Mitra, Ashim K

2007-01-01

Multidrug resistance associated protein (MRP) is a major family of efflux transporters involved in drug efflux leading to drug resistance. The objective of this study was to explore physical barriers for ocular drug absorption and to verify if the role of efflux transporters. MRP-2 is a major homologue of MRP family and found to express on the apical side of cell membrane. Cultured Rabbit Corneal Epithelial Cells (rCEC) were selected as an in vitro model for corneal epithelium. [14C]-erythromycin which is a proven substrate for MRP-2 was selected as a model drug for functional expression studies. MK-571, a known specific and potent inhibitor for MRP-2 was added to inhibit MRP mediated efflux. Membrane fraction of rCEC was used for western blot analysis. Polarized transport of [14C]-erythromycin was observed in rCEC and transport from B-->A was significantly high than from A-->B. Permeability's increased significantly from A-->B in the presence of MK-571 and ketoconozole. Uptake of [14C]-erythromycin in the presence of MK-571 was significantly higher than control in rCEC. RT-PCR analysis indicated a unique and distinct band at approximately 498 bp corresponding to MRP-2 in rCEC and MDCK11-MRP-2 cells. Immunoprecipitation followed by Western Blot analysis indicated a specific band at approximately 190 kDa in membrane fraction of rCEC and MDCK11-MRP-2 cells. For the first time we have demonstrated high expression of MRP-2 in rabbit corneal epithelium and its functional activity causing drug efflux. RT-PCR, immunoprecipitation followed by Western blot analysis further confirms the result.

A Secondary Antibody-Detecting Molecular Weight Marker with Mouse and Rabbit IgG Fc Linear Epitopes for Western Blot Analysis

PubMed Central

Cheng, Ta-Chun; Tung, Yi-Ching; Chu, Pei-Yu; Chuang, Chih-Hung; Hsieh, Yuan-Chin; Huang, Chien-Chiao; Wang, Yeng-Tseng; Kao, Chien-Han; Roffler, Steve R.; Cheng, Tian-Lu

2016-01-01

Molecular weight markers that can tolerate denaturing conditions and be auto-detected by secondary antibodies offer great efficacy and convenience for Western Blotting. Here, we describe M&R LE protein markers which contain linear epitopes derived from the heavy chain constant regions of mouse and rabbit immunoglobulin G (IgG Fc LE). These markers can be directly recognized and stained by a wide range of anti-mouse and anti-rabbit secondary antibodies. We selected three mouse (M1, M2 and M3) linear IgG1 and three rabbit (R1, R2 and R3) linear IgG heavy chain epitope candidates based on their respective crystal structures. Western blot analysis indicated that M2 and R2 linear epitopes are effectively recognized by anti-mouse and anti-rabbit secondary antibodies, respectively. We fused the M2 and R2 epitopes (M&R LE) and incorporated the polypeptide in a range of 15–120 kDa auto-detecting markers (M&R LE protein marker). The M&R LE protein marker can be auto-detected by anti-mouse and anti-rabbit IgG secondary antibodies in standard immunoblots. Linear regression analysis of the M&R LE protein marker plotted as gel mobility versus the log of the marker molecular weights revealed good linearity with a correlation coefficient R2 value of 0.9965, indicating that the M&R LE protein marker displays high accuracy for determining protein molecular weights. This accurate, regular and auto-detected M&R LE protein marker may provide a simple, efficient and economical tool for protein analysis. PMID:27494183

A Secondary Antibody-Detecting Molecular Weight Marker with Mouse and Rabbit IgG Fc Linear Epitopes for Western Blot Analysis.

PubMed

Lin, Wen-Wei; Chen, I-Ju; Cheng, Ta-Chun; Tung, Yi-Ching; Chu, Pei-Yu; Chuang, Chih-Hung; Hsieh, Yuan-Chin; Huang, Chien-Chiao; Wang, Yeng-Tseng; Kao, Chien-Han; Roffler, Steve R; Cheng, Tian-Lu

2016-01-01

Molecular weight markers that can tolerate denaturing conditions and be auto-detected by secondary antibodies offer great efficacy and convenience for Western Blotting. Here, we describe M&R LE protein markers which contain linear epitopes derived from the heavy chain constant regions of mouse and rabbit immunoglobulin G (IgG Fc LE). These markers can be directly recognized and stained by a wide range of anti-mouse and anti-rabbit secondary antibodies. We selected three mouse (M1, M2 and M3) linear IgG1 and three rabbit (R1, R2 and R3) linear IgG heavy chain epitope candidates based on their respective crystal structures. Western blot analysis indicated that M2 and R2 linear epitopes are effectively recognized by anti-mouse and anti-rabbit secondary antibodies, respectively. We fused the M2 and R2 epitopes (M&R LE) and incorporated the polypeptide in a range of 15-120 kDa auto-detecting markers (M&R LE protein marker). The M&R LE protein marker can be auto-detected by anti-mouse and anti-rabbit IgG secondary antibodies in standard immunoblots. Linear regression analysis of the M&R LE protein marker plotted as gel mobility versus the log of the marker molecular weights revealed good linearity with a correlation coefficient R2 value of 0.9965, indicating that the M&R LE protein marker displays high accuracy for determining protein molecular weights. This accurate, regular and auto-detected M&R LE protein marker may provide a simple, efficient and economical tool for protein analysis.

Da0324, an inhibitor of nuclear factor-κB activation, demonstrates selective antitumor activity on human gastric cancer cells

PubMed Central

Jin, Rong; Xia, Yiqun; Chen, Qiuxiang; Li, Wulan; Chen, Dahui; Ye, Hui; Zhao, Chengguang; Du, Xiaojing; Shi, Dengjian; Wu, Jianzhang; Liang, Guang

2016-01-01

Background The transcription factor nuclear factor-κB (NF-κB) is constitutively activated in a variety of human cancers, including gastric cancer. NF-κB inhibitors that selectively kill cancer cells are urgently needed for cancer treatment. Curcumin is a potent inhibitor of NF-κB activation. Unfortunately, the therapeutic potential of curcumin is limited by its relatively low potency and poor cellular bioavailability. In this study, we presented a novel NF-κB inhibitor named Da0324, a synthetic asymmetric mono-carbonyl analog of curcumin. The purpose of this study is to research the expression of NF-κB in gastric cancer and the antitumor activity and mechanism of Da0324 on human gastric cancer cells. Methods The expressions between gastric cancer tissues/cells and normal gastric tissues/cells of NF-κB were evaluated by Western blot. The inhibition viability of compounds on human gastric cancer cell lines SGC-7901, BGC-823, MGC-803, and normal gastric mucosa epithelial cell line GES-1 was assessed with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Absorption spectrum method and high-performance liquid chromatography method detected the stability of the compound in vitro. The compound-induced changes of inducible NF-κB activation in the SGC-7901 and BGC-823 cells were examined by Western blot analysis and immunofluorescence methods. The antitumor activity of compound was performed by clonogenic assay, matrigel invasion assay, flow cytometric analysis, Western blot analysis, and Hoechst 33258 staining assay. Results High levels of p65 were found in gastric cancer tissues and cells. Da0324 displayed higher growth inhibition against several types of gastric cancer cell lines and showed relatively low toxicity to GES-1. Moreover, Da0324 was more stable than curcumin in vitro. Western blot analysis and immunofluorescence methods showed that Da0324 blocked NF-κB activation. In addition, Da0324 significantly inhibited tumor proliferation and invasion, arrested the cell cycle, and induced apoptosis in vitro. Conclusion The asymmetric mono-carbonyl analog of curcumin Da0324 exhibited significantly improved antigastric cancer activity. Da0324 may be a promising NF-κB inhibitor for the selective targeting of cancer cells. However, further studies are needed in animals to validate these findings for the therapeutic use of Da0324. PMID:27042000

Investigating the Regulation and Potential Role of Nonhypoxic Hypoxia-Inducible Factor 1 (HIF-1) in Aromatase Inhibitor Resistant Breast Cancer

DTIC Science & Technology

2012-10-01

digestion , prior to analysis of HIF-1 protein by western blot apaparatus. Representative blots are shown. Figure 3. The effects of cell density...mouse xenograft tumors treated for 56 weeks with letrozole, and maintained in phenol red-free (PRF) modified IMEM (Invitrogen) supplemented with 5...conditions for 2–3 days prior to cytospinning. Pelleted cells were fixed with 4 % para- formaldehyde and incubated with OCT4 antibody (Santa Cruz

Detection of anti-salmonella flgk antibodies in chickens by automated capillary immunoassay

USDA-ARS?s Scientific Manuscript database

Western blot is a very useful tool to identify specific protein, but is tedious, labor-intensive and time-consuming. An automated "Simple Western" assay has recently been developed that enables the protein separation, blotting and detection in an automatic manner. However, this technology has not ...

A Study of Rubisco through Western Blotting and Tissue Printing Techniques

ERIC Educational Resources Information Center

Ma, Zhong; Cooper, Cynthia; Kim, Hyun-Joo; Janick-Buckner, Diane

2009-01-01

We describe a laboratory exercise developed for a cell biology course for second-year undergraduate biology majors. It was designed to introduce undergraduates to the basic molecular biology techniques of Western blotting and immunodetection coupled with the technique of tissue printing in detecting the presence, relative abundance, and…

Ferulic acid enhances IgE binding to peanut allergens in western blots.

USDA-ARS?s Scientific Manuscript database

Because phenolic compounds can precipitate or complex with proteins, we postulated that interactions of phenolics with IgE antibodies help enhance IgE binding to peanut allergens in Western blots. Three different phenolics, such as, ferulic, caffeic and chlorogenic acids were examined. Each was mixe...

Progesterone receptor isoforms in the mammary gland of cats and dogs.

PubMed

Gracanin, A; de Gier, J; Zegers, K; Bominaar, M; Rutteman, G R; Schaefers-Okkens, A C; Kooistra, H S; Mol, J A

2012-12-01

Progesterone exerts its effect by binding to specific progesterone receptors (PR) within the cell. In dogs and cats, no data are available on PR isoforms as found in other species. We therefore investigated the sequence of the PR gene and encoded protein in dogs and cats, the expression of PR isoforms in mammary tissue using Western blots and the presence of PR in mammary tissue using immunohistochemistry. Comparison of the amino acid sequence of the canine and feline PR with human PR revealed major differences in the PR-B-specific upstream segment (BUS). However, the essential activation function 3 (AF3) domain was intact in the cat but mutated in the dog. The DNA and ligand-binding domains were highly similar among the species. In cats with fibroadenomatous hyperplasia (FAH), high expression of PR mRNA together with growth hormone (GH), GH receptor (GHR) and IGF-I mRNA was found in comparison with feline mammary carcinomas. Immunohistochemical analysis showed strong nuclear as well as cytoplasmic staining for PR in FAH. Western blot analysis revealed expression of the PR-A and PR-B isoforms in the feline mammary gland. In canine mammary tissue, the most abundant PR staining was found in proliferative zones of the mammary gland. Western blot analyses showed mainly staining for PR-A with lower PR-B staining. It is concluded that in dogs and cats both PR isoforms are expressed. The role of mutations found in the canine PR-B is discussed. © 2012 Blackwell Verlag GmbH.

Water avoidance stress induces frequency through cyclooxygenase-2 expression: a bladder rat model.

PubMed

Yamamoto, Keisuke; Takao, Tetsuya; Nakayama, Jiro; Kiuchi, Hiroshi; Okuda, Hidenobu; Fukuhara, Shinichiro; Yoshioka, Iwao; Matsuoka, Yasuhiro; Miyagawa, Yasushi; Tsujimura, Akira; Nonomura, Norio

2012-02-01

Water avoidance stress is a potent psychological stressor and it is associated with visceral hyperalgesia, which shows degeneration of the urothelial layer mimicking interstitial cystitis. Cyclooxygenase-2 inhibitors have been recognized to ameliorate frequency both in clinical and experimental settings. We investigated the voiding pattern and cyclooxygenase-2 expression in a rat bladder model of water avoidance stress. After being subjected to water avoidance stress or a sham procedure, rats underwent metabolic cage analysis and cystometrography. Real time reverse transcription polymerase chain reaction was carried out to examine cyclooxygenase-2 messenger ribonucleic acid in bladders of rats. Protein expression of cyclooxygenase-2 was analyzed with immunohistochemistry and western blotting. Furthermore, the effects of the cyclooxygenase-2 inhibitor, etodolac, were investigated by carrying out cystometrography, immunohistochemistry and western blotting. Metabolic cage analysis and cystometrography showed significantly shorter intervals and less volume of voiding in water avoidance stress rats. Significantly higher expression of cyclooxygenase-2 messenger ribonucleic acid was verified by reverse transcription polymerase chain reaction. Immunohistochemistry and western blotting showed significantly higher cyclooxygenase-2 protein levels in water avoidance stress bladders. Furthermore, immunohistochemistry showed high cyclooxygenase-2 expression exclusively in smooth muscle cells. All water avoidance stress-induced changes were reduced by cyclooxygenase-2 inhibitor pretreatment. Chronic stress might cause frequency through cyclooxygenase-2 gene upregulation in bladder smooth muscle cells. Further study of cyclooxygenase-2 in the water avoidance stress bladder might provide novel therapeutic modalities for interstitial cystitis. © 2011 The Japanese Urological Association.

A Spore Coat Protein, CotS, of Bacillus subtilis Is Synthesized under the Regulation of ςK and GerE during Development and Is Located in the Inner Coat Layer of Spores

PubMed Central

Takamatsu, Hiromu; Chikahiro, Yukari; Kodama, Takeko; Koide, Hidekatsu; Kozuka, Satoshi; Tochikubo, Kunio; Watabe, Kazuhito

1998-01-01

The spore coat of Bacillus subtilis has a unique morphology and consists of polypeptides of different sizes, whose synthesis and assembly are precisely regulated by a cascade of transcription factors and regulatory proteins. We examined the factors that regulate cotS gene expression and CotS assembly into the coat layer of B. subtilis by Northern blot and Western blot analysis. Transcription of cotS mRNA was not detected in sporulating cells of ςK and gerE mutants by Northern blot analysis. By Western blot analysis using anti-CotS antibody, CotS was first detected in protein samples solubilized from wild-type cells at 5 h after the start of sporulation. CotS was not detected in the vegetative cells and spores of a gerE mutant or in the spores of mutants deficient in ςE, ςF, ςG, or ςK. CotS was detected in the sporangium but not in the spores of a cotE mutant. The sequence of the promoter region of cotS was similar to the consensus sequences for binding of ςK and GerE. These results demonstrate that ςK and GerE are required for cotS expression and that CotE is essential for the assembly of CotS in the coat. Immunoelectron microscopic observation using anti-CotS antibody revealed that CotS is located within the spore coat, in particular in the inner coats of dormant spores. PMID:9603889

Etoposide induces apoptosis via the mitochondrial- and caspase-dependent pathways and in non-cancer stem cells in Panc-1 pancreatic cancer cells.

PubMed

Zhang, She-Hong; Huang, Qian

2013-12-01

Pancreatic cancer is a highly aggressive malignant tumor. In the present study, we performed several methods, including CCK-8 assay, immunofluorescence technique, western blotting and flow cytometry, to determine the effects of VP16 (etoposide) on Panc-1 pancreatic cancer cells. The results demonstrated that VP16 inhibited the growth of and induced apoptosis in Panc-1 cells. Western blot analysis showed that VP16 inhibited the expression of Bcl-2 and enhanced the expression of Bax, caspases-3 and -9, cytochrome c and PARP. Notably, a strong inhibitory effect of VP16 on Panc-1 cells mainly occurred in non-CSCs. These data provide a new strategy for the therapy of pancreatic cancer.

Actin expression in some Platyhelminthe species.

PubMed

Fagotti, A; Panara, F; Di Rosa, I; Simoncelli, F; Gabbiani, G; Pascolini, R

1994-10-01

Actin expression in some Platyhelminthe species was demonstrated by western-blotting and immunocytochemical analysis using two distinct anti-actin antibodies: the anti-total actin that reacts against all actin isoforms of higher vertebrates and the anti-alpha SM-1 that recognizes the alpha-smooth muscle (alpha SM) isotype of endothermic vertebrates (Skalli et al., 1986). Western-blotting experiments showed that all species tested, including some free-living Platyhelminthes (Tricladida and Rhabdocoela) and the parasitic Fasciola hepatica, were stained by anti-total actin antibody while only Dugesidae and Dendrocoelidae showed a positive immunoreactivity against anti-alpha SM-1. These results were confirmed by cytochemical immunolocalization using both avidin biotin conjugated peroxidase reaction on paraffin sections, and immunogold staining on Lowicryl 4KM embedded specimens. Our findings may contribute to the understanding of Platyhelminthes phylogeny.

Outbreak of larval Echinococcus multilocularis infection in Japanese monkey (Macaca fuscata) in a zoo, Hokkaido: western blotting patterns in the infected monkeys.

PubMed

Sato, Chiaki; Kawase, Shiro; Yano, Shoki; Nagano, Hideki; Fujimoto, Satoshi; Kobayashi, Nobuyuki; Miyahara, Kazuro; Yamada, Kazutaka; Sato, Motoyoshi; Kobayashi, Yoshiyasu

2005-01-01

A high prevalence of larval Echinococcus multilocularis (Em) infection was found in zoo primates in Hokkaido, Japan. In October 1997, a Japanese monkey (Macaca fuscata) died and histopathologically diagnosed as alveolar hydatidosis. Serum samples were collected from the remaining Japanese monkeys and examined for antibodies against Em by enzyme-linked immunosorbent assay and western blotting. Serological tests showed 12 more animals of the remaining 57 monkeys were possibly infected. Ultrasonography revealed that nine of these 12 animals had a cystic lesion in the liver. The band patterns of western blotting in the monkeys were very similar to those in human.

A Proteomic Approach for the Identification of Up-Regulated Proteins Involved in the Metabolic Process of the Leiomyoma.

PubMed

Ura, Blendi; Scrimin, Federica; Arrigoni, Giorgio; Franchin, Cinzia; Monasta, Lorenzo; Ricci, Giuseppe

2016-04-09

Uterine leiomyoma is the most common benign smooth muscle cell tumor of the uterus. Proteomics is a powerful tool for the analysis of complex mixtures of proteins. In our study, we focused on proteins that were upregulated in the leiomyoma compared to the myometrium. Paired samples of eight leiomyomas and adjacent myometrium were obtained and submitted to two-dimensional gel electrophoresis (2-DE) and mass spectrometry for protein identification and to Western blotting for 2-DE data validation. The comparison between the patterns revealed 24 significantly upregulated (p < 0.05) protein spots, 12 of which were found to be associated with the metabolic processes of the leiomyoma and not with the normal myometrium. The overexpression of seven proteins involved in the metabolic processes of the leiomyoma was further validated by Western blotting and 2D Western blotting. Four of these proteins have never been associated with the leiomyoma before. The 2-DE approach coupled with mass spectrometry, which is among the methods of choice for comparative proteomic studies, identified a number of proteins overexpressed in the leiomyoma and involved in several biological processes, including metabolic processes. A better understanding of the mechanism underlying the overexpression of these proteins may be important for therapeutic purposes.

A Proteomic Approach for the Identification of Up-Regulated Proteins Involved in the Metabolic Process of the Leiomyoma

PubMed Central

Ura, Blendi; Scrimin, Federica; Arrigoni, Giorgio; Franchin, Cinzia; Monasta, Lorenzo; Ricci, Giuseppe

2016-01-01

Uterine leiomyoma is the most common benign smooth muscle cell tumor of the uterus. Proteomics is a powerful tool for the analysis of complex mixtures of proteins. In our study, we focused on proteins that were upregulated in the leiomyoma compared to the myometrium. Paired samples of eight leiomyomas and adjacent myometrium were obtained and submitted to two-dimensional gel electrophoresis (2-DE) and mass spectrometry for protein identification and to Western blotting for 2-DE data validation. The comparison between the patterns revealed 24 significantly upregulated (p < 0.05) protein spots, 12 of which were found to be associated with the metabolic processes of the leiomyoma and not with the normal myometrium. The overexpression of seven proteins involved in the metabolic processes of the leiomyoma was further validated by Western blotting and 2D Western blotting. Four of these proteins have never been associated with the leiomyoma before. The 2-DE approach coupled with mass spectrometry, which is among the methods of choice for comparative proteomic studies, identified a number of proteins overexpressed in the leiomyoma and involved in several biological processes, including metabolic processes. A better understanding of the mechanism underlying the overexpression of these proteins may be important for therapeutic purposes. PMID:27070597

Profiling EGFR activity in head and neck squamous cell carcinoma by using a novel layered membrane Western blot technology.

PubMed

Patel, Vyomesh; Ramesh, Arun; Traicoff, June L; Baibakov, Galina; Emmert-Buck, Michael R; Gutkind, J Silvio; Knezevic, Vladimir

2005-05-01

Given the role of epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinomas (HNSCC), several rational approaches have now been utilized to abrogate tyrosine kinase activity and its disengagement from downstream signal transducers. Monitoring the activity of these molecules could potentially be useful to determine not only drug efficacy but also to identify HNSCC patients most likely to benefit from this type of therapy. In this study we have used a novel high throughput multi-layered Western blotting (MLWestern) method that allows the detection of multiple proteins from a single experiment in order to characterize key components in the EGFR signaling pathway in HNSCC cells. Total and activated forms of EGFR and the downstream effectors, Erk and Akt were readily detected in HNSCC cells, where in the control cells (HaCaT) these proteins could only be detected in EGF stimulated cells. Results from conventional Western blot and MLWestern were comparable. Clustering analysis of protein expression revealed similarities in cellular response between some of the cell lines indicative of similarities in their biological response. The data indicate that MLWestern can be potentially applied to identify molecular targets that could be used for rational therapeutic intervention strategies.

Western blotting revisited: critical perusal of underappreciated technical issues.

PubMed

Gorr, Thomas A; Vogel, Johannes

2015-04-01

The most commonly used semiquantitative analysis of protein expression still employs protein separation by denaturing SDS-PAGE with subsequent Western blotting and quantification of the resulting ODs of bands visualized with specific antibodies. However, many questions regarding this procedure are usually ignored, although still in need of answering: Does isolation or separation procedure harm the integrity or affect modifications (e.g., phosphorylation) of the protein of interest? Does denaturation reduce binding of antibodies used for detection? Should denaturation be performed or should a native gel be run? How can artificial degradations or aggregations be distinguished from biological relevant ones? If the antibody detects multiple bands (which is not uncommon), which one(s) should be taken into account for quantification and why? Which loading control protein should be chosen and is it really "housekeeping" and how can this be verified? Is the image acquisition system linear and does it come with a sufficient dynamic range? How to account and control for background staining? This article is intended to address these questions and raise the readers awareness to possible Western blot alternatives in the attempt of minimizing possible pitfalls that might loom anywhere from protein isolation to acquisition of final quantitative data. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Interaction of murine intestinal mast cell proteinase with inhibitors (serpins) in blood; analysis by SDS-PAGE and western blotting.

PubMed Central

Irvine, J; Newlands, G F; Huntley, J F; Miller, H R

1990-01-01

The interaction of mouse intestinal mast cell proteinase (IMCP) with serine proteinase inhibitors (serpins) in blood was analysed: (i) by examining the capacity of the inhibitors in blood to block the binding of the irreversible serine esterase inhibitor [3H]diisopropyl fluorophosphate (DFP); (ii) by Western blotting. The binding of [3H]DFP to IMCP was blocked very rapidly by inhibitors in mouse serum and, by Western blotting, this inhibition was associated with the appearance of a 73,000 MW proteinase/inhibitor complex together with a series of higher (greater than 100,000) MW complexes. IMCP was not dissociated from these complexes when electrophoresed under reducing conditions, although prior heat treatment of mouse serum (60 for 30-160 min) abolished the formation of all proteinase/inhibitor complexes. Similarly, the activity of a 48,000 MW inhibitor of chymotrypsin was abolished by heat treatment. A titration experiment established that between 0.5 and 5 mg IMCP were inhibited per ml of serum. The properties and MW of the IMCP inhibitor complexes are typical of serpins and suggest that IMCP secreted during intestinal immunological reactions would be rapidly and irreversibly inactivated by plasma-derived inhibitors. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:2312150

7, 8, 3′-Trihydroxyflavone Promotes Neurite Outgrowth and Protects Against Bupivacaine-Induced Neurotoxicity in Mouse Dorsal Root Ganglion Neurons

PubMed Central

Shi, Haohong; Luo, Xingjing

2016-01-01

Background 7, 8, 3′-trihydroxyflavone (THF) is a novel pro-neuronal small molecule that acts as a TrkB agonist. In this study, we examined the effect of THF on promoting neuronal growth and protecting anesthetics-induced neurotoxicity in dorsal root ganglion (DRG) neurons in vitro. Material/Methods Neonatal mouse DRG neurons were cultured in vitro and treated with various concentrations of THF. The effect of THF on neuronal growth was investigated by neurite outgrowth assay and Western blot. In addition, the protective effects of THF on bupivacaine-induced neurotoxicity were investigated by apoptosis TUNEL assay, neurite outgrowth assay, and Western blot, respectively. Results THF promoted neurite outgrowth of DRG neurons in dose-dependent manner, with an EC50 concentration of 67.4 nM. Western blot analysis showed THF activated TrkB signaling pathway by inducing TrkB phosphorylation. THF also rescued bupivacaine-induced neurotoxicity by reducing apoptosis and protecting neurite retraction in DRG neurons. Furthermore, the protection of THF in bupivacaine-injured neurotoxicity was directly associated with TrkB phosphorylation in a concentration-dependent manner in DRG neurons. Conclusions THF has pro-neuronal effect on DRG neurons by promoting neurite growth and protecting against bupivacaine-induced neurotoxicity, likely through TrkB activation. PMID:27371503

Comparison of a serum indirect fluorescent antibody test with two Western blot tests for the diagnosis of equine protozoal myeloencephalitis.

PubMed

Duarte, Paulo C; Daft, Barbara M; Conrad, Patricia A; Packham, Andrea E; Gardner, Ian A

2003-01-01

A serum indirect fluorescent antibody test (IFAT) was compared with a Western blot (WB) and a modified Western blot (mWB) for diagnosis of equine protozoal myeloencephalitis (EPM). Using receiver-operating characteristic (ROC) analysis, the area under the curve of the IFAT was greater than the areaunder the curves of the WB and the mWB (P = 0.025 and P = 0.044, respectively). There was no statistically significant difference between the areas under the curves of the WBs (P > 0.05). On the basis of an arbitrarily chosen cut-off titer for a positive test result of 1:80 for the IFAT and interpreting weak positive WB results as positive test results, the sensitivities and 95% confidence intervals (CI) of all 3 tests were identical and equal to 88.9% (51.8-99.7%). The specificities and 95% CIs of the IFAT, WB, and mWB test were 100% (91-100%), 87.2% (72.6-95.7%), and 69.2% (52.4-83%), respectively. The overall accuracy of the IFAT was shown to be better than that of the WBs and, therefore, the test has potential for use in the diagnosis of EPM caused by Sarcocystis neurona.

HSP-70 as a nonspecific early marker in cisplatin ototoxicity.

PubMed

Ramírez-Camacho, R; Citores, M J; Trinidad, A; Verdaguer, J M; García-Berrocal, J R; Marero, A Martín; Puente, A; González-García, J A; Vargas, J A

2007-06-01

The great variety of pathological entities related to the presence of circulating HSP-70 suggests a nonspecific cellular damage. As the present study shows, positive results decrease with respect to the time elapsed after the injection of the ototoxic agent. HSP-70 appears as an early and transient marker that could permit early detection of inner ear damage. The aim of this study was to determine the presence of HSP-70 at different time points by means of Western blot immunoassay in the sera of rats treated with cisplatin. Thirty-six Wistar rats were intraperitoneally injected with cisplatin at a dose of 5 mg/kg and blood samples were collected at 7 and 90 days. Determination of HSP-70 was made by means of a modified Western blot immunoassay kit originally used for human HSP-70 antigen detection. A control group of 18 animals was used for comparison. Western blot was positive in 77.8% of the animals in the 7 days group, decreasing to a 44.4% in the 90 days group. In the control group, Western blot was positive in 5.5%.

Multiplexed Western Blotting Using Microchip Electrophoresis.

PubMed

Jin, Shi; Furtaw, Michael D; Chen, Huaxian; Lamb, Don T; Ferguson, Stephen A; Arvin, Natalie E; Dawod, Mohamed; Kennedy, Robert T

2016-07-05

Western blotting is a commonly used protein assay that combines the selectivity of electrophoretic separation and immunoassay. The technique is limited by long time, manual operation with mediocre reproducibility, and large sample consumption, typically 10-20 μg per assay. Western blots are also usually used to measure only one protein per assay with an additional housekeeping protein for normalization. Measurement of multiple proteins is possible; however, it requires stripping membranes of antibody and then reprobing with a second antibody. Miniaturized alternatives to Western blot based on microfluidic or capillary electrophoresis have been developed that enable higher-throughput, automation, and greater mass sensitivity. In one approach, proteins are separated by electrophoresis on a microchip that is dragged along a polyvinylidene fluoride membrane so that as proteins exit the chip they are captured on the membrane for immunoassay. In this work, we improve this method to allow multiplexed protein detection. Multiple injections made from the same sample can be deposited in separate tracks so that each is probed with a different antibody. To further enhance multiplexing capability, the electrophoresis channel dimensions were optimized for resolution while keeping separation and blotting times to less than 8 min. Using a 15 μm deep × 50 μm wide × 8.6 cm long channel, it is possible to achieve baseline resolution of proteins that differ by 5% in molecular weight, e.g., ERK1 (44 kDa) from ERK2 (42 kDa). This resolution allows similar proteins detected by cross-reactive antibodies in a single track. We demonstrate detection of 11 proteins from 9 injections from a single Jurkat cell lysate sample consisting of 400 ng of total protein using this procedure. Thus, multiplexed Western blots are possible without cumbersome stripping and reprobing steps.

Retraction Statement: Anti-inflammatory properties of tianeptine on lipopolysaccharide-induced changes in microglial cells involve toll-like receptor-related pathways.

PubMed

2017-09-01

'Anti-inflammatory properties of tianeptine on lipopolysaccharide-induced changes in microglial cells involve toll-like receptor-related pathways' by Slusarczyk, J., Trojan, E., Glombik, K., Piotrowska, A., Budziszewska, B., Kubera, M., Popiolek-Barczyk, K., Lason, W., Mika, J. and Basta-Kaim, A. The above article from the Journal of Neurochemistry published on 14 February 2016 on Wiley Online Library ( www.onlinelibrary.com), and in Volume 136, pp. 958-970, is being retracted by agreement between the corresponding author Agnieszka Basta-Kaim, the Journal's Editor-in-Chief Jörg Schulz, and John Wiley & Sons Ltd. The Editorial Office was alerted by a science journalist that the same Western Blot lane had been used to represent two different proteins. The Western Blot signal of iNOS in Fig. 4a was supposedly identical to the Western Blot signal of phospho-JNK in Fig. 6b. The corresponding author stated that "on the final step of figure 6 preparation the first author made, by mistake, an incorrect attachment of representative p-JNK blots." A corrected Fig. 6b is enclosed below. The second concern reaching the Editorial Office was that the same Western Blot signal appeared to have been used to represent two different experimental conditions: the iNOS control signal (-/- LPS/TIA Fig. 4a) appears as a horizontal and vertical mirror image of the last signal in this line (+/10 LPS/TIA Fig. 4a). The raw membrane which was used to produce Fig. 4a is enclosed on the next page and highlights the steps that were undertaken during figure preparation. Although the initial concern was not proven, concerns remained regarding the question how an inadvertent flipping of the first Western blot lane could happen. A corrected Fig. 4a prepared by the corresponding author from the raw image of iNOS western blot depicted above, without flipped first lane, is presented below: Although the corresponding author provided a large amount of evidence to explain disparities in the presentation of Western Blot images, due to the number of inconsistencies that were revealed during review of the provided evidence and the inability to confirm the nature of the steps that led to them, it was felt that the above mentioned Western Blot images presented in this publication were not reliable, even if the conclusions may still be valid. The first author would like to apologize to the readers, reviewers and editors of Journal of Neurochemistry for the errors. Reference Slusarczyk J., Trojan E., Glombik K., Piotrowska A., Budziszewska B., Kubera M., Popiolek-Barczyk K., Lason W., Mika J. and Basta-Kaim A. (2016) Anti-inflammatory properties of tianeptine on lipopolysaccharide-induced changes in microglial cells involve toll-like receptor-related pathways. J. Neurochem. 136, 958-970. https://doi.org/10.1111/jnc.13452. © 2017 International Society for Neurochemistry.

Human umbilical cord mesenchymal stem cells improve the reserve function of perimenopausal ovary via a paracrine mechanism.

PubMed

Li, Jia; Mao, QiuXian; He, JingJun; She, HaoQing; Zhang, Zhi; Yin, ChunYan

2017-03-09

Human umbilical cord mesenchymal stem cells (hUCMSCs) are a type of pluripotent stem cell which are isolated from the umbilical cord of newborns. hUCMSCs have great therapeutic potential. We designed this experimental study in order to investigate whether the transplantation of hUCMSCs can improve the ovarian reserve function of perimenopausal rats and delay ovarian senescence. We selected naturally aging rats confirmed by vaginal smears as models of perimenopausal rats, divided into the control group and the treatment group, and selected young fertile female rats as normal controls. hUCMSCs were transplanted into rats of the treatment group through tail veins. Enzyme-linked immunosorbent assay (ELISA) detected serum levels of sex hormones, H&E staining showed ovarian tissue structure and allowed follicle counting, immunohistochemistry and western blot analysis revealed ovarian expression of hepatocyte growth factor (HGF), vascular endothelial cell growth factor (VEGF), and insulin-like growth factor-1 (IGF-1), polymerase chain reaction (PCR) and western blot analysis revealed hUCMSCs expression of HGF, VEGF, and IGF-1. At time points of 14, 21, and 28 days after hUCMSCs transplantation, estradiol (E 2 ) and anti-Müllerian hormone (AMH) increased while follicle-stimulating hormone (FSH) decreased; ovarian structure improved and follicle number increased; ovarian expression of HGF, VEGF, and IGF-1 protein elevated significantly. Meanwhile, PCR and western blot analysis indicated hUCMSCs have the capacity of secreting HGF, VEGF, and IGF-1 cytokines. Our results suggest that hUCMSCs can promote ovarian expression of HGF, VEGF, and IGF-1 through secreting those cytokines, resulting in improving ovarian reserve function and withstanding ovarian senescence.

[Regulatory analysis of hypoxia on innate immunity of human corneal epithelium].

PubMed

Pang, K P; Pan, H; Wu, X Y

2016-11-15

Objective: To investigate the role of hypoxia on the regulation of innate immunity of human corneal epithelium. Methods: Telomerase-immortalized human epithelial cells (THCEs) were incubated under normoxia (21% O 2 ) or hypoxic (1% O 2 ) conditions respectively. After 6, 12, 24, 48 h culture, the mRNA and protein levels of toll like receptor 4 (TLR4) were measured by real-time polymerase chain reaction (RT-PCR) and Western blot analysis. After 24 h culture, THCEs of each group were challenged respectively with TLR4 ligand lipopolysaccharide (LPS) (1 μg/ml) for 6 h. RT-PCR was used to assess the mRNA level of myeloid differentiation factor 88 (MyD88), interleukin(IL)6, IL-8 and tumor necrosis factor α (TNF-α). Western blot was used to examine the protein level of inhibitor of nuclear factor kappa-B α (IκBα) and phosphorylated IκBα (p-IκBα). Enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of the inflammatory cytokines IL-6, IL-8 and TNF-α. Results: The results of RT-PCR and Western blot analysis showed that the expression of TLR4 downregulated 90% and 55% respectively after hypoxic exposure for 48 h. Hypoxia also inhibited LPS-induced secretion of IL-6, IL-8, TNF-α, expression of MyD88 and activation of NF-κB. The mRNA level of MyD88 was diminished 63%, and the protein expression of p-IκBα was also lowered. Meanwhile, the secretions of IL-6, IL-8 and TNF-α under hypoxia were reduced (31%, 55% and 50% respectively). Conclusion: Hypoxia attenuated immune and inflammatory response of the cornea epithelium by suppressing TLR4 signaling, and could enhance cell susceptibility to microorganism infection.

The nuclear-factor kappaB pathway is activated in pterygium.

PubMed

Siak, Jay Jyh Kuen; Ng, See Liang; Seet, Li-Fong; Beuerman, Roger W; Tong, Louis

2011-01-05

Pterygium is a prevalent ocular surface disease with unknown pathogenesis. The authors investigated the role of nuclear factor kappa B (NF-κB) transcription factors in pterygium. Surgically excised primary pterygia were studied compared with uninvolved conjunctiva tissues. NF-κB activation was evaluated using Western blot analysis, ELISA, and DNA-binding assays. Primary pterygium fibroblasts were treated with TNF-α (20 ng/mL), and NF-κB activation was evaluated using immunocytochemistry, Western blot analysis, phospho-IκBα ELISA, and DNA-binding assays. TNF-α stimulation of NF-κB target genes RelB, NFKB2, RANTES, MCP-1, ENA-78, MMP-1, MMP-2, and MMP-3 in pterygium fibroblasts was compared with that in primary tenon fibroblasts by real-time PCR. Phosphorylation of IκBα (Ser32) was increased in pterygia tissues compared with uninvolved conjunctiva tissues, as determined by Western blot analysis and ELISA. IκBα expression was decreased, whereas nuclear RelA and p50 DNA-binding capacities were increased. Within 30 minutes of treatment with TNF-α, pterygium fibroblasts showed increased IκBα phosphorylation and nuclear translocation of RelA and p50. Treatment with TNF-α beyond 12 hours resulted in increased nuclear expression of RelB, p100, and p52. Furthermore, the upregulation of RANTES, MCP-1, ENA-78, MMP-1, MMP-2, and MMP-3 expression was more pronounced in TNF-α-treated pterygium fibroblasts than in tenon fibroblasts. The NF-κB pathway is shown for the first time to be activated in pterygia tissues compared with normal conjunctiva tissues. Stimulation by the inflammatory cytokine TNF-α can activate both canonical and noncanonical NF-κB pathways in pterygium fibroblasts with concomitant upregulation of NF-κB target genes.

Tanshinone IIA protects H9c2 cells from oxidative stress-induced cell death via microRNA-133 upregulation and Akt activation.

PubMed

Gu, Yunfei; Liang, Zhuo; Wang, Haijun; Jin, Jun; Zhang, Shouyan; Xue, Shufeng; Chen, Jianfeng; He, Huijuan; Duan, Kadan; Wang, Jing; Chang, Xuewei; Qiu, Chunguang

2016-08-01

The aim of the present study was to investigate the cardioprotective effect of tanshinone IIA and the underlying molecular mechanisms. An in vitro model of oxidative stress injury was established in cardiac H9c2 cells, and the effects of tanshinone IIa were investigated using cell viability, reverse transcription-quantitative polymerase chain reaction and western blotting assays. The results demonstrated that tanshinone IIA protects H9c2 cells from H 2 O 2 -induced cell death in a concentration-dependent manner, via a mechanism involving microRNA-133 (miR-133), and that treatment with TIIA alone exerted no cytotoxic effects on H9c2. In order to further elucidate the mechanisms underlying the actions of TIIA, reverse transcription-quantitative polymease chain reaction and western blot analysis were performed. Reductions in miR-133 expression levels induced by increasing concentrations of H 2 O 2 were reversed by treatment with tanshinone IIA. In addition, the inhibition of miR-133 by transfection with an miR-133 inhibitor abolished the cardioprotective effects of tanshinone IIA against H 2 O 2 -induced cell death. Furthermore, western blot analysis demonstrated that tanshinone IIA activated Akt kinase via the phosphorylation of serine 473. Inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway by pretreatment with the PI3K specific inhibitors wortmannin and LY294002 also eliminated the cardioprotective effects of tanshinone IIA against H 2 O 2 -induced cell death. Western blot analysis demonstrated that H 2 O 2 -induced reductions in B cell lymphoma 2 (Bcl-2) expression levels were reversed by tanshinone IIA. In addition, the effect of tanshinone IIA on Bcl-2 protein expression level in an oxidative environment was suppressed by a PI3K inhibitor, wortmannin, indicating that tanshinone IIA exerts cardioprotective effects against H 2 O 2 -induced cell death via the activation of the PI3K/Akt signal transduction pathway and the consequent upregulation of Bcl-2. In conclusion, the present study demonstrates that TIIA is able to protcet H9c2 cells from oxidative stress-induced cell death through signalling pathways involving miR-133 and Akt, and that tanshinone IIA is a promising natural cardioprotective agent.

A Streamlined Western Blot Exercise: An Efficient and Greener Approach in the Laboratory Classroom

ERIC Educational Resources Information Center

Ness, Traci L.; Robinson, Rebekah L.; Mojadedi, Wais; Peavy, Lydia; Weiland, Mitch H.

2015-01-01

SDS-PAGE and western blotting are two commonly taught protein detection techniques in biochemistry and molecular biology laboratory classrooms. A pitfall associated with incorporating these techniques into the laboratory is the significant wait times that do not allow students to obtain timely results. The waiting associated with SDS-PAGE comes…

CATALASE FROM A FUNGAL MICROBIAL PESTICIDE INDUCES A UNIQUE IGE RESPONSE.

EPA Science Inventory

BALB/c mice exposed by involuntary aspiration to Metarhizium anisopliae extract (MACA), a microbial pesticide, have shown responses characteristic of human allergic lung disease/asthma. IgE-binding proteins have been identified in MACA by Western blot analysis, 2-dimensio...

[Research on secretion expression in Pichia pastoris and function of the HC-pro gene of watermelon mosaic virus].

PubMed

Zhang, Jian-Xin; Wu, Yun-Feng; Wang, Xiu-Min

2007-11-01

HC-pro gene of Watermelon Mosaic virus was obtained by RT-PCR was 1371bp in length. It was cloned into pPI(9K, then the eucaryotic recombinant expression plasmid pPIC9K-WHC was constructed. After being linearized with restriction endonuclease Sal I , the recombinant plasmid was transformed into Pichia pastoris GS115 by electroporation. The high copy transformants with Mut+ /His+ phenotype were selected by RT-PCR and screening on G418, MD and MM medium. Induced by methanol for 5 days, the culture supernatant was analyzed by SDS-PAGE, the results showed that a specific protein with a molecular weight of about 66 kD was expressed. Western blot analysis proved that the expression protein could specifically bind to HC-Pro polyclonal antibody. Far western blot analysis proved that the expression protein could bind to coat protein, given support to "bridge" hypothesis that HC-Pro help aphid transmission of non-persistent viruses.

Expression of the glutamine metabolism-related proteins glutaminase 1 and glutamate dehydrogenase in canine mammary tumours.

PubMed

Ryu, J-E; Park, H-K; Choi, H-J; Lee, H-B; Lee, H-J; Lee, H; Yu, E-S; Son, W-C

2018-06-01

Glutamine metabolism is an important metabolic pathway for cancer cell survival, and there is a critical connection between tumour growth and glutamine metabolism. Because of their similarities, canine mammary carcinomas are useful for studying human breast cancer. Accordingly, we investigated the correlations between the expression of glutamine metabolism-related proteins and the pathological features of canine mammary tumours. We performed immunohistochemical and western blot analysis of 39 mammary tumour tissues. In immunohistochemical analysis, the expression of glutaminase 1 (GLS1) in the epithelial region increased according to the histological grade (P < .005). In the stromal region, complex-type tumours displayed significantly higher GLS1 intensity than simple-type tumours. However, glutamate dehydrogenase expression did not show the same tendencies as GLS1. The western blot results were consistent with the immunohistochemical findings. These results suggest that the expression of GLS1 is correlates with clinicopathological factors in canine mammary tumours and shows a similar pattern to human breast cancer. © 2017 John Wiley & Sons Ltd.

Quantitative assessment of serum-specific IgE in the diagnosis of human cystic echinococcosis.

PubMed

Marinova, I; Nikolov, G; Michova, A; Kurdova, R; Petrunov, B

2011-07-01

Anti-Echinococcus serum immunoglobulin (Ig)E was assessed by the ImmunoCAP system and compared with anti-Echinococcus serum IgG assessed by enzyme-linked immunosorbent assay (ELISA) and Western blot. The ImmunoCAP system revealed very high specificity (one false positive of 110 healthy individuals), low cross-reactivity (one false positive of 58 patients with other diseases) and decreased sensitivity (73.55%). Receiver operating characteristic analysis displayed a beneficial diagnostic value with high accuracy. Comparison of the ImmunoCAP system with ELISA and Western blot showed significantly higher specificity and significantly lower cross-reactivity compared with the ELISA. Examination of sera from 155 patients with cystic echinococcosis (CE) showed varying levels of anti-Echinococcus IgE (range, 0.01-118.33 kUA/L). However, most samples had moderately elevated IgE levels. Analysis of serum-specific IgE revealed significantly higher sensitivity of the ImmunoCAP system and significantly higher antibody levels in hepatic CE compared with pulmonary CE. © 2011 Blackwell Publishing Ltd.

Problem-Solving Test: Southwestern Blotting

ERIC Educational Resources Information Center

Szeberényi, József

2014-01-01

Terms to be familiar with before you start to solve the test: Southern blotting, Western blotting, restriction endonucleases, agarose gel electrophoresis, nitrocellulose filter, molecular hybridization, polyacrylamide gel electrophoresis, proto-oncogene, c-abl, Src-homology domains, tyrosine protein kinase, nuclear localization signal, cDNA,…

DOE Office of Scientific and Technical Information (OSTI.GOV)

Azarkh, Eugene; Robinson, Erin; Hirunkanokpun, Supanee

Mosquito densonucleosis viruses synthesize two non-structural proteins, NS1 and NS2. While NS1 has been studied relatively well, little is known about NS2. Antiserum was raised against a peptide near the N-terminus of NS2, and used to conduct Western blot analysis and immuno-fluorescence assays. Western blots revealed a prominent band near the expected size (41 kDa). Immuno-fluorescence studies of mosquito cells transfected with AeDNV indicate that NS2 has a wider distribution pattern than does NS1, and the distribution pattern appears to be a function of time post-infection. Nuclear localization of NS2 requires intact C-terminus but does not require additional viral proteins.more » Mutations ranging from complete NS2 knock-out to a single missense amino acid substitution in NS2 can significantly reduce viral replication and production of viable progeny.« less

CRALBP is a highly prevalent autoantigen for human autoimmune uveitis.

PubMed

Deeg, Cornelia A; Raith, Albert J; Amann, Barbara; Crabb, John W; Thurau, Stephan R; Hauck, Stefanie M; Ueffing, Marius; Wildner, Gerhild; Stangassinger, Manfred

2007-01-01

Cellular retinaldehyde binding protein (CRALBP) is an autoantigen in spontaneous equine recurrent uveitis. In order to test whether CRALBP contributes to human autoimmune uveitis, the specificity of antibodies from human uveitis patient's sera was first evaluated in two-dimensional (2D) Western blot analysis. Subsequent identification of the immunoreactive proteins by mass spectrometry resulted in the identification of CRALBP as a putative autoantigen. Additionally, sera from human uveitis and control patients were by Western blot using purified human recombinant CRALBP. Anti-CRALBP autoantibodies occur more frequently (P<.01) in human uveitis patients than in normal controls. Thirty out of 56 tested uveitis patient's sera contained autoantibodies reactive against CRALBP, compared to only four out of 23 normal control subjects. The presence of CRALBP autoantibodies in 54% of tested uveitis patients supports CRALBP as a possible autoantigen in human autoimmune uveitis.

Productive Infection of Bovine Papillomavirus Type 2 in the Placenta of Pregnant Cows Affected with Urinary Bladder Tumors

PubMed Central

Roperto, Sante; Borzacchiello, Giuseppe; Esposito, Iolanda; Riccardi, Marita; Urraro, Chiara; Lucà, Roberta; Corteggio, Annunziata; Tatè, Rosarita; Cermola, Michele; Paciello, Orlando; Roperto, Franco

2012-01-01

Papillomaviruses (PVs) are believed to be highly epitheliotropic as they usually establish productive infections within stratified epithelia. In vitro, various PVs appear to complete their entire life-cycle in different trophoblastic cell lines. In this study, infection by and protein expression of bovine papillomavirus type 2 (BPV-2) in the uterine and chorionic epithelium of the placenta has been described in four cows suffering from naturally occurring papillomavirus-associated urothelial bladder tumors. E5 oncoprotein was detected both by Western blot analysis and immunohistochemically. It appears to be complexed and perfectly co-localized with the activated platelet-derived growth factor ß receptor (PDGFßR) by laser scanning confocal microscopy. The activated PDGFßR might be involved in organogenesis and neo-angiogenesis rather than in cell transformation during pregnancy. The major capsid protein, L1, believed to be only expressed in productive papillomavirus infection has been detected by Western blot analysis. Immunohistochemical investigations confirmed the presence of L1 protein both in the cytoplasm and nuclei of cells of the uterine and chorionic epithelium. Trophoblastic cells appear to be the major target for L1 protein expression. Finally, the early protein E2, required for viral DNA replication and known to be expressed during a productive infection, has been detected by Western blot and immunohistochemically. Electron microscopic investigations detected viral particles in nuclei of uterine and chorionic epithelium. This study shows that both active and productive infections by BPV-2 in the placenta of pregnant cows can occur in vivo. PMID:22479413

Productive infection of bovine papillomavirus type 2 in the placenta of pregnant cows affected with urinary bladder tumors.

PubMed

Roperto, Sante; Borzacchiello, Giuseppe; Esposito, Iolanda; Riccardi, Marita; Urraro, Chiara; Lucà, Roberta; Corteggio, Annunziata; Tatè, Rosarita; Cermola, Michele; Paciello, Orlando; Roperto, Franco

2012-01-01

Papillomaviruses (PVs) are believed to be highly epitheliotropic as they usually establish productive infections within stratified epithelia. In vitro, various PVs appear to complete their entire life-cycle in different trophoblastic cell lines. In this study, infection by and protein expression of bovine papillomavirus type 2 (BPV-2) in the uterine and chorionic epithelium of the placenta has been described in four cows suffering from naturally occurring papillomavirus-associated urothelial bladder tumors. E5 oncoprotein was detected both by Western blot analysis and immunohistochemically. It appears to be complexed and perfectly co-localized with the activated platelet-derived growth factor ß receptor (PDGFßR) by laser scanning confocal microscopy. The activated PDGFßR might be involved in organogenesis and neo-angiogenesis rather than in cell transformation during pregnancy. The major capsid protein, L1, believed to be only expressed in productive papillomavirus infection has been detected by Western blot analysis. Immunohistochemical investigations confirmed the presence of L1 protein both in the cytoplasm and nuclei of cells of the uterine and chorionic epithelium. Trophoblastic cells appear to be the major target for L1 protein expression. Finally, the early protein E2, required for viral DNA replication and known to be expressed during a productive infection, has been detected by Western blot and immunohistochemically. Electron microscopic investigations detected viral particles in nuclei of uterine and chorionic epithelium. This study shows that both active and productive infections by BPV-2 in the placenta of pregnant cows can occur in vivo.

Single cell–resolution western blotting

PubMed Central

Kang, Chi-Chih; Yamauchi, Kevin A; Vlassakis, Julea; Sinkala, Elly; Duncombe, Todd A; Herr, Amy E

2017-01-01

This protocol describes how to perform western blotting on individual cells to measure cell-to-cell variation in protein expression levels and protein state. like conventional western blotting, single-cell western blotting (scWB) is particularly useful for protein targets that lack selective antibodies (e.g., isoforms) and in cases in which background signal from intact cells is confounding. scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of a polyacrylamide gel (PAG). the gel layer functions as both a molecular sieving matrix during PAGE and a blotting scaffold during immunoprobing. scWB involves five main stages: (i) gravity settling of cells into microwells; (ii) chemical lysis of cells in each microwell; (iii) PAGE of each single-cell lysate; (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of immobilized proteins. Multiplexing can be achieved by probing with antibody cocktails and using antibody stripping/reprobing techniques, enabling detection of 10+ proteins in each cell. We also describe microdevice fabrication for both uniform and pore-gradient microgels. to extend in-gel immunoprobing to gels of small pore size, we describe an optional gel de-cross-linking protocol for more effective introduction of antibodies into the gel layer. once the microdevice has been fabricated, the assay can be completed in 4–6 h by microfluidic novices and it generates high-selectivity, multiplexed data from single cells. the technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine. PMID:27466711

Do Deregulated Cas Proteins Induce Genomic Instability in Early-Stage Ovarian Cancer

DTIC Science & Technology

2006-12-01

use Western blot analysis of tumor lysates to correlate expression of HEF1, p130Cas, Aurora A, and phospho-Aurora A. This analysis is in progress. In...and importantly, evaluated a number of different detection/image analysis systems to ensure reproducible quantitative results. We have used a pilot...reproducible Interestingly, preliminary statistical analysis using Spearman and Pearson correlation indicates at least one striking correlation

Evidence of Clostridium perfringens epsilon toxin associated with multiple sclerosis.

PubMed

Wagley, Sariqa; Bokori-Brown, Monika; Morcrette, Helen; Malaspina, Andrea; D'Arcy, Caroline; Gnanapavan, Sharmilee; Lewis, Nicholas; Popoff, Michel R; Raciborska, Dominika; Nicholas, Richard; Turner, Ben; Titball, Richard W

2018-04-01

It was recently reported that, using Western blotting, some multiple sclerosis (MS) patients in the United States had antibodies against epsilon toxin (Etx) from Clostridium perfringens, suggesting that the toxin may play a role in the disease. We investigated for serum antibodies against Etx in UK patients with clinically definite multiple sclerosis (CDMS) or presenting with clinically isolated syndrome (CIS) or optic neuritis (ON) and in age- and gender-matched controls. We tested sera from CDMS, CIS or ON patients or controls by Western blotting. We also tested CDMS sera for reactivity with linear overlapping peptides spanning the amino acid sequence (Pepscan) of Etx. Using Western blotting, 24% of sera in the combined CDMS, CIS and ON groups ( n = 125) reacted with Etx. In the control group ( n = 125), 10% of the samples reacted. Using Pepscan, 33% of sera tested reacted with at least one peptide, whereas in the control group only 16% of sera reacted. Out of 61 samples, 21 (43%) were positive to one or other testing methodology. Three samples were positive by Western blotting and Pepscan. Our results broadly support the previous findings and the role of Etx in the aetiology of MS warrants further investigation.

Antigenic Protein In Microgravity-Grown Human Mixed Mullerian Tumor (LN1) Cells Preserved In RNA Stabilizing Agent

NASA Technical Reports Server (NTRS)

Hammond, Dianne K.; Becker, Jeanne; Elliott, T. F.; Holubec, K.; Baker, T. L.; Love, J. E.

2004-01-01

Cells treated with RNAlater(TradeMark) have previously been shown to contain antigenic proteins that can be visualized using Western blot analysis. These proteins seem to be stable for several months when stored in RNA stabilizer at 4 C. Antigenic protein can be recovered from cells that have been processed using an Ambion RNAqueous(Registered TradeMark) kit to remove RNA. In this set of experiments, human mixed Mullerian tumor (LNI) cells grown on the International Space Station during Expedition 3 were examined for antigenic stability after removal of RNA. The cells were stored for three months in RNAlater(TradeMark) and RNA was extracted. The RNA filtrate containing the protein was precipitated, washed, and suspended in buffer containing sodium dodecyl sulfate (SDS). Samples containing equal concentrations of protein were loaded onto SDS-polyacrylamide gels. Proteins were separated by electrophoresis and transferred by Western blot to polyvinylidene fluoride (PVDF) membrane. The Western blots were stained with an enhanced chemiluminescent ECL(Registered Trademark) Plus detection kit (Amersham) and scanned using a Storm 840 gel image analyzer (Amersham, Molecular Dynamics). ImageQuant(Registered TradeMark) software was used to quantify the densities of the protein bands. The ground control and flight LN1 cell samples showed a similar staining pattern over time with antibodies to vimentin, glyceraldehyde-3-phosphate dehydrogenase, and epithelial membrane antigens.

Antigenic Protein In Microgravity-Grown Human Mixed Mullerian Tumor (LN1) Cells Preserved In RNA Stabilizing Agent

NASA Technical Reports Server (NTRS)

Hammond, Dianne K.; Becker, Jeanne; Holubec, K.; Baker, T. L.; Love, J. E.

2004-01-01

Cells treated with RNAlater(TradeMark) have previously been shown to contain antigenic proteins that can be visualized using Western blot analysis. These proteins seem to be stable for several months when stored in RNA stabilizer at 4 C. Antigenic protein can be recovered from cells that have been processed using an Ambion RNAqueous(Registered TradeMark) kit to remove RNA. In this set of experiments, human mixed Mullerian tumor (LN1) cells grown on the International Space Station during Expedition 3 were examined for antigenic stability after removal of RNA. The cells were stored for three months in RNAlater(TradeMark) and RNA was extracted. The RNA filtrate Containing the protein was precipitated, washed, and suspended in buffer containing sodium dodecyl sulfate (SDS). Samples containing equal concentrations of protein were loaded onto SDS-polyacrylamide gels. Proteins were separated by electrophoresis and transferred by Western blot to polyvinylidene fluoride (PVDF) membrane. The Western blots were stained with an enhanced chemiluminescent ECL(Registered TradeMark)Plus detection kit (Amersham) and scanned using a Storm 840 gel image analyzer (Amersham, Molecular Dynamics). ImageQuant(Registered TradeMark)a software was used to quantify the densities of the protein bands. The ground control and flight LN1 cell samples showed a similar staining pattern over time with antibodies to vimentin, glyceraldehyde-3-phosphate dehydrogenase, and epithelial membrane antigens.

An assessment of a Western blot method for the investigation of canine cutaneous adverse food reactions.

PubMed

Maina, Elisa; Matricoti, Irina; Noli, Chiara

2018-06-01

Adverse food reaction (AFR) is diagnosed with a two month elimination diet (ED), followed by challenge with the original food. To evaluate reactivity of selected EDs and performance of a Western blot serological test for the diagnosis of AFR. Twenty five food reactive (FR) and 13 non food reactive (NFR) privately owned dogs. Sera were tested for antibodies against hydrolyzed poultry feather (RCA), hydrolyzed soy (PHA), hydrolyzed fish (FUH), limited antigen horse and potato (THP), fresh horse meat and the offending food for each FR dog as documented by provocative challenge. Fourteen sera were negative and two positive to all foods. Sera from five of 13 NFR and three of 25 FR dogs were reactive to hydrolyzed foods. The RCA diet was recognized by four of 38, FUH by six of 38 and PHA by one of 28 samples. THP was recognized by 14 of 33 and fresh horse by one of ten dogs that had never eaten horse meat. The test correctly identified one of 15 dogs allergic to FUH. Twenty of 25 FR sera were negative for the dogs' respective offending foods (20% sensitivity), whereas four of 13 NFR sera were positive to the dogs' usual diets (69% specificity). Western blot analysis cannot be considered as a valid tool for the diagnosis of AFR; it may serve as an aid in selecting an ED. © 2018 ESVD and ACVD.

Establishment of CMab-43, a Sensitive and Specific Anti-CD133 Monoclonal Antibody, for Immunohistochemistry.

PubMed

Itai, Shunsuke; Fujii, Yuki; Nakamura, Takuro; Chang, Yao-Wen; Yanaka, Miyuki; Saidoh, Noriko; Handa, Saori; Suzuki, Hiroyoshi; Harada, Hiroyuki; Yamada, Shinji; Kaneko, Mika K; Kato, Yukinari

2017-10-01

CD133, also known as prominin-1, was first described as a cell surface marker on early progenitor and hematopoietic stem cells. It is a five-domain transmembrane protein composed of an N-terminal extracellular tail, two small cytoplasmic loops, two large extracellular loops containing seven potential glycosylation sites, and a short C-terminal intracellular tail. CD133 has been used as a marker to identify cancer stem cells derived from primary solid tumors and as a prognostic marker of gliomas. Herein, we developed novel anti-CD133 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. We expressed the full length of CD133 in LN229 glioblastoma cells, immunized mice with LN229/CD133 cells, and performed the first screening using flow cytometry. After limiting dilution, we established 100 anti-CD133 mAbs, reacting with LN229/CD133 cells but not with LN229 cells. Subsequently, we performed the second and third screening with Western blot and immunohistochemical analyses, respectively. Among 100 mAbs, 11 strongly reacted with CD133 in Western blot analysis. One of 11 clones, CMab-43 (IgG 2a , kappa), showed a sensitive and specific reaction against colon cancer cells, warranting the use of CMab-43 in detecting CD133 in pathological analyses of CD133-expressing cancers.

Monoclonal Antibody L1Mab-13 Detected Human PD-L1 in Lung Cancers.

PubMed

Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Yanaka, Miyuki; Chang, Yao-Wen; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

2018-04-01

Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting cells. It is also expressed in several tumor cells such as melanoma and lung cancer cells. A strong correlation has been reported between human PD-L1 (hPD-L1) expression in tumor cells and negative prognosis in cancer patients. Here, a novel anti-hPD-L1 monoclonal antibody (mAb) L 1 Mab-13 (IgG 1 , kappa) was produced using a cell-based immunization and screening (CBIS) method. We investigated hPD-L1 expression in lung cancer using flow cytometry, Western blot, and immunohistochemical analyses. L 1 Mab-13 specifically reacted hPD-L1 of hPD-L1-overexpressed Chinese hamster ovary (CHO)-K1 cells and endogenous hPD-L1 of KMST-6 (human fibroblast) in flow cytometry and Western blot. Furthermore, L 1 Mab-13 reacted with lung cancer cell lines (EBC-1, Lu65, and Lu99) in flow cytometry and stained lung cancer tissues in a membrane-staining pattern in immunohistochemical analysis. These results indicate that a novel anti-hPD-L1 mAb, L 1 Mab-13, is very useful for detecting hPD-L1 of lung cancers in flow cytometry, Western blot, and immunohistochemical analyses.

Development and Application of an Indirect Enzyme-Linked Immunosorbent Assay Using Recombinant Mag1 for Serodiagnosis of Toxoplasma gondii In Dogs.

PubMed

Zhuo, Xunhui; Sun, Hongchao; Zhang, Zhi; Luo, Jiaqing; Shan, Ying; Du, Aifang

2017-06-01

Serologic tests are widely accepted and applied as means to detect anti- Toxoplasma gondii immunoglobulin G antibodies. In this study, recombinant matrix antigen (rMAG1) was induced by isopropyl-β-d-thiogalactoside and purified by nickel-nitrilotriacetic acid purification system. We then developed and optimized an indirect enzyme-linked immunosorbent assay (ELISA) through checkerboard assays using serial dilutions of antigens and sera to assess the potential use of rMAG1 in serologic detection of T. gondii infection in dogs. Serum samples from 93 domestic dogs were analyzed by western blot and rMAG1-ELISA. The results were compared with those obtained from an ELISA with the soluble Toxoplasma lysate antigens (TLA). We found that although yielding an excellent agreement (96.7%) with western blot data (κ = 0.9659), rMAG1-ELISA produced higher sensitivity (93.9% vs. 87.8%) and specificity (98.3% vs. 96.7%) than TLA-ELISA. In addition, receiver operating characteristic analysis also revealed that rMAG1-ELISA is in more agreement with western blot (area under the curve [AUC] = 0.985) relative to TLA-ELISA (AUC = 0.955). These results indicated that the rMAG1-ELISA established in this study provides a promising and reliable tool for serologic detection of T. gondii infection in dogs.

Short-term application of dexamethasone on stem cells derived from human gingiva reduces the expression of RUNX2 and β-catenin.

PubMed

Kim, Bo-Bae; Kim, Minji; Park, Yun-Hee; Ko, Youngkyung; Park, Jun-Beom

2017-06-01

Objective Next-generation sequencing was performed to evaluate the effects of short-term application of dexamethasone on human gingiva-derived mesenchymal stem cells. Methods Human gingiva-derived stem cells were treated with a final concentration of 10 -7  M dexamethasone and the same concentration of vehicle control. This was followed by mRNA sequencing and data analysis, gene ontology and pathway analysis, quantitative real-time polymerase chain reaction of mRNA, and western blot analysis of RUNX2 and β-catenin. Results In total, 26,364 mRNAs were differentially expressed. Comparison of the results of dexamethasone versus control at 2 hours revealed that 7 mRNAs were upregulated and 25 mRNAs were downregulated. The application of dexamethasone reduced the expression of RUNX2 and β-catenin in human gingiva-derived mesenchymal stem cells. Conclusion The effects of dexamethasone on stem cells were evaluated with mRNA sequencing, and validation of the expression was performed with qualitative real-time polymerase chain reaction and western blot analysis. The results of this study can provide new insights into the role of mRNA sequencing in maxillofacial areas.

CROSS REACTIVITY IN ALLERGIC ASTHMA-LIKE RESPONSES BETWEEN MOLD AND HOUSE DUST MITE IN MICE

EPA Science Inventory

Molds are ubiquitous in the environment and exposures to molds contribute to various human diseases including allergic asthma. Some mold allergens have been implicated as the causal agent for allergic asthma. Western blot analysis demonstrated IgE-binding cross-reactivity among m...

Single Chain Antibodies as Estrogen Receptor Repressors in Breast Cancer

DTIC Science & Technology

2000-06-01

differential display we identified proteinase inhibitor-9 as an mRNA upregulated by estrogen in a human hepatoblastoma cell line (HepG2) stably transfected...antiestrogen ICI 182,780 was a pure antag- human hepatoblastoma cell line (3), contained ER (4), this cell onist. Western blot analysis showed that

Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting.

PubMed

Eaton, Samantha L; Roche, Sarah L; Llavero Hurtado, Maica; Oldknow, Karla J; Farquharson, Colin; Gillingwater, Thomas H; Wishart, Thomas M

2013-01-01

Western blotting has been a key technique for determining the relative expression of proteins within complex biological samples since the first publications in 1979. Recent developments in sensitive fluorescent labels, with truly quantifiable linear ranges and greater limits of detection, have allowed biologists to probe tissue specific pathways and processes with higher resolution than ever before. However, the application of quantitative Western blotting (QWB) to a range of healthy tissues and those from degenerative models has highlighted a problem with significant consequences for quantitative protein analysis: how can researchers conduct comparative expression analyses when many of the commonly used reference proteins (e.g. loading controls) are differentially expressed? Here we demonstrate that common controls, including actin and tubulin, are differentially expressed in tissues from a wide range of animal models of neurodegeneration. We highlight the prevalence of such alterations through examination of published "-omics" data, and demonstrate similar responses in sensitive QWB experiments. For example, QWB analysis of spinal cord from a murine model of Spinal Muscular Atrophy using an Odyssey scanner revealed that beta-actin expression was decreased by 19.3±2% compared to healthy littermate controls. Thus, normalising QWB data to β-actin in these circumstances could result in 'skewing' of all data by ∼20%. We further demonstrate that differential expression of commonly used loading controls was not restricted to the nervous system, but was also detectable across multiple tissues, including bone, fat and internal organs. Moreover, expression of these "control" proteins was not consistent between different portions of the same tissue, highlighting the importance of careful and consistent tissue sampling for QWB experiments. Finally, having illustrated the problem of selecting appropriate single protein loading controls, we demonstrate that normalisation using total protein analysis on samples run in parallel with stains such as Coomassie blue provides a more robust approach.

Expression and localization of CXCL16 and CXCR6 in ovarian endometriotic tissues.

PubMed

Manabe, Shuichi; Iwase, Akira; Goto, Maki; Kobayashi, Hiroharu; Takikawa, Sachiko; Nagatomo, Yoshinari; Nakahara, Tatsuo; Bayasula; Nakamura, Tomoko; Hirokawa, Wakana; Kikkawa, Fumitaka

2011-12-01

Inflammatory mediators, including chemokines, may play crucial roles in the development of endometriosis. Therefore, we investigated the expression and localization of CXCL16 and its receptor, CXCR6, in ovarian endometriotic tissues. We also examined whether CXCL16 induces IL-8 production in endometriotic stromal cells. We performed immunohistochemical and Western blotting analyses of in vivo and in vitro samples. IL-8 production was assayed using an ELISA. Both CXCL16 and CXCR6 were expressed by endometriotic epithelial cells and stromal cells, but not normal ovarian stroma. A Western blotting analysis using primary cultured endometriotic stromal cells showed a constant expression of CXCL16 and CXCR6 in the proliferative phase, secretory phase and during gonadotropin-releasing hormone agonist therapy. CXCL16 induced IL-8 production in several endometriotic stromal cells in vitro. CXCL16 and CXCR6 might be involved in the pathophysiology of endometriosis through regulation of the inflammatory response.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chae, Jung-Il; Cho, Young Keun; Cho, Seong-Keun

The potential medical applications of animal cloning include xenotransplantation, but the complex molecular cascades that control porcine organ development are not fully understood. Still, it has become apparent that organs derived from cloned pigs may be suitable for transplantation into humans. In this study, we examined the pancreas of an adult cloned pig developed through somatic cell nuclear transfer (SCNT) using two-dimensional electrophoresis (2-DE) and Western blotting. Proteomic analysis revealed 69 differentially regulated proteins, including such apoptosis-related species as annexins, lamins, and heat shock proteins, which were unanimously upregulated in the SCNT sample. Among the downregulated proteins in SCNT pancreasmore » were peroxiredoxins and catalase. Western blot results indicate that several antioxidant enzymes and the anti-apoptotic protein were downregulated in SCNT pancreas, whereas several caspases were upregulated. Together, these data suggest that the accumulation of reactive oxygen species (ROS) in the pancreas of an adult cloned pig leads to apoptosis.« less

Expression of the tumor suppressor genes NF2, 4.1B, and TSLC1 in canine meningiomas.

PubMed

Dickinson, P J; Surace, E I; Cambell, M; Higgins, R J; Leutenegger, C M; Bollen, A W; LeCouteur, R A; Gutmann, D H

2009-09-01

Meningiomas are common primary brain tumors in dogs; however, little is known about the molecular genetic mechanisms involved in their tumorigenesis. Several tumor suppressor genes have been implicated in meningioma pathogenesis in humans, including the neurofibromatosis 2 (NF2), protein 4.1B (4.1 B), and tumor suppressor in lung cancer-1 (TSLC1) genes. We investigated the expression of these tumor suppressor genes in a series of spontaneous canine meningiomas using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) (NF2; n = 25) and western blotting (NF2/merlin, 4.1B, TSLC1; n = 30). Decreased expression of 4.1B and TSLC1 expression on western blotting was seen in 6/30 (20%) and in 15/30 (50%) tumors, respectively, with 18/30 (60%) of meningiomas having decreased or absent expression of one or both proteins. NF2 gene expression assessed by western blotting and RT-PCR varied considerably between individual tumors. Complete loss of NF2 protein on western blotting was not seen, unlike 4.1B and TSLC1. Incidence of TSLC1 abnormalities was similar to that seen in human meningiomas, while perturbation of NF2 and 4.1B appeared to be less common than reported for human tumors. No association was observed between tumor grade, subtype, or location and tumor suppressor gene expression based on western blot or RT-PCR. These results suggest that loss of these tumor suppressor genes is a frequent occurrence in canine meningiomas and may be an early event in tumorigenesis in some cases. In addition, it is likely that other, as yet unidentified, genes play an important role in canine meningioma formation and growth.

Serological differentiation of murine typhus and epidemic typhus using cross-adsorption and Western blotting.

PubMed

La Scola, B; Rydkina, L; Ndihokubwayo, J B; Vene, S; Raoult, D

2000-07-01

Differentiation of murine typhus due to Rickettsia typhi and epidemic typhus due to Rickettsia prowazekii is critical epidemiologically but difficult serologically. Using serological, epidemiological, and clinical criteria, we selected sera from 264 patients with epidemic typhus and from 44 patients with murine typhus among the 29,188 tested sera in our bank. These sera cross-reacted extensively in indirect fluorescent antibody assays (IFAs) against R. typhi and R. prowazekii, as 42% of the sera from patients with epidemic typhus and 34% of the sera from patients with murine typhus exhibited immunoglobulin M (IgM) and/or IgG titers against the homologous antigen (R. prowazekii and R. typhi, respectively) that were more than one dilution higher than those against the heterologous antigen. Serum cross-adsorption studies and Western blotting were performed on sera from 12 selected patients, 5 with murine typhus, 5 with epidemic typhus, and 2 suffering from typhus of undetermined etiology. Differences in IFA titers against R. typhi and R. prowazekii allowed the identification of the etiological agent in 8 of 12 patients. Western blot studies enabled the identification of the etiological agent in six patients. When the results of IFA and Western blot studies were considered in combination, identification of the etiological agent was possible for 10 of 12 patients. Serum cross-adsorption studies enabled the differentiation of the etiological agent in all patients. Our study indicates that when used together, Western blotting and IFA are useful serological tools to differentiate between R. prowazekii and R. typhi exposures. While a cross-adsorption study is the definitive technique to differentiate between infections with these agents, it was necessary in only 2 of 12 cases (16.7%), and the high costs of such a study limit its use.

Serological Differentiation of Murine Typhus and Epidemic Typhus Using Cross-Adsorption and Western Blotting

PubMed Central

La Scola, Bernard; Rydkina, Lena; Ndihokubwayo, Jean-Bosco; Vene, Sirkka; Raoult, Didier

2000-01-01

Differentiation of murine typhus due to Rickettsia typhi and epidemic typhus due to Rickettsia prowazekii is critical epidemiologically but difficult serologically. Using serological, epidemiological, and clinical criteria, we selected sera from 264 patients with epidemic typhus and from 44 patients with murine typhus among the 29,188 tested sera in our bank. These sera cross-reacted extensively in indirect fluorescent antibody assays (IFAs) against R. typhi and R. prowazekii, as 42% of the sera from patients with epidemic typhus and 34% of the sera from patients with murine typhus exhibited immunoglobulin M (IgM) and/or IgG titers against the homologous antigen (R. prowazekii and R. typhi, respectively) that were more than one dilution higher than those against the heterologous antigen. Serum cross-adsorption studies and Western blotting were performed on sera from 12 selected patients, 5 with murine typhus, 5 with epidemic typhus, and 2 suffering from typhus of undetermined etiology. Differences in IFA titers against R. typhi and R. prowazekii allowed the identification of the etiological agent in 8 of 12 patients. Western blot studies enabled the identification of the etiological agent in six patients. When the results of IFA and Western blot studies were considered in combination, identification of the etiological agent was possible for 10 of 12 patients. Serum cross-adsorption studies enabled the differentiation of the etiological agent in all patients. Our study indicates that when used together, Western blotting and IFA are useful serological tools to differentiate between R. prowazekii and R. typhi exposures. While a cross-adsorption study is the definitive technique to differentiate between infections with these agents, it was necessary in only 2 of 12 cases (16.7%), and the high costs of such a study limit its use. PMID:10882661

Overexpression of the Arabidopsis 10-kilodalton acyl-coenzyme A-binding protein ACBP6 enhances freezing tolerance.

PubMed

Chen, Qin-Fang; Xiao, Shi; Chye, Mee-Len

2008-09-01

Small 10-kD acyl-coenzyme A-binding proteins (ACBPs) are highly conserved proteins that are prevalent in eukaryotes. In Arabidopsis (Arabidopsis thaliana), other than the 10-kD ACBP homolog (designated Arabidopsis ACBP6), there are five larger forms of ACBPs ranging from 37.5 to 73.1 kD. In this study, the cytosolic subcellular localization of Arabidopsis ACBP6 was confirmed by analyses of transgenic Arabidopsis expressing autofluorescence-tagged ACBP6 and western-blot analysis of subcellular fractions using ACBP6-specific antibodies. The expression of Arabidopsis ACBP6 was noticeably induced at 48 h after 4 degrees C treatment by northern-blot analysis and western-blot analysis. Furthermore, an acbp6 T-DNA insertional mutant that lacked ACBP6 mRNA and protein displayed increased sensitivity to freezing temperature (-8 degrees C), while ACBP6-overexpressing transgenic Arabidopsis plants were conferred enhanced freezing tolerance. Northern-blot analysis indicated that ACBP6-associated freezing tolerance was not dependent on the induction of cold-regulated COLD-RESPONSIVE gene expression. Instead, ACBP6 overexpressors showed increased expression of mRNA encoding phospholipase Ddelta. Lipid profiling analyses of rosettes from cold-acclimated, freezing-treated (-8 degrees C) transgenic Arabidopsis plants overexpressing ACBP6 showed a decline in phosphatidylcholine (-36% and -46%) and an elevation of phosphatidic acid (73% and 67%) in comparison with wild-type plants. From our comparison, the gain in freezing tolerance in ACBP6 overexpressors that was accompanied by decreases in phosphatidylcholine and an accumulation of phosphatidic acid is consistent with previous findings on phospholipase Ddelta-overexpressing transgenic Arabidopsis. In vitro filter-binding assays indicating that histidine-tagged ACBP6 binds phosphatidylcholine, but not phosphatidic acid or lysophosphatidylcholine, further imply a role for ACBP6 in phospholipid metabolism in Arabidopsis, including the possibility of ACBP6 in the cytosolic trafficking of phosphatidylcholine.

[Knockdown of DNA-PKcs inhibits cell cycle and its mechanism of drug-resistant Bel7402/5-Fu hepatocellular carcinoma cells].

PubMed

Li, Dayu; Liu, Yun; Yu, Chunbo; Liu, Xiping; Fan, Fang

2017-12-01

Objective To study the effect of the knock-down of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) on the cell cycle of the multidrug-resistant (MDR) Bel7402/5-Fu hepatocellular carcinoma cells and its MDR mechanism. Methods After cationic liposome-mediated siDNA-PKcs oligonucleotide transfection, the drug sensitivity of Bel7402/5-Fu cells to 5-fluorouracil (5-Fu) and adriamycin (ADM) was determined by MTT assay; the cell cycle were detected by flow cytometry; meanwhile, the protein expressions of cell cycle-related proteins P21, cell cycle protein B1 (cyclin B1), cell cycle division protein 2 (CDC2) were tested by Western blotting; the expressions of ataxia telangiectasia mutated (ATM) and p53 at both mRNA and protein levels were detected by real-time PCR and Western blot analysis. Results The MTT results showed siDNA-PKcs increased the chemotherapeutic sensitivity of Bel7402/5-Fu cells to 5-Fu and ADM. The flow cytometric analysis showed siDNA-PKcs decreased the percentage of S-phase cells but increased the percentage of G2/M phase cells. Western blotting showed siDNA-PKcs increased the protein expression of P21 but decreased cyclinB1 and CDC2 proteins. In addition, siDNA-PKcs also increased the expressions of ATM and p53. Conclusion DNA-PKcs silencing increases P21 while decreases cyclin B1 and CDC2 expressions, and finally induces G2/M phase arrest in Bel7402/5-Fu cells, which may be related to ATM-p53 signaling pathway.

Diagnostic potential of Western blot analysis of sera from dogs with leishmaniasis in endemic areas and significance of the pattern.

PubMed

Aisa, M J; Castillejo, S; Gallego, M; Fisa, R; Riera, M C; de Colmenares, M; Torras, S; Roura, X; Sentis, J; Portus, M

1998-02-01

Serum samples collected from 237 dogs in Catalonia (northeastern Spain) were screened by Western blot analysis to detect the presence of antibodies specific to different Leishmania infantum polypeptide fractions. Leishmaniasis was confirmed in 72 of these dogs by direct examination and/or culture. Another 165 animals from the Priorat region were studied periodically for 2-8 years between 1987 and 1995, giving a total of 565 determinations. A control group of 93 dogs from nonendemic areas was also studied. Sera from dogs with leishmaniasis recognized antigens with molecular weights ranging from 12 to 85 kD. The most sensitive antigens were those of 70, 65, 46, 30, 28, 14, and 12 kD, which were recognized by 75%, 75%, 78%, 75%, 81%, 79%, and 75%, respectively, of the sera from dogs with positive parasitologic examination results. Antigens of 70 and 65 kD were also recognized by two dogs from nonendemic areas. Antigens of 14 and 12 kD were the first to be recognized by sera of asymptomatic dogs with titers less than the cut-off value of the dot-ELISA that increased during the longitudinal study, and the presence of antibodies specific for these fractions was observed for up to six years before seroconversion observed by dot-ELISA. These antibodies were also the first to disappear in dogs in which the disease was self-limited. The study corroborates the high sensitivity and specificity of Western blots in the diagnosis of canine leishmaniasis when the bands of low molecular weight (less than 46 kD) are considered, and indicates that fractions of 14 and 12 kD are useful in detecting early forms of the disease.

siRNA Targeting of the SNCG Gene Inhibits the Growth of Gastric Carcinoma SGC7901 Cells in vitro and in vivo by Downregulating the Phosphorylation of AKT/ERK.

PubMed

Fan, Changru; Liu, Jinju; Tian, Jianhai; Zhang, Yuliang; Yan, Maojun; Zhu, Chaoyu

2018-06-15

The aim of the study was to evaluate the effects of synuclein-γ (SNCG) silencing on gastric cancer SGC7901 cells and to elucidate the associated mechanisms. pGCSIL-lentiviral siRNA targeting of the SNCG gene was employed to inhibit SNCG expression. Several experiments such as quantitative real-time PCR, Western blotting, MTT, colony formation, migration assay, and flow cytometry were performed to investigate the biological behavior of infected SGC7901 cells. BALB/c nude mice were used as tumor xenograft models to assess the effects of SNCG silencing on tumor growth. Western blot analysis was carried out to determine the relative levels of AKT, p-AKT, ERK, and p-ERK expression. Our results showed that SNCG was overexpressed in SGC7901 cells as compared to normal gastric mucosal epithelial cells. SGC7901 cells transfected with SNCG siRNA demonstrated significantly decreased gastric cancer growth (p < 0.01), reduced cell migration, cell cycle arrest in the G0/G1 phase, promoted tumor cell apoptosis (p < 0.01), and inhibited tumorigenesis in xenograft animal models. Western blot analysis indicated that the protein levels of p-AKT and p-ERK were much lower in the SNCG siRNA group than in the control groups. The results of the present study suggest that SNCG siRNA plays a significant role in the proliferation, migration, and tumorigenesis of gastric cancer by downregulating the phosphorylation of AKT and ERK. RNA interference-mediated silencing of SNCG may provide an opportunity to develop a novel treatment strategy for gastric cancer. © 2018 S. Karger AG, Basel.

Development and characterization of mouse monoclonal antibodies reactive with chicken IL-1ß

USDA-ARS?s Scientific Manuscript database

Two mouse monoclonal antibodies (mAbs) specific for chicken interleukin-1ß (chIL-1ß) were produced and characterized. Both mAbs identified a 66.0 kDa recombinant protein expressed in Escherichia coli by Western blot analysis that corresponded to the expected molecular weight of a recombinant fusion ...

The Impact of a Common MDM2 SNP on the Sensitivity of Breast Cancer to Treatment

DTIC Science & Technology

2011-06-01

Kirchhoff T, Alexe G, Bond EE, Robins H, Bartel F, Taubert H, Wuerl P, Hait W, Toppmeyer D, Offit K, and Levine A. MDM2 SNP309 accelerates tumor...the Western blot analysis corresponding to the quantification in the upper graphs . 29 Figure 5. Effect of

Effects of Modification in the Laminin-10 Basal Lamina on Prostate Cancer Invasion

DTIC Science & Technology

2007-10-01

assays of ERK phosphorylations were performed by Western blotting with specific antibodies (14). 4. For immunohistochemistry analysis of MT1-MMP...Maquoi, E., Munaut, C., Remacle, A., and Foidart, J. M. (1997) Invasion Metastasis 17(5), 221-239 6. Foda , H. D., and Zucker, S. (2001) Drug Discov

Development and characterization of mouse monoclonal antibodies reactive with chicken IL1 Beta

USDA-ARS?s Scientific Manuscript database

Two mouse monoclonal antibodies (mAbs) specific for chicken interleukin-1 Beta (chIL-1 Beta) were produced and characterized. Both mAbs identified a 66.0 kDa recombinant protein expressed in Escherichia coli by Western blot analysis that corresponded to the expected molecular weight of a recombinant...

Characterization of a heparin-binding growth factor from adenocarcinoma of the kidney.

PubMed

Mydlo, J H; Heston, W D; Fair, W R

1988-12-01

A polypeptide isolated from tissue extracts of renal adenocarcinoma was mitogenic for BALB/c 3T3 cells and human umbilical vein (HUV) cells in culture. It also demonstrated angiogenic ability using the chorioallantoic membrane bioassay. Using heparin-sepharose affinity chromatography the purified protein eluted with a NaCl concentration between 1.4 and 1.8 M and demonstrated a molecular weight of approximately 17,000 daltons based on SDS polyacrylamide gel electrophoresis. Half maximal stimulation of tritiated thymidine incorporation into BALB/c 3T3 cells was achieved by 1.6 ng./ml. of the heparin binding material. Western blot analysis using antibodies specific to basic fibroblast growth factor (bFGF) only or acidic FGF (aFGF) only demonstrated that the purified protein binds to the former and not the latter. The characteristics of this material, in effect the elution profile off heparin-Sepharose, the molecular weight, angiogenic activity and the results of western blot analysis, suggest that this growth factor is similar to the family of basic fibroblast growth factors.

A new assay for fast, reliable CRIM status determination in infantile-onset Pompe disease.

PubMed

Wang, Zhaohui; Okamoto, Patricia; Keutzer, Joan

2014-02-01

Pompe disease is caused by a deficiency of acid α-glucosidase (GAA; EC, 3.2.1.20), and the infantile-onset form is rapidly fatal if left untreated. However, recombinant human GAA (rhGAA) enzyme replacement therapy (ERT) extends survival for infantile Pompe patients. Although cross-reactive immunologic material (CRIM)-negative patients, who lack detectable endogenous GAA, mount an immune response to rhGAA that renders the therapy ineffective, timely induction of immune tolerance in these patients may improve clinical outcomes. Previously, CRIM status has been determined by Western blot analysis in cultured skin fibroblasts, a process that can take a few weeks. We present a blood-based CRIM assay that can yield results within 48 to 72 h. Results from this assay have been confirmed by GAA Western blot analysis in fibroblasts or by GAA sequencing in a small number of Pompe disease patients. Rapid classification of CRIM status will assist in identifying the most effective treatment course and minimizing treatment delays in patients with infantile-onset Pompe disease. © 2013.

Rift Valley Fever Virus Structural and Nonstructural Proteins: Recombinant Protein Expression and Immunoreactivity Against Antisera from Sheep

PubMed Central

Faburay, Bonto; Wilson, William; McVey, D. Scott; Drolet, Barbara S.; Weingartl, Hana; Madden, Daniel; Young, Alan; Ma, Wenjun

2013-01-01

Abstract The Rift Valley fever virus (RVFV) encodes the structural proteins nucleoprotein (N), aminoterminal glycoprotein (Gn), carboxyterminal glycoprotein (Gc), and L protein, 78-kD, and the nonstructural proteins NSm and NSs. Using the baculovirus system, we expressed the full-length coding sequence of N, NSs, NSm, Gc, and the ectodomain of the coding sequence of the Gn glycoprotein derived from the virulent strain of RVFV ZH548. Western blot analysis using anti-His antibodies and monoclonal antibodies against Gn and N confirmed expression of the recombinant proteins, and in vitro biochemical analysis showed that the two glycoproteins, Gn and Gc, were expressed in glycosylated form. Immunoreactivity profiles of the recombinant proteins in western blot and in indirect enzyme-linked immunosorbent assay against a panel of antisera obtained from vaccinated or wild type (RVFV)-challenged sheep confirmed the results obtained with anti-His antibodies and demonstrated the suitability of the baculo-expressed antigens for diagnostic assays. In addition, these recombinant proteins could be valuable for the development of diagnostic methods that differentiate infected from vaccinated animals (DIVA). PMID:23962238

Bombyx mori nucleopolyhedrovirus ORF101 encodes a budded virus envelope associated protein.

PubMed

Chen, Huiqing; Li, Mei; Huang, Guoping; Mai, Weijun; Chen, Keping; Zhou, Yajing

2014-08-01

Orf101 (Bm101) of Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this study, Bm101 was characterized. Transcripts of Bm101 were detected from 24 through 96 h post infection (h p.i.) by RT-PCR. The corresponding protein was also detected from 24 to 96 h p.i. in BmNPV-infected BmN cells by Western blot analysis using a polyclonal antibody against Bm101. Western blot assay of occlusion-derived virus and budded virus (BV) preparations revealed that Bm101 encodes a 28-kDa structural protein that is associated with BV and is located in the envelope fraction of budded virions. In addition, confocal analysis showed that the protein was localized in the cytosol and cytoplasmic membrane in virus-infected cells. In conclusion, the available data suggest that Bm101 is a functional ORF of BmNPV and encodes a protein expressed in the late stage of the infection cycle that is associated with the BV envelope.

Identification of Yeast V-ATPase Mutants by Western Blots Analysis of Whole Cell Lysates

NASA Astrophysics Data System (ADS)

Parra-Belky, Karlett

2002-11-01

A biochemistry laboratory was designed for an undergraduate course to help students better understand the link between molecular engineering and biochemistry. Students identified unknown yeast strains with high specificity using SDS-PAGE and Western blot analysis of whole cell lysates. This problem-solving exercise is a common application of biochemistry in biotechnology research. Three different strains were used: a wild-type and two mutants for the proton pump vacuolar ATPase (V-ATPase). V-ATPases are multisubunit enzymes and the mutants used were deletion mutants; each lacked one structural gene of the complex. After three, three-hour labs, mutant strains were easily identified by the students and distinguished from wild-type cells analyzing the pattern of SDS-PAGE distribution of proteins. Identifying different subunits of one multimeric protein allowed for discussion of the structure and function of this metabolic enzyme, which captured the interest of the students. The experiment can be adapted to other multimeric protein complexes and shows improvement of the described methodology over previous reports, perhaps because the problem and its solution are representative of the type of techniques currently used in research labs.

Expression and function of CD8 alpha/beta chains on rat and human mast cells.

PubMed

Kim, Mi-Sun; Kim, Sung-Hoon; Lee, Hye-Jung; Kim, Hyung-Min

2004-03-01

The expression and functional role of CD8 glycoprotein, a marker of cytotoxic/suppressor T lymphocytes and NK cells, were not studied on freshly isolated connective tissue type rat peritoneal mast cells, a rat mucosal type mast cell line (RBL 2H3), or human mast cell line (HMC-1). We used the reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, immunohistochemistry and enzyme-linked immunosorbent assay. RT-PCR and Western blot analysis identified the presence of CD8 alpha/beta chains on the mast cells, and immunohistochemistry confirmed CD8alpha expression on rat or human mast cells. Functional studies demonstrated that stimulation of CD8 alpha/beta chains on rat mast cells induced the secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), which are regarded as important mediators during infection. However, co-stimulation with stem cell factor had no effect on CD8-induced mediator secretion. Our findings demonstrate novel biological roles of CD8 molecules in mast cells.

The genome-wide expression profile of Curcuma longa-treated cisplatin-stimulated HEK293 cells

PubMed Central

Sohn, Sung-Hwa; Ko, Eunjung; Chung, Hwan-Suck; Lee, Eun-Young; Kim, Sung-Hoon; Shin, Minkyu; Hong, Moochang; Bae, Hyunsu

2010-01-01

AIM The rhizome of turmeric, Curcuma longa (CL), is a herbal medicine used in many traditional prescriptions. It has previously been shown that CL treatment showed greater than 47% recovery from cisplatin-induced cell damage in human kidney HEK 293 cells. This study was conducted to evaluate the recovery mechanisms of CL that occur during cisplatin induced nephrotoxicity by examining the genome wide mRNA expression profiles of HEK 293 -cells. METHOD Recovery mechanisms of CL that occur during cisplatin-induced nephrotoxicity were determined by microarray, real-time PCR, immunofluorescent confocal microscopy and Western blot analysis. RESULTS The results of microarray analysis and real-time PCR revealed that NFκB pathway-related genes and apoptosis-related genes were down-regulated in CL-treated HEK 293 cells. In addition, immunofluorescent confocal microscopy and Western blot analysis revealed that NFκB p65 nuclear translocation was inhibited in CL-treated HEK 293 cells. Therefore, the mechanism responsible for the effects of CL on HEK 293 cells is closely associated with regulation of the NFκB pathway. CONCLUSION CL possesses novel therapeutic agents that can be used for the prevention or treatment of cisplatin-induced renal disorders. PMID:20840446

Characteristics of seroconversion and implications for diagnosis of post-treatment Lyme disease syndrome: acute and convalescent serology among a prospective cohort of early Lyme disease patients.

PubMed

Rebman, Alison W; Crowder, Lauren A; Kirkpatrick, Allison; Aucott, John N

2015-03-01

Two-tier serology is often used to confirm a diagnosis of Lyme disease. One hundred and four patients with physician diagnosed erythema migrans rashes had blood samples taken before and after 3 weeks of doxycycline treatment for early Lyme disease. Acute and convalescent serologies for Borrelia burgdorferi were interpreted according to the 2-tier antibody testing criteria proposed by the Centers for Disease Control and Prevention. Serostatus was compared across several clinical and demographic variables both pre- and post-treatment. Forty-one patients (39.4%) were seronegative both before and after treatment. The majority of seropositive individuals on both acute and convalescent serology had a positive IgM western blot and a negative IgG western blot. IgG seroconversion on western blot was infrequent. Among the baseline variables included in the analysis, disseminated lesions (p < 0.0001), a longer duration of illness (p < 0.0001), and a higher number of reported symptoms (p = 0.004) were highly significantly associated with positive final serostatus, while male sex (p = 0.05) was borderline significant. This variability, and the lack of seroconversion in a subset of patients, highlights the limitations of using serology alone in identifying early Lyme disease. Furthermore, these findings underline the difficulty for rheumatologists in identifying a prior exposure to Lyme disease in caring for patients with medically unexplained symptoms or fibromyalgia-like syndromes.

Mechanism of Tumor Metastasis Suppression by the KAI1 Gene

DTIC Science & Technology

2008-02-01

antibody to Flag covalently crosslinked to agarose beads followed by western blot with monoclonal antibody to Flag (lanes 1, 2). For coimmunoprecipitation...negative controls (lanes 4, 6). IgH appeared in lanes 5 and 6, as antibody to hemagglutinin was not crosslinked to the agarose beads during...mixed in the presence of a cell-impermeable crosslinker DTSSP for 30 min followed by immunoprecipitation with DARC antibody and western blot with

Demonstration of the salmonid humoral response to Renibacterium salmoninarum using a monoclonal antibody against salmonid immunoglobulin

USGS Publications Warehouse

Bartholomew, J.L.; Arkoosh , M.R.; Rohovec, J.S.

1991-01-01

The specificity of the antibody response of salmonids to Renibacterium salmoninarum antigens was demonstrated by western blotting techniques that utilized a monoclonal antibody against salmonid immunoglobulin. In this study, the specificity of the response in immunized chinook salmon Oncorhynchus tshawytschawas compared with the response in naturally infected chinook salmon and coho salmon O. kisutch, and immunized rabbits. The antibody response in immunized salmon and rabbits and the naturally infected fish was primarily against the 57–58kilodalton protein complex. In addition to recognizing these proteins in the extracellular fraction and whole-cell preparations, antibody from the immunized salmon and rabbits detected four proteins with lower molecular masses. Western blotting techniques allow identification of the specific antigens recognized and are a useful tool for comparing the immunogenicity of different R. salmoninarumpreparations. Immunofluorescent techniques with whole bacteria were less sensitive than western blotting in detecting salmonid anti-R. salmoninarumantibody.

Negative inotropic effect of carbachol and interaction between acetylcholine receptor-operated potassium channel (K.ACh channel) and GTP binding protein in mouse isolated atrium--a novel methodological trial.

PubMed

Okada, Muneyoshi; Noma, Chihiro; Yamawaki, Hideyuki; Hara, Yukio

2013-01-01

Interaction between acetylcholine receptor-operated potassium channel (K.ACh channel) and GTP binding protein was examined by an immunoprecipitation-Western blotting system in mouse isolated atrium. The carbachol-induced negative inotropic action in indomethacin-pretreated mouse atrium was significantly inhibited by a K.ACh channel blocker, tertiapin or atropine. Kir3.1 K.ACh channel (Kir3.1) was immunoprecipitated with a mouse anti-Kir3.1 antibody. Coprecipitating Gβ with Kir3.1, detected by Western blotting, was significantly augmented by carbachol. Atropine, but not tertiapin, significantly inhibited the carbachol-induced coprecipitating Gβ with Kir3.1. The data indicate that immunoprecipitation with Kir3.1 and Western blotting of Gβ system is a useful method for assessing interaction between K.ACh channel and GTP binding protein in mouse atrium.

Western blot (immunoblot) assay of small, round-structured virus associated with an acute gastroenteritis outbreak in Tokyo.

PubMed

Hayashi, Y; Ando, T; Utagawa, E; Sekine, S; Okada, S; Yabuuchi, K; Miki, T; Ohashi, M

1989-08-01

Small, round-structured virus (SRSV) was detected in a stool specimen of a patient during an acute gastroenteritis outbreak in Tokyo and was tentatively named SRSV-9. SRSV-9 was purified by sucrose velocity gradient centrifugation after CsCl density gradient centrifugation. The buoyant density of SRSV-9 appeared to be 1.36 g/ml in CsCl. A Western blot (immunoblot) assay using the biotin-avidin system revealed that SRSV-9 was antigenically related to the Hawaii agent but distinct from the Norwalk agent and contained a single major structural protein with a molecular size of 63.0 +/- 0.6 kilodaltons. The prevalence of SRSV-9 infection in Tokyo was surveyed by the Western blot antibody assay by using a crude virus preparation as the antigen. Seroconversion was observed in 56.5% of the patients involved in the outbreaks from which SRSV was detected by electron microscopy.

Detection of Diverse and High Molecular Weight Nesprin-1 and Nesprin-2 Isoforms Using Western Blotting.

PubMed

Carthew, James; Karakesisoglou, Iakowos

2016-01-01

Heavily utilized in cell and molecular biology, western blotting is considered a crucial technique for the detection and quantification of proteins within complex mixtures. In particular, the detection of members of the nesprin (nuclear envelope spectrin repeat protein) family has proven difficult to analyze due to their substantial isoform diversity, molecular weight variation, and the sheer size of both nesprin-1 and nesprin-2 giant protein variants (>800 kDa). Nesprin isoforms contain distinct domain signatures, perform differential cytoskeletal associations, occupy different subcellular compartments, and vary in their tissue expression profiles. This structural and functional variance highlights the need to distinguish between the full range of proteins within the nesprin protein family, allowing for greater understanding of their specific roles in cell biology and disease. Herein, we describe a western blotting protocol modified for the detection of low to high molecular weight (50-1000 kDa) nesprin proteins.

MiR-216b inhibits cell proliferation by targeting FOXM1 in cervical cancer cells and is associated with better prognosis.

PubMed

He, Shanyang; Liao, Bing; Deng, Yalan; Su, Chang; Tuo, Jiuling; Liu, Jun; Yao, Shuzhong; Xu, Lin

2017-10-04

Our previous study showed FOXM1 expression was significantly up-regulated in cervical cancer, and was associated with poor prognosis. To clarify miRNAs-FOXM1 modulation pathways, in this study, we investigated the relationships between miR-216b and FOXM1 and the role of miR-216b in cell proliferation and prognosis of cervical cancer patients. Western blotting and qPCR were used to determine expression of FOXM1, cell cycle related factors and miR-216b level. MiR-216b overexpression and inhibited cell models were constructed, and siRNA was used for FOXM1 silencing. Cell proliferation was analyzed by MTT and colony formation assay. Dual luciferase reporter assay system was used to clarify the relationships between miR-216b and FOXM1. Kaplan-Meier survival analysis was used to evaluate prognosis. MiR-216b was down-regulated in cervical cancer cells and tissues, and its ectopic expression could decrease cell proliferation. Western blotting analysis showed miR-216b can inhibit cell proliferation by regulating FOXM1-related cell cycle factors, suppressing cyclinD1, c-myc, LEF1 and p-Rb and enhancing p21 expression. Repressing of miR-216b stimulated cervical cancer cell proliferation, whereas silencing FOXM1 expression could reverse this effect. Western blotting and luciferase assay results proved FOXM1 is a direct target of miR-216b. Survival analysis showed higher level of miR-216b was associated with better prognosis in cervical cancer patients. FOXM1 expression could be suppressed by miR-216b via direct binding to FOXM1 3'-UTR and miR-216b could inhibit cell proliferation by regulating FOXM1 related Wnt/β-catenin signal pathway. MiR-216b level is related to prognosis in cervical cancer patients and may serve as a potential prognostic marker.

A Low-Level Carbon Dioxide Laser Promotes Fibroblast Proliferation and Migration through Activation of Akt, ERK, and JNK

PubMed Central

Shingyochi, Yoshiaki; Kanazawa, Shigeyuki; Tajima, Satoshi; Tanaka, Rica; Mizuno, Hiroshi; Tobita, Morikuni

2017-01-01

Background Low-level laser therapy (LLLT) with various types of lasers promotes fibroblast proliferation and migration during the process of wound healing. Although LLLT with a carbon dioxide (CO2) laser was also reported to promote wound healing, the underlying mechanisms at the cellular level have not been previously described. Herein, we investigated the effect of LLLT with a CO2 laser on fibroblast proliferation and migration. Materials and Methods Cultured human dermal fibroblasts were prepared. MTS and cell migration assays were performed with fibroblasts after LLLT with a CO2 laser at various doses (0.1, 0.5, 1.0, 2.0, or 5.0 J/cm2) to observe the effects of LLLT with a CO2 laser on the proliferation and migration of fibroblasts. The non-irradiated group served as the control. Moreover, western blot analysis was performed using fibroblasts after LLLT with a CO2 laser to analyze changes in the activities of Akt, extracellular signal-regulated kinase (ERK), and Jun N-terminal kinase (JNK), which are signaling molecules associated with cell proliferation and migration. Finally, the MTS assay, a cell migration assay, and western blot analysis were performed using fibroblasts treated with inhibitors of Akt, ERK, or JNK before LLLT with a CO2 laser. Results In MTS and cell migration assays, fibroblast proliferation and migration were promoted after LLLT with a CO2 laser at 1.0 J/cm2. Western blot analysis revealed that Akt, ERK, and JNK activities were promoted in fibroblasts after LLLT with a CO2 laser at 1.0 J/cm2. Moreover, inhibition of Akt, ERK, or JNK significantly blocked fibroblast proliferation and migration. Conclusions These findings suggested that LLLT with a CO2 laser would accelerate wound healing by promoting the proliferation and migration of fibroblasts. Activation of Akt, ERK, and JNK was essential for CO2 laser-induced proliferation and migration of fibroblasts. PMID:28045948

[Construction and functional identification of eukaryotic expression vector carrying Sprague-Dawley rat MSX-2 gene].

PubMed

Yang, Xian-Xian; Zhang, Mei; Yan, Zhao-Wen; Zhang, Ru-Hong; Mu, Xiong-Zheng

2008-01-01

To construct a high effective eukaryotic expressing plasmid PcDNA 3.1-MSX-2 encoding Sprague-Dawley rat MSX-2 gene for the further study of MSX-2 gene function. The full length SD rat MSX-2 gene was amplified by PCR, and the full length DNA was inserted in the PMD1 8-T vector. It was isolated by restriction enzyme digest with BamHI and Xhol, then ligated into the cloning site of the PcDNA3.1 expression plasmid. The positive recombinant was identified by PCR analysis, restriction endonudease analysis and sequence analysis. Expression of RNA and protein was detected by RT-PCR and Western blot analysis in PcDNA3.1-MSX-2 transfected HEK293 cells. Sequence analysis and restriction endonudease analysis of PcDNA3.1-MSX-2 demonstrated that the position and size of MSX-2 cDNA insertion were consistent with the design. RT-PCR and Western blot analysis showed specific expression of mRNA and protein of MSX-2 in the transfected HEK293 cells. The high effective eukaryotic expression plasmid PcDNA3.1-MSX-2 encoding Sprague-Dawley Rat MSX-2 gene which is related to craniofacial development can be successfully reconstructed. It may serve as the basis for the further study of MSX-2 gene function.

Problem-solving test: Southwestern blotting.

PubMed

Szeberényi, József

2014-01-01

Terms to be familiar with before you start to solve the test: Southern blotting, Western blotting, restriction endonucleases, agarose gel electrophoresis, nitrocellulose filter, molecular hybridization, polyacrylamide gel electrophoresis, proto-oncogene, c-abl, Src-homology domains, tyrosine protein kinase, nuclear localization signal, cDNA, deletion mutants, expression plasmid, transfection, RNA polymerase II, promoter, Shine-Dalgarno sequence, polyadenylation element, affinity chromatography, Northern blotting, immunoprecipitation, sodium dodecylsulfate, autoradiography, tandem repeats. Copyright © 2014 The International Union of Biochemistry and Molecular Biology.

Biochemical Analysis of Autophagy in Algae and Plants by Monitoring the Electrophoretic Mobility of ATG8.

PubMed

Pérez-Pérez, María Esther; Andrés-Garrido, Ascensión; Crespo, José L

2016-01-01

Identification of specific autophagy markers has been fundamental to investigate autophagy as catabolic process. Among them, the ATG8 protein turned out to be one of the most widely used and specific molecular markers of autophagy both in higher and lower eukaryotes. Here, we describe how ATG8 can be used to monitor autophagy in Chlamydomonas and Arabidopsis by western blot analysis.

The Dot Blot ELISA.

ERIC Educational Resources Information Center

Gerbig, Donald G., Jr.; Fenk, Christopher J.; Goodhart, Amy S.

2000-01-01

Uses two laboratory techniques, Enzyme Linked Immunosorbent Assay (ELISA) and Western Blot, to demonstrate antibody-antigen binding concepts. Includes a list of required materials and directions for the procedure, and makes suggestions for classroom applications. (Contains 13 references.) (YDS)

Identification of a serotype-independent linear epitope of foot-and-mouth disease virus.

PubMed

Yang, Baolin; Wang, Mingxia; Liu, Wenming; Xu, Zhiqiang; Wang, Haiwei; Yang, Decheng; Ma, Wenge; Zhou, Guohui; Yu, Li

2017-12-01

Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus (FMDV), is a highly contagious infectious disease that affects domestic and wild cloven-hoofed animals worldwide. VP2 is a structural protein of FMDV. In this study, an FMDV serotype-independent monoclonal antibody (MAb), 10B10, against the viral capsid protein VP2 was generated, and a series of GST fusion proteins expressing a truncated peptide of VP2 was subjected to Western blot analysis using MAb 10B10. Their results indicated that the peptide 8 TLLEDRILT 16 of VP2 is the minimal requirement of the epitope recognized by MAb 10B10. Importantly, this linear epitope was highly conserved among all seven serotypes of FMDV in a sequence alignment analysis. Subsequent alanine-scanning mutagenesis analysis revealed that the residues Thr 8 and Asp 12 of the epitope were crucial for MAb-10B10 binding. Furthermore, Western blot analysis also revealed that the MAb 10B10-directed epitope could be recognized by positive sera from FMDV-infected cattle. The discovery that MAb 10B10 recognizes a serotype-independent linear epitope of FMDV suggests potential applications for this MAb in the development of serotype-independent tests for FMDV.

Extraordinary Diversity of Immune Response Proteins among Sea Urchins: Nickel-Isolated Sp185/333 Proteins Show Broad Variations in Size and Charge

PubMed Central

Sherman, Lauren S.; Schrankel, Catherine S.; Brown, Kristy J.; Smith, L. Courtney

2015-01-01

Effective protection against pathogens requires the host to produce a wide range of immune effector proteins. The Sp185/333 gene family, which is expressed by the California purple sea urchin Strongylocentrotus purpuratus in response to bacterial infection, encodes a highly diverse repertoire of anti-pathogen proteins. A subset of these proteins can be isolated by affinity to metal ions based on multiple histidines, resulting in one to four bands of unique molecular weight on standard Western blots, which vary depending on the individual sea urchin. Two dimensional gel electrophoresis (2DE) of nickel-isolated protein samples followed by Western blot was employed to detect nickel-isolated Sp185/333 (Ni-Sp185/333) proteins and to evaluate protein diversity in animals before and after immune challenge with marine bacteria. Ni-Sp185/333 proteins of the same molecular weight on standard Western blots appear as a broad complex of variants that differ in pI on 2DE Western blots. The Ni-Sp185/333 protein repertoire is variable among animals, and shows a variety of changes among individual sea urchins in response to immune challenges with both the same and different species of bacteria. The extraordinary diversity of the Ni-Sp185/333 proteins may provide significant anti-pathogen capabilities for sea urchins that survive solely on innate immunity. PMID:26406912

The Role of IQGAP1 in Breast Carcinoma

DTIC Science & Technology

2012-10-01

and"-tubulin expression was measured as described above. Statistical Analysis —All experiments were repeated inde- pendently at least three times...IQGAP1 Binds HER2—In vitro analysis with pure proteins was used to examine a possible interaction between IQGAP1 and HER2. GST alone or GST-HER2 was...incubated with puri- fied IQGAP1, and complexes were isolated with glutathione- Sepharose. Analysis by Western blotting reveals that IQGAP1 bindsHER2

Publisher Correction: N6-methyladenosine RNA modification regulates embryonic neural stem cell self-renewal through histone modifications.

PubMed

Wang, Yang; Li, Yue; Yue, Minghui; Wang, Jun; Kumar, Sandeep; Wechsler-Reya, Robert J; Zhang, Zhaolei; Ogawa, Yuya; Kellis, Manolis; Duester, Gregg; Zhao, Jing Crystal

2018-06-07

In the version of this article initially published online, there were errors in URLs for www.southernbiotech.com, appearing in Methods sections "m6A dot-blot" and "Western blot analysis." The first two URLs should be https://www.southernbiotech.com/?catno=4030-05&type=Polyclonal#&panel1-1 and the third should be https://www.southernbiotech.com/?catno=6170-05&type=Polyclonal. In addition, some Methods URLs for bioz.com, www.abcam.com and www.sysy.com were printed correctly but not properly linked. The errors have been corrected in the PDF and HTML versions of this article.

The Role of Osteopontin in the Malignancy of Human Breast Carcinoma

DTIC Science & Technology

1999-07-01

specific tumors [e.g., CA 125 in the case of ovarian carcinoma (17-19), HCG and ct- fetoprotein in the Fig. 4 Western blot analysis of plasma OPN and...EGF also induced the epithelium may contribute to the invasive properties of their breast decreased expression of integrin alpha 5 beta I in 8305c

A New Method for Quantitative Immunoblotting of Endogenous α-Synuclein

PubMed Central

Newman, Andrew J.; Selkoe, Dennis; Dettmer, Ulf

2013-01-01

β-Sheet-rich aggregates of α-synuclein (αSyn) are the hallmark neuropathology of Parkinson’s disease and related synucleinopathies, whereas the principal native structure of αSyn in healthy cells - unfolded monomer or α-helically folded oligomer - is under debate. Our recent crosslinking analysis of αSyn in intact cells showed that a large portion of endogenous αSyn can be trapped as oligomers, most notably as apparent tetramers. One challenge in such studies is accurately quantifying αSyn Western blot signals among samples, as crosslinked αSyn trends toward increased immunoreactivity. Here, we analyzed this phenomenon in detail and found that treatment with the reducible amine-reactive crosslinker DSP strongly increased αSyn immunoreactivity even after cleavage with the reducing agent β-mercaptoethanol. The effect was observed with all αSyn antibodies tested and in all sample types from human brain homogenates to untransfected neuroblastoma cells, permitting easy detection of endogenous αSyn in the latter, which had long been considered impossible. Coomassie staining of blots before and after several hours of washing revealed complete retention of αSyn after DSP/β-mercaptoethanol treatment, in contrast to a marked loss of αSyn without this treatment. The treatment also enhanced immunodetection of the homologs β- and γ-synuclein and of histones, another group of small, lysine-rich proteins. We conclude that by neutralizing positive charges and increasing protein hydrophobicity, amine crosslinker treatment promotes adhesion of αSyn to blotting membranes. These data help explain the recent report of fixing αSyn blots with paraformaldehyde after transfer, which we find produces similar but weaker effects. DSP/β-mercaptoethanol treatment of Western blots should be particularly useful to quantify low-abundance αSyn forms such as extracellular and post-translationally modified αSyn and splice variants. PMID:24278419

[Expression of DKKL1 in spermatozoa of men with asthenospermia].

PubMed

Yan, Qiu-Xia; Ma, Yi; Chen, Run-Qiang; Zhou, Xiu-Qin; Qiao, Jing; Xian, Ying-Jie; Feng, Ling; Chen, Cai-Rong

2018-03-20

To compare the expression of DKKL1 in ejaculated spermatozoa of normal fertile men and men with asthenospermia and investigate the role of DKKL1 in the pathogenesis of asthenospermia. The characteristics of semen samples collected from normal fertile men and men with asthenospermia were analyzed using computer-assisted sperm analysis according to WHO criteria. The ejaculated sperms were isolated by Percoll discontinuous density gradients to detect the expression of DKKL1 mRNA and protein using real-time PCR and Western blotting. The expression of DKKL1 mRNA was significantly down-regulated by 11.1 times in asthenospermic men as compared with that in normal fertile men (P<0.01). Western blotting showed that the expression of DKKL1 protein was down-regulated by 2.4 times in asthenospermic men compared to normal fertile men. The expression of DKKL1, which may play an important role in sperm motility,is significantly decreased in ejaculated spermatozoa of men with asthenospermia.

Influence of processing on the allergenic properties of pistachio nut assessed in vitro.

PubMed

Noorbakhsh, Reihaneh; Mortazavi, Seyed Ali; Sankian, Mojtaba; Shahidi, Fakhri; Maleki, Soheila J; Nasiraii, Leila Roozbeh; Falak, Reza; Sima, Hamid Reza; Varasteh, AbdolReza

2010-09-22

Pistachio (Pistacia vera) is a tree nut that has been reported to cause IgE-mediated allergic reactions. This study was undertaken to investigate the distinctions between different cultivars of pistachio nut and the influence of different processing on the IgE-binding capacity of whole pistachio protein extracts. The influence of different processes on allergenicity was investigated using competitive inhibition ELISA and Western blotting assays. The Western blotting results of extracts from pistachio cultivars showed no marked difference among them. The IgE-binding capacity was significantly lower for the protein extract prepared from steam-roasted than from raw and dry-roasted pistachio nuts. The results of sensory evaluation analysis and hedonic rating proved no significant differences in color, taste, flavor, and overall quality of raw, roasted, and steam-roasted pistachio nut treatments. The most significant finding of the present study was the successful reduction of IgE-binding by pistachio extracts using steam-roast processing without any significant changes in sensory quality of product.

Genetic and physical analyses of Methylobacterium organophilum XX genes encoding methanol oxidation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Machlin, S.M.; Tam, P.E.; Bastien, C.A.

When allyl alcohol was used as a suicide substrate, spontaneous mutants and UV light- and nitrous acid-generated mutants of Methylobacterium organophilum XX were selected which grew on methylamine but not on methanol. There was no detectable methanol dehydrogenase (MDH) activity in crude extracts of these mutants, yet Western blots revealed that some mutants still produced MDH protein. Complementation of 50 mutants by a cosmid gene bank of M. organophilum XX demonstrated that three major regions of the genome, each of which was separated by a minimum of 40 kilobases, were required for expression of active MDH. By subcloning and Tn5more » insertion mutagenesis of subcloned fragments, at least 11 genes clustered within these three regions were subsequently identified. The identity of the MDH structural gene, which was initially determined by hybridization to the structural gene of Methylobacterium sp. strain AM1, was confirmed by Western blot analysis of an MDH-..beta..-galactosidase fusion protein.« less

α-ENaC in bullfrog embryo: expression in cement gland, gills and skin.

PubMed

Fujimaki-Aoba, Kayo; Tanaka, Kayoko; Inomata, Reiko; Jensik, Philip J; Takada, Makoto

2014-01-01

The epithelial sodium channel (ENaC) is involved in Na(+) responses such as Na(+) absorption and salt taste. The alpha ENaC subunit (α-ENaC) is expressed in the skin of both the adult and larval (tadpole) bullfrog. α-ENaC expression in the developing bullfrog embryo has not been previously investigated. In this study, the expression of α-ENaC at various stages (Sts.) of bullfrog embryonic development is assessed by western blot and immunofluorescence analysis. Bullfrog α-ENaC (α-fENaC) protein was detected by western blot in embryos at Sts. (Gosner/Shumway) 19, 21 and 25. Immunofluorescence studies indicate that α-fENaC was localized to the embryonic cement glands at St. 18 (muscular response), St. 19 (heart beat) and St. 21 (mouth open and/or cornea transparent), to the external gills at St. 21 and to the outermost cell-layer of the skin at St. 25 (operculum complete). The function(s) of ENaC in these embryonic structures remain to be elucidated.

Serological cross-reactions between Bartonella and Chlamydia species: implications for diagnosis.

PubMed Central

Maurin, M; Eb, F; Etienne, J; Raoult, D

1997-01-01

Diagnosis of Chlamydia or Bartonella infections continues to rely mainly on serology. However, serological cross-reactions between members of these genera have recently been described. Sera from eight patients originally diagnosed as having Chlamydia pneumoniae endocarditis reacted with both Chlamydia sp. and Bartonella quintana antigens (microimmunofluorescence technique). Adsorption of sera with B. quintana or C. pneumoniae antigens removed anti-C. pneumoniae antibodies, whereas adsorption with C. pneumoniae antigens did not change antibody titers to B. quintana. Western blot analysis confirmed the presence of cross-reacting antigens and showed antibody patterns in all sera to be compatible with a Bartonella infection. These patients were therefore probably suffering from Bartonella-induced rather than Chlamydia-induced endocarditis. In contrast, sera from 10 patients presumed to be suffering from C. pneumoniae pneumonia did not display anti-B. quintana antibodies, although cross-reacting antigens were revealed by Western blotting. This work highlights the possibility that cases of infective Bartonella endocarditis are erroneously diagnosed as chlamydial infections. PMID:9276403

Appearance of Sodium Dodecyl Sulfate-Stable Amyloid β-Protein (Aβ) Dimer in the Cortex During Aging

PubMed Central

Enya, Miho; Morishima-Kawashima, Maho; Yoshimura, Masahiro; Shinkai, Yasuhisa; Kusui, Kaoru; Khan, Karen; Games, Dora; Schenk, Dale; Sugihara, Shiro; Yamaguchi, Haruyasu; Ihara, Yasuo

1999-01-01

We previously noted that some aged human cortical specimens containing very low or negligible levels of amyloid β-protein (Aβ) by enzyme immunoassay (EIA) provided prominent signals at 6∼8 kd on the Western blot, probably representing sodium dodecyl sulfate (SDS)-stable Aβ dimer. Re-examination of the specificity of the EIA revealed that BAN50- and BNT77-based EIA, most commonly used for the quantitation of Aβ, capture SDS-dissociable Aβ but not SDS-stable Aβ dimer. Thus, all cortical specimens in which the levels of Aβ were below the detection limits of EIA were subjected to Western blot analysis. A fraction of such specimens contained SDS-stable dimer at 6∼8 kd, but not SDS-dissociable Aβ monomer at ∼4 kd, as judged from the blot. This Aβ dimer is unlikely to be generated after death, because (i) specimens with very short postmortem delay contained the Aβ dimer, and (ii) until 12 hours postmortem, such SDS-stable Aβ dimer is detected only faintly in PDAPP transgenic mice. The presence of Aβ dimer in the cortex may characterize the accumulation of Aβ in the human brain, which takes much longer than that in PDAPP transgenic mice. PMID:9916941

Seroprevalence of toxocariasis in hypereosinophilic individuals in Ahwaz, south-western Iran.

PubMed

Maraghi, S; Rafiei, A; Hajihossein, R; Sadjjadi, S M

2012-06-01

Eosinophilia in human peripheral blood is caused by different agents, including toxocariasis. The present study aimed to evaluate the prevalence of toxocariasis in hypereosinophilic individuals in the city of Ahwaz, located in south-western Iran, using enzyme-linked immunosorbent assay and Western blot techniques. Serum samples were examined from 100 individuals with peripheral blood eosinophilia and also from another 100 individuals without eosinophilia as the control group. In hypereosinophilic individuals seroprevalence antibodies against Toxocara were found in 19 (19%), of whom 12 (63.15%) were female and 7 (36.85%) were male. Positive sera were subsequently confirmed by Western blot. All of the observed bands ranged from 24 to 100 kDa. Antibodies against Toxocara were found in 1% of the control group, but were not confirmed by Western blot. The results showed significant differences between the frequency of infection within age and gender (P < 0.05); the highest prevalence of infection was observed in adults. Differences between the hypereosinophilic and healthy individuals, in terms of Toxocara infection frequency, also proved significant (P < 0.05).The present study thus confirmed the significant prevalence of toxocariasis as a hygienic problem among hypereosinophilic individuals in this area. It is, therefore, necessary to examine these individuals for toxocariasis.

Far Western: probing membranes.

PubMed

Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

2007-08-01

INTRODUCTIONThe far-Western technique described in this protocol is fundamentally similar to Western blotting. In Western blots, an antibody is used to detect a query protein on a membrane. In contrast, in a far-Western blot (also known as an overlay assay) the antibody is replaced by a recombinant GST fusion protein (produced and purified from bacteria), and the assay detects the interaction of this protein with target proteins on a membrane. The membranes are washed and blocked, incubated with probe protein, washed again, and subjected to autoradiography. The GST fusion (probe) proteins are often labeled with (32)P; alternatively, the membrane can be probed with unlabeled GST fusion protein, followed by detection using commercially available GST antibodies. The nonradioactive approach is substantially more expensive (due to the purchase of antibody and detection reagents) than using radioactively labeled proteins. In addition, care must be taken to control for nonspecific interactions with GST alone and a signal resulting from antibody cross-reactivity. In some instances, proteins on the membrane are not able to interact after transfer. This may be due to improper folding, particularly in the case of proteins expressed from a phage expression library. This protocol describes a way to overcome this by washing the membrane in denaturation buffer, which is then serially diluted to permit slow renaturation of the proteins.

In-Depth Proteomic Analysis of the Hippocampus in a Rat Model after Cerebral Ischaemic Injury and Repair by Danhong Injection (DHI)

PubMed Central

Cui, Yiran; Liu, Xin; Li, Xianyu; Yang, Hongjun

2017-01-01

Stroke is the second most common cause of death worldwide. A systematic description and characterization of the strokes and the effects induced in the hippocampus have not been performed so far. Here, we analysed the protein expression in the hippocampus 24 h after cerebral ischaemic injury and repair. Drug intervention using Danhong injection (DHI), which has been reported to have good therapeutic effects in a clinical setting, was selected for our study of cerebral ischaemia repair in rat models. A larger proteome dataset and total 4091 unique proteins were confidently identified in three biological replicates by combining tissue extraction for rat hippocampus and LC-MS/MS analysis. A label-free approach was then used to quantify the differences among the four experimental groups (Naive, Sham, middle cerebral artery occlusion (MCAO) and MCAO + DHI groups) and showed that about 2500 proteins on average were quantified in each of the experiment group. Bioinformatics analysis revealed that in total 280 unique proteins identified above were differentially expressed (P < 0.05). By combining the subcellular localization, hierarchical clustering and pathway information with the results from injury and repair phase, 12 significant expressed proteins were chosen and verified with respect to their potential as candidates for cerebral ischaemic injury by Western blot. The primary three signalling pathways of the candidates related may be involved in molecular mechanisms related to cerebral ischaemic injury. In addition, a glycogen synthase kinase-3β (Gsk-3β) inhibitor of the candidates with the best corresponding expression trends between western blotting (WB) and label-free quantitative results were chosen for further validation. The results of Western blot analysis of protein expression and 2,3,5- chloride three phenyl tetrazole (TTC) staining of rat brains showed that DHI treatment and Gsk-3β inhibitor are both able to confer protection against ischaemic injury in rat MCAO model. The observations of the present study provide a novel understanding regarding the regulatory mechanism of cerebral ischaemic injury. PMID:28672812

Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts.

PubMed

Milovancev, Milan; Hilgart-Martiszus, Ian; McNamara, Michael J; Goodall, Cheri P; Seguin, Bernard; Bracha, Shay; Wickramasekara, Samanthi I

2013-06-13

Osteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for development of novel diagnostic, prognostic, and therapeutic tools. The surface-exposed proteome (SEP) of a cancerous cell includes a multifarious array of proteins critical to cellular processes such as proliferation, migration, adhesion, and inter-cellular communication. The specific aim of this study was to define a SEP profile of two validated canine OSA cell lines and a normal canine osteoblast cell line utilizing a biotinylation/streptavidin system to selectively label, purify, and identify surface-exposed proteins by mass spectrometry (MS) analysis. Additionally, we sought to validate a subset of our MS-based observations via quantitative real-time PCR, Western blot and semi-quantitative immunocytochemistry. Our hypothesis was that MS would detect differences in the SEP composition between the OSA and the normal osteoblast cells. Shotgun MS identified 133 putative surface proteins when output from all samples were combined, with good consistency between biological replicates. Eleven of the MS-detected proteins underwent analysis of gene expression by PCR, all of which were actively transcribed, but varied in expression level. Western blot of whole cell lysates from all three cell lines was effective for Thrombospondin-1, CYR61 and CD44, and indicated that all three proteins were present in each cell line. Semi-quantitative immunofluorescence indicated that CD44 was expressed at much higher levels on the surface of the OSA than the normal osteoblast cell lines. The results of the present study identified numerous differences, and similarities, in the SEP of canine OSA cell lines and normal canine osteoblasts. The PCR, Western blot, and immunocytochemistry results, for the subset of proteins evaluated, were generally supportive of the mass spectrometry data. These methods may be applied to other cell lines, or other biological materials, to highlight unique and previously unrecognized differences between samples. While this study yielded data that may prove useful for OSA researchers and clinicians, further refinements of the described techniques are expected to yield greater accuracy and produce a more thorough SEP analysis.

The SDF-1-CXCR4 Axis Functions Through p38-MAPK Signaling to Drive Breast Cancer Progression and Metastasis

DTIC Science & Technology

2009-09-01

line) could induce proliferation and lead to hormone independent tumors in vivo. Upon analysis of these tumors by real-time PCR, it was found that the... analysis we have shown that overexpression of CXCR4 leads to increased levels of ER-mediated gene expression; specifically we found increased levels of...SDF-1 and, the classic ER-mediated gene, Progesterone receptor (PgR). 1.B Determine if CXCR4 activates p38. Western blot analysis of breast

Incomplete IgG response to HIV-1 proteins and low avidity levels in recently converted HIV patients treated with early antiretroviral therapy.

PubMed

Re, Maria Carla; Schiavone, Pasqua; Bon, Isabella; Vitone, Francesca; De Crignis, Elisa; Biagetti, Carlo; Gibellini, Davide

2010-11-01

To evaluate the evolution of antibody avidity and Western blot reactivity in recently infected HIV-1 subjects and to study the impact of highly active antiretroviral therapy (HAART) on avidity maturation of HIV-1-specific immunoglobulin G (IgG) in patients with recent HIV-1 infection. Thirty-six HIV-1 seroconverters were enrolled in this study and followed longitudinally over 24 months to evaluate if the administration of antiretroviral therapy during primary infection affects Western blot reactivity and the evolution of antibody avidity. The patients were divided into two groups; group A consisted of 19 HIV-1-untreated patients who did not receive any drug treatment during our follow-up period; group B consisted of 17 subjects who were treated early with an association of two nucleoside reverse transcriptase inhibitors (NRTI) and one non-nucleoside reverse transcriptase inhibitor (NNRTI) within 3 months after seroconversion. At diagnosis, Western blot analysis and avidity index (mean value) were exactly matched in untreated and treated patients; subsequently, however, a significantly lower reactivity to HIV-1 pol and gag proteins and a lower avidity index (mean values) were observed in HAART-treated patients up until the end of the follow-up period. The impaired production and maturation of the humoral immunological response in antiretroviral-treated patients might be related to a rapid suppression of HIV replication, driven by HAART. These results could have important implications in understanding the complex mechanism of the immune response during HIV infection. Copyright © 2010 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Molecular analysis of human T cell lymphotropic virus type 1 and 2 (HTLV-1/2) seroindeterminate blood donors from Northeast Iran: evidence of proviral tax, env, and gag sequences.

PubMed

Zanjani, Delaram Sayadpour; Shahabi, Majid; Talaei, Nasim; Afzalaghaee, Monavar; Tehranian, Farahnaz; Bazargani, Reihane

2011-02-01

Human T cell lymphotropic virus type 1 and 2 (HTLV-1/2) Western blot indeterminate results are a problem for blood banks in endemic areas. To determine the prevalence of HTLV-1/2 infection among indeterminate donors, we analyzed 130 cases from Mashhad, an HTLV-1/2 endemic area in Northeast Iran. The most frequent Western blot bands were GD21 alone (37.2%) followed by rgp46-2 alone (32.1%). We further tested 40 available DNA samples of these cases by PCR for viral sequences, tax, gag, and pol, and found five cases (12.5%) to be positive for two or three HTLV-1 genes. There were no significant age, sex, and blood group differences between PCR-positive and PCR-negative cases. Among PCR-positive individuals, the most prevalent Western blot bands were variable combinations of rgp46-1, GD21, and gp21. The mean of the optical density (OD) of the enzyme-linked immunosorbent assay (ELISA) test was significantly higher in PCR-positive individuals. The frequency of the rgp46-1 band was also significantly higher in PCR-positive cases compared to PCR-negative ones. In conclusion, the majority of HTLV-indeterminate donors lack the HTLV provirus and therefore are not considered infected. However, in some cases with higher ODs in the ELISA test and seroreactivity to env proteins, rgp46-1 and GD21 in particular may be indicative of infection and need further evaluation by molecular methods.

Coriolus versicolor mushroom polysaccharides exert immunoregulatory effects on mouse B cells via membrane Ig and TLR-4 to activate the MAPK and NF-κB signaling pathways.

PubMed

Yang, Shu-fa; Zhuang, Tai-feng; Si, Yan-mei; Qi, Ke-yan; Zhao, Juan

2015-03-01

This study aimed to characterize the immunopotentiating effects and immune receptors for Coriolus versicolor mushroom polysaccharides (CVP), a Chinese medicinal fungus that exerts anti-tumor activities by enhancing host immunity. Proliferation assays were used to determine whether CVP could activate splenocytes. Flow cytometry analysis and IgM and IgG detection were used to characterize CVP-binding cells. Immune receptors were analyzed in immunoprecipitation and western blot assays. The downstream signaling pathways were identified by western blotting or immunostaining. CVP significantly stimulated the proliferation of mouse splenocytes. Fluorescence-labeled CVP (fl-CVP) selectively stained mouse B cells, but not T cells. CVP induced the production of IgM and IgG1 with or without exogenous IL-4. Membrane Ig (B cell antigen-receptor, BCR) was identified as a CVP-binding protein in immunoprecipitation and western blot experiments. CVP-induced B cell proliferation could be significantly inhibited by anti-mouse immunoglobulin (Ig) blocking antibody (Fab) or in cells from TLR4-mutant mice (C3H/HeJ). Phosphorylation of ERK-1/2 and p38 MAPK were clearly increased in a time-dependent manner, as was the nuclear translocation of the cytosolic NF-κB p65 subunit after CVP stimulation. Together, we demonstrate that CVP can bind and induce B cell activation using membrane Ig and TLR-4 as potential immune receptors. CVP activates mouse B cells through the MAPK and NF-κB signaling pathways. Copyright © 2014 Elsevier Ltd. All rights reserved.

Advanced glycation end product (AGE) modified proteins in tears of diabetic patients.

PubMed

Zhao, Zhenjun; Liu, Jingfang; Shi, Bingyin; He, Shuixiang; Yao, Xiaoli; Willcox, Mark D P

2010-08-11

High glucose level in diabetic patients may lead to advanced glycation end product (AGE) modified proteins. This study investigated AGE modified proteins in tears and compared their levels in diabetic patients (DM) with non-diabetic controls (CTL). Basal tears were collected from DM with (DR) or without (DNR) retinopathy and CTL. Total AGE modified proteins were detected quantitatively by a dot immunobinding assay. The AGE modified proteins were separated in 1D- and 2D-SDS gels and detected by western-blotting. The individual AGE modified proteins were also compared between groups using densitometry. Compared with the CTL group, tear concentrations of AGE modified proteins were significantly elevated in DR and DNR groups. The concentration of AGE modified proteins in diabetic tears were positively correlated with AGE modified hemoglobin (HbA1c) and postprandial blood glucose level (PBG). Western blotting of AGE modified proteins from 1D-SDS gels showed several bands, the major one at around 60 kDa. The intensities of AGE modified protein bands were higher in DM tears than in CTL tears. Western blotting from 2D-SDS gels showed a strongly stained horizontal strip, which corresponded to the major band in 1D-SDS gels. Most of the other AGE modified protein species were within molecular weight of 30-60 kDa, PI 5.2-7.0. Densitometry analysis demonstrated several AGE modified proteins were elevated in DR or DNR tears. Total and some individual AGE modified proteins were elevated in DM tears. AGE modified proteins in tears may be used as biomarkers to diagnose diabetes and/or diabetic retinopathy.

Overexpression of B7-H3 augments anti-apoptosis of colorectal cancer cells by Jak2-STAT3.

PubMed

Zhang, Ting; Jiang, Bo; Zou, Shi-Tao; Liu, Fen; Hua, Dong

2015-02-14

To investigate the role of the overexpression of B7-H3 in apoptosis in colorectal cancer cell lines and the underlying molecular mechanisms. SW620 cells that highly overexpressed B7-H3 (SW620-B7-H3-EGFP) and HCT8 cells stably transfected with B7-H3 shRNA (HCT8-shB7-H3) were previously constructed in our laboratory. Cells transfected with pIRES2-EGFP were used as negative controls (SW620-NC and HCT8-NC). Real-time PCR and western blotting analysis were used to detect the mRNA and protein expressions of the apoptosis regulator proteins Bcl-2, Bcl-xl and Bax. A cell proliferation assay was used to evaluate the survival rate and drug sensitivity of the cells. The effect of drug resistance was detected by a cell cycle assay. Active caspase-3 western blotting was used to reflect the anti-apoptotic ability of cells. Western blotting was also performed to determine the expression of proteins associated with the Jak2-STAT3 signaling pathway and the apoptosis regulator proteins after the treatment with AG490, a Jak2 specific inhibitor, in B7-H3 overexpressing cells. The data were analyzed by GraphPad Prism 6 using a non-paired t-test. Whether by overexpression in SW620 cells or downregulation in HCT8, B7-H3 significantly affected the expression of anti- and pro-apoptotic proteins, at both the transcriptional and translational levels, compared with the negative control (P < 0.05). A cell proliferation assay revealed that B7-H3 overexpression increased the drug resistance of cells and resulted in a higher survival rate (P < 0.05). In addition, the results of cell cycle and active caspase-3 western blotting proved that B7-H3 overexpression inhibited apoptosis in colorectal cancer cell lines (P < 0.05). B7-H3 overexpression improved Jak2 and STAT3 phosphorylation and, in turn, increased the expression of the downstream anti-apoptotic proteins B-cell CLL/lymphoma 2 (Bcl-2) and Bcl-xl, based on western blotting (P < 0.05). After treating B7-H3 overexpressing cells with the Jak2-specific inhibitor AG490, the phosphorylation of Jak2 and STAT3, and the expression of Bcl-2 and Bcl-xl, decreased accordingly (P < 0.05). This finding suggested that the Jak2-STAT3 pathway is involved in the mechanism mediating the anti-apoptotic ability of B7-H3. The overexpression of B7-H3 induces resistance to apoptosis in colorectal cancer cell lines by upregulating the Jak2-STAT3 signaling pathway, potentially providing new approaches to the treatment of colorectal cancer.

Checking transfer efficiency and equal loading via qualitative optical way in western blotting.

PubMed

Gong, Jun-Hua; Gong, Jian-Ping; Zheng, Kai-Wen

2017-11-01

The ability to determine that successful transfer and equal loading occur prior to using primary antibodies is important. And total protein staining is commonly used to check transfer efficiency and normalization, which play a crucial role in western blotting. Ponceau S and coomassie blue are commonly used, but there are disadvantages reported in recent years. Therefore, we are interested in finding another method, which is cheap, easy and fast. As we know, protein binding region of PVDF membrane is still hydrophilic when carbinol volatilizes, however, the non-protein binding region of PVDF membrane became hydrophobic again. And this different wettability between non-protein binding region and protein binding region of Polyvinylidene difluoride membrane may be used to check transfer efficiency and equal loading in western blotting. Based on the principle above, we describe an optical approach where an experimenter can observe that the proteins have been transferred to the membrane without any staining within minutes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Methods to uncover an antibody epitope in the KPI domain of Alzheimer's amyloid precursor protein for immunohistochemistry in human brain.

PubMed

Campbell, E; Pearson, R C; Parkinson, D

1999-11-15

A novel polyclonal antibody (Ab993), specific for a KPI domain epitope of APP, was characterised for use in immunoprecipitation, Western blotting and immunohistochemistry. Conditioned medium from NTera2/D1 cells was used for immunoprecipitation and Western blots. Paraffin-embedded human brain sections were used for immunohistochemistry. The antibody recognised KPI-containing APP on Western blots after standard solubilisation but immunoprecipitation of soluble APP required reduction with 2-mercaptoethanol followed by alkylation of reduced sulphydryl bonds with sodium iodoacetate. Immunohistochemical staining of human brain sections was significantly enhanced by this pre-treatment. Microwaving of sections also increased immunolabelling, by a mechanism that was additive to reduction and alkylation. Incubation in 80% formic acid did not confer any enhancement of immunoreactivity. Ab993, applied with the methods reported here, is expected to be valuable in investigations of the pathogenesis of Alzheimer's disease to determine the source of the beta-amyloid peptide.

Detection of proteins on blot transfer membranes.

PubMed

Sasse, Joachim; Gallagher, Sean R

2003-11-01

In the basic and alternate protocols of this unit, proteins are stained after electroblotting from polyacrylamide gels to blot transfer membranes. If the samples of interest are electrophoresed in duplicate and transferred to a blot transfer membrane, half of the membrane can be stained to determine the efficiency of transfer to the membrane and the other half can be used for immunoblotting (i.e., western blotting). Detection limits of each staining method are given along with a list of compatible blot transfer membranes and gels. A support protocol describes a method for alkali treatment that enhances subsequent staining of bound proteins.

Upregulation of 14-3-3 eta in chronic liver fluke infection is a potential diagnostic marker of cholangiocarcinoma.

PubMed

Haonon, Ornuma; Rucksaken, Rucksak; Pinlaor, Porntip; Pairojkul, Chawalit; Chamgramol, Yaovalux; Intuyod, Kitti; Onsurathum, Sudarat; Khuntikeo, Narong; Pinlaor, Somchai

2016-03-01

To discover protein markers in chronic/advanced opisthorchiasis for the early detection of Opisthorchis viverrini (OV)-associated cholangiocarcinoma (CCA). Liver tissues derived from normal hamsters and those with chronic/advanced opisthorchiasis (n = 5 per group) were subjected to 2DE and LC-MS/MS. Candidate protein expression was confirmed in hamster models and human CCA tissue microarray (TMA) using immunohistochemistry and Western blot. Proteomics analysis detected 14-3-3 eta only in infected hamsters, not in uninfected controls. Immunohistochemistry and Western blot analysis confirmed low expression of 14-3-3 eta in normal hamster livers and demonstrated increased expression through time in infected livers. This protein was also observed in parasite organs, especially during the chronic phase of opisthorchiasis. Moreover, increased expression of 14-3-3 eta, relative to normal hamster livers, was observed during the early stage of CCA induced by OV infection and administration of N-nitrosodimethylamine. Immunohistochemical analysis of human TMA revealed that 14-3-3 eta was highly expressed in CCA (84.23%, 187/222 cases) but was not found in hepatocellular carcinoma or healthy liver tissues. 14-3-3 eta protein has potential as a screening and early diagnostic marker for CCA. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Construction of eukaryotic recombinant vector and expression in COS7 cell of LipL32-HlyX fusion gene from Leptospira serovar Lai].

PubMed

Huang, Bi; Bao, Lang; Zhong, Qi; Zhang, Huidong; Zhang, Ying

2009-04-01

This study was conducted to construct eukaryotic recombinant vector of LipL32-HlyX fusion gene from Leptospira serovar Lai and express it in mammalian cell. Both of LipL32 gene and HlyX gene were amplified from Leptospira strain O17 genomic DNA by PCR. Then with the two genes as template, LipL32-HlyX fusion gene was obtained by SOE PCR (gene splicing by overlap extension PCR). The fusion gene was then cloned into pcDNA3.1 by restriction nuclease digestion. Having been transformed into E. coli DH5alpha, the recombiant plasmid was identified by restriction nuclease digestion, PCR analysis and sequencing. The recombinant plasmid was then transfected into COS7 cell whose expression was detected by RT-PCR and Western blotting analysis. RT-PCR amplified a fragment about 2000 bp and Western blotting analysis found a specific band about 75 KD which was consistent with the expected fusion protein size. In conclusion, the successful construction of eukaryotic recombinant vector containing LipL32-HlyX fusion gene and the effective expression in mammalian have laid a foundation for the application of Leptospira DNA vaccine.

Localization of the ANG II type 2 receptor in the microcirculation of skeletal muscle

NASA Technical Reports Server (NTRS)

Nora, E. H.; Munzenmaier, D. H.; Hansen-Smith, F. M.; Lombard, J. H.; Greene, A. S.; Cowley, A. W. (Principal Investigator)

1998-01-01

Only functional studies have suggested the presence of the ANG II type 2 (AT2) receptor in the microcirculation. To determine the distribution of this receptor in the rat skeletal muscle microcirculation, a polyclonal rabbit anti-rat antiserum was developed and used for immunohistochemistry and Western blot analysis. The antiserum was prepared against a highly specific and antigenic AT2-receptor synthetic peptide and was validated by competition and sensitivity assays. Western blot analysis demonstrated a prominent, single band at approximately 40 kDa in cremaster and soleus muscle. Immunohistochemical analysis revealed a wide distribution of AT2 receptors throughout the skeletal muscle microcirculation in large and small microvessels. Microanatomic studies displayed an endothelial localization of the AT2 receptor, whereas dual labeling with smooth muscle alpha-actin also showed colocalization of the AT2 receptor with vascular smooth muscle cells. Other cells associated with the microvessels also stained positive for AT2 receptors. Briefly, this study confirms previous functional data and localizes the AT2 receptor to the microcirculation. These studies demonstrate that the AT2 receptor is present on a variety of vascular cell types and that it is situated in a fashion that would allow it to directly oppose ANG II type 1 receptor actions.

Comparative Proteomic Analysis of Yak Follicular Fluid during Estrus

PubMed Central

Guo, Xian; Pei, Jie; Ding, Xuezhi; Chu, Min; Bao, Pengjia; Wu, Xiaoyun; Liang, Chunnian; Yan, Ping

2016-01-01

The breeding of yaks is highly seasonal, there are many crucial proteins involved in the reproduction control program, especially in follicular development. In order to isolate differential proteins between mature and immature follicular fluid (FF) of yak, the FF from yak follicles with different sizes were sampled respectively, and two-dimensional gel electrophoresis (2-DE) of the proteins was carried out. After silver staining, the Image Master 2D platinum software was used for protein analysis and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) was performed for differential protein identification. The expression level of transferrin and enolase superfamily member 1 (ENOSF1) was determined by Western blotting for verification analysis. The results showed that 2-DE obtained an electrophoresis map of proteins from mature and immature yak FF with high resolution and repeatability. A comparison of protein profiles identified 12 differently expressed proteins, out of which 10 of them were upregulated while 2 were downregulated. Western blotting showed that the expression of transferrin and ENOSF1 was enhanced with follicular development. Both the obtained protein profiles and the differently expressed proteins identified in this study provided experimental data related to follicular development during yak breeding seasons. This study also laid the foundation for understanding the microenvironment during oocyte development. PMID:26954118

Induction of Parkinson disease-related proteins in motor neurons after transient spinal cord ischemia in rabbits.

PubMed

Sakurai, Masahiro; Kawamura, Takae; Nishimura, Hidekazu; Suzuki, Hiroyoshi; Tezuka, Fumiaki; Abe, Koji

2009-04-01

The mechanism of spinal cord injury has been thought to be related to the vulnerability of spinal motor neuron cells against ischemia. However, the mechanisms of such vulnerability are not fully understood. We investigated a possible mechanism of neuronal death by immunohistochemical analysis for DJ-1, PINK1, and alpha-Synuclein. We used a 15-min rabbit spinal cord ischemia model, with use of a balloon catheter. Western blot analysis for DJ-1, PINK1, and alpha-Synuclein; temporal profiles of DJ-1, PINK1, and alpha-Synuclein immunoreactivity; and double-label fluorescence immunocytochemical studies were performed. Western blot analysis revealed scarce immunoreactivity for DJ-1, PINK1, and alpha-Synuclein in the sham-operated spinal cords. However, they became apparent at 8 h after transient ischemia, which returned to the baseline level at 1 day. Double-label fluorescence immunocytochemical study revealed that both DJ-1 and PINK1, and DJ-1 and alpha-Synuclein were positive at 8 h of reperfusion in the same motor neurons, which eventually die. The induction of DJ-1 and PINK1 proteins in the motor neurons at the early stage of reperfusion may indicate oxidative stress, and the induction of alpha-Synuclein may be implicated in the programmed cell death change after transient spinal cord ischemia.

Structural analysis of photosystem I polypeptides using chemical crosslinking

NASA Technical Reports Server (NTRS)

Armbrust, T. S.; Odom, W. R.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

1994-01-01

Thylakoid membranes, obtained from leaves of 14 d soybean (Glycine max L. cv. Williams) plants, were treated with the chemical crosslinkers glutaraldehyde or 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) to investigate the structural organization of photosystem I. Polypeptides were resolved using lithium dodecyl sulfate polyacrylamide gel electrophoresis, and were identified by western blot analysis using a library of polyclonal antibodies specific for photosystem I subunits. An electrophoretic examination of crosslinked thylakoids revealed numerous crosslinked products, using either glutaraldehyde or EDC. However, only a few of these could be identified by western blot analysis using subunit-specific polyclonal antibodies. Several glutaraldehyde dependent crosslinked species were identified. A single band was identified minimally composed of PsaC and PsaD, documenting the close interaction between these two subunits. The most interesting aspect of these studies was a crosslinked species composed of the PsaB subunit observed following EDC treatment of thylakoids. This is either an internally crosslinked species, which will provide structural information concerning the topology of the complex PsaB protein, a linkage with a polypeptide for which we do not yet have an immunological probe, or a masking of epitopes by the EDC linkage at critical locations in the peptide which is linked to PsaB.

Lifetime fluorescence spectroscopy for in situ investigation of osteogenic differentiation

NASA Astrophysics Data System (ADS)

Marcu, Laura; Elbarbary, Amir; Zuk, Patricia; De Ugarte, Daniel A.; Benhaim, Prosper; Kurt, Hamza; Hedrick, Marc H.; Ashjian, Peter

2003-07-01

Time-Resolved Laser-Induced Fluorescence Spectroscopy (TR-LIFS) represents a potential tool for the in-situ characterization of bioengineered tissues. In this study, we evaluate the application of TR-LIFS to non-intrusive monitoring of matrix composition during osteogenetic differentiation. Human adipose-derived stem cells, harvested from 3 patients, were induced in osteogenic media for 3, 5, and 7 weeks. Samples were subsequently collected and probed for time-resolved fluorescence emission with a pulsed nitrogen laser. Fluorescence parameters, derived from both spectral- and time-domain, were used for sample characterization. The samples were further analyzed using Western blot analysis and computer-based densitometry. A significant change in the fluorescence parameters was detected for samples beyond 3 weeks of osteogenic differentiation. The spectroscopic observations: 1) show increase of collagen I when contrasted against the time-resolved fluorescence spectra of commercially available collagens; and 2) are in agreement with Western blot analysis that demonstrated significant increase in collagen I content between 3- vs. 5-weeks and 3- vs. 7-weeks and no changes for collagens III, IV, and V. Our results suggest that TR-LIFS can be used as a non-invasive means for the detection of specific collagens in maturing connective tissues.

Tangeritin inhibits adipogenesis by down-regulating C/EBPα, C/EBPβ, and PPARγ expression in 3T3-L1 fat cells.

PubMed

He, Y F; Liu, F Y; Zhang, W X

2015-10-29

The treatment of obese patients is a topic investigated by an increasing number of researchers. This study aimed to elucidate the possible inhibitory effect of tangeritin on the development and function of fat cells. 3T3-L1 fat cells were grown to confluence and subjected to different concentrations of tangeritin. The most effective tangeritin inhibition concentration was determined by the MTT assay. The treated cells were subjected to real-time reverse transcriptase PCR and western blot analysis, to detect changes in the CCAAT/enhancer binding protein (C/EBP)α, C/EBPβ, and peroxisome proliferator activated receptor (PPAR)γ expression levels. The MTT assay revealed that the fat cell growth was inhibited at a 20 ng/mL concentration of tangeritin. The results of real-time PCR revealed a significant decrease in the expression of C/EBPα, C/EBPβ, and PPARγ mRNA, following the treatment with tangeritin. Western blot analysis also presented similar results at a protein level. Therefore, we concluded that tangeritin inhibits adipogenesis via the down-regulation of C/EBPα, C/EBPβ, and PPARγ mRNA and protein expression in 3T3-L1 cells.

Preservation of pathological tissue specimens by freeze-drying for immunohistochemical staining and various molecular biological analyses.

PubMed

Matsuo, S; Sugiyama, T; Okuyama, T; Yoshikawa, K; Honda, K; Takahashi, R; Maeda, S

1999-05-01

Conditions of preserving DNA, RNA and protein in pathological specimens are of great importance as degradation of such macromolecules would critically affect results of molecular biological analysis. The feasibility of freeze-drying as a means of preserving pathological tissue samples for molecular analysis has previously been shown. In the present study, further tests on long-term storage conditions and analyses of freeze-dried samples by polymerase chain reaction (PCR), reverse transcriptase (RT)-PCR, western blotting and immunohistochemistry are reported. Rat chromosomal DNA of freeze-dried samples stored for 4 years showed slight degradation while RNA degradation was more prominently seen at an earlier stage of storage. However, these 4 year DNA and RNA samples were still able to serve as a template for some PCR and RT-PCR analyses, respectively. Overexpression of c-erbB-2 and p53 protein was demonstrated by western blotting and immunohistochemical staining using freeze-dried human breast cancer tissues. Although macromolecules in freeze-dried samples degrade to some extent during the preservation period, they should still be of value for certain molecular biological analyses and morphological examination; hence, providing more convenient and inexpensive ways of pathological tissue storage.

Severe acute respiratory syndrome (SARS) S protein production in plants: Development of recombinant vaccine

PubMed Central

Pogrebnyak, Natalia; Golovkin, Maxim; Andrianov, Vyacheslav; Spitsin, Sergei; Smirnov, Yuriy; Egolf, Richard; Koprowski, Hilary

2005-01-01

In view of a recent spread of severe acute respiratory syndrome (SARS), there is a high demand for production of a vaccine to prevent this disease. Recent studies indicate that SARS-coronavirus (CoV) spike protein (S protein) and its truncated fragments are considered the best candidates for generation of the recombinant vaccine. Toward the development of a safe, effective, and inexpensive vaccine candidate, we have expressed the N-terminal fragment of SARS-CoV S protein (S1) in tomato and low-nicotine tobacco plants. Incorporation of the S1 fragment into plant genomes as well as its transcription was confirmed by PCR and RT-PCR analyses. High levels of expression of recombinant S1 protein were observed in several transgenic lines by Western blot analysis using specific antibodies. Plant-derived antigen was evaluated to induce the systemic and mucosal immune responses in mice. Mice showed significantly increased levels of SARS-CoV-specific IgA after oral ingestion of tomato fruits expressing S1 protein. Sera of mice parenterally primed with tobacco-derived S1 protein revealed the presence of SARS-CoV-specific IgG as detected by Western blot and ELISA analysis. PMID:15956182

Severe acute respiratory syndrome (SARS) S protein production in plants: development of recombinant vaccine.

PubMed

Pogrebnyak, Natalia; Golovkin, Maxim; Andrianov, Vyacheslav; Spitsin, Sergei; Smirnov, Yuriy; Egolf, Richard; Koprowski, Hilary

2005-06-21

In view of a recent spread of severe acute respiratory syndrome (SARS), there is a high demand for production of a vaccine to prevent this disease. Recent studies indicate that SARS-coronavirus (CoV) spike protein (S protein) and its truncated fragments are considered the best candidates for generation of the recombinant vaccine. Toward the development of a safe, effective, and inexpensive vaccine candidate, we have expressed the N-terminal fragment of SARS-CoV S protein (S1) in tomato and low-nicotine tobacco plants. Incorporation of the S1 fragment into plant genomes as well as its transcription was confirmed by PCR and RT-PCR analyses. High levels of expression of recombinant S1 protein were observed in several transgenic lines by Western blot analysis using specific antibodies. Plant-derived antigen was evaluated to induce the systemic and mucosal immune responses in mice. Mice showed significantly increased levels of SARS-CoV-specific IgA after oral ingestion of tomato fruits expressing S1 protein. Sera of mice parenterally primed with tobacco-derived S1 protein revealed the presence of SARS-CoV-specific IgG as detected by Western blot and ELISA analysis.

Expression of Fra-1 in human hepatocellular carcinoma and its prognostic significance.

PubMed

Gao, Xiao-Qiang; Ge, Yong-Sheng; Shu, Qing-Hua; Ma, Hua-Xing

2017-06-01

This study aimed to explore the clinical significance and prognostic value of Fra-1 in hepatocellular carcinoma patients after curative resection. Fra-1 expression was investigated using a combination of techniques: immunohistochemistry for 66 samples of hepatocellular carcinoma and quantitative real-time polymerase chain reaction and western blotting assays for 19 matched hepatocellular carcinoma specimens. Fra-1 was present in 38 of 66 (57.6%) tumor tissues, with intense staining in the nuclei. There was also positive staining in 14 of 66 (21.2%) adjacent peritumoral tissues, with weak staining in the cytoplasm. Quantitative real-time polymerase chain reaction and western blotting assays confirmed higher expression of Fra-1 messenger RNA and Fra-1 protein in tumor tissues than adjacent non-tumor tissues for 19 hepatocellular carcinoma samples (p < 0.001). Positive expression of Fra-1 was significantly related to vascular invasion and serum alpha-fetoprotein. Kaplan-Meier survival analysis found that overexpressed Fra-1 was correlated with poor overall survival and disease-free survival. Multivariate analysis identified Fra-1 as an independent prognostic factor. Fra-1 may be involved in the progress of hepatocellular carcinoma and could be a promising molecular candidate in the diagnosis and treatment of hepatocellular carcinoma.

Transport of nattokinase across the rat intestinal tract.

PubMed

Fujita, M; Hong, K; Ito, Y; Misawa, S; Takeuchi, N; Kariya, K; Nishimuro, S

1995-09-01

Intraduodenal administration of nattokinase (NK) at a dose of 80 mg/kg, resulted in the degradation of fibrinogen in plasma suggesting transport of NK across the intestinal tract in normal rats. The action of NK on the cleavage of fibrinogen in the plasma from blood samples drawn at intervals after intraduodenal administration of the enzyme was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis with an anti-fibrinogen gamma chain antibody. The 270 kDa fragment carrying antigenic sites for the binding of the anti-fibrinogen gamma chain antibody appeared within 0.5 h and was then degraded gradually to a 105 kDa fragment via a 200 kDa fragment. This suggests that fibrinogen was degraded to a 105 kDa fragment via several intermediates (270 and 200 kDa). In parallel with the degradation process, plasma recalcification times were remarkably prolonged NK was also detected in the plasma from blood samples drawn 3 and 5 h after administration of the enzyme by SDS-PAGE and Western blotting analysis with an anti-NK antibody. The results indicate that NK is absorbed from the rat intestinal tract and that NK cleaves fibrinogen in plasma after intraduodenal administration of the enzyme.

Change in the antioxidative capacity of extraocular muscles in patients with exotropia.

PubMed

Jung, Su Kyung; Choi, Jun Sob; Shin, Sun Young

2015-04-01

We compared the oxidative stress and antioxidant capacities of the medial rectus muscles (MRMs) between the patients with constant exotropia and control subjects. A total of 40 MRMs from patients with constant exotropia and 40 MRMs from normal donor eyes (controls) were assessed. Neuronal nitric oxide synthase (nNOS), superoxide dismutase (SOD), and catalase levels were compared between the two groups using western blot analysis. In addition, the lipofuscin accumulation level was compared between the exotropic and control groups. According to western blot analysis, the nNOS level was significantly higher in the exotropic group than in the control group (P < 0.05). On the other hand, a catalase expression level was higher in the control group than in the exotropic group (P < 0.05). The SOD1 level did not show a significant difference between the two groups. The relative lipofuscin fluorescence intensity was higher in the exotropic group than in the control group (P < 0.001). Based on these findings, the MRMs of patients with exotropia had a redox imbalance status. Further study is needed to investigate whether this imbalance in antioxidant capacity is present in the extraocular muscles of patients with other strabismus.

Novel flow cytometric analysis of the progress and route of internalization of a monoclonal anti-carcinoembryonic antigen (CEA) antibody.

PubMed

Ford, C H; Tsaltas, G C; Osborne, P A; Addetia, K

1996-03-01

A flow cytometric method of studying the internalization of a monoclonal antibody (Mab) directed against carcinoembryonic antigen (CEA) has been compared with Western blotting, using three human colonic cancer cell lines which express varying amounts of the target antigen. Cell samples incubated for increasing time intervals with fluoresceinated or unlabelled Mab were analyzed using flow cytometry or polyacrylamide gel electrophoresis and Western blotting. SDS/PAGE analysis of cytosolic and membrane components of solubilized cells from the cell lines provided evidence of non-degraded internalized anti-CEA Mab throughout seven half hour intervals, starting at 5 min. Internalized anti-CEA was detected in the case of high CEA expressing cell lines (LS174T, SKCO1). Very similar results were obtained with an anti-fluorescein flow cytometric assay. Given that these two methods consistently provided comparable results, use of flow cytometry for the detection of internalized antibody is suggested as a rapid alternative to most currently used methods for assessing antibody internalization. The question of the endocytic route followed by CEA-anti-CEA complexes was addressed by using hypertonic medium to block clathrin mediated endocytosis.

Formulation, Characterization, and Antitumor Properties of Trans- and Cis-Citral in the 4T1 Breast Cancer Xenograft Mouse Model

PubMed Central

Zeng, San; Kapur, Arvinder; Patankar, Manish S.; Xiong, May P.

2015-01-01

Purpose Citral is composed of a random mixture of two geometric stereoisomers geranial (trans-citral) and neral (cis-citral) yet few studies have directly compared their in vivo antitumor properties. A micelle formulation was therefore developed. Methods Geranial and neral were synthesized. Commercially-purchased citral, geranial, and neral were formulated in PEG-b-PCL (block sizes of 5000:10000, Mw/Mn 1.26) micelles. In vitro degradation, drug release, cytotoxicity, flow cytometry, and western blot studies were conducted. The antitumor properties of drug formulations (40 mg/kg and 80 mg/kg based on MTD studies) were evaluated on the 4T1 xenograft mouse model and tumor tissues were analyzed by western blot. Results Micelles encapsulated drugs with >50% LE at 5-40% drug to polymer (w/w), displayed sustained release (t1/2 of 8-9 hours), and improved drug stability at pH 5.0. The IC50 of drug formulations against 4T1 cells ranged from 1.4-9.9 μM. Western blot revealed that autophagy was the main cause of cytotoxicity. Geranial at 80 mg/kg was most effective at inhibiting tumor growth. Conclusions Geranial is significantly more potent than neral and citral at 80 mg/kg (p<0.001) and western blot of tumor tissues confirms that autophagy and not apoptosis is the major mechanism of tumor growth inhibition in p53-null 4T1 cells. PMID:25673043

Formulation, Characterization, and Antitumor Properties of Trans- and Cis-Citral in the 4T1 Breast Cancer Xenograft Mouse Model.

PubMed

Zeng, San; Kapur, Arvinder; Patankar, Manish S; Xiong, May P

2015-08-01

Citral is composed of a random mixture of two geometric stereoisomers geranial (trans-citral) and neral (cis-citral) yet few studies have directly compared their in vivo antitumor properties. A micelle formulation was therefore developed. Geranial and neral were synthesized. Commercially-purchased citral, geranial, and neral were formulated in PEG-b-PCL (block sizes of 5000:10,000, Mw/Mn 1.26) micelles. In vitro degradation, drug release, cytotoxicity, flow cytometry, and western blot studies were conducted. The antitumor properties of drug formulations (40 and 80 mg/kg based on MTD studies) were evaluated on the 4T1 xenograft mouse model and tumor tissues were analyzed by western blot. Micelles encapsulated drugs with >50% LE at 5-40% drug to polymer (w/w), displayed sustained release (t1/2 of 8-9 h), and improved drug stability at pH 5.0. The IC50 of drug formulations against 4T1 cells ranged from 1.4 to 9.9 μM. Western blot revealed that autophagy was the main cause of cytotoxicity. Geranial at 80 mg/kg was most effective at inhibiting tumor growth. Geranial is significantly more potent than neral and citral at 80 mg/kg (p < 0.001) and western blot of tumor tissues confirms that autophagy and not apoptosis is the major mechanism of tumor growth inhibition in p53-null 4T1 cells.

Canine Lat1: molecular structure, distribution and its expression in cancer samples.

PubMed

Ochiai, Hideharu; Morishita, Taiki; Onda, Ken; Sugiyama, Hiroki; Maruo, Takuya

2012-07-01

A full-length cDNA sequence of canine L-type amino acid transporter 1 (Lat1) was determined from a canine brain. The sequence was 1828 bp long and was predicted to encode 485 amino acid polypeptides. The deduced amino acid sequence of canine Lat1 showed 93.2% and 91.1% similarities to those of humans and rats, respectively. Northern blot analysis detected Lat1 expression in the cerebellum at 4 kb, and Western blot analysis showed a single band at 40 kDa. RT-PCR analysis revealed a distinct expression of Lat1 in the pancreas and testis in addition to the cerebrum and cerebellum. Notably, Lat1 expression was observed in the tissues of thyroid cancer, melanoma and hemangiopericytoma. Although the cancer samples examined were not enough, Lat1 may serve as a useful biomarker of cancer cells in veterinary clinic.

The combination of quantitative PCR and western blot detecting CP4-EPSPS component in Roundup Ready soy plant tissues and commercial soy-related foodstuffs.

PubMed

Xiao, Xiao; Wu, Honghong; Zhou, Xinghu; Xu, Sheng; He, Jian; Shen, Wenbiao; Zhou, Guanghong; Huang, Ming

2012-06-01

With the widespread use of Roundup Ready soy (event 40-3-2) (RRS), the comprehensive detection of genetically modified component in foodstuffs is of significant interest, but few protein-based approaches have been found useful in processed foods. In this report, the combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different RRS plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing both meat and plant protein concentrates. The validity of the 2 methods was confirmed first. We also showed that the CP4-EPSPS protein existed in different RRS plant tissues. In certain cases, the results from the western blot and the qPCR were not consistent. To be specific, at least 2 degraded fragments of CP4-EPSPS protein (35.5 and 24.6 kDa) were observed. For dried bean curd crust and deep-fried bean curd, a degraded protein fragment with the size of 24.6 kDa appeared, while cp4-epsps gene could not be traced by qPCR. In contrast, we found a signal of cp4-epsps DNA in 3 foodstuffs, including soy-containing ham cutlet product, meat ball, and sausage by qPCR, while CP4-EPSPS protein could not be detected by western blot in such samples. Our study therefore concluded that the combination of DNA- and protein-based methods would compensate each other, thus resulting in a more comprehensive detection from nucleic acid and protein levels. The combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different Roundup Ready soy (event 40-3-2) plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing a combination of both meat and plant protein concentrates. This study indicated that the combination of DNA- and protein-based methods would supplement each other for genetically modified detection from nucleic acid and protein levels. Accordingly, qPCR and western blot could be used in CP4-EPSPS detection in a wide variety of soy-related foodstuffs. © 2012 Institute of Food Technologists®

Redox Proteomic Profiling of Specifically Carbonylated Proteins in the Serum of Triple Transgenic Alzheimer's Disease Mice.

PubMed

Shen, Liming; Chen, Youjiao; Yang, Aochu; Chen, Cheng; Liao, Liping; Li, Shuiming; Ying, Ming; Tian, Jing; Liu, Qiong; Ni, Jiazuan

2016-04-12

Oxidative stress is a key event in the onset and progression of neurodegenerative diseases, including Alzheimer's disease (AD). To investigate the role of oxidative stress in AD and to search for potential biomarkers in peripheral blood, serums were collected in this study from the 3-, 6-, and 12-month-old triple transgenic AD mice (3×Tg-AD mice) and the age- and sex-matched non-transgenic (non-Tg) littermates. The serum oxidized proteins were quantified by slot-blot analysis and enzyme-linked immunosorbent assay (ELISA) to investigate the total levels of serum protein carbonyl groups. Western blotting, in conjunction with two-dimensional gel electrophoresis (2D-Oxyblot), was employed to identify and quantify the specifically-carbonylated proteins in the serum of 3×Tg-AD mice. The results showed that the levels of serum protein carbonyls were increased in the three month old 3×Tg-AD mice compared with the non-Tg control mice, whereas no significant differences were observed in the six and 12 months old AD mice, suggesting that oxidative stress is an early event in AD progression. With the application of 2D-Oxyblot analysis, (immunoglobin) Ig gamma-2B chain C region (IGH-3), Ig lambda-2 chain C region (IGLC2), Ig kappa chain C region (IGKC), and Ig kappa chain V-V region HP R16.7 were identified as significantly oxidized proteins compared with the control. Among them IGH-3 and IGKC were validated via immunoprecipitation and Western blot analysis. Identification of oxidized proteins in the serums of 3×Tg-AD mice can not only reveal potential roles of those proteins in the pathogenesis of AD but also provide potential biomarkers of AD at the early stage.

Hypoxia and Prx1 in Malignant Progression of Prostate Cancer

DTIC Science & Technology

2006-09-01

Species (ROS) Formation The rate of ROS formation was determined by flow cytometry analysis using the probe 20,70-dichlorofluorescin diacetate (DCFH-DA...DA were subjected to 4-h hypoxia treatment. After the indicated time, fluorescent cells were analyzed by flow cytometry . Western Blot Analysis Equal...species (ROS) generation was measured by flow cytometry at 0.5, 1, 2, 3, 6, 12, or 24 h after hypoxia treatment. The rate of ROS generation increased

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Wei; Man, Xiao-Yong; Li, Chun-Ming

Vascular endothelial growth factor (VEGF) is one of the strongest regulators of physiological and pathological angiogenesis. VEGF receptor 2 (VEGFR-2), the primary receptor for VEGF, is thought to mediate major functional effects of VEGF. Previously, we have localized both VEGF and VEGFR-2 in human hair follicles. In this study, we further defined the expression and roles of VEGFR-2 on human hair follicle dermal papilla (DP) cells. The expression of VEGFR-2 on DP cells was examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis separately, and localization of VEGFR-2 was defined by immunofluorescence. The effect of VEGF on DPmore » cells was analyzed by MTT assays and specific inhibitors. Finally, the role of VEGF involved in the signaling pathways was investigated by Western blot. RT-PCR and Western blot analysis demonstrated the expression of VEGFR-2 on DP cells. Immunostaining for VEGFR-2 showed strong signal on cultured human DP cells in vitro. Exogenous VEGF{sub 165} stimulated proliferation of DP cells in a dose-dependent manner. Furthermore, this stimulation was blocked by a VEGFR-2 neutralizing antibody (MAB3571) and an ERK inhibitor (PD98059). VEGF{sub 165}-induced phosphorylation of ERK1/2 was abolished by MAB3571 and PD98059, while the phosphorylation of p38, JNK and AKT were not changed by VEGF{sub 165}. Taken together, VEGFR-2 is expressed on primary human hair follicle DP cells and VEGF induces proliferation of DP cells through VEGFR-2/ERK pathway, but not p38, JNK or AKT signaling. -- Highlights: Black-Right-Pointing-Pointer We examine the expression of VEGFR-2 on cultured human dermal papilla (DP) cells. Black-Right-Pointing-Pointer VEGF{sub 165} stimulated proliferation of human DP cells in a dose-dependent manner. Black-Right-Pointing-Pointer This stimulation was through VEGFR-2-mediated activation of ERK.« less

Regulation of sheep α-TTP by dietary vitamin E and preparation of monoclonal antibody for sheep α-TTP.

PubMed

Liu, Kun; Luo, Hai-Ling; Zuo, Zhao-Yun; Jia, Hui-Na; Zhang, Yu-Wei; Chang, Yan-Fei; Jiao, Li-Juan

2014-04-25

α-Tocopherol transfer protein (α-TTP) is a cytosolic protein that plays an important role in regulating concentrations of plasma α-tocopherol (the most bio-active form of vitamin E). Despite the central roles that α-TTP plays in maintaining vitamin E adequacy, we have only recently proved the existence of the α-TTP gene in sheep and, for the first time, cloned its full-length cDNA. However, the study of sheep α-TTP is still in its infancy. In the present study, thirty-five local male lambs of Tan sheep with similar initial body weight were randomly divided into five groups and fed with diets supplemented with 0 (control group), 20, 100, 200, 2000IU·sheep(-1)·d(-1) vitamin E for 120 days. At the end of the experiment, the plasma and liver vitamin E contents were analyzed first and then α-TTP mRNA and protein expression levels were determined by quantitative real-time PCR (qRT-PCR) and Western-blot analysis, respectively. In addition, as no sheep α-TTP antibody was available, a specific monoclonal antibody (McAb) against the ovine α-TTP protein was prepared. The effect of vitamin E supplementation was confirmed by the significant changes in the concentrations of vitamin E in the plasma and liver. As shown by qRT-PCR and Western-blot analysis, dietary vitamin E does not affect sheep α-TTP gene expression, except for high levels of vitamin E supplementation, which significantly increased expression at the protein level. Importantly, the specific sheep anti-α-TTP McAb we generated could provide optimal recognition in ELISA, Western-blot and immunohistochemistry assays, which will be a powerful tool in future studies of the biological functions of sheep α-TTP. Copyright © 2014 Elsevier B.V. All rights reserved.

The utility of vitamin K3 (menadione) against pancreatic cancer.

PubMed

Osada, Shinji; Tomita, Hiroyuki; Tanaka, Yoshihiro; Tokuyama, Yasuharu; Tanaka, Hidenori; Sakashita, Fumio; Takahashi, Takao

2008-01-01

To evaluate the efficacy of vitamin K3 (VK3) against pancreatic cancer, the molecular mechanism of VK3 or gemcitabine (GEM)-induced inhibition of proliferation was characterized. The cell viability was determined using the 3-[4,5-dimethylthiazol]-2,5-diphenyl tetrazolium bromide (MTT) test method. The expressions of cellular proteins were evaluated by Western blot analysis. For morphological studies of the in vivo transplanted cancer cells, the tissues were stained with hematoxylin and eosin. The IC50 of VK3 for pancreatic cancer cells was calculated for 42.1 +/- 3.5 microM. Western blot analysis showed that VK3 induced rapid phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) 30 minutes after application. ERK but not JNK phosphorylation was maintained for at least 12 hours. Activation of apoptosis by VK3, as shown by molecular weight shifts of the pro-activated 32-kDa form of caspase-3 and poly(ADP-ribose)polymerase (PARP) cleavage of the 112-kDa form, was found. Treatment with the thiol antioxidant, L-cysteine (>0.2 mM), completely abrogated the VK3-induced phosphorylation of ERK, but not the JNK, and inhibition of proliferation. A caspase-3 inhibitor antagonized caspase-3 activation, but had no inhibitory effect on the proliferative activity of VK3. GEM at concentrations >0.1 microg/ml was found to inhibit cell proliferation after 24 hours. GEM also induced phosphorylation of JNK, activation of caspase-3 and accumulation of cyclin B1. Local application of VK3 was found to induce extensive tumor tissue necrosis, but slight hematemesis without necrosis was observed 48 hours after GEM injection. In Western blot, ERK but not JNK phosphorylation, was clearly detected in response to VK3 injection into the tumor tissue. The action of VK3 may lead to a favorable outcome against pancreatic cancer, and the detection of ERK phosphorylation in the tissue is important for predicting this effect.

Involvement of p63 in the herpes simplex virus-1-induced demise of corneal cells.

PubMed

Orosz, László; Gallyas, Eva; Kemény, Lajos; Mándi, Yvette; Facskó, Andrea; Megyeri, Klára

2010-06-07

The transcription factor p63 plays a pivotal role in the development and maintenance of epithelial tissues, including the ocular surface. In an effort to gain insight into the pathogenesis of keratitis caused by HSV-1, we determined the expression patterns of the p63 and Bax proteins in the Staatens Seruminstitute Rabbit Cornea cell line (SIRC). SIRC cells were infected with HSV-1 at various multiplicities and maintained for different periods of time. Virus replication was measured by indirect immunofluorescence assay and Western blot analysis. Cell viability was determined by MTT assay. The apoptotic response of the infected cells was quantified by ELISA detecting the enrichment of nucleosomes in the cytoplasm. Western blot analysis was used to determine the levels of p63 and Bax proteins. Indirect immunofluorescence assays and Western blot analyses demonstrated the presence of HSV-1 glycoprotein D (gD) in the infected SIRC cell line, and the pattern of gD expression was consistent with efficient viral replication. The results of MTT and ELISA assays showed that HSV-1 elicited a strong cytopathic effect, and apoptosis played an important role in the demise of the infected cells. Mock-infected SIRC cells displayed the constitutive expression of DeltaNp63alpha. The expressions of the Bax-beta and TAp63gamma isoforms were considerably increased, whereas the level of DeltaNp63alpha was decreased in the HSV-1-infected SIRC cells. Experiments involving the use of acyclovir showed that viral DNA replication was necessary for the accumulation of TAp63gamma. These data suggest that a direct, virus-mediated cytopathic effect may play an important role in the pathogenic mechanism of herpetic keratitis. By disturbing the delicate balance between the pro-survival DeltaN and the pro-apoptotic TA isoforms, HSV-1 may cause profound alterations in the viability of the ocular cells and in the tissue homeostasis of the ocular surface.

Quantitative Structure-Cytotoxicity Relationship of Cinnamic Acid Phenetyl Esters.

PubMed

Uesawa, Yoshihiro; Sakagami, Hiroshi; Okudaira, Noriyuki; Toda, Kazuhiro; Takao, Koichi; Kagaya, Hajime; Sugita, Yoshiaki

2018-02-01

Many phenolic acid phenethyl esters possess diverse biological effects including antioxidant, cytoprotective, anti-inflammation and anti-tumor activities. However, most previous antitumor studies have not considered the cytotoxicity against normal cells. Ten cinnamic acid phenetyl esters were subjected to quantitative structure-activity relationship (QSAR) analysis, based on their cytotoxicity and tumor-specificity, in order to find their new biological activities. Cytotoxicity against four human oral squamous cell carcinoma cell lines and three oral normal mesenchymal cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Tumor specificity (TS) was evaluated by the ratio of the mean 50% cytotoxic concentration (CC 50 ) against normal oral cells to that against human oral squamous cell carcinoma cell lines. Potency-selectivity expression (PSE) value was calculated by dividing the TS value by CC 50 against tumor cells. Apoptosis markers were detected by western blot analysis. Physicochemical, structural and quantum-chemical parameters were calculated based on the conformations optimized by force-field minimization. Western blot analysis demonstrated that [ 9 ] stimulated the cleavage of caspase-3, suggesting the induction of apoptosis. QSAR analysis demonstrated that TS values were correlated with shape, size and ionization potential. Chemical modification of the lead compound may be a potential choice for designing a new type of anticancer drugs. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Characterization of the Bm61 of the Bombyx mori nucleopolyhedrovirus.

PubMed

Shen, Hongxing; Chen, Keping; Yao, Qin; Zhou, Yang

2009-07-01

orf61 (bm61) of Bombyx mori Nucleopolyhedrovirus (BmNPV) is a highly conserved baculovirus gene, suggesting that it performs an important role in the virus life cycle whose function is unknown. In this study, we describe the characterization of bm61. Quantitative polymerase chain reaction (qPCR) and western blot analysis demonstrated that bm61 was expressed as a late gene. Immunofluorescence analysis by confocal microscopy showed that BM61 protein was localized on nuclear membrane and in intranuclear ring zone of infected cells. Structure localization of the BM61 in BV and ODV by western analysis demonstrated that BM61 was the protein of both BV and ODV. In addition, our data indicated that BM61 was a late structure protein localized in nucleus.

Loss of PTEN as a Predictive Biomarker of Response to Lithium Chloride, A Potential Targeted Treatment for Breast Cancer

DTIC Science & Technology

2012-06-01

infected cells, we were unable to produce HCC712 and HCC1187 cell lines with knocked out PTEN. We hypothesize that this is due to the high level of...Growth Factor Receptor in MCF-10A human breast epithelial cells. Western blot demonstrating levels of total EGFR in parental MCF-10A, and three stably...overexpression of EGFR. We performed western blot analyses to determine the degree of MAPK and PI3K pathway activation by comparing relative levels of

Characterization of an Adhesion-Associated Tumor Suppressor in Breast Cancer

DTIC Science & Technology

2001-08-01

Western blot analysis were invasive and associated with fibrous connective tissue (Fig. 4, B of whole cell lysates resolved by SDS-PAGE was...of breast cancer. Immunohistochemical analyses of archival, formalin-fixed paraffin-embedded specimens of benign and malignant breast tissues confirm...10A cells. In particular, EphA2 destabilizes cell-cell attachments while increasing cell interactions with extracellular matrix (ECM proteins). We have

Heterologous expression of laccase cDNA from Ceriporiopsis subvermispora yields copper-activated apoprotein and complex isoform patterns

Treesearch

Luis F. Larrondo; Marcela Avila; Loreto Salas; Dan Cullen; Rafael Vicuna

2003-01-01

Analysis of genomic clones encoding a putative laccase in homokaryon strains of Ceriporiopsis subvermispora led to the identification of an allelic variant of the previously described lcs-1 gene. A cDNA clone corresponding to this gene was expressed in Aspergillus nidulans and in Aspergillus niger. Enzyme assays and Western blots showed that both hosts secreted active...

Downregulation of p16(ink4a) inhibits cell proliferation and induces G1 cell cycle arrest in cervical cancer cells.

PubMed

Zhang, Chu-Yue; Bao, Wei; Wang, Li-Hua

2014-06-01

Studies have suggested that p16(ink4a) may be a surrogate biomarker for the diagnosis of cervical cancer; however, the function of p16(ink4a) in human cervical cancer cells remains largely unknown. Therefore, in this study, we aimed to investigate the role of p16(ink4a) in human cervical cancer cells. Immunocytochemistry was used to examine invasive squamous cell carcinoma and its precancerous lesions. p16(ink4a)-siRNA was transfected into SiHa and HeLa cells to deplete its expression. The cellular levels of p16(ink4a) mRNA and protein were detected by qRT-PCR and western blot analysis. Proliferation rates were assessed by methyl thiazolyl tetrazolium (MTT) and plate colony formation assays. Cellular migration and invasion ability were assessed by a wound healing assay and Transwell assay. Cellular apoptosis and the cell cycle were measured by flow cytometry. The protein levels of retinoblastoma (Rb), phosphorylated Rb (phospho-Rb), cyclin D1 and caspase-3 were determined by western blot analysis. The results revealed that p16(ink4a) was overexpressed in the cervical cancer and precancerous lesions (P<0.05). The downregulation of p16(ink4a) in the SiHa and HeLa cells inhibited their proliferation, migration and invasion. In the SiHa cells, p16(ink4a)-siRNA also induced G1 cell cycle arrest and apoptosis. Western blot analysis revealed that the downregulation of p16(ink4a) in the SiHa cells markedly induced caspase-3 activation and decreased cyclin D1 expression. These data suggest that the overexpression of p16(ink4a) appears to be useful in monitoring cervical precancerous lesions, which supports that the hypothesis that p16(ink4a) is a surrogate biomarker for the diagnosis of cervical cancer. The therapeutic targeting of overexpressed p16(ink4a) in the p16(ink4a)-cyclin-Rb pathway may be a useful strategy in the treatment of cervical cancer.

Slit2 ameliorates renal inflammation and fibrosis after hypoxia-and lipopolysaccharide-induced epithelial cells injury in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Xiangjun; Yao, Qisheng, E-mail: yymcyqs@126.com; Sun, Xinbo

Hypoxic acute kidney injury (AKI) is often incompletely repaired and leads to chronic kidney disease (CKD), which is characterized by tubulointerstitial inflammation and fibrosis. The Slit2 family of secreted glycoproteins is expressed in the kidney, it has been shown to exert an anti-inflammatory activity and prevent ischemic renal injury in vivo. However, whether Slit2 reduces renal fibrosis and inflammation after hypoxic and inflammatory epithelial cells injury in vitro remains unknown. In this study, we aimed to evaluate whether Slit2 ameliorated fibrosis and inflammation in two renal epithelial cells line challenged with hypoxia and lipopolysaccharide (LPS). Renal epithelial cells were treatedmore » with hypoxia and LPS to induce cell injury. Hoechst staining and Western blot analysis was conducted to examine epithelial cells injury. Immunofluorescence staining and Western blot analysis was performed to evaluate tubulointerstitial fibrosis. Real-time polymerase chain reaction (PCR) tested the inflammatory factor interleukin (IL)−1β and tumor necrosis factor (TNF)-α, and Western blot analysis determined the hypoxia-inducible factor (HIF)−1α, Toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB. Results revealed that hypoxia induced epithelial cells apoptosis, inflammatory factor IL-1β and TNF-α release and tubulointerstitial fibrosis. LPS could exacerbate hypoxia -induced epithelial cells apoptosis, IL-1β and TNF-α release and fibrosis. Slit2 reduced the expression of fibronectin, the rate of epithelial cell apoptosis, and the expression of inflammatory factor. Slit2 could also inhibit the expression of TLR4 and NF-κB, but not the expression of HIF-1α. Therefore, Slit2 attenuated inflammation and fibrosis after LPS- and hypoxia-induced epithelial cells injury via the TLR4/NF-κB signaling pathway, but not depending on the HIF-1α signaling pathway. - Highlights: • Slit2 ameliorates inflammation after hypoxia-and LPS-induced epithelial cells injury. • Slit2 ameliorates fibrosis after hypoxia-and LPS-induced epithelial cells injury. • Slit2 ameliorates inflammation and fibrosis after hypoxia-and LPS-induced renal epithelial cells injury via TLR4/NF-κB.« less

Interrogation of Multidrug Resistance (MDR1) P-glycoprotein (Pgp, ABCB1) Expression in Human Pancreatic Carcinoma Cells: Correlation of 99mTc-Sestamibi Uptake with Western Blot Analysis

PubMed Central

Harpstrite, Scott E.; Gu, Hannah; Natarajan, Radhika; Sharma, Vijay

2014-01-01

Objective Histopathological studies indicate approximately 63% of pancreatic tumors express MDR1 Pgp and its polymorphic variants. However, Pgp expression detected at the messenger RNA or protein level does not always correlate with functional transport activity. Because Pgp transport activity is affected by specific mutations as well as the phosphorylation state of the protein, altered or less active forms of Pgp may also be detected by PCR or immunohistochemistry, which do not accurately reflect the status of tumor cell resistance. To interrogate status of functional expression of MDR1 Pgp in MiaPaCa-2 and PANC-1 cells, cellular transport studies using 99mTc-Sestamibi were performed and correlated with western blot analysis. Methods Biochemical transport assays in human pancreatic carcinoma MiaPaCa-2 and PANC-1 cells, human epidermal carcinoma drug sensitive KB-3-1 cells and human breast carcinoma MCF-7 cells (negative controls), and human epidermal carcinoma drug resistant KB-8-5 cells, human breast carcinoma stably transfected with Pgp MCF-7/MDR1Pgp cells, and liver carcinoma HepG2 cells (positive controls) were performed. Protein levels were determined using a monoclonal antibody C219. Results 99mTc-Sestamibi demonstrates accumulation in human pancreatic carcinoma MiaPaCa-2 and PANC-1 cells. Uptake profiles are not affected by treatment with LY335979, a Pgp-inhibitor, and correlate to Western blot analysis. Conclusions These cellular transport studies indicate an absence of Pgp at a functional level in MiaPaCa-2 and PANC-1 cells. Because major pancreatic tumors originate from pancreatic duct and 99mTc-Sestamibi undergoes a dominant hepatobiliary mode of excretion, it would not be a sensitive probe for imaging pancreatic adenocarcinomas. Following interrogation of the functional status of Pgp in other pancreatic carcinoma cells, chemotherapeutic drugs that are also MDR1 substrates could offer alternative therapeutics for treating pancreatic adenocarcinomas. PMID:25036383

The detection of hemorrhagic proteins in snake venoms using monoclonal antibodies against Virginia opossum (Didelphis virginiana) serum.

PubMed

Sánchez, E E; García, C; Pérez, J C; De La Zerda, S J

1998-10-01

Most snakes and a few warm-blooded animals have a resistance to snake venoms because of naturally occurring antihemorrhagins found in their sera. The antihemorrhagins in serum of Virginia opossum (Didelphis virginiana) neutralize hemorrhagic activity by binding to hemorrhagins in snake venoms. The binding characteristic of antihemorrhagins in D. virginiana serum was used to develop a five-step western blot. The detection of hemorrhagic proteins were measured indirectly with antihemorrhagins in Virginia opossum serum and with DV-2LD#2, a monoclonal antibody specific for Virginia opossum antihemorrhagins. Snake venoms were separated by native-PAGE, transferred to a Millipore Immobilon-P membrane and then incubated with crude Virginia opossum serum. The hemorrhagins in snake venom bind to antihemorrhagins in Virginia opossum serum which react with DV-2LD#2 a monoclonal antibody that is specific for Virginia opossum antihemorrhagins. DV-2LD#2 monoclonal antibody inhibits antihemorrhagic activity in Virginia opossum serum when mixed in equal amounts. The inhibition of antihemorrhagins by DV-2LD#2 monoclonal antibody suggests specificity. DV-2LD#2 monoclonal antibody does not recognize antihemorrhagins in gray woodrat (Neotoma micropus) serum. The five-step western blot reveals two well-defined bands which represent hemorrhagins found in Western diamondback rattlesnake (Crotalus atrox) venom. Venoms from 15 different snake species were examined to determine the usefulness of the five-step western blot. Other hemorrhagic venoms (Great Basin rattlesnake (C. viridis lutosus), Prairie rattlesnake (C. viridis viridis), Tancitaran dusky rattlesnake (C. pusillus), Northern Mojave rattlesnake (C. scutulatus scutulatus type B) and Northern Pacific rattlesnake (C. v. oreganus)) had one single band in the five-step western blot. DV-2LD#2 did not bind to the non-hemorrhagic venoms and reacted with 50% of the hemorrhagic venoms used in this study. The monoclonal antibody, CAH, reacted with all the hemorrhagic venoms except for the venom of the King cobra (Ophiophagus hannah) and did not react with the non-hemorrhagic venoms. The hemorrhagic binding site of CAH monoclonal antibody and the antihemorrhagin in Virginia opossum are different binding sites. The five-step western blot will be a very useful assay for determining hemorrhagic activity without using live animals.

The relationship of ASE-1 and NOR-90 in autoimmune sera.

PubMed

Whitehead, C M; Fritzler, M J; Rattner, J B

1998-11-01

The nucleolar proteins ASE-1 and NOR-90 can become confused because they have similar cytological and Western blot features. We investigated the frequency and relationship between these 2 proteins and identified clinical features of patients with ASE-1 antibodies. The characteristics of ASE-1 and NOR-90 are shown by indirect immunofluorescence (IIF) and Western blot data. The sera are characterized by their ability to immunoprecipitate the in vitro transcription and translation (TnT) product of either the ASE-1 or NOR-90 cDNA. Clinical features were obtained by retrospective chart review. Of the 15 sera identified as potentially NOR-90 positive by IIF and Western blot 8/15 (53%) were able to immunoprecipitate a NOR-90 TnT product. Of the remaining 7 sera, 4 (57%) were only able to immunoprecipitate an ASE-1 TnT product. Four (57%) of the remaining 7 sera were able to immunoprecipitate an ASE-1 TnT product. In a second cohort of confirmed NOR-90 positive sera, 2/8 (25%) were able to immunoprecipitate an ASE-1 TnT product. In total, ASE-1 autoantibodies were found in 6/16 (37.5%) of confirmed NOR-90 sera from both cohorts. There were no common clinical features found in seven ASE-1 positive patients; however, 3 (43%) had a malignancy and 3 (43%) had slowly progressive systemic sclerosis. Autoantibodies to ASE-1 and NOR-90 can occur alone or together in autoimmune sera. Due to their similar IIF and Western blot profile the only way to correctly characterize these sera is by immunoprecipitation of the appropriate TnT product.

S100A8 as potential salivary biomarker of oral squamous cell carcinoma using nanoLC-MS/MS.

PubMed

Jou, Yu-Jen; Hua, Chun-Hung; Lin, Chia-Der; Lai, Chih-Ho; Huang, Su-Hua; Tsai, Ming-Hsui; Kao, Jung-Yie; Lin, Cheng-Wen

2014-09-25

Oral squamous cell carcinoma (OSCC) shows low 5-year survival; early treatment greatly reduces mortality and morbidity. Saliva is a non-invasive sample, with good potential to discover biomarkers for early detection. NanoLC-MS/MS served to analyze saliva proteome from control subjects (n=35) and OSCC patients T1 (n=29), T2 (n=36), T3 (n=14) and T4 (n=21) stages. Identified biomarkers were verified by Western blot and ELISA assays. NanoLC-MS/MS analysis of salivary proteins between 10 and 15kDa identified S100A8, hemoglobin delta and gamma-G globin in T3 and T4 stage OSCC as well as S100A7 in T1 and T2 stage OSCC. Western blot and ELISA indicated positive correlation between salivary S100A8 increment and tumor size stage. High level of S100A8 appeared in 3.4, 13.9, 92.9, and 100% of saliva OSCC patients with T1, T2, T3, and T4 stages, respectively. Significant increase of salivary S100A7 was observed in 20.7% and 11.1% of those with T1 and T2, respectively. AUROC curve indicated high sensitivity, specificity and accuracy of S100A8-based ELISA as a detector. NanoLC-MS/MS, Western blot and ELISA manifested salivary S100A8 as a specific and sensitive marker for detection of OSCC patients. Salivary S100A8 protein could be applicable in developing OSCC diagnostics. Copyright © 2014 Elsevier B.V. All rights reserved.

Evaluation of an enzyme immunoassay system for measuring herpes simplex virus (HSV) type 1-specific and HSV type 2-specific IgG antibodies.

PubMed

Prince, H E; Ernst, C E; Hogrefe, W R

2000-01-01

MRL Diagnostics has developed a dual enzyme immunoassay (EIA) system that employs the recombinant Herpes Simplex Virus (HSV) type-specific glycoproteins G1 (HSV1) and G2 (HSV2) to detect HSV type-specific IgG antibodies. This system was evaluated using 155 consecutive sera previously tested in a conventional dual EIA system (Zeus) that employs multiple HSV1 and HSV2 proteins to detect type-common as well as type-specific antibodies. Sera were also analyzed by Western blot to determine the true HSV type-specific IgG reactivity pattern. Of 110 sera giving concordant reactivity patterns in the MRL and Zeus EIA systems, 108 (98%) also displayed concordant Western blot patterns; two sera gave false positive HSV2 reactivity in both EIA systems. Of 45 sera giving discordant MRL and Zeus EIA reactivity patterns, 41 (91%) displayed a Western blot reactivity pattern that matched the MRL reactivity pattern. Both the HSV1 IgG component and the HSV2 IgG component of the MRL EIA system were 100% sensitive and > 95% specific. In contrast, the Zeus HSV1 IgG EIA was 98% sensitive and 79% specific, and the Zeus HSV2 IgG EIA was 85% sensitive and 79% specific. An analysis of the distribution of index values in the MRL EIA system showed that low-positive values (1.0-3.0) were rare, but, when detected, often represented false positive results; only 11 MRL low-positive results were observed, but all 6 MRL false positive results were found within this low-positive subgroup. These findings show that the MRL dual EIA system effectively detects HSV type-specific IgG antibodies. Copyright 2000 Wiley-Liss, Inc.

Safety and efficacy of intravitreal injection of recombinant erythropoietin for protection of photoreceptor cells in a rat model of retinal detachment

PubMed Central

Xie, Z; Chen, F; Wu, X; Zhuang, C; Zhu, J; Wang, J; Ji, H; Wang, Y; Hua, X

2012-01-01

Purpose To elucidate the safety and efficacy of exogenous erythropoietin (EPO) for the protection of photoreceptor cells in a rat model of retinal detachment (RD). Methods Recombinant rat EPO (400 ng) was injected into the vitreous cavity of normal rats to observe the eye manifestations. Retinal function was assessed by flash electroretinograms. Histopathological examination of retinal tissue was performed at 14 days and 2 months after injection, respectively. To investigate the inhibitory effect of EPO on photoreceptor cell apoptosis in RD rats, 100, 200, or 400 ng EPO was injected into the vitreous cavity immediately after RD model establishment. Apoptosis of photoreceptor cells was determined at 3 days after injection. Caspase-3 activation was measured by western blot analysis and immunofluorescence, respectively, and the level of Bcl-XL expression was analyzed by western blot. Results Intravitreal injection of EPO 400 ng into normal rats had no significant impact on retinal function, morphology, or structure. Apoptosis of retinal photoreceptor cells apparently increased after RD and was significantly reduced following EPO treatment. The thickness of the outer nuclear layer in the RD+400 ng group was significantly thicker than that in other experimental RD groups both at 14 days and at 2 months after RD (P<0.05). Western blot and immunofluorescence analyses showed decreased caspase-3 activation and increased Bcl-XL expression following EPO treatment. Conclusion Intravitreal injection of EPO 400 ng is safe, and EPO may suppress caspase-3 activation and enhance Bcl-XL expression, resulting in inhibition of apoptosis and protection of photoreceptor cells. PMID:22020175

Purified human breast milk MUC1 and MUC4 inhibit human immunodeficiency virus.

PubMed

Mthembu, Yolanda; Lotz, Zoe; Tyler, Marilyn; de Beer, Corena; Rodrigues, Jeronimo; Schoeman, Leann; Mall, Anwar Suleman

2014-01-01

The HIV-AIDS pandemic is prevalent in sub-Saharan Africa. Breastfeeding is a risk factor, with transmission from mother to child being as high as 40%. To determine the antiviral activity of crude breast milk and its purified mucins MUC1 and MUC4 against HIV-1 in patients who were HIV positive compared to those who were not. Twenty-one human milk samples were taken from both groups. Breast milk mucins were purified by density-gradient ultracentrifugation in caesium chloride and analyzed by SDS-PAGE, Western blotting and amino acid content. The inhibition of the virus by crude milk and purified mucin was assayed by an in vitro HIV-1 p24 assay. SDS-PAGE for purified mucin showed several high-molecular-weight bands for the HIV-negative group and prominently stained single bands on the stacking gel with faintly periodic acid Schiff-positive glycoprotein bands observed in some cases in the running gel for the HIV-positive mucins. Western blot analysis identified the mucins in both groups to be MUC1 and MUC4. Both mucins showed more intensity on Western blotting for the HIV-positive group. There was no difference in the content of serine, threonine and proline of purified mucins for both groups. HIV-1 was not inhibited by crude breast milk from normal (13/14 samples) and infected individuals (19/19 samples). Fifteen of 20 and 16/18 samples of purified mucin from the uninfected and HIV-positive groups, respectively, inhibited the virus. Crude breast milk does not inhibit HIV-1, whilst purified mucins do in an in vitro assay. © 2014 S. Karger AG, Basel.

Contact lens care solutions downregulate membrane-associated mucins 1 and 16 in cultured human corneal epithelial cells and at the rat corneal surface in vivo.

PubMed

Tchedre, Kissaou; Imayasu, Masaki; Hori, Yuichi; Cavanagh, H Dwight

2013-11-01

The purpose of this study was first to evaluate the effect of multipurpose contact lens care solutions (MPSs) on the expression of membrane-associated mucins (MUC1 and MUC16) in SV40-transformed human corneal epithelial (HCE-T) cells and in vivo rat cornea. The second aim of this study was to determine the role of the common MPS additive boric acid in reducing mucin expression and release. The HCE-T cells were exposed to different concentrations of MPS-F, MPS-G, MPS-H, MPS-I, and MPS-J with 100% treatment for 30 minutes and 10% treatment for 24 hours. MUC1 and MUC16 expressions were subsequently analyzed by Western blotting. Wister rats were also subjected to MPS-A, MPS-B, MPS-C, MPS-D, and MPS-E and received phosphate-buffered saline exposure (1 drop in the right eye every 10 minutes for 1 hour). The left eye was used as control. Cornea sections and lysates were used for the immunohistochemical assay of MUC1 and MUC16 expressions. Conditioned media from treated HCE-T cells were also analyzed using Western blotting. The MPSs containing boric acid downregulated MUC1 and MUC16 in the rat cornea, whereas MPSs without boric acid had no effect as demonstrated by the Western blotting and immunohistochemical analysis. Conditioned media from MPS-containing boric acid revealed some trace of MUC16. The clinical use of MPSs containing boric acid that reduce MUC1 and MUC16 availability should be avoided. Additionally, the presence of MUC16 in the conditioned media suggests that boric acid may have enhanced cleavage of MUC16 at the cell membrane surface.

Combined use of leptin and mechanical stress has osteogenic effects on ossification of the posterior longitudinal ligament.

PubMed

Chen, Shuai; Zhu, Haifeng; Wang, Gangliang; Xie, Ziang; Wang, Jiying; Chen, Jian

2018-06-16

To evaluate the effects of leptin/leptin receptor (LepR) combined with mechanical stress on the development of ossification of the posterior longitudinal ligament (OPLL), which is a disease characterized by ectopic bone formation of the posterior longitudinal ligament (PLL) and can lead to radiculopathy and myelopathy. Six human samples of the PLL were analyzed for the expression of leptin and LepR by RT-PCR and western blotting. PLL cells were stimulated with leptin and mechanical stress delivered via a Flexcell tension system, and osteogenic differentiation was evaluated by RT-PCR and western blotting analysis of osteogenic marker expression as well as by alkaline phosphatase (ALP) staining and alizarin red S staining. Activation of mitogen-activated protein kinase (MAPK), Janus kinase (JAK) 2-signal transducer, activator of transcription (STAT) 3 and phosphatidylinositol 3-kinase (PI3K)-Akt was evaluated by western blotting. Samples from the OPLL group had higher LepR mRNA and protein levels and lower leptin levels than those from healthy controls. Exposure to leptin and Flexcell increased the number of ALP-positive cells and calcium nodules in a dose-dependent manner; this effect was accompanied by upregulation of the osteogenic markers osteocalcin, runt-related transcription factor 2 (RUNX2) and osteopontin. Extracellular signal-regulated kinase, P38 MAPK, JAK2, STAT3, PI3K and Akt signaling, was also activated by the combined effects of leptin and mechanical stress. Leptin and LepR are differentially expressed in OPLL tissues, and the combined use of leptin/LepR and mechanical stress promotes osteogenic differentiation of PLL cells via MAPK, JAK2-STAT3 and PI3K/Akt signaling. These slides can be retrieved under Electronic Supplementary Material.

Obstructing Androgen Receptor Activation in Prostate Cancer Cells Through Post-translational Modification by NEDD8

DTIC Science & Technology

2012-11-01

FACS flow cytometer analysis . In addition, we will measure the steady state protein level of p53, p21, p27, and pRb. In the Jab1 silencing cell...affected by DHT treatment, and the endogenous AR level was not affected by Jab1 silencing. Interestingly, Western blot analysis of immunoprecipitated AR...Avantaggiati, and R. G. Pestell . 2003. Acetylation of androgen receptor enhances coactivator binding and promotes prostate cancer cell growth. Mol

Role of Adenosine Receptor A2A in Traumatic Optic Neuropathies (Addendum)

DTIC Science & Technology

2016-03-01

inflammation was evaluated using Western blot, Real-Time PCR and immuno-staining analyses. Role of A2AAR signaling in the anti-inflammation effect of ABT...Neuroimmunology 277 (2014) 96–104were evaluated by analysis of variance (one-way ANOVA), and the significance of differences between groups was assessed by the...Ahmad et al. / Journal of Neuroimmunology 277 (2014) 96–104were evaluated by analysis of variance (one-way ANOVA), and the significance of differences

Algorithms for detecting antibodies to HIV-1: results from a rural Ugandan cohort.

PubMed

Nunn, A J; Biryahwaho, B; Downing, R G; van der Groen, G; Ojwiya, A; Mulder, D W

1993-08-01

To evaluate an algorithm using two enzyme immunoassays (EIA) for anti-HIV-1 antibodies in a rural African population and to assess alternative simplified algorithms. Sera obtained from 7895 individuals in a rural population survey were tested using an algorithm based on two different EIA systems: Recombigen HIV-1 EIA and Wellcozyme HIV-1 Recombinant. Alternative algorithms were assessed using negative or confirmed positive sera. None of the 227 sera classified as unequivocably negative by the two assays were positive by Western blot. Of 192 sera unequivocably positive by both assays, four were seronegative by Western blot. The possibility of technical error cannot be ruled out in three of these. One of the alternative algorithms assessed classified all borderline or discordant assay results as negative had a specificity of 100% and a sensitivity of 98.4%. The cost of this algorithm is one-third that of the conventional algorithm. Our evaluation suggests that high specificity and sensitivity can be obtained without using Western blot and at a considerable reduction in cost.

Expression of retinoblastoma gene product (pRb) in mantle cell lymphomas. Correlation with cyclin D1 (PRAD1/CCND1) mRNA levels and proliferative activity.

PubMed Central

Jares, P.; Campo, E.; Pinyol, M.; Bosch, F.; Miquel, R.; Fernandez, P. L.; Sanchez-Beato, M.; Soler, F.; Perez-Losada, A.; Nayach, I.; Mallofré, C.; Piris, M. A.; Montserrat, E.; Cardesa, A.

1996-01-01

Mantle cell lymphomas (MCLs) are molecularly characterized by bcl-1 rearrangement and constant cyclin D1 (PRAD-1/CCND1) gene overexpression. Cyclin D1 is a G1 cyclin that participates in the control of the cell cycle progression by interacting with the retinoblastoma gene product (pRb). Inactivation of the Rb tumor suppressor gene has been implicated in the development of different types of human tumors including some high grade non-Hodgkin's lymphomas. To determine the role of the retinoblastoma gene in the pathogenesis of MCLs and its possible interaction with cyclin D1, pRb expression was examined in 23 MCLs including 17 typical and 6 blastic variants by immunohistochemistry and Western blot. Rb gene structure was studied in 13 cases by Southern blot. Cytogenetic analysis was performed in 5 cases. The results were compared with the cyclin D1 mRNA levels examined by Northern analysis, and the proliferative activity of the tumors was measured by Ki-67 growth fraction and flow cytometry. pRb was expressed in all MCLs. The expression varied from case to case (mean, 14.1% of positive cells; range, 1.3 to 42%) with a significant correlation with the proliferative activity of the tumors (mitotic index r = 0.85; Ki-67 r = 0.7; S phase = 0.73). Blastic variants showed higher numbers of pRb-positive cells (mean, 29%) than the typical cases (10%; P < 0.005) by immunohistochemistry and, concordantly, higher levels of expression by Western blot. In addition, the blastic cases also had an increased expression of the phosphorylated protein. No alterations in Rb gene structure were observed by Southern blot analysis. Cyclin D1 mRNA levels were independent of pRb expression and the proliferative activity of the tumors. These findings suggest that pRb in MCLs is normally regulated in relation to the proliferative activity of the tumors. Cyclin D1 overexpression may play a role in the maintenance of cell proliferation by overcoming the suppressive growth control of pRb. Images Figure 1 Figure 2 Figure 4 PMID:8623927

Osthole induces G2/M arrest and apoptosis in lung cancer A549 cells by modulating PI3K/Akt pathway

PubMed Central

2011-01-01

Background To explore the effects of Osthole on the proliferation, cell cycle and apoptosis of human lung cancer A549 cells. Methods Human lung cancer A549 cells were treated with Osthole at different concentrations. Cell proliferation was measured using the MTT assay. Cell cycle was evaluated using DNA flow cytometry analysis. Induction of apoptosis was determined by flow cytometry and fluorescent microscopy. The expressions of Cyclin B1, p-Cdc2, Bcl-2, Bax, t-Akt and p-Akt were evaluated by Western blotting. Results Osthole inhibited the growth of human lung cancer A549 cells by inducing G2/M arrest and apoptosis. Western blotting demonstrated that Osthole down-regulated the expressions of Cyclin B1, p-Cdc2 and Bcl-2 and up-regulated the expressions of Bax in A549 cells. Inhibition of PI3K/Akt signaling pathway was also observed after treating A549 cells with Osthole. Conclusions Our findings suggest that Osthole may have a therapeutic application in the treatment of human lung cancer. PMID:21447176

Expression of P53 protein after exposure to ionizing radiation

NASA Astrophysics Data System (ADS)

Salazar, A. M.; Salvador, C.; Ruiz-Trejo, C.; Ostrosky, P.; Brandan, M. E.

2001-10-01

One of the most important tumor suppressor genes is p53 gene, which is involved in apoptotic cell death, cell differentiation and cell cycle arrest. The expression of p53 gene can be evaluated by determining the presence of P53 protein in cells using Western Blot assay with a chemiluminescent method. This technique has shown variabilities that are due to biological factors. Film developing process can influence the quality of the p53 bands obtained. We irradiated tumor cell lines and human peripheral lymphocytes with 137Cs and 60Co gamma rays to standardize irradiation conditions, to compare ionizing radiation with actinomycin D and to reduce the observed variability of P53 protein induction levels. We found that increasing radiation doses increase P53 protein induction while it decreases viability. We also conclude that ionizing radiation could serve as a positive control for Western Blot analysis of protein P53. In addition, our results show that the developing process may play an important role in the quality of P53 protein bands and data interpretation.

Myricetin inhibits UVB-induced angiogenesis by regulating PI-3 kinase in vivo

PubMed Central

Jung, Sung Keun; Lee, Ki Won; Byun, Sanguine; Lee, Eun Jung; Kim, Jong-Eun; Bode, Ann M.; Dong, Zigang

2010-01-01

Myricetin is one of the principal phytochemicals in onions, berries and red wine. Previous studies showed that myricetin exhibits potent anticancer and chemopreventive effects. The present study examined the effect of myricetin on ultraviolet (UV) B-induced angiogenesis in an SKH-1 hairless mouse skin tumorigenesis model. Topical treatment with myricetin inhibited repetitive UVB-induced neovascularization in SKH-1 hairless mouse skin. The induction of vascular endothelial growth factor, matrix metalloproteinase (MMP)-9 and MMP-13 expression by chronic UVB irradiation was significantly suppressed by myricetin treatment. Immunohistochemical and western blot analyses revealed that myricetin inhibited UVB-induced hypoxia inducible factor-1α expression in mouse skin. Western blot analysis and kinase assay data revealed that myricetin suppressed UVB-induced phosphatidylinositol-3 (PI-3) kinase activity and subsequently attenuated the UVB-induced phosphorylation of Akt/p70S6K in mouse skin lysates. A pull-down assay revealed the direct binding of PI-3 kinase and myricetin in mouse skin lysates. Our results indicate that myricetin suppresses UVB-induced angiogenesis by regulating PI-3 kinase activity in vivo in mouse skin. PMID:20008033

Identification of a protein associated with the activity of cytokine-induced killer cells

PubMed Central

Cao, Jingsong; Chen, Cong; Gao, Yongqiang; Hu, Li; Liang, Yu; Xiao, Jianhua

2017-01-01

Cytokine-induced killer cells (CIKs) adoptive immunotherapy for efficient antitumor ability is used clinically, but details regarding the proteins associated with CIK activity remain unclear. In the current study, the cytotoxicity of CIKs on hepatoma was identified to be significantly downregulated by 1.61-fold following gentamincin treatment. Further research revealed that a differentially expressed protein (P43) was significantly downregulated by 1.22-fold using one-dimensional gel electrophoresis analysis. Of these, the P43 was identified as human haptoglobin using liquid chromatography-mass spectrometry. Western blotting demonstrated that the haptoglobin specifically reacted with rabbit anti-human-haptoglobin. Furthermore, western blotting results verified that the haptoglobin was significantly downregulated by 1.17-fold compared with the control group. In addition, the expression of haptoglobin mRNA was significantly downregulated by 1.73-fold following gentamincin treatment. Taken together, the results of the present study demonstrated that the expression of haptoglobin protein was associated with the activity of CIKs, and the results will be beneficial to the further investigation of CIK activity-enhancement mechanism. PMID:29163711

Vascular smooth muscle cells exhibit a progressive loss of rigidity with serial culture passaging.

PubMed

Dinardo, Carla Luana; Venturini, Gabriela; Omae, Samantha Vieira; Zhou, Enhua H; da Motta-Leal-Filho, Joaquim Maurício; Dariolli, Rafael; Krieger, José Eduardo; Alencar, Adriano Mesquita; Costa Pereira, Alexandre

2012-01-01

One drawback of in vitro cell culturing is the dedifferentiation process that cells experience. Smooth muscle cells (SMC) also change molecularly and morphologically with long term culture. The main objective of this study was to evaluate if culture passages interfere in vascular SMC mechanical behavior. SMC were obtained from five different porcine arterial beds. Optical magnetic twisting cytometry (OMTC) was used to characterize mechanically vascular SMC from different cultures in distinct passages and confocal microscopy/western blotting, to evaluate cytoskeleton and extracellular matrix proteins. We found that vascular SMC rigidity or viscoelastic complex modulus (G) decreases with progression of passages. A statistically significant negative correlation between G and passage was found in four of our five cultures studied. Phalloidin-stained SMC from higher passages exhibited lower mean signal intensity per cell (confocal microscopy) and quantitative western blotting analysis showed a decrease in collagen I content throughout passages. We concluded that vascular SMC progressively lose their stiffness with serial culture passaging. Thus, limiting the number of passages is essential for any experiment measuring viscoelastic properties of SMC in culture.

Large scale systematic proteomic quantification from non-metastatic to metastatic colorectal cancer

NASA Astrophysics Data System (ADS)

Yin, Xuefei; Zhang, Yang; Guo, Shaowen; Jin, Hong; Wang, Wenhai; Yang, Pengyuan

2015-07-01

A systematic proteomic quantification of formalin-fixed, paraffin-embedded (FFPE) colorectal cancer tissues from stage I to stage IIIC was performed in large scale. 1017 proteins were identified with 338 proteins in quantitative changes by label free method, while 341 proteins were quantified with significant expression changes among 6294 proteins by iTRAQ method. We found that proteins related to migration expression increased and those for binding and adherent decreased during the colorectal cancer development according to the gene ontology (GO) annotation and ingenuity pathway analysis (IPA). The integrin alpha 5 (ITA5) in integrin family was focused, which was consistent with the metastasis related pathway. The expression level of ITA5 decreased in metastasis tissues and the result has been further verified by Western blotting. Another two cell migration related proteins vitronectin (VTN) and actin-related protein (ARP3) were also proved to be up-regulated by both mass spectrometry (MS) based quantification results and Western blotting. Up to now, our result shows one of the largest dataset in colorectal cancer proteomics research. Our strategy reveals a disease driven omics-pattern for the metastasis colorectal cancer.

[Prokaryotic expression, purification and antigenicity identification of recombinant human survivin protein].

PubMed

Yin, Xiaotao; Wang, Wei; Tian, Renli; Xu, Yuanji; Yan, Jinqi; Zhang, Wei; Gao, Jiangping; Yu, Jiyun

2013-08-01

To construct a prokaryotic expression plasmid pET28a-survivin, optimize the recombinant protein expression conditions in E.coli, and purify the survivin recombinant protein and identify its antigenicity. Survivin cDNA segment was amplified by PCR and cloned into prokaryotic expression vector pET28a(+) to construct the recombinant expression vector pET28a-survivin. The expression vector was transformed into BL21 (DE3) and the fusion protein survivin/His was induced by IPTG. The fusion protein was purified through Ni affinity chromatography. The antigenicity of the purified survivin protein was identified by Western blotting and ELISA. The recombinant expression vector was verified successfully by BamHI and HindIII. The fusion protein induced by IPTG was obtained with Mr; about 24 000. The purity of the purified protein reached 90% by SDS-PAGE analysis. And the antigenicity of the survivin protein was validated by Western blotting and ELISA. The prokaryotic expression plasmid pET28a-survivin was successfully constructed and the survivin protein was expressed and purified in E.coli. The antigenicity of the purified survivin protein was demonstrated desirable.

Antimetastatic effects of Rheum palmatum L. extract on oral cancer cells.

PubMed

Chen, Yang-Yu; Hsieh, Ming-Ju; Hsieh, Yih-Shou; Chang, Yu-Chao; Chen, Pei-Ni; Yang, Shun-Fa; Ho, Hsin-Yu; Chou, Ying-Erh; Lin, Chiao-Wen

2017-10-01

Rheum palmatum L., a traditional Chinese medication, has been used for the treatment of various disorders. However, the detailed impacts and underlying mechanisms of R. palmatum L. extracts (RLEs) on human oral cancer cell metastasis are still unclear. Here, we tested the hypothesis that an RLE has antimetastatic effects on SCC-9 and SAS human oral cancer cells. Gelatin zymography, Western blot, real-time polymerase chain reaction, and luciferase assay were used to explore the underlying mechanisms involved in the antimetastatic effects on oral cancer cells. Our results revealed that the RLE (up to 20 μg/mL, without cytotoxicity) attenuated SCC-9 and SAS cell motility, invasiveness, and migration by reducing matrix metalloproteinase (MMP)-2 enzyme activities. Western blot analysis of the MAPK signaling pathway indicated that the RLE significantly decreased phosphorylated ERK1/2 levels but not p38 and JNK levels. In conclusion, RLEs exhibit antimetastatic activity against oral cancer cells through the transcriptional repression of MMP-2 via the Erk1/2 signaling pathways. Thus, RLEs may be potentially useful as antimetastatic agents for oral cancer chemotherapy. © 2017 Wiley Periodicals, Inc.

Resveratrol inhibits LPS-induced mice mastitis through attenuating the MAPK and NF-κB signaling pathway.

PubMed

Zhang, Xu; Wang, Yanan; Xiao, Chong; Wei, Zhengkai; Wang, Jingjing; Yang, Zhengtao; Fu, Yunhe

2017-06-01

Resveratrol is a natural polyphenol extracted from mangy plants. It has been reported that resveratrol show multitudinous positive role in biology such as anti-oxidant, anti-nociception and anti-inflammatory effects. Therefore, the present study devotes to test the effect of resveratrol on LPS-induced mastitis in mice. Resveratrol was administered intraperitoneally 1 h before LPS treatment. And the anti-inflammatory effect of resveratrol was measured by histopathological examination, MPO assay, real-time PCR and western blotting analysis. The results showed that resveratrol significantly reduced the LPS-induced mammary histopathological changes. Meanwhile, it sharply attenuated the activity of MPO. The result also indicated that the resveratrol can decrease the expression of pro-inflammatory cytokines TNF-α and IL-1β. From the results of western blotting, resveratrol suppressed the expression of phosphorylation of p65 and IκB from NF-κB signal pathway and phosphorylation of p38 and ERK from MAPK signal pathway. These findings suggested that resveratrol may inhibit the inflammatory response in the mastitis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Proteomic analysis of mature and immature ejaculated spermatozoa from fertile men

PubMed Central

Cui, Zhihong; Sharma, Rakesh; Agarwal, Ashok

2016-01-01

Dysfunctional spermatozoa maturation is the main reason for the decrease in sperm motility and morphology in infertile men. Ejaculated spermatozoa from healthy fertile men were separated into four fractions using three-layer density gradient. Proteins were extracted and bands were digested on a LTQ-Orbitrap Elite hybrid mass spectrometer system. Functional annotations of proteins were obtained using bioinformatics tools and pathway databases. Western blotting was performed to verify the expression levels of the proteins of interest. 1469 proteins were identified in four fractions of spermatozoa. The number of detected proteins decreased according to the maturation level of spermatozoa. During spermatozoa maturation, proteins involved in gamete generation, cell motility, energy metabolism and oxidative phosphorylation processes showed increasing expression levels and those involved in protein biosynthesis, protein transport, protein ubiquitination, and response to oxidative stress processes showed decreasing expression levels. We validated four proteins (HSP 70 1A, clusterin, tektin 2 and tektin 3) by Western blotting. The study shows protein markers that may provide insight into the ejaculated spermatozoa proteins in different stages of sperm maturation that may be altered or modified in infertile men. PMID:26510506

Expression and characterization of human CB1 cannabinoid receptor in methylotrophic yeast Pichia pastoris.

PubMed

Kim, Tae-Kang; Zhang, Rundong; Feng, Wenke; Cai, Jian; Pierce, William; Song, Zhao-Hui

2005-03-01

For the purpose of purification and structural characterization, the CB1 cannabinoid receptors are expressed in methylotrophic yeast Pichia pastoris. The expression plasmid was constructed in which the CB1 gene is under the control of the highly inducible promoter of P. pastoris alcohol oxidase I gene. To facilitate easy detection and purification, a FLAG tag was introduced at the N-terminal, a c-myc epitope and a hexahistidine tag were introduced at the C-terminal of the CB1. In membrane preparations of CB1 gene transformed yeast cells, Western blot analysis detected the expression of CB1 proteins. Radioligand binding assays demonstrated that the tagged CB1 receptors expressed in P. pastoris have a pharmacological profile similar to that of the untagged CB1 receptors expressed in mammalian systems. Furthermore, the tagged CB1 receptors were purified by anti-FLAG M2 affinity chromatography and the identity of the purified CB1 receptor proteins was confirmed by Western blot analysis. MALDI/TOF mass spectrometry analysis of the peptides extracted from tryptic digestions of purified CB1 preparations detected 17 peptide fragments derived from the CB1, thus further confirming the identity of the purified receptor. In conclusion, these data demonstrated for the first time that epitope tagged, functional CB1 cannabinoid receptors can be expressed in P. pastoris for purification and mass spectrometry characterization.

Endothelial nitric oxide synthase expression in systemic and pulmonary circulation of streptozotocin induced diabetic rats: comparison using image analysis.

PubMed

Sridulyakul, P; Chakraphan, D; Bhattarakosol, P; Patumraj, S

2003-01-01

To compare the level of endothelial nitric oxide synthase (eNOS) expression produced in heart and lung vascular tissue, the protein content was determined using Western blot analysis with the enhancement of image processing. Heart and lung extracts from 12 and 24 weeks from control (CON) and streptozotocin-induced diabetic (DM) rats were collected for Western blot analysis. Using monoclonal antibody against rat eNOS protein (140 kDa), the eNOS-protein bands were detected with enhanced chemiluminescence (ECL; Amersham) and exposured to film (Hyperfilm-ECL; Amersham). Images of eNOS bands on each film were then scanned and saved to digital files. Using Global Lab Image software, the number of pixels in each digital file was counted and calibrated for eNOS-protein content. For the CON and DM groups, the mean values of eNOS-protein contents were calculated and expressed as a percentage of total protein content, 5 micrograms. It was found that the eNOS level in DM hearts was significantly decreased, as compared to age-matched CON hearts. On the other hand, eNOS levels in DM lungs was increased, compared to CON lungs. Therefore, it may be concluded that high, not low, flow-mediated eNOS expression is a good measure of hyperglycemic-induced endothelial dysfunction.

Glyceraldehyde-3-phosphate dehydrogenase: a universal internal control for Western blots in prokaryotic and eukaryotic cells.

PubMed

Wu, Yonghong; Wu, Min; He, Guowei; Zhang, Xiao; Li, Weiguang; Gao, Yan; Li, Zhihui; Wang, Zhaoyan; Zhang, Chenggang

2012-04-01

In the current study, we examined the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in a number of organisms and the stability of GAPDH under various conditions. Our results revealed that GAPDH is present in multiple Escherichia coli strains, the yeast strain GS115, Caenorhabditis elegans, rat PC12 cells, and both mouse and rat brain. Furthermore, GAPDH was stably expressed under different concentrations of inducer and at different times of induction in E. coli (BL21) cells and yeast GS115 cells. Stable expression of GAPDH protein was also observed in C.elegans and PC12 cells that were treated with different concentrations of paraquat or sodium sulfite, respectively. In addition, we were able to detect and identify the endogenous gapA protein in E.coli via immunoprecipitation and MALDI-TOF-MS analysis. Endogenous gapA protein and exogenously expressed (subcloned) GAPDH proteins were detected in E. coli BL21 but not for gapC. With the exception of gapC in E. coli, the various isoforms of GAPDH possessed enzymatic activity. Finally, sequence analysis revealed that the GAPDH proteins were 76% identical, with the exception of E. coli gapC. Taken together, our results indicate that GAPDH could be universally used as an internal control for the Western blot analysis of prokaryotic and eukaryotic samples. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

CNS development under altered gravity: cerebellar glial and neuronal protein expression in rat neonates exposed to hypergravity

NASA Astrophysics Data System (ADS)

Nguon, K.; Li, G.-H.; Sajdel-Sulkowska, E. M.

2004-01-01

The future of space exploration depends on a solid understanding of the developmental process under microgravity, specifically in relation to the central nervous system (CNS). We have previously employed a hypergravity paradigm to assess the impact of altered gravity on the developing rat cerebellum [Exp. Biol. Med. 226 (2000) 790]. The present study addresses the molecular mechanisms involved in the cerebellar response to hypergravity. Specifically, the study focuses on the expression of selected glial and neuronal cerebellar proteins in rat neonates exposed to hypergravity (1.5 G) from embryonic day (E)11 to postnatal day (P)6 or P9 (the time of maximal cerebellar changes) comparing them against their expression in rat neonates developing under normal gravity. Proteins were analyzed by quantitative Western blots of cerebellar homogenates; RNA analysis was performed in the same samples using quantitative PCR. Densitometric analysis of Western blots suggested a reduction in glial (glial acidic protein, GFAP) and neuronal (neuronal cell adhesion moiecule, NCAM-L1, synaptophysin) proteins, but the changes in individual cerebellar proteins in hypergravity-exposed neonates appeared both age- and gender-specific. RNA analysis suggested a reduction in GFAP and synaptophysin mRNAs on P6. These data suggest that exposure to hypergravity may interfere with the expression of selected cerebellar proteins. These changes in protein expression may be involved in mediating the effect of hypergravity on the developing rat cerebellum.

Chronic corticosterone administration reduces dendritic complexity in mature, but not young granule cells in the rat dentate gyrus.

PubMed

Yau, Suk-Yu; Li, Ang; Tong, Jian-Bin; Bostrom, Crystal; Christie, Brian R; Lee, Tatia M C; So, Kwok-Fai

2016-09-21

Our previous work has shown that exposure to the stress hormone corticosterone (40 mg/kg CORT) for two weeks induces dendritic atrophy of pyramidal neurons in the hippocampal CA3 region and behavioral deficits. However, it is unclear whether this treatment also affects the dentate gyrus (DG), a subregion of the hippocampus comprising a heterogeneous population of young and mature neurons. We examined the effect of CORT treatment on the dendritic complexity of mature and young granule cells in the DG. We utilized a Golgi staining method to investigate the dendritic morphology and spine density of young neurons in the inner granular cell layer (GCL) and mature neurons in the outer GCL in response to CORT application. The expressions of glucocorticoid receptors during neuronal maturation were examined using Western blot analysis in a primary hippocampal neuronal culture. Sholl analysis revealed that CORT treatment decreased the number of intersections and shortened the dendritic length in mature, but not young, granule cells. However, the spine density of mature and young neurons was not affected. Western blot analysis showed a progressive increase in the protein levels of glucocorticoid receptors (GRs) in the cultured primary hippocampal neurons during neuronal maturation. These data suggest that mature neurons are likely more vulnerable to chronic exposure to CORT; this may be due to their higher expression of GRs when compared to younger DG neurons.

N-nitrosations of basic amino acid residues in polypeptide.

PubMed

Kuo, Wu-Nan; Ivy, Dynisha; Guruvadoo, Luvina; White, Atavia; Graham, Latia

2004-09-01

Changes in the electrophoretic pattern were noted in the products of polypeptides of identical basic amino acids preincubated with reactive or degraded PN, suggesting the occurrence of N-nitrosation of the epsilon-amino group of lysine, the guanido group of arginine and the imidazole group of histidine. Additionally, increase in the N-nitroso immunoreactivity of preincubated histones H2A and H2B was detected by Western blot analysis.

Protein Changes in Macrophages Induced by Plasma from Rats Exposed to 35-GHz Millimeter Waves

DTIC Science & Technology

2010-12-01

HumanEffectiveness Directorate, Air Force Research Laboratory, Brooks City-Base,Texas A macrophage assay and proteomic screening were used to...mW/cm2 until core temperature reached 41.0 8C. Two-dimensional polyacrylamide gel electrophoresis, image analysis, and Western blotting were used to...stimulation. Proteins of interest were identified using peptide mass fingerprinting. Compared to plasma from sham- exposed rats, plasma from

Screening of Organophosphorus Acid Anhydrases from different Sources by Western Blot Analysis

DTIC Science & Technology

1991-01-01

agents) have been found3 in both procaryotic and eukaryotic organisms. These enzymes, referred to as organophosphorous acid anhydrases (OPA anhydrases...of organisms, both procaryotic and eukaryotic. Previously, these enzymes were known as DFPases, somanases, paraoxonases or parathion hydrolases, based...cell (SLM Aminco) at 16,000 psi. Cellular debris was removed by centrifugation (46,000xg) for 30 minutes at 40C. The crude cell supernatant which

Acid-growth response and alpha-expansins in suspension cultures of bright yellow 2 tobacco

NASA Technical Reports Server (NTRS)

Link, B. M.; Cosgrove, D. J.

1998-01-01

The possibility that Bright Yellow 2 (BY2) tobacco (Nicotiana tabacum L.) suspension-cultured cells possess an expansin-mediated acid-growth mechanism was examined by multiple approaches. BY2 cells grew three times faster upon treatment with fusicoccin, which induces an acidification of the cell wall. Exogenous expansins likewise stimulated BY2 cell growth 3-fold. Protein extracted from BY2 cell walls possessed the expansin-like ability to induce extension of isolated walls. In western-blot analysis of BY2 wall protein, one band of 29 kD was recognized by anti-expansin antibody. Six different classes of alpha-expansin mRNA were identified in a BY2 cDNA library. Northern-blot analysis indicated moderate to low abundance of multiple alpha-expansin mRNAs in BY2 cells. From these results we conclude that BY2 suspension-cultured cells have the necessary components for expansin-mediated cell wall enlargement.

Secretagogin is a novel marker for neuroendocrine differentiation.

PubMed

Birkenkamp-Demtröder, Karin; Wagner, Ludwig; Brandt Sørensen, Flemming; Bording Astrup, Lone; Gartner, Wolfgang; Scherübl, Hans; Heine, Bernhard; Christiansen, Peer; Ørntoft, Torben Falck

2005-01-01

Our previous microarray-based studies identified secretagogin to be highly expressed in normal colon mucosa compared to basal expression in colon adenocarcinomas. The aim of this study was to analyze the differential expression of secretagogin in normal mucosa, adenocarcinomas, and neuroendocrine tumors. Western blotting, immunohistochemistry, immunofluorescence microscopy and ELISA were applied. Western blot analysis detected a 32-kDa secretagogin band in samples from normal mucosa. Immunohistochemical analyses on tissue specimens showed that secretagogin is exclusively expressed in neuroendocrine cells and nerve cells in normal mucosa of the digestive tract. Tissues adjacent to benign hyperplasic polyps and adenomas showed a decreased number of secretagogin-expressing neuroendocrine cells. Secretagogin co-localized with neuroendocrine markers (chromogranin A, neuron-specific enolase, synaptophysin) in neuroendocrine cells in crypts of normal mucosa, and in tumor cells of carcinoids. Secretagogin was strongly expressed in the cytosol and the nucleus of 19 well-differentiated neuroendocrine carcinoids and carcinoid metastases, as well as in neuroendocrine tumors from the lung, pancreas and adrenal gland. Secretagogin was detected in plasma from carcinoid patients with distant metastasis. Combined immunohistochemical analysis of secretagogin and FK506-binding protein 65, a protein de novo synthesized in adenocarcinomas, distinguished well-differentiated carcinoids, adenocarcinoids and undifferentiated carcinomas. We conclude that secretagogin is a novel marker for neuroendocrine differentiation.

Comparative proteomics of umbilical vein blood plasma from normal and gestational diabetes mellitus patients reveals differentially expressed proteins associated with childhood obesity.

PubMed

Miao, Zhijing; Wang, Jianqing; Wang, Fuqiang; Liu, Lan; Ding, Hongjuan; Shi, Zhonghua

2016-11-01

Offspring obesity is one of long-term complications of gestational diabetes mellitus (GDM). The aim of this study is to identify proteins differentially expressed in the umbilical vein blood plasma, which could become markers for early diagnosis of childhood obesity. Umbilical vein plasma samples were collected from 30 control and 30 GDM patients in 2007-2008 whose offspring were suffering from obesity at 6-7 years old. Multiplexed isobaric tandem mass tag labeling combined with LC-MS/MS was used to identify differentially expressed proteins. Ingenuity pathway analysis was performed to identify canonical pathways, biological functions, and networks of interacting proteins. Western blotting was used to verify the expression of three selected proteins. A total of 318 proteins were identified, of which 12 proteins were upregulated in GDM group while 24 downregulated. Lipid metabolism was the top category identified by ingenuity pathway analysis. Three randomly chosen proteins were validated by Western blotting, which were consistent with LC-MS. There are significant differences of protein profile in the umbilical vein blood plasma between normal and GDM patients with obese offspring. The results indicate that a variety of proteins and biological mechanisms may contribute to childhood obesity. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Advanced glycation end products promote ChREBP expression and cell proliferation in liver cancer cells by increasing reactive oxygen species.

PubMed

Chen, Hanbei; Li, Yakui; Zhu, Yemin; Wu, Lifang; Meng, Jian; Lin, Ning; Yang, Dianqiang; Li, Minle; Ding, WenJin; Tong, Xuemei; Su, Qing

2017-08-01

The aim of the study was to elucidate the mechanism by which advanced glycation end products (AGEs) promote cell proliferation in liver cancer cells.We treated liver cancer HepG2 cells with 200 mg/L AGEs or bovine serum albumin (BSA) and assayed for cell viability, cell cycle, and apoptosis. We performed real-time PCR and Western blot analysis for RNA and protein levels of carbohydrate responsive element-binding protein (ChREBP) in AGEs- or BSA-treated HepG2 cells. We analyzed the level of reactive oxygen species (ROS) in HepG2 cells treated with AGEs or BSA.We found that increased S-phase cell percentage and decreased apoptosis contributed to AGEs-induced liver cancer cell proliferation. Real-time PCR and Western blot analysis showed that AGEs stimulated RNA and protein levels of ChREBP, a transcription factor promoting glycolysis and maintaining cell proliferation in liver cancer cells. Intriguingly, the level of ROS was higher in AGEs-treated liver cancer cells. Treating liver cancer cells with antioxidant N-acetyl cystein (NAC) partly blocked AGEs-induced ChREBP expression and cell proliferation.Our results suggest that the AGEs-ROS-ChREBP pathway plays a critical role in promoting ChREBP expression and liver cancer cell proliferation.

Solanine induced apoptosis and increased chemosensitivity to Adriamycin in T-cell acute lymphoblastic leukemia cells.

PubMed

Yi, Ying-Jie; Jia, Xiu-Hong; Wang, Jian-Yong; Chen, Jie-Ru; Wang, Hong; Li, You-Jie

2018-05-01

Solanine is an alkaloid and is the main extract of the traditional Chinese herb, Solanum nigrum Linn . It has been reported that Solanine has anti-inflammatory and antitumor properties. The present study aimed to investigate the antitumor effect of Solanine in Jurkat cells and demonstrate the molecular mechanism of antitumor activity of Solanine. A Cell Counting Kit-8 assay demonstrated that Solanine inhibited the proliferation of Jurkat cells in a dose-and time-dependent manner. Cell apoptosis was measured by flow cytometry. Flow cytometry revealed that Solanine induced apoptosis in a dose-dependent manner in Jurkat cells. Reverse transcription-quantitative polymerase chain reaction demonstrated that Solanine modulated the mRNA levels of B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax). Additionally, Bcl-2 and Bax expression was measured using western blot analysis. Western blot analysis revealed a significant increase in the expression of Bax and decrease in the expression of Bcl-2. Solanine increased the chemosensitivity of Jurkat cells to Adriamycin. In summary, the present results indicated that the antitumor activity of Solanine was associated with inhibition of cell proliferation, induction of apoptosis and increasing cytotoxicity of Adriamycin. Therefore, Solanine may have potential as a novel agent for the treatment of acute lymphocytic leukemia.

Fisetin Protects against Intracerebral Hemorrhage-Induced Neuroinflammation in Aged Mice.

PubMed

Chen, Cheng; Yao, Li; Cui, Jing; Liu, Bao

2018-01-01

Fisetin is commonly used as an anti-inflammatory and neuroprotective drug. In this study, we aimed to investigate the efficacy of fisetin in alleviating intracerebral hemorrhage (ICH)-induced brain injury. Mouse ICH models were constructed using the collagenase-induction method. ICH mice received fisetin treatment at the dose of 10-90 mg/kg, followed by the evaluation of neurological deficit through neurologic severity scores (mNSS), brain water content and terminal deoxynucleotidyl transferase dUTP nick end labeling analysis of cell apoptosis. Cytokine levels were also assessed with enzyme-linked immunosorbent assay. The activation of astrocytes and microglia was evaluated through S100 staining and Western blot analysis of ionized calcium-binding adaptor molecule 1 respectively. Nuclear factor kappa-B (NF-κB) signaling was also evaluated by Western blot. ICH mice demonstrated dramatic increase in mNSS, brain edema and cell apoptosis, indicating severe brain deficit. Fisetin treatment lowered these parameters, suggesting the alleviation of brain injury. Levels of proinflammatory cytokines were reduced, accompanied by a prominent decrease in activated astrocytes and microglia. NF-κB signaling was also attenuated by fisetin treatment. Fisetin effectively alleviates ICH by downregulating proinflammatory cytokines and attenuating NF-κB signaling. These data suggest fisetin as a valuable natural flavonol for clinical management of ICH-induced brain injury. © 2018 S. Karger AG, Basel.

Ferritin L and Ferritin H are differentially located within hepatic and extra hepatic organs under physiological and acute phase conditions.

PubMed

Ahmad, Shakil; Moriconi, Federico; Naz, Naila; Sultan, Sadaf; Sheikh, Nadeem; Ramadori, Giuliano; Malik, Ihtzaz Ahmed

2013-01-01

Ferritin L (FTL) and Ferritin H (FTH) subunits are responsible for intercellular iron storage. We previously reported increasing amounts of liver cytoplasmic and nuclear iron content during acute phase response (APR). Aim of the present study is to demonstrate intracellular localization of ferritin subunits in liver compared with extra hepatic organs of rat under physiological and acute phase conditions. Rats were administered turpentine-oil (TO) intramuscularly to induce a sterile abscess (acute-phase-model) and sacrificed at different time points. Immunohistochemistry was performed utilizing horse-reddish-peroxidise conjugated secondary antibody on 4μm thick section. Liver cytoplasmic and nuclear protein were used for Western blot analysis. By means of immunohistology, FTL was detected in cytoplasm while a strong nuclear positivity for FTH was evident in the liver. Similarly, in heart, spleen and brain FTL was detected mainly in the cytoplasm while FTH demonstrated intense nuclear and a weak cytoplasmic expression. Western blot analysis of cytoplasmic and nuclear fractions from liver, heart, spleen and brain further confirmed mainly cytoplasmic expression of FTL in contrast to the nuclear and cytoplasmic expression of FTH. The data presented demonstrate the differential localization of FTL and FTH within hepatic and extra hepatic organs being FTL predominantly in the cytoplasm while FTH predominantly in nucleus.

miR-340 alleviates chemoresistance of osteosarcoma cells by targeting ZEB1.

PubMed

Yan, Haibin; Zhang, Bingyun; Fang, Chongbin; Chen, Liqiu

2018-06-01

Chemoresistance during treatment of osteosarcoma (OS) is attracting more and more attention as the main clinical obstacle. The purpose of this study was to elucidate the role of miR-340 in chemoresistance of OS. Plasmid construction and transfection, miRNA arrays, PCR analyses, and western blot analysis, as well as MTT, apoptosis, and luciferase assays were carried out in MG-63 cells and MG-63/cisplatin (DDP)-resistant cells. The results showed that miR-340 was downregulated in OS tissues and drug-resistant OS cells. Moreover, a negative correlation was observed between miR-340 and ZEB1 expression in OS tissues. Forced expression of miR-340 in drug-resistant OS cells significantly reduced multidrug resistance-1 and P-gp expression. Overexpression of miR-340 enhanced sensitivity to DDP by inhibiting viability and promoting apoptosis. The luciferase assay and western blot analysis identified ZEB1 as a direct target of miR-340, and miR-340 negatively regulated ZEB1 expression. Ectopic expression of ZEB1 reversed the effects of miR-340 on P-gp expression, cell viability, and apoptosis. miR-340 alleviated chemoresistance of OS cells by targeting ZEB1. Our results indicate that targeting miR-340 may be a potential therapeutic approach to treat drug-resistant OS.

Quantitative protein expression analysis of CLL B cells from mutated and unmutated IgV(H) subgroups using acid-cleavable isotope-coded affinity tag reagents.

PubMed

Barnidge, David R; Jelinek, Diane F; Muddiman, David C; Kay, Neil E

2005-01-01

Relative protein expression levels were compared in leukemic B cells from two patients with chronic lymphocytic leukemia (CLL) having either mutated (M-CLL) or unmutated (UM-CLL) immunoglobulin variable heavy chain genes (IgV(H)). Cells were separated into cytosol and membrane protein fractions then labeled with acid-cleavable ICAT reagents (cICAT). Labeled proteins were digested with trypsin then subjected to SCX and affinity chromatography followed by LC-ESI-MS/MS analysis on a linear ion trap mass spectrometer. A total of 9 proteins from the cytosol fraction and 4 from the membrane fraction showed a 3-fold or greater difference between M-CLL and UM-CLL and a subset of these were examined by Western blot where results concurred with cICAT abundance ratios. The abundance of one of the proteins in particular, the mitochondrial membrane protein cytochrome c oxidase subunit COX G was examined in 6 M-CLL and 6 UM-CLL patients using western blot and results showed significantly greater levels (P < 0.001) in M-CLL patients vs UM-CLL patients. These results demonstrate that stable isotope labeling and mass spectrometry can complement 2D gel electrophoresis and gene microarray technologies for identifying putative and perhaps unique prognostic markers in CLL.

Carbonic anhydrase I in a cartilaginous fish, the shortspine spurdog ( Squalus mitsukurii)

NASA Astrophysics Data System (ADS)

Kim, Soo Cheol; Sumi, Kanij Rukshana; Kim, Jung Woo; Choi, Myeong Rak; Min, Byung Hwa; Kho, Kang Hee

2016-09-01

Carbonic anhydrase (CA), a ubiquitous enzyme found in many species, including fishes, is involved in physiological functions such as pH homeostasis, calcification, photosynthesis, and ionic regulation. CA I, a member of the α-CA family, is a cytoplasmic isozyme involved in carbon dioxide transport, ion exchange, and acid-base balance. Approximately half of the extant shark species occur only in deep waters; however, few published studies on sharks include these taxa. As fisheries worldwide enter deeper waters, the provision of biological data for these little-known taxa is critical to their management and conservation. To address this limitation, we aimed to detect CA I in various tissues of the shortspine spurdog ( Squalus mitsukurii) and characterize its physicochemical properties by using sodium dodecyl-sulfate polyacrylamide gel electrophoresis and isoelectric focusing, together with immunohistochemistry. CA I was detected on SDS-PAGE and western blot analysis as a specific band at 29 kDa in various tissues of the shortspine spurdog, and as a specific band at pI 6.5 in various tissues of the shortspine spurdog by IEF and western blot analysis. CA I immunoreactivity in various tissues of the shortspine spurdog was detected in intracellular locations. To our knowledge, this is the first report of the localization of CA isozymes in various tissues of S. mitsukurii.

EB1 protein alteration characterizes sporadic but not ulcerative colitis associated colorectal cancer.

PubMed

Gemoll, Timo; Kollbeck, Sophie L; Karstens, Karl F; Hò, Gia G; Hartwig, Sonja; Strohkamp, Sarah; Schillo, Katharina; Thorns, Christoph; Oberländer, Martina; Kalies, Kathrin; Lehr, Stefan; Habermann, Jens K

2017-08-15

While carcinogenesis in Sporadic Colorectal Cancer (SCC) has been thoroughly studied, less is known about Ulcerative Colitis associated Colorectal Cancer (UCC). This study aimed to identify and validate differentially expressed proteins between clinical samples of SCC and UCC to elucidate new insights of UCC/SCC carcinogenesis and progression. Multiplex-fluorescence two-dimensional gel electrophoresis (2-D DIGE) and mass spectrometry identified 67 proteoforms representing 43 distinct proteins. After analysis by Ingenuity Pathway Analysis ® (IPA), subsequent Western blot validation proofed the differential expression of Heat shock 27 kDA protein 1 (HSPB1) and Microtubule-associated protein R/EB family, member 1 (EB1) while the latter one showed also expression differences by immunohistochemistry. Fresh frozen tissue of UCC ( n = 10) matched with SCC ( n = 10) was investigated. Proteins of cancerous intestinal mucosal cells were obtained by Laser Capture Microdissection (LCM) and compared by 2-D DIGE. Significant spots were identified by mass spectrometry. After IPA, three proteins [EB1, HSPB1, and Annexin 5 (ANXA5)] were chosen for further validation by Western blotting and tissue microarray-based immunohistochemistry. This study identified significant differences in protein expression of colorectal carcinoma cells from UCC patients compared to patients with SCC. Particularly, EB1 was validated in an independent clinical cohort.

Endoplasmic reticulum stress induces secretion of high-mobility group proteins and is associated with tumor-infiltrating lymphocytes in triple-negative breast cancer

PubMed Central

Park, In Ah; Heo, Sun-Hee; Song, In Hye; Kim, Young-Ae; Park, Hye Seon; Bang, Won Seon; Park, Suk Young; Jo, Jeong-Hyon; Lee, Hee Jin; Gong, Gyungyub

2016-01-01

Background Although the prognostic and predictive significance of tumor-infiltrating lymphocytes (TILs) in triple-negative breast cancer (TNBC) have been shown, the cause of the TIL influx is unclear. Here, we investigated whether extracellular secretion of HMGN1 is associated with TIL influx, as well as increased endoplasmic reticulum stress (ERS), in human TNBC. Methods We reviewed the slides of 767 patients with TNBC and evaluated the TIL levels. We also assessed the expression of HMGs and several ERS-associated molecules using immunohistochemical staining. Western blot analysis of human TNBC cell lines and pharmacological ERS inducers was used to determine if HMGN1 migrates from the nucleus to the extracellular space in response to ERS. Results On immunohistochemical staining, either higher nuclear or cytoplasmic expression of both HMGB1 and HMGN1 was significantly associated with ERS. TILs showed a positive correlation with the cytoplasmic expression of the HMGs. Western blot analysis of TNBC cell lines showed that ERS induction resulted in the secretion of HMG proteins. Conclusions This is the first study to elucidate the associations among ERS, secretion of HMGs, and degree of TILs in TNBCs. Understanding the mechanisms of TIL influx will help in the development of effective immunotherapeutic agents for TNBC. PMID:27494867

Network Analysis of Epidermal Growth Factor Signaling Using Integrated Genomic, Proteomic and Phosphorylation Data

PubMed Central

Waters, Katrina M.; Liu, Tao; Quesenberry, Ryan D.; Willse, Alan R.; Bandyopadhyay, Somnath; Kathmann, Loel E.; Weber, Thomas J.; Smith, Richard D.; Wiley, H. Steven; Thrall, Brian D.

2012-01-01

To understand how integration of multiple data types can help decipher cellular responses at the systems level, we analyzed the mitogenic response of human mammary epithelial cells to epidermal growth factor (EGF) using whole genome microarrays, mass spectrometry-based proteomics and large-scale western blots with over 1000 antibodies. A time course analysis revealed significant differences in the expression of 3172 genes and 596 proteins, including protein phosphorylation changes measured by western blot. Integration of these disparate data types showed that each contributed qualitatively different components to the observed cell response to EGF and that varying degrees of concordance in gene expression and protein abundance measurements could be linked to specific biological processes. Networks inferred from individual data types were relatively limited, whereas networks derived from the integrated data recapitulated the known major cellular responses to EGF and exhibited more highly connected signaling nodes than networks derived from any individual dataset. While cell cycle regulatory pathways were altered as anticipated, we found the most robust response to mitogenic concentrations of EGF was induction of matrix metalloprotease cascades, highlighting the importance of the EGFR system as a regulator of the extracellular environment. These results demonstrate the value of integrating multiple levels of biological information to more accurately reconstruct networks of cellular response. PMID:22479638

Is Oxidative Stress in Mice Brain Regions Diminished by 2-[(2,6-Dichlorobenzylidene)amino]-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carbonitrile?

PubMed Central

Fortes, A. C.; Almeida, A. A. C.; Oliveira, G. A. L.; Santos, P. S.; De Lucca Junior, W.; Mendonça Junior, F. J. B.; Freitas, R. M.; Soares-Sobrinho, J. L.; Soares, M. F. R.

2013-01-01

2-[(2,6-Dichlorobenzylidene)amino]-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carbonitrile, 5TIO1, is a new 2-aminothiophene derivative with promising pharmacological activities. The aim of this study was to evaluate its antioxidant activity in different areas of mice central nervous system. Male Swiss adult mice were intraperitoneally treated with Tween 80 dissolved in 0.9% saline (control group) and 5TIO1 (0.1, 1, and 10 mg kg−1). Brain homogenates—hippocampus, striatum, frontal cortex, and cerebellum—were obtained after 24 h of observation. Superoxide dismutase and catalase activities, lipid peroxidation and nitrite content were measured using spectrophotometrical methods. To clarify the 5TIO1's mechanism on oxidative stress, western blot analysis of superoxide dismutase and catalase was also performed. 5TIO1 decreased lipid peroxidation and nitrite content in all brain areas and increased the antioxidant enzymatic activities, specially, in cerebellum. The data of Western blot analysis did not demonstrate evidence of the upregulation of these enzymes after the administration of this compound. Our findings strongly support that 5TIO1 can protect the brain against neuronal damages regularly observed during neuropathologies. PMID:23577220

Parkin overexpression protects retinal ganglion cells against glutamate excitotoxicity.

PubMed

Hu, Xinxin; Dai, Yi; Sun, Xinghuai

2017-01-01

To investigate the role of parkin in regulating mitochondrial homeostasis of retinal ganglion cells (RGCs) under glutamate excitotoxicity. Rat RGCs were purified from dissociated retinal tissue with a modified two-step panning protocol. Cultured RGCs were transfected with parkin using an adenovirus system. The distribution and morphology of mitochondria in the RGCs were assessed with MitoTracker. The expression and distribution of parkin and optineurin proteins were measured with western blot analysis and immunofluorescence. Cytotoxicity of RGCs was evaluated by measuring lactate dehydrogenase (LDH) activity. Mitochondrial membrane potential was determined with the JC-1 assay. The expression of Bax and Bcl-2 were measured with western blot analysis. In the presence of glutamate-induced excitotoxicity, the number of mitochondria in the axons of the RGCs was predominantly increased, and the mitochondrial membrane potential in RGCs was depolarized. The expression of the parkin and optineurin proteins was upregulated and distributed mostly in the axons of the RGCs. Overexpression of parkin stabilized the mitochondrial membrane potential of RGCs, decreased cytotoxicity and apoptosis, attenuated the expression of Bax, and promoted the expression of optineurin under glutamate excitotoxicity. Overexpression of parkin exerted a significant protective effect on cultured RGCs against glutamate excitotoxicity. Interventions to alter the parkin-mediated mitochondria pathway may be useful in protecting RGCs against excitotoxic RGC damage.

Periostin, a matrix specific protein, is associated with proliferation and invasion of pancreatic cancer.

PubMed

Ben, Qi-Wen; Jin, Xiao-Long; Liu, Jun; Cai, Xia; Yuan, Fei; Yuan, Yao-Zong

2011-03-01

Overexpression of periostin is present in various malignant tumors and correlates with disease progression. However, its clinicopathological significance in pancreatic cancer is currently not known. Expression of periostin was analyzed by RT-PCR and western blotting in pancreatic cancers and cell lines. Using immunohistochemistry, expression of periostin in pancreatic cancers was evaluated according to factors influencing overall survival with Kaplan-Meier analysis. Ectopic expression of periostin was used to examine the effects of periostin on proliferation and invasiveness of pancreatic cancer cells in vitro. There was no detectable periostin mRNA and protein expression in the 4 pancreatic cell lines. Expression of periostin was found to be up-regulated in pancreatic cancer compared to the adjacent tumor free (TF) tissues by western blotting. The positive ratio of periostin expression in the neoplastic stroma was significantly correlated with the depth of invasion (p=0.007) and lymph node metastasis (p=0.027). Survival analysis showed that stromal or epithelium expression of periostin was associated with poor survival (p=0.035, p=0.022, log-rank test, respectively). In vitro studies showed that periostin was able to promote proliferation and invasiveness of pancreatic cancer cells. These results suggest that periostin may be involved in the progression and invasion of pancreatic cancer.

A modified method using the SonoPrep ultrasonic skin permeation system for sampling human interstitial fluid is compatible with proteomic techniques.

PubMed

Lecomte, Marie M J; Atkinson, Kelly R; Kay, Daniel P; Simons, Joanne L; Ingram, John R

2013-02-01

The use of biomarkers in skin is a novel diagnostic tool. Interstitial fluid (ISF) from skin provides a snapshot of proteins secreted at the time of sampling giving insights into the patient's health status. A minimally invasive technique for the transdermal collection of human ISF proteins. A low frequency ultrasonic skin permeation device (SonoPrep ultrasonic skin permeation system) was used to produce micropores in the stratum corneum through which ISF was extracted using a portable pulsed vacuum ISF collection device. On average, protein concentrations recovered ranged between 0.064 and 4.792 μg/μL (mean 1.258 μg/μL). Two-dimensional gel electrophoresis revealed that this sample type was amenable to this type of analysis. Gel images indicated that both highly abundant proteins and lower abundance proteins were isolated from the skin. Western blot analysis confirmed the presence of proteins commonly found in plasma and the epidermis. A minimally invasive method for the transdermal recovery of ISF proteins has been developed. We have demonstrated that ISF samples obtained using this approach can be analysed with proteomic techniques, such as two-dimensional gel electrophoresis and western blots, providing another tool for the identification of disease specific protein biomarkers. © 2012 John Wiley & Sons A/S.

Differential Gene Expression in Primary Human Skin Keratinocytes and Fibroblasts in Response to Ionizing Radiation

PubMed Central

Warters, Raymond L.; Packard, Ann T.; Kramer, Gwen F.; Gaffney, David K.; Moos, Philip J.

2009-01-01

Although skin is usually exposed during human exposures to ionizing radiation, there have been no thorough examinations of the transcriptional response of skin fibroblasts and keratinocytes to radiation. The transcriptional response of quiescent primary fibroblasts and keratinocytes exposed to from 10 cGy to 5 Gy and collected 4 h after treatment was examined. RNA was isolated and examined by microarray analysis for changes in the levels of gene expression. Exposure to ionizing radiation altered the expression of 279 genes across both cell types. Changes in RNA expression could be arranged into three main categories: (1) changes in keratinocytes but not in fibroblasts, (2) changes in fibroblasts but not in keratinocytes, and (3) changes in both. All of these changes were primarily of p53 target genes. Similar radiation-induced changes were induced in immortalized fibroblasts or keratinocytes. In separate experiments, protein was collected and analyzed by Western blotting for expression of proteins observed in microarray experiments to be overexpressed at the mRNA level. Both Q-PCR and Western blot analysis experiments validated these transcription changes. Our results are consistent with changes in the expression of p53 target genes as indicating the magnitude of cell responses to ionizing radiation. PMID:19580510

Comparative Depigmentation Effects of Resveratrol and Its Two Methyl Analogues in α-Melanocyte Stimulating Hormone-Triggered B16/F10 Murine Melanoma Cells

PubMed Central

Yoon, Hoon-Seok; Hyun, Chang-Gu; Lee, Nam-Ho; Park, Sung-Soo; Shin, Dong-Bum

2016-01-01

Previous research showed that resveratrol (trans-3,4′,5-trihydroxystilbene) and pinostilbene (trans-3-methoxy-4′,5-dihydroxystilbene) were able to inhibit tyrosinase directly; however, anti-melanogenic effects of pterostilbene (trans-3,5-dimethoxy-4′-hydroxystilbene) and resveratrol trimethyl ether (RTE) have not been compared. To investigate the hypopigmentation effects of pterostilbene and RTE, melanin contents and intracellular tyrosinase activity were determined by western blot analysis. Firstly, pterostilbene showed the inhibitory effects on α-melanocyte stimulating hormone (MSH)-induced melanin synthesis stronger than RTE, resveratrol, and arbutin. Pterostilbene inhibited melanin biosynthesis in a dose-dependent manner in α-MSH-stimulated B16/F10 murine melanoma cells. Specifically, melanin content and intracellular tyrosinase activity were inhibited by 63% and 58%, respectively, in response to treatment with 10 μM of pterostilbene. The results of western blot analysis indicated that pterostilbene induced downregulation of tyrosinase protein expression and suppression of α-MSH-stimulated melan-A protein expression stronger than RTE or resveratrol. Based on these results, our study suggests that pterostilbene can induce hypopigmentation effects more effectively than resveratrol and RTE, and it functions via downregulation of protein expression associated with hyperpigmentation in α-MSH-triggered B16/F10 murine melanoma cells. PMID:27390733

Expression of Vesicular Glutamate Transporters VGLUT1 and VGLUT2 in the Rat Dental Pulp and Trigeminal Ganglion following Inflammation

PubMed Central

Hong, Jae Hyun; Kim, Yun Sook; Choi, So Young; Kim, Tae Heon; Cho, Yi Sul; Bae, Yong Chul

2014-01-01

Background There is increasing evidence that peripheral glutamate signaling mechanism is involved in the nociceptive transmission during pathological conditions. However, little is known about the glutamate signaling mechanism and related specific type of vesicular glutamate transporter (VGLUT) in the dental pulp following inflammation. To address this issue, we investigated expression and protein levels of VGLUT1 and VGLUT2 in the dental pulp and trigeminal ganglion (TG) following complete Freund’s adjuvant (CFA) application to the rat dental pulp by light microscopic immunohistochemistry and Western blot analysis. Results The density of VGLUT2− immunopositive (+) axons in the dental pulp and the number of VGLUT2+ soma in the TG increased significantly in the CFA-treated group, compared to control group. The protein levels of VGLUT2 in the dental pulp and TG were also significantly higher in the CFA-treated group than control group by Western blot analysis. The density of VGLUT1+ axons in the dental pulp and soma in the TG remained unchanged in the CFA-treated group. Conclusions These findings suggest that glutamate signaling that is mediated by VGLUT2 in the pulpal axons may be enhanced in the inflamed dental pulp, which may contribute to pulpal axon sensitization leading to hyperalgesia following inflammation. PMID:25290694

Expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in the rat dental pulp and trigeminal ganglion following inflammation.

PubMed

Yang, Eun Sun; Jin, Myoung Uk; Hong, Jae Hyun; Kim, Yun Sook; Choi, So Young; Kim, Tae Heon; Cho, Yi Sul; Bae, Yong Chul

2014-01-01

There is increasing evidence that peripheral glutamate signaling mechanism is involved in the nociceptive transmission during pathological conditions. However, little is known about the glutamate signaling mechanism and related specific type of vesicular glutamate transporter (VGLUT) in the dental pulp following inflammation. To address this issue, we investigated expression and protein levels of VGLUT1 and VGLUT2 in the dental pulp and trigeminal ganglion (TG) following complete Freund's adjuvant (CFA) application to the rat dental pulp by light microscopic immunohistochemistry and Western blot analysis. The density of VGLUT2- immunopositive (+) axons in the dental pulp and the number of VGLUT2+ soma in the TG increased significantly in the CFA-treated group, compared to control group. The protein levels of VGLUT2 in the dental pulp and TG were also significantly higher in the CFA-treated group than control group by Western blot analysis. The density of VGLUT1+ axons in the dental pulp and soma in the TG remained unchanged in the CFA-treated group. These findings suggest that glutamate signaling that is mediated by VGLUT2 in the pulpal axons may be enhanced in the inflamed dental pulp, which may contribute to pulpal axon sensitization leading to hyperalgesia following inflammation.

Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts

PubMed Central

2013-01-01

Background Osteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for development of novel diagnostic, prognostic, and therapeutic tools. The surface-exposed proteome (SEP) of a cancerous cell includes a multifarious array of proteins critical to cellular processes such as proliferation, migration, adhesion, and inter-cellular communication. The specific aim of this study was to define a SEP profile of two validated canine OSA cell lines and a normal canine osteoblast cell line utilizing a biotinylation/streptavidin system to selectively label, purify, and identify surface-exposed proteins by mass spectrometry (MS) analysis. Additionally, we sought to validate a subset of our MS-based observations via quantitative real-time PCR, Western blot and semi-quantitative immunocytochemistry. Our hypothesis was that MS would detect differences in the SEP composition between the OSA and the normal osteoblast cells. Results Shotgun MS identified 133 putative surface proteins when output from all samples were combined, with good consistency between biological replicates. Eleven of the MS-detected proteins underwent analysis of gene expression by PCR, all of which were actively transcribed, but varied in expression level. Western blot of whole cell lysates from all three cell lines was effective for Thrombospondin-1, CYR61 and CD44, and indicated that all three proteins were present in each cell line. Semi-quantitative immunofluorescence indicated that CD44 was expressed at much higher levels on the surface of the OSA than the normal osteoblast cell lines. Conclusions The results of the present study identified numerous differences, and similarities, in the SEP of canine OSA cell lines and normal canine osteoblasts. The PCR, Western blot, and immunocytochemistry results, for the subset of proteins evaluated, were generally supportive of the mass spectrometry data. These methods may be applied to other cell lines, or other biological materials, to highlight unique and previously unrecognized differences between samples. While this study yielded data that may prove useful for OSA researchers and clinicians, further refinements of the described techniques are expected to yield greater accuracy and produce a more thorough SEP analysis. PMID:23758893

Proteomic Identification of Non-Gal Antibody Targets After Pig-to-Primate Cardiac Xenotransplantation

PubMed Central

Byrne, Guerard W.; Stalboerger, Paul G.; Davila, Eduardo; Heppelmann, Carrie J.; Gazi, Mozammel H.; McGregor, Hugh C. J.; LaBreche, Peter T.; Davies, William R.; Rao, Vinay P.; Oi, Keiji; Tazelaar, Henry D.; Logan, John S.; McGregor, Christopher G. A.

2008-01-01

Background Experience with non-antigenic galactose α1,3 galactose (αGal) polymers and development of αGal deficient pigs has reduced or eliminated the significance of this antigen in xenograft rejection. Despite these advances, delayed xenograft rejection (DXR) continues to occur most likely due to antibody responses to non-Gal endothelial cell (EC) antigens. Methods To gauge the diversity of the non-Gal antibody response we used antibody derived from CD46 transgenic heterotopic cardiac xenografts performed without T-cell immunosuppression, Group A (n = 4) and Gal knockout (GT-KO) heart transplants under tacrolimus and sirolimus immunosuppression, Group B (n = 8). Non-Gal antibody was measured by flow cytometry and by Western blots using GT-KO EC membrane antigens. A nanoLC/MS/MS analysis of proteins recovered from 2D gels was used to identify target antigens. Results Group A recipients exhibited a mixed cellular and humoral rejection. Group B recipients mainly exhibited classical DXR. Western blot analysis showed a non-Gal antibody response induced by GT+ and GT-KO hearts to an overlapping set of pig aortic EC membrane antigens. Proteomic analysis identified 14 potential target antigens but failed to define several immunodominant targets. Conclusions These experiments indicate that the non-Gal antibody response is directed to a number of stress response and inflammation related pig EC antigens and a few undefined targets. Further analysis of these antibody specificities using alternative methods is required to more fully define the repertoire of non-Gal antibody responses. PMID:18957049

Is the Cry1Ab protein from Bacillus thuringiensis (Bt) taken up by plants from soils previously planted with Bt corn and by carrot from hydroponic culture?

PubMed

Icoz, I; Andow, D; Zwahlen, C; Stotzky, G

2009-07-01

The uptake of the insecticidal Cry1Ab protein from Bacillus thuringiensis (Bt) by various crops from soils on which Bt corn had previously grown was determined. In 2005, the Cry1Ab protein was detected by Western blot in tissues (leaves plus stems) of basil, carrot, kale, lettuce, okra, parsnip, radish, snap bean, and soybean but not in tissues of beet and spinach and was estimated by enzyme-linked immunosorbent assay (ELISA) to be 0.05 +/- 0.003 ng g(-1) of fresh plant tissue in basil, 0.02 +/- 0.014 ng g(-1) in okra, and 0.34 +/- 0.176 ng g(-1) in snap bean. However, the protein was not detected by ELISA in carrot, kale, lettuce, parsnip, radish, and soybean or in the soils by Western blot. In 2006, the Cry1Ab protein was detected by Western blot in tissues of basil, carrot, kale, radish, snap bean, and soybean from soils on which Bt corn had been grown the previous year and was estimated by ELISA to be 0.02 +/- 0.014 ng g(-1) of fresh plant tissue in basil, 0.19 +/- 0.060 ng g(-1) in carrot, 0.05 +/- 0.018 ng g(-1) in kale, 0.04 +/- 0.022 ng g(-1) in radish, 0.53 +/- 0.170 ng g(-1) in snap bean, and 0.15 +/- 0.071 ng g(-1) in soybean. The Cry1Ab protein was also detected by Western blot in tissues of basil, carrot, kale, radish, and snap bean but not of soybean grown in soil on which Bt corn had not been grown since 2002; the concentration was estimated by ELISA to be 0.03 +/- 0.021 ng g(-1) in basil, 0.02 +/- 0.008 ng g(-1) in carrot, 0.04 +/- 0.017 ng g(-1) in kale, 0.02 +/- 0.012 ng g(-1) in radish, 0.05 +/- 0.004 ng g(-1) in snap bean, and 0.09 +/- 0.015 ng g(-1) in soybean. The protein was detected by Western blot in 2006 in most soils on which Bt corn had or had not been grown since 2002. The Cry1Ab protein was detected by Western blot in leaves plus stems and in roots of carrot after 56 days of growth in sterile hydroponic culture to which purified Cry1Ab protein had been added and was estimated by ELISA to be 0.08 +/- 0.021 and 0.60 +/- 0.148 ng g(-1) of fresh leaves plus stems and roots, respectively. No Cry1Ab protein was detected in the tissues of carrot grown in hydroponic culture to which no Cry1Ab protein had been added. Because of the different results obtained with different commercial Western blot (i.e., from Envirologix and Agdia) and ELISA kits (i.e., from Envirologix, Agdia, and Abraxis), it is not clear whether the presence of the Cry1Ab protein in the tissues of some plants under field condition and in carrot in sterile hydroponic culture was the result of the uptake of the protein by the plants or of the accuracy and sensitivity of the different commercial kits used. More detailed studies with additional techniques are obviously needed to confirm the uptake of Cry proteins from soil by plants subsequently planted after a Bt crop.

Evaluation of the Recombinant Protein TpF1 of Treponema pallidum for Serodiagnosis of Syphilis

PubMed Central

Jiang, Chuanhao; Zhao, Feijun; Xiao, Jinhong; Zeng, Tiebing; Yu, Jian; Ma, Xiaohua; Wu, Haiying

2013-01-01

Syphilis is a chronic infection caused by Treponema pallidum subsp. pallidum, and diagnosis with sensitive and specific methods is a challenging process that is important for its prevention and treatment. In the present study, we established a recombinant protein TpF1-based indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and a Western blot assay for human and rabbit sera. The 20-kDa recombinant protein TpF1 was detected by Western blotting performed with sera from rabbits immunized with recombinant TpF1 and infected with the T. pallidum Nichols strain and T. pallidum clinical isolates but was not detected by Western blotting with sera from uninfected rabbits. The sensitivity of the recombinant protein was determined by screening sera from individuals with primary, secondary, latent, and congenital syphilis (n = 82). The specificity of the recombinant protein was determined by screening sera from uninfected controls (n = 30) and individuals with potentially cross-reactive infections, including Lyme disease (n = 30) and leptospirosis (n = 5). The sensitivities of TpF1-based ELISAs were 93.3%, 100%, 100%, and 100% for primary, secondary, latent, and congenital syphilis, respectively, and the specificities were all 100% for sera from uninfected controls and individuals with potentially cross-reactive infections. In Western blot assays, the sensitivities and specificities of TpF1 for human sera were all 100%. The reactivities of TpF1 with syphilitic sera were proportional to the titers of the T. pallidum particle agglutination (TPPA) assay. These data indicate that the recombinant protein TpF1 is a highly immunogenic protein in human and rabbit infections and a promising marker for the screening of syphilis. PMID:23945159

Evaluation of Modified 2-Tiered Serodiagnostic Testing Algorithms for Early Lyme Disease

PubMed Central

Strle, Klemen; Nigrovic, Lise E.; Lantos, Paul M.; Lepore, Timothy J.; Damle, Nitin S.; Ferraro, Mary Jane; Steere, Allen C.

2017-01-01

Abstract Background. The conventional 2-tiered serologic testing protocol for Lyme disease (LD), an enzyme immunoassay (EIA) followed by immunoglobulin M and immunoglobulin G Western blots, performs well in late-stage LD but is insensitive in patients with erythema migrans (EM), the most common manifestation of the illness. Western blots are also complex, difficult to interpret, and relatively expensive. In an effort to improve test performance and simplify testing in early LD, we evaluated several modified 2-tiered testing (MTTT) protocols, which use 2 assays designed as first-tier tests sequentially, without the need of Western blots. Methods. The MTTT protocols included (1) a whole-cell sonicate (WCS) EIA followed by a C6 EIA; (2) a WCS EIA followed by a VlsE chemiluminescence immunoassay (CLIA); and (3) a variable major protein-like sequence, expressed (VlsE) CLIA followed by a C6 EIA. Sensitivity was determined using serum from 55 patients with erythema migrans; specificity was determined using serum from 50 patients with other illnesses and 1227 healthy subjects. Results. Sensitivity of the various MTTT protocols in patients with acute erythema migrans ranged from 36% (95% confidence interval [CI], 25%–50%) to 54% (95% CI, 42%–67%), compared with 25% (95% CI, 16%–38%) using the conventional protocol (P = .003–0.3). Among control subjects, the 3 MTTT protocols were similarly specific (99.3%–99.5%) compared with conventional 2-tiered testing (99.5% specificity; P = .6–1.0). Conclusions. Although there were minor differences in sensitivity and specificity among MTTT protocols, each provides comparable or greater sensitivity in acute EM, and similar specificity compared with conventional 2-tiered testing, obviating the need for Western blots. PMID:28329259

Multiplex Immunoassay for Lyme Disease Using VlsE1-IgG and pepC10-IgM Antibodies: Improving Test Performance through Bioinformatics ▿

PubMed Central

Porwancher, Richard B.; Hagerty, C. Greg; Fan, Jianqing; Landsberg, Lisa; Johnson, Barbara J. B.; Kopnitsky, Mark; Steere, Allen C.; Kulas, Karen; Wong, Susan J.

2011-01-01

The Centers for Disease Control and Prevention currently recommends a 2-tier serologic approach to Lyme disease laboratory diagnosis, comprised of an initial serum enzyme immunoassay (EIA) for antibody to Borrelia burgdorferi followed by supplementary IgG and IgM Western blotting of EIA-positive or -equivocal samples. Western blot accuracy is limited by subjective interpretation of weakly positive bands, false-positive IgM immunoblots, and low sensitivity for detection of early disease. We developed an objective alternative second-tier immunoassay using a multiplex microsphere system that measures VlsE1-IgG and pepC10-IgM antibodies simultaneously in the same sample. Our study population comprised 79 patients with early acute Lyme disease, 82 patients with early-convalescent-phase disease, 47 patients with stage II and III disease, 34 patients post-antibiotic treatment, and 794 controls. A bioinformatic technique called partial receiver-operator characteristic (ROC) regression was used to combine individual antibody levels into a single diagnostic score with a single cutoff; this technique enhances test performance when a high specificity is required (e.g., ≥95%). Compared to Western blotting, the multiplex assay was equally specific (95.6%) but 20.7% more sensitive for early-convalescent-phase disease (89.0% versus 68.3%, respectively; 95% confidence interval [95% CI] for difference, 12.1% to 30.9%) and 12.5% more sensitive overall (75.0% versus 62.5%, respectively; 95% CI for difference, 8.1% to 17.1%). As a second-tier test, a multiplex assay for VlsE1-IgG and pepC10-IgM antibodies performed as well as or better than Western blotting for Lyme disease diagnosis. Prospective validation studies appear to be warranted. PMID:21367982

Triton-polyacrylamide gel electrophoresis and leucine aminopeptidase activity staining detect Triton-slowed bands including high-molecular-mass aminopeptidase N (CD13) isoform in cholestatic patient sera.

PubMed

Kawai, Makoto; Hara, Yukichi

2006-02-01

Western blotting of aminopeptidase N (APN) detects a high-molecular-mass isoform (260 kDa) [M. Kawai, Y. Otake, Y. Hara High-molecular-mass isoform of aminopeptidase N/CD13 in serum from cholestatic patients. Clin Chim Acta 330 (2003) 141-149] in cholestatic patient serum but is time-consuming. Human sera were electrophoresed on polyacrylamide gel containing Triton-X100 (Triton-PAGE) and stained with leucine-B-naphthylamide (LAP-staining). The stained bands were eluted from the gel, treated with N- and O-glycosidase if necessary, and analyzed by Western blotting [M. Kawai, Y. Otake, Y. Hara High-molecular-mass isoform of aminopeptidase N/CD13 in serum from cholestatic patients. Clin Chim Acta 330 (2003) 141-149]. Triton-PAGE and LAP-staining clearly detected fast bands in all the sera examined. Almost parallel with leucine aminopeptidase activity, slow bands were strongly stained in all 11 cholestatic patients but clearly stained in 3 out of 14 patients with hepatobiliary diseases other than cholestasis. PAGE with various concentrations of Triton showed that Triton slows down slow bands but not fast bands. Western blotting showed that Triton-PAGE-slow bands of cholestasis contained 140 and 260-kDa APN and that fast bands were slightly smaller than monomer-size slow bands after glycosidase treatment. Less time-consuming than Western blotting, Triton-PAGE and LAP-staining detect novel APN bands slowed by Triton and partly composed of the high-molecular-mass isoform in cholestasis. The slow bands seem to be homodimers of APN with transmembrane anchors. The polypeptide of the fast band seems to be processed differently from that of the slow band.

Diagnostic-test evaluation of immunoassays for anti-Toxoplasma gondii IgG antibodies in a random sample of Mexican population.

PubMed

Caballero-Ortega, Heriberto; Castillo-Cruz, Rocío; Murieta, Sandra; Ortíz-Alegría, Luz Belinda; Calderón-Segura, Esther; Conde-Glez, Carlos J; Cañedo-Solares, Irma; Correa, Dolores

2014-05-14

There are few articles on evaluation of Toxoplasma gondii serological tests. Besides, commercially available tests are not always useful and are expensive for studies in open population. The aim of this study was to evaluate in-house ELISA and western blot for IgG antibodies in a representative sample of people living in Mexico. Three hundred and five serum samples were randomly selected from two national seroepidemiological survey banks; they were taken from men and women of all ages and from all areas of the country. ELISA cut-off was established using the mean plus three standard deviations of negative samples. Western blots were analysed by two experienced technicians and positivity was established according to the presence of at least three diagnostic bands. A commercial ELISA kit was used as a third test. Two reference standards were built up: one using concordant results of two assays leaving the evaluated test out and the other in which the evaluated test was included (IN) with at least two concordant results to define diagnosis. the lowest values of diagnostic parameters were obtained with the OUT reference standards: in-house ELISA had 96.9% sensitivity, 62.1% specificity, 49.6% PPV, 98.1% NPV and 71.8% accuracy, while western blot presented 81.8%, 89.7%, 84.0%, 88.2% and 86.6% values and the best kappa coefficient (0.72-0.82). The in-house ELISA is useful for screening people of Mexico, due to its high sensitivity, while western blot may be used to confirm diagnosis. These techniques might prove useful in other Latin American countries.

Albumin Overload and PINK1/Parkin Signaling-Related Mitophagy in Renal Tubular Epithelial Cells.

PubMed

Tan, Jin; Xie, Qi; Song, Shuling; Miao, Yuyang; Zhang, Qiang

2018-03-01

BACKGROUND Albumin, as a major urinary protein component, is a risk factor for chronic kidney disease progression. Mitochondrial dysfunction is one of the main causes of albumin-induced proximal tubule cells injury. Mitophagy is considered as a pivotal protective mechanism for the elimination of dysfunctional mitochondria. The objective of this research was to determine whether albumin overload-induced mitochondrial dysfunction can activate PINK1/Parkin-mediated mitophagy in renal tubular epithelial cells (TECs). MATERIAL AND METHODS Immunofluorescence assay and Western blot assay were used to detect the effects of albumin overload on autophagy marker protein LC3. Transmission electron microscopy and Western blot assay were used to investigate the role of albumin in mitochondrial injury. Western blot assay and co-localization of acidic lysosomes and mitochondria assay were employed to detect the activation of mitophagy induced by albumin. Finally, we explored the role of PINK1/Parkin signaling in albumin-induced mitophagy by inhibiting mitophagy by knockdown of PARK2 (Parkin) level. RESULTS Immunofluorescence and Western blot results showed that the expression level of LC3-II increased, and the maximum increase point was observed after 8 h of albumin treatment. Transmission electron microscopy results demonstrated that albumin overload-induced mitochondrial injury and quantity of autophagosomes increased. Additionally, expression of PINK1 and cytosolic cytochrome C increased and mitochondria cytochrome C decreased in the albumin group. The co-localization of acidic lysosomes and mitochondria demonstrated that the number of albumin overload-induced mitophagy-positive dots increased. The transient transfection of PARK2 siRNA result showed knockdown of the expression level of PARK2 can inhibit mitophagy induced by albumin. CONCLUSIONS In conclusion, our study suggests that mitochondrial dysfunction activates the PINK1/Parkin signaling and mitophagy in renal tubular epithelial cells under albumin overload condition.

Regulation of DMT1 on autophagy and apoptosis in osteoblast

PubMed Central

Liu, Fei; Zhang, Wei-Lin; Meng, Hong-Zheng; Cai, Zheng-Yu; Yang, Mao-Wei

2017-01-01

Iron overload has recently been associated with the changes in the bone microstructure that occur in osteoporosis. However, the effect of iron overload on osteoblasts is unclear. The purpose of this study was to explore the function of divalent metal transporter 1 (DMT1) in the pathological processes of osteoporosis. Osteoblast hFOB1.19 cells were cultured in medium supplemented with different concentrations (0, 50, 100, 200, 300, 400, 500 μmol/L) of ferric ammonium citrate (FAC) as a donor of ferric ions. We used western blotting and immunofluorescence to determine the levels of DMT1 after treatment with FAC. Apoptosis was evaluated by detecting the levels of cleaved caspase 3, BCL2, and BAX with western blotting. Autophagy was evaluated by detecting the levels of LC3 with western blotting and immunofluorescence. Beclin-1 expression was also assessed with western blotting. The autophagy inhibitor 3-methyladenine was used to determine whether autophagy affects the apoptosis induced by FAC. Our results show that FAC increased the levels of DMT1, upregulated the expression of BCL2, and downregulated the apoptosis-related proteins cleaved caspase 3 and BAX. Both LC3I/LC3II levels and beclin-1 were also increased, indicating that FAC increases the accumulation of autophagosomes in hFOB1.19 cells. FAC-induced autophagy was increased by the apoptosis inhibitor 3-MA but was reduced in DMT1 shRNA hFOB1.19 cells. These results suggest that the increased expression of DMT1 induces iron overload and iron overload induces osteoblast autophagy and apoptosis, thus affecting the pathological processes of osteoporosis. Clarifying the mechanisms underlying the effects of DMT1 will allow the identification of novel targets for the prevention and treatment of osteoporosis. PMID:28367088

Effects of antibodies to phosphorylated and non-phosphorylated tau on in vitro tau phosphorylation at Serine-199: Preliminary report.

PubMed

Loeffler, David A; Smith, Lynnae M; Klaver, Andrea C; Martić, Sanela

2015-07-01

Phosphorylation of multiple amino acids on tau protein ("hyperphosphorylation") is required for the development of tau pathology in Alzheimer's disease. Administration of anti-tau antibodies to transgenic "tauopathy mice" has been shown to reduce their tau pathology but the mechanisms responsible are unclear. To examine the effects of anti-tau antibodies on tau phosphorylation, we used western blots to study the effects of three antibodies to phosphorylated tau (pTau), namely anti-pTau S199, T231, and S396, and three antibodies to non-phosphorylated tau on in vitro phosphorylation of recombinant human tau-441 at S199. Inclusion of an anti-pTau T231 antibody in the phosphorylation reaction reduced the intensity of monomeric pTau S199 in western blots of denaturing gels, but the other antibodies had no apparent effects on this process. Surprisingly, including all three anti-phospho-tau antibodies in the reaction did not reduce the intensity of the monomer band, possibly due to steric hindrance between the antibodies. These preliminary findings suggest that anti-tau antibodies may have minimal direct effects on tau phosphorylation. Limitations of using western blots to examine the effects of anti-tau antibodies on this process were found to include between-experiment variability in pTau band densities and poor resolution of high molecular weight pTau oligomers. The presence of bands representing immunoglobulins as well as pTau may also complicate interpretation of the western blots. Further studies are indicated to examine the effects of anti-pTau antibodies on phosphorylation of other tau amino acids in addition to S199. Copyright © 2015 Elsevier Inc. All rights reserved.

[The detection of antibodies against HIV-1 24-kd protein. A clinico-serological correlation].

PubMed

Díaz Torres, H; Silva Cabrera, E; Rodríguez García, O; Bárcenas Moses, J; Lubián Caballero, A L

1996-01-01

The presence of antibodies against the HIV protein of 24 kd was studies by the parallel use of the DAVIH BLOT western blot and of the DAVIH AC P24 ELISA in serum samples from 176 patients at different HIV-1 infection stages. The results were correlated with the clinical classification of the patient at the moment of taking the sample and with the further evolution during 6 months. 57% of the patients with opportunistic minor infections and 96% of AIDS patients had low antibodies titres. Dead patients showed no reactivity or presented very low titres in samples taken before dying. Different titrations were observed in serum groups with an apparently uniform reactivity in the western blot. The results show and adequate clinical and serological correlation. Therefore, the DAVIH AC P24 ELISA could be useful in the clinical follow-up of HIV-1 infected persons.

Stable chloroplast transformation of immature scutella and inflorescences in wheat (Triticum aestivum L.).

PubMed

Cui, Cuiju; Song, Fei; Tan, Yi; Zhou, Xuan; Zhao, Wen; Ma, Fengyun; Liu, Yunyi; Hussain, Javeed; Wang, Yuesheng; Yang, Guangxiao; He, Guangyuan

2011-04-01

Chloroplast transformation in wheat was achieved by bombardment of scutella from immature embryos and immature inflorescences, respectively. A wheat chloroplast site-specific expression vector, pBAGNRK, was constructed by placing an expression cassette containing neomycin phosphotransferase II (nptII) and green fluorescent protein (gfp) as selection and reporter genes, respectively, in the intergenic spacer between atpB and rbcL of wheat chloroplast genome. Integration of gfp gene in the plastome was identified by polymerase chain reaction (PCR) analysis and Southern blotting using gfp gene as a probe. Expression of GFP protein was examined by western blot. Three positive transformants were obtained and the Southern blot of partial fragment of atpB and rbcL (targeting site) probes verified that one of them was homoplasmic. Stable expression of GFP fluorescence was confirmed by confocal microscopy in the leaf tissues from T(1) progeny seedlings. PCR analysis of gfp gene also confirmed the inheritance of transgene in the T(1) progeny. These results strengthen the feasibility of wheat chloroplast transformation and also give a novel method for the introduction of important agronomic traits in wheat through chloroplast transformation.

Diversity, Replication, Pathogenicity and Cell Biology of Crimean Congo Hemorrhagic Fever Virus

DTIC Science & Technology

2008-10-01

author. Mailing address: Department of Microbi - ology, University of Pennsylvania, 225 Johnson Pavilion, 3610 Hamil- ton Walk, Philadelphia, PA 19104...to 15% Tris-HCl gels (Bio-Rad, Hercules, CA), followed by Western blot analysis with mouse anti-V5 (Invitrogen) as the primary antibody and sheep anti...bovine serum. In addition, TGN46, a sheep antibody specific for a heavily glycosylated protein localized primarily in the trans-Golgi network, was

Induced Accelerated Aging in Induced Pluripotent Stem Cell Lines from Patients with Parkinson’s Disease

DTIC Science & Technology

2013-11-01

clones . Western blot analysis will be used to detect the protein expression after selection. 2. Differentiation into oligoprecursor cells (OPCs... monkey and mouse which will be tested in iPSC derived neurons aged with progerin. 13 Key Research Accomplishments: • Milestone 1 (month 1-2...iPSC clones with drug-inducible progerin construct we established the plasmid transfection for iPSC induced neural stem cells, the retroviral

Escherichia coli gamma-glutamyltranspeptidase mutants deficient in processing to subunits.

PubMed

Hashimoto, W; Suzuki, H; Nohara, S; Kumagai, H

1992-11-30

Arginyl residues 513 and 571 of Escherichia coli K-12 gamma-glutamyl-transpeptidase (EC 2.3.2.2) were substituted with alanyl and glycyl residues, respectively, by oligonucleotide-directed in vitro mutagenesis. Both mutants were devoid of the enzymatic activity. On Western blot analysis, we found that both mutants accumulated a gamma-glutamyltranspeptidase precursor which was not processed into large and small subunits in the periplasmic space of Escherichia coli.

Dissecting Antibodies with Regards to Linear and Conformational Epitopes

PubMed Central

Forsström, Björn; Bisławska Axnäs, Barbara; Rockberg, Johan; Danielsson, Hanna; Bohlin, Anna; Uhlen, Mathias

2015-01-01

An important issue for the performance and specificity of an antibody is the nature of the binding to its protein target, including if the recognition involves linear or conformational epitopes. Here, we dissect polyclonal sera by creating epitope-specific antibody fractions using a combination of epitope mapping and an affinity capture approach involving both synthesized peptides and recombinant protein fragments. This allowed us to study the relative amounts of antibodies to linear and conformational epitopes in the polyclonal sera as well as the ability of each antibody-fraction to detect its target protein in Western blot assays. The majority of the analyzed polyclonal sera were found to have most of the target-specific antibodies directed towards linear epitopes and these were in many cases giving Western blot bands of correct molecular weight. In contrast, many of the antibodies towards conformational epitopes did not bind their target proteins in the Western blot assays. The results from this work have given us insights regarding the nature of the antibody response generated by immunization with recombinant protein fragments and has demonstrated the advantage of using antibodies recognizing linear epitopes for immunoassay involving wholly or partially denatured protein targets. PMID:25816293

Evaluation of Recombinant Plasmodium knowlesi Merozoite Surface Protein-133 for Detection of Human Malaria

PubMed Central

Cheong, Fei Wen; Lau, Yee Ling; Fong, Mun Yik; Mahmud, Rohela

2013-01-01

Plasmodium knowlesi is now known as the fifth Plasmodium species that can cause human malaria. The Plasmodium merozoite surface protein (MSP) has been reported to be potential target for vaccination and diagnosis of malaria. MSP-133 has been shown to be immunogenic and its T cell epitopes could mediate cellular immune protection. However, limited studies have focused on P. knowlesi MSP-133. In this study, an approximately 28-kDa recombinant P. knowlesi MSP-133 (pkMSP-133) was expressed by using an Escherichia coli system. The purified pkMSP-133 reacted with serum samples of patients infected with P. knowlesi (31 of 31, 100%) and non-P. knowlesi malaria (27 of 28, 96.43%) by Western blotting. The pkMSP-133 also reacted with P. knowlesi (25 of 31, 80.65%) and non-P. knowlesi malaria sera (20 of 28, 71.43%) in an enzyme-linked immunosorbent assay (ELISA). Most of the non-malarial infection (49 of 52 in by Western blotting and 46 of 52 in the ELISA) and healthy donor serum samples (65 of 65 by Western blotting and ELISA) did not react with recombinant pkMSP-133. PMID:23509118

The upregulation of receptor activator NF-kappaB ligand expression by interleukin-1alpha and Porphyromonas endodontalis in human osteoblastic cells.

PubMed

Chen, S-C; Huang, F-M; Lee, S-S; Li, M-Z; Chang, Y-C

2009-04-01

To investigate the receptor activator of nuclear factor-kappa B (NF-kappaB) ligand (RANKL) in osteoblastic cells stimulated with inflammatory mediators. The expression of RANKL in human osteoblastic cell line U2OS stimulated by pro-inflammatory cytokine interleukin (IL)-1alpha and black-pigmented bacteria Porphyromonas endodontalis was investigated by Western blot and enzyme-linked immunosorbent assay (ELISA). The significance of the results obtained from control and treated groups was statistically analysed by the paired Student's t-test. IL-1alpha was found to upregulate RANKL production in U2OS cells (P < 0.05). Investigations of the time dependence of RANKL expression in IL-1alpha-treated cells revealed a rapid accumulation of RANKL protein after 1 h of exposure; it remained elevated throughout the 24-h incubation period shown by Western blot and ELISA. In addition, P. endodontalis also increased RANKL expression in U2OS cells after 4-h incubation period demonstrated by Western blot and ELISA (P < 0.05). IL-1alpha and P. endodontalis may be involved in developing apical periodontitis through the stimulation of RANKL production.

The Use of Biotin to Demonstrate Immunohistochemistry, Western Blotting, and Dot Blots in University Practical Classes

ERIC Educational Resources Information Center

Millar, Thomas James; Knighton, Ronald; Chuck, Jo-Anne

2012-01-01

Immunological detection of proteins is an essential method to demonstrate to undergraduate biology students, however, is often difficult in resource and time poor student laboratory sessions. This method describes a failsafe method to rapidly and economically demonstrate this technique using biotinylated proteins or biotin itself as targets for…

Protection Conferred by recombinant Yersinia pestis Antigens Produced by a Rapid and Highly Scalable Plant Expression System

DTIC Science & Technology

2006-01-24

translational fusions with dsRED (lanes 8), and cytosol-targeted GFP (lanes 9). RbcL, large subunit of Rubisco . 862 ! www.pnas.org"cgi"doi൒.1073...analysis of F1-V expression with SDS"PAGE-Coomassie staining was difficult because the chimeric protein comigrates with the large subunit of Rubisco , a...contaminated by the Rubisco large subunit, which is very similar in size to F1-V. Analysis of Purified Plant-Produced Antigens. Western blots were

CYP1A protein expression and catalytic activity in double-crested cormorants experimentally exposed to Deepwater Horizon Mississippi Canyon 252 oil

USGS Publications Warehouse

Alexander, Courtney R.; Hooper, Michael J.; Cacela, Dave; Smelker, Kim D.; Calvin, Caleshia S.; Dean, Karen M.; Bursian, Steve J.; Cunningham, Fred L.; Hanson-Dorr, Katie C.; Horak, Katherine E.; Isanhart, John P.; Link, Jane E.; Shriner, Susan A.; Godard-Codding, Céline A.J.

2017-01-01

Double-crested cormorants (Phalacrocorax auritus, DCCO) were orally exposed to Deepwater Horizon Mississippi Canyon 252 (DWH) oil to investigate oil-induced toxicological impacts. Livers were collected for multiple analyses including cytochrome P4501A (CYP1A) enzymatic activity and protein expression. CYP1A enzymatic activity was measured by alkoxyresorufin O-dealkylase (AROD) assays. Activities specific to the O-dealkylation of four resorufin ethers are reported: benzyloxyresorufin O-debenzylase (BROD), ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and pentoxyresorufin O-depentylase (PROD). CYP1A protein expression was measured by western blot analysis with a CYP1A1 mouse monoclonal antibody. In study 1, hepatic BROD, EROD, and PROD activities were significantly induced in DCCO orally exposed to 20 ml/kg body weight (bw) oil as a single dose or daily for 5 days. Western blot analysis revealed hepatic CYP1A protein induction in both treatment groups. In study 2 (5 ml/kg bw oil or 10 ml/kg bw oil, 21 day exposure), all four hepatic ARODs were significantly induced. Western blots showed an increase in hepatic CYP1A expression in both treatment groups with a significant induction in birds exposed to 10 ml/kg oil. Significant correlations were detected among all 4 AROD activities in both studies and between CYP1A protein expression and both MROD and PROD activities in study 2. EROD activity was highest for both treatment groups in both studies while BROD activity had the greatest fold-induction. While PROD activity values were consistently low, the fold-induction was high, usually 2nd highest to BROD activity. The observed induced AROD profiles detected in the present studies suggest both CYP1A4/1A5 DCCO isoforms are being induced after MC252 oil ingestion. A review of the literature on avian CYP1A AROD activity levels and protein expression after exposure to CYP1A inducers highlights the need for species-specific studies to accurately evaluate avian exposure to oil.

Anti-Peptide Monoclonal Antibodies Generated for Immuno-Multiple Reaction Monitoring-Mass Spectrometry Assays Have a High Probability of Supporting Western blot and ELISA*

PubMed Central

Schoenherr, Regine M.; Saul, Richard G.; Whiteaker, Jeffrey R.; Yan, Ping; Whiteley, Gordon R.; Paulovich, Amanda G.

2015-01-01

Immunoaffinity enrichment of peptides coupled to targeted, multiple reaction monitoring-mass spectrometry (immuno-MRM) has recently been developed for quantitative analysis of peptide and protein expression. As part of this technology, antibodies are generated to short, linear, tryptic peptides that are well-suited for detection by mass spectrometry. Despite its favorable analytical performance, a major obstacle to widespread adoption of immuno-MRM is a lack of validated affinity reagents because commercial antibody suppliers are reluctant to commit resources to producing anti-peptide antibodies for immuno-MRM while the market is much larger for conventional technologies, especially Western blotting and ELISA. Part of this reluctance has been the concern that affinity reagents generated to short, linear, tryptic peptide sequences may not perform well in traditional assays that detect full-length proteins. In this study, we test the feasibility and success rates of generating immuno-MRM monoclonal antibodies (mAbs) (targeting tryptic peptide antigens) that are also compatible with conventional, protein-based immuno-affinity technologies. We generated 40 novel, peptide immuno-MRM assays and determined that the cross-over success rates for using immuno-MRM monoclonals for Western blotting is 58% and for ELISA is 43%, which compare favorably to cross-over success rates amongst conventional immunoassay technologies. These success rates could most likely be increased if conventional and immuno-MRM antigen design strategies were combined, and we suggest a workflow for such a comprehensive approach. Additionally, the 40 novel immuno-MRM assays underwent fit-for-purpose analytical validation, and all mAbs and assays have been made available as a resource to the community via the Clinical Proteomic Tumor Analysis Consortium's (CPTAC) Antibody (http://antibodies.cancer.gov) and Assay Portals (http://assays.cancer.gov), respectively. This study also represents the first determination of the success rate (92%) for generating mAbs for immuno-MRM using a recombinant B cell cloning approach, which is considerably faster than the traditional hybridoma approach. PMID:25512614

Chronic changes in pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulation in rats.

PubMed

Han, Xun; Ran, Ye; Su, Min; Liu, Yinglu; Tang, Wenjing; Dong, Zhao; Yu, Shengyuan

2017-01-01

Background Preclinical experimental studies revealed an acute alteration of pituitary adenylate cyclase-activating polypeptide in response to a single activation of the trigeminovascular system, which suggests a potential role of pituitary adenylate cyclase-activating polypeptide in the pathogenesis of migraine. However, changes in pituitary adenylate cyclase-activating polypeptide after repeated migraine-like attacks in chronic migraine are not clear. Therefore, the present study investigated chronic changes in pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulations in the rat. Methods A rat model of chronic migraine was established by repeated chemical dural stimulations using an inflammatory soup for a different numbers of days. The pituitary adenylate cyclase-activating polypeptide levels were quantified in plasma, the trigeminal ganglia, and the trigeminal nucleus caudalis using radioimmunoassay and Western blotting in trigeminal ganglia and trigeminal nucleus caudalis tissues. Western blot analysis and real-time polymerase chain reaction were used to measure the protein and mRNA expression of pituitary adenylate cyclase-activating polypeptide-related receptors (PAC1, VPAC1, and VPAC2) in the trigeminal ganglia and trigeminal nucleus caudalis to identify changes associated with repetitive applications of chemical dural stimulations. Results All rats exhibited significantly decreased periorbital nociceptive thresholds to repeated inflammatory soup stimulations. Radioimmunoassay and Western blot analysis demonstrated significantly decreased pituitary adenylate cyclase-activating polypeptide levels in plasma and trigeminal ganglia after repetitive chronic inflammatory soup stimulation. Protein and mRNA analyses of pituitary adenylate cyclase-activating polypeptide-related receptors demonstrated significantly increased PAC1 receptor protein and mRNA expression in the trigeminal ganglia, but not in the trigeminal nucleus caudalis, and no significant differences were found in the expression of the VPAC1 and VPAC2 receptors. Conclusions This study demonstrated the chronic alteration of pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulation in the rat, which suggests the crucial involvement of pituitary adenylate cyclase-activating polypeptide in the development of migraine. The selective increase in pituitary adenylate cyclase-activating polypeptide-related receptors suggests that the PAC1 receptor pathway is a novel target for the treatment of migraine.

Flavonoid fraction of guava leaf extract attenuates lipopolysaccharide-induced inflammatory response via blocking of NF-κB signalling pathway in Labeo rohita macrophages.

PubMed

Sen, Shib Sankar; Sukumaran, V; Giri, Sib Sankar; Park, Se Chang

2015-11-01

Psidium guajava L. is a well-known traditional medicinal plant widely used in folk medicine. To explore the anti-inflammatory activity of the flavonoid fraction of guava leaf extract (FGLE), we investigated its ability to suppress the levels of inflammatory mediators elevated by lipopolysaccharide (LPS) in Labeo rohita head-kidney (HK) macrophages. HK macrophages of L. rohita were treated with LPS in the presence or absence of the FGLE. We examined the inhibitory effect of FGLE on LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production. The inhibitory effect of FGLE on nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were investigated by RT-PCR and western blot. The effect of FGLE on proinflammatory cytokines tumour necrosis factor alpha (TNF-α) or interleukin-1β (IL-1β) was also investigated by ELISA and RT-PCR. The phosphorylation of three mitogen activated protein kinases (MAPK) molecules ERK, JNK and p38 was analysed by western blot analysis. FGLE inhibited LPS-induced NO and PGE2 production. It also effectively inhibited TNF-α, IL-1β, IL-10, iNOS, and COX-2 production in a concentration-dependent manner. In addition, FGLE suppressed the mRNA expression levels of TNF-α and IL-1β in LPS-stimulated HK macrophages. RT-PCR and western blot analysis showed that FGLE decreased both the mRNA and protein expression levels of LPS-induced iNOS and COX-2 in HK macrophages. FGLE suppresses the phosphorylation of MAPK molecules in LPS-stimulated HK macrophages. FGLE also significantly inhibited LPS-induced NF-κB transcriptional activity. The molecular mechanism by which FGLE suppresses the expression of inflammatory mediators appears to involve the inhibition of NF-κB activation, through the suppression of LPS-induced IκB-α degradation. Together these results suggest that FGLE contains potential therapeutic agent(s), which regulate NF-κB activation, for the treatment of inflammatory conditions in L. rohita macrophages. Copyright © 2015 Elsevier Ltd. All rights reserved.

Role of troponin I proteolysis in the pathogenesis of stunned myocardium.

PubMed

Gao, W D; Atar, D; Liu, Y; Perez, N G; Murphy, A M; Marban, E

1997-03-01

Myocardial stunning is characterized by decreased myofilament Ca2+ responsiveness. To investigate the molecular basis of stunned myocardium, we performed PAGE and Western immunoblot analysis of the contractile proteins. Isolated rat hearts were retrogradely perfused at 37 degrees C for either 50 minutes (control group) or for 10 minutes, followed by 20-minute global ischemia and 20-minute reperfusion (stunned group), or for 20-minute ischemia without reflow. Another group consisted of hearts subjected to 20-minute ischemia in which stunning was mitigated by 10-minute reperfusion with low Ca2+/low pH solution. Myocardial tissue samples subjected to PAGE revealed no obvious differences among groups. Western immunoblots for actin, tropomyosin, troponin C, troponin T, myosin light chain-1, and myosin light chain-2 showed highly selective recognition of the appropriate full-length molecular weight bands in all groups. Troponin I (TnI) Western blots revealed an additional band (approximately 26 kD, compared with 32 kD for the full-length protein) in stunned myocardial samples only. In parallel experiments, skinned trabeculae were treated with calpain I for 20 minutes; Western blots showed a TnI degradation pattern similar to that observed in stunned myocardium. Such TnI degradation was prevented by calpastatin, a naturally occurring calpain inhibitor. The results show that (1) TnI is partially and selectively degraded in stunned myocardium; (2) this degradation could be prevented by low Ca2+/low pH reperfusion, which also prevented the contractile dysfunction of stunning; and (3) calpain I could similarly degrade TnI, supporting the idea that Ca(2+)-dependent myofilament proteolysis underlies myocardial stunning.

Expression and characterization of an M cell-specific ligand-fused dengue virus tetravalent epitope using Saccharomyces cerevisiae.

PubMed

Nguyen, Ngoc-Luong; So, Kum-Kang; Kim, Jung-Mi; Kim, Sae-Hae; Jang, Yong-Suk; Yang, Moon-Sik; Kim, Dae-Hyuk

2015-01-01

A fusion construct (Tet-EDIII-Co1) consisting of an M cell-specific peptide ligand (Co1) at the C-terminus of a recombinant tetravalent gene encoding the amino acid sequences of dengue envelope domain III (Tet-EDIII) from four serotypes was expressed and tested for binding activity to the mucosal immune inductive site M cells for the development of an oral vaccine. The yeast episomal expression vector, pYEGPD-TER, which was designed to direct gene expression using the glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, a functional signal peptide of the amylase 1A protein from rice, and the GAL7 terminator, was used to clone the Tet-EDIII-Co1 gene and resultant plasmids were then used to transform Saccharomyces cerevisiae. PCR and back-transformation into Escherichia coli confirmed the presence of the Tet-EDIII-Co1 gene-containing plasmid in transformants. Northern blot analysis of transformed S. cerevisiae identified the presence of the Tet-EDIII-Co1-specific transcript. Western blot analysis indicated that the produced Tet-EDIII-Co1 protein with the expected molecular weight was successfully secreted into the culture medium. Quantitative Western blot analysis and ELISA revealed that the recombinant Tet-EDIII-Co1 protein comprised approximately 0.1-0.2% of cell-free extracts (CFEs). In addition, 0.1-0.2 mg of Tet-EDIII-Co1 protein per liter of culture filtrate was detected on day 1, and this quantity peaked on day 3 after cultivation. In vivo binding assays showed that the Tet-EDIII-Co1 protein was delivered specifically to M cells in Peyer's patches (PPs) while the Tet-EDIII protein lacking the Co1 ligand did not, which demonstrated the efficient targeting of this antigenic protein through the mucosal-specific ligand. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Extracellular vesicles mediate signaling between the aqueous humor producing and draining cells in the ocular system

PubMed Central

Lerner, Natalie; Avissar, Sofia

2017-01-01

Purpose Canonical Wnt signaling is associated with glaucoma pathogenesis and intraocular pressure (IOP) regulation. Our goal was to gain insight into the influence of non-pigmented ciliary epithelium (NPCE)-derived exosomes on Wnt signaling by trabecular meshwork (TM) cells. The potential impact of exosomes on Wnt signaling in the ocular drainage system remains poorly understood. Methods Exosomes isolated from media collected from cultured NPCE cells by differential ultracentrifugation were characterized by dynamic light scattering (DLS), tunable resistive pulse sensing (TRPS), and nanoparticle tracking analysis (NTA), sucrose density gradient migration and transmission electron microscopy (TEM). The cellular target specificity of the NPCE-derived exosomes was investigated by confocal microscopy-based monitoring of the uptake of DiD-labeled exosomes over time, as compared to uptake by various cell lines. Changes in Wnt protein levels in TM cells induced by NPCE exosomes were evaluated by Western blot. Results Exosomes derived from NPCE cells were purified and detected as small rounded 50–140 nm membrane vesicles, as defined by DLS, NTA, TRPS and TEM. Western blot analysis indicated that the nanovesicles were positive for classic exosome markers, including Tsg101 and Alix. Isolated nanoparticles were found in sucrose density fractions typical of exosomes (1.118–1.188 g/mL sucrose). Using confocal microscopy, we demonstrated time-dependent specific accumulation of the NPCE-derived exosomes in NTM cells. Other cell lines investigated hardly revealed any exosome uptake. We further showed that exosomes induced changes in Wnt signaling protein expression in the TM cells. Western blot analysis further revealed decreased phosphorylation of GKS3β and reduced β-catenin levels. Finally, we found that treatment of NTM cells with exosomes resulted in a greater than 2-fold decrease in the level of β-catenin in the cytosolic fraction. In contrast, no remarkable difference in the amount of β-catenin in the nuclear fraction was noted, relative to the control. Conclusions The data suggest that NPCE cells release exosome-like vesicles and that these nanoparticles affect canonical Wnt signaling in TM cells. These findings may have therapeutic relevance since canonical Wnt pathway is involved in intra-ocular pressure regulation. Further understanding of NPCE-derived exosome-responsive signaling pathways may reveal new targets for pharmacological intervention within the drainage system as a target for glaucoma therapy. PMID:28241021

Extracellular vesicles mediate signaling between the aqueous humor producing and draining cells in the ocular system.

PubMed

Lerner, Natalie; Avissar, Sofia; Beit-Yannai, Elie

2017-01-01

Canonical Wnt signaling is associated with glaucoma pathogenesis and intraocular pressure (IOP) regulation. Our goal was to gain insight into the influence of non-pigmented ciliary epithelium (NPCE)-derived exosomes on Wnt signaling by trabecular meshwork (TM) cells. The potential impact of exosomes on Wnt signaling in the ocular drainage system remains poorly understood. Exosomes isolated from media collected from cultured NPCE cells by differential ultracentrifugation were characterized by dynamic light scattering (DLS), tunable resistive pulse sensing (TRPS), and nanoparticle tracking analysis (NTA), sucrose density gradient migration and transmission electron microscopy (TEM). The cellular target specificity of the NPCE-derived exosomes was investigated by confocal microscopy-based monitoring of the uptake of DiD-labeled exosomes over time, as compared to uptake by various cell lines. Changes in Wnt protein levels in TM cells induced by NPCE exosomes were evaluated by Western blot. Exosomes derived from NPCE cells were purified and detected as small rounded 50-140 nm membrane vesicles, as defined by DLS, NTA, TRPS and TEM. Western blot analysis indicated that the nanovesicles were positive for classic exosome markers, including Tsg101 and Alix. Isolated nanoparticles were found in sucrose density fractions typical of exosomes (1.118-1.188 g/mL sucrose). Using confocal microscopy, we demonstrated time-dependent specific accumulation of the NPCE-derived exosomes in NTM cells. Other cell lines investigated hardly revealed any exosome uptake. We further showed that exosomes induced changes in Wnt signaling protein expression in the TM cells. Western blot analysis further revealed decreased phosphorylation of GKS3β and reduced β-catenin levels. Finally, we found that treatment of NTM cells with exosomes resulted in a greater than 2-fold decrease in the level of β-catenin in the cytosolic fraction. In contrast, no remarkable difference in the amount of β-catenin in the nuclear fraction was noted, relative to the control. The data suggest that NPCE cells release exosome-like vesicles and that these nanoparticles affect canonical Wnt signaling in TM cells. These findings may have therapeutic relevance since canonical Wnt pathway is involved in intra-ocular pressure regulation. Further understanding of NPCE-derived exosome-responsive signaling pathways may reveal new targets for pharmacological intervention within the drainage system as a target for glaucoma therapy.

1082-39, an analogue of sorafenib, inhibited human cancer cell growth more potently than sorafenib.

PubMed

Chu, Jia-Hui; Zhao, Cui-Rong; Song, Zhi-Yu; Wang, Rui-Qi; Qin, Yi-Zhuo; Li, Wen-Bao; Qu, Xian-Jun

2014-04-01

1082-39, an analogue of sorafenib, is a derivative of indazole diarylurea. We evaluated the activity of 1082-39 against human cancer cell growth. Its effects and mechanisms of action were then compared with those of sorafenib. The experiments were performed in human melanoma M21 cells. Cell viability was estimated by using the colorimetric assay. Annexin V-FITC/PI staining assay was used to recognize the apoptotic cells. Further analysis of the mitochondria membrane potential (MMP) was performed by the JC-1 fluorescence probe staining. The levels of apoptotic proteins and kinases related to cancer proliferation were determined by western blotting assay. 1082-39 possessed the activity against cancer cell proliferation with time- and dose-dependent manner. 1082-39 induced M21 cell to apoptosis, showing the increase of annexin V-FITC/PI staining cells, the MMP collapse and releasing cytochrome c from mitochondria. Western blotting analysis showed the activation of the mitochondria-mediated intrinsic pathway, showing the increase of cleaved caspase-9, cleaved caspase-3 and cleaved PARP. Statistical analysis suggested that 1082-39 possessed greater activities than sorafenib in the inhibition of M21 proliferation and induction of apoptosis. These effects of 1082-39 might arise from its activity of regulation the PI3K/Akt and Wnt/β-catenin signaling pathways. 1082-39 is a promising candidate compound which could develop as a potent anticancer agent. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Exosome secretion by eosinophils: A possible role in asthma pathogenesis.

PubMed

Mazzeo, Carla; Cañas, José Antonio; Zafra, Maria Paz; Rojas Marco, Ainara; Fernández-Nieto, Mar; Sanz, Veronica; Mittelbrunn, María; Izquierdo, Manuel; Baixaulli, Francesc; Sastre, Joaquín; Del Pozo, Victoria

2015-06-01

Eosinophils secrete several granules that are involved in the propagation of inflammatory responses in patients with pathologies such as asthma. We hypothesized that some of these granules are exosomes, which, when transferred to the recipient cells, could modulate asthma progression. Eosinophils were purified from peripheral blood and cultured with or without IFN-γ or eotaxin. Multivesicular bodies (MVBs) in eosinophils were studied by using fluorescence microscopy, transmission electron microscopy (TEM), and flow cytometry. Exosome secretion was measured and exosome characterization was performed with TEM, Western blotting, and NanoSight analysis. Generation of MVBs in eosinophils was confirmed by using fluorescence microscopy and flow cytometry and corroborated by means of TEM. Having established that eosinophils contain MVBs, our aim was to demonstrate that eosinophils secrete exosomes. To do this, we purified exosomes from culture medium of eosinophils and characterized them. Using Western blot analysis, we demonstrated that eosinophils secreted exosomes and that the discharge of exosomes to extracellular media increases after IFN-γ stimulation. We measured exosome size and quantified exosome production from healthy and asthmatic subjects using nanotracking analysis. We found that exosome production was augmented in asthmatic patients. Our findings are the first to demonstrate that eosinophils contain functional MVBs and secrete exosomes and that their secretion is increased in asthmatic patients. Thus exosomes might play an important role in the progression of asthma and eventually be considered a biomarker. Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Synergism between NF-kappa B inhibitor, celastrol, and XIAP inhibitor, embelin, in an acute myeloid leukemia cell line, HL-60.

PubMed

Pazhang, Yaghub; Jaliani, Hossein Zarei; Imani, Mehdi; Dariushnejad, Hassan

2016-01-01

Embelin and celastrol, inhibitors of XIAP and NF-κB proteins respectively, have been derived from natural sources and shown anti-tumor activities against different cancer cell lines. Some interactions have recently been discovered between XIAP and NF-κB pathways, but the effects of these inhibitors in combination have not been investigated yet. We have studied possible synergistic effects of embelin in combination with celastrol, in an acute myeloid leukemia model, HL-60 cell line. Cytotoxicity of embelin and celastrol, separately and in combination, was determined by MTT assay and flow cytometry. Chou-Talalay's method was used to assess the synergistic effect of two components. Immunocytochemistry and western blot analysis of the two tumor marker proteins. (survivin and COX-2) was also performed to investigate downstream effects of two components. Analysis of MTT assay and flow cytometry showed that there is a substantial synergistic effect in some affected fractions of drug-treated HL-60. cells, while in other affected fractions a mild synergism or additive effect was observed. Immunocytochemistry and western blot analysis revealed that the expression of survivin and COX-2 proteins was reduced in treated cells. Embelin and celastrol showed potent antitumor activity and synergistic effects in combination. Therefore targeting XIAP and NF-κB pathways simultaneously can be investigated in more detail to make use of embelin and celastrol as a combination therapy of cancer.

Molecular identification and expression analysis of lipocalins from blood feeding taiga tick, Ixodes persulcatus Schulze.

PubMed

Konnai, Satoru; Nishikado, Hideto; Yamada, Shinji; Imamura, Saiki; Ito, Takuya; Onuma, Misao; Murata, Shiro; Ohashi, Kazuhiko

2011-02-01

Lipocalins have been known for their several biological activities in blood-sucking arthropods. Recently, the identification and characterization of lipocalins from Ixodes ricinus (LIRs) have been reported and functions of lipocalins are well documented. In this study, we have characterized four Ixodes persulcatus lipocalins that were discovered while analyzing I. persulcatus tick salivary gland EST library. We show that the four I. persulcatus lipocalins, here after named LIPERs (lipocalin from I. persulcatus) are 28.8-94.4% identical to LIRs from I. ricinus. Reverse transcriptase-PCR analysis revealed that lipocalin genes were expressed specifically in the salivary glands throughout life cycle stages of the ticks and were up-regulated by blood feeding. The specific expressions were also confirmed by Western blotting analysis. Furthermore, to investigate whether native lipocalins are secreted into the host during tick feeding, the reactivity of anti-serum raised against saliva of adult ticks to recombinant lipocalins was tested by Western blotting. The lipocalins are potentially secreted into the host during tick feeding as revealed by specific reactivity of recombinant lipocalins with mouse antibodies to I. persulcatus tick saliva. Preliminary vaccination of mice with recombinant lipocalins elicited that period to reach engorgement was significantly delayed and the engorgement weight was significantly reduced as compared to the control. Further elucidation of the biological functions of LIPERs are required to fully understand the pathways involved in the modulation of host immune responses. Copyright © 2010 Elsevier Inc. All rights reserved.

[Knockdown of dopamine receptor D2 upregulates the expression of adiogenic genes in mouse primary mesencephalic neurons].

PubMed

Ding, Jiaqi; Chen, Xiaoli; Lin, Jiaji; Zhu, Junling; Li, Zhuyi

2018-01-01

Objective To study the effects of dopamine receptor D2 (DRD2) on the adipogenesis genes in mouse primary mesencephalic neurons. Methods The lentiviral vectors which expressed specific shRNA targeting DRD2 were constructed to decrease DRD2 expression in mouse primary mesencephalic neurons. High throughput sequencing (HTS) analysis was used to investigate gene expression changes between the DRD2 knock-down group and the negative control group. Real-time quantitative PCR (qRT-PCR) and Western blot analysis were applied to verify the differently expressed genes. Fatty acids were measured by fatty acid detection kit. Results DRD2 expression was effectively down-regulated in mouse primary mesencephalic neurons by lentiviral vectors. HTS revealed adipogenesis genes were significantly up-regulated after DRD2 down-regulation, mainly including delta(14)-sterol reductase, acetyl-coenzyme A synthetase, insulin-induced gene 1 protein and especially stearoyl-coenzyme A desaturase 1 (SCD1, 4-fold upregulated). The qRT-PCR and Western blot analysis verified that SCD1 was upregulated 2.6 folds and 2 folds respectively by lentiviral DRD2-shRNA vectors. Moreover, the SCD1-related free fatty acids were significantly more increased than the negative control group. Conclusion DRD2 in primary mesencephalic neurons had a significant regulative effect on the adipogenesis genes. The up-regulation of SCD1 can accelerate the conversion of saturated fatty acids to monounsaturated fatty acids and prevent the damage of lipid toxicity to cells.

Secretome analysis of rat osteoblasts during icariin treatment induced osteogenesis

PubMed Central

Qian, Weiqing; Su, Yan; Zhang, Yajie; Yao, Nianwei; Gu, Nin; Zhang, Xu; Yin, Hong

2018-01-01

Osteoporosis is a serious public health problem and icariin (ICA) is the active component of the Epimedium sagittatum, a traditional Chinese medicinal herb. The present study aimed to investigate the effects and underlying mechanisms of ICA as a potential therapy for osteoporosis. Calvaria osteoblasts were isolated from newborn rats and treated with ICA. Cell viability, apoptosis, alkaline phosphatase activity and calcium deposition were analyzed. Bioinformatics analyses were performed to identify differentially expressed proteins (DEPs) in response to ICA treatment. Western blot analysis was performed to validate the expression of DEPs. ICA administration promoted osteoblast viability, alkaline phosphatase activity, calcium deposition and inhibited osteoblast apoptosis. Secretome analysis of ICA-treated cells was performed using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A total of 56 DEPs were identified, including serpin family F member 1 (PEDF), protein disulfide isomerase family A, member 3 (PDIA3), nuclear protein, co-activator of histone transcription (NPAT), c-Myc and heat shock protein 70 (HSP70). These proteins were associated with signaling pathways, including Fas and p53. Bioinformatics and western blot analyses confirmed that the expression levels of the six DEPs were upregulated following ICA treatment. These genes may be directly or indirectly involved in ICA-mediated osteogenic differentiation and osteogenesis. It was demonstrated that ICA treatment promoted osteogenesis by modulating the expression of PEDF, PDIA3, NPAT and HSP70 through signaling pathways, including Fas and p53. PMID:29532868

CNS development under altered gravity: cerebellar glial and neuronal protein expression in rat neonates exposed to hypergravity

NASA Technical Reports Server (NTRS)

Nguon, K.; Li, G-H; Sajdel-Sulkowska, E. M.

2004-01-01

The future of space exploration depends on a solid understanding of the developmental process under microgravity, specifically in relation to the central nervous system (CNS). We have previously employed a hypergravity paradigm to assess the impact of altered gravity on the developing rat cerebellum. The present study addresses the molecular mechanisms involved in the cerebellar response to hypergravity. Specifically, the study focuses on the expression of selected glial and neuronal cerebellar proteins in rat neonates exposed to hypergravity (1.5 G) from embryonic day (E)11 to postnatal day (P)6 or P9 (the time of maximal cerebellar changes) comparing them against their expression in rat neonates developing under normal gravity. Proteins were analyzed by quantitative Western blots of cerebellar homogenates; RNA analysis was performed in the same samples using quantitative PCR. Densitometric analysis of Western blots suggested a reduction in glial (glial acidic protein, GFAP) and neuronal (neuronal cell adhesion molecule, NCAM-L1, synaptophysin) proteins, but the changes in individual cerebellar proteins in hypergravity-exposed neonates appeared both age- and gender-specific. RNA analysis suggested a reduction in GFAP and synaptophysin mRNAs on P6. These data suggest that exposure to hypergravity may interfere with the expression of selected cerebellar proteins. These changes in protein expression may be involved in mediating the effect of hypergravity on the developing rat cerebellum. c2003 COSPAR. Published by Elsevier Ltd. All rights reserved.

Activation of PPARγ mediates icaritin-induced cell cycle arrest and apoptosis in glioblastoma multiforme.

PubMed

Liu, Yongji; Shi, Ling; Liu, Yuan; Li, Peng; Jiang, Guoping; Gao, Xiaoning; Zhang, Yongbin; Jiang, Chuanwu; Zhu, Weiping; Han, Hongxing; Ju, Fang

2018-04-01

Glioblastoma multiforme (GBM) is the most prevalent primary malignancy of the brain. This study was designed to investigate whether icaritin exerts anti-neoplastic activity against GBM in vitro. Cell Counting Kit-8 (CCK-8) assay was utilized to examine the viability of GBM cells. The apoptotic cell population was measured by flow cytometry analysis. Cell cycle distribution was detected by flow cytometry as well. Western blot analysis was performed to examine the level of biomarker proteins in GBM cells. Levels of PPARγ mRNA and protein were detected by qPCR and western blot analysis, respectively. To examine the role of PPARγ in the anti-neoplastic activity of icaritin, PPARγ antagonist GW9662 or PPARγ siRNA was used. The activity of PPARγ was determined by DNA binding and luciferase assays. Our findings revealed that icaritin markedly suppresses cell growth in a dose-dependent and time-dependent fashion. The cell population at the G0/G1 phase of the cell cycle was significantly increased following icaritin treatment. Meanwhile, icaritin promoted apoptotic cell death in T98G and U87MG cells. Further investigation showed upregulation of PPARγ played a key role in the anti-neoplastic activities of icaritin. Moreover, our result demonstrated activation of AMPK signaling by icaritin mediated the modulatory effect of icaritin on PPARγ. Our results suggest the PPARγ may mediate anti-neoplastic activities against GBM. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

[Expression in E.coli and bioactivity assay of Micrococcus luteus resuscitation promoting factor domain and its mutants].

PubMed

Yue, Chen-Li; Shi, Jie-Ran; Shi, Chang-Hong; Zhang, Hai; Zhao, Lei; Zhang, Ting-Fen; Zhao, Yong; Xi, Li

2008-10-01

To express Micrococcus luteus resuscitation promoting factor (Rpf) domain and its mutants in prokaryotic cells, and to investigate their bioactivity. The gene of Rpf domain and its mutants (E54K, E54A) were amplified by polymerase chain reaction (PCR) from the genome of Micrococcus luteus and cloned into pMD18-T vector. After sequenced, the Rpf domain and its mutant gene were subcloned into expression vector PGEX-4T-1, and transfected into E. coli DH5alpha. The expressed product was purified by affinity chromatography using GST Fusion Protein Purification bead. The aim proteins were identified by SDS-PAGE analysis and by Western blot with monoclonal antibodies against Rpf domain (mAb). The bioactivity of the proteins was analyzed by stimulating the resuscitation of Mycobacterium smegmatis. The sequences of the PCR products were identical to those of the Rpf domain and its mutant gene in GenBank. The relative molecular mass identified by SDS-PAGE analysis was consistent with that had been reported, which was also confirmed by Western blot analysis that there were specific bindings at 32 000 with Rpf domain mAb. The purified GST-Rpf domain could stimulate resuscitation of Mycobacterium smegmatis. Replacements E54A and especially E54K resulted in inhibition of Rpf resuscitation activity. Rpf domain and two kinds of its mutant protein were obtained, and its effects on the resuscitation of dormant Mycobacterium smegmatis were clarified.

Nonenzymatic chemiluminescent detection and quantitation of total protein on Western and slot blots allowing subsequent immunodetection and sequencing.

PubMed

Alba, F J; Daban, J R

1997-10-01

We have studied the light emission efficiency of proteins labeled with different fluorescent dyes chemically excited by the bis(2,4,6-trichlorophenyl)oxalate (TCPO)-H2O2 reaction. Using this peroxyoxalate chemiluminescence system, the best results were obtained with proteins covalently labeled with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF). Blotted proteins on polyvinylidene difluoride (PVDF) membranes can be labeled rapidly with MDPF. Our results demonstrate that energy from the excited intermediate produced in the TCPO-H2O2 reaction can be efficiently transferred to MDPF-labeled proteins in solution and on PVDF membranes. Although this nonenzymatic chemiluminescent system produces a background emission that reduces the sensitivity, the method developed in this work allows detection of 5 ng of protein in blots after 5 min exposure to X-ray film. Chemiluminescence of MDPF-labeled proteins on Western and slot blots may also be detected and quantified using a charge-coupled device (CCD) camera or a storage phosphor imaging system. This chemiluminescent method allows the staining of the total electrophoretic pattern but does not preclude further N-terminal sequencing and immunodetection of specific bands.

Gemcitabine inhibits proliferation and induces apoptosis in human pancreatic cancer PANC-1 cells.

PubMed

Yong-Xian, Gui; Xiao-Huan, Li; Fan, Zhang; Guo-Fang, Tian

2016-10-01

The aim of the study is to investigate the underlying molecular mechanisms by which gemcitabine (gem) inhibits proliferation and induces apoptosis in human pancreatic cancer PANC-1 cells in vitro. After PANC-1 cells had been treated by indicated concentration (0, 5, and 25 mg/L) of gem for 48 h, cell proliferation was evaluated by 3'-(4, 5 dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay; cell morphology was observed by transmission electron microscopy; Expression of c-IAP2 and Bcl-2 proteins was analyzed by Western blot; the activity of caspase-3 and -9 was detected by spectrophotometry. Gem significantly inhibited cell proliferation and could induce apoptosis of human pancreatic cancer PANC-1 cells, with a dose-dependent manner. Western blot analysis showed that gem significantly reduced c-IAP2 and Bcl-2 proteins expression level (P < 0.05). Spectrophotometric assay showed that gem significantly increased caspase-3 and -9 activity in PANC-1 cells. Gem could induce apoptosis of human pancreatic cancer PANC-1 cells, probably through downregulating c-IAP2 and Bcl-2 expression levels, and at the same time activating caspase-3 and -9.

Evaluation of Toxocara cati Excretory-Secretory Larval Antigens in Serodiagnosis of Human Toxocariasis.

PubMed

Zibaei, Mohammad; Sadjjadi, Seyed Mahmoud; Sarkari, Bahador; Uga, Shoji

2016-05-01

Toxocariasis is the clinical term that is applied to infection in the human host with Toxocara species larvae. Serological tests are important tools for the diagnosis of toxocariasis. The aim of this study was to evaluate the excretory-secretory (ES) antigens of T. cati larvae using enzyme-linked immunosorbent assay (ELISA) and also Western blotting for serodiagnosis of human toxocariasis. The ES antigens were prepared from T. cati third-stage larvae. Serum samples were obtained from 33 confirmed cases of toxocariasis, 35 patients infected with other parasitic diseases, and 30 from healthy individuals tested with ELISA and immunoblotting. The ELISA showed appropriate performance in term of specificity (96.7%) and sensitivity (97.0%). Electrophoretic analysis of T. cati ES antigens revealed a range of 20- to 150-kDa fractions. The highest sensitivity was achieved with 42- and 50-kDa fractions. The ELISA analyses using T. cati ES antigens demonstrated good sensitivity and specificity compared to T. canis ES as antigens for diagnosis of human toxocariasis. Accordingly, application of Western blotting, based on 42- and 50-kDa fractions of ES antigens, can be recommended for the accurate diagnosis of toxocariasis. © 2015 Wiley Periodicals, Inc.

SJL-1, a C-type lectin, acts as a surface defense molecule in Japanese sea cucumber, Apostichopus japonicus.

PubMed

Ono, Keisuke; Suzuki, Takuya Alan; Toyoshima, Youichi; Suzuki, Tomoya; Tsutsui, Shigeyuki; Odaka, Tomoyuki; Miyadai, Toshiaki; Nakamura, Osamu

2018-05-01

The surface defense molecules of aquatic invertebrates against infectious microorganisms have remained largely unexplored. In the present study, hemagglutinins were isolated from an extract of body surface layer of Japanese sea cucumber, Apostichopus japonicus, by affinity chromatography with fixed rabbit erythrocyte membranes. The N-terminal sequence of a 15-kDa agglutinin was almost identical with that of SJL-1, a C-type lectin formerly identified in this species. Because cDNA sequence and tissue distribution of SJL-1 have not been reported, we performed cDNA sequencing, gene expression analysis, and western blotting and immunohistochemical evaluation with anti-recombinant SJL-1 (rSJL-1) antibodies. The hemagglutinin gene was transcribed mainly in the integument, tentacles, and respiratory tree. Western blotting revealed that SJL-I is present in a body surface rinse, indicating that SJL-1 is secreted onto the body surface. SJL-1-positive cells scattered beneath the outermost layer of the integument were detected by immunohistochemistry. Furthermore, rSJL-1 agglutinated Gram-positive and Gram-negative bacteria, and yeast. These results indicate that SJL-1 acts as a surface defense molecule in A. japonicus. Copyright © 2018 Elsevier Ltd. All rights reserved.

Pregnancy-associated glycoprotein, chymosin and pepsinogen immunoreactivity of proteins extracted from fetal gastric tissue in bovine species.

PubMed

Bella, A; Sousa, N M; Dehimi, M L; Beckers, J F

2012-06-01

The objective of this work was to investigate the expression of gastric aspartic proteinases in fundic and pyloric mucosa removed from bovine fetuses. For this purpose, fractions issued from classical biochemical protocols were analyzed by proteolytic method, by PAG-RIA and by Western blot with the use of antisera raised against both pepsinogens and PAG. A strong reaction of proteins extracted from the fundic mucosa collected at the beginning of pregnancy was revealed with both anti-bPAG-I and anti-bPAG-II antisera, suggesting the expression of pepsinogen F in bovine species. Concerning pyloric mucosa, the analysis by Western blot highlighted a very strong immunoreaction with the anti-bovine chymosin serum. Amino-terminal sequencing allowed to identify bovine fetuin and albumin in fundic extracts, chymosin in the pyloric mucosa extracts, as well as some unknown proteins in both mucosa. Despite no N-terminal microsequence corresponding to the hypothetical pepsinogen F could be identified, it cannot be excluded that an existing bovine pepsinogen F-like molecule could be degraded during the purification procedure or that co-purified proteins could be responsible for masking its N-terminal microsequence. Copyright © 2011 Elsevier Ltd. All rights reserved.

Experimental transmission and tissue tropism of white spot syndrome virus (WSSV) in two species of lobsters, Panulirus homarus and Panulirus ornatus.

PubMed

Syed Musthaq, S; Sudhakaran, R; Balasubramanian, G; Sahul Hameed, A S

2006-10-01

The susceptibility of two species of lobsters, Panulirus homarus and Panulirus ornatus to white spot syndrome virus (WSSV) was tested by oral route and intramuscular injection. The results revealed that these lobsters were as highly susceptible as marine shrimp when the WSSV was administered intramuscularly. The WSSV caused 100% mortality in both Panulirus homarus and Panulirus ornatus, at 168 and 120 h, respectively, after intramuscular injection and failed to cause mortality when given orally. The presence of WSSV in moribund lobsters was confirmed by single-step and nested PCR, Western blot, histology, and bioassay test. It was found in eyestalk, gill, head muscle, tail muscle, hemolymph, appendages, and stomach. In lobsters with oral route infection, all tested organs except stomach and head muscle was negative for WSSV by nested PCR at 120 h post-inoculation. The stomach and head muscle was positive by nested PCR at 120 h p.i., but negative at 168 h p.i. Western blot analysis was negative in all the tested organs of both species of lobster at 120 h post-inoculation by oral route.

A quantitative image cytometry technique for time series or population analyses of signaling networks.

PubMed

Ozaki, Yu-ichi; Uda, Shinsuke; Saito, Takeshi H; Chung, Jaehoon; Kubota, Hiroyuki; Kuroda, Shinya

2010-04-01

Modeling of cellular functions on the basis of experimental observation is increasingly common in the field of cellular signaling. However, such modeling requires a large amount of quantitative data of signaling events with high spatio-temporal resolution. A novel technique which allows us to obtain such data is needed for systems biology of cellular signaling. We developed a fully automatable assay technique, termed quantitative image cytometry (QIC), which integrates a quantitative immunostaining technique and a high precision image-processing algorithm for cell identification. With the aid of an automated sample preparation system, this device can quantify protein expression, phosphorylation and localization with subcellular resolution at one-minute intervals. The signaling activities quantified by the assay system showed good correlation with, as well as comparable reproducibility to, western blot analysis. Taking advantage of the high spatio-temporal resolution, we investigated the signaling dynamics of the ERK pathway in PC12 cells. The QIC technique appears as a highly quantitative and versatile technique, which can be a convenient replacement for the most conventional techniques including western blot, flow cytometry and live cell imaging. Thus, the QIC technique can be a powerful tool for investigating the systems biology of cellular signaling.

Effect of LED light spectra on starvation-induced oxidative stress in the cinnamon clownfish Amphiprion melanopus.

PubMed

Choi, Cheol Young; Shin, Hyun Suk; Choi, Young Jae; Kim, Na Na; Lee, Jehee; Kil, Gyung-Suk

2012-11-01

The present study aimed to test starvation-induced oxidative stress in the cinnamon clownfish Amphiprion melanopus illuminated by light-emitting diodes (LEDs): red (peak at 630 nm), green (peak at 530 nm), and blue (peak at 450 nm) within a visible light. We investigated the oxidative stress induced by starvation for 12 days during illumination with 3 LED light spectra through measuring antioxidant enzyme (superoxide dismutase [SOD] and catalase [CAT]) mRNA expression and activity; CAT western blotting; and measuring lipid peroxidation [LPO]), plasma H(2)O(2), lysozyme, glucose, alanine aminotransferase (AlaAT), aspartate aminotransferase (AspAT), and melatonin levels. In green and blue lights, expression and activity of antioxidant enzyme mRNA were significantly lower than those of other light spectra, results that are in agreement with CAT protein expression level by western blot analysis. Also, in green and blue lights, plasma H(2)O(2), lysozyme, glucose, AlaAT, AspAT, and melatonin levels were significantly lower than those in other light spectra. These results indicate that green and blue LEDs inhibit oxidative stress and enhance immune function in starved cinnamon clownfish. Copyright © 2012 Elsevier Inc. All rights reserved.

Comparison of Plasma Exosomes by Differential Ultracentrifugation and Solvent Precipitation Methods.

PubMed

Peng, Qiao; Zhang, Jing; Zhou, Gang

2018-06-01

Emerging evidence has identified that exosomes play a pivotal role in intercellular signal transmission. However, the standardized purification techniques to isolate high quality exosomes are still deficient at present. This study was to evaluate reproducibility and efficiency of differential ultracentrifugation and solvent precipitation-based kits by isolating plasma-derived exosomes from oral lichen planus patients. Morphology, exosomal biomarkers, particle size distribution, proteomic components, and protein yield of isolated exosomes were evaluated by transmission electron microscope, western blot, laser diffraction instrument, Coomassie staining, and BCA protein assay kit, respectively. TEM displayed representative cup-shaped morphology of exosomes and western blot identified exosomal biomarkers CD9 and CD63. The size distribution showed that particles by differential ultracentrifugation were mainly from 26.15 nm to 166.5 nm, while some of the particles obtained by solvent precipitation kits were larger than 1,000 nm. In addition, exosomes isolated by solvent precipitation kits showed a significantly higher amount of protein yield due to plasma albumin contamination. Both differential ultracentrifugation and precipitation based kits could successfully isolate plasma exosomes, and exosomes by differential ultracentrifugation were purer and more appropriate for further proteomic analysis.

Alpha lipoic acid (ALA) modulates expression of apoptosis associated proteins in hippocampus of rats exposed during postnatal period to sodium arsenite (NaAsO2).

PubMed

Dixit, Shilpi; Dhar, Pushpa; Mehra, Raj D

2015-01-01

The present study focused on the role of exogenous alpha lipoic acid (ALA) in amelioration of inorganic arsenic ( iAs ) induced effects on apoptosis and apoptosis associated proteins in developing rat hippocampus. NaAsO 2 (1.5/2.0 mg/kg bw) alone or along with ALA (70 mg/kg bw) was administered to rat pups (experimental groups) by intraperitoneal (i.p.) route from postnatal day (PND) 4-15. Controls received no treatment/distilled water/ALA. On PND 16, the animals were perfusion fixed and the brains were processed for paraffin embedding (CV and TUNEL staining) and cryopreservation (immunohistochemistry). The fresh brain tissue was used for Western blotting. Significant increase was observed in TUNEL positive cells and Bax (pro-apoptotic protein) expression in hippocampal sub-regions of iAs alone treated groups, whereas Bcl-2 expression was intensified in animals receiving ALA with iAs . Densitometric analysis (Western blots) revealed optimal restoration of Bax and Bcl-2 ratio in animals receiving ALA with iAs , thereby suggesting the protective role of ALA in iAs induced developmental neurotoxicity.

Identification of the interaction region between hemagglutinin components of the botulinum toxin complex.

PubMed

Suzuki, Tomonori; Miyashita, Shin-Ichiro; Hayashi, Shintaro; Miyata, Keita; Inui, Ken; Kondo, Yosuke; Miyazaki, Satoru; Ohyama, Tohru; Niwa, Koichi; Watanabe, Toshihiro; Sagane, Yoshimasa

2014-04-01

The large toxin complex (L-TC) produced by Clostridium botulinum is formed from the M-TC (BoNT/NTNHA complex) by conjugation of M-TC with HA-33/HA-17 trimer consists of two HA-33 proteins and a single HA-17 protein. This association is mediated by HA-70, which interacts with HA-17. The current study aims to identify the regions of the HA-70 molecule that adhere to the HA-33/HA-17 complex. Products from limited proteolysis of HA-70 were resolved by SDS-PAGE and transferred onto PVDF membranes, where they were probed with HA-33/HA-17 in a far-western blot. Among the HA-70 fragments, HA-33/HA-17 bound to those containing at least the C-terminal half of the HA-70 molecule, but not those carrying the N-terminal half. Additional docking simulation analysis indicated that the HA-70 region Gln420-Tyr575 is responsible for binding to HA-17, which is consistent with the far-western blot data. The findings here reveal additional details concerning the three-dimensional structure of the functional HA sub-complex in the botulinum toxin complex. Copyright © 2014 Elsevier B.V. All rights reserved.

Minocycline Attenuates Iron-Induced Brain Injury.

PubMed

Zhao, Fan; Xi, Guohua; Liu, Wenqaun; Keep, Richard F; Hua, Ya

2016-01-01

Iron plays an important role in brain injury after intracerebral hemorrhage (ICH). Our previous study found minocycline reduces iron overload after ICH. The present study examined the effects of minocycline on the subacute brain injury induced by iron. Rats had an intracaudate injection of 50 μl of saline, iron, or iron + minocycline. All the animals were euthanized at day 3. Rat brains were used for immunohistochemistry (n = 5-6 per each group) and Western blotting assay (n = 4). Brain swelling, blood-brain barrier (BBB) disruption, and iron-handling proteins were measured. We found that intracerebral injection of iron resulted in brain swelling, BBB disruption, and brain iron-handling protein upregulation (p < 0.05). The co-injection of minocycline with iron significantly reduced iron-induced brain swelling (n = 5, p < 0.01). Albumin, a marker of BBB disruption, was measured by Western blot analysis. Minocycline significantly decreased albumin protein levels in the ipsilateral basal ganglia (p < 0.01). Iron-handling protein levels in the brain, including ceruloplasmin and transferrin, were reduced in the minocycline co-injected animals. In conclusion, the present study suggests that minocycline attenuates brain swelling and BBB disruption via an iron-chelation mechanism.

Role of the Conserved Oligomeric Golgi Complex in the Abnormalities of Glycoprotein Processing in Breast Cancer Cells

DTIC Science & Technology

2005-05-01

AD Award Number: DAMD17-03-1-0243 TITLE: Role of the Conserved Oligomeric Golgi Complex in the Abnormalities of Glycoprotein Processing in Breast...Glycoprotein Processing in Breast Cancer 5b.GRANTNUMBER Cells DAAD17-03-1-0243 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Sergey N... processing of glycoproteins, exocytosis, protein delivery systems, gene expression, western and northern blot analysis, immunotiuorescence, gradient

Western Blot Analysis of the Exotoxin Components From Bacillus anthracis Separated by Isoelectric Focusing Gel Electrophoresis

DTIC Science & Technology

2004-02-14

measured by using in vitro cell-based assays. This study provides another method of characterizing various isolates of B . anthracis by determining the...isoelectric points of the exotoxin components and may be useful in the development of protective vaccines against B . anthracis infection. 15. SUBJECT...LIMITATION OF ABSTRACT SAR 18. NUMBER OF PAGES 11 19a. NAME OF RESPONSIBLE PERSON a. REPORT unclassified b . ABSTRACT unclassified c. THIS PAGE

Effects of Extremely Low-Frequency Electromagnetic Fields on Neurogenesis and Cognitive Behavior in an Experimental Model of Hippocampal Injury

PubMed Central

Sakhaie, Mohammad Hassan; Pourheydar, Bagher; Majd, Zahra; Atefimanesh, Pezhman

2017-01-01

Exposure to extremely low-frequency electromagnetic fields may induce constant modulation in neuronal plasticity. In recent years, tremendous efforts have been made to design a suitable strategy for enhancing adult neurogenesis, which seems to be deterred due to brain senescence and several neurodegenerative diseases. In this study, we evaluated the effects of ELF-EMF on neurogenesis and memory, following treatment with trimethyltin chloride (TMT) as a neurotoxicant. The mice in all groups (n = 56) were injected with BrdU during the experiment for seven consecutive days to label newborn cells. Spatial memory was assessed by the Morris water maze (MWM) test. By the end of the experiment, neurogenesis and neuronal differentiation were assessed in the hippocampus, using immunohistochemistry and Western blot analysis. Based on the findings, exposure to ELF-EMF enhanced spatial learning and memory in the MWM test. ELF-EMF exposure significantly enhanced the number of BrdU+ and NeuN+ cells in the dentate gyrus of adult mice (P < 0.001 and P < 0.05, resp.). Western blot analysis revealed significant upregulation of NeuroD2 in ELF-EMF-exposed mice compared to the TMT-treated group (P < 0.05). These findings suggest that ELF-EMF might have clinical implications for the improvement of neurodegenerative processes and could help develop a novel therapeutic approach in regenerative medicine. PMID:29259353

Additive Effects of Rebamipide Plus Proton Pump Inhibitors on the Expression of Tight Junction Proteins in a Rat Model of Gastro-Esophageal Reflux Disease.

PubMed

Gweon, Tae-Geun; Park, Jong-Hyung; Kim, Byung-Wook; Choi, Yang Kyu; Kim, Joon Sung; Park, Sung Min; Kim, Chang Whan; Kim, Hyung-Gil; Chung, Jun-Won

2018-01-15

The aim of this study was to investigate the effects of rebamipide on tight junction proteins in the esophageal mucosa in a rat model of gastroesophageal reflux disease (GERD). GERD was created in rats by tying the proximal stomach. The rats were divided into a control group, a proton pump inhibitor (PPI) group, and a PPI plus rebamipide (PPI+R) group. Pantoprazole (5 mg/kg) was administered intraperitoneally to the PPI and PPI+R groups. An additional dose of rebamipide (100 mg/kg) was administered orally to the PPI+R group. Mucosal erosions, epithelial thickness, and leukocyte infiltration into the esophageal mucosa were measured in isolated esophagi 14 days after the procedure. A Western blot analysis was conducted to measure the expression of claudin-1, -3, and -4. The mean surface area of mucosal erosions, epithelial thickness, and leukocyte infiltration were lower in the PPI group and the PPI+R group than in the control group. Western blot analysis revealed that the expression of claudin-3 and -4 was significantly higher in the PPI+R group than in the control group. Rebamipide may exert an additive effect in combination with PPI to modify the tight junction proteins of the esophageal mucosa in a rat model of GERD. This treatment might be associated with the relief of GERD symptoms.

Exosome enrichment of human serum using multiple cycles of centrifugation.

PubMed

Kim, Jeongkwon; Tan, Zhijing; Lubman, David M

2015-09-01

In this work, we compared the use of repeated cycles of centrifugation at conventional speeds for enrichment of exosomes from human serum compared to the use of ultracentrifugation (UC). After removal of cells and cell debris, a speed of 110 000 × g or 40 000 × g was used for the UC or centrifugation enrichment process, respectively. The enriched exosomes were analyzed using the bicinchoninic acid assay, 1D gel separation, transmission electron microscopy, Western blotting, and high-resolution LC-MS/MS analysis. It was found that a five-cycle repetition of UC or centrifugation is necessary for successful removal of nonexosomal proteins in the enrichment of exosomes from human serum. More significantly, 5× centrifugation enrichment was found to provide similar or better performance than 5× UC enrichment in terms of enriched exosome protein amount, Western blot band intensity for detection of CD-63, and numbers of identified exosome-related proteins and cluster of differentiation (CD) proteins. A total of 478 proteins were identified in the LC-MS/MS analyses of exosome proteins obtained from 5× UCs and 5× centrifugations including many important CD membrane proteins. The presence of previously reported exosome-related proteins including key exosome protein markers demonstrates the utility of this method for analysis of proteins in human serum. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A meiotic DNA polymerase from a mushroom, Agaricus bisporus.

PubMed Central

Takami, K; Matsuda, S; Sono, A; Sakaguchi, K

1994-01-01

A meiotic DNA polymerase [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7], which likely has a role in meiotic DNA repair, was isolated from a mushroom, Agaricus bisporus. The purified fraction displays three bands in SDS/PAGE, at molecular masses of 72 kDa, 65 kDa and 36 kDa. Optimal activity is at pH 7.0-8.0 in the presence of 5 mM Mg2+ and 50 mM KCl and at 28-30 degrees C, which is the temperature for meiosis. This enzyme is resistant to N-ethylmaleimide and sensitive to 2',3'-dideoxythymidine 5'-triphosphate, suggesting that it is a beta-like DNA polymerase. These characteristics are similar to those of Coprinus DNA polymerase beta [Sakaguchi and Lu (1982) Mol. Cell. Biol. 2, 752-757]. In Western-blot analysis, the antiserum against the Coprinus polymerase reacts only with the 65 kDa band, which coincides with the molecular mass of the Coprinus polymerase. Western-blot analysis also showed that the antiserum could react with crude extracts not only from the Agaricales family, to which Agaricus and Coprinus belong, but also from different mushroom families and Saccharomyces. The Agaricus polymerase activity can be found only in the meiotic-cell-rich fraction, but the enzyme is also present in the somatic cells in an inactive state. Images Figure 2 Figure 5 Figure 6 PMID:8172591

[Construction and expression of six deletion mutants of human astrovirus C-terminal nsP1a/4 protein].

PubMed

Zhao, Wei; Niu, Ke; Zhao, Jian; Jin, Yi-ming; Sui, Ting-ting; Wang, Wen

2013-09-01

Human astrovirus (HAstV) is one of the leading causes of actue virual diarrhea in infants. HAstV-induced epithdlial cell apoptosis plays an important role in the pathogenesis of HAstV infection. Our previous study indicated that HAstV non-structural protein nsPla C-terminal protein nsPla/4 was the major apoptosis functional protein and probably contained the main apoptosis domains. In order to screen for astrovirus encoded apoptotic protien, nsPla/4 and six turncated proteins, which possessed nsPla/4 protein different function domain ,were cloned into green fluorescent protein (GFP) vector pEG-FP-N3. After 24-72 h transfection, the fusion protein expression in BHK21 cells, was analysis by fluorescence microscope and Western blot. The results indicated seven fusion proteins were observed successfully in BHK21 cell after transfected for 24 h. Western blot analysis showed that the level of fusion protein expressed in BHK21 cells was increased significantly at 72h compared to 48h in transfected cells. The successful expression of deletion mutants of nsPla/4 protein was an important foundation to gain further insights into the function of apoptosis domains of nsPla/4 protein and it would also provide research platform to further confirm the molecule pathogenic mechanism of human astrovirus.

Cardiotoxic Effects of Short-Term Doxorubicin Administration: Involvement of Connexin 43 in Calcium Impairment.

PubMed

Pecoraro, Michela; Rodríguez-Sinovas, Antonio; Marzocco, Stefania; Ciccarelli, Michele; Iaccarino, Guido; Pinto, Aldo; Popolo, Ada

2017-10-11

The use of Doxorubicin (DOXO), a potent antineoplastic agent, is limited by the development of cardiotoxicity. DOXO-induced cardiotoxicity is multifactorial, although alterations in calcium homeostasis, seem to be involved. Since even the Connexin43 (Cx43) plays a pivotal role in these two phenomena, in this study we have analyzed the effects of DOXO on Cx43 expression and localization. Damage caused by anthracyclines on cardiomyocytes is immediate after each injection, in the present study we used a short-term model of DOXO-induced cardiomyopathy. C57BL/6j female mice were randomly divided in groups and injected with DOXO (2 or 10 mg/kg i.p.) for 1-3 or 7 days once every other day. Cardiac function was assessed by Echocardiography. Sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCAII) and phospholamban (PLB) expression were assessed by Western blot analysis, intracellular [Ca 2+ ] were detected spectrofluorometrically by means of Fura-2 pentakis (acetoxymethyl) ester (FURA-2AM), and Cx43 and pCx43 expression and localization was analyzed by Western blot and confirmed by immunofluorescence analysis. DOXO induces impairment in Ca 2+ homeostasis, already evident after a single administration, and affects Cx43 expression and localization. Our data suggest that DOXO-induced alterations in Ca 2+ homeostasis causes in the cells the induction of compensatory mechanisms until a certain threshold, above which cardiac injury is triggered.

Fisetin and polymeric micelles encapsulating fisetin exhibit potent cytotoxic effects towards ovarian cancer cells.

PubMed

Xiao, Xue; Zou, Juan; Fang, Yin; Meng, Yibo; Xiao, Chao; Fu, Jiaxin; Liu, Shiyu; Bai, Peng; Yao, Yuan

2018-03-15

The anti-tumor activities of Natural compounds and their derivatives are of great interest to pharmaceutical industries. Fisetin is one of prospective natural compounds in this regard but unfortunately with poor hydrophilicity. The effects of unmodified and modified fisetin in cultured ovarian cancer cells were compared by transmission electronmicroscopy to determine apoptotic bodies, MTT assay to quantitate cell numbers, and fluorescence activated cell sorting analyse of various markers to determine the apoptotic state. In addition, the efficacy of fisetin and fisetin-micelles in vivo was determined by using immunocompromised mice. Apoptosis was measured by established markers using both western blot analysis and immunochemistry. Angiogenesis in a xenograft mouse model carring SKOV3 cells was evaluated by color Doppler ultrasound and immunohistochemistry. Multiple lines of evidence indicated that fisetin and fisetin micelles induce apoptosis in ovarian cancer cells in a dose-dependent manner. Histological analysis, terminal deoxynucleotidyltransferase-mediated nick-end labeling assay, western blot, immunohistochemical detection and microvessel density detection demonstrated that fisetin and fisetin micelles induced increased tumor apoptosis, proliferation suppression and antiangiogenesis activities. As far as we know, the present study is the first time to demonstrate the potency of both fisetin and fisetin micelles inducing apoptosis in ovarian cancer cells. Further studies will be needed to validate the therapeutic potential of fisetin and fisetin micelles in ovarian cancer treatment.

Cervical Cancer Cell Line Secretome Highlights the Roles of Transforming Growth Factor-Beta-Induced Protein ig-h3, Peroxiredoxin-2, and NRF2 on Cervical Carcinogenesis.

PubMed

Kontostathi, Georgia; Zoidakis, Jerome; Makridakis, Manousos; Lygirou, Vasiliki; Mermelekas, George; Papadopoulos, Theofilos; Vougas, Konstantinos; Vlamis-Gardikas, Alexios; Drakakis, Peter; Loutradis, Dimitrios; Vlahou, Antonia; Anagnou, Nicholas P; Pappa, Kalliopi I

2017-01-01

Cancer cells acquire unique secretome compositions that contribute to tumor development and metastasis. The aim of our study was to elucidate the biological processes involved in cervical cancer, by performing a proteomic analysis of the secretome from the following informative cervical cell lines: SiHa (HPV16+), HeLa (HPV18+), C33A (HPV-), and HCK1T (normal). Proteins were analyzed by 2D gel electrophoresis coupled to MALDI-TOF-MS. Enrichment of secreted proteins with characteristic profiles for each cell line was followed by the identification of differentially expressed proteins. Particularly, transforming growth factor-beta-induced protein ig-h3 (Beta ig-h3) and peroxiredoxin-2 (PRDX2) overexpression in the secretome of cancer cell lines was detected and confirmed by Western blot. Bioinformatics analysis identified the transcription factor NRF2 as a regulator of differentially expressed proteins in the cervical cancer secretome. NRF2 levels were measured by both Western blot and Multiple Reaction Monitoring (MRM) in the total cell extract of the four cell lines. NRF2 was upregulated in SiHa and C33A compared to HCK1T. In conclusion, the secreted proteins identified in cervical cancer cell lines indicate that aberrant NRF2-mediated oxidative stress response (OSR) is a prominent feature of cervical carcinogenesis.

MicroRNA-21 promotes bone mesenchymal stem cells migration in vitro by activating PI3K/Akt/MMPs pathway.

PubMed

Lv, Chen; Yang, Shengwu; Chen, Xin; Zhu, Xiongbai; Lin, Wenjun; Wang, Lu; Huang, Zhengxiang; Wang, Mingyue; Tu, Guanjun

2017-12-01

MicroRNA-21 (miR-21) contributes to anti-apoptosis in bone marrow mesenchymal stem cells (BMSC), but its role in the migration of BMSCs remains vague. The aim of this study was to determine the possible effect of miR-21 on regulating BMSCs directional migration and the expression of MMP-2/MMP-9 in BMSCs in vitro. BMSCs were successfully infected with miR-21-up lentivirus. Cell migration using Transwell assay indicated that upregulated expression of miR-21 could significantly promote BMSCs migration. Western blot analysis indicated that miR-21 significantly upregulated the expression of MMP-2 and MMP-9, which were related to metastasis-associated genes. GM6001, the specific MMPs inhibitor, abrogated the upregulated expression of MMP-2/MMP-9 and abolished the positive effect of miR-21 on promoting BMSCs migration. Meanwhile, miR-21 significantly enhanced Akt phosphorylation, as measured by Western blot analysis. LY294002, an inhibitor of Akt activation, abrogated the phosphorylation of Akt and abolished the positive effect of miR-21 on promoting BMSCs migration and upregulating MMP-2/MMP-9 expression. These results suggest that miR-21 contributes to BMSCs migration by upregulating MMP-2/MMP-9, potentially via the PI3K/Akt pathway. Copyright © 2017 Elsevier Ltd. All rights reserved.

Distribution profiles of transient receptor potential melastatin- and vanilloid-related channels in rat spermatogenic cells and sperm.

PubMed

Li, Shilin; Wang, Xinghuan; Ye, Haixia; Gao, Weicheng; Pu, Xiaoyong; Yang, Zhonghua

2010-03-01

In the present study, we aimed to investigate the expression and distribution of transient receptor potential melastatin (TRPM)- and vanilloid (TRPV)- related channels in rat spermatogenic cells and spermatozoa. Spermatogenic cells and spermatozoa were obtained from male Sprague-Dawley rats. Reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of all TRPM and TRPV channel members with specific primers. Western blot analysis was applied for detecting the expression of TRPM and TRPV channel proteins. Immunohistochemistry staining for TRPM4, TRPM7 and TRPV5 was also performed in rat testis. The mRNAs of TRPM3, TRPM4, TRPM7 and TRPV5 were detected in the spermatogenic cells and spermatozoa in rat. Western blot analysis verified the expression of TRPM4, TRPM7 and TRPV5 in the rat spermatogenic cells and spermatozoa. Immunocytochemistry staining for TRPM and TRPV channel families indicated that TRPM4 and TRPM7 proteins were highly expressed in different stages of spermatogenic cells and spermatozoa, while TRPV5 protein was lowly expressed in these cells. Our results demonstrate that mRNAs or proteins for TRPM3, TRPM4, TRPM7 and TRPV5 exist in rat spermatogenic cells and spermatozoa. These data presented here may assist in elucidating the possible physiological function of TRPM and TRPV channels in spermatogenic cells and spermatozoa.

Duck hepatitis A virus structural proteins expressed in insect cells self-assemble into virus-like particles with strong immunogenicity in ducklings.

PubMed

Wang, Anping; Gu, Lingling; Wu, Shuang; Zhu, Shanyuan

2018-02-01

Duck hepatitis A virus (DHAV), a non-enveloped ssRNA virus, can cause a highly contagious disease in young ducklings. The three capsid proteins of VP0, VP1 and VP3 are translated within a single large open reading frame (ORF) and hydrolyzed by protease 3CD. However, little is known on whether the recombinant viral structural proteins (VPs) expressed in insect cells could spontaneously assemble into virus-like particles (VLPs) and whether these VLPs could induce protective immunity in young ducklings. To address these issues, the structural polyprotein precursor gene P1 and the protease gene 3CD were amplified by PCR, and the recombinant proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures and immunogenicity. The recombinant proteins expressed in Sf9 cells were detected by indirect immunofluorescence assay and Western blot analysis. Electron microscopy showed that the recombinant proteins spontaneously assembled into VLPs in insect cells. Western blot analysis of the purified VLPs revealed that the VLPs were composed with the three structural proteins. In addition, vaccination with the VLPs induced high humoral immune response and provided strong protection. Therefore, our findings may provide a framework for development of new vaccines for the prevention of duck viral hepatitis. Copyright © 2018 Elsevier B.V. All rights reserved.

Hydrogen sulfide ameliorates subarachnoid hemorrhage-induced neuronal apoptosis via the ROS-MST1 pathway

PubMed Central

Shi, Ligen; Lei, Jianwei; Xu, Hangzhe; Zheng, Jingwei; Wang, Yan; Peng, Yucong; Yu, Jun; Zhang, Jianmin

2017-01-01

Background Hydrogen sulfide (H2S) has shown a neuroprotective role in several cerebrovascular diseases. This study aimed to explore the underlying mechanisms of H2S in early brain injury after subarachnoid hemorrhage (SAH). Methods One hundred seventy-seven male Sprague-Dawley rats were employed in this study. Sodium hydrosulfide (NaHS), a donor of H2S, was injected intraperitoneally at 60 min after SAH was induced by endovascular perforation. Western blot analysis determined the expression of several proteins of interest, and an immunofluorescence assay was used to examine neuronal apoptosis. Results Exogenous NaHS markedly improved neurological scores, attenuated brain edema, and ameliorated neuronal apoptosis at 24 h after SAH induction. The underlying mechanisms of H2S in ameliorating neuronal apoptosis might be executed through inhibition of the activity of mammalian sterile 20-like kinase 1 (MST1) protein. Western blot analysis demonstrated that exogenous NaHS decreased cleaved MST1 (cl-MST1) while increasing full-length MST1 expression. This anti-apoptotic effect of H2S could be reversed by chelerythrine, which could activate MST1 via caspase-dependent cleavage. Conclusions Exogenous NaHS, as a donor of H2S, could ameliorate early brain injury after SAH by inhibiting neuronal apoptosis by reducing the activity of the MST1 protein. PMID:29088725

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Xing; Xu, Yanli; Meng, Qian

Colorectal cancer (CRC) is one of the most common types of malignant tumor worldwide. Currently, although many researchers have been devoting themselves in CRC studies, the process of locating biomarkers for CRC early diagnosis and prognostic is still very slow. Using a centrifugal proteomic reactor-based proteomic analysis of minute amount of colonic biopsies by enteroscopy sampling, 2620 protein groups were quantified between cancer mucosa and adjacent normal colorectal mucosa. Of which, 403 protein groups were differentially expressed with statistic significance between cancer and normal tissues, including 195 up-regulated and 208 down-regulated proteins in cancer tissues. Three proteins (SOD3, PRELP andmore » NGAL) were selected for further Western blot validation. And the resulting Western blot experimental results were consistent with the quantitative proteomic data. SOD3 and PRELP are down-regulated in CRC mucosa comparing to adjacent normal tissue, while NGAL is up-regulated in CRC mucosa. In conclusion, the centrifugal proteomic reactor-based label-free quantitative proteomic approach provides a highly sensitive and powerful tool for analyzing minute protein sample from tiny colorectal biopsies, which may facilitate CRC biomarkers discovery for diagnoses and prognoses. -- Highlights: •Minute amount of colonic biopsies by endoscopy is suitable for proteomic analysis. •Centrifugal proteomic reactor can be used for processing tiny clinic biopsy sample. •SOD3 and PRELP are down-regulated in CRC, while NGAL is up-regulated in CRC.« less

[Construction and expression of HSV-2gD-Hsp70 fusion protein gene].

PubMed

Fan, Jian-Yong; Yang, Hui-Lan; Wang, Ying; Guan, Lei

2006-11-01

To construct and express Hsp70-HSV2gD fusion protein. Genes of Hsp70 and HSV-2gD were subcloned into vectors pGEX-4T-1 respectively. After confirmed by DNA sequence analysis, the recombinant plasmids pGEX-4T-HSP-gD was transformed into E. coli DH5alpha and induced to express with IPTG. The expressed protein was characterized by SDS-PAGE and Western blot after purified. BALB/c mice were immunized with fusion proteins respectively via intra-m uscular injection. The proliferation of spleen lymphocytes, the level of y-IFN in culture and anti-HSV-2gD IgG antibody in serum was detected was detected. The expressed protein was analyzed by SDS-PAGE after induced with IPTG, which showed a new band with an apparent molecular mass corresponding to the predicted size (118 kD). Western Blotting analysis demonstrates that the purified Hsp70-HSV2gD fusion protein had specific binding activity. The stimulation indexes of spleen lymphocytes, the level of gamma-IFN in culture and anti-HSV-2gD IgG antibody in serum of GST-Hsp70-gD group was obviously higher than that of other groups (P < 0.05 respectively). The successful expression of the Hsp70-HSV2gD fusion protein, which can induce immune responses, laid a solid foundation for its further research.

Cardiomyocyte differentiation of rat bone marrow multipotent progenitor cells is associated with downregulation of Oct-4 expression.

PubMed

Lu, Tiewei; Pelacho, Beatriz; Hao, Hong; Luo, Min; Zhu, Jing; Verfaillie, Catherine M; Tian, Jie; Liu, Zhenguo

2010-10-01

This study was to determine if bone marrow multipotent adult progenitor cells (MAPCs) underwent cardiac specification and Oct-4 expression during their cardiomyocyte differentiation in vitro. MAPCs were isolated from rat bone marrow, treated with 5-azacytidine (5-aza, 1μM) for 24h, and cultured in a serum-free medium for cardiac differentiation for up to 35 days. The cells started to express early cardiac-specific genes Nkx2.5 and GATA-4 with a significant increase in their mRNA level within 24h after 5-aza treatment. Western blotting analysis and immunofluorescence staining revealed that the cardiac-specific proteins connexin-43 and troponin I were expressed in the cells 7 days after 5-aza treatment. Flow cytometry analysis demonstrated that over 37% of the cells were positive for troponin I by 35 days of differentiation, although the cells did not display spontaneous contraction. On the other hand, the undifferentiated MAPCs expressed a significant level of the stem-cell-specific marker Oct-4 that was dramatically decreased in the cells shortly after the initiation of cardiomyocyte differentiation as evaluated using real-time (RT)-polymerase chain reaction, Western blotting, immunofluorescence staining, and flow cytometry. These data indicated that MAPCs were able to effectively differentiate into cardiomyocyte-like cells after 5-aza induction in association with downregulation of Oct-4 expression.

Substance P Promotes the Progression of Endometrial Adenocarcinoma.

PubMed

Ma, Jing; Yuan, Shifa; Cheng, Jianxin; Kang, Shan; Zhao, Wenhong; Zhang, Jie

2016-06-01

It has been demonstrated that substance P (SP) promotes while neurokinin-1 receptor (NK-1R) antagonist inhibits the proliferation of several human cancer cells. Currently, it is still unknown whether such actions exist in human endometrial carcinoma. This study aimed to explore the role of SP/NK-1R signaling in the progression of endometrial adenocarcinoma. The expression levels of SP and NK-1R in endometrial adenocarcinoma tissues and Ishikawa cell line were detected by real-time quantitative PCR and Western blot analysis. The effects of SP on Ishikawa cells proliferation and invasion were analyzed using MTT assay and transwell matrigel invasion assay, respectively. The expression levels of matrix metalloproteinase 9 (MMP-9) and vascular endothelial growth factor C (VEGF-C) in Ishikawa cells after administration of SP were detected by real-time quantitative RCR and Western blot analysis. The expression levels of SP and NK-1R were significantly higher in endometrial adenocarcinoma tissues and Ishikawa cells than in normal endometrium. Substance P significantly enhanced the proliferation and invasion of Ishikawa cells. In addition, SP induced the expression of MMP-9 and VEGF-C in Ishikawa cells, whereas NK-1R antagonist inhibited these effects. Substance P plays an important role in the development of endometrial carcinoma by inducing the expression of MMP-9 and VEGF-C and promoting cancer cell proliferation and metastasis, which can be blocked by NK-1R antagonist.

Substance P provides neuroprotection in cerebellar granule cells through Akt and MAPK/Erk activation: evidence for the involvement of the delayed rectifier potassium current.

PubMed

Amadoro, G; Pieri, M; Ciotti, M T; Carunchio, I; Canu, N; Calissano, P; Zona, C; Severini, C

2007-05-01

In the current study, we have evaluated the ability of substance P (SP) and other neurokinin 1 receptor (NK1) agonists to protect, in a dose- and time-dependent manner, primary cultures of rat cerebellar granule cells (CGCs) from serum and potassium deprivation-induced cell death (S-K5). We also established the presence of SP high affinity NK1 transcripts and the NK1 protein localization in the membrane of a sub-population of CGCs. Moreover, SP significantly and dose-dependently reduced the Akt 1/2 and Erk1/2 dephosphorylation induced by S-K5 conditions, as demonstrated by Western blot analysis. Surprisingly, in SP-treated CGCs caspase-3 activity was not inhibited, while the calpain-1 activity was moderately reduced. Corroborating this result, SP blocked calpain-mediated cleavage of tau protein, as demonstrated by the reduced appearance of a diagnostic fragment of 17 kDa by Western blot analysis. In addition, SP induced a significant reduction of the delayed rectifier K+ currents (Ik) in about 42% of the patched neurons, when these were evoked with depolarizing potential steps. Taken together, the present results demonstrate that the activation of NK1 receptors expressed in CGCs promote the neuronal survival via pathways involving Akt and Erk activation and by inhibition of Ik which can contribute to the neuroprotective effect of the peptide.

Unraveling Molecular Differences of Gastric Cancer by Label-Free Quantitative Proteomics Analysis.

PubMed

Dai, Peng; Wang, Qin; Wang, Weihua; Jing, Ruirui; Wang, Wei; Wang, Fengqin; Azadzoi, Kazem M; Yang, Jing-Hua; Yan, Zhen

2016-01-21

Gastric cancer (GC) has significant morbidity and mortality worldwide and especially in China. Its molecular pathogenesis has not been thoroughly elaborated. The acknowledged biomarkers for diagnosis, prognosis, recurrence monitoring and treatment are lacking. Proteins from matched pairs of human GC and adjacent tissues were analyzed by a coupled label-free Mass Spectrometry (MS) approach, followed by functional annotation with software analysis. Nano-LC-MS/MS, quantitative real-time polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry were used to validate dysregulated proteins. One hundred forty-six dysregulated proteins with more than twofold expressions were quantified, 22 of which were first reported to be relevant with GC. Most of them were involved in cancers and gastrointestinal disease. The expression of a panel of four upregulated nucleic acid binding proteins, heterogeneous nuclear ribonucleoprotein hnRNPA2B1, hnRNPD, hnRNPL and Y-box binding protein 1 (YBX-1) were validated by Nano-LC-MS/MS, qRT-PCR, western blot and immunohistochemistry assays in ten GC patients' tissues. They were located in the keynotes of a predicted interaction network and might play important roles in abnormal cell growth. The label-free quantitative proteomic approach provides a deeper understanding and novel insight into GC-related molecular changes and possible mechanisms. It also provides some potential biomarkers for clinical diagnosis.

[Establishment of a human bladder cancer cell line stably co-expressing hSPRY2 and luciferase genes and its subcutaneous tumor xenograft model in nude mice].

PubMed

Yin, Xiaotao; Li, Fanglong; Jin, Yipeng; Yin, Zhaoyang; Qi, Siyong; Wu, Shuai; Wang, Zicheng; Wang, Lin; Yu, Jiyun; Gao, Jiangping

2017-03-01

Objective To establish a human bladder cancer cell line stably co-expressing human sprouty2 (hSPRY2) and luciferase (Luc) genes simultaneously, and develop its subcutaneous tumor xenograft model in nude mice. Methods The hSPRY2 and Luc gene segments were amplified by PCR, and were cloned into lentiviral vector pCDH and pLVX respectively to produce corresponding lentivirus particles. The J82 human bladder cancer cells were infected with these two kinds of lentivirus particles, and then further screened by puromycin and G418. The expressions of hSPRY2 and Luc genes were detected by bioluminescence, immunofluorescence and Western blot analysis. The screened J82-hSPRY2/Luc cells were injected subcutaneously into BALB/c nude mice, and the growth of tumor was monitored dynamically using in vivo fluorescence imaging system. Results J82-hSPRY2/Luc cell line stably expressing hSPRY2 and Luc genes was established successfully. Bioluminescence, immunofluorescence and Western blot analysis validated the expressions of hSPRY2 and Luc genes. The in vivo fluorescence imaging system showed obvious fluorescence in subcutaneous tumor xenograft in nude mice. Conclusion The J82-hSPRY2/Luc bladder cancer cell line and its subcutaneous tumor xenograft model in nude mice have been established successfully.

Coronin-1C is a novel biomarker for hepatocellular carcinoma invasive progression identified by proteomics analysis and clinical validation.

PubMed

Wu, Long; Peng, Chun-Wei; Hou, Jin-Xuan; Zhang, Yan-Hua; Chen, Chuang; Chen, Liang-Dong; Li, Yan

2010-02-24

To better search for potential markers for hepatocellular carcinoma (HCC) invasion and metastasis, proteomic approach was applied to identify potential metastasis biomarkers associated with HCC. Membrane proteins were extracted from MHCC97L and HCCLM9 cells, with a similar genetic background and remarkably different metastasis potential, and compared by SDS-PAGE and identified by ESI-MS/MS. The results were further validated by western blot analysis, immunohistochemistry (IHC) of tumor tissues from HCCLM9- and MHCC97L-nude mice, and clinical specimens. Membrane proteins were extracted from MHCC97L and HCCLM9 cell and compared by SDS-PAGE analyses. A total of 14 differentially expressed proteins were identified by ESI-MS/MS. Coronin-1C, a promising candidate, was found to be overexpressed in HCCLM9 cells as compared with MHCC97L cells, and validated by western blot and IHC from both nude mice tumor tissues and clinical specimens. Coronin-1C level showed an abrupt upsurge when pulmonary metastasis occurred. Increasing coronin-1C expression was found in liver cancer tissues of HCCLM9-nude mice with spontaneous pulmonary metastasis. IHC study on human HCC specimens revealed that more patients in the higher coronin-1C group had overt larger tumor and more advanced stage. Coronin-1C could be a candidate biomarker to predict HCC invasive behavior.

Cervical Cancer Cell Line Secretome Highlights the Roles of Transforming Growth Factor-Beta-Induced Protein ig-h3, Peroxiredoxin-2, and NRF2 on Cervical Carcinogenesis

PubMed Central

Zoidakis, Jerome; Makridakis, Manousos; Lygirou, Vasiliki; Mermelekas, George; Vougas, Konstantinos; Drakakis, Peter

2017-01-01

Cancer cells acquire unique secretome compositions that contribute to tumor development and metastasis. The aim of our study was to elucidate the biological processes involved in cervical cancer, by performing a proteomic analysis of the secretome from the following informative cervical cell lines: SiHa (HPV16+), HeLa (HPV18+), C33A (HPV−), and HCK1T (normal). Proteins were analyzed by 2D gel electrophoresis coupled to MALDI-TOF-MS. Enrichment of secreted proteins with characteristic profiles for each cell line was followed by the identification of differentially expressed proteins. Particularly, transforming growth factor-beta-induced protein ig-h3 (Beta ig-h3) and peroxiredoxin-2 (PRDX2) overexpression in the secretome of cancer cell lines was detected and confirmed by Western blot. Bioinformatics analysis identified the transcription factor NRF2 as a regulator of differentially expressed proteins in the cervical cancer secretome. NRF2 levels were measured by both Western blot and Multiple Reaction Monitoring (MRM) in the total cell extract of the four cell lines. NRF2 was upregulated in SiHa and C33A compared to HCK1T. In conclusion, the secreted proteins identified in cervical cancer cell lines indicate that aberrant NRF2-mediated oxidative stress response (OSR) is a prominent feature of cervical carcinogenesis. PMID:28261610

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

PubMed Central

Spracklen, Andrew J.; Tootle, Tina L.

2013-01-01

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis. PMID:24326735

Standardized Chinese Formula Xin-Ke-Shu inhibits the myocardium Ca2+ overloading and metabolic alternations in isoproterenol-induced myocardial infarction rats

PubMed Central

Liu, Yue-Tao; Zhou, Chao; Jia, Hong-Mei; Chang, Xing; Zou, Zhong-Mei

2016-01-01

Xin-Ke-Shu (XKS) is a traditional Chinese patent medicine used for treatment of coronary heart diseases in China. However, its mechanism of action is still unclear. In this paper, the mediation of XKS on the isoproterenol (ISO)-induced myocardial infarction (MI) rat were evaluated based on a tissue-targeted metabonomics in vitro/vivo. The result indicated that twelve metabolic pathways were involved in the therapeutic effect of XKS in vivo, where seven pathways were associated with the Ca2+ overloading mechanism. In agreement with regulation on metabolic variations, XKS markedly reversed the over-expressions of three involved proteins including phospholipase A2 IIA (PLA2 IIA), calcium/calmodulin-dependent protein kinase II (CaMK II) and Pro-Caspase-3. The metabolic regulations of XKS on H9c2 cell also partially confirmed its metabolic effect. These metabolic characteristics in vitro/vivo and western blotting analysis suggested that XKS protected from MI metabolic perturbation major via inhibition of Ca2+ overloading mechanism. Furthermore, 11 active ingredients of XKS exerted steady affinity with the three proteins through the molecular docking study. Our findings indicate that the metabonomics in vitro/vivo combined with western blotting analysis offers the opportunity to gain insight into the comprehensive efficacy of TCMs on the whole metabolic network. PMID:27457884

Regulation of membrane-associated mucins in the human corneal epithelial cells by dexamethasone.

PubMed

Seo, Kyoung Yul; Chung, So-Hyang; Lee, Joon H; Park, Mi Young; Kim, Eung Kweon

2007-07-01

To study the influence of dexamethasone on membrane-associated mucins produced by human corneal epithelial cells. Human corneal epithelial cells were cultured in medium supplemented with various concentrations of dexamethasone (ranging from 10 to 10 M). Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis using monoclonal antibodies specific for human MUC1 (HMFG-1), MUC4 (1G8), and MUC16 (OC125) were performed to evaluate the effect of dexamethasone on membrane-associated mucin expression. The effect of glucocorticoid receptor antagonist (RU38486) on dexamethasone-induced mucin expression was estimated. RT-PCR revealed that MUC1 and MUC16 gene expression were upregulated 48 hours after addition of dexamethasone and that MUC4 gene expression was downregulated in the same condition. Western blot analysis showed that MUC1 and MUC16 proteins were increased after addition of dexamethasone. However, MUC4 was not detected by anti-MUC4 monoclonal antibody (1G8) for ASGP-2 under our conditions. Treatment with RU38486 inhibited the changes of MUC1, MUC4, and MUC16 by dexamethasone; thus, the effect of dexamethasone on mucin expression is mediated by glucocorticoid receptors. This study shows that MUC1, MUC4, and MUC16 are regulated differently by dexamethasone in human corneal epithelial cells. External application of dexamethasone might affect the precorneal mucin.

Identification and Validation of Potential New Biomarkers for Prostate Cancer Diagnosis and Prognosis Using 2D-DIGE and MS

PubMed Central

Geisler, Cordelia; Gaisa, Nadine T.; Pfister, David; Fuessel, Susanne; Kristiansen, Glen; Braunschweig, Till; Gostek, Sonja; Beine, Birte; Diehl, Hanna C.; Jackson, Angela M.; Borchers, Christoph H.; Heidenreich, Axel; Meyer, Helmut E.; Knüchel, Ruth; Henkel, Corinna

2015-01-01

This study was designed to identify and validate potential new biomarkers for prostate cancer and to distinguish patients with and without biochemical relapse. Prostate tissue samples analyzed by 2D-DIGE (two-dimensional difference in gel electrophoresis) and mass spectrometry (MS) revealed downregulation of secernin-1 (P < 0.044) in prostate cancer, while vinculin showed significant upregulation (P < 0.001). Secernin-1 overexpression in prostate tissue was validated using Western blot and immunohistochemistry while vinculin expression was validated using immunohistochemistry. These findings indicate that secernin-1 and vinculin are potential new tissue biomarkers for prostate cancer diagnosis and prognosis, respectively. For validation, protein levels in urine were also examined by Western blot analysis. Urinary vinculin levels in prostate cancer patients were significantly higher than in urine from nontumor patients (P = 0.006). Using multiple reaction monitoring-MS (MRM-MS) analysis, prostatic acid phosphatase (PAP) showed significant higher levels in the urine of prostate cancer patients compared to controls (P = 0.012), while galectin-3 showed significant lower levels in the urine of prostate cancer patients with biochemical relapse, compared to those without relapse (P = 0.017). Three proteins were successfully differentiated between patients with and without prostate cancer and patients with and without relapse by using MRM. Thus, this technique shows promise for implementation as a noninvasive clinical diagnostic technique. PMID:25667921

Release of tissue inhibitor of metalloproteinase-2 from alginate microcapsule encapsulating genetically engineered cells

PubMed Central

Kim, Yeon Seong; Jeong, Young-II; Jin, Shu-Guang; Pei, Jian; Wen, Min; Kim, In-Young; Moon, Kyung-Sub; Jung, Tae-Young; Ryu, Hyang-Hwa; Jung, Shin

2013-01-01

Background In this study, 293T cells were genetically engineered to secrete tissue inhibitor of metalloproteinase-2 (TIMP2) and encapsulated into alginate microcapsules to continuously release TIMP2 protein. Methods The anti-invasive potential of the microcapsules was studied in vitro using brain tumor cells. The TIMP2 gene was transfected to 293T cells, and genetically engineered 293TIMP2 cells were encapsulated into alginate microcapsules. Release of TIMP2 protein was detected with Western blot analysis and the anti-invasive potential against U87MG cells was tested using gelatin zymography and a Matrigel assay. Results Cell viability within the alginate microcapsules was maintained at a cell density of 5 × 106. Because polycationic polymers are helpful for maintaining the mechanical strength of microcapsules with good cell viability, the alginate microcapsules were reinforced with chitosan (0.1% w/v). Expression of TIMP2 protein in cell lysates and secretion of TIMP2 into the conditioned medium was confirmed by Western blot analysis. Alginate microcapsules encapsulating 293TIMP2 cells released TIMP2 protein into the medium efficiently, where the TIMP2 protein participated in degradation of the matrix metalloproteinase-2 enzyme and inhibited invasion of U87MG cells. Conclusion Alginate microcapsules encapsulating 293TIMP2 cells are promising candidates for anti-invasive treatment of glioma. PMID:24231999

Quantitative proteomic analysis for high-throughput screening of differential glycoproteins in hepatocellular carcinoma serum

PubMed Central

Gao, Hua-Jun; Chen, Ya-Jing; Zuo, Duo; Xiao, Ming-Ming; Li, Ying; Guo, Hua; Zhang, Ning; Chen, Rui-Bing

2015-01-01

Objective Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths. Novel serum biomarkers are required to increase the sensitivity and specificity of serum screening for early HCC diagnosis. This study employed a quantitative proteomic strategy to analyze the differential expression of serum glycoproteins between HCC and normal control serum samples. Methods Lectin affinity chromatography (LAC) was used to enrich glycoproteins from the serum samples. Quantitative mass spectrometric analysis combined with stable isotope dimethyl labeling and 2D liquid chromatography (LC) separations were performed to examine the differential levels of the detected proteins between HCC and control serum samples. Western blot was used to analyze the differential expression levels of the three serum proteins. Results A total of 2,280 protein groups were identified in the serum samples from HCC patients by using the 2D LC-MS/MS method. Up to 36 proteins were up-regulated in the HCC serum, whereas 19 proteins were down-regulated. Three differential glycoproteins, namely, fibrinogen gamma chain (FGG), FOS-like antigen 2 (FOSL2), and α-1,6-mannosylglycoprotein 6-β-N-acetylglucosaminyltransferase B (MGAT5B) were validated by Western blot. All these three proteins were up-regulated in the HCC serum samples. Conclusion A quantitative glycoproteomic method was established and proven useful to determine potential novel biomarkers for HCC. PMID:26487969

[Prokaryotic expression of vp3 gene of Muscovy duck parvovirus, and its antiserum preparation for detection of virus multiplication].

PubMed

Huang, Yu; Zhu, Yumin; Dong, Shijuan; Yu, Ruisong; Zhang, Yuanshu; Li, Zhen

2015-01-01

New epidemic broke out in recent year which was suspected to be caused by variant Muscovy duck parvovirus (MDPV). For this reason, new MDPV detection methods are needed for the new virus strains. In this study, a pair of primers were designed according to the full-length genome of MDPV strain SAAS-SHNH, which were identified in 2012, and were used to amplify the vp3 gene of MDPV by polymerase chain reaction. After being sequenced, the vp3 gene was subcloned into the prokaryotic expression vector PET28a. The recombinant plasmid was transformed into E. coli BL21 and induced with IPTG. SDS-PAGE and Western blotting analysis showed the MDPV vp3 gene was successfully expressed. After being purified by Ni2+ affinity chromatography system, the recombinant protein was used as antigen to immunize rabbits to obtain antiserum. Western blotting analysis showed that the acquired antiserum could react specifically with VP3 protein of J3D6 strain and MDPV vaccine strain. The antiserum could also be used for detection of cultured MDPV from primary duck embryo fibroblasts by immune fluorescence assay (IFA). It could be concluded that the VP3 protein and its antibody prepared in the research could be used for detection of VP3 antiserum and antigen respectively.

PAd-shRNA-PTN reduces pleiotrophin of pancreatic cancer cells and inhibits neurite outgrowth of DRG

PubMed Central

Yao, Jun; Zhang, Min; Ma, Qing-Yong; Wang, Zheng; Wang, Lian-Cai; Zhang, Dong

2011-01-01

AIM: To investigate the silencing effects of pAd-shRNA-pleiotrophin (PTN) on PTN in pancreatic cancer cells, and to observe the inhibition of pAd-shRNA-PTN on neurite outgrowth from dorsal root ganglion (DRG) neurons in vitro. METHODS: PAd-shRNA-PTN was used to infect pancreatic cancer BxPC-3 cells; assays were conducted for knockdown of the PTN gene on the 0th, 1st, 3rd, 5th, 7th and 9th d after infection using immunocytochemistry, real-time quantitative polymerase chain reaction (PCR), and Western blotting analysis. The morphologic changes of cultured DRG neurons were observed by mono-culture of DRG neurons and co-culture with BXPC-3 cells in vitro. RESULTS: The real-time quantitative PCR showed that the inhibition rates of PTN mRNA expression in the BxPC-3 cells were 20%, 80%, 50% and 25% on the 1st, 3rd, 5th and 7th d after infection. Immunocytochemistry and Western blotting analysis also revealed the same tendency. In contrast to the control, the DRG neurons co-cultured with the infected BxPC-3 cells shrunk; the number and length of neurites were significantly decreased. CONCLUSION: Efficient and specific knockdown of PTN in pancreatic cancer cells and the reduction in PTN expression resulted in the inhibition of neurite outgrowth from DRG neurons. PMID:21677838

The Profile of Heparanase Expression Distinguishes Differentiated Thyroid Carcinoma from Benign Neoplasms

PubMed Central

Matos, Leandro Luongo; Suarez, Eloah Rabello; Theodoro, Thérèse Rachell; Trufelli, Damila Cristina; Melo, Carina Mucciolo; Garcia, Larissa Ferraz; Oliveira, Olivia Capela Grimaldi; Matos, Maria Graciela Luongo; Kanda, Jossi Ledo; Nader, Helena Bonciani; Martins, João Roberto Maciel; Pinhal, Maria Aparecida Silva

2015-01-01

Introduction The search for a specific marker that could help to distinguish between differentiated thyroid carcinoma and benign lesions remains elusive in clinical practice. Heparanase (HPSE) is an endo-beta-glucoronidase implicated in the process of tumor invasion, and the heparanase-2 (HPSE2) modulates HPSE activity. The aim of this study was to evaluate the role of heparanases in the development and differential diagnosis of follicular pattern thyroid lesions. Methods HPSE and HPSE2 expression by qRT-PCR, immunohistochemistry evaluation, western blot analysis and HPSE enzymatic activity were evaluated. Results The expression of heparanases by qRT-PCR showed an increase of HPSE2 in thyroid carcinoma (P = 0.001). HPSE activity was found to be higher in the malignant neoplasms than in the benign tumors (P<0.0001). On Western blot analysis, HPSE2 isoforms were detected only in malignant tumors. The immunohistochemical assay allowed us to establish a distinct pattern for malignant and benign tumors. Carcinomas showed a typical combination of positive labeling for neoplastic cells and negative immunostaining in colloid, when compared to benign tumors (P<0.0001). The proposed diagnostic test presents sensitivity and negative predictive value of around 100%, showing itself to be an accurate test for distinguishing between malignant and benign lesions. Conclusions This study shows, for the first time, a distinct profile of HPSE expression in thyroid carcinoma suggesting its role in carcinogenesis. PMID:26488476

Resistin modulates glucose uptake and glucose transporter-1 (GLUT-1) expression in trophoblast cells.

PubMed

Di Simone, Nicoletta; Di Nicuolo, Fiorella; Marzioni, Daniela; Castellucci, Mario; Sanguinetti, Maurizio; D'lppolito, Silvia; Caruso, Alessandro

2009-02-01

The adipocytokine resistin impairs glucose tolerance and insulin sensitivity. Here, we examine the effect of resistin on glucose uptake in human trophoblast cells and we demonstrate that transplacental glucose transport is mediated by glucose transporter (GLUT)-1. Furthermore, we evaluate the type of signal transduction induced by resistin in GLUT-1 regulation. BeWo choriocarcinoma cells and primary cytotrophoblast cells were cultured with increasing resistin concentrations for 24 hrs. The main outcome measures include glucose transport assay using [(3)H]-2-deoxy glucose, GLUT-1 protein expression by Western blot analysis and GLUT-1 mRNA detection by quantitative real-time RT-PCR. Quantitative determination of phospho(p)-ERK1/2 in cell lysates was performed by an Enzyme Immunometric Assay and Western blot analysis. Our data demonstrate a direct effect of resistin on normal cytotrophoblastic and on BeWo cells: resistin modulates glucose uptake, GLUT-1 messenger ribonucleic acid (mRNA) and protein expression in placental cells. We suggest that ERK1/2 phosphorylation is involved in the GLUT-1 regulation induced by resistin. In conclusion, resistin causes activation of both the ERK1 and 2 pathway in trophoblast cells. ERK1 and 2 activation stimulated GLUT-1 synthesis and resulted in increase of placental glucose uptake. High resistin levels (50-100 ng/ml) seem able to affect glucose-uptake, presumably by decreasing the cell surface glucose transporter.

Chronic nandrolone administration promotes oxidative stress, induction of pro-inflammatory cytokine and TNF-α mediated apoptosis in the kidneys of CD1 treated mice.

PubMed

Riezzo, Irene; Turillazzi, Emanuela; Bello, Stefania; Cantatore, Santina; Cerretani, Daniela; Di Paolo, Marco; Fiaschi, Anna Ida; Frati, Paola; Neri, Margherita; Pedretti, Monica; Fineschi, Vittorio

2014-10-01

Nandrolone decanoate administration and strenuous exercise increase the extent of renal damage in response to renal toxic injury. We studied the role played by oxidative stress in the apoptotic response caused by nandrolone decanoate in the kidneys of strength-trained male CD1 mice. To measure cytosolic enzyme activity, glutathione peroxidase (GPx), glutathione reductase (GR) and malondialdehyde (MDA) were determined after nandrolone treatment. An immunohistochemical study and Western blot analysis were performed to evaluate cell apoptosis and to measure the effects of renal expression of inflammatory mediators (IL-1β, TNF-α) on the induction of apoptosis (HSP90, TUNEL). Dose-related oxidative damage in the kidneys of treated mice is shown by an increase in MDA levels and by a reduction of antioxidant enzyme GR and GPx activities, resulting in the kidney's reduced radical scavenging ability. Renal specimens of the treated group showed relevant glomeruli alterations and increased immunostaining and protein expressions, which manifested significant focal segmental glomerulosclerosis. The induction of proinflammatory cytokine expression levels was confirmed by Western blot analysis. Long-term administration of nandrolone promotes oxidative injury in the mouse kidneys. TNF-α mediated injury due to nandrolone in renal cells appears to play a role in the activation of both the intrinsic and extrinsic apoptosis pathways. Copyright © 2014 Elsevier Inc. All rights reserved.

Tyrosine Kinase Inhibitor, Vatalanib, Inhibits Proliferation and Migration of Human Pterygial Fibroblasts.

PubMed

Kim, Hong Kyu; Choi, Ji-Young; Park, Sang Min; Rho, Chang Rae; Cho, Kyong Jin; Jo, Sangmee Ahn

2017-09-01

Vatalanib is a small-molecule tyrosine kinase inhibitor. We investigated the effects of vatalanib on the proliferation and migration of cultured human pterygial fibroblasts (HPFs). Pterygium tissues were obtained after pterygium excision surgery and subjected to primary culture. HPFs were treated with vatalanib at various concentrations. Mitomycin C (MMC) was used as a positive control. Cell proliferation and migration assays were used to investigate the effects of vatalanib. Cell death was measured using flow cytometry analysis. Western blot analysis was performed to identify signaling molecules associated with the response to vatalanib. Vatalanib inhibited both proliferation and migration of HPFs in a dose-dependent manner. Cell proliferation was significantly suppressed by vatalanib (10 and 100 μM) and MMC (0.004% and 0.04%) treatments. Migration assays revealed significant HPF delay when treated with vatalanib (1, 10, and 100 μM) and MMC (0.004% and 0.04%) compared with that in a negative control. Cell death analysis showed that high concentrations of vatalanib (100 μM) and MMC (0.004% and 0.04%) decreased cell numbers. Western blot analysis of vatalanib-treated cells showed vascular endothelial growth factor and transforming growth factor-β significantly reduced, but there was no alteration in p53 protein levels in HPFs. These results indicate that vatalanib significantly suppressed the proliferation and migration of HPFs by decreasing vascular endothelial growth factor and transforming growth factor-β. Vatalanib showed less toxicity than that of MMC. Based on these results, vatalanib may potentially serve as a new adjuvant treatment after pterygium excision surgery.

Molecular cloning, mRNA expression and tissue distribution analysis of Slc7a11 gene in alpaca (Lama paco) skins associated with different coat colors.

PubMed

Tian, Xue; Meng, Xiaolin; Wang, Liangyan; Song, Yunfei; Zhang, Danli; Ji, Yuankai; Li, Xuejun; Dong, Changsheng

2015-01-25

Slc7a11 encoding solute carrier family 7 member 11 (amionic amino acid transporter light chain, xCT), has been identified to be a critical genetic regulator of pheomelanin synthesis in hair and melanocytes. To better understand the molecular characterization of Slc7a11 and the expression patterns in skin of white versus brown alpaca (lama paco), we cloned the full length coding sequence (CDS) of alpaca Slc7a11 gene and analyzed the expression patterns using Real Time PCR, Western blotting and immunohistochemistry. The full length CDS of 1512bp encodes a 503 amino acid polypeptide. Sequence analysis showed that alpaca xCT contains 12 transmembrane regions consistent with the highly conserved amino acid permease (AA_permease_2) domain similar to other vertebrates. Sequence alignment and phylogenetic analysis revealed that alpaca xCT had the highest identity and shared the same branch with Camelus ferus. Real Time PCR and Western blotting suggested that xCT was expressed at significantly high levels in brown alpaca skin, and transcripts and protein possessed the same expression pattern in white and brown alpaca skins. Additionally, immunohistochemical analysis further demonstrated that xCT staining was robustly increased in the matrix and root sheath of brown alpaca skin compared with that of white. These results suggest that Slc7a11 functions in alpaca coat color regulation and offer essential information for further exploration on the role of Slc7a11 in melanogenesis. Copyright © 2014 Elsevier B.V. All rights reserved.

Occupational allergy to Spagulax®(Plantago ovata seed).

PubMed

Viñas, M; Pineda, F; Izquierdo-Domínguez, A; Castillo, M; Castillo, M J; Hernández, N; Ibero, M

2017-11-01

We report the case of a 36-year-old male pharmaceutical laboratory worker. On handling Spagulax ® sachets whose content is a laxative called Plantago ovata, he immediately presented rhinoconjunctivitis. Methods. Specific allergy study included SDS-PAGE with Western Blot and specific nasal challenge to Plantago ovata extract. Results. Prick by prick for Spagulax ® was negative. Total IgE: 126.5 U/mL. Western Blot recognized two proteins of 15 and 20 kDa in the extract of Plantago ovata and three proteins of 15, 18 and 50 kDa in the extract of Plantago lanceolata. Conclusions. We present a case of occupational allergy due to inhalation of and/or contact with Plantago ovata seeds.

Circulating anti-retinal antibodies as immune markers in age-related macular degeneration

PubMed Central

Patel, Nishal; Ohbayashi, Masahara; Nugent, Alex K; Ramchand, Kanchan; Toda, Masako; Chau, Kai-Yin; Bunce, Catey; Webster, Andrew; Bird, Alan C; Ono, Santa Jeremy; Chong, Victor

2005-01-01

Age-related macular maculopathy (ARM) and age-related macular degeneration (AMD) are the leading causes of blindness in the Western world. Despite the magnitude of this clinical problem, very little is known about the pathogenesis of the disease. In this study, we analysed the sera (using indirect immunohistochemistry and Western blot analysis) from a very large cohort of such patients and normal age-matched controls to detect circulating anti-retinal antibodies. Patients with bilateral drusen (n = 64) and with chorioretinal neovascularization (CNV) (n = 51) were recruited in addition to age-matched control subjects (n = 39). The sera were analysed for anti-retinal immunoglobulins on retinal sections. The data were then correlated with the clinical features graded according to the International Classification and Grading System of ARM and AMD. The sera of patients with drusen (93·75%) and CNV (82·27%) were found to have a significantly (P = 0·02) higher titre of autoantibodies to the retina in comparison with controls (8·69%), indicating significant evidence of involvement of the immune process in early stages of AMD. Subsequent statistical analysis of the drusen group showed significant progressive staining (P = 0·0009) in the nuclei layers from early to late stages of ARM. Western blotting confirmed the presence of anti-retinal immunoglobulins to retinal antigens. As anti-retinal immunoglobulins are present in patients with bilateral drusen and exudative AMD, these antibodies could play a significant role in the pathogenesis of AMD. Whilst we do not have evidence that these antibodies precede disease onset, the possibility that their presence might contribute to disease progression needs to be investigated. Finally, the eventual identification of the target antigens detected by these antibodies may permit the future development of new diagnostic methods for ARM and AMD. PMID:15946260

Growth and differentiation of human lens epithelial cells in vitro on matrix

NASA Technical Reports Server (NTRS)

Blakely, E. A.; Bjornstad, K. A.; Chang, P. Y.; McNamara, M. P.; Chang, E.; Aragon, G.; Lin, S. P.; Lui, G.; Polansky, J. R.

2000-01-01

PURPOSE: To characterize the growth and maturation of nonimmortalized human lens epithelial (HLE) cells grown in vitro. METHODS: HLE cells, established from 18-week prenatal lenses, were maintained on bovine corneal endothelial (BCE) extracellular matrix (ECM) in medium supplemented with basic fibroblast growth factor (FGF-2). The identity, growth, and differentiation of the cultures were characterized by karyotyping, cell morphology, and growth kinetics studies, reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence, and Western blot analysis. RESULTS: HLE cells had a male, human diploid (2N = 46) karyotype. The population-doubling time of exponentially growing cells was 24 hours. After 15 days in culture, cell morphology changed, and lentoid formation was evident. Reverse transcription-polymerase chain reaction (RT-PCR) indicated expression of alphaA- and betaB2-crystallin, fibroblast growth factor receptor 1 (FGFR1), and major intrinsic protein (MIP26) in exponential growth. Western analyses of protein extracts show positive expression of three immunologically distinct classes of crystallin proteins (alphaA-, alphaB-, and betaB2-crystallin) with time in culture. By Western blot analysis, expression of p57(KIP2), a known marker of terminally differentiated fiber cells, was detectable in exponential cultures, and levels increased after confluence. MIP26 and gamma-crystallin protein expression was detected in confluent cultures, by using immunofluorescence, but not in exponentially growing cells. CONCLUSIONS: HLE cells can be maintained for up to 4 months on ECM derived from BCE cells in medium containing FGF-2. With time in culture, the cells demonstrate morphologic characteristics of, and express protein markers for, lens fiber cell differentiation. This in vitro model will be useful for investigations of radiation-induced cataractogenesis and other studies of lens toxicity.

ET-1 Promotes Differentiation of Periodontal Ligament Stem Cells into Osteoblasts through ETR, MAPK, and Wnt/β-Catenin Signaling Pathways under Inflammatory Microenvironment

PubMed Central

Liang, Li; Zhou, Wei; Yang, Nan; Yu, Jifeng; Liu, Hongchen

2016-01-01

Periodontitis is a kind of chronic inflammatory disease that affects the tooth-supporting tissues. ET-1 is related to periodontitis and involved in the regulation of cytokines, but the mechanisms remain unclear. The aim of this study is to investigate how ET-1 affects proinflammatory cytokine expression and differentiation in human periodontal ligament stem cells (PDLSCs). PDLSCs were isolated from the periodontal ligament tissues of periodontitis patients and then treated with ET-1 (1, 10, or 100 nM) for 12 h, 24 h, or 72 h. The osteogenic potential of PDLSCs was tested using ALP staining. TNF-α, IL-1β, and IL-6 levels were evaluated by ELISA and western blot. Runx2, OCN, and COL1 mRNA and western levels were detected by RT-PCR and western blot, respectively. To examine the signaling pathways and molecular mechanisms involved in ET-1-mediated cytokine expression and osteogenic differentiation, ETR pathway, MAPKs pathway, Wnt/β-catenin pathway, and Wnt/Ca2+ pathway were detected by RT-PCR and western blot, respectively. ET-1 promoted differentiation of PDLSCs into osteoblasts by increasing secretion of TNF-α, IL-1β, and IL-6 in a dose- and time-dependent manner. ET-1 also increased expression of Runx2, OCN, and COL1. ET-1 promotes differentiation of PDLSCs into osteoblasts through ETR, MAPK, and Wnt/β-catenin signaling pathways under inflammatory microenvironment. PMID:26884650

Anti-inflammatory effect of garlic 14-kDa protein on LPS-stimulated-J774A.1 macrophages.

PubMed

Rabe, Shahrzad Zamani Taghizadeh; Ghazanfari, Tooba; Siadat, Zahra; Rastin, Maryam; Rabe, Shahin Zamani Taghizadeh; Mahmoudi, Mahmoud

2015-04-01

Garlic 14-kDa protein is purified from garlic (Allium sativum L.) which is used in traditional medicine and exerts various immunomodulatory activities. The present study investigated the suppressive effect of garlic 14-kDa protein on LPS-induced expression of pro-inflammatory mediators and underlying mechanism in inflammatory macrophages. J774A.1 macrophages were treated with 14-kDa protein (5-30 μg/ml) with/without LPS (1 μg/ml) and the production of inflammatory mediators such as prostaglandin E2 (PGE2), TNF-α, and IL-1β released were measured using ELISA. Nitric oxide (NO) production was determined using the Griess method. The anti-inflammatory activity of 14-kDa protein was examined by measuring inducible nitric oxide synthase and cyclooxygenase-2 proteins using western blot. The expression of nuclear NF-κB p65 subunit was assessed by western blot. Garlic 14-kDa protein significantly inhibited the excessive production of NO, PGE, TNF-α, and IL-1β in lipopolysaccharide (LPS)-activated J774A.1 macrophages in a concentration-related manner without cytotoxic effect. Western blot analysis demonstrated that garlic 14-kDa protein suppressed corresponding inducible NO synthase expression and activated cyclooxygenase-2 protein expression. The inhibitory effect was mediated partly by a reduction in the activity and expression of transcription factor NF-κB protein. Our results suggested, for the first time, garlic 14-kDa protein exhibits anti-inflammatory properties in macrophages possibly by suppressing the inflammatory mediators via the inhibition of transcription factor NF-κB signaling pathway. The traditional use of garlic as anti-inflammatory remedy could be ascribed partly to 14-kDa protein content. This protein might be a useful candidate for controlling inflammatory diseases and further investigations in vivo.

Novel Serum Biomarkers Detected by Protein Array in Polycystic Ovary Syndrome with Low Progesterone Level.

PubMed

Zheng, Qin; Zhou, Feifei; Cui, Xinyuan; Liu, Mulin; Li, Yulin; Liu, Shuai; Tan, Jichun; Yan, Qiu

2018-01-01

Polycystic ovary syndrome (PCOS), characterized by female infertility and metabolic abnormalities, is one of the most common endocrine disorders. The etiology of PCOS remains unknown. The comprehensive analysis of protein alterations in PCOS patients is meaningful for identifying diagnostic biomarkers of PCOS. Here, we explored the clinical value of serum proteins as novel biomarkers to detect PCOS with low progesterone level. A total of 43 patients with PCOS and 30 healthy women were enrolled. Protein array was used to detect the variations of serum proteins between PCOS patients and healthy women. The level of five serum proteins was further confirmed by ELISA and western blot. The human ovarian granulosa cells (KGN) was cultured to examine the underlying mechanism of PCOS. CCK8 assay and western blot were carried out to evaluate the alterations in proliferative ability, TUNEL assay and DAPI staining to detect the apoptosis of KGN cells. Among the 507 proteins, we identified 76 differentially expressed serum proteins (≧1.5 fold), with 40 elevated and 36 decreased proteins. Moreover, 47 proteins were newly reported in PCOS. The alterations in the five significantly decreased proteins (EREG, inhibin βA, IDE, PDGF-D and KNG1) were further confirmed by ELISA and western blot. The level of these proteins were directly associated with the low progesterone, and the expression could be upregulated by progesterone. EREG and inhibin βA also promoted the proliferation and inhibited the apoptosis of ovarian granulosa cells. The study highlights that serum proteins are differentially expressed in PCOS patients and healthy women, and EREG and inhibin βA levels are upregulated by progesterone, which are correlated with ovarian functions. The study suggests that EREG and inhibin βA may be applied as novel potential biomarkers for PCOS with low progesterone level. © 2018 The Author(s). Published by S. Karger AG, Basel.

DNA Repair Genes ERCC1 and BRCA1 Expression in Non-Small Cell Lung Cancer Chemotherapy Drug Resistance.

PubMed

Wang, Shuai; Liu, Feng; Zhu, Jingyan; Chen, Peng; Liu, Hongxing; Liu, Qi; Han, Junqing

2016-06-12

BACKGROUND Surgery combined with chemotherapy is an important therapy for non-small cell lung cancer (NSCLC). However, chemotherapy drug resistance seriously hinders the curative effect. Studies show that DNA repair genes ERCC1 and BRCA1 are associated with NSCLC chemotherapy, but their expression and mechanism in NSCLC chemotherapy drug-resistant cells has not been elucidated. MATERIAL AND METHODS NSCLC cell line A549 and drug resistance cell line A549/DDP were cultured. Real-time PCR and Western blot analyses were used to detect ERCC1 and BRCA1 mRNA expression. A549/DDP cells were randomly divided into 3 groups: the control group; the siRNA-negative control group (scramble group); and the siRNA ERCC1 and BRCA1siRNA transfection group. Real-time PCR and Western blot analyses were used to determine ERCC1 and BRCA1 mRNA and protein expression. MTT was used to detect cell proliferation activity. Caspase 3 activity was tested by use of a kit. Western blot analysis was performed to detect PI3K, AKT, phosphorylated PI3K, and phosphorylated AKT protein expression. RESULTS ERCC1 and BRCA1 were overexpressed in A549/DDP compared with A549 (P<0.05). ERCC1 and BRCA1siRNA transfection can significantly reduce ERCC1 and BRCA1 mRNA and protein expression (P<0.05). Downregulating ERCC1 and BRCA1 expression obviously inhibited cell proliferation and increased caspase 3 activity (P<0.05). Downregulating ERCC1 and BRCA1 significantly decreased PI3K and AKT phosphorylation levels (P<0.05). CONCLUSIONS ERCC1 and BRCA1 were overexpressed in NSCLC drug-resistant cells, and they regulated lung cancer occurrence and development through the phosphorylating PI3K/AKT signaling pathway.

Effects of strontium on proliferation and differentiation of rat bone marrow mesenchymal stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Yunfeng; Li, Jihua; Zhu, Songsong

Highlights: Black-Right-Pointing-Pointer Strontium ranelate (SrR) inhibits proliferation of BMMSCs. Black-Right-Pointing-Pointer SrR increases osteoblastic but decreases adipocytic differentiation of BMMSCs. Black-Right-Pointing-Pointer SrR increases expression of Runx2, BSP and OCN by BMMSCs in osteogenic medium. Black-Right-Pointing-Pointer SrR decreases expression of PPAR{gamma}, aP2/ALBP and LPL by BMMSCs in adipogenic medium. -- Abstract: Strontium ranelate (SrR) was an effective anti-osteoporotic drug to increase bone formation and decrease bone resorption. However, reports about the effect of SR on osteoblastic and adipocytic differentiation from bone marrow mesenchymal stem cells (BMMSCs) are limited. The purpose of this study is to evaluate whether SrR affects the ability ofmore » BMMSCs to differentiate into osteoblasts or adipocytes. Rat BMMSCs were identified by flow cytometry and exposed to SR (0.1 and 1.0 mM Sr{sup 2+}) under osteogenic or adipogenic medium for 1 and 2 weeks. The proliferation and differentiation of BMMSCs were analyzed by MTT, alkaline phosphatase (ALP), Oil red O staining, quantitative real-time RT-PCR and Western blot assays. SrR significantly inhibited the proliferation, increased osteoblastic but decreased adipocytic differentiation of rat BMMSCs dose-dependently. In osteogenic medium, SrR increased the expression of ALP, the mRNA levels of Cbfa1/Runx2, bone sialoprotein, and osteocalcin by RT-PCR, and the protein levels of Cbfa1/Runx2 by Western blot. In adipogenic medium, SrR decreased the mRNA levels of PPAR{gamma}2, adipocyte lipid-binding protein 2 (aP2/ALBP), and lipoprotein lipase (LPL) by RT-PCR, and the protein expression of PPAR{gamma} in Western blot analysis. These results indicated that the effects of SrR to promote osteoblastic but inhibit adipocytic differentiation of BMMSCs might contribute to its effect on osteoporosis treatment.« less

Human T-cell lymphotropic virus type 1 provirus and phylogenetic analysis in patients with mycosis fungoides and their family relatives.

PubMed

Shohat, M; Shohat, B; Mimouni, D; Pauli, G; Ellerbrok, H; David, M; Hodak, E

2006-08-01

Mycosis fungoides (MF) is a cutaneous T-cell lymphoma of unknown aetiology. A pathogenic role of human T-cell lymphotropic virus type 1 (HTLV-1) has been suggested but remains controversial. To determine whether MF is linked to HTLV-1. Blood samples were collected from 60 patients, 15 family relatives of patients with MF (MFRs), 20 healthy controls and 10 patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The presence of HTLV-1 antibodies in serum was tested by the Western blot rp21e-enhanced test. DNA was extracted from the blood with the Qiagen blood kit. We used 500 ng of DNA either in conventional HTLV-1-specific polymerase chain reaction (PCR) or in real-time PCR using primers sk43 and sk44 together with a tax-specific fluorescent probe. In Western blot, antibodies against three to four HTLV-1 antigens were detected in 52% of patients with MF. All of the patients with HAM/TSP were positive, while only 7% of the MFRs and none of the 20 healthy controls reacted with HTLV-1 antigens in Western blot. One of 60 patients with MF and one of 15 MFRs were positive in HTLV-1 PCR. These two PCR-positive samples which were quantified in real-time PCR showed that fewer than five in 10(6) cells were HTLV-1 infected. We succeeded in amplifying and sequencing the 5' end of the provirus from the blood of the PCR-positive MFR by seminested PCR. A positive result was also obtained in this test. Phylogenetic tree analyses revealed a high homology of this sequence with other HTLV-1 sequences from the Middle East. The above PCR-positive MFR was the brother of a PCR-negative patient with MF. These findings demonstrate that HTLV-1 is probably not the aetiological agent of MF. However, it may play a role in immunosuppression and in the spreading of the disease.

The downregulation of Mcl-1 via USP9X inhibition sensitizes solid tumors to Bcl-xl inhibition

PubMed Central

2012-01-01

Background It has been shown in many solid tumors that the overexpression of the pro-survival Bcl-2 family members Bcl-xL and Mcl-1 confers resistance to a variety of chemotherapeutic agents. Mcl-1 is a critical survival protein in a variety of cell lineages and is critically regulated via ubiquitination. Methods The Mcl-1, Bcl-xL and USP9X expression patterns in human lung and colon adenocarcinomas were evaluated via immunohistochemistry. Interaction between USP9X and Mcl-1 was demonstrated by immunoprecipitation-western blotting. The protein expression profiles of Mcl-1, Bcl-xL and USP9X in multiple cancer cell lines were determined by western blotting. Annexin-V staining and cleaved PARP western blotting were used to assay for apoptosis. The cellular toxicities after various treatments were measured via the XTT assay. Results In our current analysis of colon and lung cancer samples, we demonstrate that Mcl-1 and Bcl-xL are overexpressed and also co-exist in many tumors and that the expression levels of both genes correlate with the clinical staging. The downregulation of Mcl-1 or Bcl-xL via RNAi was found to increase the sensitivity of the tumor cells to chemotherapy. Furthermore, our analyses revealed that USP9X expression correlates with that of Mcl-1 in human cancer tissue samples. We additionally found that the USP9X inhibitor WP1130 promotes Mcl-1 degradation and increases tumor cell sensitivity to chemotherapies. Moreover, the combination of WP1130 and ABT-737, a well-documented Bcl-xL inhibitor, demonstrated a chemotherapeutic synergy and promoted apoptosis in different tumor cells. Conclusion Mcl-1, Bcl-xL and USP9X overexpression are tumor survival mechanisms protective against chemotherapy. USP9X inhibition increases tumor cell sensitivity to various chemotherapeutic agents including Bcl-2/Bcl-xL inhibitors. PMID:23171055

[Construction and expression of the eukaryotic expression vector carrying HSV-1 gC glycoprotein gene].

PubMed

Dang, Yin-li; Yan, Yan; Zhang, Xiao-xiao; Li, Pu-yuan; Yu, Lan; Zhang, Lei; Zhang, Fang-lin; Xu, Zhi-kai; Wu, Xing-an

2011-05-01

To stably express herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) in Chinese hamster ovary cells (CHO-K1). The eukaryotic expression vector pCI-mCMV-gC-1-IRES-DHFR-L22R was constructed and transfected into CHO-K1 cells by Lipofectamine 2000. The transfected cells were selected by G418 and methotrexate (MTX). The expression of HSV-1 gC was analyzed by Slot blot. HSV-1 gC proteins were purified with His-Ni Sepharose and then detected by Western blot. The eukaryotic expression vector pCI-mCMV-gC-1-IRES-DHFR-L22R was constructed successfully. CHO-K1 cells stably expressing HSV-1 gC proteins were established and confirmed by Western blot. The HSV-1 gC proteins have been expressed successfully and have good bioactivity. The results make it possible for further study and clinical use of HSV-1 gC.

Protein tyrosine phosphatase-1B (PTP1B) helps regulate EGF-induced stimulation of S-phase entry in human corneal endothelial cells

PubMed Central

Ishino, Yutaka; Zhu, Cheng; Harris, Deshea L.

2008-01-01

Purpose Human corneal endothelial cells (HCEC), particularly from older donors, only proliferate weakly in response to EGF. The protein tyrosine phosphatase, PTP1B, is known to negatively regulate EGF-induced signaling in several cell types by dephosphorylating the epidermal growth factor receptor (EGFR). The current studies were conducted to determine whether PTP1B plays a role in regulating cell cycle entry in HCEC in response to EGF stimulation. Methods Donor corneas were obtained from the National Disease Research Interchange and accepted for study based on established exclusion criteria. PTP1B was localized in the endothelium of ex vivo corneas and in cultured cells by immunocytochemistry. Western blot analysis verified PTP1B protein expression in HCEC and then compared the relative expression of EGFR and PTP1B in HCEC from young (<3 years old) and older donors (>60 years old). The effect of inhibiting the activity of PTP1B on S-phase entry was tested by comparing time-dependent BrdU incorporation in subconfluent HCEC incubated in the presence or absence of the PTP1B inhibitor, CinnGEL 2Me, before EGF stimulation. Results PTP1B was localized in a punctate pattern mainly within the cytoplasm of HCEC in ex vivo corneas and cultured cells. Western blots revealed the presence of three PTP1B-positive bands in HCEC and the control. Further western blot analysis showed no significant age-related difference in expression of EGFR (p=0.444>0.05); however, PTP1B expression was significantly higher in HCEC from older donors (p=0.024<0.05). Pre-incubation of HCEC with the PTP1B inhibitor significantly increased (p=0.019<0.05) the number of BrdU positive cells by 48 h after EGF stimulation. Conclusions Both immunolocalization and western blot studies confirmed that PTP1B is expressed in HCEC. Staining patterns strongly suggest that at least a subset of PTP1B is localized to the cytoplasm and most likely to the endoplasmic reticulum, the known site of EGFR/PTP1B interaction following EGF stimulation. PTP1B expression, but not EGFR expression, was elevated in HCEC from older donors, suggesting that the reduced proliferative activity of these cells in response to EGF is due, at least in part, to increased PTP1B activity. The fact that inhibition of PTP1B increased the relative number of cells entering S-phase strongly suggests that PTP1B helps negatively regulate EGF-stimulated cell cycle entry in HCEC. These results also suggest that it may be possible to increase the proliferative activity of HCEC, particularly in cells from older donors, by inhibiting the activity of this important protein tyrosine phosphatase. PMID:18253097

FOXO/TXNIP pathway is involved in the suppression of hepatocellular carcinoma growth by glutamate antagonist MK-801

PubMed Central

2013-01-01

Background Accumulating evidence has suggested the importance of glutamate signaling in cancer growth, yet the signaling pathway has not been fully elucidated. N-methyl-D-aspartic acid (NMDA) receptor activates intracellular signaling pathways such as the extracellular-signal-regulated kinase (ERK) and forkhead box, class O (FOXO). Suppression of lung carcinoma growth by NMDA receptor antagonists via the ERK pathway has been reported. However, series of evidences suggested the importance of FOXO pathways for the regulation of normal and cancer cell growth. In the liver, FOXO1 play important roles for the cell proliferation such as hepatic stellate cells as well as liver metabolism. Our aim was to investigate the involvement of the FOXO pathway and the target genes in the growth inhibitory effects of NMDA receptor antagonist MK-801 in human hepatocellular carcinoma. Methods Expression of NMDAR1 in cancer cell lines from different tissues was examined by Western blot. NMDA receptor subunits in HepG2, HuH-7, and HLF were examined by reverse transcriptase polymerase chain reaction (RT-PCR), and growth inhibition by MK-801 and NBQX was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of MK-801 on the cell cycle were examined by flow cytometry and Western blot analysis. Expression of thioredoxin-interacting protein (TXNIP) and p27 was determined by real-time PCR and Western blotting. Activation of the FOXO pathway and TXNIP induction were examined by Western blotting, fluorescence microscopy, Chromatin immunoprecipitation (ChIP) assay, and reporter gene assay. The effects of TXNIP on growth inhibition were examined using the gene silencing technique. Results NMDA receptor subunits were expressed in all cell lines examined, and MK-801, but not NBQX, inhibited cell growth of hepatocellular carcinomas. Cell cycle analysis showed that MK-801 induced G1 cell cycle arrest by down-regulating cyclin D1 and up-regulating p27. MK-801 dephosphorylated Thr24 in FOXO1 and induced its nuclear translocation, thus increasing transcription of TXNIP, a tumor suppressor gene. Knock-down of TXNIP ameliorated the growth inhibitory effects of MK-801. Conclusions Our results indicate that functional NMDA receptors are expressed in hepatocellular carcinomas and that the FOXO pathway is involved in the growth inhibitory effects of MK-801. This mechanism could be common in hepatocellular carcinomas examined, but other mechanisms such as ERK pathway could exist in other cancer cells as reported in lung carcinoma cells. Altered expression levels of FOXO target genes including cyclin D1 and p27 may contribute to the inhibition of G1/S cell cycle transition. Induction of the tumor suppressor gene TXNIP plays an important role in the growth inhibition by MK-801. Our report provides new evidence that FOXO-TXNIP pathway play a role in the inhibition of the hepatocellular carcinoma growth by MK-801. PMID:24112473

Normalized Quantitative Western Blotting Based on Standardized Fluorescent Labeling.

PubMed

Faden, Frederik; Eschen-Lippold, Lennart; Dissmeyer, Nico

2016-01-01

Western blot (WB) analysis is the most widely used method to monitor expression of proteins of interest in protein extracts of high complexity derived from diverse experimental setups. WB allows the rapid and specific detection of a target protein, such as non-tagged endogenous proteins as well as protein-epitope tag fusions depending on the availability of specific antibodies. To generate quantitative data from independent samples within one experiment and to allow accurate inter-experimental quantification, a reliable and reproducible method to standardize and normalize WB data is indispensable. To date, it is a standard procedure to normalize individual bands of immunodetected proteins of interest from a WB lane to other individual bands of so-called housekeeping proteins of the same sample lane. These are usually detected by an independent antibody or colorimetric detection and do not reflect the real total protein of a sample. Housekeeping proteins-assumed to be constitutively expressed mostly independent of developmental and environmental states-can greatly differ in their expression under these various conditions. Therefore, they actually do not represent a reliable reference to normalize the target protein's abundance to the total amount of protein contained in each lane of a blot.Here, we demonstrate the Smart Protein Layers (SPL) technology, a combination of fluorescent standards and a stain-free fluorescence-based visualization of total protein in gels and after transfer via WB. SPL allows a rapid and highly sensitive protein visualization and quantification with a sensitivity comparable to conventional silver staining with a 1000-fold higher dynamic range. For normalization, standardization and quantification of protein gels and WBs, a sample-dependent bi-fluorescent standard reagent is applied and, for accurate quantification of data derived from different experiments, a second calibration standard is used. Together, the precise quantification of protein expression by lane-to-lane, gel-to-gel, and blot-to-blot comparisons is facilitated especially with respect to experiments in the area of proteostasis dealing with highly variable protein levels and involving protein degradation mutants and treatments modulating protein abundance.

Transgenic approach to improve wheat (Triticum aestivum L.) nutritional quality.

PubMed

Tamás, Cecília; Kisgyörgy, Boglárka N; Rakszegi, Mariann; Wilkinson, Mark D; Yang, Moon-Sik; Láng, László; Tamás, László; Bedo, Zoltán

2009-07-01

An amaranth (Amaranthus hypochondriacus) albumin gene, encoding the 35-kDa AmA1 protein of the seed, with a high content of essential amino acids, was used in the biolistic transformation of bread wheat (Triticum aestivum L.) variety Cadenza. The transformation cassette carried the ama1 gene under the control of a powerful wheat endosperm-specific promoter (1Bx17 HMW-GS). Southern-blot analysis of T(1) lines confirmed the integration of the foreign gene, while RT-PCR and Western-blot analyses of the samples confirmed the transcription and translation of the transgene. The effects of the extra albumin protein on the properties of flour, produced from bulked T(2) seeds, were calculated using total protein and essential amino acid content analysis, polymeric/monomeric protein and HMW/LMW glutenin subunit ratio measurements. The results indicated that not only can essential amino acid content be increased, but some parameters associated with functional quality may also be improved because of the expression of the AmA1 protein.

Uridine 5'-Monophosphate Synthase Is Transcriptionally Regulated by Pyrimidine Levels in Nicotiana plumbaginifolia

PubMed

Santoso; Thornburg

1998-02-01

To understand the regulation and expression of pyrimidine biosynthesis in plants, we have examined the effect of the metabolic inhibitor 5-fluoroorotic acid (FOA) on uridine-5'-monophosphate synthase (UMPSase) expression in cell cultures of Nicotiana plumbaginifolia. UMPSase is the rate-limiting step of pyrimidine biosynthesis in plants. Addition of FOA causes an up-regulation of UMPSase enzyme activity in cell cultures after a lag phase of several days. Western-blot analysis demonstrated that the up-regulation in enzyme activity was caused by increased expression of the UMPSase protein. Northern-blot analysis demonstrated a higher level of UMPSase mRNA in the FOA-induced tissues than in control tissues. Run-on transcriptional assays showed that the UMPSase gene was transcriptionally activated after FOA treatment. The mechanism of toxicity of FOA is through thymine starvation. We found that addition of thymine abrogated the FOA-mediated up-regulation of UMPSase. In addition, methotrexate and aminopterin, which affect thymine levels by inhibiting dihydrofolate reductase, also up-regulate UMPSase in N. plumbaginifolia cells.

Oviductal extracellular vesicles (oviductosomes, OVS) are conserved in humans: murine OVS play a pivotal role in sperm capacitation and fertility.

PubMed

Bathala, Pradeepthi; Fereshteh, Zeinab; Li, Kun; Al-Dossary, Amal A; Galileo, Deni S; Martin-DeLeon, Patricia A

2018-03-01

Are extracellular vesicles (EVs) in the murine oviduct (oviductosomes, OVS) conserved in humans and do they play a role in the fertility of Pmca4-/- females? OVS and their fertility-modulating proteins are conserved in humans, arise via the apocrine pathway, and mediate a compensatory upregulation of PMCA1 (plasma membrane Ca2+-ATPase 1) in Pmca4-/- female mice during proestrus/estrus, to account for their fertility. Recently murine OVS were identified and shown during proestrus/estrus to express elevated levels of PMCA4 which they can deliver to sperm. PMCA4 is the major Ca2+ efflux pump in murine sperm and Pmca4 deletion leads to loss of sperm motility and male infertility as there is no compensatory upregulation of the remaining Ca2+ pump, PMCA1. Of the four family members of PMCAs (PMCA1-4), PMCA1 and PMCA4 are ubiquitous, and to date there have been no reports of one isoform being upregulated to compensate for another in any organ/tissue. Since Pmca4-/- females are fertile, despite the abundant expression of PMCA4 in wild-type (WT) OVS, we propose that OVS serve a role of packaging and delivering to sperm elevated levels of PMCA1 in Pmca4-/- during proestrus/estrus to compensate for PMCA4's absence. Fallopian tubes from pre-menopausal women undergoing hysterectomy were used to study EVs in the luminal fluid. Oviducts from sexually mature WT mice were sectioned after perfusion fixation to detect EVs in situ. Oviducts were recovered from WT and Pmca4-/- after hormonally induced estrus and sectioned for PMCA1 immunofluorescence (IF) (detected with confocal microscopy) and hematoxylin and eosin staining. Reproductive tissues, luminal fluids and EVs were recovered after induced estrus and after natural cycling for western blot analysis of PMCA1 and qRT-PCR of Pmca1 to compare expression levels in WT and Pmca4-/-. OVS, uterosomes, and epididymal luminal fluid were included in the comparisons. WT and Pmca4-/- OVS were analyzed for the presence of known PMCA4 partners in sperm and their ability to interact with PMCA1, via co-immunoprecipitation. In vitro uptake of PMCA1 from OVS was analyzed in capacitated and uncapacitated sperm via quantitative western blot analysis, IF localization and flow cytometry. Caudal sperm were also assayed for uptake of tyrosine-phosphorylated proteins which were shown to be present in OVS. Finally, PMCA1 and PMCA4 in OVS and that delivered to sperm were assayed for enzymatic activity. Human fallopian tubes were flushed to recover luminal fluid which was processed for OVS via ultracentrifugation. Human OVS were negatively stained for transmission electron microscopy (TEM) and subjected to immunogold labeling, to detect PMCA4. Western analysis was used to detect HSC70 (an EV biomarker), PMCA1 and endothelial nitric oxide synthase (eNOS) which is a fertility-modulating protein delivered to human sperm by prostasomes. Oviducts of sexually mature female mice were sectioned after perfusion fixation for TEM tomography to obtain 3D information and to distinguish cross-sections of EVs from those of microvilli and cilia. Murine tissues, luminal fluids and EVs were assayed for PMCA1 (IF and western blot) or qRT-PCR. PMCA1 levels from western blots were quantified, using band densities and compared in WT and Pmca4-/- after induced estrus and in proestrus/estrus and metestrus/diestrus in cycling females. In vitro uptake of PMCA1 and tyrosine-phosphorylated proteins was quantified with flow cytometry and/or quantitative western blot. Ca2+-ATPase activity in OVS and sperm before and after PMCA1 and PMCA4 uptake was assayed, via the enzymatic hydrolysis rate of ATP. TEM revealed that human oviducts contain EVs (exosomal and microvesicular). These EVs contain PMCA4 (immunolabeling), eNOS and PMCA1 (western blot) in their cargo. TEM tomography showed the murine oviduct with EV-containing blebs which typify the apocrine pathway for EV biogenesis. Western blots revealed that during proestrus/estrus PMCA1 was significantly elevated in the oviductal luminal fluid (OLF) (P = 0.02) and in OVS (P = 0.03) of Pmca4-/-, compared to WT. Further, while PMCA1 levels did not fluctuate in OLF during the cycle in WT, they were significantly (P = 0.02) higher in proestrus/estrus than at metestrus/diestrus in Pmca4-/-. The elevated levels of PMCA1 in proestrus/estrus, which mimics PMCA4 in WT, is OLF/OVS-specific, and is not seen in oviductal tissues, uterosomes or epididymal luminal fluid of Pmca4-/-. However, qRT-PCR revealed significantly elevated levels of Pmca1 transcript in Pmca4-/- oviductal tissues, compared to WT. PMCA1 could be transferred from OVS to sperm and the levels were significantly higher for capacitated vs uncapacitated sperm, as assessed by flow cytometry (P = 0.001) after 3 h co-incubation, quantitative western blot (P < 0.05) and the frequency of immuno-labeled sperm (P < 0.001) after 30 min co-incubation. Tyrosine phosphorylated proteins were discovered in murine OVS and could be delivered to sperm after their co-incubation with OVS, as detected by western, immunofluorescence localization, and flow cytometry. PMCA1 and PMCA4 in OVS were shown to be enzymatically active and this activity increased in sperm after OVS interaction. None. Although oviductal tissues of WT and Pmca4-/- showed no significant difference in PMCA1 levels, Pmca4-/- levels of OVS/OLF during proestrus/estrus were significantly higher than in WT. We have attributed this enrichment or upregulation of PMCA1 in Pmca4-/- partly to selective packaging in OVS to compensate for the lack of PMCA4. However, in the absence of a difference between WT and Pmca4-/- in the PMCA1 levels in oviductal tissues as a whole, we cannot rule out significantly higher PMCA1 expression in the oviductal epithelium that gives rise to the OVS as significantly higher Pmca1 transcripts were detected in Pmca4-/-. Since OVS and fertility-modulating cargo components are conserved in humans, it suggests that murine OVS role in regulating the expression of proteins required for capacitation and fertility is also conserved. Secondly, OVS may explain some of the differences in in vivo and in vitro fertilization for mouse mutants, as seen in mice lacking the gene for FER which is the enzyme required for sperm protein tyrosine phosphorylation. Our observation that murine OVS carry and can modulate sperm protein tyrosine phosphorylation by delivering them to sperm provides an explanation for the in vivo fertility of Fer mutants, not seen in vitro. Finally, our findings have implications for infertility treatment and exosome therapeutics. The work was supported by National Institute of Health (RO3HD073523 and 5P20RR015588) grants to P.A.M.-D. There are no conflicts of interests.

Generation and Characterization of Anti-CD34 Monoclonal Antibodies that React with Hematopoietic Stem Cells

PubMed Central

Aghebati Maleki, Leili; Majidi, Jafar; Baradaran, Behzad; Movassaghpour, Aliakbar; Abdolalizadeh, Jalal

2014-01-01

CD34 is a type I membrane protein with a molecular mass of approximately 110 kDa. This antigen is associated with human hematopoietic progenitor cells and is a differentiation stage-specific leukocyte antigen. In this study we have generated and characterized monoclonal antibodies (mAbs) directed against a CD34 marker. Mice were immunized with two keyhole lympet hemocyanin (KLH)-conjugated CD34 peptides. Fused cells were grown in hypoxanthine, aminopterine and thymidine (HAT) selective medium and cloned by the limiting dilution (L.D) method. Several monoclones were isolated by three rounds of limited dilutions. From these, we chose stable clones that presented sustained antibody production for subsequent characterization. Antibodies were tested for their reactivity and specificity to recognize the CD34 peptides and further screened by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. One of the mAbs (3D5) was strongly reactive against the CD34 peptide and with native CD34 from human umbilical cord blood cells (UCB) in ELISA and Western blotting analyses. The results have shown that this antibody is highly specific and functional in biomedical applications such as ELISA and Western blot assays. This monoclonal antibodies (mAb) can be a useful tool for isolation and purification of human hematopoietic stem cells (HSCs). PMID:24611141

Validation of endothelin B receptor antibodies reveals two distinct receptor-related bands on Western blot.

PubMed

Barr, Travis P; Kornberg, Daniel; Montmayeur, Jean-Pierre; Long, Melinda; Reichheld, Stephen; Strichartz, Gary R

2015-01-01

Antibodies are important tools for the study of protein expression but are often used without full validation. In this study, we used Western blots to characterize antibodies targeted to the N or C terminal (NT or CT, respectively) and the second or third intracellular loop (IL2 or IL3, respectively) of the endothelin B receptor (ETB). The IL2-targeted antibody accurately detected endogenous ETB expression in rat brain and cultured rat astrocytes by labeling a 50-kDa band, the expected weight of full-length ETB. However, this antibody failed to detect transfected ETB in HEK293 cultures. In contrast, the NT-targeted antibody accurately detected endogenous ETB in rat astrocyte cultures and transfected ETB in HEK293 cultures by labeling a 37-kDa band but failed to detect endogenous ETB in rat brain. Bands detected by the CT- or IL3-targeted antibody were found to be unrelated to ETB. Our findings show that functional ETB can be detected at 50 or 37kDa on Western blot, with drastic differences in antibody affinity for these bands. The 37-kDa band likely reflects ETB processing, which appears to be dependent on cell type and/or culture condition. Copyright © 2014 Elsevier Inc. All rights reserved.

Immunodiagnosis of Echinococcus Infections: Confirmatory Testing and Species Differentiation by a New Commercial Western Blot

PubMed Central

Liance, Martine; Janin, Veronique; Bresson-Hadni, Solange; Vuitton, Dominique-Angele; Houin, Rene; Piarroux, Renaud

2000-01-01

The Echinococcus Western Blot IgG (LDBIO Diagnostics, Lyon, France), using a whole larval antigen from Echinococcus multilocularis, was evaluated for serodiagnosis and differentiation between two human parasitic infections of worldwide importance: cystic echinococcosis, due to Echinococcus granulosus, and alveolar echinococcosis, due to E. multilocularis. Fifty and 61 serum samples from patients with cystic and alveolar echinococcosis, respectively, were used for assessing diagnostic sensitivity. The sensitivity of the assay was compared with those of screening tests used for these applications. Sera used for assessing cross-reactivities were from 154 patients with other diseases, either parasitic or not. The assay allowed the detection of serum immunoglobulin G antibodies in 97% of Echinococcus-infected patients. It had a higher sensitivity than screening assays for the detection for each echinococcosis. The assay allowed us to correctly distinguish between E. granulosus- and E. multilocularis-infected patients in 76% of cases. It did not allow us to distinguish active from inactive forms of both echinococcoses. The occurrence of cross-reactivities with neurocysticercosis indicates the necessity for retesting sera with species-specific antigens, for rare patients with neurologic disorders. This study shows the usefulness of the commercially available Echinococcus Western Blot IgG for the serological confirmation of human echinococcosis. PMID:11015390

H2Mab-77 is a Sensitive and Specific Anti-HER2 Monoclonal Antibody Against Breast Cancer.

PubMed

Itai, Shunsuke; Fujii, Yuki; Kaneko, Mika K; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Chang, Yao-Wen; Handa, Saori; Takahashi, Maki; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

2017-08-01

Human epidermal growth factor receptor 2 (HER2) plays a critical role in the progression of breast cancers, and HER2 overexpression is associated with poor clinical outcomes. Trastuzumab is an anti-HER2 humanized antibody that leads to significant survival benefits in patients with HER2-positive metastatic breast cancers. In this study, we developed novel anti-HER2 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. Initially, we expressed the full length or ectodomain of HER2 in LN229 glioblastoma cells and then immunized mice with ectodomain of HER2 or LN229/HER2, and performed the first screening by enzyme-linked immunosorbent assays using ectodomain of HER2. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical analyses (fourth screening). Among 100 mAb clones, only three mAbs reacted with HER2 in Western blot, and clone H 2 Mab-77 (IgG 1 , kappa) was selected. Finally, immunohistochemical analyses with H 2 Mab-77 showed sensitive and specific reactions against breast cancer cells, warranting the use of H 2 Mab-77 to detect HER2 in pathological analyses of breast cancers.

Lactoferrin Expression in Human and Murine Ocular Tissue.

PubMed

Rageh, Abrar A; Ferrington, Deborah A; Roehrich, Heidi; Yuan, Ching; Terluk, Marcia R; Nelson, Elizabeth F; Montezuma, Sandra R

2016-07-01

Lactoferrin (LF) is a multifunctional protein known to provide innate defense due to its antimicrobial and anti-inflammatory properties. In the eye, LF has been identified in the tears and vitreous humor. Its presence in other ocular tissues has not been determined. Our aim is to assess the presence of LF in the cornea, iris, retina and retinal pigment epithelium (RPE) of humans and mice. To test for the endogenous production of LF, reverse transcription polymerase chain reaction was performed in cultured human cells from the cornea and RPE and in murine tissues. To confirm LF localization in specific ocular tissue, immunohistochemistry was performed on flat mounts of cornea, retina and RPE in human donor eyes. The presence of LF was assessed by western blotting in human and mouse ocular tissue and human culture cells (cornea and RPE). To verify antibody specificity, purified human LF and transferrin (TF) were used on 1D and 2D western blots. LF gene expression was confirmed in the cornea and RPE cell cultures from humans, suggesting that LF is an endogenously produced protein. PCR results from mouse ocular tissue showed LF expression in cornea, iris, RPE, but not in retina. These results were also consistent with immunohistochemical localization of LF in human donor tissue. Antibody reaction for human LF was specific and western blotting showed its presence in the cornea, iris and RPE tissues. A faint reaction for the retina was observed but was likely due to contamination from other ocular tissues. Multiple commercially available antibodies for murine LF cross-reacted with TF, so no reliable results were obtained for murine western blot. LF is expressed in multiple eye tissues of humans and mice. This widespread expression and multifunctional activity of LF suggests that it may play an important role in protecting eye tissues from inflammation-associated diseases.

Type specificity of complement-fixing antibody against herpes simplex virus type 2 AG-4 early antigen in patients with asymptomatic infection.

PubMed Central

Sherlock, C H; Ashley, R L; Shurtleff, M L; Mack, K D; Corey, L

1986-01-01

We evaluated the type specificity of complement-fixing (CF) antibody against the AG-4 early antigen of herpes simplex virus (HSV) type 2 (HSV-2) by comparing a commercial AG-4 CF kit (Simplex-2; Gene Link Australia, Inc., Princeton, N.J.) with quantal microneutralization (MN) and absorption-Western blotting in testing sera from patients with and without a history of genital herpes. Sera characterized as HSV type 1 (HSV-1) or HSV-2 positive or negative by MN were selected and tested by CF, and those with discordant results were further analyzed for specific antibodies by absorption with HSV-1 or HSV-2 antigen and Western blotting with heterologous HSV proteins. A total of 34 of 42 (81%) sera HSV-2 positive by MN, 19 of 43 (44%) sera HSV-1 positive by MN, and 0 of 19 sera negative by MN were positive by CF. Absorption-Western blotting showed that 12 of 18 (67%) sera HSV-1 positive by MN but positive by CF had no HSV-2-specific antibody and that all 7 sera HSV-2 positive by MN but negative by CF had HSV-2-specific antibody. When MN and absorption-Western blotting data were combined to analyze patients with no history of genital herpes, 7 of 19 (37%) with no HSV-2-specific antibody were positive by CF, and 7 of 27 (26%) with HSV-2-specific antibody were negative by CF. The positive and negative predictive values for the CF test were 78 and 75%, respectively, in this group. The presence of antibody to the HSV AG-4 antigen does not discriminate sufficiently between HSV-1- and HSV-2-infected patients to be of value in predicting HSV-2 infection in the absence of symptomatic disease. Images PMID:3023439

Cloning and expression of clostridium acetobutylicum ATCC 824 acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase in Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cary, J.W.; Petersen, D.J.; Bennett, G.N.

1990-06-01

Coenzyme A (CoA)-transferase (acetoacetyl-CoA:acetate/butyrate:CoA-transferase (butyrate-acetoacetate CoA-transferase) (EC 2.8.3.9)) of Clostridium acetobutylicum ATCC 824 is an important enzyme in the metabolic shift between the acid-producing and solvent-forming states of this organism. The genes encoding the two subunits of this enzyme have been cloned and subsequent subcloning experiments established the position of the structural genes for CoA-transferase. Complementation of Escherichia coli ato mutants with the recombinant plasmid pCoAT4 (pUC19 carrying a 1.8-kilobase insert of C. acetobutylicum DNA encoding CoA-transferase activity) enabled the transformants to grow on butyrate as a sole carbon source. Despite the ability of CoA-transferase to complement the ato defectmore » in E. coli mutants, Southern blot and Western blot (immunoblot) analyses showed showed that neither the C. acetobutylicum genes encoding CoA-transferase nor the enzyme itself shared any apparent homology with its E. coli counterpart. Polypeptides of M{sub r} of the purified CoA-transferase subunits were observed by Western blot and maxicell analysis of whole-cell extracts of E.coli harboring pCoAT4. The proximity and orientation of the genes suggest that the genes encoding the two subunits of CoA-transferase may form an operon similar to that found in E. coli. In the plasmid, however, transcription appears to be primarily from the lac promoter of the vector.« less

Therapy of Prostate Cancer Using a Human Antibody Targeting the Type 1 Insulin-Like Growth Factor Receptor (IGF-IR)

DTIC Science & Technology

2009-09-01

euthanized, tumors harvested and portions processed for IHC, Western blot, flow cytometry , culture, and RNA analysis. If not enough tissue is available...temperature for 60 minutes. Samples were analyzed by flow cytometry using a BD FACScan. Data were analyzed with CellQuestPRO software. Evaluation of BrdUrd...were approved by the University of Washington Institutional Animal Care and Use Committee (IACUC). Flow cytometry . To measure tumor IGF-IR expression

Diversity, Replication, Pathogenicity and Cell Biology of Crimean Congo Hemorrhagic Fever Virus

DTIC Science & Technology

2010-10-01

in CCHFV pathogenesis or whether it is even O glycosylated. A third unusual feature is * Corresponding author. Mailing address: Department of Microbi ...CA), followed by Western blot analysis with mouse anti-V5 (Invitrogen) as the primary antibody and sheep anti-mouse horseradish peroxidase conjugate as...250) or with mouse anti-V5 MAb (diluted 1:500) (Invitrogen) in PBS containing 0.5 mM MgCl2 and 4% fetal bovine serum. In addition, TGN46, a sheep

Elucidating the Role of cAbl and the Abi-Family of cAbl Target Proteins in Cancer Development and Progression

DTIC Science & Technology

1999-07-01

patients with Ph’-positive leukemias also revealed loss of Abi proteins. We determined by RNase protection assay and reverse transcriptase polymerase...myelogenous leukemia . Abi protein levels also appeared unaltered by Western blot analysis of human lung, liver, colon, and breast carcinoma tissues as...generated in the presence of Bcr-Abl • Abi protein degradation was observed in Ph’+ leukemia -derived cells, but not in Ph1- leukemias or in human breast

Homogentisate 1,2 dioxygenase is expressed in human osteoarticular cells: implications in alkaptonuria.

PubMed

Laschi, Marcella; Tinti, Laura; Braconi, Daniela; Millucci, Lia; Ghezzi, Lorenzo; Amato, Loredana; Selvi, Enrico; Spreafico, Adriano; Bernardini, Giulia; Santucci, Annalisa

2012-09-01

Alkaptonuria (AKU) results from defective homogentisate1,2-dioxygenase (HGD), causing degenerative arthropathy. The deposition of ochronotic pigment in joints is so far attributed to homogentisic acid produced by the liver, circulating in the blood and accumulating locally. Human normal and AKU osteoarticular cells were tested for HGD gene expression by RT-PCR, mono- and 2D-Western blotting. HGD gene expression was revealed in chondrocytes, synoviocytes, osteoblasts. Furthermore, HGD expression was confirmed by Western blotting, that also revealed the presence of five enzymatic molecular species. Our findings indicate that AKU osteoarticular cells produce the ochronotic pigment in loco and this may strongly contribute to induction of ochronotic arthropathy. Copyright © 2011 Wiley Periodicals, Inc.

[Preparation and application of monoclonal antibodies against DR region of Na+-K+-ATPase α1 subunit].

PubMed

Yan, Xiaofei; Wu, Litao; DU, Xiaojuan; Li, Jing; Zhang, Fujun; Han, Yan; Lyu, Shemin; Li, Dongmin

2016-12-01

Objective To prepare monoclonal antibodies against DR region (897DVEDSYGQQWTYEQR911) of Na + -K + -ATPase α1 subunit and identify their properties. Methods BALB/c mice were immunized with DR-keyholelimpet hemocyanin (KLH). Splenocytes from the immunized mice were collected and subsequently fused with SP2/0 mouse myeloma cells. Positive hybridoma clones were obtained after cell fusion and selection. ELISA was used to detect DR antibody titer in the cell supernatants. DR region-specific monoclonal antibodies were analyzed by dot blotting, Western blotting and immunofluorescence assay. Na + -K + -ATPase activity was detected by SensoLyte R FDP Protein Phosphatase Assay Kit and the protective effect of the monoclonal antibody against high glucose-induced cell injury was assessed in H9c2 cells. Results Three hybridoma cell lines which secreted stable DR monoclonal antibody were obtained. The strongest positive cell line, named DRm217, was selected to prepare ascites. Dot blotting, Western blotting and immunofluorescence assay showed that DRm217 recognized specially DR region of Na + -K + -ATPase and bound on H9c2 cell membranes. DRm217 stimulated Na + -K + -ATPase activity and alleviated high glucose-induced H9c2 cells injury. Conclusion The monoclonal antibodies against DR region of Na + -K + -ATPase α1 subunit is prepared.

Diagnostic potential of low molecular weight excretory secretory proteins of Paramphistomum epiclitum for caprine amphistomosis.

PubMed

Jaiswal, Amit Kumar; Shanker, Daya; Sudan, Vikrant; Singh, Amit

2018-06-15

In the present study, the 75% alcoholic fractionation of excretory-secretory (ES) antigen isolated from 200 to 300 live P. epiclitum was assessed for its diagnostic potential for the detection of caprine amphistomosis by using antibody detection enzyme immunoassay. Prior to enzyme immunoassay, 75% alcoholic fractionation of excretory-secretory (ES) antigen was subjected to SDS- PAGE and western blot analysis for the presence of immunoreactive polypeptides. SDS-PAGE analysis of ES antigen resolved a total 7 polypeptides bands of size 56, 27, 25, 22.5, 12, 11 and 10 kDa. Western blot analysis revealed only two immunoreactive polypeptides (11 kDa and 12 kDa) when polypeptides resolved in SDS-PAGE were probed with known positive pooled serum. None of the polypeptides showed reactions with pooled known negative serum. The working dilutions of antigen, sera and conjugates were determined by checkerboard titration for employing ELISA and cut-off O.D. was calculated 0.616 by using the mean absorbance value of 11 negative kid sera. The sensitivity and specificity of ELISA was found to be 100% and 86.76%, respectively. As per kappa value estimation, the strength of agreement was found to be good. Antibodies to 75% alcoholic fractionation of ES antigen was detected in 20% goats (n = 160) of either sex, although faecal examination detected 10.6% goats to be infected with amphistomosis. The study confirmed that 75% alcoholic fractionation of ES antigen of P. epiclitum based ELISA had good value for serodiagnosis of caprine amphistomosis. Copyright © 2018 Elsevier B.V. All rights reserved.

Expression of insulin-like growth factor-1 and insulin-like growth factor-1 receptors in EL4 lymphoma cells overexpressing growth hormone.

PubMed

Weigent, Douglas A; Arnold, Robyn E

2005-03-01

Almost all of the previous studies with growth hormone (GH) have been done with exogenously supplied GH and, therefore, involve actions of the hormone through its receptor. However, the actions of endogenous or lymphocyte GH are still unclear. In a previous study, we showed that overexpression of GH (GHo) in a lymphoid cell line resulted in protection of the cells to apoptosis mediated by nitric oxide (NO). In the present study, we show that the protection from apoptosis could be transferred to control cells with culture fluids obtained from GHo cells and blocked by antibodies to the insulin-like growth factor-1 (IGF-1) or antibodies to the IGF-1-receptor (IGF-1R). Northern and Western blot analysis detected significantly higher levels of IGF-1 in cells overexpressing GH. An increase in the expression of the IGF-1R in GHo cells was also detected by Western blot analysis, (125)I-IGF-1 binding and analysis of IGF-1R promoter luciferase constructs. Transfection of GHo cells with a dominant negative IGF-1R mutant construct blocked the generation of NO and activation of Akt seen in GHo cells compared to vector alone control EL4 cells. The results suggest that one of the consequences of the overexpression of GH, in cells lacking the GH receptor, is an increase in the expression of IGF-1 and the IGF-1R which mediate the protection of EL4 lymphoma cells from apoptosis.

Identification, expression and phylogenetic analysis of EgG1Y162 from Echinococcus granulosus.

PubMed

Zhang, Fengbo; Ma, Xiumin; Zhu, Yuejie; Wang, Hongying; Liu, Xianfei; Zhu, Min; Ma, Haimei; Wen, Hao; Fan, Haining; Ding, Jianbing

2014-01-01

This study was to clone, identify and analyze the characteristics of egG1Y162 gene from Echinococcus granulosus. Genomic DNA and total RNAs were extracted from four different developmental stages of protoscolex, germinal layer, adult and egg of Echinococcus granulosus, respectively. Fluorescent quantitative PCR was used for analyzing the expression of egG1Y162 gene. Prokaryotic expression plasmid of pET41a-EgG1Y162 was constructed to express recombinant His-EgG1Y162 antigen. Western blot analysis was performed to detect antigenicity of EgG1Y162 antigen. Gene sequence, amino acid alignment and phylogenetic tree of EgG1Y162 were analyzed by BLAST, online Spidey and MEGA4 software, respectively. EgG1Y162 gene was expressed in four developmental stages of Echinococcus granulosus. And, egG1Y162 gene expression was the highest in the adult stage, with the relative value of 19.526, significantly higher than other three stages. Additionally, Western blot analysis revealed that EgG1Y162 recombinant protein had good reaction with serum samples from Echinococcus granulosus infected human and dog. Moreover, EgG1Y162 antigen was phylogenetically closest to EmY162 antigen, with the similarity over 90%. Our study identified EgG1Y162 antigen in Echinococcus granulosus for the first time. EgG1Y162 antigen had a high similarity with EmY162 antigen, with the genetic differences mainly existing in the intron region. And, EgG1Y162 recombinant protein showed good antigenicity.

Prognostic value of transformer 2β expression in prostate cancer.

PubMed

Diao, Yan; Wu, Dong; Dai, Zhijun; Kang, Huafeng; Wang, Ziming; Wang, Xijing

2015-01-01

Deregulation of transformer 2β (Tra2β) has been implicated in several cancers. However, the role of Tra2β expression in prostate cancer (PCa) is unclear. Therefore, this study was to investigate the expression of Tra2β in PCa and evaluated its association with clinicopathological variables and prognosis. Thirty paired fresh PCa samples were analyzed for Tra2β expression by Western blot analysis. Immunohistochemistry (IHC) assay was performed in 160 PCa samples after radical prostatectomy and adjacent non-cancerous tissues. Tra2β protein expression was divided into high expression group and low expression group by IHC. We also investigated the association of Tra2β expression with clinical and pathologic parameters. Kaplan-Meier plots and Cox proportional hazards regression model were used to analyze the association between Tra2β protein expression and prognosis of PCa patients. Our results showed that Tra2β was significantly upregulated in PCa tissues by western blot and IHC. Our data indicated that high expression of Tra2β was significantly associated with lymph node metastasis (P=0.002), clinical stage (P=0.015), preoperative prostate-specific antigen (P=0.003), Gleason score (P=0.001), and biochemical recurrence (P=0.021). High Tra2β expression was a significant predictor of poor biochemical recurrence free survival and overall survival both in univariate and multivariate analysis. We show that Tra2β was significantly upregulated in PCa patients after radical prostatectomy, and multivariate analysis confirmed Tra2β as an independent prognostic factor.

Altered glycosylation profile of purified plasma ACT from Alzheimer's disease.

PubMed

Ianni, Manuela; Manerba, Marcella; Di Stefano, Giuseppina; Porcellini, Elisa; Chiappelli, Martina; Carbone, Ilaria; Licastro, Federico

2010-12-16

Alzheimer's disease (AD) is one of the most frequent cause of neurodegenerative disorder in the elderly. Inflammation has been implicated in brain degenerative processes and peripheral markers of brain AD related impairment would be useful. Plasma levels of alpha-1-antichymotrypsin (ACT), an acute phase protein and a secondary component of amyloid plaques, are often increased in AD patients and high blood ACT levels correlate with progressive cognitive deterioration. During inflammatory responses changes in the micro-heterogeneity of ACT sugar chains have been described. N-Glycanase digestion from Flavobacterium meningosepticum (PNGase F) was performed on both native and denatured purified ACT condition and resolved to Western blot with the purpose to revealed the ACT de-glycosylation pattern.Further characterization of the ACT glycan profile was obtained by a glycoarray; each lectin group in the assay specifically recognizes one or two glycans/epitopes. Lectin-bound ACT produced a glyco-fingerprint and mayor differences between AD and controls samples were assessed by a specific algorithms. Western blot analysis of purified ACT after PNGase F treatment and analysis of sugar composition of ACT showed significantly difference in "glyco-fingerprints" patterns from controls (CTR) and AD; ACT from AD showing significantly reduced levels of sialic acid. A difference in terminal GlcNac residues appeared to be related with progressive cognitive deterioration. Low content of terminal GlcNac and sialic acid in peripheral ACT in AD patients suggests that a different pattern of glycosylation might be a marker of brain inflammation. Moreover ACT glycosylation analysis could be used to predict AD clinical progression and used in clinical trials as surrogate marker of clinical efficacy.

Vascular endothelial growth factor and nitric oxide synthase expression in human tooth germ development.

PubMed

Mastrangelo, F; Sberna, M T; Tettamanti, L; Cantatore, G; Tagliabue, A; Gherlone, E

2016-01-01

Vascular Endothelia Growth Factor (VEGF) and Nitric Oxide Synthase (NOS) expression, were evaluated in human tooth germs at two different stages of embryogenesis, to clarify the role of angiogenesis during tooth tissue differentiation and growth. Seventy-two third molar germ specimens were selected during oral surgery. Thirty-six were in the early stage and 36 in the later stage of tooth development. The samples were evaluated with Semi-quantitative Reverse Transcription-Polymerase chain Reaction analyses (RT-PcR), Western blot analysis (WB) and immunohistochemical analysis. Western blot and immunohistochemical analysis showed a VEGF and NOS 1-2-3 positive reaction in all samples analysed. VEGF high positive decrease reaction was observed in stellate reticulum cells, ameloblast and odontoblast clusters in early stage compared to later stage of tooth germ development. Comparable VEGF expression was observed in endothelial cells of early and advanced stage growth. NOS1 and NOS3 expressions showed a high increased value in stellate reticulum cells, and ameloblast and odontoblast clusters in advanced stage compared to early stage of development. The absence or only moderate positive reaction of NOS2 was detected in all the different tissues. Positive NOS2 expression showed in advanced stage of tissue development compared to early stage. The action of VEGF and NOS molecules are important mediators of angiogenesis during dental tissue development. VEGF high positive expression in stellate reticulum cells in the early stage of tooth development compared to the later stage and the other cell types, suggests a critical role of the stellate reticulum during dental embryo-morphogenesis.

Muscadine Grape Skin Extract Induces an Unfolded Protein Response-Mediated Autophagy in Prostate Cancer Cells: A TMT-Based Quantitative Proteomic Analysis

PubMed Central

Burton, Liza J.; Rivera, Mariela; Hawsawi, Ohuod; Zou, Jin; Hudson, Tamaro; Wang, Guangdi; Zhang, Qiang; Cubano, Luis; Boukli, Nawal; Odero-Marah, Valerie

2016-01-01

Muscadine grape skin extract (MSKE) is derived from muscadine grape (Vitis rotundifolia), a common red grape used to produce red wine. Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR) that serves as a survival mechanism to relieve ER stress and restore ER homeostasis. However, when persistent, ER stress can alter the cytoprotective functions of the UPR to promote autophagy and cell death. Although MSKE has been documented to induce apoptosis, it has not been linked to ER stress/UPR/autophagy. We hypothesized that MSKE may induce a severe ER stress response-mediated autophagy leading to apoptosis. As a model, we treated C4-2 prostate cancer cells with MSKE and performed a quantitative Tandem Mass Tag Isobaric Labeling proteomic analysis. ER stress response, autophagy and apoptosis were analyzed by western blot, acridine orange and TUNEL/Annexin V staining, respectively. Quantitative proteomics analysis indicated that ER stress response proteins, such as GRP78 were greatly elevated following treatment with MSKE. The up-regulation of pro-apoptotic markers PARP, caspase-12, cleaved caspase-3, -7, BAX and down-regulation of anti-apoptotic marker BCL2 was confirmed by Western blot analysis and apoptosis was visualized by increased TUNEL/Annexin V staining upon MSKE treatment. Moreover, increased acridine orange, and LC3B staining was detected in MSKE-treated cells, suggesting an ER stress/autophagy response. Finally, MSKE-mediated autophagy and apoptosis was antagonized by co-treatment with chloroquine, an autophagy inhibitor. Our results indicate that MSKE can elicit an UPR that can eventually lead to apoptosis in prostate cancer cells. PMID:27755556

Thioredoxin-1 attenuates sepsis-induced cardiomyopathy after cecal ligation and puncture in mice.

PubMed

Wilson, Rickesha L; Selvaraju, Vaithinathan; Lakshmanan, Rajesh; Thirunavukkarasu, Mahesh; Campbell, Jacob; McFadden, David W; Maulik, Nilanjana

2017-12-01

Sepsis is a leading cause of mortality among patients in intensive care units across the USA. Thioredoxin-1 (Trx-1) is an essential 12 kDa cytosolic protein that, apart from maintaining the cellular redox state, possesses multifunctional properties. In this study, we explored the possibility of controlling adverse myocardial depression by overexpression of Trx-1 in a mouse model of severe sepsis. Adult C57BL/6J and Trx-1 Tg/+ mice were divided into wild-type sham (WTS), wild-type cecal ligation and puncture (WTCLP), Trx-1 Tg/+ sham (Trx-1 Tg/+ S), and Trx-1 Tg/+ CLP groups. Cardiac function was evaluated before surgery, 6 and 24 hours after CLP surgery. Immunohistochemical and Western blot analysis were performed after 24 hours in heart tissue sections. Echocardiography analysis showed preserved cardiac function in the Trx-1 Tg/+ CLP group compared with the WTCLP group. Similarly, Western blot analysis revealed increased expression of Trx-1, heme oxygenase-1 (HO-1), survivin (an inhibitor of apoptosis [IAP] protein family), and decreased expression of thioredoxin-interacting protein (TXNIP), caspase-3, and 3- nitrotyrosine in the Trx-1 Tg/+ CLP group compared with the WTCLP group. Immunohistochemical analysis showed reduced 4-hydroxynonenal, apoptosis, and vascular leakage in the cardiac tissue of Trx-1 Tg/+ CLP mice compared with mice in the WTCLP group. Our results indicate that overexpression of Trx-1 attenuates cardiac dysfunction during CLP. The mechanism of action may involve reduction of oxidative stress, apoptosis, and vascular permeability through activation of Trx-1/HO-1 and anti-apoptotic protein survivin. Copyright © 2017 Elsevier Inc. All rights reserved.

Regulation of matrix metalloproteinases (MMPs) expression and secretion in MDA-MB-231 breast cancer cells by LIM and SH3 protein 1 (LASP1).

PubMed

Endres, Marcel; Kneitz, Susanne; Orth, Martin F; Perera, Ruwan K; Zernecke, Alma; Butt, Elke

2016-09-27

The process of tumor invasion requires degradation of extracellular matrix by proteolytic enzymes. Cancer cells form protrusive invadopodia, which produce and release matrix metalloproteinases (MMPs) to degrade the basement membrane thereby enabling metastasis. We investigated the effect of LASP1, a newly identified protein in invadopodia, on expression, secretion and activation of MMPs in invasive breast tumor cell lines.By analyzing microarray data of in-house generated control and LASP1-depleted MDA-MB-231 breast cancer cells, we observed downregulation of MMP1, -3 and -9 upon LASP1 depletion. This was confirmed by Western blot analysis. Conversely, rescue experiments restored in part MMP expression and secretion. The regulatory effect of LASP1 on MMP expression was also observed in BT-20 breast cancer cells as well as in prostate and bladder cancer cell lines.In line with bioinformatic FunRich analysis of our data, which mapped a high regulation of transcription factors by LASP1, public microarray data analysis detected a correlation between high LASP1 expression and enhanced c-Fos levels, a protein that is part of the transcription factor AP-1 and known to regulate MMP expression. Compatibly, in luciferase reporter assays, AP-1 showed a decreased transcriptional activity after LASP1 knockdown.Zymography assays and Western blot analysis revealed an additional promotion of MMP secretion into the extracellular matrix by LASP1, thus, most likely, altering the microenvironment during cancer progression.The newly identified role of LASP1 in regulating matrix degradation by affecting MMP transcription and secretion elucidated the migratory potential of LASP1 overexpressing aggressive tumor cells in earlier studies.

Prion Infected Meat-and-Bone Meal Is Still Infectious after Biodiesel Production

PubMed Central

Bruederle, Cathrin E.; Hnasko, Robert M.; Kraemer, Thomas; Garcia, Rafael A.; Haas, Michael J.; Marmer, William N.; Carter, John Mark

2008-01-01

The epidemic of bovine spongiform encephalopathy (BSE) has led to a world-wide drop in the market for beef by-products, such as Meat-and-Bone Meal (MBM), a fat-containing but mainly proteinaceaous product traditionally used as an animal feed supplement. While normal rendering is insufficient, the production of biodiesel from MBM has been suggested to destroy infectivity from transmissible spongiform encephalopathies (TSEs). In addition to producing fuel, this method simultaneously generates a nutritious solid residue. In our study we produced biodiesel from MBM under defined conditions using a modified form of alkaline methanolysis. We evaluated the presence of prion in the three resulting phases of the biodiesel reaction (Biodiesel, Glycerol and Solid Residue) in vitro and in vivo. Analysis of the reaction products from 263K scrapie infected MBM led to no detectable immunoreactivity by Western Blot. Importantly, and in contrast to the biochemical results the solid MBM residue from the reaction retained infectivity when tested in an animal bioassay. Histochemical analysis of hamster brains inoculated with the solid residue showed typical spongiform degeneration and vacuolation. Re-inoculation of these brains into a new cohort of hamsters led to onset of clinical scrapie symptoms within 75 days, suggesting that the specific infectivity of the prion protein was not changed during the biodiesel process. The biodiesel reaction cannot be considered a viable prion decontamination method for MBM, although we observed increased survival time of hamsters and reduced infectivity greater than 6 log orders in the solid MBM residue. Furthermore, results from our study compare for the first time prion detection by Western Blot versus an infectivity bioassay for analysis of biodiesel reaction products. We could show that biochemical analysis alone is insufficient for detection of prion infectivity after a biodiesel process. PMID:18698417

Deleted in malignant brain tumor 1 is secreted in the oviduct and involved in the mechanism of fertilization in equine and porcine species.

PubMed

Ambruosi, Barbara; Accogli, Gianluca; Douet, Cécile; Canepa, Sylvie; Pascal, Géraldine; Monget, Philippe; Moros Nicolás, Carla; Holmskov, Uffe; Mollenhauer, Jan; Robbe-Masselot, Catherine; Vidal, Olivier; Desantis, Salvatore; Goudet, Ghylène

2013-08-01

Oviductal environment affects preparation of gametes for fertilization, fertilization itself, and subsequent embryonic development. The aim of this study was to evaluate the effect of oviductal fluid and the possible involvement of deleted in malignant brain tumor 1 (DMBT1) on IVF in porcine and equine species that represent divergent IVF models. We first performed IVF after pre-incubation of oocytes with or without oviductal fluid supplemented or not with antibodies directed against DMBT1. We showed that oviductal fluid induces an increase in the monospermic fertilization rate and that this effect is canceled by the addition of antibodies, in both porcine and equine species. Moreover, pre-incubation of oocytes with recombinant DMBT1 induces an increase in the monospermic fertilization rate in the pig, confirming an involvement of DMBT1 in the fertilization process. The presence of DMBT1 in the oviduct at different stages of the estrus cycle was shown by western blot and confirmed by immunohistochemical analysis of ampulla and isthmus regions. The presence of DMBT1 in cumulus-oocyte complexes was shown by western blot analysis, and the localization of DMBT1 in the zona pellucida and cytoplasm of equine and porcine oocytes was observed using immunofluorescence analysis and confocal microscopy. Moreover, we showed an interaction between DMBT1 and porcine spermatozoa using surface plasmon resonance studies. Finally, a bioinformatic and phylogenetic analysis allowed us to identify the DMBT1 protein as well as a DMBT1-like protein in several mammals. Our results strongly suggest an important role of DMBT1 in the process of fertilization.

Muscadine Grape Skin Extract Induces an Unfolded Protein Response-Mediated Autophagy in Prostate Cancer Cells: A TMT-Based Quantitative Proteomic Analysis.

PubMed

Burton, Liza J; Rivera, Mariela; Hawsawi, Ohuod; Zou, Jin; Hudson, Tamaro; Wang, Guangdi; Zhang, Qiang; Cubano, Luis; Boukli, Nawal; Odero-Marah, Valerie

2016-01-01

Muscadine grape skin extract (MSKE) is derived from muscadine grape (Vitis rotundifolia), a common red grape used to produce red wine. Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR) that serves as a survival mechanism to relieve ER stress and restore ER homeostasis. However, when persistent, ER stress can alter the cytoprotective functions of the UPR to promote autophagy and cell death. Although MSKE has been documented to induce apoptosis, it has not been linked to ER stress/UPR/autophagy. We hypothesized that MSKE may induce a severe ER stress response-mediated autophagy leading to apoptosis. As a model, we treated C4-2 prostate cancer cells with MSKE and performed a quantitative Tandem Mass Tag Isobaric Labeling proteomic analysis. ER stress response, autophagy and apoptosis were analyzed by western blot, acridine orange and TUNEL/Annexin V staining, respectively. Quantitative proteomics analysis indicated that ER stress response proteins, such as GRP78 were greatly elevated following treatment with MSKE. The up-regulation of pro-apoptotic markers PARP, caspase-12, cleaved caspase-3, -7, BAX and down-regulation of anti-apoptotic marker BCL2 was confirmed by Western blot analysis and apoptosis was visualized by increased TUNEL/Annexin V staining upon MSKE treatment. Moreover, increased acridine orange, and LC3B staining was detected in MSKE-treated cells, suggesting an ER stress/autophagy response. Finally, MSKE-mediated autophagy and apoptosis was antagonized by co-treatment with chloroquine, an autophagy inhibitor. Our results indicate that MSKE can elicit an UPR that can eventually lead to apoptosis in prostate cancer cells.

Null missense ABCR (ABCA4) mutations in a family with stargardt disease and retinitis pigmentosa.

PubMed

Shroyer, N F; Lewis, R A; Yatsenko, A N; Lupski, J R

2001-11-01

To determine the type of ABCR mutations that segregate in a family that manifests both Stargardt disease (STGD) and retinitis pigmentosa (RP), and the functional consequences of the underlying mutations. Direct sequencing of all 50 exons and flanking intronic regions of ABCR was performed for the STGD- and RP-affected relatives. RNA hybridization, Western blot analysis, and azido-adenosine triphosphate (ATP) labeling was used to determine the effect of disease-associated ABCR mutations in an in vitro assay system. Compound heterozygous missense mutations were identified in patients with STGD and RP. STGD-affected individual AR682-03 was compound heterozygous for the mutation 2588G-->C and a complex allele, [W1408R; R1640W]. RP-affected individuals AR682-04 and-05 were compound heterozygous for the complex allele [W1408R; R1640W] and the missense mutation V767D. Functional analysis of the mutation V767D by Western blot and ATP binding revealed a severe reduction in protein expression. In vitro analysis of ABCR protein with the mutations W1408R and R1640W showed a moderate effect of these individual mutations on expression and ATP-binding; the complex allele [W1408R; R1640W] caused a severe reduction in protein expression. These data reveal that missense ABCR mutations may be associated with RP. Functional analysis reveals that the RP-associated missense ABCR mutations are likely to be functionally null. These studies of the complex allele W1408R; R1640W suggest a synergistic effect of the individual mutations. These data are congruent with a model in which RP is associated with homozygous null mutations and with the notion that severity of retinal disease is inversely related to residual ABCR activity.

Effects of Granulocyte-Macrophage Colony-Stimulating (GM-CSF) Factor on Corneal Epithelial Cells in Corneal Wound Healing Model

PubMed Central

Rho, Chang Rae; Park, Mi-young; Kang, Seungbum

2015-01-01

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that activates granulocyte and macrophage cell lineages. It is also known to have an important function in wound healing. This study investigated the effect of GM-CSF in wound healing of human corneal epithelial cells (HCECs). We used human GM-CSF derived from rice cells (rice cell-derived recombinant human GM-CSF; rhGM-CSF). An in vitro migration assay was performed to investigate the migration rate of HCECs treated with various concentrations of rhGM-CSF (0.1, 1.0, and 10.0 μg/ml). MTT assay and flow cytometric analysis were used to evaluate the proliferative effect of rhGM-CSF. The protein level of p38MAPK was analyzed by western blotting. For in vivo analysis, 100 golden Syrian hamsters were divided into four groups, and their corneas were de-epithelialized with alcohol and a blade. The experimental groups were treated with 10, 20, or 50 μg/ml rhGM-CSF four times daily, and the control group was treated with phosphate-buffered saline. The corneal wound-healing rate was evaluated by fluorescein staining at the initial wounding and 12, 24, 36, and 48 hours after epithelial debridement. rhGM-CSF accelerated corneal epithelial wound healing both in vitro and in vivo. MTT assay and flow cytometric analysis revealed that rhGM-CSF treatment had no effects on HCEC proliferation. Western blot analysis demonstrated that the expression level of phosphorylated p38MAPK increased with rhGM-CSF treatment. These findings indicate that rhGM-CSF enhances corneal wound healing by accelerating cell migration. PMID:26376304

Effects of Granulocyte-Macrophage Colony-Stimulating (GM-CSF) Factor on Corneal Epithelial Cells in Corneal Wound Healing Model.

PubMed

Rho, Chang Rae; Park, Mi-young; Kang, Seungbum

2015-01-01

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that activates granulocyte and macrophage cell lineages. It is also known to have an important function in wound healing. This study investigated the effect of GM-CSF in wound healing of human corneal epithelial cells (HCECs). We used human GM-CSF derived from rice cells (rice cell-derived recombinant human GM-CSF; rhGM-CSF). An in vitro migration assay was performed to investigate the migration rate of HCECs treated with various concentrations of rhGM-CSF (0.1, 1.0, and 10.0 μg/ml). MTT assay and flow cytometric analysis were used to evaluate the proliferative effect of rhGM-CSF. The protein level of p38MAPK was analyzed by western blotting. For in vivo analysis, 100 golden Syrian hamsters were divided into four groups, and their corneas were de-epithelialized with alcohol and a blade. The experimental groups were treated with 10, 20, or 50 μg/ml rhGM-CSF four times daily, and the control group was treated with phosphate-buffered saline. The corneal wound-healing rate was evaluated by fluorescein staining at the initial wounding and 12, 24, 36, and 48 hours after epithelial debridement. rhGM-CSF accelerated corneal epithelial wound healing both in vitro and in vivo. MTT assay and flow cytometric analysis revealed that rhGM-CSF treatment had no effects on HCEC proliferation. Western blot analysis demonstrated that the expression level of phosphorylated p38MAPK increased with rhGM-CSF treatment. These findings indicate that rhGM-CSF enhances corneal wound healing by accelerating cell migration.

Adenosine 5'-monophosphate-induced hypothermia inhibits the activation of ERK1/2, JNK, p38 and NF-κB in endotoxemic rats.

PubMed

Wang, Yunlong; Zhang, Aihua; Lu, Shulai; Pan, Xinting; Jia, Dongmei; Yu, Wenjuan; Jiang, Yanxia; Li, Xinde; Wang, Xuefeng; Zhang, Jidong; Hou, Lin; Sun, Yunbo

2014-11-01

Many studies have shown that LPS mainly activates four signal transduction pathways to induce inflammation, namely the p38, ERK1/2, JNK and IKK/NF-κB pathways. Studies have demonstrated that 5'-AMP-induced hypothermia (AIH) exhibits high anti-inflammatory capabilities. In this study, we explore that how AIH inhibits the inflammatory response. Wistar rats were divided into five groups: a control group, an LPS group, a 5'-AMP pre-treatment group, a 5'-AMP post-treatment group and a 5'-AMP group. For each group, plasma and lung were collected from the rats at 6h and 12h after LPS injection. ELISA assays were used to detect plasma levels of CD14, CRP and MCP-1. Inflammatory pathway activation and TLR4 expression were assayed separately by Western blot analysis and immunohistochemistry. Our results showed that rats treated with AIH either before or after an LPS-challenge had a significant decrease in plasma levels of CD14, CRP and TLR4 compared with rats that received LPS only. Western blot analysis showed that AIH inhibited the activation of extracellular signal-regulated kinases (ERK) 1/2, p38, c-Jun N-terminal kinase (JNK) and NF-κB in inflammatory rats. Our study concluded that AIH attenuated LPS-induced inflammation mainly by inhibiting activation on the ERK1/2, p38, JNK and NF-κB signaling pathways. Copyright © 2014 Elsevier B.V. All rights reserved.

Protective effect of forsythiaside A on OVA-induced asthma in mice.

PubMed

Qian, Jin; Ma, Xiaorong; Xun, Yali; Pan, Lei

2017-10-05

Forsythiaside A (FSA), an active constituent isolated from the Chinese medicinal herb Forsythia suspensa, has been known to have anti-inflammatory effect. However, the effect of FSA on allergic airway inflammation remains unclear. The aim of this study was to investigate the effect of FSA on OVA-induced asthma in mice. Mice model of asthma was induced by OVA. OVA-induced airway hyperresponsiveness (AHR) and inflammatory cells in BALF were detected. The production of IgE, IL-4, IL-5, IFN-γ, and IL-13 were detected by ELISA. The effects of FSA on Nrf2 and NF-κB signaling pathways were detected by western blot analysis. The results showed that treatment of FSA significantly attenuated OVA-induced lung histopathological changes. FSA inhibited OVA-induced AHR and inflammatory cells in BALF. OVA-induced IgE, IL-4, IL-5, and IL-13 production were also inhibited by FSA. Western blot analysis showed that treatment of FSA inhibited OVA-induced NF-κB activation. Treatment of FSA dose-dependently up-regulated the expression of Nrf2 and HO-1. In addition, we found that FSA up-regulated the expression of Nrf2 and HO-1 both in A549 cells and MS-H cells. Taken together, FSA suppressed inflammatory responses in OVA-induced asthma through activating Nrf2/HO-1 signaling pathway. FSA may be a promising potential preventive agent for asthma treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

Prostaglandin metabolizing enzymes in correlation with vitamin D receptor in benign and malignant breast cell lines.

PubMed

Thill, Marc; Fischer, Dorothea; Becker, Steffi; Cordes, Tim; Dittmer, Christine; Diedrich, Klaus; Salehin, Darius; Friedrich, Michael

2009-09-01

The antiproliferative effects of calcitriol (1,25(OH)2D3) mediated via the vitamin D receptor (VDR), render the biologically active form of vitamin D a promising target in breast cancer therapy. Furthermore, breast cancer is associated with inflammatory processes based on an up-regulation of cyclooxygenase-2 (COX-2) expression, the prostaglandin E2 (PGE2) synthesizing enzyme. The PGE2 metabolizing enzyme, 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is described as a tumor suppressor in cancer. First references suggest a correlation between vitamin D and prostaglandin metabolism through the impact of 1,25(OH)2D3 on the expression of COX-2 and 15-PGDH. The expression of VDR, COX-2 and 15-PGDH in benign MCF-10F and malignant MCF-7 breast cells was determined by real-time PCR (RT-PCR) and Western blot analysis. Although the RT-PCR data were divergent from those obtained from the Western blot analysis, the COX-2 protein expression was MCF-7 2-fold higher in the MCF-7 compared to the MCF-10F cells. Moreover, a correlation of 15-PGDH to VDR by RT-PCR was found in both cell lines. The VDR protein levels were inversely correlated to the 15-PGDH protein levels and revealed that the MCF-10F cells had the highest VDR expression. A possible link between VDR-associated target genes and prostaglandin metabolism is suggested.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Riezzo, Irene; Turillazzi, Emanuela; Bello, Stefania

Nandrolone decanoate administration and strenuous exercise increase the extent of renal damage in response to renal toxic injury. We studied the role played by oxidative stress in the apoptotic response caused by nandrolone decanoate in the kidneys of strength-trained male CD1 mice. To measure cytosolic enzyme activity, glutathione peroxidase (GPx), glutathione reductase (GR) and malondialdehyde (MDA) were determined after nandrolone treatment. An immunohistochemical study and Western blot analysis were performed to evaluate cell apoptosis and to measure the effects of renal expression of inflammatory mediators (IL-1β, TNF-α) on the induction of apoptosis (HSP90, TUNEL). Dose-related oxidative damage in the kidneysmore » of treated mice is shown by an increase in MDA levels and by a reduction of antioxidant enzyme GR and GPx activities, resulting in the kidney's reduced radical scavenging ability. Renal specimens of the treated group showed relevant glomeruli alterations and increased immunostaining and protein expressions, which manifested significant focal segmental glomerulosclerosis. The induction of proinflammatory cytokine expression levels was confirmed by Western blot analysis. Long-term administration of nandrolone promotes oxidative injury in the mouse kidneys. TNF-α mediated injury due to nandrolone in renal cells appears to play a role in the activation of both the intrinsic and extrinsic apoptosis pathways. - Highlights: • We analyze abuse of nandrolone decanoate in strength-trained male CD1 mice. • Nandrolone decanoate administration increases oxidative stress. • Increased cytokine expressions were observed. • Renal apoptosis was described. • Long-term administration of nandrolone promotes oxidative injury in mice kidney.« less

Morphological and molecular variations induce mitochondrial dysfunction as a possible underlying mechanism of athletic amenorrhea.

PubMed

Xiong, Ruo-Hong; Wen, Shi-Lei; Wang, Qiang; Zhou, Hong-Ying; Feng, Shi

2018-01-01

Female athletes may experience difficulties in achieving pregnancy due to athletic amenorrhea (AA); however, the underlying mechanisms of AA remain unknown. The present study focuses on the mitochondrial alteration and its function in detecting the possible mechanism of AA. An AA rat model was established by excessive swimming. Hematoxylin and eosin staining, and transmission electron microscopic methods were performed to evaluate the morphological changes of the ovary, immunohistochemical examinations and radioimmunoassays were used to detect the reproductive hormones and corresponding receptors. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to test the mtDNA copy number. PCR and western blot analysis were used to test the expression of ND2. The change of morphological features of the rat ovaries revealed evident abnormalities. Particularly, the features of the mitochondria were markedly altered. In addition, reproductive hormones in the serum and tissues of AA rats were also detected to evaluate the function of the ovaries, and the levels of these hormones were significantly decreased. Furthermore, the mitochondrial DNA copy number (mtDNA) and expression of NADH dehydrogenase subunit 2 (ND2) were quantitated by qPCR or western blot analysis. Accordingly, the mtDNA copy number and expression of ND2 expression were markedly reduced in the AA rats. In conclusion, mitochondrial dysfunction in AA may affect the cellular energy supply and, therefore, result in dysfunction of the ovary. Thus, mitochondrial dysfunction may be considered as a possible underlying mechanism for the occurrence of AA.

Betaine supplement enhances skeletal muscle differentiation in murine myoblasts via IGF-1 signaling activation.

PubMed

Senesi, Pamela; Luzi, Livio; Montesano, Anna; Mazzocchi, Nausicaa; Terruzzi, Ileana

2013-07-19

Betaine (BET) is a component of many foods, including spinach and wheat. It is an essential osmolyte and a source of methyl groups. Recent studies have hypothesized that BET might play a role in athletic performance. However, BET effects on skeletal muscle differentiation and hypertrophy are still poorly understood. We examined BET action on neo myotubes maturation and on differentiation process, using C2C12 murine myoblastic cells. We used RT2-PCR array, Western blot and immunofluorescence analysis to study the BET effects on morphological features of C2C12 and on signaling pathways involved in muscle differentiation and hypertrophy. We performed a dose-response study, establishing that 10 mM BET was the dose able to stimulate morphological changes and hypertrophic process in neo myotubes. RT2-PCR array methodology was used to identify the expression profile of genes encoding proteins involved in IGF-1 pathway. A dose of 10 mM BET was found to promote IGF-1 receptor (IGF-1 R) expression. Western blot and immunofluorescence analysis, performed in neo myotubes, pointed out that 10 mM BET improved IGF-1 signaling, synthesis of Myosin Heavy Chain (MyHC) and neo myotubes length. Our findings provide the first evidence that BET could promote muscle fibers differentiation and increase myotubes size by IGF-1 pathway activation, suggesting that BET might represent a possible new drug/integrator strategy, not only in sport performance but also in clinical conditions characterized by muscle function impairment.

Dexamethasone attenuates oxidation of extracellular matrix proteins by human monocytes.

PubMed

Ahmed, Shahid; Adamidis, Ananea; Jan, Louis C; Gibbons, Nora; Mattana, Joseph

2003-10-01

In response to infection or in immune complex-mediated diseases, inflammatory cells may oxidatively damage extracellular matrix (ECM) proteins. In this study we evaluated whether human monocytes could oxidize ECM and whether this could be modulated by exposure to LPS, IgG complexes, and dexamethasone (DEX). Wells in tissue culture plates were coated with the ECM preparation Matrigel. Porous inserts with or without the human monocyte cell line THP-1 were placed into ECM-containing wells and cells were exposed to control conditions or to LPS (10 ng/ml), IgG complexes (200 and 500 microg/ml), or DEX (10(-7) and 10(-6) M). ECM was then subjected to Western blot analysis using an antibody to oxidized protein. In addition, Western blot analysis was carried out on DEX-treated cells to evaluate expression of the NADPH oxidase components p67-phox and gp91-phox. THP-1 cells enhanced ECM oxidation and this effect was augmented by LPS and by IgG aggregates. Preincubation of cells with DEX attenuated ECM oxidation and was also associated with decreased expression of p67-phox and gp91-phox. These findings suggest that human monocytes can oxidize ECM proteins and that this may be modulated by IgG complexes and LPS. Dexamethasone appears to attenuate ECM oxidation and a better understanding of this mechanism might allow for interventions to minimize oxidative damage to ECM proteins by monocytes in infectious and inflammatory states.

S100a8/NF-κB signal pathway is involved in the 800-nm diode laser-induced skin collagen remodeling.

PubMed

Ren, Xiaolin; Ge, Minggai; Qin, Xiaofeng; Xu, Peng; Zhu, Pingya; Dang, Yongyan; Gu, Jun; Ye, Xiyun

2016-05-01

The 800-nm diode laser is widely used for hair removal and also promotes collagen synthesis, but the molecular mechanism by which dermis responses to the thermal damage induced by the 800-nm diode laser is still unclear. Ten 2-month-old mice were irradiated with the 800-nm diode laser at 20, 40, and 60 J/cm(2), respectively. Skin samples were taken for PCR, Western blot analysis, and histological study at day 3 or 30 after laser irradiation. The expression of S100a8 and its two receptors (advanced glycosylation end product-specific receptor, RAGE and toll-like receptor 4, TRL4) was upregulated at day 3 after laser treatments. P-p65 levels were also elevated, causing the increase of cytokine (tumor necrosis factor, TNF-α and interleukin 6, IL-6) and MMPs (MMP1a, MMP9). At day 30, PCR and Western blot analysis showed significant increase of type I and III procollagen in the dermis treated with laser. Importantly, skin structure was markedly improved in the laser-irradiated skin compared with the control. Thus, it seemed that S100a8 upregulation triggered NF-κB signal pathway through RAGE and TLR4, responding to laser-induced dermis wound healing. The involvement of the NF-κB pathway in MMP gene transcription promoted the turnover of collagen in the skin, accelerating new collagen synthesis.

The Role of Constitutive Androstane Receptor in Oxazaphosphorine-mediated Induction of Drug-metabolizing Enzymes in Human Hepatocytes

PubMed Central

Wang, Duan; Li, Linhao; Fuhrman, Jennifer; Ferguson, Stephen; Wang, Hongbing

2013-01-01

Purpose The objective of this study was to investigate the roles of the constitutive androstane receptor (CAR) in cyclophosphamide (CPA)- and ifosfamide (IFO)-mediated induction of hepatic drug-metabolizing enzymes (DME). Methods Induction of DMEs was evaluated using real-time RT-PCR and Western blotting analysis in human primary hepatocyte (HPH) cultures. Activation of CAR, pregnane X receptor (PXR), and aryl hydrocarbon receptor by CPA and IFO was assessed in cell-based reporter assays in HepG2 cells and/or nuclear translocation assays in HPHs. Results CYP2B6 reporter activity was significantly enhanced by CPA and IFO in HepG2 cells co-transfected with CYP2B6 reporter plasmid and a chemical-responsive human CAR variant (CAR1+A) construct. Real-time RT-PCR and Western blotting analysis in HPHs showed that both CPA and IFO induced the expressions of CYP2B6 and CYP3A4. Notably, treatment of HPHs with CPA but not IFO resulted in significant nuclear accumulation of CAR, which represents the initial step of CAR activation. Further studies in HPHs demonstrated that selective inhibition of PXR by sulforaphane preferentially repressed IFO- over CPA-mediated induction of CYP2B6. Conclusion These results provide novel insights into the differential roles of CAR in the regulation of CPA- and IFO-induced DME expression and potential drug-drug interactions. PMID:21487929

Cardioprotective effects of BMS-180448, a prototype mitoK(ATP) channel opener, and the role of salvage kinases, in the rat model of global ischemia and reperfusion heart injury.

PubMed

Lee, Ju-Han; Jung, In-Sang; Lee, Sung-Hun; Yang, Min-Kyu; Hwang, Ji-Hye; Lee, Hak-Dong; Cho, Yu-Sun; Song, Min-Jin; Yi, Kyu-Yang; Yoo, Sung-Eun; Kwon, Suk-Hyung; Kim, Bokyung; Lee, Chang-Soo; Shin, Hwa-Sup

2007-05-01

To investigate the involvement of reperfusion-induced salvage kinases (RISK) as possible signaling molecules for the cardioprotective effects of BMS-180448, a prototype mitochondrial ATP-sensitive K+ (mitoK(ATP)) channel opener, we measured its cardioprotective effects in a rat model of ischemia/reperfusion (I/R) heart injury, together with western blotting analysis of five different signaling proteins. In isolated rat hearts subjected to 30-min global ischemia followed by 30-min reperfusion, BMS-180448 (1, 3 and 10 microM) significantly increased reperfusion left ventricular developed pressure (LVDP) and 30-min reperfusion double product (heart rate x LVDP) in a concentration-dependent manner, while decreasing left ventricular end-diastolic pressure (LVEDP) throughout reperfusion period in a concentration-dependent manner. SDS-PAGE/western blotting analysis of left ventricle reperfused for 30 min revealed that BMS-180448 significantly decreased phospho-GSK3beta at high concentration, whereas it tended to increase slightly phospho-eNOS and phospho-p70S6K with concentration. However, BMS-180448 had no effect on phospho-Akt and phospho-Bad. These results suggest that the cardioprotective effects of BMS-180448 against I/R heart injury may result from direct activation of mitoK(ATP) channel in cardiomyocytes, with the minimal role of RISK pathway in the activation of this channel and the cardioprotective effects of BMS-180448.

Connexin 43 expression of foreign body giant cells after implantation of nanoparticulate hydroxyapatite.

PubMed

Herde, Katja; Hartmann, Sonja; Brehm, Ralph; Kilian, Olaf; Heiss, Christian; Hild, Anne; Alt, Volker; Bergmann, Martin; Schnettler, Reinhard; Wenisch, Sabine

2007-11-01

In bone a role of connexin 43 has been implicated with the fusion of mononuclear precursors of the monocyte/macrophage lineage into multinucleated cells. In order to investigate the putative role of connexin 43 in formation of bone osteoclast-like foreign body giant cells which are formed in response to implantation of biomaterials, nanoparticulate hydroxyapatite had been implanted into defects of minipig femura. After 20 days the defect areas were harvested and connexin 43 expression and synthesis were investigated by using immunohistochemistry, Western Blot, and in situ hybridization within macrophages and osteoclast-like foreign body giant cells. Morphological analysis of gap junctions is performed ultrastructurally. As shown on protein and mRNA level numerous connexin 43 positive macrophages and foreign body giant cells (FBGC) were localized within the granulation tissue and along the surfaces of the implanted hydroxyapatite (HA). Besides, the formation of FBGC by fusion of macrophages could be shown ultrastructurally. Connexin 43 labeling observed on the protein and mRNA level could be attributed to gap junctions identified ultrastructurally between macrophages, between FBGC, and between FBGC and macrophages. Annular gap junctions in the cytoplasm of FBGC pointed to degradation of the channels, and the ubiquination that had occurred in the course of degradation was confirmed by Western blot analysis. All in all, the presently observed pattern of connexin 43 labeling refers to an functional role of gap junctional communication in the formation of osteoclast-like foreign body giant cells formed in response to implantation of the nanoparticulate HA.

Intestinal flora of FAP patients containing APC-like sequences.

PubMed

Hainova, K; Adamcikova, Z; Ciernikova, S; Stevurkova, V; Tyciakova, S; Zajac, V

2014-01-01

Colorectal cancer mortality is one of the most common cause of cancer-related mortality. A multiple risk factors are associated with colorectal cancer, including hereditary, enviromental and inflammatory syndromes affecting the gastrointestinal tract. Familial adenomatous polyposis (FAP) is characterized by the emergence of hundreds to thousands of colorectal adenomatous polyps and FAP syndrome is caused by mutations within the adenomatous polyposis coli (APC) tumor suppressor gene. We analyzed 21 rectal bacterial subclones isolated from FAP patient 41-1 with confirmed 5bp ACAAA deletion within codons 1060-1063 for the presence of APC-like sequences in longest exon 15. The studied section was defined by primers 15Efor-15Erev, what correlates with mutation cluster region (MCR) in which the 75% of all APC germline mutations were detected. More than 90% homology was showed by sequencing and subsequent software comparison. The expression of APC-like sequences was demostrated by Western blot analysis using monoclonal and polyclonal antibodies against APC protein. To study missing link between the DNA analysis (PCR, DNA sequencing) and protein expresion experiments (Western blotting) we analyzed bacterial transcripts containing the 15Efor-15Erev sequence of APC gene by reverse transcription-PCR, what indicated that an APC gene derived fragment may be produced. We observed 97-100 % homology after computer comparison of cDNA PCR products. Our results suggest that presence of APC-like sequences in intestinal/rectal bacteria is enrichment of bacterial genetic information in which horizontal gene transfer between humans and microflora play an important role.

Adolescent social isolation affects schizophrenia-like behavior and astrocyte biomarkers in the PFC of adult rats.

PubMed

Sun, Lan; Min, Li; Zhou, Hao; Li, Man; Shao, Feng; Wang, Weiwen

2017-08-30

Social isolation is regarded as a cause of schizophrenia spectrum disorders. Animal models of schizophrenia are constructed by repeated early environment deprivation as an important paradigm to reveal its pathological mechanism. Male Sprague Dawley rats were assigned to either social-rearing (SR) or isolated-rearing (IR) groups during postnatal days (PNDs) 21-34. On PND 56, all rats underwent behavioral testing including locomotor activity, anxiety-related behaviors in an open field and prepulse inhibition (PPI). Then, the rats were sacrificed and prefrontal cortex (PFC) tissues were separated for high-throughput proteomics analysis and Western blot validation. Rats of the IR group showed increased spontaneous locomotion, increased anxiety-like behavior and disrupted PPI compared with rats of the SR group. Based on proteomics analysis, a total of 124 PFC proteins were found to be significantly differentially expressed between the SR group and the IR group, the most remarkable of which were glial fibrillary acidic protein (GFAP), Annexin A2 (ANXA2) and vimentin (VIM), three astrocyte biomarkers. Further Western blot measurement confirmed that the levels of GFAP, ANXA2 and VIM were increased significantly in IR rats. Adolescent social isolation induced schizophrenia-like behaviors and significantly different expression of 124 PFC proteins in adult rats, especially GFAP, ANXA2 and VIM, which suggests that astrocyte development might be involved in the neural mechanism of schizophrenia. Copyright © 2017. Published by Elsevier B.V.

Proteins related to the functions of fibroblast-like synoviocytes identified by proteomic analysis.

PubMed

Zhang, Hui; Fan, Lie Ying; Zong, Ming; Sun, Li Shan; Lu, Liu

2012-01-01

It is well known that the fibroblast-like synoviocytes (FLS) play a key role in pathogenesis of rheumatoid arthritis (RA). This study was performed to separate the differentially expressed proteins of FLS from the patients with RA or osteoarthritis (OA) by two-dimensional electrophoresis (2-DE), and found proteins associated with the functions of FLS by mass spectrometry (MS). Total proteins were extracted and quantified from the primary cultured FLS from patients of RA (n=8) or OA (n=6). Proteins were separated by high-resolution 2-DE, and identified the differentially expressed proteins by MS. Western blot analyses was used to validated the expression of candidate proteins. The mRNA of these proteins was detected by semi-quantitative fluorescent PCR. There are 1147 protein spots from RA and 1324 protein spots from OA showed on 2-DE graphs, respectively. We have selected 84 protein spots for MS analysis, and 27 protein spots were successfully identified. We have found that protein isoaspartyl methyltransferase (PIMT) and pirin (iron-binding nuclear protein, PIR) with lower expression in RA, and thioredoxin 1(Trx-1) only expressed in RA may be associated with functions of FLS. Western Blot confirmed the expression of PIMT and pirin lower in RA, and Trx-1 expressed only in RA. The results of semi-quantitative fluorescent PCR are also consistent with 2-DE graphs. PIMT, pirin and Trx-1 affect the functions of FLS in some style and can be the drug targets of RA.

Distinctive expression patterns of glycoprotein non-metastatic B and folliculin in renal tumors in patients with Birt–Hogg–Dubé syndrome

PubMed Central

Furuya, Mitsuko; Hong, Seung-Beom; Tanaka, Reiko; Kuroda, Naoto; Nagashima, Yoji; Nagahama, Kiyotaka; Suyama, Takahito; Yao, Masahiro; Nakatani, Yukio

2015-01-01

Birt–Hogg–Dubé syndrome (BHD) is an inherited disorder associated with a germline mutation of the folliculin gene (FLCN). The affected families have a high risk for developing multiple renal cell carcinomas (RCC). Diagnostic markers that distinguish between FLCN-related RCC and sporadic RCC have not been investigated, and many patients with undiagnosed BHD fail to receive proper medical care. We investigated the histopathology of 27 RCCs obtained from 18 BHD patients who were diagnosed by genetic testing. Possible somatic mutations of RCC lesions were investigated by DNA sequencing. Western blotting and immunohistochemical staining were used to compare the expression levels of FLCN and glycoprotein non-metastatic B (GPNMB) between FLCN-related RCCs and sporadic renal tumors (n = 62). The expression of GPNMB was also evaluated by quantitative RT-PCR. Histopathological analysis revealed that the most frequent histological type was chromophobe RCC (n = 12), followed by hybrid oncocytic/chromophobe tumor (n = 6). Somatic mutation analysis revealed small intragenic mutations in six cases and loss of heterozygosity in two cases. Western blot and immunostaining analyses revealed that FLCN-related RCCs showed overexpression of GPNMB and underexpression of FLCN, whereas sporadic tumors showed inverted patterns. GPNMB mRNA in FLCN-related RCCs was 23-fold more abundant than in sporadic tumors. The distinctive expression patterns of GPNMB and FLCN might identify patients with RCCs who need further work-up for BHD. PMID:25594584

The effect of thalidomide on spinal cord ischemia/reperfusion injury in a rabbit model.

PubMed

Lee, C-J; Kim, K-W; Lee, H-M; Nahm, F S; Lim, Y-J; Park, J-H; Kim, C-S

2007-02-01

Randomized study. To evaluate the effects of thalidomide on spinal cord ischemia/reperfusion injury via reduced TNF-alpha production. Animal experimental laboratory, Clinical Research Institute of Seoul National University Hospital, Seoul, Korea. Spinal cord ischemia was induced in rabbits by occluding the infrarenal aorta. Rabbits in group N did not undergo ischemic insult, but rabbits in groups C (the untreated group), THA, and THB underwent ischemic insult for 15 min. The THA and THB groups received thalidomide (20 mg/kg) intraperitoneally (i.p.) before ischemia, but only the THB group received thalidomide (i.p., 20 mg/kg) after 24 and 48 h of reperfusion. After evaluating neurologic functions at 1.5 h, 3, and 5 days of reperfusion, rabbits were killed for histopathologic examination and Western blot analysis of TNF-alpha. The THA and THB groups showed significantly less neurologic dysfunction than the C group at 1.5 h, 3, and 5 days of reperfusion. The number of normal spinal motor neurons in ventral gray matter was higher in THA and THB than in C, but no difference was observed between THA and THB. Western blot analysis showed a significantly higher level of TNF-alpha in C than in THA and THB at 1.5 h of reperfusion, but no difference was observed between C, THA, or THB at 3 or 5 days of reperfusion. Thalidomide treatment before ischemic insult reduces early phase ischemia/reperfusion injury of the spinal cord in rabbits.

Effects of demethoxycurcumin on the viability and apoptosis of skin cancer cells.

PubMed

Wu, Yaoqun; Zhang, Pei; Yang, Hongyun; Ge, Yong; Xin, Yong

2017-07-01

The present study investigated the effects and mechanisms of demethoxycurcumin (DMC) on a human skin squamous cell carcinoma cell line, A431, and a human keratinocyte cell line, HaCaT. A431 and HaCaT cells were cultured in vitro. The effects of DMC treatment on cell viability were analyzed using the Cell Counting kit‑8 (CCK‑8) assay; cell cycle distribution was analyzed by flow cytometry; apoptosis was assessed by flow cytometry and Hoechst 33258 staining; and the protein expression levels of cytochrome c, B‑cell lymphoma 2 (Bcl‑2), Bcl‑2‑associated X protein (BAX), caspase‑9 and caspase‑3 were evaluated by western blotting. CCK‑8 assay results demonstrated that DMC treatment significantly inhibited viability of A431 and HaCaT cells in a dose‑dependent manner. Flow cytometric analysis confirmed that DMC treatment induced apoptosis in a dose‑dependent manner, and significantly increased the proportion of cells in G2/M phase. Western blot analysis indicated that the protein expression levels of Bcl‑2 were decreased, whereas the expression levels of BAX, caspase‑9, caspase‑3 and cytochrome c were increased following DMC treatment compared with in untreated cells. In conclusion, DMC treatment significantly inhibited viability of A431 and HaCaT cells, and induced cell cycle arrest in G2/M phase. The present study indicated that DMC may induce apoptosis of skin cancer cells through a caspase‑dependent pathway.

Additive Effects of Rebamipide Plus Proton Pump Inhibitors on the Expression of Tight Junction Proteins in a Rat Model of Gastro-Esophageal Reflux Disease

PubMed Central

Gweon, Tae-Geun; Park, Jong-Hyung; Kim, Byung-Wook; Choi, Yang Kyu; Kim, Joon Sung; Park, Sung Min; Kim, Chang Whan; Kim, Hyung-Gil; Chung, Jun-Won; Incheon

2018-01-01

Background/Aims The aim of this study was to investigate the effects of rebamipide on tight junction proteins in the esophageal mucosa in a rat model of gastroesophageal reflux disease (GERD). Methods GERD was created in rats by tying the proximal stomach. The rats were divided into a control group, a proton pump inhibitor (PPI) group, and a PPI plus rebamipide (PPI+R) group. Pantoprazole (5 mg/kg) was administered intraperitoneally to the PPI and PPI+R groups. An additional dose of rebamipide (100 mg/kg) was administered orally to the PPI+R group. Mucosal erosions, epithelial thickness, and leukocyte infiltration into the esophageal mucosa were measured in isolated esophagi 14 days after the procedure. A Western blot analysis was conducted to measure the expression of claudin-1, -3, and -4. Results The mean surface area of mucosal erosions, epithelial thickness, and leukocyte infiltration were lower in the PPI group and the PPI+R group than in the control group. Western blot analysis revealed that the expression of claudin-3 and -4 was significantly higher in the PPI+R group than in the control group. Conclusions Rebamipide may exert an additive effect in combination with PPI to modify the tight junction proteins of the esophageal mucosa in a rat model of GERD. This treatment might be associated with the relief of GERD symptoms. PMID:29069891

Role of connexin 43 in the maintenance of spontaneous activity in the guinea pig prostate gland

PubMed Central

Dey, Anupa; Kusljic, Snezana; Lang, Richard J; Exintaris, Betty

2010-01-01

BACKGROUND AND PURPOSE To investigate the role of connexin 43 in the maintenance of spontaneous activity in prostate tissue from young and old guinea pigs. EXPERIMENTAL APPROACH Conventional intracellular microelectrode and tension recording techniques, coupled with Western blot analysis and immunohistochemistry for connexin 43 (CX43) were used. The effects of three gap junction uncouplers, 18β glycyrrhetinic acid (10 µM, 40 µM), carbenoxolone (10 µM, 50 µM) and octanol (0.5 mM, 1 mM), were studied in cells displaying slow wave activity and on spontaneously contracting tissue from prostate glands of young (2–5 months) and old (9–16 months) guinea pigs. KEY RESULTS 18β Glycyrrhetinic acid (40 µM), carbenoxolone (50 µM) or octanol (0.5 mM) abolished slow wave activity in prostate tissue from young and old guinea pigs and depolarized membrane potential by approximately 5 mV. These treatments also abolished all contractions in both sets of prostate tissue. These effects were reversed upon washout. Western blot analysis and CX43 immunohistochemistry showed that there was no age-related difference in the expression and distribution of CX43 in prostate tissues. CONCLUSION AND IMPLICATIONS When gap junctional communication via CX43 was disrupted, spontaneous activity was abolished at a cellular and whole tissue level; CX43 is therefore essential for the maintenance of spontaneous slow wave activity and subsequent contractile activity in the guinea pig prostate gland. PMID:20735413

Cytotoxicity of flavones and flavonols to a human esophageal squamous cell carcinoma cell line (KYSE-510) by induction of G2/M arrest and apoptosis.

PubMed

Zhang, Qiang; Zhao, Xin-Huai; Wang, Zhu-Jun

2009-08-01

In this study, cytotoxic effects of structurally related flavones and flavonols on a human esophageal squamous cell carcinoma cell line (KYSE-510) were determined, and the molecular mechanisms responsible for their cytotoxic effects were studied. The results of MTT assay showed that flavones (luteolin, apigenin, chrysin) and flavonols (quercetin, kaempferol, myricetin) were able to induce cytotoxicity in KYSE-510 cells in a dose- and time-dependent manner, and the cytotoxic potency of these compounds was in the order of: luteolin>quercetin>chrysin>kaempferol>apigenin>myricetin. Flow cytometry and DNA fragmentation analysis indicated that the cytotoxicity induced by flavones and flavonols was mediated by G(2)/M cell cycle arrest and apoptosis. Furthermore, the expression of genes related to cell cycle arrest and apoptosis was assessed by oligonucleotide microarray, real-time RT-PCR and Western blot. It was shown that the treatment of KYSE-510 cells with these compounds caused G(2)/M arrest through up-regulation of p21(waf1) and down-regulation of cyclin B1 at the mRNA and protein levels, and induced p53-independent mitochondrial-mediated apoptosis through up-regulation of PIG3 and cleavage of caspase-9 and caspase-3. The results of western blot analysis further showed that increases of p63 and p73 protein translation or stability might be contributed to the regulation of p21(waf1), cyclin B1 and PIG3.

Triptolide inhibits TGF-β1-induced cell proliferation in rat airway smooth muscle cells by suppressing Smad signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Ming; Lv, Zhiqiang; Huang, Linjie

Background: We have reported that triptolide can inhibit airway remodeling in a murine model of asthma via TGF-β1/Smad signaling. In the present study, we aimed to investigate the effect of triptolide on airway smooth muscle cells (ASMCs) proliferation and the possible mechanism. Methods: Rat airway smooth muscle cells were cultured and made synchronized, then pretreated with different concentration of triptolide before stimulated by TGF-β1. Cell proliferation was evaluated by MTT assay. Flow cytometry was used to study the influence of triptolide on cell cycle and apoptosis. Signal proteins (Smad2, Smad3 and Smad7) were detected by western blotting analysis. Results: Triptolidemore » significantly inhibited TGF-β1-induced ASMC proliferation (P<0.05). The cell cycle was blocked at G1/S-interphase by triptolide dose dependently. No pro-apoptotic effects were detected under the concentration of triptolide we used. Western blotting analysis showed TGF-β1 induced Smad2 and Smad3 phosphorylation was inhibited by triptolide pretreatment, and the level of Smad7 was increased by triptolide pretreatment. Conclusions: Triptolide may function as an inhibitor of asthma airway remodeling by suppressing ASMCs proliferation via negative regulation of Smad signaling pathway. - Highlights: • In this study, rat airway smooth muscle cells were cultured and made synchronized. • Triptolide inhibited TGF-β1-induced airway smooth muscle cells proliferation. • Triptolide inhibited ASMCs proliferation via negative regulation of Smad signaling pathway.« less

Silibinin treatment prevents endotoxin-induced uveitis in rats in vivo and in vitro.

PubMed

Chen, Ching-Long; Chen, Jiann-Torng; Liang, Chang-Min; Tai, Ming-Cheng; Lu, Da-Wen; Chen, Yi-Hao

2017-01-01

Uveitis, an intraocular inflammatory disease, occurs mostly in young people and can result in the loss of socioeconomic capabilities. Silibinin has been shown to exert anti-inflammatory effects in human retinal pigment epithelial (RPE) cells. The present study investigated the anti-inflammatory effect of silibinin pretreatment on endotoxin-induced uveitis (EIU) in rats and the mechanisms by which it exerts these effects. Uveitis was induced via injection of lipopolysaccharides (LPS) into Lewis rats. Twenty-four hours after the LPS injection, histological examination showed that silibinin decreased inflammatory cell infiltration in the anterior segment of the eyes of LPS-treated rats. Analyses of the aqueous humor showed that silibinin decreased cell infiltration, protein concentration, nitric oxide (NO), and prostaglandin (PG)-E2 production. Western blot analysis indicated that silibinin decreased the expression of inducible NO synthase (iNOS), cyclooxygenase (COX-2), and phosphorylated IkB in the iris-ciliary body (ICB). Immunohistochemistry showed that silibinin decreased intercellular adhesion molecule (ICAM-1) expression in the ICB. In addition, western blot analysis showed that silibinin attenuated the expression of iNOS, COX-2, ICAM-1, and nuclear p65 in LPS-treated RAW cells. In conclusion, silibinin pretreatment prevents EIU and the subsequent production of proinflammatory mediators and ICAM-1, at least in part, by blocking the NF-κB-dependent signaling pathway both in vivo and in vitro. These effects may contribute to the silibinin-mediated preventive effects on intraocular inflammatory diseases such as acute uveitis.

Silibinin treatment prevents endotoxin-induced uveitis in rats in vivo and in vitro

PubMed Central

Chen, Ching-Long; Chen, Jiann-Torng; Liang, Chang-Min; Tai, Ming-Cheng; Lu, Da-Wen; Chen, Yi-Hao

2017-01-01

Uveitis, an intraocular inflammatory disease, occurs mostly in young people and can result in the loss of socioeconomic capabilities. Silibinin has been shown to exert anti-inflammatory effects in human retinal pigment epithelial (RPE) cells. The present study investigated the anti-inflammatory effect of silibinin pretreatment on endotoxin-induced uveitis (EIU) in rats and the mechanisms by which it exerts these effects. Uveitis was induced via injection of lipopolysaccharides (LPS) into Lewis rats. Twenty-four hours after the LPS injection, histological examination showed that silibinin decreased inflammatory cell infiltration in the anterior segment of the eyes of LPS-treated rats. Analyses of the aqueous humor showed that silibinin decreased cell infiltration, protein concentration, nitric oxide (NO), and prostaglandin (PG)-E2 production. Western blot analysis indicated that silibinin decreased the expression of inducible NO synthase (iNOS), cyclooxygenase (COX-2), and phosphorylated IkB in the iris-ciliary body (ICB). Immunohistochemistry showed that silibinin decreased intercellular adhesion molecule (ICAM-1) expression in the ICB. In addition, western blot analysis showed that silibinin attenuated the expression of iNOS, COX-2, ICAM-1, and nuclear p65 in LPS-treated RAW cells. In conclusion, silibinin pretreatment prevents EIU and the subsequent production of proinflammatory mediators and ICAM-1, at least in part, by blocking the NF-κB–dependent signaling pathway both in vivo and in vitro. These effects may contribute to the silibinin-mediated preventive effects on intraocular inflammatory diseases such as acute uveitis. PMID:28376126

Prokaryotic expression of CP gene of Fritillary virus Y infecting Thunberg fritillary and antiserum preparation.

PubMed

Wei, Chuan-Bao; Wei, Yang-Yang; Yang, Yu; Liu, Shi-Liang; Hu, Hao-Yu; He, Yue

2011-10-01

To prepare antiserum against Fritillary virus Y (FVY) CP for detecting FVY and study serological relationships with other viruses. Specific primer was designed according to Genbank (accession: AM039800) to amplify CP gene of FVY infecting Thunberg fritillary. Sequence relationship with other potyviruses was made by Blast. The CP gene was inserted into pSBET and expressed in Escherichia coli BL21 (DE3) plys E strain. The object protein was purified by 12% SDS-PAGE firstly and subsequently 5% - 20% gradient SDS-PAGE. The antiserum against the CP was raised in mouse and its specificity was confirmed by Western blot analysis. The reactivity of the antiserum produced to FVY CP was tested by Western blot against the over-expressed coat proteins of 17 potyviruses. The ability to combine with nature FVY particles was confirmed by ELISA analysis. It shared 81.2% nucleotide acids identities with TrVY (Tricyrtis virus Y, AY 864850) CP gene, 68.1% with SMV-P (Soybean mosaic virus Pinellia strain, AJ507388. 2) CP gene and 67.2% with ZYMV (Zucchini yellow mosaic virus Luan isolate) CP gene. The prepared antiserum was special to FVY CP, also reacted moderately to the expressed CP of SMV-P (Soybean mosaic virus Pinellia strain) and weakly to that of ZYMV (Zucchini yellow mosaic virus Luan isolate). The antibody could combine to nature FVY particles and the antiserum is suitable for FVY detection by ELISA in large scale.

Knockdown of HOXA10 reverses the multidrug resistance of human chronic mylogenous leukemia K562/ADM cells by downregulating P-gp and MRP-1.

PubMed

Yi, Ying-Jie; Jia, Xiu-Hong; Wang, Jian-Yong; Li, You-Jie; Wang, Hong; Xie, Shu-Yang

2016-05-01

Multidrug resistance (MDR) of leukemia cells is a major obstacle in chemotherapeutic treatment. The high expression and constitutive activation of P-glycoprotein (P-gp) and multidrug resistance protein-1 (MRP-1) have been reported to play a vital role in enhancing cell resistance to anticancer drugs in many tumors. The present study aimed to investigate the reversal of MDR by silencing homeobox A10 (HOXA10) in adriamycin (ADR)-resistant human chronic myelogenous leukemia (CML) K562/ADM cells by modulating the expression of P-gp and MRP-1. K562/ADM cells were stably transfected with HOXA10-targeted short hairpin RNA (shRNA). The results of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis showed that the mRNA and protein expression of HOXA10 was markedly suppressed following transfection with a shRNA-containing vector. The sensitivity of the K562/ADM cells to ADR was enhanced by the silencing of HOXA10, due to the increased intracellular accumulation of ADR. The accumulation of ADR induced by the silencing of HOXA10 may be due to the downregulation of P-gp and MRP-1. Western blot analysis revealed that downregulating HOXA10 inhibited the protein expression of P-gp and MRP-1. Taken together, these results suggest that knockdown of HOXA10 combats resistance and that HOXA10 is a potential target for resistant human CML.

Expression of SHANK3 in the Temporal Neocortex of Patients with Intractable Temporal Epilepsy and Epilepsy Rat Models.

PubMed

Zhang, Yanke; Gao, Baobing; Xiong, Yan; Zheng, Fangshuo; Xu, Xin; Yang, Yong; Hu, Yida; Wang, Xuefeng

2017-07-01

SH3 and multiple ankyrin (ANK) repeat domain 3 (SHANK3) is a synaptic scaffolding protein enriched in the postsynaptic density of excitatory synapses. SHANK3 plays an important role in the formation and maturation of excitatory synapses. In the brain, SHANK3 directly or indirectly interacts with various synaptic molecules including N-methyl-D-aspartate receptor, the metabotropic glutamate receptor (mGluR), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. Previous studies have shown that Autism spectrum disorder is a result of mutations of the main SHANK3 isoforms, which may be due to deficit in excitatory synaptic transmission and plasticity. Recently, accumulating evidence has demonstrated that overexpression of SHANK3 could induce seizures in vivo. However, little is known about the role of SHANK3 in refractory temporal lobe epilepsy (TLE). Therefore, we investigated the expression pattern of SHANK3 in patients with intractable temporal lobe epilepsy and in pilocarpine-induced models of epilepsy. Immunofluorescence, immunohistochemistry, and western blot analysis were used to locate and determine the expression of SHANK3 in the temporal neocortex of patients with epilepsy, and in the hippocampus and temporal lobe cortex of rats in a pilocarpine-induced epilepsy model. Double-labeled immunofluorescence showed that SHANK3 was mainly expressed in neurons. Western blot analysis confirmed that SHANK3 expression was increased in the neocortex of TLE patients and rats. These results indicate that SHANK3 participates in the pathology of epilepsy.

Nucleotide-binding oligomerization domain 2 (NOD2) activation induces apoptosis of human oral squamous cell carcinoma cells.

PubMed

Yoon, Hyo-Eun; Ahn, Mee-Young; Kwon, Seong-Min; Kim, Dong-Jae; Lee, Jun; Yoon, Jung-Hoon

2016-04-01

Microbial Pattern-recognition receptors (PRRs), such as nucleotide-binding oligomerization domains (NODs), are essential for mammalian innate immune response. This study was designed to determine the effect of NOD1 and NOD2 agonist on innate immune responses and antitumor activity in oral squamous cell carcinoma (OSCC) cells. NODs expression was examined by RT-PCR, and IL-8 production by NODs agonist was examined by ELISA. Western blot analysis was performed to determine the MAPK activation in response to their agonist. Cell proliferation was determined by MTT assay. Flow cytometry and Western blot analysis were performed to determine the MDP-induced cell death. The levels of NODs were apparently expressed in OSCC cells. NODs agonist, Tri-DAP and MDP, led to the production of IL-8 and MAPK activation. NOD2 agonist, MDP, inhibited the proliferation of YD-10B cells in a dose-dependent manner. Also, the ratio of Annexin V-positive cells and cleaved PARP was increased by MDP treatment in YD-10B cells, suggesting that MDP-induced cell death in YD-10B cells may be owing to apoptosis. Our results indicate that NODs are functionally expressed in OSCC cells and can trigger innate immune responses. In addition, NOD2 agonist inhibited cell proliferation and induced apoptosis. These findings provide the potential value of MDP as novel candidates for antitumor agents of OSCC. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

TCDD disrupts posterior palatogenesis and causes cleft palate.

PubMed

Yamada, Tomohiro; Hirata, Azumi; Sasabe, Eri; Yoshimura, Tomohide; Ohno, Seiji; Kitamura, Naoya; Yamamoto, Tetsuya

2014-01-01

Dioxins (e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) cause cleft palate at a high rate. A post-fusional split may contribute to the pathogenesis, and tissue fragility may be a concern. The objective of this study was to investigate the effects of TCDD on the palatal epithelium, bone and muscle, which contribute to tissue integrity. ICR mice (10-12 weeks old) were used. TCDD was administered on E12.5 at 40 mg/kg. Immunohistochemical staining for AhR, ER-α, laminin, collagen IV, osteopontin, Runx2, MyoD, and desmin were performed. Furthermore, western blot analysis for osteopontin, Runx2, MyoD, and desmin were performed to evaluate protein expression in the palatal tissue. Immunohistologically, there was little difference in the collagen IV and laminin localization in the palatal epithelium between control versus TCDD-treated mice. Runx2 and osteopontin immunoreactivity decreased in the TCDD-treated palatal bone, and MyoD and desmin decreased in the TCDD-treated palatal muscle. AhR and ER-α immunoreactivity were localized to the normal palatal bone, but ER-α was diminished in the TCDD-treated palate. On western blot analysis, Runx2, MyoD, and desmin were all downregulated in the TCDD-treated palate. TCDD may suppress palatal osteogenesis and myogenesis via AhR, and cause cleft palates via a post-fusional split mechanism, in addition to a failure of palatal fusion. Copyright © 2013 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

Knockdown of RhoA expression alters ovarian cancer biological behavior in vitro and in nude mice.

PubMed

Wang, Xiaoxia; Jiang, Wenyan; Kang, Jiali; Liu, Qicai; Nie, Miaoling

2015-08-01

RhoA regulates cell proliferation, migration, angiogenesis and gene expression. Altered RhoA activity contributes to cancer progression. The present study investigated the effects of RhoA knockdown on the regulation of ovarian cancer biological behavior in vitro and in nude mice. The expression of RhoA was knocked down using a lentivirus carrying RhoA short hairpin RNA (shRNA) in ovarian cancer cells and was confirmed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The altered ovarian cancer biological behaviors were assayed by cell viability, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL), migration, invasion, and nude mice tumorigenicity assays, while the altered gene expression was detected by RT-qPCR and western blot analysis. The results showed that lentivirus-carrying RhoA shRNA significantly suppressed RhoA expression in ovarian cancer cells, which suppressed tumor cell viability, migration, invasion and adhesion in vitro. RhoA silencing also inhibited the tumorigenicity of ovarian cancer cells in nude mice, which was characterized by the suppression of tumor xenograft formation and growth and induction of tumor cell apoptosis. The results of the present study demonstrated that knockdown of RhoA expression had a significant antitumor effect on ovarian cancer cells in vitro and in nude mice, suggesting that RhoA may be a target for the development of a novel therapeutic strategy in the control of ovarian cancer.

Coronin-1C is a novel biomarker for hepatocellular carcinoma invasive progression identified by proteomics analysis and clinical validation

PubMed Central

2010-01-01

Background To better search for potential markers for hepatocellular carcinoma (HCC) invasion and metastasis, proteomic approach was applied to identify potential metastasis biomarkers associated with HCC. Methods Membrane proteins were extracted from MHCC97L and HCCLM9 cells, with a similar genetic background and remarkably different metastasis potential, and compared by SDS-PAGE and identified by ESI-MS/MS. The results were further validated by western blot analysis, immunohistochemistry (IHC) of tumor tissues from HCCLM9- and MHCC97L-nude mice, and clinical specimens. Results Membrane proteins were extracted from MHCC97L and HCCLM9 cell and compared by SDS-PAGE analyses. A total of 14 differentially expressed proteins were identified by ESI-MS/MS. Coronin-1C, a promising candidate, was found to be overexpressed in HCCLM9 cells as compared with MHCC97L cells, and validated by western blot and IHC from both nude mice tumor tissues and clinical specimens. Coronin-1C level showed an abrupt upsurge when pulmonary metastasis occurred. Increasing coronin-1C expression was found in liver cancer tissues of HCCLM9-nude mice with spontaneous pulmonary metastasis. IHC study on human HCC specimens revealed that more patients in the higher coronin-1C group had overt larger tumor and more advanced stage. Conclusions Coronin-1C could be a candidate biomarker to predict HCC invasive behavior. PMID:20181269

shRNA-mediated EMMPRIN silencing inhibits human leukemic monocyte lymphoma U937 cell proliferation and increases chemosensitivity to adriamycin.

PubMed

Gao, Hui; Jiang, Qixiao; Han, Yantao; Peng, Jianjun; Wang, Chunbo

2015-03-01

EMMPRIN is a widely distributed cell surface glycoprotein, which plays an important role in tumor progression and confers resistance to some chemotherapeutic drugs. Recent studies have shown that EMMPRIN overexpression indicates poor prognosis in acute myeloid leukemia (AML). However, little was known on the role of EMMPRIN in leukemia. Human leukemia cell line U937 was stably transfected with a EMMPRIN-targeted shRNA-containing vector to investigate the effect of EMMPRIN on cellular functions. EMMPRIN expression was monitored by qRT-PCR and Western blotting. Cell viability and proliferation were determined by trypan blue exclusion and BrdU labeling, respectively. Cell cycle and apoptosis were analyzed by flow cytometry. Cytotoxicity of chemotherapeutic agent adriamycin on cells was assessed by MTT assay. Knockdown of EMMPRIN gene significantly inhibited cell viability and decreased cell proliferation. Fluorescence-activated cell-sorting analysis revealed that the reduced EMMPRIN expression resulted in cell cycle arrest at G1 phase and induced apoptosis. Meanwhile, western blotting analysis showed that EMMPRIN knockdown was associated with downregulation of cell cycle- and apoptosis-related molecules including cyclin D1, cyclin E, as well as increase in cleavage of caspase-3 and PARP. This study also showed that silencing of EMMPRIN sensitized U937 cells to Adriamycin. EMMPRIN is involved in proliferation, growth, and chemosensitivity of human AML line U937, indicating that EMMPRIN may be a promising therapeutic target for AML.

Activity of taspine isolated from Radix et Rhizoma Leonticis against estrogen-receptor-positive breast cancer.

PubMed

Zhan, Yingzhuan; Zhang, Yanmin; Chen, Yinnan; Wang, Nan; Zheng, Lei; He, Langchong

2011-09-01

The aim of our study was to investigate the effect of taspine isolated from Radix et Rhizoma Leonticis on the growth of oestrogen-receptor-positive breast cancer xenografts in vivo and the possible mechanism for this action. In vivo taspine studies were conducted with ZR-75-30 human breast cancer xenografts in athymic mice, and then tumors tissue lysates were subjected to Western blotting analysis of estrogen receptor (ER) and progesterone receptor (PR), which was related to inhibition of tumor growth. For in vitro study, cell proliferation, cell cycle and apoptosis of ZR-75-30 cell treated with or without taspine were detected. ER and PR expression were detected by Western blotting, ER and PR mRNA were verified by reverse transcription polymerase chain reaction (RT-PCR). The results showed that treatment over 14 days resulted in a sustained and significant reduction in xenograft weight compared with untreated controls. Cell cycle and apoptosis analysis documented that taspine could change cell cycle and induce cell apoptosis. There was a significant decrease observed in the expression of ER and PR both in tumor tissue and cells after treatment with taspine, RT-PCR also showed a reduction in the expression of mRNA for ER and PR in the group treated with taspine. Taken together, these results suggested that taspine might serve as a promising candidate of ER antagonist in the treatment of oestrogen-independent breast cancer. Copyright © 2011 Elsevier B.V. All rights reserved.

The Anticancer Effects of Radachlorin-mediated Photodynamic Therapy in the Human Endometrial Adenocarcinoma Cell Line HEC-1-A.

PubMed

Kim, Su-Mi; Rhee, Yun-Hee; Kim, Jong-Soo

2017-11-01

We investigated the effect of photodynamic therapy (PDT) using radachlorin on invasion, vascular formation and apoptosis by targeting epidermal growth factor receptor (EGFR)/vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathways in the HEC-1-A endometrial adenocarcinoma cell line. To investigate the apoptotic pathway, we performed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, and western blot analysis. We also evaluated the effects of PDT on tubular capillary formation in and invasion by HEC-1-A cells with a tube formation assay, invasion assay, prostaglandin E2 (PGE2) assay, and western blot analysis. PDT had anticancer effects on HEC-1-A through activation of the intrinsic pathway of apoptosis via caspase-9 and poly-(ADP-ribose) polymerase (PARP). PDT also inhibited tubular capillary formation in and invasion by HEC-1-A under VEGF pretreatment, that resulted from down-regulation of VEGFR2, EGFR, Ras homolog gene family/ member A (RhoA) and PGE2. These results are indicative of the specificity of radachlorin-mediated PDT to VEGF. The major advantage of radachlorin-mediated PDT is its selectivity for cancer tissue while maintaining adjacent normal endometrial tissue. Therefore, radachlorin-mediated PDT might offer high anticancer efficacy for endometrial adenocarcinoma and an especially useful modality for preserving fertility. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Chlorogenic acid attenuates lipopolysaccharide-induced mice mastitis by suppressing TLR4-mediated NF-κB signaling pathway.

PubMed

Ruifeng, Gao; Yunhe, Fu; Zhengkai, Wei; Ershun, Zhou; Yimeng, Li; Minjun, Yao; Xiaojing, Song; Zhengtao, Yang; Naisheng, Zhang

2014-04-15

Chlorogenic acid (CGA), one of the most abundant polyphenols in the diet, has been reported to have potent anti-inflammatory properties. However, the effect of CGA on lipopolysaccharide (LPS)-induced mice mastitis has not been investigated. The purpose of the present study was to elucidate whether CGA could ameliorate the inflammation response in LPS-induced mice mastitis and to clarify the possible mechanism. The mouse model of mastitis was induced by injection of LPS through the duct of mammary gland. CGA was administered intraperitoneally with the dose of 12.5, 25, and 50mg/kg respectively 1h before and 12h after induction of LPS. In this study, the effect of CGA on LPS-induced mice mastitis was assessed through histopathological examination, ELISA assay, and western blot analysis. The results showed that CGA significantly reduced TNF-α, IL-1β, and IL-6 production compared with LPS group. Besides, western blot analysis showed that CGA could inhibit the expression of TLR4 and the phosphorylation of NF-κB and IκB induced by LPS. These results suggested that anti-inflammatory effects of CGA against LPS-induced mastitis may be due to its ability to inhibit TLR4-mediated NF-κB signaling pathway. Therefore, CGA may be a potent therapeutic reagent for the prevention of the immunopathology encountered during Escherichia coli elicited mastitis. Copyright © 2014 Elsevier B.V. All rights reserved.

Rapamycin preserves the follicle pool reserve and prolongs the ovarian lifespan of female rats via modulating mTOR activation and sirtuin expression.

PubMed

Zhang, Xing-mei; Li, Li; Xu, Jin-jie; Wang, Na; Liu, Wei-juan; Lin, Xuan-hao; Fu, Yu-cai; Luo, Li-li

2013-07-01

To maintain the normal length of female reproductive life, the majority of primordial follicles must be maintained in a quiescent state for later use. In this study, we aimed to study the effects of rapamycin on primordial follicle development and investigate the role of mTOR and sirtuin signaling. Rats were treated every other day with an intraperitoneal injection of rapamycin (5mg/kg) or vehicle. After 10weeks of treatment, ovaries were harvested for hematoxylin and eosin (HE) staining, and analysis by immunohistochemistry and Western blotting. HE staining showed that the number and percentage of primordial follicles in the rapamycin-treated group were twice the control group (P<0.001). Immunohistochemical analysis showed that mTOR and phosphorylated-p70S6K were extensively expressed in surviving follicles with strong staining observed in the cytoplasm of the oocyte. Western blotting showed decreased expression of phosphorylated mTOR and phosphorylated p70S6K in the rapamycin-treated group, and increased the expression of both SIRT1 and SIRT6 compared to the control group (P<0.05). Taken together, these results suggest that rapamycin may inhibit the transition from primordial to developing follicles and preserve the follicle pool reserve, thus extending the ovarian lifespan of female rats via the modulation of mTOR and sirtuin signalings. Copyright © 2013 Elsevier B.V. All rights reserved.

Expression, purification and characterization of two truncated peste des petits ruminants virus matrix proteins in Escherichia coli, and production of polyclonal antibodies against this protein.

PubMed

Liu, Fuxiao; Wu, Xiaodong; Li, Lin; Liu, Zengshan; Wang, Zhiliang

2013-09-01

Peste des petits ruminants virus (PPRV), the etiological agent of peste des petits ruminants, is classified into the genus Morbillivirus in the family Paramyxoviridae. The PPRV matrix (M) gene is composed of 1483 base pairs, encoding a 335 amino acids M protein with a molecular weight of approximately 38kD. We have demonstrated previously that the full-length M protein was expressed at an extremely low level or not even expressed in Escherichia coli BL21 (DE3). In this study, the M protein was split into two truncated forms to be successfully expressed in E. coli at a high level using the pET30a (+) vector, respectively, by analysis of SDS-PAGE, western blot and MALDI-TOF-MS. The optimization of culture conditions led us to perform the recombinant protein induction with 0.2mM IPTG at 28°C for 12h, whereby both proteins nevertheless were expressed in the insoluble form. Therefore, both His-tagged proteins were purified under the denaturing condition using a commercially available kit. Balb/c mice were immunized with the complex of purified proteins and then effectively produced polyclonal antibodies, which reached to a relatively high titer by the analysis of ELISA. The specificity of the prepared polyclonal antibodies was checked by western blot and immunofluorescence, revealing them with the desirable specificity against both non-denatured and denatured M proteins. Copyright © 2013 Elsevier Inc. All rights reserved.

High expression of 23 kDa protein of augmenter of liver regeneration (ALR) in human hepatocellular carcinoma

PubMed Central

Yu, Hai-Ying; Zhu, Man-Hua; Xiang, Dai-Rong; Li, Jun; Sheng, Ji-Fang

2014-01-01

Background Augmenter of liver regeneration (ALR) is an important polypeptide that participates in the process of liver regeneration. Two forms of ALR proteins are expressed in hepatocytes. Previous data have shown that ALR is essential for cell survival and has potential antimetastatic properties in hepatocellular carcinoma (HCC). Aims The study aimed to evaluate the expression levels of two forms of ALR proteins in HCC and their possible significance in HCC development. Methods Balb/c mouse monoclonal antibody against ALR protein was prepared in order to detect the ALR protein in HCC by Western blotting and immunohistochemistry. ALR mRNA expression levels were measured by real-time polymerase chain reaction in HCC tissues and compared to paracancerous liver tissues in 22 HCC patients. Results ALR mRNA expression in HCC liver tissues (1.51×106 copies/μL) was higher than in paracancerous tissues (1.04×104 copies/μL). ALR protein expression was also enhanced in HCC liver tissues. The enhanced ALR protein was shown to be 23 kDa by Western blotting. Immunohistochemical analysis showed that the 23 kDa ALR protein mainly existed in the hepatocyte cytosol. Conclusion The 23 kDa ALR protein was highly expressed in HCC and may play an important role in hepatocarcinogenesis. PMID:24940072

miR-23a promotes IKKα expression but suppresses ST7L expression to contribute to the malignancy of epithelial ovarian cancer cells

PubMed Central

Yang, Zhen; Wang, Xiang-ling; Bai, Ru; Liu, Wei-ying; Li, Xin; Liu, Min; Tang, Hua

2016-01-01

Background: Dysregulation of microRNAs (miRNAs) has been found in human epithelial ovarian cancer (EOC). However, the role and mechanism of action of miR-23a in EOC remain unclear. Methods: The roles of miR-23a, IKKα, and ST7L in EOC were determined by MTT, colony formation, wounding healing, transwell, flow cytometry, immunofluorescence, RT–qPCR, and western blotting experiments. miR-23a target genes were validated by EGFP reporter assays, RT–qPCR, and western blotting analysis. Results: miR-23a is upregulated and promotes tumorigenic activity by facilitating the progress of cell cycle and EMT and repressing apoptosis in EOC cells. miR-23a enhances the expression of IKKα but suppresses the expression of ST7L by binding the 3′UTR of each transcript in EOC cells. The proliferation, migration, and invasion of EOC cells are increased by IKKα and inhibited by ST7L. Furthermore, miR-23a activates NF-κB by upregulating IKKα and WNT/MAPK pathway by downregulating ST7L. Conclusions: miR-23a functions as an oncogene by targeting IKKα and ST7L, thus contributing to the malignancy of EOC cells. PMID:27537390

Involvement of RhoGDI2 in the resistance of colon cancer cells to 5-fluorouracil.

PubMed

Zheng, Zhong; Li, Jianfang; He, Xiangyi; Chen, Xuehua; Yu, Beiqin; Ji, Jun; Zhang, Jianian; Wang, Tingfeng; Gu, Qinlong; Zhu, Zhenggang; Liu, Bingya

2010-01-01

The acquisition of resistance to 5-FU is one of the most prominent obstacles to successful chemotherapy, and the mechanisms underlying the resistance are not fully understood. The aim of this study is to identify novel mediators of 5-FU resistance in colon cancer cells. LoVo colon cancer cells were induced to 5-FU resistance in vitro. The global protein profiles between LoVo and its 5-FU resistant derivative cell line LoVo/5-FU were analyzed by two dimensional gel electrophoresis-based comparative proteomics. The identified proteins expression was confirmed by Western blot analysis. The cytotoxicity of 5-FU was measured in LoVo/5-FU after knockdown of RhoGDI2 (one of the identified protien). Three differentially expressed proteins were identified. RhoGDI2 and CapG were upregulated, whereas proapoptotic protein Maspin was down-regulated in LoVo/5-FU and validated by Western blotting. Furthermore, knockdown of RhoGDI2 expression by transfection with the RhoGDI2-specific siRNA significantly reduced the resistance to 5-FU in LoVo/5-FU (p < 0.05). These novel data suggest that these differentially expressed proteins may contribute to the development of 5-FU resistance in colon cancer cells.

Peroxiredoxin 1 is a tumor-associated antigen in esophageal squamous cell carcinoma

PubMed Central

REN, PENGFEI; YE, HUA; DAI, LIPING; LIU, MEI; LIU, XINXIN; CHAI, YURONG; SHAO, QING; LI, YANG; LEI, NINGJING; PENG, BO; YAO, WU; ZHANG, JIANYING

2013-01-01

Peroxiredoxin 1 (Prdx1) is an antioxidant and plays an important role in H2O2-mediated cell signaling. We previously found that the expression level of Prdx1 was elevated in esophagus squamous cell carcinoma (ESCC) tissue using a proteomics approach. Since overexpressed protein can induce an autoimmune response, to further examine whether serum from ESCC patients exhibits immunoreactivity against Prdx1, autoantibody responses to Prdx1 were evaluated by ELISA, western blotting and indirect immunofluorescence assay in sera from patients with ESCC and normal individuals. Immunohistochemical study with tissue array slides and western blot analysis with cancer cell lines were also performed to analyze the protein expression profiles of Prdx1 in ESCC tissues and cancer cell lines. The results demonstrated that the positive rate of autoantibody against Prdx1 in ESCC sera was 13.2% (9/68), whereas this rate was 0% (0/89) in normal individuals. Data also showed that expression of Prdx1 was significantly increased in ESCC tissues when compared to expression in paired adjacent normal tissues (P<0.05). The data indicate that Prdx1 may contribute to malignant transformation of the esophagus, and may be used as a biomarker in the immunodiagnosis of ESCC. PMID:24009050

Expression of the monocarboxylate transporter 1 (MCT1) in cells of the porcine intestine.

PubMed

Welter, Harald; Claus, Rolf

2008-06-01

Uptake of energy into cells and its allocation to individual cellular compartments by transporters are essential for tissue homeostasis. The present study gives an analysis of MCT1 expression and its cellular occurrence in the porcine intestine. Tissue portions from duodenum, jejunum, ileum, colon ascendens, colon transversum and colon descendens were collected and prepared for immunohistochemistry, Western blot and real time RT-PCR. A 169bp porcine MCT1 cDNA fragment was amplified and published. MCT1 mRNA expression in the large intestine was 20 fold higher compared to the small intestine. Western blot detected a single protein band of 41kDa at a much higher amount of MCT1 protein in the large intestine vs. the small intestine. MCT1 protein was detected in mitochondrial fractions of the large but not the small intestine. Immunohistochemistry in the small intestine showed that immune cells in the lamina propria and in the lymphoid follicles primarily expressed MCT1 while in the colon epithelial cells were the main source of MCT1. In summary, cellular expression of MCT1 differs between epithelial cells in the colon and small intestine. A possible role of MCT1 for uptake of butyrate into immune cells and the overall role of MCT1 for intestinal immune cell function remains elusive.

Cy5 total protein normalization in Western blot analysis.

PubMed

Hagner-McWhirter, Åsa; Laurin, Ylva; Larsson, Anita; Bjerneld, Erik J; Rönn, Ola

2015-10-01

Western blotting is a widely used method for analyzing specific target proteins in complex protein samples. Housekeeping proteins are often used for normalization to correct for uneven sample loads, but these require careful validation since expression levels may vary with cell type and treatment. We present a new, more reliable method for normalization using Cy5-prelabeled total protein as a loading control. We used a prelabeling protocol based on Cy5 N-hydroxysuccinimide ester labeling that produces a linear signal response. We obtained a low coefficient of variation (CV) of 7% between the ratio of extracellular signal-regulated kinase (ERK1/2) target to Cy5 total protein control signals over the whole loading range from 2.5 to 20.0μg of Chinese hamster ovary cell lysate protein. Corresponding experiments using actin or tubulin as controls for normalization resulted in CVs of 13 and 18%, respectively. Glyceraldehyde-3-phosphate dehydrogenase did not produce a proportional signal and was not suitable for normalization in these cells. A comparison of ERK1/2 signals from labeled and unlabeled samples showed that Cy5 prelabeling did not affect antibody binding. By using total protein normalization we analyzed PP2A and Smad2/3 levels with high confidence. Copyright © 2015 Elsevier Inc. All rights reserved.

Quercetin Inhibits the Migration and Invasion of HCCLM3 Cells by Suppressing the Expression of p-Akt1, Matrix Metalloproteinase (MMP) MMP-2, and MMP-9.

PubMed

Lu, Jun; Wang, Zhiqiang; Li, Shuyan; Xin, Qi; Yuan, Miaomiao; Li, Huanping; Song, Xiaoxia; Gao, Haijun; Pervaiz, Nabeel; Sun, Xudong; Lv, Wei; Jing, Tao; Zhu, Yanmei

2018-04-27

BACKGROUND Quercetin is a natural bioactive flavonoid that is present in a wide variety of vegetables and fruits and exhibits a promising anti-metastasis property in various human cancer cells. However, the effect of quercetin on human HCCLM3 cells is unclear. MATERIAL AND METHODS In the current study, a wound-healing assay was performed using quercetin-treated HCCLM3 cells to further explore whether quercetin affects the motility of human HCCLM3 cells. Transwell assay was used to explore the potential effect of quercetin in HCCLM3 cells on cell migration and cell invasion. Western blotting analysis was used to explore the expression of p-Akt1, MMP-2, and MMP-9 in quercetin-treated HCCLM3 cells. RESULTS The wound-healing time was delayed in quercetin-treated HCCLM3 cells, and the ability to migrate and invade was inhibited in quercetin-treated human HCCLM3 cells. Moreover, the protein levels of p-Akt1, MMP-2, and MMP-9 were down-regulated in quercetin-treated HCCLM3 cells, as detected by Western blotting. CONCLUSIONS Our data show that quercetin attenuated cell migration and invasion by suppressing the protein levels of p-Akt1, MMP-2, and MMP-9 in HCCLM3 cells.

Ixora coccinea Enhances Cutaneous Wound Healing by Upregulating the Expression of Collagen and Basic Fibroblast Growth Factor

PubMed Central

Upadhyay, Aadesh; Chattopadhyay, Pronobesh; Goyary, Danswrang; Mitra Mazumder, Papiya; Veer, Vijay

2014-01-01

Background. Ixora coccinea L. (Rubiaceae) has been documented for traditional use in hypertension, menstrual irregularities, sprain, chronic ulcer, and skin diseases. In the present study, I. coccinea was subjected to in vitro and in vivo wound healing investigation. Methods. Petroleum ether, chloroform, methanol, and water sequential I. coccinea leaves extracts were evaluated for in vitro antioxidant, antimicrobial, and fibroblast proliferation activities. The promising I. coccinea methanol extract (IxME) was screened for in vivo wound healing activity in Wistar rat using circular excision model. Wound contraction measurement, hydroxyproline quantification, and western blot for collagen type III (COL3A1), basic fibroblast growth factor (bFGF), and Smad-2, -3, -4, and -7 was performed with 7-day postoperative wound granulation tissue. Gentamicin sulfate (0.01% w/w) hydrogel was used as reference standard. Results. IxME showed the potent antimicrobial, antioxidant activities, with significant fibroblast proliferation inducing activity, as compared to all other extracts. In vivo study confirmed the wound healing accelerating potential of IxME, as evidenced by faster wound contraction, higher hydroxyproline content, and improved histopathology of granulation tissue. Western blot analysis revealed that the topical application of I. coccinea methanol extract stimulates the fibroblast growth factor and Smad mediated collagen production in wound tissue. PMID:24624303

Calpain: a molecule to induce AIF-mediated necroptosis in RGC-5 following elevated hydrostatic pressure

PubMed Central

2014-01-01

Background RIP3 (Receptor-interacting protein 3) pathway was mainly described as the molecular mechanism of necroptosis (programmed necrosis). But recently, non-RIP3 pathways were found to mediate necroptosis. We deliberate to investigate the effect of calpain, a molecule to induce necroptosis as reported (Cell Death Differ 19:245–256, 2012), in RGC-5 following elevated hydrostatic pressure. Results First, we identified the existence of necroptosis of RGC-5 after insult by using necrostatin-1 (Nec-1, necroptosis inhibitor) detected by flow cytometry. Immunofluorescence staining and western blot were used to detect the expression of calpain. Western blot analysis was carried out to describe the truncated AIF (tAIF) expression with or without pretreatment of ALLN (calpain activity inhibitor). Following elevated hydrostatic pressure, necroptotic cells pretreated with or without ALLN was stained by Annexin V/PI, The activity of calpain was also examined to confirm the inhibition effect of ALLN. The results showed that after cell injury there was an upregulation of calpain expression. Upon adding ALLN, the calpain activity was inhibited, and tAIF production was reduced upon injury along with the decreased number of necroptosis cells. Conclusion Our study found that calpain may induce necroptosis via tAIF-modulation in RGC-5 following elevated hydrostatic pressure. PMID:24884644

Low-level laser treatment stimulates hair growth via upregulating Wnt10b and β-catenin expression in C3H/HeJ mice.

PubMed

Zhang, Tiran; Liu, Liqiang; Fan, Jincai; Tian, Jia; Gan, Cheng; Yang, Zengjie; Jiao, Hu; Han, Bing; Liu, Zheng

2017-07-01

This study was conducted in order to evaluate the role of low-level laser treatment (LLLT) in hair growth in C3H/HeJ mice. Healthy C57BL/6 mice were randomly divided into two groups: with and without low-level laser treatment. The skin color of each mouse was observed each day. Skin samples were collected for H&E, immunofluorescence, PCR, and western blot analysis, to observe the morphology of hair follicles and detect the expression levels of Wnt10b and β-catenin. Observation of skin color demonstrated that black pigmentation started significantly earlier in the laser group than in the control group. Hair follicle number in both groups showed no difference; however, the hair follicle length presented a significant difference. Wnt10b protein was detected on the second day in hair matrix cells in the LLLT group but not in the control group. PCR and western blot results both illustrated that expression of Wnt10b and β-catenin was significantly higher in the LLLT group than in the control group. Our study illustrated that low-level laser treatment can promote hair regrowth by inducing anagen phase of hair follicles via initiating the Wnt10b/β-catenin pathway.

[Cloning, expression and identification of functional fragment rC3B of human complement C3 in E. Coli].

PubMed

Gan, Hui; Zhou, Yong; Sun, Ping; Zhu, Xiao-Xia; Wang, Quan-Li; Zhan, Lin-Sheng

2007-08-01

This study was purposed to verify the binding part of human complement C3 to complement receptor III (CRIII) in monocytes, the peptide rC3B, including the binding-site, was expressed, purified and identified. rC3B, the binding part of human complement C3 to CRIII, was selected by computer-aided modeling and summarizing researches published. Then, rC3B gene fragment was amplified by PCR, and cloned into prokaryotic vector pQE30a. The fusion protein rC3B was expressed in E.coli M15 and purified by Ni(2+)-chelating affinity chromatography. The activity of rC3B was identified by Western blot and adherence assay with monocytes. The results showed that rC3B fragment was obtained, and a prokaryotic expression vector pQE30-rC3B was constructed. rC3B was efficiently expressed and purified. In Western blot, the target protein showed the activity of binding with C3 antibody, while the purified protein showed the activity of adherence with monocytes. It is concluded that the recombinant C3B was obtained and identified, and this study lay the basis for the further functional analysis of C3.

Altered expression of zonula occludens-2 precedes increased blood-brain barrier permeability in a murine model of fulminant hepatic failure.

PubMed

Shimojima, Naoki; Eckman, Christopher B; McKinney, Michael; Sevlever, Daniel; Yamamoto, Satoshi; Lin, Wenlang; Dickson, Dennis W; Nguyen, Justin H

2008-01-01

Brain edema secondary to increased blood-brain barrier (BBB) permeability is a lethal complication in fulminant hepatic failure (FHF). Intact tight junctions (TJ) between brain capillary endothelial cells are critical for normal BBB function. However, the role of TJ in FHF has not been explored. We hypothesized that alterations in the composition of TJ proteins would result in increased BBB permeability in FHF. In this study, FHF was induced in C57BL/6J mice by using azoxymethane. BBB permeability was assessed with sodium fluorescein. Expression of TJ proteins was determined by Western blot, and their cellular distribution was examined using immunofluorescent microscopy. Comatose FHF mice had significant cerebral sodium fluorescein extravasation compared with control and precoma FHF mice, indicating increased BBB permeability. Western blot analysis showed a significant decrease in zonula occludens (ZO)-2 expression starting in the precoma stage. Immunofluorescent microscopy showed a significantly altered distribution pattern of ZO-2 in isolated microvessels from precoma FHF mice. These changes were more prominent in comatose FHF animals. Significant alterations in ZO-2 expression and distribution in the tight junctions preceded the increased BBB permeability in FHF mice. These results suggest that ZO-2 may play an important role in the pathogenesis of brain edema in FHF.

Thread Embedding Acupuncture Inhibits Ultraviolet B Irradiation-Induced Skin Photoaging in Hairless Mice.

PubMed

Kim, Yoon-Jung; Kim, Ha-Neui; Shin, Mi-Sook; Choi, Byung-Tae

2015-01-01

Thread embedding acupuncture (TEA) is an acupuncture treatment applied to many diseases in Korean medical clinics because of its therapeutic effects by continuous stimulation to tissues. It has recently been used to enhance facial skin appearance and antiaging, but data from evidence-based medicine are limited. To investigate whether TEA therapy can inhibit skin photoaging by ultraviolet B (UVB) irradiation, we performed analyses for histology, histopathology, in situ zymography and western blot analysis in HR-1 hairless mice. TEA treatment resulted in decreased wrinkle formation and skin thickness (Epidermis; P = 0.001 versus UV) in UVB irradiated mice and also inhibited degradation of collagen fibers (P = 0.010 versus normal) by inhibiting proteolytic activity of gelatinase matrix-metalloproteinase-9 (MMP-9). Western blot data showed that activation of c-Jun N-terminal kinase (JNK) induced by UVB (P = 0.002 versus normal group) was significantly inhibited by TEA treatment (P = 0.005 versus UV) with subsequent alleviation of MMP-9 activation (P = 0.048 versus UV). These results suggest that TEA treatment can have anti-photoaging effects on UVB-induced skin damage by maintenance of collagen density through regulation of expression of MMP-9 and related JNK signaling. Therefore, TEA therapy may have potential roles as an alternative treatment for protection against skin damage from aging.

Thread Embedding Acupuncture Inhibits Ultraviolet B Irradiation-Induced Skin Photoaging in Hairless Mice

PubMed Central

Kim, Yoon-Jung; Kim, Ha-Neui; Shin, Mi-Sook; Choi, Byung-Tae

2015-01-01

Thread embedding acupuncture (TEA) is an acupuncture treatment applied to many diseases in Korean medical clinics because of its therapeutic effects by continuous stimulation to tissues. It has recently been used to enhance facial skin appearance and antiaging, but data from evidence-based medicine are limited. To investigate whether TEA therapy can inhibit skin photoaging by ultraviolet B (UVB) irradiation, we performed analyses for histology, histopathology, in situ zymography and western blot analysis in HR-1 hairless mice. TEA treatment resulted in decreased wrinkle formation and skin thickness (Epidermis; P = 0.001 versus UV) in UVB irradiated mice and also inhibited degradation of collagen fibers (P = 0.010 versus normal) by inhibiting proteolytic activity of gelatinase matrix-metalloproteinase-9 (MMP-9). Western blot data showed that activation of c-Jun N-terminal kinase (JNK) induced by UVB (P = 0.002 versus normal group) was significantly inhibited by TEA treatment (P = 0.005 versus UV) with subsequent alleviation of MMP-9 activation (P = 0.048 versus UV). These results suggest that TEA treatment can have anti-photoaging effects on UVB-induced skin damage by maintenance of collagen density through regulation of expression of MMP-9 and related JNK signaling. Therefore, TEA therapy may have potential roles as an alternative treatment for protection against skin damage from aging. PMID:26185518

The antigens contributing to the serological cross-reactions of Proteus antisera with Klebsiella representatives.

PubMed

Palusiak, Agata

2015-03-01

Proteus sp. and Klebsiella sp. mainly cause infections of the urinary and respiratory tracts or wounds in humans. The representatives of both genera produce virulence factors like lipopolysaccharide (LPS) or outer membrane proteins (OMPs) having much in common in the structures and/or functions. To check how far this similarity is revealed in the serological cross-reactivity, the bacterial masses of 24 tested Klebsiella sp. strains were tested in ELISA with polyclonal rabbit antisera specific to the representatives of 79 Proteus O serogroups. The strongest reacting systems were selected to Western blot, where the majority of Klebsiella masses reacted in a way characteristic for electrophoretic patterns of proteins. The strongest reactions were obtained for proteins of near 67 and 40 kDa and 12.5 kDa. Mass spectrometry analysis of the proteins samples of one Proteus sp. and one Klebsiella sp. strain showed the GroEL like protein of a sequence GI number 2980926 to be similar for both strains. In Western blot some Klebsiella sp. masses reacted similarly to the homologous Proteus LPSs. The LPS contribution in the observed reactions of the high molecular-mass LPS species was confirmed for Klebsiella oxytoca 0.062. Copyright © 2014 Elsevier Ltd. All rights reserved.

Molecular characterization and functional analysis of Eimeria tenella malate dehydrogenase.

PubMed

Chen, Ting; Huang, Bing; Zhao, Qiping; Dong, Hui; Zhu, Shunhai; Zhao, Zongping; Lv, Ling; Yan, Ming; Han, Hongyu

2018-05-08

Eimeria tenella is a serious intracellular parasite that actively invades cecal epithelial cells of chickens. The widespread use of drugs causes severe resistance to Eimeria tenella. We detected that malate dehydrogenase (MDH), one of the differentially expressed genes, was upregulated in diclazuril-resistant and maduramicin-resistant strains through transcriptome sequencing. In this study, we cloned and expressed MDH of E. tenella (EtMDH). Quantitative real-time polymerase chain reactions (qPCR) and Western blots were used to analyze the expression of EtMDH in resistant and sensitive strains, indicating EtMDH was upregulated in two resistant strains at the messenger RNA and protein levels. Enzyme activity was tested through absorbance measurement and the EtMDH activity increased in two resistant strains. Expression levels of EtMDH in four developmental stages of E. tenella were tested through qPCR and Western blot. Invasion inhibition assays explored if EtMDH was involved in invasion of DF-1 cells by E. tenella sporozoites. Indirect immunofluorescence assays investigated EtMDH distribution during parasite development in DF-1 cells invaded by E. tenella sporozoites. Experimental results showed that EtMDH may be related to drug resistance of E. tenella during its development and invasion. EtMDH may be an effective molecular marker for detection of E. tenella drug resistance.

Transcorneal electrical stimulation rescues axotomized retinal ganglion cells by activating endogenous retinal IGF-1 system.

PubMed

Morimoto, Takeshi; Miyoshi, Tomomitsu; Matsuda, Satoshi; Tano, Yasuo; Fujikado, Takashi; Fukuda, Yutaka

2005-06-01

To investigate the effect of transcorneal electrical stimulation (TES) on the survival of axotomized RGCs and the mechanism underlying the TES-induced neuroprotection in vivo. Adult male Wistar rats received TES after optic nerve (ON) transection. Seven days after the ON transection, the density of the surviving RGCs was determined, to evaluate the neuroprotective effect of TES. The levels of the mRNA and protein of insulin-like growth factor (IGF)-1 in the retina after TES were determined by RT-PCR and Northern and Western blot analyses. The localization of IGF-1 protein in the retina was examined by immunohistochemistry. TES after ON transection increased the survival of axotomized RGCs in vivo, and the degree of rescue depended on the strength of the electric charge. RT-PCR and Northern and Western blot analyses revealed a gradual upregulation of intrinsic IGF-1 in the retina after TES. Immunohistochemical analysis showed that IGF-1 immunoreactivity was localized initially in the endfeet of Muller cells and then diffused into the inner retina. TES can rescue the axotomized RGCs by increasing the level of IGF-1 production by Muller cells. These findings provide a new therapeutic approach to prevent or delay the degeneration of retinal neurons without the administration of exogenous neurotrophic factors.

Estimation of low-dose radiation-responsive proteins in the absence of genomic instability in normal human fibroblast cells.

PubMed

Yim, Ji-Hye; Yun, Jung Mi; Kim, Ji Young; Nam, Seon Young; Kim, Cha Soon

2017-11-01

Low-dose radiation has various biological effects such as adaptive responses, low-dose hypersensitivity, as well as beneficial effects. However, little is known about the particular proteins involved in these effects. Here, we sought to identify low-dose radiation-responsive phosphoproteins in normal fibroblast cells. We assessed genomic instability and proliferation of fibroblast cells after γ-irradiation by γ-H2AX foci and micronucleus formation analyses and BrdU incorporation assay, respectively. We screened fibroblast cells 8 h after low-dose (0.05 Gy) γ-irradiation using Phospho Explorer Antibody Microarray and validated two differentially expressed phosphoproteins using Western blotting. Cell proliferation proceeded normally in the absence of genomic instability after low-dose γ-irradiation. Phospho antibody microarray analysis and Western blotting revealed increased expression of two phosphoproteins, phospho-NFκB (Ser536) and phospho-P70S6K (Ser418), 8 h after low-dose radiation. Our findings suggest that low-dose radiation of normal fibroblast cells activates the expression of phospho-NFκB (Ser536) and phospho-P70S6K (Ser418) in the absence of genomic instability. Therefore, these proteins may be involved in DNA damage repair processes.

Development and characterization of monoclonal antibody to the lymphocystis disease virus of Japanese flounder Paralichthys olivaceus isolated from China.

PubMed

Cheng, Shunfeng; Zhan, Wenbin; Xing, Jing; Sheng, Xiuzhen

2006-08-01

Lymphocystis disease virus (LCDV) can infect, both naturally and experimentally, about 100 different teleost fish species. In this study, LCDV was purified using differential and gradient centrifugation from skin tumours of Japanese flounder Paralichthys olivaceus. A panel of five monoclonal antibodies (Mabs) against LCDV were produced by immunization of Balb/c mice with purified virus preparations. Analysed by the indirect enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), Western blot and immunogold electron microscopy (IEM), they showed specificity for LCDV. Immunofluorescent studies showed that the specific fluorescence signals appeared at the peripheral zone of hypertrophied cells cytoplasm where was the cytoplasmic inclusion bodies location and many of them formed ribbon-shaped. Western blot analysis demonstrated that two Mabs 1D7 and 2B6 reacted specifically to a single protein with an approximately molecular weight of 116kDa, Mab 3G3 reacted with two LCDV proteins at molecular mass of approximately 116 and 90kDa. Immunogold transmission electron-microscopy provided visualized evidence that the epitopes recognized by these Mabs were located on the outer surface of virions. The Mabs characterized should prove useful for developing LCDV diagnostic assays and for studying the biology of infection and pathogenesis of disease.

Development of an Anti-HER2 Monoclonal Antibody H2Mab-139 Against Colon Cancer.

PubMed

Kaneko, Mika K; Yamada, Shinji; Itai, Shunsuke; Kato, Yukinari

2018-02-01

Human epidermal growth factor receptor 2 (HER2) expression has been reported in several cancers, such as breast, gastric, lung, pancreatic, and colorectal cancers. HER2 is overexpressed in those cancers and is associated with poor clinical outcomes. Trastuzumab, a humanized anti-HER2 antibody, provides significant survival benefits for patients with HER2-overexpressing breast cancers and gastric cancers. In this study, we developed a novel anti-HER2 monoclonal antibody (mAb), H 2 Mab-139 (IgG 1 , kappa) and investigated it against colon cancers using flow cytometry, western blot, and immunohistochemical analyses. Flow cytometry analysis revealed that H 2 Mab-139 reacted with colon cancer cell lines, such as Caco-2, HCT-116, HCT-15, HT-29, LS 174T, COLO 201, COLO 205, HCT-8, SW1116, and DLD-1. Although H 2 Mab-139 strongly reacted with LN229/HER2 cells on the western blot, we did not observe a specific signal for HER2 in colon cancer cell lines. Immunohistochemical analyses revealed sensitive and specific reactions of H 2 Mab-139 against colon cancers, indicating that H 2 Mab-139 is useful in detecting HER2 overexpression in colon cancers using flow cytometry and immunohistochemical analyses.

Retinal glutamate transporter changes in experimental glaucoma and after optic nerve transection in the rat.

PubMed

Martin, Keith R G; Levkovitch-Verbin, Hana; Valenta, Danielle; Baumrind, Lisa; Pease, Mary Ellen; Quigley, Harry A

2002-07-01

High levels of glutamate can be toxic to retinal ganglion cells. Effective buffering of extracellular glutamate by retinal glutamate transporters is therefore important. This study was conducted to investigate whether glutamate transporter changes occur with two models of optic nerve injury in the rat. Glaucoma was induced in one eye of 35 adult Wistar rats by translimbal diode laser treatment to the trabecular meshwork. Twenty-five more rats underwent unilateral optic nerve transection. Two glutamate transporters, GLAST (EAAT-1) and GLT-1 (EAAT-2), were studied by immunohistochemistry and quantitative Western blot analysis. Treated and control eyes were compared 3 days and 1, 4, and 6 weeks after injury. Optic nerve damage was assessed semiquantitatively in epoxy-embedded optic nerve cross sections. Trabecular laser treatment resulted in moderate intraocular pressure (IOP) elevation in all animals. After 1 to 6 weeks of experimental glaucoma, all treated eyes had significant optic nerve damage. Glutamate transporter changes were not detected by immunohistochemistry. Western blot analysis demonstrated significantly reduced GLT-1 in glaucomatous eyes compared with control eyes at 3 days (29.3% +/- 6.7%, P = 0.01), 1 week (55.5% +/- 13.6%, P = 0.02), 4 weeks (27.2% +/- 10.1%, P = 0.05), and 6 weeks (38.1% +/- 7.9%, P = 0.01; mean reduction +/- SEM, paired t-tests, n = 5 animals per group, four duplicate Western blot analyses per eye). The magnitude of the reduction in GLT-1 correlated significantly with mean IOP in the glaucomatous eye (r(2) = 0.31, P = 0.01, linear regression). GLAST was significantly reduced (33.8% +/- 8.1%, mean +/- SEM) after 4 weeks of elevated IOP (P = 0.01, paired t-test, n = 5 animals per group). In contrast to glaucoma, optic nerve transection resulted in an increase in GLT-1 compared with the control eye (P = 0.01, paired t-test, n = 15 animals). There was no significant change in GLAST after transection. GLT-1 and GLAST were significantly reduced in an experimental rat glaucoma model, a response that was not found after optic nerve transection. Reductions in GLT-1 and GLAST may increase the potential for glutamate-induced injury to RGC in glaucoma.

Involvement of Resveratrol and ω-3 Polyunsaturated Fatty Acids on Sirtuin 1 Gene Expression in THP1 Cells.

PubMed

Tsuchiya, Takafumi; Endo, Ayano; Tsujikado, Kyoko; Inukai, Toshihiko

2017-10-01

Resveratrol, a kind of polyphenol, has the potential to activate the longevity gene in several cells, in the same manner as calorie restriction. We investigated the effect of resveratrol and ω-3-line polyunsaturated fatty acid on surtuin 1 (SIRT1) gene expression in human monocytes (THP1) cells. We examined the gene expression of THP1 cells using real-time polymerase chain reaction and Western blotting analysis. Resveratol, eicosapentaenoic acid (EPA) and docosahexaeanoic acid (DHA) as n-3 polyunsaturated fatty acid were added on THP1 cells. We observed the changes in the SIRT1 gene expression in those cells, under various doses of agents and in time courses. Then, we examined the interaction of glucose and mannitol on those agents׳ effect of the gene expression. The concentration range of glucose and mannitol was from 5-20mM, respectively. The SIRT1 gene expression could be defined in 24 and 48 hours both in real-time polymerase chain reaction analysis and in Western blotting. Resveratrol showed SIRT1 gene expression in a dose-dependent manner in the range of 0-20μM in both analyses. Although EPA at 10μM showed marked increase in SIRT1 gene expression compared to control condition in Western blotting, this phenomenon was not in dose-dependent manner. DHA did not exhibit any augmentation of SIRT1 gene expression in a dose-dependent manner in the range of 0-20μM in both analyses. We refined the dose-dependent inhibition of the SIRT1 gene expression within 20mM glucose medium. Although 20mM did not exhibit any inhibition, 10μM resveratrol induced the gene expression compared to control medium. Both 5 and 15mM mannitol medium did not significantly alter basic gene expression and 10μM resveratrol-induced gene expression. The present results suggest that resveratrol and EPA, but not DHA, markedly activated the SIRT1 gene expression in THP1 cells, and that high glucose medium could inhibit the basic gene expression, but not powerful resveratrol-induced gene expression, in those cells. Copyright © 2017 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.

MiR-214 inhibits cell migration, invasion and promotes the drug sensitivity in human cervical cancer by targeting FOXM1.

PubMed

Wang, Jian-Mei; Ju, Bao-Hui; Pan, Cai-Jun; Gu, Yan; Li, Meng-Qi; Sun, Li; Xu, Yan-Ying; Yin, Li-Rong

2017-01-01

MicroRNAs (miRNAs) play key roles in progression of cervical cancer. In the present study, we investigated the role of miR-214 in the process of migration, invasion and drug sensitivity to cisplatin in cervical cancer. We detected the differential expression of miR-214 in 19 cases cervical cancer tissues and normal tissues as well as 4 cervical cancer cells and one normal cervical cells by Real-time PCR. Then, wound healing assay, transwell invasion assay and MTT were used to detect the effects of migration, invasion and sensitivity to cisplatin of cervical cancer when miR-214 was overexpressed. Western blot, immunofluorescence and Flow Cytometry were used to detect the mechanism of migration, invasion and sensitivity to cisplatin. Next, bioinformatics analysis was used to find the target of miR-214. Through the luciferase reporter assay, Real-time PCR and western blot, we confirmed the binding relationship of miR-214 and FOXM1. In cervical cancer tissues, the expression of FOXM1 was detected by western blot and Immunohistochemistry. We also knocked down FOXM1 in cervical cancer cells, wound healing assay, transwell invasion assay and MTT were performed to detect the migration, invasion and sensitivity to cisplatin abilities of FOXM1. Western blot and Flow Cytometry were used to detect the mechanism of migration, invasion and sensitivity to cisplatin by FOXM1. Finally, we performed rescue expriments to confirm the function relationship between miR-214 and FOXM1. 1. Our results showed that miR-214 was frequently downregulated in tumor tissues and cancer cells especially in CIN III and cervical cancer stages. 2. Overexpression of miR-214 significantly inhibited migration and invasion of cervical cancer cells and prompted the sensitivity to cisplatin. 3. FOXM1 was identified as a target of miR-214 and down-regulated by miR-214. 4. Knocking down FOXM1 could inhibited migration and invasion of cervical cancer cells and prompted the sensitivity to cisplatin. 5. FOXM1 was upregulated in tumor tissues. 6. The mechanism of migration, invasion and sensitivity to cisplatin were the resluts of changes of EMT and apoptosis. 7. The restoration of FOXM1 expression can counteract the effect of miR-214 on cell migration, invasion and sensitivity to cisplatin of cervical cancer cells. These findings indicate that miR-214 acts as a tumor suppressor during the process of migration, invasion and drug sensitivity through targeting FOXM1, suggesting miR-214 as a potential new diagnostic and therapeutic target for the treatment of cervical cancer.

The change of nuclear LC3 distribution in acute myeloid leukemia cells.

PubMed

Guo, Wenjian; Jin, Jingrui; Pan, Jiajia; Yao, Rongxing; Li, Xia; Huang, Xin; Ma, Zhixing; Huang, Sujuan; Yan, Xiao; Jin, Jie; Dong, Aishu

2018-05-09

Making sure the change of nuclear LC3 distribution in the autophagy of acute myeloid leukemia (AML) cell and finding out the regulation mechanism may lead to a breakthrough for killing AML cells. Western blots were performed to assess the expression of autophagy proteins. Changes in the LC3 distribution were monitored by immunofluorescence assays together with western blots, and the expression levels of Sirt1, DOR, Beclin1, HMGB1, and AMPK mRNA were detected via fluorescent quantitative PCR. The effects of Sirt1 and DOR on cell proliferation and survival were analyzed by MTT, flow cytometry, and western blotting assays. We found that treating AML cells with Ara-c or Sorafenib resulted in autophagy enhancement, and when autophagy was enhanced, nuclear LC3 moved into the cytoplasm. Notably, when autophagy was inhibited by blocking the nuclear LC3 shift, the cytotoxicity of drugs was enhanced. Our results also identified Sirt1 and DOR as regulatory molecules for the observed nuclear LC3 shift, and these molecules further affected the expression of Beclin1, HMGB1, and AMPK. Our results suggest the distribution of nuclear LC3 can be a novel way for further studying death of AML cells,and the regulatory molecules may be new targets for treating AML. Copyright © 2018 Elsevier Inc. All rights reserved.

Detection of Iss and Bor on the surface of Escherichia coli.

PubMed

Lynne, A M; Skyberg, J A; Logue, C M; Nolan, L K

2007-03-01

To confirm the presence of Iss and Bor on the outer membrane of Escherichia coli using Western blots of outer membrane protein (OMP) preparations and fluorescence microscopy, and explore the use of fluorescence microscopy for the detection of avian pathogenic E. coli (APEC) and diagnosis of avian colibacillosis. Knockout mutants of iss and bor were created using a one-step recombination of target genes with PCR-generated antibiotic resistance cassettes. Anti-Iss monoclonal antibodies (Mabs) that cross-react with Bor protein were used to study the mutants relative to the wild-type organism. These Mabs were used as reagents to study OMP preparations of the mutants with Western blotting and intact E. coli cells with fluorescence microscopy. Iss and Bor were detected in Western blots of OMP preparations of the wild type. Also, Iss was detected on Deltabor mutants, and Bor was detected on Deltaiss mutants. Iss and Bor were also detected on the surface of the intact, wild-type cells and mutants using fluorescence microscopy. These results demonstrate that Bor and Iss are exposed on E. coli's outer membrane where they may be recognized by the host's immune system. To our knowledge, this is the first report confirming Iss' location in the outer membrane of an E. coli isolate. Such surface exposure has implications for the use of these Mabs for APEC detection and colibacillosis control.

The novel protein C3orf43 accelerates hepatocyte proliferation.

PubMed

Zhang, Chunyan; Chang, Cuifang; Li, Deming; Zhang, Fuchun; Xu, Cunshuan

2017-01-01

Our previous study found that single-pass membrane protein with coiled-coil domains 1 (C3orf43; XM_006248472.3) was significantly upregulated in the proliferative phase during liver regeneration. This indicates that C3orf43 plays a vital role in liver cell proliferation. However, its physiological functions remains unclear. The expressions of C3orf43 in BRL-3A cells transfected with C3orf43-siRNA (C3-siRNA) or overexpressing the vector plasmid pCDH-C3orf43 (pCDH-C3) were measured via RT-qPCR and western blot. Cell growth and proliferation were determined using MTT and flow cytometry. Cell proliferation-related gene expression was measured using RT-qPCR and western blot. It was found that upregulation of C3orf43 by pCDH-C3 promoted hepatocyte proliferation, and inhibition of C3orf43 by C3-siRNA led to the reduction of cell proliferation. The results of qRT-PCR and western blot assay showed that the C3-siRNA group downregulated the expression of cell proliferation-related genes like JUN, MYC, CCND1 and CCNA2, and the pCDH-C3 group upregulated the expression of those genes. These findings reveal that C3orf43 may contribute to hepatocyte proliferation and may have the potential to promote liver repair and regeneration.

Clinical application of a rapid method using agarose gel electrophoresis and Western blotting to evaluate von Willebrand factor protease activity.

PubMed

Kirzek, D M; Rick, M E

2001-03-01

A method for evaluating the activity of the von Willebrand factor (vWF) protease is described, and a clinical application is illustrated. The procedure utilizes gel electrophoresis, Western blotting, and luminographic detection methods to evaluate the distribution of vWF multimers before and after incubation of clinical samples under conditions that favor proteolysis by this enzyme. Physiologically, the high-molecular-weight multimers of vWF are cleaved by the vWF protease under conditions of high shear stress in parts of the arterial circulation; cleavage of vWF multimers is also observed after exposure of vWF to denaturing agents in vitro and thus can serve as a laboratory test for the activity of the protease. vWF protease activity is decreased or absent in patients with thrombotic thrombocytopenic purpura due to an inhibiting autoantibody, and this leads to high levels of noncleaved vWF and to life-threatening thrombosis, thrombocytopenia and anemia. The assay evaluates the activity of the protease by assessing the cleavage of vWF multimers after patient plasmas are incubated in vitro under denaturing conditions. With the use of these electrophoresis and Western blotting techniques, patient plasmas can be rapidly assessed for the activity of the vWF protease which may aid in the treatment strategy for these patients.

Exendin-4 enhances the migration of adipose-derived stem cells to neonatal rat ventricular cardiomyocyte-derived conditioned medium via the phosphoinositide 3-kinase/Akt-stromal cell-derived factor-1α/CXC chemokine receptor 4 pathway

PubMed Central

ZHOU, HAO; YANG, JUNJIE; XIN, TING; ZHANG, TAO; HU, SHUNYIN; ZHOU, SHANSHAN; CHEN, GUANGHUI; CHEN, YUNDAI

2015-01-01

Adipose-derived stem cells (ADSCs) are considered a suitable source of cells for the repair of tissue following acute myocardial infarction (AMI); however, the transplantation efficiency of ADSCs remains low. Therefore, identification of an efficient method to enhance the migration of engrafted cells to the target site is required. The present study used exendin-4 (Ex-4), a glucagon-like peptide-1 receptor agonist, to optimize the migratory capacity of ADSCs. The aim was to determine the effect and mechanisms of Ex-4 on the migration of ADSCs to neonatal rat ventricular cardiomyocyte-derived conditioned medium (NRVC-CM). The ADSCs and cardiomyocytes were cultured in vitro. Following incubation of the ADSCs with Ex-4, cell proliferation was measured using an MTT assay and the expression levels of CXC chemokine receptor 4 (CXCR4) were investigated by reverse transctiption quantitative polymerase chain reaction (RT-qPCR), western blot analysis and flow cytometry. In addition, the expression levels of stromal cell-derived factor-1α (SDF-1α) were evaluated in the NRVC-CM treated with Ex-4 by ELISA, RT-qPCR and western blot analysis. The migration of the ADSCs to the NRVC-CM was examined using a Transwell assay. Changes in the protein expression levels of phosphorylated (p−)Akt were examined in the two types of cell by western blot analysis. The results suggested that Ex-4 promoted the proliferation and expression of CXCR4 in the ADSCs, increased the secretion of SDF-1α in the cardiomyocytes and increased the expression levels of p-Akt in both cells. However, the alterations to the SDF-1α/C XC R4 cascade in the cells were abrogated following pretreatment with LY-294002, a phosphoinositide 3-kinase(PI3K) inhibitor. Furthermore, a Transwell migration assay revealed marked translocation of the ADSCs through the membranes, towards the NRVC-CM, following treatment with Ex-4. However, these effects were reduced significantly by pretreatment of the cells with the SDF-1α/CXCR4 cascade antagonist, AMD3100, and the PI3K inhibitor, LY-294002. These results indicated that Ex-4 augmented the SDF-1α/CXCR4 cascade by activating the PI3K/Akt pathways in the ADSCs and NRVCs. Furthermore, enhancement of the PI3K/Akt-SDF-1α/CXCR4 pathway may be important in the migratory response of ADSCs to NRVC-CM in vitro. PMID:25625935

Molecular characterization and functional analysis of pteridine reductase in wild-type and antimony-resistant Leishmania lines.

PubMed

de Souza Moreira, Douglas; Ferreira, Rafael Fernandes; Murta, Silvane M F

2016-01-01

Pteridine reductase (PTR1) is an NADPH-dependent reductase that participates in the salvage of pteridines, which are essential to maintain growth of Leishmania. In this study, we performed the molecular characterization of ptr1 gene in wild-type (WTS) and SbIII-resistant (SbR) lines from Leishmania guyanensis (Lg), Leishmania amazonensis (La), Leishmania braziliensis (Lb) and Leishmania infantum (Li), evaluating the chromosomal location, mRNA levels of the ptr1 gene and PTR1 protein expression. PFGE results showed that the ptr1 gene is located in a 797 kb chromosomal band in all Leishmania lines analyzed. Interestingly, an additional chromosomal band of 1070 kb was observed only in LbSbR line. Northern blot results showed that the levels of ptr1 mRNA are increased in the LgSbR, LaSbR and LbSbR lines. Western blot assays using the polyclonal anti-LmPTR1 antibody demonstrated that PTR1 protein is more expressed in the LgSbR, LaSbR and LbSbR lines compared to their respective WTS counterparts. Nevertheless, no difference in the level of mRNA and protein was observed between the LiWTS and LiSbR lines. Functional analysis of PTR1 enzyme was performed to determine whether the overexpression of ptr1 gene in the WTS L. braziliensis and L. infantum lines would change the SbIII-resistance phenotype of transfected parasites. Western blot results showed that the expression level of PTR1 protein was increased in the transfected parasites compared to the non-transfected ones. IC50 analysis revealed that the overexpression of ptr1 gene in the WTS L. braziliensis line increased 2-fold the SbIII-resistance phenotype compared to the non-transfected counterpart. Furthermore, the overexpression of ptr1 gene in the WTS L. infantum line did not change the SbIII-resistance phenotype. These results suggest that the PTR1 enzyme may be implicated in the SbIII-resistance phenotype in L. braziliensis line. Copyright © 2015 Elsevier Inc. All rights reserved.

Identification of three duplicated Spin genes in medaka (Oryzias latipes).

PubMed

Wang, Xiao-Lei; Mei, Jie; Sun, Min; Hong, Yun-Han; Gui, Jian-Fang

2005-05-09

Gene and genomic duplications are very important and frequent events in fish evolution, and the divergence of duplicated genes in sequences and functions is a focus of research on gene evolution. Here, we report the identification and characterization of three duplicated Spindlin (Spin) genes from medaka (Oryzias latipes): OlSpinA, OlSpinB, and OlSpinC. Molecular cloning, genomic DNA Blast analysis and phylogenetic relationship analysis demonstrated that the three duplicated OlSpin genes should belong to gene duplication. Furthermore, Western blot analysis revealed significant expression differences of the three OlSpins among different tissues and during embryogenesis in medaka, and suggested that sequence and functional divergence might have occurred in evolution among them.

Homogentisate 1,2 Dioxygenase is Expressed in Human Osteoarticular Cells: Implications in Alkaptonuria

PubMed Central

Laschi, Marcella; Tinti, Laura; Braconi, Daniela; Millucci, Lia; Ghezzi, Lorenzo; Amato, Loredana; Selvi, Enrico; Spreafico, Adriano; Bernardini, Giulia; Santucci, Annalisa

2012-01-01

Alkaptonuria (AKU) results from defective homogentisate1,2-dioxygenase (HGD), causing degenerative arthropathy. The deposition of ochronotic pigment in joints is so far attributed to homogentisic acid produced by the liver, circulating in the blood and accumulating locally. Human normal and AKU osteoarticular cells were tested for HGD gene expression by RT-PCR, mono- and 2D-Western blotting. HGD gene expression was revealed in chondrocytes, synoviocytes, osteoblasts. Furthermore, HGD expression was confirmed by Western blotting, that also revealed the presence of five enzymatic molecular species. Our findings indicate that AKU osteoarticular cells produce the ochronotic pigment in loco and this may strongly contribute to induction of ochronotic arthropathy. J. Cell. Physiol. 227: 3254–3257, 2012. © 2011 Wiley Periodicals, Inc. PMID:22105303

Analysis of Sigma Receptor (σR1) expression in retinal ganglion cells cultured under hyperglycemic conditions and in diabetic mice

PubMed Central

Ola, M. Shamsul; Moore, Pamela; Maddox, Dennis; El-Sherbeny, Amira; Huang, Wei; Roon, Penny; Agarwal, Neeraj; Ganapathy, Vadivel; Smith, Sylvia B.

2013-01-01

Summary The type 1 sigma receptor (σR1) is a nonopiate and nonphencyclidine binding site that has numerous pharmacological and physiological functions. In some studies, agonists for σR1 have been shown to afford neuroprotective against overstimulation of the NMDA receptor. σR1 expression has been demonstrated recently in retinal ganglion cells (RGC). RGCs undergo apoptosis early in diabetic retinopathy via NMDA receptor overstimulation. In the present study we asked whether RGCs cultured under hyperglycemic conditions and RGCs of diabetic mice continue to express σ1. RGCs were cultured 48 h in RPMI medium containing either 45 mM glucose or 11 mM glucose plus 34 mM mannitol (osmolar control). C57BL/6 mice were made diabetic using streptozotocin. The retina was dissected from normal and streptozotocin-induced diabetic mice 3, 6 and 12 weeks post-onset of diabetes. σR1 was analyzed in cells using semiquantitative RT-PCR and in tissues σR1 by semiquantitative RT-PCR, in situ hybridization, western blot analysis and immunolocalization. The RT-PCR analysis of cultured RGCs showed that σR1 mRNA is expressed under hyperglycemic conditions at levels similar to control cells. Similarly, analysis of retinas of diabetic mice showed no difference in levels of mRNA encoding σR1 compared to retinas of control mice. In situ hybridization analysis showed that expression patterns of σR1 mRNA in the ganglion cell layer were similar between diabetic and control mice. Western blot analysis suggested that levels of σR1 in retina were similar between diabetic and control retinas. Immunohistochemical analysis of σR1 showed a similar pattern of σR1 protein expression between control and diabetic retina. These studies demonstrate that σR1 is expressed under hyperglycemic conditions in vitro and in vivo. PMID:12425939

Analysis of sigma receptor (sigmaR1) expression in retinal ganglion cells cultured under hyperglycemic conditions and in diabetic mice.

PubMed

Ola, M Shamsul; Moore, Pamela; Maddox, Dennis; El-Sherbeny, Amira; Huang, Wei; Roon, Penny; Agarwal, Neeraj; Ganapathy, Vadivel; Smith, Sylvia B

2002-11-15

The type 1 sigma receptor (sigmaR1) is a nonopiate and nonphencyclidine binding site that has numerous pharmacological and physiological functions. In some studies, agonists for sigmaR1 have been shown to afford neuroprotection against overstimulation of the NMDA receptor. sigmaR1 expression has been demonstrated recently in retinal ganglion cells (RGC). RGCs undergo apoptosis early in diabetic retinopathy via NMDA receptor overstimulation. In the present study we asked whether RGCs cultured under hyperglycemic conditions and RGCs of diabetic mice continue to express sigmaR1. RGCs were cultured 48 h in RPMI medium containing either 45 mM glucose or 11 mM glucose plus 34 mM mannitol (osmolar control). C57BL/6 mice were made diabetic using streptozotocin. The retina was dissected from normal and streptozotocin-induced diabetic mice 3, 6 and 12 weeks post-onset of diabetes. sigmaR1 was analyzed in cells using semiquantitative RT-PCR and in tissues by semiquantitative RT-PCR, in situ hybridization, Western blot analysis and immunolocalization. The RT-PCR analysis of cultured RGCs showed that sigmaR1 mRNA is expressed under hyperglycemic conditions at levels similar to control cells. Similarly, analysis of retinas of diabetic mice showed no difference in levels of mRNA encoding sigmaR1 compared to retinas of control mice. In situ hybridization analysis showed that expression patterns of sigmaR1 mRNA in the ganglion cell layer were similar between diabetic and control mice. Western blot analysis suggested that levels of sigmaR1 in retina were similar between diabetic and control retinas. Immunohistochemical analysis of sigmaR1 showed a similar pattern of sigmaR1 protein expression between control and diabetic retina. These studies demonstrate that sigmaR1 is expressed under hyperglycemic conditions in vitro and in vivo.

[FANCA gene mutation analysis in Fanconi anemia patients].

PubMed

Chen, Fei; Peng, Guang-Jie; Zhang, Kejian; Hu, Qun; Zhang, Liu-Qing; Liu, Ai-Guo

2005-10-01

To screen the FANCA gene mutation and explore the FANCA protein function in Fanconi anemia (FA) patients. FANCA protein expression and its interaction with FANCF were analyzed using Western blot and immunoprecipitation in 3 cases of FA-A. Genomic DNA was used for MLPA analysis followed by sequencing. FANCA protein was undetectable and FANCA and FANCF protein interaction was impaired in these 3 cases of FA-A. Each case of FA-A contained biallelic pathogenic mutations in FANCA gene. No functional FANCA protein was found in these 3 cases of FA-A, and intragenic deletion, frame shift and splice site mutation were the major pathogenic mutations found in FANCA gene.

Characterization of Bombyx mori nucleopolyhedrovirus Bm17.

PubMed

Shen, Hongxing; Wang, Rudu; Han, Qinggong; Zhang, Wen; Nin, Bin; Zhou, Yang; Shao, Shihe; Yao, Qin; Chen, Keping; Liu, Xiaoyong

2013-10-01

Open reading frame17 (Bm17) of Bombyx mori nucleopolyhedrovirus is a highly conserved gene in lepidopteran nucleopolyhedroviruses, suggesting that it performs an important role in the virus life cycle whose function is unknown. In this report, we describe the characterization of Bm17. Reversed transcriptive-PCR (RT-PCR) and Western blot analysis demonstrated that Bm17 was expressed as a late gen. Immunofluorescence analysis by confocal microscopy showed that BM17 protein was localized on cytoplasm and nucleus of infected cells. These results show that BM17 was a late protein localized in cytoplasm and nucleus. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Schizosaccharomyces pombe Polysome Profile Analysis and RNA Purification.

PubMed

Wolf, Dieter A; Bähler, Jürg; Wise, Jo Ann

2017-04-03

Polysome profile analysis is widely used by investigators studying the mechanism and regulation of translation. The method described here uses high-velocity centrifugation of whole cell extracts on linear sucrose gradients to separate 40S and 60S ribosomal subunits from 80S monosomes and polysomes. Cycloheximide is included in the lysis buffer to "freeze" polysomes by blocking translation. After centrifugation, the gradient is fractionated and RNA (and/or protein) is prepared from each fraction for subsequent analysis of individual species using northern or western blots. The entire RNA population in each fraction can be analyzed by hybridization to microarrays or by high-throughput RNA sequencing, and the proteins present can be identified by mass spectrometry analysis. © 2017 Cold Spring Harbor Laboratory Press.

Novel Role of Merlin Tumor Suppressor in Autophagy and its Implication in Treating NF2-Associated Tumors

DTIC Science & Technology

2014-04-01

with the idea that Merlin serves as an adaptor linking LC3 to DIC. Moreover, Merlin-KD led to reduce levels of p62 degradation and LC3 -II accumulation...autophagy flux In response to nutrient starvation (Stv), Merlin-knockdown (KD) MEFs show attenuated levels of LC3 -II accumulation or p62 ...degradation, as compared with control MEFs (Cr-KD). Graphs: Densitometric analysis of LC3 -II and p62 on Western blots. Pixel intensities of LC3 -II and p62

[Toxoplasma gondii: the characterization of an anti-P30 monoclonal antibody].

PubMed

Fachado, A; Fernández, N; Hernández, E; Fonseca, L

1996-01-01

A specific monoclonal antibody was characterized to Toxoplasma gondii. The hybridoma produced IgG immunoglobulins. The western blot analysis showed that the monoclonal antibody was specific for the antigen of an apparent molecular mass of 30 kd, which was present on the antigen surface. The monoclonal antibody was purified starting from mouse's ascitic fluid and it was matched with sepharose 4B. This immunoabsorbent was used to purify the specific parasitic antigen. The monoclonal antibody studied may be useful for those techniques contributing to the toxoplasmosis diagnosis.

Evidence for Phex haploinsufficiency in murine X-linked hypophosphatemia.

PubMed

Wang, L; Du, L; Ecarot, B

1999-04-01

Mutations in the PHEX gene (phosphate-regulating gene with homology to endopeptidases on the X-chromosome) are responsible for X-linked hypophosphatemia (HYP). We previously reported the full-length coding sequence of murine Phex cDNA and provided evidence of Phex expression in bone and tooth. Here, we report the cloning of the entire 3.5-kb 3'UTR of the Phex gene, yielding a total of 6248 bp for the Phex transcript. Southern blot and RT-PCR analyses revealed that the 3' end of the coding sequence and the 3'UTR of the Phex gene, spanning exons 16 to 22, are deleted in Hyp, the mouse model for HYP. Northern blot analysis of bone revealed lack of expression of stable Phex mRNA from the mutant allele and expression of Phex transcripts from the wild-type allele in Hyp heterozygous females. Expression of the Phex protein in heterozygotes was confirmed by Western analysis with antibodies raised against a COOH-terminal peptide of the mouse Phex protein. Taken together, these results indicate that the dominant pattern of Hyp inheritance in mice is due to Phex haploinsufficiency.

Ugene, a newly identified protein that is commonly over-expressed in cancer, and that binds uracil DNA-glycosylase

PubMed Central

Guo, Chunguang; Zhang, Xiaodong; Fink, Stephen P; Platzer, Petra; Wilson, Keith; Willson, James K. V.; Wang, Zhenghe; Markowitz, Sanford D

2008-01-01

Expression microarrays identified a novel transcript, designated as Ugene, whose expression is absent in normal colon and colon adenomas, but that is commonly induced in malignant colon cancers. These findings were validated by real-time PCR and Northern blot analysis in an independent panel of colon cancer cases. In addition, Ugene expression was found to be elevated in many other common cancer types, including, breast, lung, uterus, and ovary. Immunofluorescence of V5-tagged Ugene revealed it to have a nuclear localization. In a pull-down assay, uracil DNA-glycosylase 2 (UNG2), an important enzyme in the base excision repair pathway, was identified as a partner protein that binds to Ugene. Co-immunoprecipitation and Western blot analysis confirmed the binding between the endogenous Ugene and UNG2 proteins. Using deletion constructs, we find that Ugene binds to the first 25 amino acids of the UNG2 NH2-terminus. We suggest Ugene induction in cancer may contribute to the cancer phenotype by interacting with the base excision repair pathway. PMID:18676834

Uridine 5′-Monophosphate Synthase Is Transcriptionally Regulated by Pyrimidine Levels in Nicotiana plumbaginifolia1

PubMed Central

Santoso, Djoko; Thornburg, Robert

1998-01-01

To understand the regulation and expression of pyrimidine biosynthesis in plants, we have examined the effect of the metabolic inhibitor 5-fluoroorotic acid (FOA) on uridine-5′-monophosphate synthase (UMPSase) expression in cell cultures of Nicotiana plumbaginifolia. UMPSase is the rate-limiting step of pyrimidine biosynthesis in plants. Addition of FOA causes an up-regulation of UMPSase enzyme activity in cell cultures after a lag phase of several days. Western-blot analysis demonstrated that the up-regulation in enzyme activity was caused by increased expression of the UMPSase protein. Northern-blot analysis demonstrated a higher level of UMPSase mRNA in the FOA-induced tissues than in control tissues. Run-on transcriptional assays showed that the UMPSase gene was transcriptionally activated after FOA treatment. The mechanism of toxicity of FOA is through thymine starvation. We found that addition of thymine abrogated the FOA-mediated up-regulation of UMPSase. In addition, methotrexate and aminopterin, which affect thymine levels by inhibiting dihydrofolate reductase, also up-regulate UMPSase in N. plumbaginifolia cells. PMID:9490773

Quantification of CSF cystatin C using liquid chromatography tandem mass spectrometry.

PubMed

Matsuda, Chikashi; Shiota, Yuri; Sheikh, Abdullah Md; Okazaki, Ryota; Yamada, Kazuo; Yano, Shozo; Minohata, Toshikazu; Matsumoto, Ken-Ichi; Yamaguchi, Shuhei; Nagai, Atsushi

2018-03-01

Cystatin C (CST3), a ubiquitously expressed cysteine protease inhibitor, is implicated in several neurological diseases. Here, we have developed an accurate CST3 measurement system based on liquid chromatography tandem mass spectrometry (LC-MS/MS). LC-MS/MS based measurement for CSF CST3 was validated by determination of assay precision, accuracy and recovery. The values were compared with those measured by immunoassay. Glycosylation of CST3 in CSF was analyzed by Western blotting and lectin blotting. Measuring standard CST3 by LC-MS/MS produced a linear standard curve that correlated with assigned values (r 2 =0.99). Both intra- and inter-assay variation was <10%. Although showed a correlation, the average CST3 concentration measured by LC-MS/MS was significantly higher than that of immunoassay. Western blotting showed the presence of a 25KDa species along with CST3 monomer (14KDa) in CSF. The volume of 25KDa species was decreased by deglycosylation. Lectin blotting revealed a 25KDa glycosylated protein in sialidase-treated CSF, which was decreased by deglycosylation. However, deglycosylation did not alter CST3 concentration measured by immunoassay. Our results suggest that LC-MS/MS-based CST3 measurement is a robust method with higher detection ability. Such method could be useful for the diagnosis and monitoring of neurological diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of proacrosin/acrosin system after liquid storage and cryopreservation of turkey semen (Meleagris gallopavo).

PubMed

Słowińska, M; Liszewska, E; Dietrich, G J; Ciereszko, A

2012-09-15

This study was designed to identify the effect of liquid storage at 4 °C for 48 h and cryopreservation on the proacrosin/acrosin system of turkey spermatozoa. Anti-acrosin I antibodies were produced and used to demonstrate Western blot analysis profile of the proacrosin/acrosin system of sperm and seminal plasma and possible changes in the proacrosin/acrosin system of turkey sperm stored for 2.5, 24, and 48 h or cryopreserved. At the same time acrosin-like activity was examined by the measurement of amidase activity of sperm extracts, sperm suspension, and seminal plasma of turkey semen. A computer-assisted sperm analysis system was used to monitor the sperm motility characteristics of turkey sperm stored for 48 h or cryopreserved. Different profiles of the sperm proacrosin/acrosin system were observed regarding the presence or absence of inhibitors (p-nitrophenyl-p'-guanidine benzoate [NPGB] and Kazal family inhibitor) during the extraction process. When NPGB was present three main bands were observed with the molecular weight ranging from 66 to 35 kDa. Bands corresponding to acrosin I and II were not observed. In sperm extract without NPGB, three or four bands were observed with the molecular weight ranging from 41 to 30 kDa. The bands corresponding to acrosin I and II were observed. During liquid storage a decrease in sperm motility and an increase in sperm-extracted amidase activity were observed. After 24 and 48 h of storage, extracted amidase activity was higher than at 2.5 h by 24% and 31%, respectively. However, no changes in the Western blot analysis profiles of sperm extract and seminal plasma were visible during liquid storage. After cryopreservation a decrease in sperm motility and all sperm motility parameters were observed. In contrast to liquid storage, cryopreservation did not increase extracted amidase activity. However, changes in Western blot analysis profiles were visible in sperm extract and seminal plasma after cryopreservation. After freezing-thawing, additional bands appeared in sperm extract and seminal plasma. These bands were of different molecular weight regarding the presence or absence of NPGB. These data suggest that the mechanism of damage to the proacrosin/acrosin system is different for liquid storage and cryopreservation. Liquid storage seems to increase in the susceptibility of the proacrosin/acrosin system to be activated during extraction. Kazal inhibitors of turkey seminal plasma are involved in the control of proacrosin activation. The disturbances of the proacrosin/acrosin system of turkey spermatozoa can be related to a disturbance in the induction of the acrosome reaction. Our results may be important for a better understanding of the proacrosin/acrosin system of turkey spermatozoa and disturbance to this system during liquid storage and cryopreservation. Copyright © 2012 Elsevier Inc. All rights reserved.

Acid-Sensing Ion Channel 1a Regulates Fate of Rat Nucleus Pulposus Cells in Acid Stimulus Through Endoplasmic Reticulum Stress.

PubMed

Xie, Zhi-Yang; Chen, Lu; Zhang, Cong; Liu, Lei; Wang, Feng; Cai, Feng; Wang, Xiao-Hu; Shi, Rui; Sinkemani, Arjun; Yu, Hao-Min; Hong, Xin; Wu, Xiao-Tao

2018-01-01

Acid-sensing ion channel 1a (ASIC1a) participates in human intervertebral disc degeneration (IVDD) and regulates the destiny of nucleus pulposus cells (NPCs) in acid stimulus. However, the mechanism of ASIC1a activation and its downstream pathway remain unclear. Endoplasmic reticulum (ER) stress also participates in the acid-induced apoptosis of NPCs. The main purpose of this study was to investigate whether there is any connection between ASIC1a and ER stress in an acid-induced nucleus pulposus degeneration model. The IVDs of Sprague-Dawley rats were stained by immunohistochemical staining to evaluate the expression of ASIC1a in normal and degenerated rat nucleus pulposus. ASIC1a expression was also quantified by quantitative real-time-polymerase chain reaction and Western blotting analysis. NPCs were exposed to the culture media with acidity at pH 7.2 and 6.5 for 24 h, with or without 4-phenylbutyrate (4-PBA, a blocker of the ER stress pathway). Cell apoptosis was examined by Annexin V/Propidium Iodide (PI) staining and was quantified using flow cytometry analysis. ASIC1a-mediated intracellular calcium was determined by Ca 2+ imaging using Fura-2-AM. Acidity-induced changes in ER stress markers were studied using Western blotting analysis. In vivo , ASIC1a expression was upregulated in natural degeneration. In vitro , acid stimulus increased intracellular calcium levels, but this effect was blocked by knockdown of ASIC1a, and this reversal was partly inhibited by 4-PBA. In addition, blockade of ASIC1a reduced expression of ER stress markers, especially the proapoptotic markers. ASIC1a partly regulates ER stress and promotes apoptosis of NPCs under acid stimulus and may be a novel therapeutic target in IVDD.