Estimation of hepatitis E virus (HEV) pig seroprevalence using ELISA and Western blot and comparison between human and pig HEV sequences in Belgium.

PubMed

Thiry, Damien; Mauroy, Axel; Saegerman, Claude; Thomas, Isabelle; Wautier, Magali; Miry, Cora; Czaplicki, Guy; Berkvens, Dirk; Praet, Nicolas; van der Poel, Wim; Cariolet, Roland; Brochier, Bernard; Thiry, Etienne

2014-08-27

Zoonotic transmission of hepatitis E virus (HEV) is of special concern, particularly in high income countries were waterborne infections are less frequent than in developing countries. High HEV seroprevalences can be found in European pig populations. The aims of this study were to obtain prevalence data on HEV infection in swine in Belgium and to phylogenetically compare Belgian human HEV sequences with those obtained from swine. An ELISA screening prevalence of 73% (95% CI 68.8-77.5) was determined in Belgian pigs and a part of the results were re-evaluated by Western blot (WB). A receiver operating characteristic curve analysis was performed and scenarios varying the ELISA specificity relative to WB were analysed. The seroprevalences estimated by the different scenarios ranged between 69 and 81% and are in agreement with the high exposure of the European pig population to HEV. Pig HEV sequences were genetically compared to those detected in humans in Belgium and a predominance of genotype 3 subtype f was shown in both swine and humans. The high HEV seroprevalence in swine and the close phylogenetic relationships between pig and human HEV sequences further support the risk for zoonotic transmission of HEV between humans and pigs. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

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.

[Mass spectrometry identification and immune cross-reactivity of a minor shrimp allergen-sarcoplasmic calcium binding protein from Litopenaeus vannamei].

PubMed

Wang, Cai-xia; Huang, Jian-fang; Xiang, Jun-jian; Sun, Yi-fan; Lv, Si; Guo, Jie

2012-08-01

To identify sarcoplasmic calcium-binding protein (SCP) as a minor shrimp allergen by mass spectrometry, and to analyze the immune cross-reactivity among crustacean SCPs. The M(r); 21 000 allergen from Litopenaeus vannamei was identified by MALDI-TOF/TOF-MS. BLAST and ClustalW were used to compare amino acid sequence identity of the allergen among crustaceans. The puritifed M(r); 21 000 allergen was injected subcutaneously in mice to produce the specific polyclonal antibodies to analyze immune cross-reactivity of the allergen with proteins from 8 other species of crustaceans by Western blotting. The M(r); 21 000 shrimp allergen was identified as SCP. Sequence comparison revealed that SCP had 81%-100% amino acid identity among crustaceans. Western blotting showed that the proteins with M(r); about 21 000, corresponding to SCP from Metapenaeus ensis, Penaeus monodon, Oratosquilla oratoria, Macrobrachium rosenbergii, Procambarus clarkii, Portunus pelagicus, Charybdis feriatus, Eriocheir sinensis were recognized by polyclonal antibodies against SCP of Litopenaeus vannamei. SCP is a minor shrimp allergen, and SCPs have a high sequence homology and strong immune cross-reactivity among crustaceans, which can be used as detective, diagnostic and safe immunotherapeutic agents for subjects with shrimp allergy.

Structure-Related Roles for the Conservation of the HIV-1 Fusion Peptide Sequence Revealed by Nuclear Magnetic Resonance.

PubMed

Serrano, Soraya; Huarte, Nerea; Rujas, Edurne; Andreu, David; Nieva, José L; Jiménez, María Angeles

2017-10-17

Despite extensive characterization of the human immunodeficiency virus type 1 (HIV-1) hydrophobic fusion peptide (FP), the structure-function relationships underlying its extraordinary degree of conservation remain poorly understood. Specifically, the fact that the tandem repeat of the FLGFLG tripeptide is absolutely conserved suggests that high hydrophobicity may not suffice to unleash FP function. Here, we have compared the nuclear magnetic resonance (NMR) structures adopted in nonpolar media by two FP surrogates, wtFP-tag and scrFP-tag, which had equal hydrophobicity but contained wild-type and scrambled core sequences LFLGFLG and FGLLGFL, respectively. In addition, these peptides were tagged at their C-termini with an epitope sequence that folded independently, thereby allowing Western blot detection without interfering with FP structure. We observed similar α-helical FP conformations for both specimens dissolved in the low-polarity medium 25% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), but important differences in contact with micelles of the membrane mimetic dodecylphosphocholine (DPC). Thus, whereas wtFP-tag preserved a helix displaying a Gly-rich ridge, the scrambled sequence lost in great part the helical structure upon being solubilized in DPC. Western blot analyses further revealed the capacity of wtFP-tag to assemble trimers in membranes, whereas membrane oligomers were not observed in the case of the scrFP-tag sequence. We conclude that, beyond hydrophobicity, preserving sequence order is an important feature for defining the secondary structures and oligomeric states adopted by the HIV FP in membranes.

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

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.

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).

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.

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.

A Laboratory Exercise Illustrating the Sensitivity and Specificity of Western Blot Analysis

ERIC Educational Resources Information Center

Chang, Ming-Mei; Lovett, Janice

2011-01-01

Western blot analysis, commonly known as "Western blotting," is a standard tool in every laboratory where proteins are analyzed. It involves the separation of polypeptides in polyacrylamide gels followed by the electrophoretic transfer of the separated polypeptides onto a nitrocellulose or polyvinylidene fluoride membrane. A replica of the…

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.

β-actin as a loading control for plasma-based Western blot analysis of major depressive disorder patients.

PubMed

Zhang, Rufang; Yang, Deyu; Zhou, Chanjuan; Cheng, Ke; Liu, Zhao; Chen, Liang; Fang, Liang; Xie, Peng

2012-08-15

Western blot analysis is a commonly used technique for determining specific protein levels in clinical samples. For normalization of protein levels in Western blot, a suitable loading control is required. On account of its relatively high and constant expression, β-actin has been widely employed in Western blot of cell cultures and tissue extracts. However, β-actin's presence in human plasma and this protein's putative role as a plasma-based loading control for Western blot analysis remain unknown. In this study, an enzyme-linked immunosorbent assay was used to determine the concentration of β-actin in human plasma, which is 6.29±0.54 ng/ml. In addition, the linearity of β-actin immunostaining and loaded protein amount was evaluated by Western blot, and a fine linearity (R²=0.974±0.012) was observed. Furthermore, the expression of plasma β-actin in major depressive disorder subjects and healthy controls was compared. The data revealed no statistically significant difference between these two groups. Moreover, the total coefficient of variation for β-actin expression in the two groups was 9.2±1.2%. These findings demonstrate that β-actin is present in human plasma and may possibly be used as a suitable loading control for plasma-based Western blot analysis in major depressive disorder. Copyright © 2012 Elsevier Inc. All rights reserved.

Protein analysis through Western blot of cells excised individually from human brain and muscle tissue

PubMed Central

Koob, A.O.; Bruns, L.; Prassler, C.; Masliah, E.; Klopstock, T.; Bender, A.

2016-01-01

Comparing protein levels from single cells in tissue has not been achieved through Western blot. Laser capture microdissection allows for the ability to excise single cells from sectioned tissue and compile an aggregate of cells in lysis buffer. In this study we analyzed proteins from cells excised individually from brain and muscle tissue through Western blot. After we excised individual neurons from the substantia nigra of the brain, the accumulated surface area of the individual cells was 120,000, 24,000, 360,000, 480,000, 600,000 μm2. We used an optimized Western blot protocol to probe for tyrosine hydroxylase in this cell pool. We also took 360,000 μm2 of astrocytes (1700 cells) and analyzed the specificity of the method. In muscle we were able to analyze the proteins of the five complexes of the electron transport chain through Western blot from 200 human cells. With this method, we demonstrate the ability to compare cell-specific protein levels in the brain and muscle and describe for the first time how to visualize proteins through Western blot from cells captured individually. PMID:22402104

Protein analysis through Western blot of cells excised individually from human brain and muscle tissue.

PubMed

Koob, A O; Bruns, L; Prassler, C; Masliah, E; Klopstock, T; Bender, A

2012-06-15

Comparing protein levels from single cells in tissue has not been achieved through Western blot. Laser capture microdissection allows for the ability to excise single cells from sectioned tissue and compile an aggregate of cells in lysis buffer. In this study we analyzed proteins from cells excised individually from brain and muscle tissue through Western blot. After we excised individual neurons from the substantia nigra of the brain, the accumulated surface area of the individual cells was 120,000, 24,000, 360,000, 480,000, 600,000 μm2. We used an optimized Western blot protocol to probe for tyrosine hydroxylase in this cell pool. We also took 360,000 μm2 of astrocytes (1700 cells) and analyzed the specificity of the method. In muscle we were able to analyze the proteins of the five complexes of the electron transport chain through Western blot from 200 human cells. With this method, we demonstrate the ability to compare cell-specific protein levels in the brain and muscle and describe for the first time how to visualize proteins through Western blot from cells captured individually. Copyright © 2012 Elsevier Inc. All rights reserved.

The western blot

USDA-ARS?s Scientific Manuscript database

Western blotting is a technique that involves the separation of proteins by gel electrophoresis, their blotting or transfer to a membrane, and selective immunodetection of an immobilized antigen. This is an important and routine method for protein analysis that depends on the specificity of antibod...

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.

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.

A new background subtraction method for Western blot densitometry band quantification through image analysis software.

PubMed

Gallo-Oller, Gabriel; Ordoñez, Raquel; Dotor, Javier

2018-06-01

Since its first description, Western blot has been widely used in molecular labs. It constitutes a multistep method that allows the detection and/or quantification of proteins from simple to complex protein mixtures. Western blot quantification method constitutes a critical step in order to obtain accurate and reproducible results. Due to the technical knowledge required for densitometry analysis together with the resources availability, standard office scanners are often used for the imaging acquisition of developed Western blot films. Furthermore, the use of semi-quantitative software as ImageJ (Java-based image-processing and analysis software) is clearly increasing in different scientific fields. In this work, we describe the use of office scanner coupled with the ImageJ software together with a new image background subtraction method for accurate Western blot quantification. The proposed method represents an affordable, accurate and reproducible approximation that could be used in the presence of limited resources availability. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

Peritransplant Treg-Based Immunomodulation to Improve VCA Outcomes

DTIC Science & Technology

2017-10-01

function as assessed in vitro assays (mean ± SD, n=4/group) using cells analyzed at day 5. (D) Western blots of Foxp3 protein expression in Tregs from...mice and undertaking bisulphite conversion, cloning and sequencing . WT Tregs were largely demethylated at the TSDR site (open circles, Fig. 2...term murine limb vascularized composite allotransplantation (VCA) survival. • Aim 2 - Determine if histone/ protein deacetylase (HDAC) inhibitor

[Construction and selection of effective mouse Smad6 recombinant lenti-virus interference vectors].

PubMed

Yu, Jing; Qi, Mengchun; Deng, Jiupeng; Liu, Gang; Chen, Huaiqing

2010-10-01

This experiment was designed to construct mouse Smad6 recombinant RNA interference vectors and determine their interference effects on bone marrow mesenchymal stem cells (BMSCs). Three recombinant Smad6 RNA interference vectors were constructed by molecular clone techniques with a lenti-virus vector expressing green fluorescent protein (GFP), and the correctness of recombinant vectors was verified by DNA sequencing. Mouse BMSCs were used for transfection experiments and BMP-2 was in use for osteogenic induction of MSCs. The transfection efficiency of recombinant vectors was examined by Laser confocal scanning microscope and the interference effect of recombinant vectors on Smad6 gene expression was determined by real-time RT-PCR and Western blot, respectively. Three Smad6 recombinant RNA interference vectors were successfully constructed and their correctness was proved by DNA sequencing. After transfection, GFPs were effectively expressed in MSCs and all of three recombinant vectors gained high transfection efficiency (> 95%). Both real-time PCR and Western blot examination indicated that among three recombinant vectors, No. 2 Svector had the best interference effect and the interference effect was nearly 91% at protein level. In conclusion, Mouse recombinant Smad6 RNA interference (RNAi) vector was successfully constructed and it provided an effective tool for further studies on BMP signal pathways.

[Construction and expression of recombinant lentiviral vectors of AKT2,PDK1 and BAD].

PubMed

Zhu, Jing; Chen, Bo-Jiang; Huang, Na; Li, Wei-Min

2014-03-01

To construct human protein kinase B (ATK2), phosphoinositide-dependent kinase 1 (PDK1) and bcl-2-associated death protein (BAD) lentiviral expression vector, and to determine their expressions in 293T cells. Total RNA was extracted from lung cancer tissues. The full-length coding regions of human ATK2, BAD and PDK1 cDNA were amplified via RT-PCR using specific primers, subcloned into PGEM-Teasy and then sequenced for confirmation. The full-length coding sequence was cut out with a specific restriction enzyme digest and subclone into pCDF1-MCS2-EF1-copGFP. The plasmids were transfected into 293T cells using the calcium phosphate method. The over expression of AKT2, BAD and PDK1 were detected by Western blot. AKT2, PDK1 and BAD were subcloned into pCDF1-MCS2-EF1-copGFP, with an efficiency of transfection of 100%, 95%, and 90% respectively. The virus titers were 6.7 x 10(6) PFU/mL in the supernatant. After infection, the proteins of AKT2, PDK1 and BAD were detected by Western blot. The lentivial vector pCDF1-MCS2-EF1-copGFP containing AKT2, BAD and PDK1 were successfully constructed and expressed in 293T cells.

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.

78 FR 6120 - Government-Owned Inventions; Availability for Licensing

Federal Register 2010, 2011, 2012, 2013, 2014

2013-01-29

..., including immunoprecipitation, western blot analysis, immunohistochemistry, ELISA, etc. Competitive... immunoprecipitation, western blot analysis, immunohistochemistry, ELISA, etc. Competitive Advantages: Binding of a new...

Fluorescence-based Western blotting for quantitation of protein biomarkers in clinical samples.

PubMed

Zellner, Maria; Babeluk, Rita; Diestinger, Michael; Pirchegger, Petra; Skeledzic, Senada; Oehler, Rudolf

2008-09-01

Since most high throughput techniques used in biomarker discovery are very time and cost intensive, highly specific and quantitative analytical alternative application methods are needed for the routine analysis. Conventional Western blotting allows detection of specific proteins to the level of single isotypes while its quantitative accuracy is rather limited. We report a novel and improved quantitative Western blotting method. The use of fluorescently labelled secondary antibodies strongly extends the dynamic range of the quantitation and improves the correlation with the protein amount (r=0.997). By an additional fluorescent staining of all proteins immediately after their transfer to the blot membrane, it is possible to visualise simultaneously the antibody binding and the total protein profile. This allows for an accurate correction for protein load. Applying this normalisation it could be demonstrated that fluorescence-based Western blotting is able to reproduce a quantitative analysis of two specific proteins in blood platelet samples from 44 subjects with different diseases as initially conducted by 2D-DIGE. These results show that the proposed fluorescence-based Western blotting is an adequate application technique for biomarker quantitation and suggest possibilities of employment that go far beyond.

Multistrip Western blotting: a tool for comparative quantitative analysis of multiple proteins.

PubMed

Aksamitiene, Edita; Hoek, Jan B; Kiyatkin, Anatoly

2015-01-01

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 data output per single blotting cycle up to tenfold; allows concurrent measurement of up to nine different total and/or posttranslationally modified protein expression obtained 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 advantageous to apply in biomedical diagnostics, systems biology, and cell signaling research.

Puberty, Differentiation and Breast Cancer Risk

DTIC Science & Technology

1999-09-01

tetracycline or doxocycline to culture medium. We designed hammerhead ribozymes which recognize four different regions of mBrca2, 6 Proprietary Data DAMD17...containing a ribozyme directed against the 5’ end of mBrca2 appear to show dox-dependent regulation of Brca2 levels (as measured by Western blot...for possible modest regulation), we have designed ribozymes which recognize human and mouse BRCA2 sequence, and we intend to create tet-inducible

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).

Analysis of the 3’ untranslated regions of α-tubulin and S-crystallin mRNA and the identification of CPEB in dark- and light-adapted octopus retinas

PubMed Central

Kelly, Shannan; Yamamoto, Hideki

2008-01-01

Purpose We previously reported the differential expression and translation of mRNA and protein in dark- and light-adapted octopus retinas, which may result from cytoplasmic polyadenylation element (CPE)–dependent mRNA masking and unmasking. Here we investigate the presence of CPEs in α-tubulin and S-crystallin mRNA and report the identification of cytoplasmic polyadenylation element binding protein (CPEB) in light- and dark-adapted octopus retinas. Methods 3’-RACE and sequencing were used to isolate and analyze the 3’-UTRs of α-tubulin and S-crystallin mRNA. Total retinal protein isolated from light- and dark-adapted octopus retinas was subjected to western blot analysis followed by CPEB antibody detection, PEP-171 inhibition of CPEB, and dephosphorylation of CPEB. Results The following CPE-like sequence was detected in the 3’-UTR of isolated long S-crystallin mRNA variants: UUUAACA. No CPE or CPE-like sequences were detected in the 3’-UTRs of α-tubulin mRNA or of the short S-crystallin mRNA variants. Western blot analysis detected CPEB as two putative bands migrating between 60-80 kDa, while a third band migrated below 30 kDa in dark- and light-adapted retinas. Conclusions The detection of CPEB and the identification of the putative CPE-like sequences in the S-crystallin 3’-UTR suggest that CPEB may be involved in the activation of masked S-crystallin mRNA, but not in the regulation of α-tubulin mRNA, resulting in increased S-crystallin protein synthesis in dark-adapted octopus retinas. PMID:18682811

Familial Mediterranean fever with a single MEFV mutation: Where is the second hit?

PubMed Central

Booty, Matthew G.; Chae, Jae Jin; Masters, Seth L.; Remmers, Elaine F.; Barham, Beverly; Lee, Julie M.; Barron, Karyl S.; Holland, Steve; Kastner, Daniel L.; Aksentijevich, Ivona

2009-01-01

Objective FMF has traditionally been considered an autosomal recessive disease; however, it has been observed that a substantial number of patients with clinical FMF possess only one demonstrable MEFV mutation. Here, an extensive search for a second MEFV mutation was performed in 46 patients clinically diagnosed with FMF and carrying only one high-penetrance FMF mutation. Methods MEFV and other candidate genes were sequenced by standard capillary electrophoresis. The entire 15 kb MEFV genomic region was re-sequenced in 10 patients using a hybridization-based chip technology. MEFV gene expression levels were determined by qRT-PCR and pyrin protein levels were examined by Western blotting. Results A second MEFV mutation was not identified in any of the screened patients. Haplotype analysis did not identify a common haplotype that might be associated with the transmission of a second FMF allele. Western blots did not demonstrate a significant difference in pyrin levels between single and double variant patients; however, FMF patients of both types showed higher protein expression compared to controls and non-FMF patients with active inflammation. Screening of genes encoding pyrin-interacting proteins identified rare variants in a small number of patients, suggesting the possibility of digenic inheritance. Conclusion Our data underscore the existence of a significant subset of FMF patients who are carriers of only one MEFV mutation and demonstrate that complete MEFV sequencing is not likely to yield a second mutation. Screening for the set of most common mutations appears sufficient in the presence of clinical symptoms to diagnose FMF and initiate a trial of colchicine. PMID:19479870

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.

[Recombinant OspC identification and antigenicity detection from Borrelia burgdorferi PD91 in China].

PubMed

Chen, Jian; Wan, Kang-Lin

2003-10-01

To recombine OspC gene from Borrelia burgdorferi PD91 of China and expressed it in E. coli for early diagnosis of Lyme disease. The OspC gene was amplified from the genome of Borrelia burgdorferi PD91 strain by polymerase chain reaction and recombined with plasmid PET-11D. The recombinant plasmid PET-11D-OspC was identified with PCR, restriction endonuclease analysis and sequencing. The antigenicity was verified with Western Blot. OspC gene was cloned correctly into vector PET-11D. The resultant sequence was definitely different from the published sequence. The recombinant OspC seemed to have had strong antigenicity. The findings laid basis for the studies on early diagnosis of Lyme disease.

[The expression of interferon-lambda1 in CHO cell].

PubMed

Yuan, Wu-Mei; Ma, Fen-Lian; Zhang, Qian; Zheng, Wen-Zhi; Zheng, Li-Shu

2013-06-01

To construct the eukaryotic expression vector PCI-dhfr-lambda1 and PCI-dhfr-SP163-lambda1 which linked the enhancer SP163 with interferon lambda1. Then express the interferon lambda1 in CHO (dhfr-) cells. Using PCR method to introduce the restriction enzyme sites and through the fusion PCR binding the enhancer with the interferon Lambda1. After sequenced, lambda1 and SP163-lambda1 was inserted into PCI-dhfr forming the expression vector PCI-dhfr-lambda1 and PCI-dhfr-SP163-lambda1 which was constructed successfully confirming by sequencing. Then the expressing vectors were transfected into CHO (dhfr-) cells using liposome transfection method and interferon lambda1 protein was assayed with indirect immunofluorescence and Western Blot. Using cytopathic effect inhibition evaluated the antiviral activity of interferon lambda1. Successfully constructing the eukaryotic expression vectors of interferon lambda and the vectors could express interferon lambda1. The result of immunofluorescence showed the enhancer developed the expression of interferon lambda1. Detecting the interferon lambda1 in CHO (dhfr-) cells after transfecting 48 hour using Western Blot. The cytopathic effect inhibition showed the expressed interferon lambda1 has the antiviral activity. Successfully expressed the interferon lambda1 in CHO (dhfr-) cells and the protein possesses antiviral activity, which may supply a valuable basis for building the stable cell line of interferon lambda1.

[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.

Characterization of R132H mutation-specific IDH1 antibody binding in brain tumors.

PubMed

Capper, David; Weissert, Susanne; Balss, Jörg; Habel, Antje; Meyer, Jochen; Jäger, Diana; Ackermann, Ulrike; Tessmer, Claudia; Korshunov, Andrey; Zentgraf, Hanswalter; Hartmann, Christian; von Deimling, Andreas

2010-01-01

Heterozygous point mutations of isocitrate dehydrogenase (IDH)1 codon 132 are frequent in grade II and III gliomas. Recently, we reported an antibody specific for the IDH1R132H mutation. Here we investigate the capability of this antibody to differentiate wild type and mutated IDH1 protein in central nervous system (CNS) tumors by Western blot and immunohistochemistry. Results of protein analysis are correlated to sequencing data. In Western blot, anti-IDH1R132H mouse monoclonal antibody mIDH1R132H detected a specific band only in mutated tumors. Immunohistochemistry of 345 primary brain tumors demonstrated a strong cytoplasmic and weaker nuclear staining in 122 cases. Correlation with direct sequencing of 186 cases resulted in consensus of 177 cases. Genetic retesting of cases with conflicting findings resulted in a match of 186/186 cases, with all discrepancies resolving in favor of immunohistochemistry. Intriguing is the ability of mIDH1R132H to detect single infiltrating tumor cells. The very high frequency and the distribution of this mutation among specific brain tumor entities allow the highly sensitive and specific discrimination of various tumors by immunohistochemistry, such as anaplastic astrocytoma from primary glioblastoma or diffuse astrocytoma World Health Organization (WHO) grade II from pilocytic astrocytoma or ependymoma. Noteworthy is the discrimination of the infiltrating edge of tumors with IDH1 mutation from reactive gliosis.

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.

Identification and Characterization of Novel Surface Proteins in Lactobacillus johnsonii and Lactobacillus gasseri

PubMed Central

Ventura, Marco; Jankovic, Ivana; Walker, D. Carey; Pridmore, R. David; Zink, Ralf

2002-01-01

We have identified and sequenced the genes encoding the aggregation-promoting factor (APF) protein from six different strains of Lactobacillus johnsonii and Lactobacillus gasseri. Both species harbor two apf genes, apf1 and apf2, which are in the same orientation and encode proteins of 257 to 326 amino acids. Multiple alignments of the deduced amino acid sequences of these apf genes demonstrate a very strong sequence conservation of all of the genes with the exception of their central regions. Northern blot analysis showed that both genes are transcribed, reaching their maximum expression during the exponential phase. Primer extension analysis revealed that apf1 and apf2 harbor a putative promoter sequence that is conserved in all of the genes. Western blot analysis of the LiCl cell extracts showed that APF proteins are located on the cell surface. Intact cells of L. johnsonii revealed the typical cell wall architecture of S-layer-carrying gram-positive eubacteria, which could be selectively removed with LiCl treatment. In addition, the amino acid composition, physical properties, and genetic organization were found to be quite similar to those of S-layer proteins. These results suggest that APF is a novel surface protein of the Lactobacillus acidophilus B-homology group which might belong to an S-layer-like family. PMID:12450842

Activation of Antitumorigenic Stat3beta in Breast Cancer by Splicing Redirection

DTIC Science & Technology

2013-07-01

putative mapped ESEs (shown in green). (B) (Top) RT-PCR and (Bottom) Western Blot analysis of STAT3 a/b levels in MDA-MB-435s cells treated with...codon (PTC), ultimately causing RNA degradation following nonsense mediated decay (NMD). (B) RT-PCR and Western Blot analysis of STAT3 α/β levels in MDA...MB-435s cells treated with 16µM of ST6, ST7 or INV for 4 days. α-tubulin was used as loading control. (C) RT-PCR and Western Blot analysis of STAT3

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...

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.

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.

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.

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.

Effects of Reusing Gel Electrophoresis and Electrotransfer Buffers on Western Blotting.

PubMed

Heda, Ghanshyam D; Omotola, Oluwabukola B; Heda, Rajiv P; Avery, Jamie

2016-09-01

SDS-PAGE and Western blotting are 2 of the most commonly used biochemical methods for protein analysis. Proteins are electrophoretically separated based on their MWs by SDS-PAGE and then electrotransferred to a solid membrane surface for subsequent protein-specific analysis by immunoblotting, a procedure commonly known as Western blotting. Both of these procedures use a salt-based buffer, with the latter procedure consisting of methanol as an additive known for its toxicity. Previous reports present a contradictory view in favor or against reusing electrotransfer buffer, also known as Towbin's transfer buffer (TTB), with an aim to reduce the toxic waste. In this report, we present a detailed analysis of not only reusing TTB but also gel electrophoresis buffer (EB) on proteins of low to high MW range. Our results suggest that EB can be reused for at least 5 times without compromising the electrophoretic separation of mixture of proteins in an MW standard, BSA, and crude cell lysates. Additionally, reuse of EB did not affect the quality of subsequent Western blots. Successive reuse of TTB, on the other hand, diminished the signal of proteins of different MWs in a protein standard and a high MW membrane protein cystic fibrosis transmembrane-conductance regulator (CFTR) in Western blotting.

Cytokine Gene Expression in Response to SnSAG1 in Horses with Equine Protozoal Myeloencephalitis

PubMed Central

Spencer, Jennifer A.; Deinnocentes, Patricia; Moyana, Edith M.; Guarino, Anthony J.; Ellison, Siobhan E.; Bird, R. Curtis; Blagburn, Byron L.

2005-01-01

Equine protozoal myeloencephalitis (EPM) is a neurologic syndrome seen in horses from the Americas and is mainly caused by Sarcocystis neurona. Recently, a 29-kDa surface antigen from S. neurona merozoites was identified as being highly immunodominant on a Western blot. This antigen has been sequenced and cloned, and the expressed protein has been named SnSAG1. In a previous study, cell-mediated immune responses to SnSAG1 were shown to be statistically significantly reduced in horses with EPM in comparison to EPM-negative control horses. It therefore appears as though the parasite is able to induce immunosuppression towards parasite-derived antigens as parasite-specific responses are decreased. Isolated peripheral blood lymphocytes from 21 EPM (cerebrospinal fluid [CSF] Western blot)-negative horses with no clinical signs and 21 horses with clinical signs of EPM (CSF Western blot positive) were cocultured with SnSAG1 for 48 and 72 h, and the effect on cytokine production was investigated by means of reverse transcriptase PCR. Cytokines assayed include gamma interferon (IFN-γ), tumor necrosis factor alpha, interleukin (IL)-2, IL-4, and IL-6. β-Actin was used as the housekeeping gene. A Wilcoxon signed-rank test of the findings indicated that there was a statistically significant decrease in IFN-γ production after 48 h in culture for samples from horses with clinical disease. There was also a statistically significant increase in IL-4 production after 72 h in culture for samples from horses with EPM. These results further support the notion that this parasite is able to subvert the immune system in horses with clinical disease. PMID:15879026

Immunological characteristics of outer membrane protein omp31 of goat Brucella and its monoclonal antibody.

PubMed

Zheng, W Y; Wang, Y; Zhang, Z C; Yan, F

2015-10-05

We examined the immunological characteristics of outer membrane protein omp31 of goat Brucella and its monoclonal antibody. Genomic DNA from the M5 strain of goat Brucella was amplified by polymerase chain reaction and cloned into the prokaryotic expression vector pGEX-4T-1. The expression and immunological characteristics of the fusion protein GST-omp31 were subjected to preliminary western blot detection with goat Brucella rabbit immune serum. The Brucella immunized BALB/c mouse serum was detected using purified protein. The high-potency mouse splenocytes and myeloma Sp2/0 cells were fused. Positive clones were screened by enzyme-linked immunosorbent assay to establish a hybridoma cell line. Mice were inoculated intraperitoneally with hybridoma cells to prepare ascites. The mAb was purified using the n-caprylic acid-ammonium sulfate method. The characteristics of mAb were examined using western blotting and enzyme-linked immunosorbent assay. A 680-base pair band was observed after polymerase chain reaction. Enzyme digestion identification and sequencing showed that the pGEX-4T-1-omp31 prokaryotic expression vector was successfully established; a target band of approximately 57 kDa with an apparent molecular weight consistent with the size of the target fusion protein. At 25°C, the expression of soluble expression increased significantly; the fusion protein GST-omp31 was detected by western blotting. Anti-omp31 protein mAb was obtained from 2 strains of Brucella. The antibody showed strong specificity and sensitivity and did not cross-react with Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Mycobacterium tuberculosis, or Bacillus pyocyaneus. The pGEX-4T-1-omp31 prokaryotic expression vector was successfully established and showed good immunogenicity. The antibody also showed strong specificity and good sensitivity.

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.

SnSAG5 is an alternative surface antigen of Sarcocystis neurona strains that is mutually exclusive to SnSAG1.

PubMed

Crowdus, Carolyn A; Marsh, Antoinette E; Saville, Willliam J; Lindsay, David S; Dubey, J P; Granstrom, David E; Howe, Daniel K

2008-11-25

Sarcocystis neurona is an obligate intracellular parasite that causes equine protozoal myeloencephalitis (EPM). Previous work has identified a gene family of paralogous surface antigens in S. neurona called SnSAGs. These surface proteins are immunogenic in their host animals, and are therefore candidate molecules for development of diagnostics and vaccines. However, SnSAG diversity exists in strains of S. neurona, including the absence of the major surface antigen gene SnSAG1. Instead, sequence for an alternative SnSAG has been revealed in two of the SnSAG1-deficient strains. Herein, we present data characterizing this new surface protein, which we have designated SnSAG5. The results indicated that the protein encoded by the SnSAG5 sequence is indeed a surface-associated molecule that has characteristics consistent with the other SAGs identified in S. neurona and related parasites. Importantly, Western blot analyses of a collection of S. neurona strains demonstrated that 6 of 13 parasite isolates express SnSAG5 as a dominant surface protein instead of SnSAG1. Conversely, SnSAG5 was not detected in SnSAG1-positive strains. One strain, which was isolated from the brain of a sea otter, did not express either SnSAG1 or SnSAG5. Genetic analysis with SnSAG5-specific primers confirmed the presence of the SnSAG5 gene in Western blot-positive strains, while also suggesting the presence of a novel SnSAG sequence in the SnSAG1-deficient, SnSAG5-deficient otter isolate. The findings provide further indication of S. neurona strain diversity, which has implications for diagnostic testing and development of vaccines against EPM as well as the population biology of Sarcocystis cycling in the opossum definitive host.

[Better performance of Western blotting: quick vs slow protein transfer, blotting membranes and the visualization methods].

PubMed

Kong, Ling-Quan; Pu, Ying-Hui; Ma, Shi-Kun

2008-01-01

To study how the choices of the quick vs slow protein transfer, the blotting membranes and the visualization methods influence the performance of Western blotting. The cellular proteins were abstracted from human breast cell line MDA-MB-231 for analysis with Western blotting using quick (2 h) and slow (overnight) protein transfer, different blotting membranes (nitrocellulose, PVDF and nylon membranes) and different visualization methods (ECL and DAB). In Western blotting with slow and quick protein transfer, the prestained marker presented more distinct bands on nitrocellulose membrane than on the nylon and PVDF membranes, and the latter also showed clear bands on the back of the membrane to very likely cause confusion, which did not occur with nitrocellulose membrane. PVDF membrane allowed slightly clearer visualization of the proteins with DAB method as compared with nitrocellulose and nylon membranes, and on the latter two membranes, quick protein transfer was likely to result in somehow irregular bands in comparison with slow protein transfer. With slow protein transfer and chemiluminescence for visualization, all the 3 membranes showed clear background, while with quick protein transfer, nylon membrane gave rise to obvious background noise but the other two membranes did not. Different membranes should be selected for immunoblotting according to the actual needs of the experiment. Slow transfer of the proteins onto the membranes often has better effect than quick transfer, and enhanced chemiluminescence is superior to DAB for protein visualization and allows highly specific and sensitive analysis of the protein expressions.

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.

Expression and copy number gains of the RET gene in 631 early and mid stage non‐small cell lung cancer cases

PubMed Central

Tan, Ling; Hu, Yerong; Tao, Yongguang; Wang, Bin; Xiao, Jun; Tang, Zhenjie; Lu, Ting

2018-01-01

Background To identify whether RET is a potential target for NSCLC treatment, we examined the status of the RET gene in 631 early and mid stage NSCLC cases from south central China. Methods RET expression was identified by Western blot. RET‐positive expression samples were verified by immunohistochemistry. RET gene mutation, copy number variation, and rearrangement were analyzed by DNA Sanger sequencing, TaqMan copy number assays, and reverse transcription‐PCR. ALK and ROS1 expression levels were tested by Western blot and EGFR mutation using Sanger sequencing. Results The RET‐positive rate was 2.5% (16/631). RET‐positive expression was related to poorer tumor differentiation (P < 0.05). In the 16 RET‐positive samples, only two samples of moderately and poorly differentiated lung adenocarcinomas displayed RET rearrangement, both in RET‐KIF5B fusion partners. Neither ALK nor ROS1 translocation was found. The EGFR mutation rate in RET‐positive samples was significantly lower than in RET‐negative samples (P < 0.05). Conclusion RET‐positive expression in early and mid stage NSCLC cases from south central China is relatively low and is related to poorer tumor differentiation. RET gene alterations (copy number gain and rearrangement) exist in all RET‐positive samples. RET‐positive expression is a relatively independent factor in NSCLC patients, which indicates that the RET gene may be a novel target site for personalized treatment of NSCLC. PMID:29473341

[Construction, identification and expression of three kinds of shuttle plasmids of adenovirus expression vector of hepatitis C virus structure gene].

PubMed

Cao, Yi-zhan; Hao, Chun-qiu; Feng, Zhi-hua; Zhou, Yong-xing; Li, Jin-ge; Jia, Zhan-sheng; Wang, Ping-zhong

2003-02-01

To construct three recombinant shuttle plasmids of adenovirus expression vector which can express hepatitis C virus(HCV) different structure genes(C, C+E1, C+E1+E2) in order to pack adenovirus expression vectors which can express HCV different structure gene effectively. The different HCV structure genes derived from the plasmid pBRTM/HCV1-3011 by using polymerase chain reaction (PCR) were inserted into the backward position of cytomegalovirus(CMV) immediate early promotor element of shuttle plasmid(pAd.CMV-Link.1) of adenovirus expression vector respectively, then the three recombinant plasmids (pAd.HCV-C, pAd.HCV-CE1, pAd.HCV-S) were obtained. The recombinant plasmids were identified by endonuclease, PCR and sequencing. HCV structure genes were expressed transiently with Lipofectamine 2000 coated in HepG2 cells which were confirmed by immunofluorescence and Western-Blot. Insert DNAs of the three recombinant plasmids' were confirmed to be HCV different structure genes by endonuclease, PCR and sequencing. The three recombinant plasmids can express HCV structure gene (C, C+E1, C+E1+E2) transiently in HepG2 cells which were confirmed by immunofluorescence and Western-Blot. The three recombinant shuttle plasmids of adenovirus expression vector can express HCV structure gene(C, C+E1, C+E1+E2) transiently. This should be useful to pack adenovirus expression vector which can express HCV structure genes.

[Construction of BAD Lentivirus Vector and Its Effect on Proliferation in A549 Cell Lines].

PubMed

Huang, Na; He, Yan-qi; Zhu, Jing; Li, Wei-min

2015-05-01

To construct the recombinant lentivirus expressing vector BAD (Bcl-2-associated death protein) gene and to study its effect on A549 cell proliferation. The BAD gene was amplified from plasmid pAV-MCMV-BAD-GFP by PCR. The purified BAD gene fragment was inserted into a lentivirus vector (pLVX-IRES-ZsGreen 1), and the insertion was identified by PCR, restriction endonuclease analysis and DNA sequencing. A549 cells were then transfected with the packaged recombinant lentivirus, and resistant cell clones were selected with flow cytometry. The expression of BAD in A549 cell lines stably transduction with a lentivirus was examined using Western blot. The effect of BAD overexpression on proliferation of A549 cells was evaluated by using CCK-8 kit. Restriction enzyme digestion and DNA sequencing showed that the full-length BAD gene (507 bp) had been successfully subcloned into the lentiviral vector to result in the recombinant vector pLVX-IRES-ZsGreen 1. Monoclonal cell lines BAD-A549 was produced after transfection with the recombinant lentivirus and selected with flow cytometry. Stable expression of BAD protein was verified by Western blot. In vitro, the OD value in BAD group was significantly lower than that of control groups from 120-144 h (P<0. 05). A549 cell lines stably transduced with a lentivirus expressing the BAD gene had been successfully generated. In vitro, BAD overexpression significantly inhibited A549 cells proliferation.

Phytomonas iron superoxide dismutase: a possible molecular marker.

PubMed

Marín, Clotilde; Hitos, Ana B; Rodríguez-González, Isabel; Dollet, Michel; Sánchez-Moreno, Manuel

2004-05-01

We have isolated and biochemically characterized two iron superoxide dismutases activities (SODI and SODII) from a plant trypanosomatid isolated from Euphorbia characias. The isoenzyme FeSODII has immunogenic capacity, and the positivity of the anti-SODII serum persists to a dilution of 1/40,000, by Western blot. In addition, Western blot has been used to test the positivity of the anti-SODII serum against antigen fractions (SOD) from 17 isolates belonging to the family Trypanosomatidae and for which we had previously determined the isoenzymatic profile. The reaction proved positive only with those plant isolates considered to belong to the genus Phytomonas, whereas there was no reaction of the anti-SODII serum, against the antigen fractions from the species Trypanosoma cruzi, Leishmania donovani, Herpetomonas samuelpessoai, Herpetomonas davidi, Crithidia luciliae and Leptomonas collosoma. FeSODII is located mainly over the entire surface of the parasite, as well as in the nucleus, glycosomes and membranes. The above makes FeSODII promising as a molecular tool for diagnosis and identification, and as a potential chemotherapeutic target for designing drugs aimed at controlling not only of the diseases caused by Phytomonas species, but also for the great metabolic similarity to other trypanosomatids of animals and humans, it may be possible for these results to be extrapolated. Moreover, the sequencing of the amino-terminal end of the FeSODII enables the design of primers that in the near future will make it possible to sequence the gene of this isoenzyme.

Generation and characterization of anti-MUC4 monoclonal antibodies reactive with normal and cancer cells in humans.

PubMed

Moniaux, Nicolas; Varshney, Grish Chandra; Chauhan, Subhash Chand; Copin, Marie Christine; Jain, Maneesh; Wittel, Uwe A; Andrianifahanana, Mahefatiana; Aubert, Jean-Pierre; Batra, Surinder Kumar

2004-02-01

We have previously cloned the full-length cDNA (approximately 28 Kb) and established the complete genomic organization (25 exons/introns over 100 kb) of the human MUC4 mucin. This large molecule is predicted to protrude over 2 microm above the cell surface, in which MUC4alpha is an extracellular mucin-type glycoprotein subunit and MUC4beta is the transmembrane subunit. Over two thirds of the encoded protein sequence consists of 16-amino-acid tandem repeats (TR), which are flanked by unique sequences. In this study we generated and characterized monoclonal antibodies (MAbs) directed against the TR region of MUC4. Mice were immunized with a KLH-conjugated MUC4 TR peptide, STGDTTPLPVTDTSSV. Several clones were purified by three rounds of limited dilutions and stable clones presenting a sustained antibody production were selected for subsequent characterization. Antibodies were tested for their reactivity and specificity to recognize the MUC4 peptide and further screened by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. One of the MAbs (8G7) was strongly reactive against the MUC4 peptide and with native MUC4 from human tissues or pancreatic cancer cells in Western blotting, immunohistochemistry, and confocal analysis. Anti-MUC4 MAb may represent a powerful tool for the study of MUC4 function under normal and pathological conditions and for diagnosis of solid tumors including those in the breast, pancreas, lungs, and ovaries.

[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.

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...

Twin-arginine translocase may have a role in the chaperone function of NarJ from Escherichia coli

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

Chan, Catherine S.; Howell, Jenika M.; Workentine, Matthew L.

2006-04-28

NarJ is a chaperone involved in folding, maturation, and molybdenum cofactor insertion of nitrate reductase A from Escherichia coli. It has also been shown that NarJ exhibits sequence homology to a family of chaperones involved in maturation and cofactor insertion of E. coli redox enzymes that are mediated by twin-arginine translocase (Tat) dependent translocation. In this study, we show that NarJ binds the N-terminal region of NarG through Far Western studies and isothermal titration calorimetry, and the binding event occurs towards a short peptide sequence that contains a homologous twin-arginine motif. Fractionation experiments also show that the interaction of NarJmore » to the cytoplasmic membrane exhibits Tat-dependence. Upon further investigation through Far Western blots, the interactome of NarJ also exhibits Tat-dependence. Together the data suggest that the Tat system may play a role in the maturation pathway of nitrate reductase A.« less

[Knockdown of STAT3 inhibits proliferation and migration of HepG2 hepatoma cells induced by IFN1].

PubMed

Li, Xiaofang; Wang, Yuqi; Yan, Ben; Fang, Peipei; Ma, Chao; Xu, Ning; Fu, Xiaoyan; Liang, Shujuan

2018-02-01

Objective To prepare lentiviruses expressing shRNA sequences targeting human signal transducer and activator of transcription 3 (STAT3) and detect the effect of STAT3 knockdown on type I interferon (IFN1)-induced proliferation and migration in HepG2 cells. Methods Four STAT3-targeting shRNA sequences (shRNA1-shRNA4) and one control sequence (Ctrl shRNA) were selected and cloned respectively into pLKO.1-sp6-pgk-GFP to construct shRNA-expressing vectors. Along with backbone psPAX2 and pMD2.G vectors, they were separately transfected into HEK293T cells to prepare lentiviruses. HepG2 cells were infected with the lentiviruses. Cytoplastic STAT3 level was detected by Western blotting to screen effective shRNA sequence(s) targeting STAT3. Proliferation and migration of HepG2 cells were analyzed by CCK-8 assay and Transwell TM migration and scratching assay, respectively. To detect the effect of IFN1 on cell proliferation and migration of HepG2 cells, the cells were treated with 2000 U/mL IFNα2b for indicated time and the activation of IFN-triggered STAT1 signal transduction was assayed by Western blotting. Results Two most effective STAT3-targeting shRNA sequences shRNA1 and shRNA2 were selected, and the expression of both STAT3 shRNA significantly decreased proliferation and migration of HepG2 cells. When treated with IFNα2b, 2000 U/mL of IFN1 showed more competent in attenuating growth and migration of HepG2 cells. Our data further proved that knockdown of STAT3 increased the phosphorylation of STAT1, and IFNα2b further enhanced the activation of STAT1 signaling in HepG2 cells. Conclusion Knockdown of STAT3 inhibits cell migration and growth, and rescues IFN response through up-regulating STAT1 signal transduction in HepG2 hepatoma cells.

Cross-sectional study of serum antibodies against Sarcocystis neurona in cats tested for antibodies against Toxoplasma gondii.

PubMed

Rossano, Mary G; Murphy, Alice J; Vrable, Ruth A; Vanzo, Nicole E; Lewis, Stacy K; Sheline, Katherine D; Kaneene, John B; Mansfield, Linda S

2002-08-15

To determine apparent seroprevalence of antibodies against Sarcocystis neurona in a population of domestic cats previously tested for antibodies against Toxoplasma gondii. Cross-sectional study. Serum from 196 domestic cats. Banked serum samples submitted to the Michigan State University Animal Health Diagnostic Laboratory for T. gondii diagnostic testing were tested for antibodies against S. neurona by use of an indirect fluorescent antibody (IFA) test and a western blot test. Submission records were analyzed to determine descriptive statistics and test for associations between positive results of a test for S. neurona and other variables in the data set. 10 of 196 (5%) samples yielded positive results for antibodies against S. neurona by use of western blot analysis, whereas 27 samples yielded positive results by use of the IFA. No association was found between S. neurona western blot test results and T. gondii test results, age, sex, or the reason for T. gondii testing. The S. neurona IFA titer was positively and significantly associated with positive results of western blot analysis. Domestic cats are not likely to play a substantial role as intermediate hosts in the natural life cycle of S. neurona. Results indicate that natural infection of domestic cats may occur, and small animal practitioners should be aware of this fact when evaluating cats with neurologic disease. The S. neurona IFA test had lower specificity than western blot analysis.

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...

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.

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.

Regulation of Adhesion and Migration by the Rsu1- and PINCH1-mediated Inhibition of Focal Adhesion Formation and Actin Polymerization

DTIC Science & Technology

2012-06-08

RT-PCR and sequencing of the Rsu1 open reading frame revealed that exon 8 is missing in the alternatively spliced Rsu1 RNA in human gliomas and...concentration of 75 nM. Cells were collected 72-96 hours post-transfection. 16 Western blotting Cell lysates were collected in RIPA buffer...cell lysates were collected and bound to the glutathione beads loaded with the GST-fusion of the Rac1/cdc42 binding domain of Pak. The bound Rac1

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.

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…

Evidence for a Pneumocystis carinii Flo8-like transcription factor: insights into organism adhesion.

PubMed

Kottom, Theodore J; Limper, Andrew H

2016-02-01

Pneumocystis carinii (Pc) adhesion to alveolar epithelial cells is well established and is thought to be a prerequisite for the initiation of Pneumocystis pneumonia. Pc binding events occur in part through the major Pc surface glycoprotein Msg, as well as an integrin-like molecule termed PcInt1. Recent data from the Pc sequencing project also demonstrate DNA sequences homologous to other genes important in Candida spp. binding to mammalian host cells, as well as organism binding to polystyrene surfaces and in biofilm formation. One of these genes, flo8, a transcription factor needed for downstream cAMP/PKA-pathway-mediated activation of the major adhesion/flocculin Flo11 in yeast, was cloned from a Pc cDNA library utilizing a partial sequence available in the Pc genome database. A CHEF blot of Pc genomic DNA yielded a single band providing evidence this gene is present in the organism. BLASTP analysis of the predicted protein demonstrated 41 % homology to the Saccharomyces cerevisiae Flo8. Northern blotting demonstrated greatest expression at pH 6.0-8.0, pH comparable to reported fungal biofilm milieu. Western blot and immunoprecipitation assays of PcFlo8 protein in isolated cyst and tropic life forms confirmed the presence of the cognate protein in these Pc life forms. Heterologous expression of Pcflo8 cDNA in flo8Δ-deficient yeast strains demonstrated that the Pcflo8 was able to restore yeast binding to polystyrene and invasive growth of yeast flo8Δ cells. Furthermore, Pcflo8 promoted yeast binding to HEK293 human epithelial cells, strengthening its functional classification as a Flo8 transcription factor. Taken together, these data suggest that PcFlo8 is expressed by Pc and may exert activity in organism adhesion and biofilm formation.

Evidence for a Pneumocystis carinii Flo8-like Transcription Factor: Insights into Organism Adhesion

PubMed Central

Kottom, Theodore J.; Limper, Andrew H.

2015-01-01

Pneumocystis carinii (Pc) adhesion to alveolar epithelial cells is well established and is thought to be a prerequisite for initiation of Pneumocystis pneumonia. Pc binding events occur in part through the major Pc surface glycoprotein Msg, as well as an integrin-like molecule termed PcInt1. Recent data from the Pc sequencing project also demonstrate DNA sequences homologous to other genes important in Candida spp. binding to mammalian host cells, as well as organism binding to polystyrene surfaces and in biofilm formation. One of these genes, flo8, a transcription factor needed for downstream cAMP/PKA-pathway-mediated activation of the major adhesin/flocculin Flo11 in yeast, was cloned from a Pc cDNA library utilizing a partial sequence available in the Pc genome database. A CHEF blot of Pc genomic DNA yielded a single band providing evidence this gene is present in the organism. BLASTP analysis of the predicted protein demonstrated 41% homology to the Saccharomyces cerevisiae Flo8. Northern blotting demonstrated greatest expression at pH 6.0–8.0, pH comparable to reported fungal biofilm milieu. Western blot and immunoprecipitation assays of PcFlo8 protein in isolated cyst and tropic life forms confirmed the presence of the cognate protein in these Pc life forms. Heterologous expression of Pcflo8 cDNA in flo8Δ (deficient) yeast strains demonstrated the Pcflo8 was able to restore yeast binding to polystyrene and invasive growth of yeast flo8Δ cells. Furthermore, Pcflo8 promoted yeast binding to HEK293 human epithelial cells, strengthening its functional classification as a Flo8 transcription factor. Taken together these data suggests that PcFlo8 is expressed by Pc and may exert activity in organism adhesion and biofilm formation. PMID:26215665

The fixABCX genes in Rhodospirillum rubrum encode a putative membrane complex participating in electron transfer to nitrogenase.

PubMed

Edgren, Tomas; Nordlund, Stefan

2004-04-01

In our efforts to identify the components participating in electron transport to nitrogenase in Rhodospirillum rubrum, we used mini-Tn5 mutagenesis followed by metronidazole selection. One of the mutants isolated, SNT-1, exhibited a decreased growth rate and about 25% of the in vivo nitrogenase activity compared to the wild-type values. The in vitro nitrogenase activity was essentially wild type, indicating that the mutation affects electron transport to nitrogenase. Sequencing showed that the Tn5 insertion is located in a region with a high level of similarity to fixC, and extended sequencing revealed additional putative fix genes, in the order fixABCX. Complementation of SNT-1 with the whole fix gene cluster in trans restored wild-type nitrogenase activity and growth. Using Western blotting, we demonstrated that expression of fixA and fixB occurs only under conditions under which nitrogenase also is expressed. SNT-1 was further shown to produce larger amounts of both ribulose 1,5-bisphosphate carboxylase/oxygenase and polyhydroxy alkanoates than the wild type, indicating that the redox status is affected in this mutant. Using Western blotting, we found that FixA and FixB are soluble proteins, whereas FixC most likely is a transmembrane protein. We propose that the fixABCX genes encode a membrane protein complex that plays a central role in electron transfer to nitrogenase in R. rubrum. Furthermore, we suggest that FixC is the link between nitrogen fixation and the proton motive force generated in the photosynthetic reactions.

Molecular identification, immunolocalization, and characterization of Clonorchis sinensis triosephosphate isomerase.

PubMed

Zhou, Juanjuan; Liao, Hua; Li, Shan; Zhou, Chenhui; Huang, Yan; Li, Xuerong; Liang, Chi; Yu, Xinbing

2015-08-01

Clonorchis sinensis triosephosphate isomerase (CsTIM) is a key regulatory enzyme of glycolysis and gluconeogenesis, which catalyzes the interconversion of glyceraldehyde 3-phosphate to dihydroxyacetone phosphate. In this study, the biochemical characterizations of CsTIM have been examined. A full-length complementary DNA (cDNA; Cs105350) sequence encoding CsTIM was obtained from our C. sinensis cDNA library. The open reading frame of CsTIM contains 759 bp which encodes 252 amino acids. The amino acid sequence of CsTIM shares 60-65% identity with other species. Western blot analysis displayed that recombinant CsTIM (rCsTIM) can be probed by anti-rCsTIM rat serum and anti-C. sinensis excretory/secretory products (anti-CsESPs) rat serum. Quantitative reverse transcription (RT)-PCR and western blotting analysis revealed that CsTIM messenger RNA (mRNA) and protein were differentially expressed in development cycle stages of the parasite, including adult worm, metacercaria, excysted metacercaria, and egg. In addition, immunolocalization assay showed that CsTIM was located in the seminal vesicle, eggs, and testicle. Moreover, rCsTIM exhibited active enzyme activity in catalytic reactions. The Michaelis constant (K m) of rCsTIM was 0.33 mM, when using glyceraldehyde 3-phosphate as the substrate. The optimal temperature and pH of CsTIM were 37 °C and 7.5-9.5, respectively. Collectively, these results suggest that CsTIM is an important protein involved in glycometabolism, and CsTIM possibly take part in many biological functions in the growth and development of C. sinensis.

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,…

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.

[Cloning, sequencing and prokaryotic expression of cDNAs for the antifreeze protein family from the beetle Tenebrio molitor].

PubMed

Liu, Zhong-Yuan; Wang, Yun; Lü, Guo-Dong; Wang, Xian-Lei; Zhang, Fu-Chun; Ma, Ji

2006-12-01

The partial cDNA sequence coding for the antifreeze proteins in the Tenebrio molitor was obtained by RT-PCR. Sequence analysis revealed nine putative cDNAs with a high degree of homology to Tenebrio molitor antifreeze proteins. The recombinant pGEX-4T-1-tmafp-XJ430 was introduced into E. coli BL21 to induce a GST fusion protein by IPTG. SDS-PAGE of the fusion protein demonstrated that the antifreeze protein migrated at a size of 38 kDa. The immunization was performed by intra-muscular injection of pCDNA3-tmafp-XJ430, and then antiserum was detected by ELISA. The titer of the antibody was 1:2,000. Western blotting analysis showed the antiserum was specific against the antifreeze protein. This finding could lead to further investigation of the properties and function of antifreeze proteins.

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.

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

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.

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.

[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.

Antipeptide antibodies that can distinguish specific subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.)

NASA Technical Reports Server (NTRS)

Cai, X.; Henry, R. L.; Takemoto, L. J.; Guikema, J. A.; Wong, P. P.; Spooner, B. S. (Principal Investigator)

1992-01-01

The amino acid sequences of the beta and gamma subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.) root nodules are very similar. However, there are small regions within the sequences that are significantly different between the two polypeptides. The sequences between amino acids 2 and 9 and between 264 and 274 are examples. Three peptides (gamma 2-9, gamma 264-274, and beta 264-274) corresponding to these sequences were synthesized. Antibodies against these peptides were raised in rabbits and purified with corresponding peptide-Sepharose affinity chromatography. Western blot analysis of polyacrylamide gel electrophoresis of bean nodule proteins demonstrated that the anti-beta 264-274 antibodies reacted specifically with the beta polypeptide and the anti-gamma 264-274 and anti-gamma 2-9 antibodies reacted specifically with the gamma polypeptide of the native and denatured glutamine synthetase. These results showed the feasibility of using synthetic peptides in developing antibodies that are capable of distinguishing proteins with similar primary structures.

Short communication: Conservation of Streptococcus uberis adhesion molecule and the sua gene in strains of Streptococcus uberis isolated from geographically diverse areas.

PubMed

Yuan, Ying; Dego, Oudessa Kerro; Chen, Xueyan; Abadin, Eurife; Chan, Shangfeng; Jory, Lauren; Kovacevic, Steven; Almeida, Raul A; Oliver, Stephen P

2014-12-01

The objective was to identify and sequence the sua gene (GenBank no. DQ232760; http://www.ncbi.nlm.nih.gov/genbank/) and detect Streptococcus uberis adhesion molecule (SUAM) expression by Western blot using serum from naturally S. uberis-infected cows in strains of S. uberis isolated in milk from cows with mastitis from geographically diverse areas of the world. All strains evaluated yielded a 4.4-kb sua-containing PCR fragment that was subsequently sequenced. Deduced SUAM AA sequences from those S. uberis strains evaluated shared >97% identity. The pepSUAM sequence located at the N terminus of SUAM was >99% identical among strains of S. uberis. Streptococcus uberis adhesion molecule expression was detected in all strains of S. uberis tested. These results suggest that sua is ubiquitous among strains of S. uberis isolated from diverse geographic locations and that SUAM is immunogenic. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Prokaryotic expression and allergenicity assessment of hygromycin B phosphotransferase protein derived from genetically modified plants.

PubMed

Lu, Y; Xu, W; Kang, A; Luo, Y; Guo, F; Yang, R; Zhang, J; Huang, K

2007-09-01

The hygromycin B phosphotransferase gene (hpt) has been widely used in the process of plant genetic engineering to produce plants that can secrete the HPT protein. As part of a safety assessment, sufficient quantities of the protein were produced in Escherichia coli to conduct in vitro digestibility and animal studies. Western blotting analysis showed that the HPT protein was digested by simulated gastric fluid within 40 s. ELISA demonstrated that the protein did not induce detectable levels of specific IgE antibodies or histamine in test animals. Alignment of the amino acid sequence of HPT with those of known allergens did not produce evidence of sequence similarities between these allergens and the HPT protein. We conclude that HPT has a low probability to induce allergenicity.

Verification of 2A peptide cleavage.

PubMed

Szymczak-Workman, Andrea L; Vignali, Kate M; Vignali, Dario A A

2012-02-01

The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. It is now possible to express multiple proteins from a single open reading frame (ORF) using 2A peptide-linked multicistronic vectors. These small sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence. The easiest and most effective way to assess 2A cleavage is to perform transient transfection of 293T cells (human embryonic kidney cells) followed by western blot analysis, as described in this protocol. 293T cells are easy to grow and can be efficiently transfected with a variety of vectors. Cleavage can be assessed by detection with antibodies against the target proteins or anti-2A serum.

Natural and cross-inducible anti-SIV antibodies in Mauritian cynomolgus macaques

PubMed Central

Li, Hongzhao; Nykoluk, Mikaela; Li, Lin; Liu, Lewis R.; Omange, Robert W.; Soule, Geoff; Schroeder, Lukas T.; Toledo, Nikki; Kashem, Mohammad Abul; Correia-Pinto, Jorge F.; Liang, Binhua; Schultz-Darken, Nancy; Alonso, Maria J.; Whitney, James B.; Plummer, Francis A.

2017-01-01

Cynomolgus macaques are an increasingly important nonhuman primate model for HIV vaccine research. SIV-free animals without pre-existing anti-SIV immune responses are generally needed to evaluate the effect of vaccine-induced immune responses against the vaccine epitopes. Here, in order to select such animals for vaccine studies, we screened 108 naïve female Mauritian cynomolgus macaques for natural (baseline) antibodies to SIV antigens using a Bio-Plex multiplex system. The antigens included twelve 20mer peptides overlapping the twelve SIV protease cleavage sites (-10/+10), respectively (PCS peptides), and three non-PCS Gag or Env peptides. Natural antibodies to SIV antigens were detected in subsets of monkeys. The antibody reactivity to SIV was further confirmed by Western blot using purified recombinant SIV Gag and Env proteins. As expected, the immunization of monkeys with PCS antigens elicited anti-PCS antibodies. However, unexpectedly, antibodies to non-PCS peptides were also induced, as shown by both Bio-Plex and Western blot analyses, while the non-PCS peptides do not share sequence homology with PCS peptides. The presence of natural and vaccine cross-inducible SIV antibodies in Mauritian cynomolgus macaques should be considered in animal selection, experimental design and result interpretation, for their best use in HIV vaccine research. PMID:28982126

[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.

The nuclear matrix protein NMP-1 is the transcription factor YY1.

PubMed Central

Guo, B; Odgren, P R; van Wijnen, A J; Last, T J; Nickerson, J; Penman, S; Lian, J B; Stein, J L; Stein, G S

1995-01-01

NMP-1 was initially identified as a nuclear matrix-associated DNA-binding factor that exhibits sequence-specific recognition for the site IV regulatory element of a histone H4 gene. This distal promoter domain is a nuclear matrix interaction site. In the present study, we show that NMP-1 is the multifunctional transcription factor YY1. Gel-shift and Western blot analyses demonstrate that NMP-1 is immunoreactive with YY1 antibody. Furthermore, purified YY1 protein specifically recognizes site IV and reconstitutes the NMP-1 complex. Western blot and gel-shift analyses indicate that YY1 is present within the nuclear matrix. In situ immunofluorescence studies show that a significant fraction of YY1 is localized in the nuclear matrix, principally but not exclusively associated with residual nucleoli. Our results confirm that NMP-1/YY1 is a ubiquitous protein that is present in both human cells and in rat osteosarcoma ROS 17/2.8 cells. The finding that NMP-1 is identical to YY1 suggests that this transcriptional regulator may mediate gene-matrix interactions. Our results are consistent with the concept that the nuclear matrix may functionally compartmentalize the eukaryotic nucleus to support regulation of gene expression. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7479833

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.

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.

[Prokaryotic expression and immunological activity of human neutrophil gelatinase associated lipocalin].

PubMed

Wu, Jianwei; Cai, Lei; Qian, Wei; Jiao, Liyuan; Li, Jiangfeng; Song, Xiaoli; Wang, Jihua

2015-07-01

To construct a prokaryotic expression vector of human neutrophil gelatinase associated lipocalin (NGAL) and identify the bioactivity of the fusion protein. The cDNA of human NGAL obtained from GenBank was linked to a cloning vector to construct the prokaryotic expression vector pCold-NGAL. Then the vector was transformed into E.coli BL21(DE3) plysS. Under the optimal induction condition, the recombinant NGAL (rNGAL) was expressed and purified by Ni Sepharose 6 Fast Flow affinity chromatography. The purity and activity of the rNGAL were respectively identified by SDS-PAGE and Western blotting combined with NGAL reagent (Latex enhanced immunoturbidimetry). Restriction enzyme digestion and nucleotide sequencing proved that the expression vector pCold-NGAL was successfully constructed. Under the optimal induction condition that we determined, the rNGAL was expressed in soluble form in E.coli BL21(DE3) plysS. The relative molecular mass of the rNGAL was 25 000, and its purity was more than 98.0%. Furthermore, Western blotting and immunoturbidimetry indicated that the rNGAL reacted with NGAL mAb specifically. Human rNGAL of high purity and bioactivity was successfully constructed in E.coli BL21(DE3) plysS using the expression vector pCold-NGAL.

The Dermatophagoides farinae group 22 allergen: cloning and expression in Escherichia coli.

PubMed

Cui, Yu-bao; Cai, Hong-xing; Zhou, Ying; Wang, Nan; Yu, Li-li; Yang, Li; Zhang, Cheng-bo

2015-09-01

Dermatophagoides farinae (Hughes) (Acari: Pyroglyphidae) and other domestic mites produce allergens that affect people worldwide. Here, the complementary DNA (cDNA) coding for group 22 allergen of D. farinae (Der f 22) from China was cloned, sequenced, and expressed successfully. The cDNA encoding Der f 22 was synthesized by reverse transcription polymerase chain reaction (RT-PCR), then ligated to the pCold-TF for expression in Escherichia coli BL21 cells. The purified recombinant fusion protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western-blotting, and tandem matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF/TOF). The full-length cDNA comprised 468 nucleotides and was 99.57% (466/468) identical with the reference sequence (GenBank: DQ643992). After the plasmid pCold-TF-Der f 22 was transformed into E. coli BL21 and expressed with the induction of IPTG, SDS-PAGE showed a specific band for the recombinant fusion protein. The recombinant fusion protein, which was purified by chromatography, bound with a His-tagged antibody by Western blotting. MALDI-TOF/TOF mass spectrometry revealed that the structure of the recombinant protein was identical to the predicted Der f 22 structure. The hydrophilic protein contains a signal peptide of 20 amino acids, and the mature Der f 22 consists of 135 amino acid residues with a molecular weight of 14.7 kDa and theoretical isoelectric points (pI) of 6.38. Its secondary structure comprises an alpha helix (38.5%), beta-sheet (45.9%), random coils (11.85%), and beta-turns (11.1%). This work represents the first reported full-length sequence and successful cloning of Der f 22 from D. farinae in China; bioinformatics analysis can be used to further study the allergenicity and clinical utility of the recombinant Der f 22. © 2015 ARS-AAOA, LLC.

Development of an IP-Free Biotechnology Platform for Constitutive Production of HPV16 L1 Capsid Protein Using the Pichia pastoris PGK1 Promoter.

PubMed

Mariz, F C; Coimbra, E C; Jesus, A L S; Nascimento, L M; Torres, F A G; Freitas, A C

2015-01-01

The human papillomavirus (HPV) L1 major capsid protein, which forms the basis of the currently available vaccines against cervical cancer, self-assembles into virus-like particles (VLPs) when expressed heterologously. We report the development of a biotechnology platform for HPV16 L1 protein expression based on the constitutive PGK1 promoter (PPGK1) from the methylotrophic yeast Pichia pastoris. The L1 gene was cloned under regulation of PPGK1 into pPGKΔ3 expression vector to achieve intracellular expression. In parallel, secretion of the L1 protein was obtained through the use of an alternative vector called pPGKΔ3α, in which a codon optimized α-factor signal sequence was inserted. We devised a work-flow based on the detection of the L1 protein by dot blot, colony blot, and western blot to classify the positive clones. Finally, intracellular HPV VLPs assembly was demonstrated for the first time in yeast cells. This study opens up perspectives for the establishment of an innovative platform for the production of HPV VLPs or other viral antigens for vaccination purposes, based on constitutive expression in P. pastoris.

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

A single-step simultaneous protein staining procedure for polyacrylamide gels and nitrocellulose membranes by Alta during western blot analysis.

PubMed

Pal, Jayanta K; Berwal, Sunil K; Soni, Rupali N

2012-01-01

A simple method for staining of proteins simultaneously on sodium dodecyl sulfate (SDS) polyacrylamide gels and nitrocellulose membranes by Alta during western blot analysis is described. A 5% solution of Alta, a commercially available cosmetic preparation, is added in the upper tank buffer during electrophoresis. On completion of electrophoresis, the gel is washed in distilled water and viewed on a white light plate and a transilluminator to photograph the protein profiles. The gel is processed for western blot transfer of proteins onto a nitrocellulose membrane, and upon completion, the protein profiles on the membrane are viewed and photographed as stated above. The membrane can then be processed for immunostaining as per the standard procedure. Thus, the staining procedure using Alta is simple, rapid (without any need of destaining), and cost-effective.

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

Caenorhabditis elegans syndecan (SDN-1) is required for normal egg laying and associates with the nervous system and the vulva.

PubMed

Minniti, Alicia N; Labarca, Mariana; Hurtado, Claudia; Brandan, Enrique

2004-10-01

In Caenorhabditis elegans, the identification of many enzymes involved in the synthesis and modification of glycosaminoglycans (GAGs), essential components of proteoglycans, has attained special attention in recent years. Mutations in all the genes that encode for GAG biosynthetic enzymes show defects in the development of the vulva, specifically in the invagination of the vulval epithelium. Mutants for certain heparan sulfate modifying enzymes present axonal and cellular guidance defects in specific neuronal classes. Although most of the enzymes involved in the biosynthesis and modification of heparan sulfate have been characterized in C. elegans, little is known regarding the core proteins to which these GAGs covalently bind in proteoglycans. A single syndecan homologue (sdn-1) has been identified in the C. elegans genome through sequence analysis. In the present study, we show that C. elegans synthesizes sulfated proteoglycans, seen as three distinct species in western blot analysis. In the sdn-1 (ok449) deletion mutant allele we observed the lack of one species, which corresponds to a 50 kDa product after heparitinase treatment. The expression of sdn-1 mRNA and sequencing revealed that sdn-1 (ok449) deletion mutants lack two glycosylation sites. Hence, the missing protein in the western blot analysis probably corresponds to SDN-1. In addition, we show that SDN-1 localizes to the C. elegans nerve ring, nerve cords and to the vulva. SDN-1 is found specifically phosphorylated in nerve ring neurons and in the vulva, in both wild-type worms and sdn-1 (ok449) deletion mutants. These mutants show a defective egg-laying phenotype. Our results show for the first time, the identification, localization and some functional aspects of syndecan in the nematode C. elegans.

Familial Mediterranean fever with a single MEFV mutation: where is the second hit?

PubMed

Booty, Matthew G; Chae, Jae Jin; Masters, Seth L; Remmers, Elaine F; Barham, Beverly; Le, Julie M; Barron, Karyl S; Holland, Steve M; Kastner, Daniel L; Aksentijevich, Ivona

2009-06-01

Familial Mediterranean fever (FMF) has traditionally been considered an autosomal-recessive disease; however, it has been observed that a substantial number of patients with clinical FMF possess only 1 demonstrable MEFV mutation. The purpose of this study was to perform an extensive search for a second MEFV mutation in 46 patients diagnosed clinically as having FMF and carrying only 1 high-penetrance FMF mutation. MEFV and other candidate genes were sequenced by standard capillary electrophoresis. In 10 patients, the entire 15-kb MEFV genomic region was resequenced using hybridization-based chip technology. MEFV gene expression levels were determined by quantitative reverse transcription-polymerase chain reaction. Pyrin protein levels were examined by Western blotting. A second MEFV mutation was not identified in any of the patients who were screened. Haplotype analysis did not identify a common haplotype that might be associated with the transmission of a second FMF allele. Western blots did not demonstrate a significant difference in pyrin levels between patients with a single mutation and those with a double mutation; however, FMF patients of both types showed higher protein expression as compared with controls and with non-FMF patients with active inflammation. Screening of genes encoding pyrin-interacting proteins identified rare mutations in a small number of patients, suggesting the possibility of digenic inheritance. Our data underscore the existence of a significant subset of FMF patients who are carriers of only 1 MEFV mutation and demonstrate that complete MEFV sequencing is not likely to yield a second mutation. Screening for the set of the most common mutations and detection of a single mutation appears to be sufficient in the presence of clinical symptoms for the diagnosis of FMF and the initiation of a trial of colchicine.

[Construction of Plasmodium falciparum signal peptide peptidase-GFP mutant and its expression analysis in the malaria parasite].

PubMed

Li, Xue-rong; Wu, Yin-juan; Shang, Mei; Li, Ye; Xu, Jin; Yu, Xin-bing; Athar, Chishti

2014-08-01

To construct recombinant plasmid pSPPcGT which contains signal peptide peptidase gene of Plasmodium falciparum (PJSPP) and GFP, and transfect into P. falciparum (3D7 strain) to obtain mutant parasites which can express PJSPP-GFP. Plasmodium falciparum(3D7 strain) genomic DNA was extracted from cultured malaria parasites. The C-terminal region of PJSPP, an 883 bp gene fragment was amplified by PCR, and then cloned into pPM2GT vector to get recombinant vector pSPPcGT. The recombinant vectors were identified by PCR, double restriction enzyme digestion and DNA sequencing. pSPPcGT vector was transfected into malaria parasites. The positive clones were selected by adding inhibitor of Plasmodium falciparum dihydrofolate reductase WR99210 to the culture medium. The pSPP-GFP-transfected parasites were fixed with methanol, stained with DAPI, and observed under immunofluorescence microscope. The PJSPP-GFP expression in P. falciparum was identified by SDS-PAGE and Western blotting. The C-terminal region of PJSPP was amplified from P.falciparum (3D7 strain) genomic DNA by PCR with the length of 883 bp. The constructed recombinant vectors were identified by PCR screening, double restriction enzyme digestion and DNA sequencing. The pSPPcGT vector was transfected into P. falciparum and the positive clones were selected by WR99210. GFP fluorescence was observed in transfected parasites by immunofluorescence microscopy, and mainly located in the cytoplasm. The PJSPP-GFP expression in malaria parasites was confirmed by Western blotting with a relative molecular mass of Mr 64,000. Recombinant vector PJSPP-GFP is constructed and transfected into P. falciparum to obtain P. falciparum mutant clone which can express PfSPP-GFP.

Sera of children with hepatitis C infection and anti-liver-kidney microsome-1 antibodies recognize different CYP2D6 epitopes than adults with LKM+/HCV+ sera.

PubMed

Herzog, D; Yamamoto, A M; Jara, P; Maggiore, G; Sarles, J; Alvarez, F

1999-11-01

Liver-kidney microsome type 1 (LKM1) antibodies are specific markers of autoimmune hepatitis (AIH) type 2. Antibodies to LKM1 have been found in 2% to 3% of adults infected with hepatitis C virus (HCV) without AIH. Thirty percent of these antibodies are directed against linear sequences of CYP2D6 protein. LKM1 antibodies in HCV+/LKM1+ sera and in sera of AIH patients do not recognize the same CYP2D6 epitopes. The current study was conducted to determine whether LKM1 antibodies in HCV+/LKM1+ children's sera are the result of the same immune response as the antibodies described in AIH type 2 and in HCV+/LKM1+ adult patients. Sera from 10 HCV+/LKM1+ children were tested against human liver microsomal and cytosolic proteins by Western blot analysis and against synthetic peptides of the CYP2D6 sequence between amino acids 200 and 429 by dot blot. The same sera were tested against radiolabeled CYP2D6 by immunoprecipitation. Four of 10 sera tested by Western blot analysis showed immunoglobulin (Ig) G-type antibodies against CYP2D6, and 2 had antibodies against proteins of 58, 66, and 84 kDa. One of the sera also contained IgM-type anti-66-kDa and 84-kDa proteins. The radioligand test detected anti-CYP2D6 antibodies in 9 of 10 patients. Five of the anti-CYP2D6-positive sera recognized a peptide between amino acids 200 and 429 including amino acids 254-271. Most HCV+/LKM1+ sera from children recognize conformational epitopes of the CYP2D6 antigen, and half recognize linear epitopes. Some HCV+/LKM1+ sera demonstrated antibodies against the AIH type 2 main antigenic site of the CYP2D6. Screening of HCV RNA should be performed before starting treatment of presumed autoimmune hepatitis associated with LKM1.

Reexamination of human T cell lymphotropic virus (HTLV-I/II) prevalence.

PubMed

Zucker-Franklin, D; Pancake, B A; Marmor, M; Legler, P M

1997-06-10

In the United States, blood donors are being screened for infection with human T cell lymphotropic viruses I and II (HTLV-I/II) by serologic means, which detect antibodies to the structural proteins of these viruses. Because patients with mycosis fungoides (MF) usually do not have such antibodies even though their cells harbor HTLV-I Tax and/or pol proviral sequences, it was questioned whether the prevalence of HTLV infection among healthy blood donors may also be underestimated by current means of testing. To examine this possibility, a study on specimens of relatives of mycosis fungoides patients (MFR) was begun. In addition, to collect data more expeditiously, a cohort of former injection drug users (IDUs) was tested by routine serologic methods, as well as by PCR/Southern blot analysis for Tax, pol, and gag proviral sequences and Western blot analysis for antibodies to the Tax gene product. To date, 6/8 MFRs and 42/81 (51.8%) of HIV-negative IDUs proved to be positive for HTLV, whereas routine serology identified none of the MFR and only 18/81 (22.2%) of the IDUs. Among the latter test subjects, the incidence of HTLV-I also proved to be 10 times higher than expected. Therefore, it is likely that among healthy blood donors infection with HTLV-I/II is more prevalent than is currently assumed. Since Tax is the transforming sequence of HTLV-I/II, testing for Tax sequences and antibodies to its gene product may be desirable in blood transfusion and tissue donor facilities.

Reexamination of human T cell lymphotropic virus (HTLV-I/II) prevalence

PubMed Central

Zucker-Franklin, Dorothea; Pancake, Bette A.; Marmor, Michael; Legler, Patricia M.

1997-01-01

In the United States, blood donors are being screened for infection with human T cell lymphotropic viruses I and II (HTLV-I/II) by serologic means, which detect antibodies to the structural proteins of these viruses. Because patients with mycosis fungoides (MF) usually do not have such antibodies even though their cells harbor HTLV-I Tax and/or pol proviral sequences, it was questioned whether the prevalence of HTLV infection among healthy blood donors may also be underestimated by current means of testing. To examine this possibility, a study on specimens of relatives of mycosis fungoides patients (MFR) was begun. In addition, to collect data more expeditiously, a cohort of former injection drug users (IDUs) was tested by routine serologic methods, as well as by PCR/Southern blot analysis for Tax, pol, and gag proviral sequences and Western blot analysis for antibodies to the Tax gene product. To date, 6/8 MFRs and 42/81 (51.8%) of HIV-negative IDUs proved to be positive for HTLV, whereas routine serology identified none of the MFR and only 18/81 (22.2%) of the IDUs. Among the latter test subjects, the incidence of HTLV-I also proved to be 10 times higher than expected. Therefore, it is likely that among healthy blood donors infection with HTLV-I/II is more prevalent than is currently assumed. Since Tax is the transforming sequence of HTLV-I/II, testing for Tax sequences and antibodies to its gene product may be desirable in blood transfusion and tissue donor facilities. PMID:9177230

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

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.

Purification and immunolocalization of an annexin-like protein in pea seedlings

NASA Technical Reports Server (NTRS)

Clark, G. B.; Dauwalder, M.; Roux, S. J.

1992-01-01

As part of a study to identify potential targets of calcium action in plant cells, a 35-kDa, annexin-like protein was purified from pea (Pisum sativum L.) plumules by a method used to purify animal annexins. This protein, called p35, binds to a phosphatidylserine affinity column in a calcium-dependent manner and binds 45Ca2+ in a dot-blot assay. Preliminary sequence data confirm a relationship for p35 with the annexin family of proteins. Polyclonal antibodies have been raised which recognize p35 in Western and dot blots. Immunofluorescence and immunogold techniques were used to study the distribution and subcellular localization of p35 in pea plumules and roots. The highest levels of immunostain were found in young developing vascular cells producing wall thickenings and in peripheral root-cap cells releasing slime. This localization in cells which are actively involved in secretion is of interest because one function suggested for the animal annexins is involvement in the mediation of exocytosis.

Factor IX expression in skeletal muscle of a severe hemophilia B patient 10 years after AAV-mediated gene transfer.

PubMed

Buchlis, George; Podsakoff, Gregory M; Radu, Antonetta; Hawk, Sarah M; Flake, Alan W; Mingozzi, Federico; High, Katherine A

2012-03-29

In previous work we transferred a human factor IX-encoding adeno-associated viral vector (AAV) into skeletal muscle of men with severe hemophilia B. Biopsy of injected muscle up to 1 year after vector injection showed evidence of gene transfer by Southern blot and of protein expression by IHC and immunofluorescent staining. Although the procedure appeared safe, circulating F.IX levels remained subtherapeutic (< 1%). Recently, we obtained muscle tissue from a subject injected 10 years earlier who died of causes unrelated to gene transfer. Using Western blot, IHC, and immunofluorescent staining, we show persistent factor IX expression in injected muscle tissue. F.IX transcripts were detected in injected skeletal muscle using RT-PCR, and isolated whole genomic DNA tested positive for the presence of the transferred AAV vector sequence. This is the longest reported transgene expression to date from a parenterally administered AAV vector, with broad implications for the future of muscle-directed gene transfer.

Factor IX expression in skeletal muscle of a severe hemophilia B patient 10 years after AAV-mediated gene transfer

PubMed Central

Buchlis, George; Podsakoff, Gregory M.; Radu, Antonetta; Hawk, Sarah M.; Flake, Alan W.; Mingozzi, Federico

2012-01-01

In previous work we transferred a human factor IX–encoding adeno-associated viral vector (AAV) into skeletal muscle of men with severe hemophilia B. Biopsy of injected muscle up to 1 year after vector injection showed evidence of gene transfer by Southern blot and of protein expression by IHC and immunofluorescent staining. Although the procedure appeared safe, circulating F.IX levels remained subtherapeutic (< 1%). Recently, we obtained muscle tissue from a subject injected 10 years earlier who died of causes unrelated to gene transfer. Using Western blot, IHC, and immunofluorescent staining, we show persistent factor IX expression in injected muscle tissue. F.IX transcripts were detected in injected skeletal muscle using RT-PCR, and isolated whole genomic DNA tested positive for the presence of the transferred AAV vector sequence. This is the longest reported transgene expression to date from a parenterally administered AAV vector, with broad implications for the future of muscle-directed gene transfer. PMID:22271447

Molecular characterization and expression of the M6 gene of grass carp hemorrhage virus (GCHV), an aquareovirus.

PubMed

Qiu, T; Lu, R H; Zhang, J; Zhu, Z Y

2001-07-01

The complete nucleotide sequence of M6 gene of grass carp hemorrhage virus (GCHV) was determined. It is 2039 nucleotides in length and contains a single large open reading frame that could encode a protein of 648 amino acids with predicted molecular mass of 68.7 kDa. Amino acid sequence comparison revealed that the protein encoded by GCHV M6 is closely related to the protein mu1 of mammalian reovirus. The M6 gene, encoding the major outer-capsid protein, was expressed using the pET fusion protein vector in Escherichia coli and detected by Western blotting using chicken anti-GCHV immunoglobulin (IgY). The result indicates that the protein encoded by M6 may share a putative Asn-42-Pro-43 proteolytic cleavage site with mu1.

Research in Undergraduate Instruction: A Biotech Lab Project for Recombinant DNA Protein Expression in Bacteria

NASA Astrophysics Data System (ADS)

Brockman, Mark; Ordman, Alfred B.; Campbell, A. Malcolm

1996-06-01

In the sophomore-level Molecular Biology and Biotechnology course at Beloit College, students learn basic methods in molecular biology in the context of pursuing a semester-long original research project. We are exploring how DNA sequence affects expression levels of proteins. A DNA fragment encoding all or part of the guanylate monokinase (gmk) sequence is cloned into pSP73 and expressed in E. coli. A monoclonal antibody is made to gmk. The expression level of gmk is determined by SDS gel elctrophoresis, a Western blot, and an ELISA assay. Over four years, an increase in enrollment in the course from 9 to 34 students, the 85% of majors pursuing advanced degrees, and course evaluations all support the conclusion that involving students in research during undergraduate courses encourages them to pursue careers in science.

[Cloning, expression and transcriptional analysis of biotin carboxyl carrier protein gene (accA) from Amycolatopsis mediterranei U32 ].

PubMed

Lu, Jie; Yao, Yufeng; Jiang, Weihong; Jiao, Ruishen

2003-02-01

Acetyl CoA carboxylase (EC 6.4.1.2, ACC) catalyzes the ATP-dependent carboxylation of acetyl CoA to yield malonyl CoA, which is the first committed step in fatty acid synthesis. A pair of degenerate PCR primers were designed according to the conserved amino acid sequence of AccA from M. tuberculosis and S. coelicolor. The product of the PCR amplification, a DNA fragment of 250bp was used as a probe for screening the U32 genomic cosmid library and its gene, accA, coding the biotinylated protein subunit of acetyl CoA carboxylase, was successfully cloned from U32. The accA ORF encodes a 598-amino-acid protein with the calculated molecular mass of 63.7kD, with 70.1% of G + C content. A typical Streptomyces RBS sequence, AGGAGG, was found at the - 6 position upstream of the start codon GTG. Analysis of the deduced amino acid sequence showed the presence of biotin-binding site and putative ATP-bicarbonate interaction region, which suggested the U32 AccA may act as a biotin carboxylase as well as a biotin carrier protein. Gene accA was then cloned into the pET28 (b) vector and expressed solubly in E. coli BL21 (DE3) by 0.1 mmol/L IPTG induction. Western blot confirmed the covalent binding of biotin with AccA. Northern blot analyzed transcriptional regulation of accA by 5 different nitrogen sources.

[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.

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)

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

Interferon-gamma of the giant panda (Ailuropoda melanoleuca): complementary DNA cloning, expression, and phylogenetic analysis.

PubMed

Tao, Yaqiong; Zeng, Bo; Xu, Liu; Yue, Bisong; Yang, Dong; Zou, Fangdong

2010-01-01

Interferon-gamma (IFN-gamma) is the only member of type II IFN and is vital in the regulation of immune and inflammatory responses. Herein we report the cloning, expression, and sequence analysis of IFN-gamma from the giant panda (Ailuropoda melanoleuca). The open reading frame of this gene is 501 base pair in length and encodes a polypeptide consisting of 166 amino acids. All conserved N-linked glycosylation sites and cysteine residues among carnivores were found in the predicted amino acid sequence of the giant panda. Recombinant giant panda IFN-gamma with a V5 epitope and polyhistidine tag was expressed in HEK293 host cells and confirmed by Western blotting. Phylogenetic analysis of mammalian IFN-gamma-coding sequences indicated that the giant panda IFN-gamma was closest to that of carnivores, then to ungulates and dolphin, and shared a distant relationship with mouse and human. These results represent a first step into the study of IFN-gamma in giant panda.

Myostatin inhibitors in sports drug testing: Detection of myostatin-neutralizing antibodies in plasma/serum by affinity purification and Western blotting.

PubMed

Walpurgis, Katja; Thomas, Andreas; Schänzer, Wilhelm; Thevis, Mario

2016-02-01

Myostatin is a key regulator of skeletal muscle growth and inhibition of its signaling pathway results in an increased muscle mass and function. The aim of this study was to develop a qualitative detection assay for myostatin-neutralizing antibodies for doping control purposes by using immunological approaches. To detect different types of myostatin-neutralizing antibodies irrespective of their amino acid sequence, an immunological assay specific for antibodies directed against myostatin and having a human Fc domain was established. Affinity purification and Western blotting strategies were combined to allow extracting and identifying relevant analytes from 200 μL of plasma/serum in a non-targeted approach. The assay was characterized regarding specificity, linearity, precision, robustness, and recovery. The assay was found to be highly specific, robust, and linear from 0.1 to 1 μg/mL. The precision was successfully specified at three different concentrations and the recovery of the affinity purification was 58%. Within this study, an immunological detection assay for myostatin-neutralizing antibodies present in plasma/serum specimens was developed and successfully characterized. The presented approach can easily be modified to include other therapeutic antibodies and serves as proof-of-concept for the detection of antibody-based myostatin inhibitors in doping control samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

[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.

[Construction and expression of recombinant human serum albumin-EPO fusion protein].

PubMed

Huang, Ying-Chun; Gou, Xing-Hua; Han, Lei; Li, De-Hua; Zhao, Lan-Ying; Wu, Qia-Qing

2011-05-01

OBJECTIVE To construct the recombinant plasmid pCI-HLE encoding human serum album-EPO (HSA-EPO) fusion protein and to express it in CHO cell. The cDNA encoding human serum album and EPO were amplified by PCR, and then spliced with the synsitic DNA fragment encoding GS (GGGGS), by overlap PCR extension to form LEPO. After BamH I digestion, the HSA and LEPO was ligated to generate the fusion HSA-EPO gene and was then cloned into the expression vector pCI-neo to generate the recombinant plasmid pCI-HLE. The plasmid pCI-HLE was transfected into CHO cell by liposome protocol. Then, the recombinant cells were screened by G418 and identified by PCR and Western blot. Expression of fusion protein was evaluated by Enzyme Linked Immunosorbent Assay (ELISA). Restrictive enzymes digestion and DNA sequencing revealed that HSA-EPO fusion gene was cloned into expression vector pCI-neo successfully. PCR and Western blot analysis confirmed that the fusion gene was integrated in the genome of CHO cells and expressed successfully. The HSA-EPO production varied from 86 Iu/(mL x 10(6) x 72 h) to 637 IU/(mLx 10(6) x 72 h). The results confirmed that HSA-EPO fusion gene can be expressed in the CHO cells, with EPO immunogenicity, which could serve as foundation for the development of long-lasting recombinant HSA-EPO protein.

[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.

Sequestration of cAMP response element-binding proteins by transcription factor decoys causes collateral elaboration of regenerating Aplysia motor neuron axons.

PubMed

Dash, P K; Tian, L M; Moore, A N

1998-07-07

Axonal injury increases intracellular Ca2+ and cAMP and has been shown to induce gene expression, which is thought to be a key event for regeneration. Increases in intracellular Ca2+ and/or cAMP can alter gene expression via activation of a family of transcription factors that bind to and modulate the expression of CRE (Ca2+/cAMP response element) sequence-containing genes. We have used Aplysia motor neurons to examine the role of CRE-binding proteins in axonal regeneration after injury. We report that axonal injury increases the binding of proteins to a CRE sequence-containing probe. In addition, Western blot analysis revealed that the level of ApCREB2, a CRE sequence-binding repressor, was enhanced as a result of axonal injury. The sequestration of CRE-binding proteins by microinjection of CRE sequence-containing plasmids enhanced axon collateral formation (both number and length) as compared with control plasmid injections. These findings show that Ca2+/cAMP-mediated gene expression via CRE-binding transcription factors participates in the regeneration of motor neuron axons.

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.

Line scanning system for direct digital chemiluminescence imaging of DNA sequencing blots

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

Karger, A.E.; Weiss, R.; Gesteland, R.F.

A cryogenically cooled charge-coupled device (CCD) camera equipped with an area CCD array is used in a line scanning system for low-light-level imaging of chemiluminescent DNA sequencing blots. Operating the CCD camera in time-delayed integration (TDI) mode results in continuous data acquisition independent of the length of the CCD array. Scanning is possible with a resolution of 1.4 line pairs/mm at the 50% level of the modulation transfer function. High-sensitivity, low-light-level scanning of chemiluminescent direct-transfer electrophoresis (DTE) DNA sequencing blots is shown. The detection of DNA fragments on the blot involves DNA-DNA hybridization with oligonucleotide-alkaline phosphatase conjugate and 1,2-dioxetane-based chemiluminescence.more » The width of the scan allows the recording of up to four sequencing reactions (16 lanes) on one scan. The scan speed of 52 cm/h used for the sequencing blots corresponds to a data acquisition rate of 384 pixels/s. The chemiluminescence detection limit on the scanned images is 3.9 [times] 10[sup [minus]18] mol of plasmid DNA. A conditional median filter is described to remove spikes caused by cosmic ray events from the CCD images. 39 refs., 9 refs.« less

Role of Obesity in Prostate Cancer Development

DTIC Science & Technology

2011-04-01

Western blot analysis . Representative staining from the immunohistochemistry is shown in Figure 6. Expression of AdipoR1 was found in all prostate tumor...with goat serum instead of primary antibody was negative (Fig 6D). Western blot analysis of frozen tissue from the same mice was also performed and...TRAMP) model. Cancer Res., 57, 3325-3330. 41. Williams,T.M., Hassan,G.S., Li,J., Cohen,A.W., Medina,F., Frank,P.G., Pestell ,R.G., Di Vizio,D., and

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

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.

17{beta}-Hydroxysteroid dehydrogenase type 13 is a liver-specific lipid droplet-associated protein

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

Horiguchi, Yuka; Araki, Makoto; Motojima, Kiyoto

2008-05-30

17{beta}-Hydroxysteroid dehydrogenase (17{beta}HSD) type 13 is identified as a new lipid droplet-associated protein. 17{beta}HSD type 13 has an N-terminal sequence similar to that of 17{beta}HSD type 11, and both sequences function as an endoplasmic reticulum and lipid droplet-targeting signal. Localization of native 17{beta}HSD type 13 on the lipid droplets was confirmed by subcellular fractionation and Western blotting. In contrast to 17{beta}HSD type 11, however, expression of 17{beta}HSD type 13 is largely restricted to the liver and is not enhanced by peroxisome proliferator-activated receptor {alpha} and its ligand. Instead the expression level of 17{beta}HSD type 13 in the receptor-null mice wasmore » increased several-fold. 17{beta}HSD type 13 may have a distinct physiological role as a lipid droplet-associated protein in the liver.« less

HIV type 1 diversity in the Seychelles.

PubMed

Razafindratsimandresy, Richter; Hollanda, Justina; Soares, Jean-Louis; Rousset, Dominique; Chetty, Agnes P; Reynes, Jean-Marc

2007-06-01

Subtype determination and drug resistance-associated mutations (DRM) detection were performed on 40 HIV-1 Western blot-positive sera detected, obtained from consecutive patients resident in the Seychelles and consulting the Communicable Disease Control Unit, HIV reference center, in Victoria Hospital (Mahe) from October 2005 to June 2006. Amplification and sequencing of at least two of the partial reverse transcriptase, protease, and partial envelope genes were successful for all strains. All three genes sequences were obtained for 39 strains. A high degree of subtype or circulating recombinant forms (CRF) was observed for these 39 strains: A-A1 (17 cases), C (10 cases), B (8 cases), CRF02_AG (2 cases), D (1 case) and CRF01_AE (1 case). According to the ANRS 2006 DRM list and algorithm, none of the 40 isolates was found to be resistant to any protease or reverse transcriptase inhibitors.

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.

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.

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.

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

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.

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®

Analysis of the complete genome of peach chlorotic mottle virus: identification of non-AUG start codons, in vitro coat protein expression, and elucidation of serological cross-reactions.

PubMed

James, D; Varga, A; Croft, H

2007-01-01

The entire genome of peach chlorotic mottle virus (PCMV), originally identified as Prunus persica cv. Agua virus (4N6), was sequenced and analysed. PCMV cross-reacts with antisera to diverse viruses, such as plum pox virus (PPV), genus Potyvirus, family Potyviridae; and apple stem pitting virus (ASPV), genus Foveavirus, family Flexiviridae. The PCMV genome consists of 9005 nucleotides (nts), excluding a poly(A) tail at the 3' end of the genome. Five open reading frames (ORFs) were identified with four untranslated regions (UTR) including a 5', a 3', and two intergenic UTRs. The genome organisation of PCMV is similar to that of ASPV and the two genomes share a nucleotide (nt) sequence identity of 58%. PCMV ORF1 encodes the replication-associated protein complex (Mr 241,503), ORF2-ORF4 code for the triple gene block proteins (TGBp; Mr 24,802, 12,370, and 7320, respectively), and ORF5 encodes the coat protein (CP) (Mr 42,505). Two non-AUG start codons participate in the initiation of translation: 35AUC and 7676AUA initiate translation of ORF1 and ORF5. In vitro expression with subsequent Western blot analysis confirmed ORF5 as the CP-encoding gene and confirmed that the codon AUA is able to initiate translation of the CP. Expression of a truncated CP fragment (Mr 39, 689) was demonstrated, and both proteins are expressed in vivo, since both were observed in Western blot analysis of PCMV-infected peach and Nicotiana occidentalis. The expressed proteins cross-reacted with an antiserum against ASPV. The amino acid sequences of the CPs of PCMV and ASPV CP share only 37% identity, but there are 11 shared peptides 4-8 aa residues long. These may constitute linear epitopes responsible for ASPV antiserum cross reactions. No significant common linear epitopes were associated with PPV. Extensive phylogenetic analysis indicates that PCMV is closely related to ASPV and is a new and distinct member of the genus Foveavirus.

Identification of the gene encoding a 38-kilodalton immunogenic and protective antigen of Streptococcus suis.

PubMed

Okwumabua, Ogi; Chinnapapakkagari, Sharmila

2005-04-01

In our continued effort to search for a Streptococcus suis protein(s) that can serve as a vaccine candidate or a diagnostic reagent, we constructed and screened a gene library with a polyclonal antibody raised against the whole-cell protein of S. suis type 2. A clone that reacted with the antibody was identified and characterized. Analysis revealed that the gene encoding the protein is localized within a 2.0-kbp EcoRI DNA fragment. The nucleotide sequence contained an open reading frame that encoded a polypeptide of 445 amino acid residues with a calculated molecular mass of 46.4 kDa. By in vitro protein synthesis and Western blot experiments, the protein exhibited an electrophoretic mobility of approximately 38 kDa. At the amino acid level the deduced primary sequence shared homology with sequences of unknown function from Streptococcus pneumoniae (89%), Streptococcus mutans (86%), Lactococcus lactis (80%), Listeria monocytogenes (74%), and Clostridium perfringens (64%). Except for strains of serotypes 20, 26, 32, and 33, Southern hybridization analysis revealed the presence of the gene in strains of other S. suis serotypes and demonstrated restriction fragment length differences caused by a point mutation in the EcoRI recognition sequence. We confirmed expression of the 38-kDa protein in the hybridization-positive isolates using specific antiserum against the purified protein. The recombinant protein was reactive with serum from pigs experimentally infected with virulent strains of S. suis type 2, suggesting that the protein is immunogenic and may serve as an antigen of diagnostic importance for the detection of most S. suis infections. Pigs immunized with the recombinant 38-kDa protein mounted antibody responses to the protein and were completely protected against challenge with a strain of a homologous serotype, the wild-type virulent strain of S. suis type 2, suggesting that it may be a good candidate for the development of a vaccine that can be used as protection against S. suis infection. Analysis of the cellular fractions of the bacterium by Western blotting revealed that the protein was present in the surface and cell wall extracts. The functional role of the protein with respect to pathogenesis and whether antibodies against the antigen confer protective immunity against diseases caused by strains of other pathogenic S. suis capsular types remains to be determined.

Identification and characterization of avian retroviruses in chicken embryo-derived yellow fever vaccines: investigation of transmission to vaccine recipients.

PubMed

Hussain, Althaf I; Johnson, Jeffrey A; Da Silva Freire, Marcos; Heneine, Walid

2003-01-01

All currently licensed yellow fever (YF) vaccines are propagated in chicken embryos. Recent studies of chick cell-derived measles and mumps vaccines show evidence of two types of retrovirus particles, the endogenous avian retrovirus (EAV) and the endogenous avian leukosis virus (ALV-E), which originate from the chicken embryonic fibroblast substrates. In this study, we investigated substrate-derived avian retrovirus contamination in YF vaccines currently produced by three manufacturers (YF-vax [Connaught Laboratories], Stamaril [Aventis], and YF-FIOCRUZ [FIOCRUZ-Bio-Manguinhos]). Testing for reverse transcriptase (RT) activity was not possible because of assay inhibition. However, Western blot analysis of virus pellets with anti-ALV RT antiserum detected three distinct RT proteins in all vaccines, indicating that more than one source is responsible for the RTs present in the vaccines. PCR analysis of both chicken substrate DNA and particle-associated RNA from the YF vaccines showed no evidence of the long terminal repeat sequences of exogenous ALV subgroups A to D in any of the vaccines. In contrast, both ALV-E and EAV particle-associated RNA were detected at equivalent titers in each vaccine by RT-PCR. Quantitative real-time RT-PCR revealed 61,600, 348,000, and 1,665,000 ALV-E RNA copies per dose of Stamaril, YF-FIOCRUZ, and YF-vax vaccines, respectively. ev locus-specific PCR testing of the vaccine-associated chicken substrate DNA was positive both for the nondefective ev-12 locus in two vaccines and for the defective ev-1 locus in all three vaccines. Both intact and ev-1 pol sequences were also identified in the particle-associated RNA. To investigate the risks of transmission, serum samples from 43 YF vaccine recipients were studied. None of the samples were seropositive by an ALV-E-based Western blot assay or had detectable EAV or ALV-E RNA sequences by RT-PCR. YF vaccines produced by the three manufacturers all have particles containing EAV genomes and various levels of defective or nondefective ALV-E sequences. The absence of evidence of infection with ALV-E or EAV in 43 YF vaccine recipients suggests low risks for transmission of these viruses, further supporting the safety of these vaccines.

Structural analysis, plastid localization, and expression of the biotin carboxylase subunit of acetyl-coenzyme A carboxylase from tobacco.

PubMed

Shorrosh, B S; Roesler, K R; Shintani, D; van de Loo, F J; Ohlrogge, J B

1995-06-01

Acetyl-coenzyme A carboxylase (ACCase, EC 6.4.1.2) catalyzes the synthesis of malonyl-coenzyme A, which is utilized in the plastid for de novo fatty acid synthesis and outside the plastid for a variety of reactions, including the synthesis of very long chain fatty acids and flavonoids. Recent evidence for both multifunctional and multisubunit ACCase isozymes in dicot plants has been obtained. We describe here the isolation of a tobacco (Nicotiana tabacum L. cv bright yellow 2 [NT1]) cDNA clone (E3) that encodes a 58.4-kD protein that shares 80% sequence similarity and 65% identity with the Anabaena biotin carboxylase subunit of ACCase. Similar to other biotin carboxylase subunits of acetyl-CoA carboxylase, the E3-encoded protein contains a putative ATP-binding motif but lacks a biotin-binding site (methionine-lysine-methionine or methionine-lysine-leucine). The deduced protein sequence contains a putative transit peptide whose function was confirmed by its ability to direct in vitro chloroplast uptake. The subcellular localization of this biotin carboxylase has also been confirmed to be plastidial by western blot analysis of pea (Pisum sativum), alfalfa (Medicago sativa L.), and castor (Ricinus communis L.) plastid preparations. Northern blot analysis indicates that the plastid biotin carboxylase transcripts are expressed at severalfold higher levels in castor seeds than in leaves.

Structural analysis, plastid localization, and expression of the biotin carboxylase subunit of acetyl-coenzyme A carboxylase from tobacco.

PubMed Central

Shorrosh, B S; Roesler, K R; Shintani, D; van de Loo, F J; Ohlrogge, J B

1995-01-01

Acetyl-coenzyme A carboxylase (ACCase, EC 6.4.1.2) catalyzes the synthesis of malonyl-coenzyme A, which is utilized in the plastid for de novo fatty acid synthesis and outside the plastid for a variety of reactions, including the synthesis of very long chain fatty acids and flavonoids. Recent evidence for both multifunctional and multisubunit ACCase isozymes in dicot plants has been obtained. We describe here the isolation of a tobacco (Nicotiana tabacum L. cv bright yellow 2 [NT1]) cDNA clone (E3) that encodes a 58.4-kD protein that shares 80% sequence similarity and 65% identity with the Anabaena biotin carboxylase subunit of ACCase. Similar to other biotin carboxylase subunits of acetyl-CoA carboxylase, the E3-encoded protein contains a putative ATP-binding motif but lacks a biotin-binding site (methionine-lysine-methionine or methionine-lysine-leucine). The deduced protein sequence contains a putative transit peptide whose function was confirmed by its ability to direct in vitro chloroplast uptake. The subcellular localization of this biotin carboxylase has also been confirmed to be plastidial by western blot analysis of pea (Pisum sativum), alfalfa (Medicago sativa L.), and castor (Ricinus communis L.) plastid preparations. Northern blot analysis indicates that the plastid biotin carboxylase transcripts are expressed at severalfold higher levels in castor seeds than in leaves. PMID:7610168

Nucleotide sequence of the coat protein gene of Lettuce big-vein virus.

PubMed

Sasaya, T; Ishikawa, K; Koganezawa, H

2001-06-01

A sequence of 1425 nt was established that included the complete coat protein (CP) gene of Lettuce big-vein virus (LBVV). The LBVV CP gene encodes a 397 amino acid protein with a predicted M(r) of 44486. Antisera raised against synthetic peptides corresponding to N-terminal or C-terminal parts of the LBVV CP reacted in Western blot analysis with a protein with an M(r) of about 48000. RNA extracted from purified particles of LBVV by using proteinase K, SDS and phenol migrated in gels as two single-stranded RNA species of approximately 7.3 kb (ss-1) and 6.6 kb (ss-2). After denaturation by heat and annealing at room temperature, the RNA migrated as four species, ss-1, ss-2 and two additional double-stranded RNAs (ds-1 and ds-2). The Northern blot hybridization analysis using riboprobes from a full-length clone of the LBVV CP gene indicated that ss-2 has a negative-sense nature and contains the LBVV CP gene. Moreover, ds-2 is a double-stranded form of ss-2. Database searches showed that the LBVV CP most resembled the nucleocapsid proteins of rhabdoviruses. These results indicate that it would be appropriate to classify LBVV as a negative-sense single-stranded RNA virus rather than as a double-stranded RNA virus.

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

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

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

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

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.

Gene within gene configuration and expression of the Drosophila melanogaster genes lethal(2) neighbour of tid [l(2)not] and lethal(2) relative of tid[l(2)rot].

PubMed

Kurzik-Dumke, U; Kaymer, M; Gundacker, D; Debes, A; Labitzke, K

1997-10-24

In this paper, we describe the structure and temporal expression pattern of the Drosophila melanogaster genes l(2)not and l(2)rot located at locus 59F5 vis à vis the tumor suppressor gene l(2)tid described previously and exhibiting a gene within gene configuration. The l(2)not protein coding region, 1530 nt, is divided into two exons by an intron, 2645 nt, harboring the genes l(2)rot, co-transcribed from the same DNA strand, and l(2)tid, co-transcribed from the opposite DNA strand, located vis à vis. To determine proteins encoded by the genes described in this study polyclonal rabbit antibodies (Ab), anti-Not and anti-Rot, were generated. Immunostaining of developmental Western blots with the anti-Not Ab resulted in the identification of a 45-kDa protein, Not45, which is smaller than the Not56 protein predicted from the sequence. Its localization in endoplasmic reticulum (ER) was established by immunoelectron microscopy of Drosophila melanogaster Schneider 2 cells. Not45 shows significant homology to yeast ALG3 protein acting as a dolichol mannosyltransferase in the asparagine-linked glycosylation. It is synthesized ubiquitously throughout embryonic life. The protein predicted from the l(2)rot sequence, Rot57, shows a homology to the NS2B protein of the yellow fever virus1 (yefv1). The results of l(2)rot RNA analysis by developmental Northern blot and by in situ RNA localization, as well as the results of the protein analysis via Western blot and immunohistochemistry suggest that l(2)rot is transcribed but not translated. Since RNAs encoded by the genes l(2)tid and l(2)rot are complementary and l(2)rot is presumably not translated we performed preliminary experiments on the function of the l(2)rot RNA as a natural antisense RNA (asRNA) regulator of l(2)tid expression, expressed in the same temporal and spatial manner as the l(2)tid- and l(2)not RNA. l(2)tid knock-out by antisense RNA yielded late embryonic lethality resulting from multiple morphogenetic defects.

Stable expression of the hepatitis B virus surface antigen containing pre-S2 protein in mouse cells using a bovine papillomavirus vector.

PubMed

Yoneyama, T; Akatsuka, T; Miyamura, T

1988-08-01

The large BglII fragment (2.8 kilobases) of hepatitis B virus DNA including the transcription unit for the hepatitis B surface antigen (HBsAg) was inserted into a bovine papillomavirus vector containing the neomycin resistance gene. The recombinant DNA was transfected into mouse C127 cells. A stable transformed cell line (MS128) secreting a large amount of 22 nm HBsAg particles containing pre-S2 protein was established. The secreted HBsAg particles had the receptor for polymerized human serum albumin. Immunoprecipitation and Western blot analyses showed that HBsAg particles consisted of two major proteins of 22K and 26K encoded by the S gene and a minor protein of 35K encoded by the pre-S2 and S genes. Southern blot analysis revealed that the transfected plasmid was integrated into the host chromosomal DNA and that most of the plasmid sequences were present. These results suggest that the stable expression of the HBsAg in MS128 cells is related to the integrated state of the recombinant DNA.

Characterization of ribonuclease III from Brucella.

PubMed

Wu, Chang-Xian; Xu, Xian-Jin; Zheng, Ke; Liu, Fang; Yang, Xu-Dong; Chen, Chuang-Fu; Chen, Huan-Chun; Liu, Zheng-Fei

2016-04-01

Bacterial ribonuclease III (RNase III) is a highly conserved endonuclease, which plays pivotal roles in RNA maturation and decay pathways by cleaving double-stranded structure of RNAs. Here we cloned rncS gene from the genomic DNA of Brucella melitensis, and analyzed the cleavage properties of RNase III from Brucella. We identified Brucella-encoding small RNA (sRNA) by high-throughput sequencing and northern blot, and found that sRNA of Brucella and Homo miRNA precursor (pre-miRNA) can be bound and cleaved by B.melitensis ribonuclease III (Bm-RNase III). Cleavage activity of Bm-RNase III is bivalent metal cations- and alkaline buffer-dependent. We constructed several point mutations in Bm-RNase III, whose cleavage activity indicated that the 133th Glutamic acid residue was required for catalytic activity. Western blot revealed that Bm-RNase III was differently expressed in Brucella virulence strain 027 and vaccine strain M5-90. Collectively, our data suggest that Brucella RNase III can efficiently bind and cleave stem-loop structure of small RNA, and might participate in regulation of virulence in Brucella. Copyright © 2016 Elsevier B.V. All rights reserved.

Large-scale production of functional human lysozyme from marker-free transgenic cloned cows.

PubMed

Lu, Dan; Liu, Shen; Ding, Fangrong; Wang, Haiping; Li, Jing; Li, Ling; Dai, Yunping; Li, Ning

2016-03-10

Human lysozyme is an important natural non-specific immune protein that is highly expressed in breast milk and participates in the immune response of infants against bacterial and viral infections. Considering the medicinal value and market demand for human lysozyme, an animal model for large-scale production of recombinant human lysozyme (rhLZ) is needed. In this study, we generated transgenic cloned cows with the marker-free vector pBAC-hLF-hLZ, which was shown to efficiently express rhLZ in cow milk. Seven transgenic cloned cows, identified by polymerase chain reaction, Southern blot, and western blot analyses, produced rhLZ in milk at concentrations of up to 3149.19 ± 24.80 mg/L. The purified rhLZ had a similar molecular weight and enzymatic activity as wild-type human lysozyme possessed the same C-terminal and N-terminal amino acid sequences. The preliminary results from the milk yield and milk compositions from a naturally lactating transgenic cloned cow 0906 were also tested. These results provide a solid foundation for the large-scale production of rhLZ in the future.

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.

[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.

Genetic relatedness of orbiviruses by RNA-RNA blot hybridization

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

Bodkin, D.K.

1985-01-01

RNA-RNA blot hybridization was developed in order to identify type-specific genes among double-stranded (ds) RNA viruses, to assess the genetic relatedness of dsRNA viruses and to classify new strains. Viral dsRNA segments were electrophoresed through 10% polyacrylamide gels, transferred to membranes, and hybridized to (5'/sup 32/P)-pCp labeled genomic RNA from a related strain. Hybridization was performed at 52/sup 0/C, 50% formamide, 5X SSC. Under these conditions heterologous RNA species must share greater than or equal to 74% sequence homology in order to form stable dsRNA hybrids. Cognate genes of nine members of the Palyam serogroup of orbiviruses were identified andmore » their sequence relatedness to the prototype. Palyam virus, was determined. Reciprocal blot hybridizations were performed using radiolabeled genomic RNA of all members of the Palyam serogroup. Unique and variant genes were identified by lack of cross-homology or by weak homology between segments. Since genes 2 and 6 exhibited the highest degree of sequence variability, response to the vertebrate immune system may be a major cause of sequence divergence among members of a single serogroup. Changuinola serogroup isolates were compared by dot-blot hybridization, while Colorado tick fever (CTF) serogroup isolates were compared by the RNA-RNA blot hybridization procedure described for reovirus and Palyam serogroup isolates. Preliminary blot hybridization data were also obtained on the relatedness of members of different Orbivirus serogroups.« less

[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.

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

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.

NF-kappaB mediates mitogen-activated protein kinase pathway-dependent iNOS expression in human melanoma.

PubMed

Uffort, Deon G; Grimm, Elizabeth A; Ellerhorst, Julie A

2009-01-01

Tumor expression of inducible nitric oxide synthase (iNOS) predicts poor outcomes for melanoma patients. We have reported the regulation of melanoma iNOS by the mitogen-activated protein kinase (MAPK) pathway. In this study, we test the hypothesis that NF-kappaB mediates this regulation. Western blotting of melanoma cell lysates confirmed the constitutive expression of iNOS. Western blot detected baseline levels of activated nuclear extracellular signal-regulated kinase and NF-kappaB. Indirect immunofluorescence confirmed the presence of NF-kappaB p50 and p65 in melanoma cell nuclei, with p50 being more prevalent. Electrophoretic mobility shift assay demonstrated baseline NF-kappaB activity, the findings confirmed by supershift analysis. Treatment of melanoma cells with the MEK inhibitor U0126 decreased NF-kappaB binding to its DNA recognition sequence, implicating the MAPK pathway in NF-kappaB activation. Two specific NF-kappaB inhibitors suppressed iNOS expression, demonstrating regulation of iNOS by NF-kappaB. Several experiments indicated the presence of p50 homodimers, which lack a transactivation domain and rely on the transcriptional coactivator Bcl-3 to carry out this function. Bcl-3 was detected in melanoma cells and co-immunoprecipitated with p50. These data suggest that the constitutively activated melanoma MAPK pathway stimulates activation of NF-kappaB hetero- and homodimers, which, in turn, drive iNOS expression and support melanoma tumorigenesis.

[Recombinant expression of Schistosoma japonicum fructose-1, 6-bisphos- phate aldolase and its expression in different developmental stages of S. japonicum].

PubMed

Yan, Ke; Zhong, Zheng-rong; Xu, Yun-xia; Ding, Shu-qin; Hu, Jian-guo; Xu, Yuan-hong; Luo, Qing-lie; Shen, Ji-long

2015-06-01

To clone, express and purify Schistosoma japonicum fructose-1, 6-bisphosphate aldolase (SjFBPA) in E. coli and observe its expression in different developmental stages of S. japonicum. FBPA gene was amplified from S. japonicum adult worm cDNA by using PCR. The amplified product was recombined into pET28a plasmid, and inducibly expressed with IPTG in E. coli BL21. SDS-PAGE and Western blotting were employed to analyze and identify the recombinant protein SjFBPA (rSjFBPA). Then, rSjFBPA was purified by chromatographic purification and its purity was analyzed by SDS- PAGE. The protein concentration of rSjFBPA purified was measured by the BCA method. Furthermore, SjFBPA mRNA was ana- lyzed in different developmental stages of S. japonicum by RT-PCR. SjFBPA was successfully amplified by using PCR and identified by restriction enzyme digestion and sequencing. The Western blotting analysis confirmed that the recombinant pro- tein could specifically reactive to the anti-His-tag monoclonal antibody. The concentration of the purified recombinant protein was about 4 mg/ml. The result of RT-PCR showed that SjFBPA mRNA was expressed in cercaria, schistosomulum, adult worm and egg of S. japonicum. SjFBPA is successfully recombined and expressed in a prokaryotic system, and SjFBPA mRNA is expressed in cercaria, schistosomulum, adult worm and egg of S. japonicum.

Isolation and characterization of Cu/Zn-superoxide dismutase in Fasciola gigantica.

PubMed

Lalrinkima, H; Raina, O K; Chandra, Dinesh; Jacob, Siju Susan; Bauri, R K; Chandra, Subhash; Yadav, H S; Singh, M N; Rialch, A; Varghese, A; Banerjee, P S; Kaur, Navneet; Sharma, Arvind

2015-01-01

A full-length complementary DNA (cDNA) encoding Cu/Zn-superoxide dismutase was isolated from Fasciola gigantica that on nucleotide sequencing showed a close homology (98.9%) with Cu/Zn-superoxide dismutase (SOD) of the temperate liver fluke, F. hepatica. Expression of the gene was found in all the three developmental stages of the parasite viz. adult, newly excysted juvenile and metacercaria at transcriptional level by reverse transcription-polymerase chain reaction (RT-PCR) and at the protein level by Western blotting. F. gigantica Cu/Zn-SOD cDNA was cloned and expressed in Escherichia coli. Enzyme activity of the recombinant protein was determined by nitroblue tetrazolium (NBT)-polyacrylamide gel electrophoresis (PAGE) and this activity was inactivated by hydrogen peroxide but not by sodium azide, indicating that the recombinant protein is Cu/Zn-SOD. The enzyme activity was relatively stable at a broad pH range of pH 4.0-10.0. Native Cu/Zn-superoxide dismutase protein was detected in the somatic extract and excretory-secretory products of the adult F. gigantica by Western blotting. NBT-PAGE showed a single Cu/Zn-SOD present in the somatic extract while three SODs are released ex vivo by the adult parasite. The recombinant superoxide dismutase did not react with the serum from buffaloes infected with F. gigantica. The role of this enzyme in defense by the parasite against the host reactive oxygen species is discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

Linear ion-trap mass spectrometric characterization of human pituitary nitrotyrosine-containing proteins

NASA Astrophysics Data System (ADS)

Zhan, Xianquan; Desiderio, Dominic M.

2007-01-01

The nitric oxide-mediated Tyr-nitration of endogenous proteins is associated with several pathological and physiological processes. In order to investigate the presence - and potential roles - of Tyr-nitration in the human pituitary, a large-format two-dimensional gel separation plus a Western blot against a specific anti-3-nitrotyrosine antibody were used to separate and detect nitroproteins from a human pituitary proteome. The nitroproteins were subjected to in-gel trypsin digestion, and high-sensitivity vacuum matrix-assisted laser desorption/ionization (vMALDI) linear ion-trap tandem mass spectrometry was used to analyze the tryptic peptides. Those MS/MS data were used to determine the amino acid sequence and the specific nitration site of each tryptic nitropeptide, and were matched to corresponding proteins with Bioworks TuboSEQUEST software. Compared to our previous study, 16 new nitrotyrosine-immunoreactive positive Western blot spots were found within the area pI 3.0-10 and Mr 10-100 kDa. Four new nitroproteins were discovered: the stanniocalcin 1 precursor--involved in calcium and phosphate metabolism; mitochondrial co-chaperone protein HscB, which might act as a co-chaperone in iron-sulfur cluster assembly in mitochrondria; progestin and adipoQ receptor family member III--a seven-transmembrane receptor; proteasome subunit alpha type 2--involved in an ATP/ubiquitin-dependent non-lysosomal proteolytic pathway. Those data demonstrate that nitric oxide-mediated Tyr-nitration might participate in various biochemical, metabolic, and pathological processes in the human pituitary.

[Cloning of human CD45 gene and its expression in Hela cells].

PubMed

Li, Jie; Xu, Tianyu; Wu, Lulin; Zhang, Liyun; Lu, Xiao; Zuo, Daming; Chen, Zhengliang

2015-11-01

To clone human CD45 gene PTPRC and establish Hela cells overexpressing recombinant human CD45 protein. The intact cDNA encoding human CD45 amplified using RT-PCR from the total RNA extracted from peripheral blood mononuclear cells (PBMCs) of a healthy donor was cloned into pMD-18T vector. The CD45 cDNA fragment amplified from the pMD-18T-CD45 by PCR was inserted to the coding region of the PcDNA3.1-3xflag vector, and the resultant recombinant expression vector PcDNA3.1-3xflag-CD45 was transfected into Hela cells. The expression of CD45 in Hela cells was detected by flow cytometry and Western blotting, and the phosphastase activity of CD45 was quantified using an alkaline phosphatase assay kit. The cDNA fragment of about 3 900 bp was amplified from human PBMCs and cloned into pMD-18T vector. The recombinant expression vector PcDNA3.1-3xflag-CD45 was constructed, whose restriction maps and sequence were consistent with those expected. The expression of CD45 in transfected Hela cells was detected by flow cytometry and Western blotting, and the expressed recombinant CD45 protein in Hela cells showed a phosphastase activity. The cDNA of human CD45 was successfully cloned and effectively expressed in Hela cells, which provides a basis for further exploration of the functions of CD45.

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.

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.

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.

Influence of silencing soluble epoxide hydrolase with RNA interference on cardiomyocytes apoptosis induced by doxorubicin.

PubMed

Du, Guangsheng; Lv, Jiagao; He, Li; Ma, Yexin

2011-06-01

In order to investigate the influence of silencing soluble epoxide hydrolase (sEH) with double-stranded small interfering RNA (siRNA) on cardiomyocytes apoptosis induced by doxorubicin (DOX), two plasmids containing siRNA sequences specific to sEH were constructed and transfected into the primary cultured cardiomyocytes by using FuGENE HD transfection agents. The mRNA and protein expression levels of sEH were detected by semiquantitative RT-PCR and Western blotting respectively, and the plasmids that silenced sEH most significantly were selected, and renamed EH-R. The plasmids carrying a nonspecific siRNA coding sequence (PCN) served as the negative control. Cardiomyocytes were divided into four groups: control group, DOX group, PCN+DOX group, and EH-R+DOX group. Apoptosis of cardiomyocytes was induced by DOX at a concentration of 1 μmol/L. Apoptosis rate of cardiomyocytes was determined by flow cytometery. The protein expression levels of Bcl-2 and Bax were detected by Western blotting. The results showed that the expression of sEH was down-regulated by EH-R plasmid. The expression levels of sEH mRNA and protein in the EH-R+DOX group were significantly decreased as compared with other groups (P<0.01). As compared with the control group, the apoptosis rate of cardiomyocytes in three DOX-treated groups was obviously increased, the expression levels of Bax increased, and those of Bcl-2 decreased (P<0.01). However, the expression levels of Bax were decreased, those of Bcl-2 increased and the apoptosis rate of cardiomyocytes obviously decreased in EH-R+DOX group when compared with those in the DOX group and the PCN+DOX group (P<0.01 for each). It was concluded that the recombinant plasmids could be successfully constructed, and transfected into the primary cultured cardiomyocytes. They could ameliorate the DOX-induced cardiomyocytes apoptosis by selectively inhibiting the expression of sEH with RNAi and increasing the expression of Bcl-2.

Identification of antigens by monoclonal antibody PD4 and its expression in Escherichia coli

PubMed Central

Ning, Jin-Ying; Sun, Guo-Xun; Huang, Su; Ma, Hong; An, Ping; Meng, Lin; Song, Shu-Mei; Wu, Jian; Shou, Cheng-Chao

2003-01-01

AIM: To clone and express the antigen of monoclonal antibody (MAb) PD4 for further investigation of its function. METHODS: MGC803 cDNA expression library was constructed and screened with PD4 as probes to clone the antigen. After failed in the library screening, immunoprecipitation and SDS-polyacrylamide gel electrophoresis were applied to purify the antigen for sequence analysis. The antigen coming from Mycoplasma hyorhinis (M. hyorhinis) was further confirmed with Western blot analysis by infecting M. hyorhinis -free HeLa cells and eliminating the M. hyorhinis from MGC803 cells. The full p37 gene was cloned by PCR and expressed successfully in Escherichia coli after site-directed mutations. Immunofluorescence assay was used to demonstrate if p37 protein could directly bind to gastric tumor cell AGS. RESULTS: The cDNA library constructed with MGC803 cells was screened by MAb PD4 as probes. Unfortunately, the positive clones identified with MAb PD4 were also reacted with unrelated antibodies. Then, immunoprecipitation was performed and the purified antigen was identified to be a membrane protein of Mycoplasma hyorhinis (M. hyorhinis) by sequencing of N-terminal amino acid residues. The membrane protein was intensively verified with Western blot by eliminating M. hyorhinis from MGC803 cells and by infecting M. hyorhinis-free HeLa cells. The full p37 gene was cloned and expressed successfully in Escherichia coli after site-directed mutations. Immunofluorescence demonstrated that p37 protein could directly bind to gastric tumor cell AGS. CONCLUSION: The antigen recognized by MAb PD4 is from M. hyorhinis, which suggests the actions involved in MAb PD4 is possibly mediated by p37 protein or M. hyorhinis. As p37 protein can bind directly to tumor cells, the pathogenic role of p37 involved in tumorigenesis justifies further investigation. PMID:14562370

Molecular cloning and functional characterization of peptidoglycan recognition protein 6 in grass carp Ctenopharyngodon idella.

PubMed

Li, Jun Hua; Yu, Zhang Long; Xue, Na Na; Zou, Peng Fei; Hu, Jing Yu; Nie, P; Chang, Ming Xian

2014-02-01

Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules of innate immunity. In this study, a long-form PGRP, designated as gcPGRP6, was identified from grass carp Ctenopharyngodon idella. The deduced amino acid sequence of gcPGRP6 is composed of 464 residues with a conserved PGRP domain at the C-terminus. The gcPGRP6 gene consists of four exons and three introns, spacing approximately 2.7 kb of genomic sequence. Phylogenetic analysis demonstrated that gcPGRP6 is clustered closely with zebrafish PGLYRP6, and formed a long-type PGRP subfamily together with PGLYRP2 members identified in teleosts and mammals. Real-time PCR and Western blotting analyses revealed that gcPGRP6 is constitutively expressed in organs/tissues examined, and its expression was significantly induced in liver and intestine of grass carp in response to PGN stimulation and in CIK cells treated with lipoteichoic acid (LTA), polyinosinic polycytidylic acid (Poly I:C) and peptidoglycan (PGN). Immunofluorescence microscopy and Western blotting analyses revealed that gcPGRP6 is effectively secreted to the exterior of CIK cells. The over-expression of gcPGRP6 in CIK cells leads to the activation of NF-κB and the inhibition of intracellular bacterial growth. Moreover, cell lysates from CIK cells transfected with pTurbo-gcPGRP6-GFP plasmid display the binding activity towards Lys-type PGN from Staphylococcus aureus and DAP-type PGN from Bacillus subtilis. Furthermore, proinflammatory cytokine IL-2 and intracellular PGN receptor NOD2 had a significantly increased expression in CIK cells overexpressed with gcPGRP6. It is demonstrated that the PGRP6 in grass carp has a role in binding PGN, in inhibiting the growth of intracellular bacteria, and in activating NF-κB, as well as in regulating innate immune genes. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cloning and expression of Bartonella henselae sucB gene encoding an immunogenic dihydrolipoamide succinyltransferase homologous protein.

PubMed

Kabeya, Hidenori; Maruyama, Soichi; Hirano, Kouji; Mikami, Takeshi

2003-01-01

Immunoscreening of a ZAP genomic library of Bartonella henselae strain Houston-1 expressed in Escherichia coli resulted in the isolation of a clone containing 3.5 kb BamHI genomic DNA fragment. This 3.5 kb DNA fragment was found to contain a sequence of a gene encoding a protein with significant homology to the dihydrolipoamide succinyltransferase of Brucella melitensis (sucB). Subsequent cloning and DNA sequence analysis revealed that the deduced amino acid sequence from the cloned gene showed 66.5% identity to SucB protein of B. melitensis, and 43.4 and 47.2% identities to those of Coxiella burnetii and E. coli, respectively. The gene was expressed as a His-Nus A-tagged fusion protein. The recombinant SucB protein (rSucB) was shown to be an immunoreactive protein of about 115 kDa by Western blot analysis with sera from B. henselae-immunized mice. Therefore the rSucB may be a candidate antigen for a specific serological diagnosis of B. henselae infection.

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.

Identification of the full-length β-actin sequence and expression profiles in the tree shrew (Tupaia belangeri).

PubMed

Zheng, Yu; Yun, Chenxia; Wang, Qihui; Smith, Wanli W; Leng, Jing

2015-02-01

The tree shrew (Tupaia belangeri) diverges from the primate order (Primates) and is classified as a separate taxonomic group of mammals - Scandentia. It has been suggested that the tree shrew can be used as an animal model for studying human diseases; however, the genomic sequence of the tree shrew is largely unidentified. In the present study, we reported the full-length cDNA sequence of the housekeeping gene, β-actin, in the tree shrew. The amino acid sequence of β-actin in the tree shrew was compared to that of humans and other species; a simple phylogenetic relationship was discovered. Quantitative polymerase chain reaction (qPCR) and western blot analysis further demonstrated that the expression profiles of β-actin, as a general conservative housekeeping gene, in the tree shrew were similar to those in humans, although the expression levels varied among different types of tissue in the tree shrew. Our data provide evidence that the tree shrew has a close phylogenetic association with humans. These findings further enhance the potential that the tree shrew, as a species, may be used as an animal model for studying human disorders.

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.

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.

The bglA Gene of Aspergillus kawachii Encodes Both Extracellular and Cell Wall-Bound β-Glucosidases

PubMed Central

Iwashita, Kazuhiro; Nagahara, Tatsuya; Kimura, Hitoshi; Takano, Makoto; Shimoi, Hitoshi; Ito, Kiyoshi

1999-01-01

We cloned the genomic DNA and cDNA of bglA, which encodes β-glucosidase in Aspergillus kawachii, based on a partial amino acid sequence of purified cell wall-bound β-glucosidase CB-1. The nucleotide sequence of the cloned bglA gene revealed a 2,933-bp open reading frame with six introns that encodes an 860-amino-acid protein. Based on the deduced amino acid sequence, we concluded that the bglA gene encodes cell wall-bound β-glucosidase CB-1. The amino acid sequence exhibited high levels of homology with the amino acid sequences of fungal β-glucosidases classified in subfamily B. We expressed the bglA cDNA in Saccharomyces cerevisiae and detected the recombinant β-glucosidase in the periplasm fraction of the recombinant yeast. A. kawachii can produce two extracellular β-glucosidases (EX-1 and EX-2) in addition to the cell wall-bound β-glucosidase. A. kawachii in which the bglA gene was disrupted produced none of the three β-glucosidases, as determined by enzyme assays and a Western blot analysis. Thus, we concluded that the bglA gene encodes both extracellular and cell wall-bound β-glucosidases in A. kawachii. PMID:10584016

Molecular Characterization of Bombyx mori Cytoplasmic Polyhedrosis Virus Genome Segment 4

PubMed Central

Ikeda, Keiko; Nagaoka, Sumiharu; Winkler, Stefan; Kotani, Kumiko; Yagi, Hiroaki; Nakanishi, Kae; Miyajima, Shigetoshi; Kobayashi, Jun; Mori, Hajime

2001-01-01

The complete nucleotide sequence of the genome segment 4 (S4) of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) was determined. The 3,259-nucleotide sequence contains a single long open reading frame which spans nucleotides 14 to 3187 and which is predicted to encode a protein with a molecular mass of about 130 kDa. Western blot analysis showed that S4 encodes BmCPV protein VP3, which is one of the outer components of the BmCPV virion. Sequence analysis of the deduced amino acid sequence of BmCPV VP3 revealed possible sequence homology with proteins from rice ragged stunt virus (RRSV) S2, Nilaparvata lugens reovirus S4, and Fiji disease fijivirus S4. This may suggest that plant reoviruses originated from insect viruses and that RRSV emerged more recently than other plant reoviruses. A chimeric protein consisting of BmCPV VP3 and green fluorescent protein (GFP) was constructed and expressed with BmCPV polyhedrin using a baculovirus expression vector. The VP3-GFP chimera was incorporated into BmCPV polyhedra and released under alkaline conditions. The results indicate that specific interactions occur between BmCPV polyhedrin and VP3 which might facilitate BmCPV virion occlusion into the polyhedra. PMID:11134312

Characterization of the synthesis and expression of the GTA-kinase from transformed and normal rodent cells.

PubMed

Kerr, M; Fischer, J E; Purushotham, K R; Gao, D; Nakagawa, Y; Maeda, N; Ghanta, V; Hiramoto, R; Chegini, N; Humphreys-Beher, M G

1994-08-02

The murine transformed cell line YC-8 and beta-adrenergic receptor agonist (isoproternol) treated rat and mouse parotid gland acinar cells ectopically express cell surface beta 1-4 galactosyltransferase during active proliferation. This activity is dependent upon the expression of the GTA-kinase (p58) in these cells. Using total RNA, cDNA clones for the protein coding region of the kinase were isolated by reverse transcriptase-PCR cloning. DNA sequence analysis failed to show sequence differences with the normal homolog from mouse cells although Southern blot analysis of YC-8, and a second cell line KI81, indicated changes in the restriction enzyme digestion profile relative to murine cell lines which do not express cell surface galactosyltransferase. The rat cDNA clone from isoproterenol-treated salivary glands showed a high degree of protein and nucleic acid sequence homology to the GTA-kinase from both murine and human sources. Northern blot analysis of YC-8 and a control cell line LSTRA revealed the synthesis of a major 3.0 kb mRNA from both cell lines plus the unique expression of a 4.5 kb mRNA in the YC-8 cells. Reverse transcriptase-PCR of LSTRA and YC-8 confirmed the increased steady state levels of the GTA-kinase mRNA in YC-8. In the mouse, induction of cell proliferation by isoproterenol resulted in a 50-fold increase in steady state mRNA levels for the kinase over the low level of expression in quiescent cells. Expression of the rat 3' untranslated region in rat parotid cells in vitro led to an increased rate of DNA synthesis, cell number an ectopic expression of cell surface galactosyltransferase in the sense orientation. Antisense expression or vector alone did not alter growth characteristics of acinar cells. A polyclonal antibody monospecific to a murine amino terminal peptide sequence revealed a uniform distribution of GTA-kinase over the cytoplasm of acinar and duct cells of control mouse parotid glands. However, upon growth stimulation, kinase was detected primarily in a perinuclear and nuclear immunostaining pattern. Western blot analysis confirmed a translocation from a cytoplasmic localization in both LSTRA and quiescent salivary cells to a membrane-associated localization in YC-8 and proliferating salivary cells.

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.

Cloning and expression of phosphoglycerate mutase from the psychrophilic yeast, Glaciozyma antarctica PI12

NASA Astrophysics Data System (ADS)

Jaafar, Nardiah Rizwana; Bakar, Farah Diba Abu; Murad, Abdul Munir Abdul; Mahadi, Nor Muhammad

2015-09-01

The conversion of 3-phosphoglycerate to 2-phosphoglycerate during glycolysis and gluconeogenesis is catalyzed by phosphoglycerate mutase (PGM). Better understanding of metabolic reactions performed by this enzyme has been studied extensively in prokaryotes and eukaryotes. Here, we report a phosphoglycerate mutase from the psychrophilic yeast, Glaciozyma antarctica. cDNA encoding for PGM from G. antarctica PI12, a psychrophilic yeast isolated from sea ice at Casey Station, Antarctica was amplified. The gene was then cloned into a cloning vector and sequenced, which verified its identity as the gene putatively encoding for PGM. The recombinant protein was expressed in Escherichia coli BL21 (DE3) as inclusion bodies and this was confirmed by SDS-PAGE and Western blot.

Zebra-borne equine herpesvirus type 1 (EHV-1) infection in non-African captive mammals.

PubMed

Abdelgawad, Azza; Azab, Walid; Damiani, Armando M; Baumgartner, Katrin; Will, Hermann; Osterrieder, Nikolaus; Greenwood, Alex D

2014-02-21

Equine herpesvirus type 1 (EHV-1) was detected in an Indian rhinoceros (Rhinoceros unicornis), which was euthanized because of severe neurological disease. Encephalitis was suspected and EHV-1 DNA was detected in brain, lung, and spleen tissues. The viral IR6 protein was detected in lung tissues by Western blot analysis. Phylogenetic analyses of EHV-1 sequences amplified from various tissues was nearly identical to one recently described that resulted in both non-fatal and fatal encephalitis in polar bears. This represents transmission of EHV-1 to a species that is not naturally sympatric with the natural host of the virus and broadens the host range to Asian non-equid perissodactyls. Copyright © 2013 Elsevier B.V. All rights reserved.

Post-staining electroblotting for efficient and reliable peptide blotting.

PubMed

Lee, Der-Yen; Chang, Geen-Dong

2015-01-01

Post-staining electroblotting has been previously described to transfer Coomassie blue-stained proteins from polyacrylamide gel onto polyvinylidene difluoride (PVDF) membranes. Actually, stained peptides can also be efficiently and reliably transferred. Because of selective staining procedures for peptides and increased retention of stained peptides on the membrane, even peptides with molecular masses less than 2 kDa such as bacitracin and granuliberin R are transferred with satisfactory results. For comparison, post-staining electroblotting is about 16-fold more sensitive than the conventional electroblotting for visualization of insulin on the membrane. Therefore, the peptide blots become practicable and more accessible to further applications, e.g., blot overlay detection or immunoblotting analysis. In addition, the efficiency of peptide transfer is favorable for N-terminal sequence analysis. With this method, peptide blotting can be normalized for further analysis such as blot overlay assay, immunoblotting, and N-terminal sequencing for identification of peptide in crude or partially purified samples.

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

[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.

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.

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

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.

Structure and transcription of the Helicoverpa armigera densovirus (HaDV2) genome and its expression strategy in LD652 cells.

PubMed

Xu, Pengjun; Graham, Robert I; Wilson, Kenneth; Wu, Kongming

2017-02-07

Densoviruses (DVs) are highly pathogenic to their hosts. However, we previously reported a mutualistic DV (HaDV2). Very little was known about the characteristics of this virus, so herein we undertook a series of experiments to explore the molecular biology of HaDV2 further. Phylogenetic analysis showed that HaDV2 was similar to members of the genus Iteradensovirus. However, compared to current members of the genus Iteradensovirus, the sequence identity of HaDV2 is less than 44% at the nucleotide-level, and lower than 36, 28 and 19% at the amino-acid-level of VP, NS1 and NS2 proteins, respectively. Moreover, NS1 and NS2 proteins from HaDV2 were smaller than those from other iteradensoviruses due to their shorter N-terminal sequences. Two transcripts of about 2.2 kb coding for the NS proteins and the VP proteins were identified by Northern Blot and RACE analysis. Using specific anti-NS1 and anti-NS2 antibodies, Western Blot analysis revealed a 78 kDa and a 48 kDa protein, respectively. Finally, the localization of both NS1 and NS2 proteins within the cell nucleus was determined by using Green Fluorescent Protein (GFP) labelling. The genome organization, terminal hairpin structure, transcription and expression strategies as well as the mutualistic relationship with its host, suggested that HaDV2 was a novel member of the genus Iteradensovirus within the subfamily Densovirinae.

Generation and validation of homozygous fluorescent knock-in cells using CRISPR-Cas9 genome editing.

PubMed

Koch, Birgit; Nijmeijer, Bianca; Kueblbeck, Moritz; Cai, Yin; Walther, Nike; Ellenberg, Jan

2018-06-01

Gene tagging with fluorescent proteins is essential for investigations of the dynamic properties of cellular proteins. CRISPR-Cas9 technology is a powerful tool for inserting fluorescent markers into all alleles of the gene of interest (GOI) and allows functionality and physiological expression of the fusion protein. It is essential to evaluate such genome-edited cell lines carefully in order to preclude off-target effects caused by (i) incorrect insertion of the fluorescent protein, (ii) perturbation of the fusion protein by the fluorescent proteins or (iii) nonspecific genomic DNA damage by CRISPR-Cas9. In this protocol, we provide a step-by-step description of our systematic pipeline to generate and validate homozygous fluorescent knock-in cell lines.We have used the paired Cas9D10A nickase approach to efficiently insert tags into specific genomic loci via homology-directed repair (HDR) with minimal off-target effects. It is time-consuming and costly to perform whole-genome sequencing of each cell clone to check for spontaneous genetic variations occurring in mammalian cell lines. Therefore, we have developed an efficient validation pipeline of the generated cell lines consisting of junction PCR, Southern blotting analysis, Sanger sequencing, microscopy, western blotting analysis and live-cell imaging for cell-cycle dynamics. This protocol takes between 6 and 9 weeks. With this protocol, up to 70% of the targeted genes can be tagged homozygously with fluorescent proteins, thus resulting in physiological levels and phenotypically functional expression of the fusion proteins.

Selectable antibiotic resistance marker gene-free transgenic rice harbouring the garlic leaf lectin gene exhibits resistance to sap-sucking planthoppers.

PubMed

Sengupta, Subhadipa; Chakraborti, Dipankar; Mondal, Hossain A; Das, Sampa

2010-03-01

Rice, the major food crop of world is severely affected by homopteran sucking pests. We introduced coding sequence of Allium sativum leaf agglutinin, ASAL, in rice cultivar IR64 to develop sustainable resistance against sap-sucking planthoppers as well as eliminated the selectable antibiotic-resistant marker gene hygromycin phosphotransferase (hpt) exploiting cre/lox site-specific recombination system. An expression vector was constructed containing the coding sequence of ASAL, a potent controlling agent against green leafhoppers (GLH, Nephotettix virescens) and brown planthopper (BPH, Nilaparvata lugens). The selectable marker (hpt) gene cassette was cloned within two lox sites of the same vector. Alongside, another vector was developed with chimeric cre recombinase gene cassette. Reciprocal crosses were performed between three single-copy T(0) plants with ASAL- lox-hpt-lox T-DNA and three single-copy T(0) plants with cre-bar T-DNA. Marker gene excisions were detected in T(1) hybrids through hygromycin sensitivity assay. Molecular analysis of T(1) plants exhibited 27.4% recombination efficiency. T(2) progenies of L03C04(1) hybrid parent showed 25% cre negative ASAL-expressing plants. Northern blot, western blot and ELISA showed significant level of ASAL expression in five marker-free T(2) progeny plants. In planta bioassay of GLH and BPH performed on these T(2) progenies exhibited radical reduction in survivability and fecundity compared with the untransformed control plants.

Nectinepsin: a new extracellular matrix protein of the pexin family. Characterization of a novel cDNA encoding a protein with an RGD cell binding motif.

PubMed

Blancher, C; Omri, B; Bidou, L; Pessac, B; Crisanti, P

1996-10-18

We report the isolation and characterization of a novel cDNA from quail neuroretina encoding a putative protein named nectinepsin. The nectinepsin cDNA identifies a major 2.2-kilobase mRNA that is detected from ED 5 in neuroretina and is increasingly abundant during embryonic development. A nectinepsin mRNA is also found in quail liver, brain, and intestine and in mouse retina. The deduced nectinepsin amino acid sequence contains the RGD cell binding motif of integrin ligands. Furthermore, nectinepsin shares substantial homologies with vitronectin and structural protein similarities with most of the matricial metalloproteases. However, the presence of a specific sequence and the lack of heparin and collagen binding domains of the vitronectin indicate that nectinepsin is a new extracellular matrix protein. Furthermore, genomic Southern blot studies suggest that nectinepsin and vitronectin are encoded by different genes. Western blot analysis with an anti-human vitronectin antiserum revealed, in addition to the 65- and 70-kDa vitronectin bands, an immunoreactive protein of about 54 kDa in all tissues containing nectinepsin mRNA. It seems likely that the form of vitronectin found in chick egg yolk plasma by Nagano et al. ((1992) J. Biol. Chem. 267, 24863-24870) is the protein that corresponds to the nectinepsin cDNA. This new protein could be an important molecule involved in the early steps of the development.

MicroRNA-21 promotes proliferation of rat hepatocyte BRL-3A by targeting FASLG.

PubMed

Li, J J; Chan, W H; Leung, W Y; Wang, Y; Xu, C S

2015-04-27

Rat liver regeneration (RLR) induced by partial hepatectomy involves cell proliferation regulated by numerous factors, including microRNAs (miRNAs). miRNA high-throughput sequencing has been established and used to analyze miRNA expression profiles. This study showed that 39 miRNAs were related to RLR through the analysis of miRNA high-throughput sequencing. Their role toward rat normal hepatocyte line BRL-3A was studied by gain- and loss-of-function analyses, and one of them, microRNA-21 (miR-21), obviously upregulated and promoted BRL-3A cell proliferation. Using bioinformatics to search for miR-21 targets revealed that Fas ligand (FASLG) is one of miR-21's target genes. A dual-luciferase report assay and Western blot assay showed that miR-21 directly targeted the 3'-untranslated region of FASLG and inhibited the expression of FASLG, which suggests that miR-21 promoted BRL-3A cell proliferation by reducing FASLG expression.

Effects of a Proline Endopeptidase on the Detection and Quantitation of Gluten by Antibody-Based Methods during the Fermentation of a Model Sorghum Beer.

PubMed

Panda, Rakhi; Fiedler, Katherine L; Cho, Chung Y; Cheng, Raymond; Stutts, Whitney L; Jackson, Lauren S; Garber, Eric A E

2015-12-09

The effectiveness of a proline endopeptidase (PEP) in hydrolyzing gluten and its putative immunopathogenic sequences was examined using antibody-based methods and mass spectrometry (MS). Based on the results of the antibody-based methods, fermentation of wheat gluten containing sorghum beer resulted in a reduction in the detectable gluten concentration. The addition of PEP further reduced the gluten concentration. Only one sandwich ELISA was able to detect the apparent low levels of gluten present in the beers. A competitive ELISA using a pepsin-trypsin hydrolysate calibrant was unreliable because the peptide profiles of the beers were inconsistent with that of the hydrolysate calibrant. Analysis by MS indicated that PEP enhanced the loss of a fragment of an immunopathogenic 33-mer peptide in the beer. However, Western blot results indicated partial resistance of the high molecular weight (HMW) glutenins to the action of PEP, questioning the ability of PEP in digesting all immunopathogenic sequences present in gluten.

Jellyfish Bioactive Compounds: Methods for Wet-Lab Work

PubMed Central

Frazão, Bárbara; Antunes, Agostinho

2016-01-01

The study of bioactive compounds from marine animals has provided, over time, an endless source of interesting molecules. Jellyfish are commonly targets of study due to their toxic proteins. However, there is a gap in reviewing successful wet-lab methods employed in these animals, which compromises the fast progress in the detection of related biomolecules. Here, we provide a compilation of the most effective wet-lab methodologies for jellyfish venom extraction prior to proteomic analysis—separation, identification and toxicity assays. This includes SDS-PAGE, 2DE, gel chromatography, HPLC, DEAE, LC-MS, MALDI, Western blot, hemolytic assay, antimicrobial assay and protease activity assay. For a more comprehensive approach, jellyfish toxicity studies should further consider transcriptome sequencing. We reviewed such methodologies and other genomic techniques used prior to the deep sequencing of transcripts, including RNA extraction, construction of cDNA libraries and RACE. Overall, we provide an overview of the most promising methods and their successful implementation for optimizing time and effort when studying jellyfish. PMID:27077869

Jellyfish Bioactive Compounds: Methods for Wet-Lab Work.

PubMed

Frazão, Bárbara; Antunes, Agostinho

2016-04-12

The study of bioactive compounds from marine animals has provided, over time, an endless source of interesting molecules. Jellyfish are commonly targets of study due to their toxic proteins. However, there is a gap in reviewing successful wet-lab methods employed in these animals, which compromises the fast progress in the detection of related biomolecules. Here, we provide a compilation of the most effective wet-lab methodologies for jellyfish venom extraction prior to proteomic analysis-separation, identification and toxicity assays. This includes SDS-PAGE, 2DE, gel chromatography, HPLC, DEAE, LC-MS, MALDI, Western blot, hemolytic assay, antimicrobial assay and protease activity assay. For a more comprehensive approach, jellyfish toxicity studies should further consider transcriptome sequencing. We reviewed such methodologies and other genomic techniques used prior to the deep sequencing of transcripts, including RNA extraction, construction of cDNA libraries and RACE. Overall, we provide an overview of the most promising methods and their successful implementation for optimizing time and effort when studying jellyfish.

Molecular cloning, sequence characterization and recombinant expression of Nanog gene in goat fibroblast cells using lentiviral based expression system.

PubMed

Singhal, Dinesh K; Singhal, Raxita; Malik, Hruda N; Kumar, Surender; Kumar, Sudarshan; Mohanty, Ashok K; Kaushik, Jai K; Malakar, Dhruba

2014-01-01

Nanog is a homeodomain containing protein which plays important roles in regulation of signaling pathways for maintenance and induction of pluripotency in stem cells. Because of its unique expression in stem cells it is also regarded as pluripotency marker. In this study goat Nanog (gNanog) gene has been amplified, cloned and characterized at sequence level with successful over-expression in CHO-K1 cell line using a lentiviral based system. gNanog ORF is 903 bp long which codes for Nanog protein of size 300 amino acids (aas). Complete nucleotide sequence shows some evolutionary mutation in goat in comparision to other species. Protein sequence of goat is highly similar to other species. Overall, gNanog nucleotide sequence and predicted protein sequence showed high similarity and minimum divergence with cattle (96 % identity/4 % divergence) and buffalo (94/5 %) while low similarity and high divergence with pig (84/15 %), human (81/23 %) and mouse (69/40 %) indicating evolutionary closeness of gNanog to cattle and buffalo. gNanog lentiviral expression construct was prepared for over-expression of Nanog gene in adult goat fibroblast cells. Lentiviral expression construct of Nanog enabled continuous protein expression for induction and maintenance of pluripotency. Western blotting revealed the expression of Nanog gene at protein level which supported that the lentiviral expression system is highly promising for Nanog protein expression in differentiated goat cell.

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

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

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.

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.

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

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.

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…

Preeclampsia is associated with hypermethylation of IGF-1 promoter mediated by DNMT1.

PubMed

Ma, Min; Zhou, Qiong-Jie; Xiong, Yu; Li, Bin; Li, Xiao-Tian

2018-01-01

Previous studies have demonstrated a dynamic epigenetic regulation of genes expression in placenta trophoblasts and a dynamic imbalance of DNA methylation and hydroxymethylation. Reduced IGF-1 has been observed in preeclampsia. This study was to investigate the interactive roles between IGF-1 and the global DNA methylation/hydroxymethylation, and the status of DNA methylation/hydroxymethylation and associated enzymes such as DNMTs and TETs in peeeclamptic placentas and hypoxic trophoblasts. It was found that IGF-1 was decreased in preeclamptic placentas and hypoxic trophoblasts when compared to the control group using immunohistochemisty, western blot, qRT-PCR and ELISA. Pyrophosphate sequencing showed IGF-1 promoter was significantly hypermethylated in preeclamptic placentas, which was responsible for reduced IGF-1 expression. Preeclamptic placentas and hypoxic trophoblasts were hypermethylated and hypohydroxymethylated accompanied by remarkably higher 5mC, DNMT1 and DNMT3b, and lower DNMT3a, 5hmC, TET1, TET2 and TET3 detected by immunohistochemisty, western blot, qRT-PCR and ELISA. Pearson's correlation confirmed a statistically significant negative correlation between IGF-1 and DNMT1. Furthermore, both treatment with 5-Aza-dc and DNMT1-siRNA significantly increased the expression of IGF-1 in HTR8 cells, indicating the potential mechanism of DNMT1-mediated DNA methylation in IGF-1 regulation. However, IGF-1 didn't change DNA methylation or hydroxymethylation. These findings suggest that preeclampsia is associated with hypermethylation of IGF-1 promoter mediated by DNMT1 and provide new insights into the diagnosis and treatment of preeclampsia.

Preeclampsia is associated with hypermethylation of IGF-1 promoter mediated by DNMT1

PubMed Central

Ma, Min; Zhou, Qiong-Jie; Xiong, Yu; Li, Bin; Li, Xiao-Tian

2018-01-01

Previous studies have demonstrated a dynamic epigenetic regulation of genes expression in placenta trophoblasts and a dynamic imbalance of DNA methylation and hydroxymethylation. Reduced IGF-1 has been observed in preeclampsia. This study was to investigate the interactive roles between IGF-1 and the global DNA methylation/hydroxymethylation, and the status of DNA methylation/hydroxymethylation and associated enzymes such as DNMTs and TETs in peeeclamptic placentas and hypoxic trophoblasts. It was found that IGF-1 was decreased in preeclamptic placentas and hypoxic trophoblasts when compared to the control group using immunohistochemisty, western blot, qRT-PCR and ELISA. Pyrophosphate sequencing showed IGF-1 promoter was significantly hypermethylated in preeclamptic placentas, which was responsible for reduced IGF-1 expression. Preeclamptic placentas and hypoxic trophoblasts were hypermethylated and hypohydroxymethylated accompanied by remarkably higher 5mC, DNMT1 and DNMT3b, and lower DNMT3a, 5hmC, TET1, TET2 and TET3 detected by immunohistochemisty, western blot, qRT-PCR and ELISA. Pearson’s correlation confirmed a statistically significant negative correlation between IGF-1 and DNMT1. Furthermore, both treatment with 5-Aza-dc and DNMT1-siRNA significantly increased the expression of IGF-1 in HTR8 cells, indicating the potential mechanism of DNMT1-mediated DNA methylation in IGF-1 regulation. However, IGF-1 didn’t change DNA methylation or hydroxymethylation. These findings suggest that preeclampsia is associated with hypermethylation of IGF-1 promoter mediated by DNMT1 and provide new insights into the diagnosis and treatment of preeclampsia. PMID:29422991

[The effect of Foxc2 overexpression on the osteogenic properties of C3H10T1/2 cells].

PubMed

Wang, Min-Jiao; Si, Jia-Wen; Li, Hong-Liang; Ouyang, Ning-Juan; Shen, Guo-Fang

2016-08-01

To investigate the effect of Foxc2 overexpression on osteogenic and adipogenic differentiation of C3H10T1/2 cells. C3H10T1/2 cells were transfected with plenti-Foxc2 and selected with puromycin for stable clones. The expression of Foxc2 was determined by real-time PCR and Western blot. Cell proliferation was detected by CCK-8 kit. Cell cycle and apoptosis were detected by flow cytometry. The level of osteogenic biomarkers Runx2, OPN, OCN and adipogenic biomarker PPARγ were quantified by real-time PCR and Western blot. Alkaline phosphatase (ALP) staining and oil red staining were conducted to evaluate the effect of Foxc2 overexpression on osteogenic and adipogenic differentiation. Statistical analysis was performed using SPSS 17.0 software package. C3H10T1/2-Foxc2 cell line was successfully constructed and verified by direct sequencing and Foxc2 overexpression in vitro. Cell proliferation was reduced and cell cycle was blocked in G1/G0 phase. Enhanced ALP staining and reduced oil red staining were observed in C3H10T1/2-Foxc2 cells as compared with the control. Foxc2 overexpression up-regulated Runx2, OPN, OCN during osteogenic differentiation and down-regulated PPARγduring adipogenic differentiation. C3H10T1/2 cell line stably expressing Foxc2 gene was successfully established, cell proliferation was reduced, osteogenesis biomarkers were up-regulated during the osteogenesis by overexpression Foxc2, PPARγwas down-regulated during adipogenesis.

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.

Humoral response of captive zebra sharks Stegostoma fasciatum to salivary gland proteins of the leech Branchellion torpedinis.

PubMed

Marancik, David P; Leary, John H; Fast, Mark M; Flajnik, Martin F; Camus, Alvin C

2012-10-01

Parasitism by the marine leech Branchellion torpedinis is known to cause disease and mortality in captive elasmobranchs and is difficult to control when inadvertently introduced into public aquaria. Preliminary characterization of the salivary gland transcriptome of B. torpedinis has identified anticoagulants, proteases, and immunomodulators that may be secreted into host tissues to aid leech feeding. This retrospective study examined antigen-specific serum IgM responses in captive zebra sharks Stegostoma fasciatum to leech salivary gland extract. Antibody response was examined by ELISA and Western blot assays in 20 serum samples from six zebra sharks, with a 5 year history of leech infection, and 18 serum samples from 8 captive bred zebra sharks, with no history of leech exposure. ELISA demonstrated significantly higher serum IgM titers to salivary gland extract in exposed zebra sharks compared to the non-exposed population. No obvious trends in antibody titers were appreciated in exposed zebra sharks over a four-year period. One-dimensional and two-dimensional Western blot assays revealed IgM targeted specific salivary gland proteins within the 40, 55, 70 and 90 kD range. Antigenic proteins identified by liquid chromatography-tandem mass spectrometry and de novo peptide sequencing include a secreted disintegrin, metalloproteinase and thrombospondin motif containing protein (ADAMTS), tubulin, aldehyde dehydrogenase and two unknown proteins. Humoral immune responses to leech salivary gland proteins warrants further investigation as there may be options to exploit immune mechanisms to reduce parasite burdens in aquaria. Copyright © 2012 Elsevier Ltd. All rights reserved.

Bidirectional binding property of high glycine-tyrosine keratin-associated protein contributes to the mechanical strength and shape of hair.

PubMed

Matsunaga, Ryo; Abe, Ryota; Ishii, Daisuke; Watanabe, Shun-Ichi; Kiyoshi, Masato; Nöcker, Bernd; Tsuchiya, Masaru; Tsumoto, Kouhei

2013-09-01

Since their first finding in wool 50years ago, keratin-associated proteins (KAPs), which are classified into three groups; high sulfur (HS) KAPs, ultra high sulfur (UHS) KAPs, and high glycine-tyrosine (HGT) KAPs, have been the target of curiosity for scientists due to their characteristic amino acid sequences. While HS and UHS KAPs are known to function in disulfide bond crosslinking, the function of HGT KAPs remains unknown. To clarify the function as well as the binding partners of HGT KAPs, we prepared KAP8.1 and other KAP family proteins, the trichocyte intermediate filament proteins (IFP) K85 and K35, the head domain of K85, and the C subdomain of desmoplakin C-terminus (DPCT-C) and investigated the interactions between them in vitro. Western blot analysis and isothermal titration calorimetry (ITC) indicate that KAP8.1 binds to the head domain of K85, which is helically aligned around the axis of the intermediate filament (IF). From these results and transmission electron microscopy (TEM) observations of bundled filament complex in vitro, we propose that the helical arrangement of IFs found in the orthocortex, which is uniquely distributed on the convex fiber side of the hair, is regulated by KAP8.1. Structure-dependent binding of DPCT-C to trichocyte IFP was confirmed by Western blotting, ITC, and circular dichroism. Moreover, DPCT-C also binds to some HGT KAPs. It is probable that such bidirectional binding property of HGT KAPs contribute to the mechanical robustness of hair. Copyright © 2013 Elsevier Inc. All rights reserved.

Proviral Features of Human T Cell Leukemia Virus Type 1 in Carriers with Indeterminate Western Blot Analysis Results

PubMed Central

Sekizuka, Tsuyoshi; Yamochi, Tadanori; Firouzi, Sanaz; Sato, Tomoo; Umeki, Kazumi; Sasaki, Daisuke; Hasegawa, Hiroo; Kubota, Ryuji; Sobata, Rieko; Matsumoto, Chieko; Kaneko, Noriaki; Momose, Haruka; Araki, Kumiko; Saito, Masumichi; Nosaka, Kisato; Utsunomiya, Atae; Koh, Ki-Ryang; Ogata, Masao; Uchimaru, Kaoru; Iwanaga, Masako; Sagara, Yasuko; Yamano, Yoshihisa; Okayama, Akihiko; Miura, Kiyonori; Satake, Masahiro; Saito, Shigeru; Itabashi, Kazuo; Yamaguchi, Kazunari; Kuroda, Makoto; Watanabe, Toshiki; Okuma, Kazu

2017-01-01

ABSTRACT Western blotting (WB) for human T cell leukemia virus type 1 (HTLV-1) is performed to confirm anti-HTLV-1 antibodies detected at the initial screening of blood donors and in pregnant women. However, the frequent occurrence of indeterminate results is a problem with this test. We therefore assessed the cause of indeterminate WB results by analyzing HTLV-1 provirus genomic sequences. A quantitative PCR assay measuring HTLV-1 provirus in WB-indeterminate samples revealed that the median proviral load was approximately 100-fold lower than that of WB-positive samples (0.01 versus 0.71 copy/100 cells). Phylogenic analysis of the complete HTLV-1 genomes of WB-indeterminate samples did not identify any specific phylogenetic groups. When we analyzed the nucleotide changes in 19 HTLV-1 isolates from WB-indeterminate samples, we identified 135 single nucleotide substitutions, composed of four types, G to A (29%), C to T (19%), T to C (19%), and A to G (16%). In the most frequent G-to-A substitution, 64% occurred at GG dinucleotides, indicating that APOBEC3G is responsible for mutagenesis in WB-indeterminate samples. Moreover, interestingly, five WB-indeterminate isolates had nonsense mutations in Pol and/or Tax, Env, p12, and p30. These findings suggest that WB-indeterminate carriers have low production of viral antigens because of a combination of a low proviral load and mutations in the provirus, which may interfere with host recognition of HTLV-1 antigens. PMID:28701419

Proviral Features of Human T Cell Leukemia Virus Type 1 in Carriers with Indeterminate Western Blot Analysis Results.

PubMed

Kuramitsu, Madoka; Sekizuka, Tsuyoshi; Yamochi, Tadanori; Firouzi, Sanaz; Sato, Tomoo; Umeki, Kazumi; Sasaki, Daisuke; Hasegawa, Hiroo; Kubota, Ryuji; Sobata, Rieko; Matsumoto, Chieko; Kaneko, Noriaki; Momose, Haruka; Araki, Kumiko; Saito, Masumichi; Nosaka, Kisato; Utsunomiya, Atae; Koh, Ki-Ryang; Ogata, Masao; Uchimaru, Kaoru; Iwanaga, Masako; Sagara, Yasuko; Yamano, Yoshihisa; Okayama, Akihiko; Miura, Kiyonori; Satake, Masahiro; Saito, Shigeru; Itabashi, Kazuo; Yamaguchi, Kazunari; Kuroda, Makoto; Watanabe, Toshiki; Okuma, Kazu; Hamaguchi, Isao

2017-09-01

Western blotting (WB) for human T cell leukemia virus type 1 (HTLV-1) is performed to confirm anti-HTLV-1 antibodies detected at the initial screening of blood donors and in pregnant women. However, the frequent occurrence of indeterminate results is a problem with this test. We therefore assessed the cause of indeterminate WB results by analyzing HTLV-1 provirus genomic sequences. A quantitative PCR assay measuring HTLV-1 provirus in WB-indeterminate samples revealed that the median proviral load was approximately 100-fold lower than that of WB-positive samples (0.01 versus 0.71 copy/100 cells). Phylogenic analysis of the complete HTLV-1 genomes of WB-indeterminate samples did not identify any specific phylogenetic groups. When we analyzed the nucleotide changes in 19 HTLV-1 isolates from WB-indeterminate samples, we identified 135 single nucleotide substitutions, composed of four types, G to A (29%), C to T (19%), T to C (19%), and A to G (16%). In the most frequent G-to-A substitution, 64% occurred at GG dinucleotides, indicating that APOBEC3G is responsible for mutagenesis in WB-indeterminate samples. Moreover, interestingly, five WB-indeterminate isolates had nonsense mutations in Pol and/or Tax, Env, p12, and p30. These findings suggest that WB-indeterminate carriers have low production of viral antigens because of a combination of a low proviral load and mutations in the provirus, which may interfere with host recognition of HTLV-1 antigens. Copyright © 2017 American Society for Microbiology.

[miR-25 promotes cell proliferation by targeting RECK in human cervical carcinoma HeLa cells].

PubMed

Qiu, Gang; Fang, Baoshuan; Xin, Guohong; Wei, Qiang; Yuan, Xiaoye; Wu, Dayong

2015-01-01

To investigate the effect of miR-25 on the proliferation of human cervical carcinoma HeLa cells and its association with reversion-inducing cysteine-rich protein with Kazal motifs (RECK). The recombinant plasmids of pcDNATM6.2-GW-pre-miR-25, pmirGLO-RECK-WT, pmirGLO-RECK-MT and anti-miR-25 were constructed, and their transfection efficiencies into HeLa cells were identified by real-time quantitative PCR (qRT-PCR). The potential proliferation-stimulating function of miR-25 was analyzed by MTT assay in HeLa cells. Furthermore, the target effect of miR-25 on the RECK was determined by dual-luciferase reporter assay system, qRT-PCR and Western blotting. Sequence analysis demonstrated that the recombinant plasmids of pcDNATM6.2-GW-pre-miR-25 and pmirGLO-RECK-WT, pmirGLO-RECK-MT were successfully constructed, and qRT-PCR revealed that the transfection efficiencies of pre-miR-25 and anti-miR-25 were desirable in HeLa cells. MTT assay showed that miR-25 over-expression promoted the proliferation of HeLa cells. In addition, the luciferase activity was significantly reduced in HeLa cells cotransfected with pre-miR-25 and RECK-WT. The qRT-PCR and Western blotting indicated that the expression level of RECK was up-regulated in HeLa cells transfected with anti-miR-25 at the transcriptional and posttranscriptional levels. miR-25 could promote cell proliferation by targeting RECK in HeLa cells.

Special AT-rich sequence binding protein 1 promotes tumor growth and metastasis of esophageal squamous cell carcinoma.

PubMed

Ma, Jun; Wu, Kaiming; Zhao, Zhenxian; Miao, Rong; Xu, Zhe

2017-03-01

Esophageal squamous cell carcinoma is one of the most aggressive malignancies worldwide. Special AT-rich sequence binding protein 1 is a nuclear matrix attachment region binding protein which participates in higher order chromatin organization and tissue-specific gene expression. However, the role of special AT-rich sequence binding protein 1 in esophageal squamous cell carcinoma remains unknown. In this study, western blot and quantitative real-time polymerase chain reaction analysis were performed to identify differentially expressed special AT-rich sequence binding protein 1 in a series of esophageal squamous cell carcinoma tissue samples. The effects of special AT-rich sequence binding protein 1 silencing by two short-hairpin RNAs on cell proliferation, migration, and invasion were assessed by the CCK-8 assay and transwell assays in esophageal squamous cell carcinoma in vitro. Special AT-rich sequence binding protein 1 was significantly upregulated in esophageal squamous cell carcinoma tissue samples and cell lines. Silencing of special AT-rich sequence binding protein 1 inhibited the proliferation of KYSE450 and EC9706 cells which have a relatively high level of special AT-rich sequence binding protein 1, and the ability of migration and invasion of KYSE450 and EC9706 cells was distinctly suppressed. Special AT-rich sequence binding protein 1 could be a potential target for the treatment of esophageal squamous cell carcinoma and inhibition of special AT-rich sequence binding protein 1 may provide a new strategy for the prevention of esophageal squamous cell carcinoma invasion and metastasis.

Production of Transgenic Pigs with an Introduced Missense Mutation of the Bone Morphogenetic Protein Receptor Type IB Gene Related to Prolificacy.

PubMed

Zhao, Xueyan; Yang, Qiang; Zhao, Kewei; Jiang, Chao; Ren, Dongren; Xu, Pan; He, Xiaofang; Liao, Rongrong; Jiang, Kai; Ma, Junwu; Xiao, Shijun; Ren, Jun; Xing, Yuyun

2016-07-01

In the last few decades, transgenic animal technology has witnessed an increasingly wide application in animal breeding. Reproductive traits are economically important to the pig industry. It has been shown that the bone morphogenetic protein receptor type IB (BMPR1B) A746G polymorphism is responsible for the fertility in sheep. However, this causal mutation exits exclusively in sheep and goat. In this study, we attempted to create transgenic pigs by introducing this mutation with the aim to improve reproductive traits in pigs. We successfully constructed a vector containing porcine BMPR1B coding sequence (CDS) with the mutant G allele of A746G mutation. In total, we obtained 24 cloned male piglets using handmade cloning (HMC) technique, and 12 individuals survived till maturation. A set of polymerase chain reactions indicated that 11 of 12 matured boars were transgene-positive individuals, and that the transgenic vector was most likely disrupted during cloning. Of 11 positive pigs, one (No. 11) lost a part of the terminator region but had the intact promoter and the CDS regions. cDNA sequencing showed that the introduced allele (746G) was expressed in multiple tissues of transgene-positive offspring of No.11. Western blot analysis revealed that BMPR1B protein expression in multiple tissues of transgene-positive F1 piglets was 0.5 to 2-fold higher than that in the transgene-negative siblings. The No. 11 boar showed normal litter size performance as normal pigs from the same breed. Transgene-positive F1 boars produced by No. 11 had higher semen volume, sperm concentration and total sperm per ejaculate than the negative siblings, although the differences did not reached statistical significance. Transgene-positive F1 sows had similar litter size performance to the negative siblings, and more data are needed to adequately assess the litter size performance. In conclusion, we obtained 24 cloned transgenic pigs with the modified porcine BMPR1B CDS using HMC. cDNA sequencing and western blot indicated that the exogenous BMPR1B CDS was successfully expressed in host pigs. The transgenic pigs showed normal litter size performance. However, no significant differences in litter size were found between transgene-positive and negative sows. Our study provides new insight into producing cloned transgenic livestock related to reproductive traits.

Isolation and identification of a high molecular weight protein in sow milk.

PubMed

Qin, Y; Qi, N; Tang, Y; He, J; Li, X; Gu, F; Zou, S

2015-05-01

A high molecular weight protein (HMWP) was isolated and purified from sow milk, and some of its biochemical characteristics and biological functions were identified. The origin of HMWP was also investigated. The molecular weight of HMWP was determined to be about 115 000 and 114 800 by SDS-PAGE and gel filtration, respectively. The sequence of 10 amino acids in N-terminal of HMWP was Ala-Leu-Val-Gln-Ser-Cys-Leu-Asn-Leu-Val. The sequence was blasted against GenBank. No protein showed significant similarity with this sequence suggesting the HMWP may be novel. The result of liquid chromatography mass spectrometry (LC-MS) also proved HMWP could be a novel protein. By amino acid assay, HMWP was rich in glutamate (including glutamine), cysteine, glycine, aspartic acid (including asparagines) and proline. The content of hydrophobic amino acids (Ala, Val, Leu, Ile, Met, Phe and Pro) was lower at 18.59% of the total amino acids suggesting HMWP has high solubility in water. Western blots of lectins were used to identify the kinds of carbohydrate residues attached to HMWP qualitatively. The result showed that HMWP was a kind of glycoprotein containing N-acetylneuraminic acid (NeuNAc), mannose (Man) and/or N-acetylglucosamine (GlcNAc). By isoelectric focusing, HMWP pI was found to be 5.1. Compared with milk fat globule membrane protein (MFGMP) isolated from the sow milk in SDS-PAGE, MFGMP did not contain HMWP. HMWP was assumed to be a secretory milk protein. HMWP was not found in bovine, goat, rabbit or human milk in SDS-PAGE gel suggesting HMWP may be unique to sow milk. By Western blot, HMWP could be detected in sow milk, not in sow serum, which suggests it is synthesized and secreted by the mammary gland. HMWP concentrations in sows milk were the lowest in the first day of lactation, rose significantly during lactation 1 to 7 days. The HMWP content of sows milk remained relatively constant ((1.95±0.13) g/l) during lactation 7 to 20 days. HMWP significantly inhibited Escherichia coli in a dose related manner in vitro. Overall, HMWP could be a novel sow milk protein with implications for the mammary gland and the piglet.

Comprehensive microRNA profiling reveals potential augmentation of the IL1 pathway in rheumatic heart valve disease.

PubMed

Lu, Qiyu; Sun, Yi; Duan, Yuyin; Li, Bin; Xia, Jianming; Yu, Songhua; Zhang, Guimin

2018-03-16

Valvular heart disease is a leading cause of cardiovascular mortality, especially in China. More than a half of valvular heart diseases are caused by acute rheumatic fever. microRNA is involved in many physiological and pathological processes. However, the miRNA profile of the rheumatic valvular heart disease is unknown. This research is to discuss microRNAs and their target gene pathways involved in rheumatic heart valve disease. Serum miRNA from one healthy individual and four rheumatic heart disease patients were sequenced. Specific differentially expressed miRNAs were quantified by Q-PCR in 40 patients, with 20 low-to-moderate rheumatic mitral valve stenosis patients and 20 severe mitral valve stenosis patients. The target relationship between certain miRNA and predicted target genes were analysis by Luciferase reporter assay. The IL-1β and IL1R1 expression levels were analyzed by immunohistochemistry and western blot in the mitral valve from surgery of mitral valve replacement. The results showed that 13 and 91 miRNAs were commonly upregulated or downregulated in all four patients. Nine miRNAs, 1 upregulated and 8 downregulated, that had a similar fold change in all 4 patients were selected for quantitative PCR verification. The results showed similar results from miRNA sequencing. Within these 9 tested miRNAs, hsa-miR-205-3p and hsa-miR-3909 showed a low degree of dispersion between the members of each group. Hsa miR-205-3p and hsa-miR-3909 were predicted to target the 3'UTR of IL-1β and IL1R1 respectively. This was verified by luciferase reporter assays. Immunohistochemistry and Western blot results showed that the mitral valve from rheumatic valve heart disease showed higher levels of IL- 1β and IL1R1 expression compared with congenital heart valve disease. This suggested a difference between rheumatic heart valve disease and other types of heart valve diseases, with more inflammatory responses in the former. In the present study, by next generation sequencing of miRNAs, it was revealed that interleukin 1β and interleukin 1 receptor 1 was involved in rheumatic heart diseases. And this is useful for diagnosis and understanding of mechanism of rheumatic heart disease.

A recurrent deletion mutation in OPA1 causes autosomal dominant optic atrophy in a Chinese family

NASA Astrophysics Data System (ADS)

Zhang, Liping; Shi, Wei; Song, Liming; Zhang, Xiao; Cheng, Lulu; Wang, Yanfang; Ge, Xianglian; Li, Wei; Zhang, Wei; Min, Qingjie; Jin, Zi-Bing; Qu, Jia; Gu, Feng

2014-11-01

Autosomal dominant optic atrophy (ADOA) is the most frequent form of hereditary optic neuropathy and occurs due to the degeneration of the retinal ganglion cells. To identify the genetic defect in a family with putative ADOA, we performed capture next generation sequencing (CNGS) to screen known retinal disease genes. However, six exons failed to be sequenced by CNGS in optic atrophy 1 gene (OPA1). Sequencing of those exons identified a 4 bp deletion mutation (c.2983-1_2985del) in OPA1. Furthermore, we sequenced the transcripts of OPA1 from the patient skin fibroblasts and found there is six-nucleotide deletion (c.2984-c.2989, AGAAAG). Quantitative-PCR and Western blotting showed that OPA1 mRNA and its protein expression have no obvious difference between patient skin fibroblast and control. The analysis of protein structure by molecular modeling suggests that the mutation may change the structure of OPA1 by formation of an alpha helix protruding into an existing pocket. Taken together, we identified an OPA1 mutation in a family with ADOA by filling the missing CNGS data. We also showed that this mutation affects the structural intactness of OPA1. It provides molecular insights for clinical genetic diagnosis and treatment of optic atrophy.

MOLECULAR CLONING, SEQUENCING, EXPRESSION AND BIOLOGICAL ACTIVITY OF GIANT PANDA (AILUROPODA MELANOLEUCA) INTERFERON-GAMMA.

PubMed

Zhu, Hui; Wang, Wen-Xiu; Wang, Bao-Qin; Zhu, Xiao-Fu; Wu, Xu-Jin; Ma, Qing-Yi; Chen, De-Kun

2012-06-29

The giant panda (Ailuropoda melanoleuca) is an endangered species and indigenous to China. Interferon-gamma (IFN-γ) is the only member of type □ IFN and is vital for the regulation of host adapted immunity and inflammatory response. Little is known aboutthe FN-γ gene and its roles in giant panda.In this study, IFN-γ gene of Qinling giant panda was amplified from total blood RNA by RT-CPR, cloned, sequenced and analysed. The open reading frame (ORF) of Qinling giant panda IFN-γ encodes 152 amino acidsand is highly similar to Sichuan giant panda with an identity of 99.3% in cDNA sequence. The IFN-γ cDNA sequence was ligated to the pET32a vector and transformed into E. coli BL21 competent cells. Expression of recombinant IFN-γ protein of Qinling giant panda in E. coli was confirmed by SDS-PAGE and Western blot analysis. Biological activity assay indicated that the recombinant IFN-γ protein at the concentration of 4-10 µg/ml activated the giant panda peripheral blood lymphocytes,while at 12 µg/mlinhibited. the activation of the lymphocytes.These findings provide insights into the evolution of giant panda IFN-γ and information regarding amino acid residues essential for their biological activity.

A recurrent deletion mutation in OPA1 causes autosomal dominant optic atrophy in a Chinese family.

PubMed

Zhang, Liping; Shi, Wei; Song, Liming; Zhang, Xiao; Cheng, Lulu; Wang, Yanfang; Ge, Xianglian; Li, Wei; Zhang, Wei; Min, Qingjie; Jin, Zi-Bing; Qu, Jia; Gu, Feng

2014-11-06

Autosomal dominant optic atrophy (ADOA) is the most frequent form of hereditary optic neuropathy and occurs due to the degeneration of the retinal ganglion cells. To identify the genetic defect in a family with putative ADOA, we performed capture next generation sequencing (CNGS) to screen known retinal disease genes. However, six exons failed to be sequenced by CNGS in optic atrophy 1 gene (OPA1). Sequencing of those exons identified a 4 bp deletion mutation (c.2983-1_2985del) in OPA1. Furthermore, we sequenced the transcripts of OPA1 from the patient skin fibroblasts and found there is six-nucleotide deletion (c.2984-c.2989, AGAAAG). Quantitative-PCR and Western blotting showed that OPA1 mRNA and its protein expression have no obvious difference between patient skin fibroblast and control. The analysis of protein structure by molecular modeling suggests that the mutation may change the structure of OPA1 by formation of an alpha helix protruding into an existing pocket. Taken together, we identified an OPA1 mutation in a family with ADOA by filling the missing CNGS data. We also showed that this mutation affects the structural intactness of OPA1. It provides molecular insights for clinical genetic diagnosis and treatment of optic atrophy.

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.

Actin expression in trypanosomatids (Euglenozoa: Kinetoplastea)

PubMed Central

Souza, Ligia Cristina Kalb; Pinho, Rosana Elisa Gonçalves Gonçalves; Lima, Carla Vanessa de Paula; Fragoso, Stênio Perdigão; Soares, Maurilio José

2013-01-01

Heteroxenic and monoxenic trypanosomatids were screened for the presence of actin using a mouse polyclonal antibody produced against the entire sequence of the Trypanosoma cruzi actin gene, encoding a 41.9 kDa protein. Western blot analysis showed that this antibody reacted with a polypeptide of approximately 42 kDa in the whole-cell lysates of parasites targeting mammals (T. cruzi, Trypanosoma brucei and Leishmania major), insects (Angomonas deanei, Crithidia fasciculata, Herpetomonas samuelpessoai and Strigomonas culicis) and plants (Phytomonas serpens). A single polypeptide of approximately 42 kDa was detected in the whole-cell lysates of T. cruzi cultured epimastigotes, metacyclic trypomastigotes and amastigotes at similar protein expression levels. Confocal microscopy showed that actin was expressed throughout the cytoplasm of all the tested trypanosomatids. These data demonstrate that actin expression is widespread in trypanosomatids. PMID:23903980

[Expression, purification and antibody preparation of recombinat SARS-CoV X5 protein].

PubMed

Wang, Li-Na; Kong, Jian-Qiang; Zhu, Ping; Du, Guan-Hua; Wang, Wei; Cheng, Ke-Di

2008-11-01

X5 protein is one of the putative unknown proteins of SARS-CoV. The recombinant protein has been successfully expressed in E. coli in the form of insoluble inclusion body. The inclusion body was dissolved in high concentration of urea. Affinity Chromatography was preformed to purify the denatured protein, and then the product was refolded in a series of gradient solutions of urea. The purified protein was obtained with the purity of > 95% and the yield of 93.3 mg x L(-1). Polyclonal antibody of this protein was obtained, and Western blotting assay indicated that the X5 protein has the strong property of antigen. Sixty-eight percent of the recombinant protein sequence was confirmed by LC-ESI-MS/MS analysis.

Primate-to-Human Retroviral Transmission in Asia

PubMed Central

Engel, Gregory A.; Schillaci, Michael A.; Rompis, Aida; Putra, Artha; Suaryana, Komang Gde; Fuentes, Agustin; Beer, Brigitte; Hicks, Sarah; White, Robert; Wilson, Brenda; Allan, Jonathan S.

2005-01-01

We describe the first reported transmission to a human of simian foamy virus (SFV) from a free-ranging population of nonhuman primates in Asia. The transmission of an exogenous retrovirus, SFV, from macaques (Macaca fascicularis) to a human at a monkey temple in Bali, Indonesia, was investigated with molecular and serologic techniques. Antibodies to SFV were detected by Western blotting of serum from 1 of 82 humans tested. SFV DNA was detected by nested polymerase chain reaction (PCR) from the blood of the same person. Cloning and sequencing of PCR products confirmed the virus's close phylogenetic relationship to SFV isolated from macaques at the same temple. This study raises concerns that persons who work at or live around monkey temples are at risk for infection with SFV. PMID:16022776

Definition of the complete Schistosoma mansoni hemoglobinase mRNA sequence and gene expression in developing parasites.

PubMed

el Meanawy, M A; Aji, T; Phillips, N F; Davis, R E; Salata, R A; Malhotra, I; McClain, D; Aikawa, M; Davis, A H

1990-07-01

Schistosoma mansoni uses a variety of proteases termed hemoglobinases to obtain nutrition from host globin. Previous reports have characterized cDNAs encoding 1 of these enzymes. However, these sequences did not define the primary structures of the mRNA and protein. The complete sequence of the 1390 base mRNA has now been determined. It encodes a 50 kDa primary translation product. In vitro translations coupled with immunoprecipitations and Western blots of parasite lysates allowed visualization of the 50 kDa form. Production of the 31 kDa mature hemoglobinase from the 50 kDa species involves removal of both NH2 and COOH terminal residues from the primary translation product. Expression of hemoglobinase mRNA and protein was examined during larval parasite development. Low levels were observed in young schistosomula. After 6-9 days in culture, high hemoglobinase levels were seen which correlated with the onset of red blood cell feeding. Immunoelectron microscopy was employed to examine hemoglobinase location and function. In adult worms the enzyme was associated with the gut lumen and gut epithelium. In cercariae, the protease was observed in the head gland, suggesting new roles for the protease.

Periplasmic expression of human interferon-alpha 2c in Escherichia coli results in a correctly folded molecule.

PubMed Central

Voss, T; Falkner, E; Ahorn, H; Krystek, E; Maurer-Fogy, I; Bodo, G; Hauptmann, R

1994-01-01

Human interferon-alpha 2c (IFN-alpha 2c) was produced in Escherichia coli under the control of the alkaline phosphatase promoter using a periplasmic expression system. Compared with other leader sequences, the heat-stable enterotoxin II leader of E. coli (STII) resulted in the highest rate of correct processing as judged by Western-blot analysis. The fermentation was designed as a batch-fed process in order to obtain a high yield of biomass. The processing rate of IFN-alpha 2c could be increased from 25% to more than 50% by shifting the fermentation pH from 7.0 to 6.7. IFN-alpha 2c extracted from the periplasm was purified by a new four-step chromatographic procedure. Whereas cytoplasmically produced IFN-alpha 2c does not have its full native structure, IFN-alpha 2c extracted from the periplasm was found to be correctly folded, as shown by c.d. spectroscopy. Peptide-map analysis in combination with m.s. revealed the correct formation of disulphide bridges. N-terminal sequence analysis showed complete removal of the leader sequence, creating the authentic N-terminus starting with cysteine. Images Figure 3 Figure 4 Figure 6 PMID:8141788

Vascular endothelial cells express isoforms of protein kinase A inhibitor.

PubMed

Lum, Hazel; Hao, Zengping; Gayle, Dave; Kumar, Priyadarsini; Patterson, Carolyn E; Uhler, Michael D

2002-01-01

The expression and function of the endogenous inhibitor of cAMP-dependent protein kinase (PKI) in endothelial cells are unknown. In this study, overexpression of rabbit muscle PKI gene into endothelial cells inhibited the cAMP-mediated increase and exacerbated thrombin-induced decrease in endothelial barrier function. We investigated PKI expression in human pulmonary artery (HPAECs), foreskin microvessel (HMECs), and brain microvessel endothelial cells (HBMECs). RT-PCR using specific primers for human PKI alpha, human PKI gamma, and mouse PKI beta sequences detected PKI alpha and PKI gamma mRNA in all three cell types. Sequencing and BLAST analysis indicated that forward and reverse DNA strands for PKI alpha and PKI gamma were of >96% identity with database sequences. RNase protection assays showed protection of the 542 nucleotides in HBMEC and HPAEC PKI alpha mRNA and 240 nucleotides in HBMEC, HPAEC, and HMEC PKI gamma mRNA. Western blot analysis indicated that PKI gamma protein was detected in all three cell types, whereas PKI alpha was found in HBMECs. In summary, endothelial cells from three different vascular beds express PKI alpha and PKI gamma, which may be physiologically important in endothelial barrier function.

An ectromelia virus profilin homolog interacts with cellular tropomyosin and viral A-type inclusion protein.

PubMed

Butler-Cole, Christine; Wagner, Mary J; Da Silva, Melissa; Brown, Gordon D; Burke, Robert D; Upton, Chris

2007-07-24

Profilins are critical to cytoskeletal dynamics in eukaryotes; however, little is known about their viral counterparts. In this study, a poxviral profilin homolog, ectromelia virus strain Moscow gene 141 (ECTV-PH), was investigated by a variety of experimental and bioinformatics techniques to characterize its interactions with cellular and viral proteins. Profilin-like proteins are encoded by all orthopoxviruses sequenced to date, and share over 90% amino acid (aa) identity. Sequence comparisons show highest similarity to mammalian type 1 profilins; however, a conserved 3 aa deletion in mammalian type 3 and poxviral profilins suggests that these homologs may be more closely related. Structural analysis shows that ECTV-PH can be successfully modelled onto both the profilin 1 crystal structure and profilin 3 homology model, though few of the surface residues thought to be required for binding actin, poly(L-proline), and PIP2 are conserved. Immunoprecipitation and mass spectrometry identified two proteins that interact with ECTV-PH within infected cells: alpha-tropomyosin, a 38 kDa cellular actin-binding protein, and the 84 kDa product of vaccinia virus strain Western Reserve (VACV-WR) 148, which is the truncated VACV counterpart of the orthopoxvirus A-type inclusion (ATI) protein. Western and far-western blots demonstrated that the interaction with alpha-tropomyosin is direct, and immunofluorescence experiments suggest that ECTV-PH and alpha-tropomyosin may colocalize to structures that resemble actin tails and cellular protrusions. Sequence comparisons of the poxviral ATI proteins show that although full-length orthologs are only present in cowpox and ectromelia viruses, an ~ 700 aa truncated ATI protein is conserved in over 90% of sequenced orthopoxviruses. Immunofluorescence studies indicate that ECTV-PH localizes to cytoplasmic inclusion bodies formed by both truncated and full-length versions of the viral ATI protein. Furthermore, colocalization of ECTV-PH and truncated ATI protein to protrusions from the cell surface was observed. These results suggest a role for ECTV-PH in intracellular transport of viral proteins or intercellular spread of the virus. Broader implications include better understanding of the virus-host relationship and mechanisms by which cells organize and control the actin cytoskeleton.

Automated hybridization/imaging device for fluorescent multiplex DNA sequencing

DOEpatents

Weiss, R.B.; Kimball, A.W.; Gesteland, R.F.; Ferguson, F.M.; Dunn, D.M.; Di Sera, L.J.; Cherry, J.L.

1995-11-28

A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, the enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots. 9 figs.

Automated hybridization/imaging device for fluorescent multiplex DNA sequencing

DOEpatents

Weiss, Robert B.; Kimball, Alvin W.; Gesteland, Raymond F.; Ferguson, F. Mark; Dunn, Diane M.; Di Sera, Leonard J.; Cherry, Joshua L.

1995-01-01

A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, then an enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots.

A prokaryotic viral sequence is expressed and conserved in mammalian brain.

PubMed

Yeh, Yang-Hui; Gunasekharan, Vignesh; Manuelidis, Laura

2017-07-03

A natural and permanent transfer of prokaryotic viral sequences to mammals has not been reported by others. Circular "SPHINX" DNAs <5 kb were previously isolated from nuclease-protected cytoplasmic particles in rodent neuronal cell lines and brain. Two of these DNAs were sequenced after Φ29 polymerase amplification, and they revealed significant but imperfect homology to segments of commensal Acinetobacter phage viruses. These findings were surprising because the brain is isolated from environmental microorganisms. The 1.76-kb DNA sequence (SPHINX 1.8), with an iteron before its ORF, was evaluated here for its expression in neural cells and brain. A rabbit affinity purified antibody generated against a peptide without homology to mammalian sequences labeled a nonglycosylated ∼41-kDa protein (spx1) on Western blots, and the signal was efficiently blocked by the competing peptide. Spx1 was resistant to limited proteinase K digestion, but was unrelated to the expression of host prion protein or its pathologic amyloid form. Remarkably, spx1 concentrated in selected brain synapses, such as those on anterior motor horn neurons that integrate many complex neural inputs. SPHINX 1.8 appears to be involved in tissue-specific differentiation, including essential functions that preserve its propagation during mammalian evolution, possibly via maternal inheritance. The data here indicate that mammals can share and exchange a larger world of prokaryotic viruses than previously envisioned.

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.

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

[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.

[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.

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

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.

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.

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

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.

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.

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.

Analysis of Chromatin Organisation

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2011-01-01

Terms to be familiar with before you start to solve the test: chromatin, nucleases, sucrose density gradient centrifugation, melting point, gel electrophoresis, ethidium bromide, autoradiography, Southern blotting, Northern blotting, Sanger sequencing, restriction endonucleases, exonucleases, linker DNA, chloroform extraction, nucleosomes,…

Transgenic loblolly pine (Pinus taeda L.) plants expressing a modified delta-endotoxin gene of Bacillus thuringiensis with enhanced resistance to Dendrolimus punctatus Walker and Crypyothelea formosicola Staud.

PubMed

Tang, Wei; Tian, Yingchuan

2003-02-01

A synthetic version of the CRY1Ac gene of Bacillus thuringiensis has been used for the transformation of loblolly pine (Pinus taeda L.) using particle bombardment. Mature zygotic embryos were used to be bombarded and to generate organogenic callus and transgenic regenerated plants. Expression vector pB48.215 DNA contained a synthetic Bacillus thuringiensis (B.t.) CRY1Ac coding sequence flanked by the double cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator sequences, and the neomycin phosphotransferase II (NPTII) gene controlled by the promoter of the nopaline synthase gene was introduced into loblolly pine tissues by particle bombardment. The transformed tissues were proliferated and selected on media with kanamycin. Shoot regeneration was induced from the kanamycin-resistant calli, and transgenic plantlets were then produced. More than 60 transformed plants from independent transformation events were obtained for each loblolly pine genotype tested. The integration and expression of the introduced genes in the transgenic loblolly pine plants was confirmed by polymerase chain reactions (PCR) analysis, by Southern hybridization, by Northern blot analysis, and by Western blot analysis. Effective resistance of transgenic plants against Dendrolimus punctatus Walker and Crypyothelea formosicola Staud was verified in feeding bioassays with the insects. The transgenic plants recovered could represent a good opportunity to analyse the impact of genetic engineering of pine for sustainable resistance to pests using a B. thuringiensis insecticidal protein. This protocol enabled the routine transformation of loblolly pine plants that were previously difficult to transform.

A kind of rd1 mouse in C57BL/6J mice from crossing with a mutated Kunming mouse.

PubMed

Yan, Weiming; Yao, Lu; Liu, Wei; Sun, Kai; Zhang, ZuoMing; Zhang, Lei

2017-04-05

We occasionally discovered a mouse with spontaneous retinitis pigmentosa (RP) from Kunming (KM) mouse breeding colony, with no obvious waveforms in ERG recordings. The aim of this study is to cross the spontaneously hereditary retinal degeneration mice (temporarily designated as KM/rd mice) derived from KM mice with C57BL/6J mice to establish a congenic inbred strain (temporarily designated as the B6/rd mice), and study the ocular phenotype and genotype of the mice. Fundus photography, tissue morphology, electroretinography (ERG), qRT-PCR, western blot and DNA sequence analysis were performed to observe the ocular phenotype and genotype of KM/rd and B6/rd mice. The fundus photography showed progressive retinal vascular degeneration and depigmentation in KM/rd and B6/rd mice. Compared to wild-type mice, the histological analysis revealed that the outer nuclear layer of the mutated mice was significantly reduced at 14days post born (P14), and almost disappeared by P21. No obvious waveforms were detected at P14 and P21 in the ERG from KM/rd and B6/rd mice. qRT-PCR results showed that the expression quantities of mRNA of pde6b gene in KM/rd and B6/rd mice were significantly lower compared with those of wild-type controls at P21. Western blot results confirmed an abnormal protein expression of pde6b gene in KM/rd and B6/rd mice with no protein products, while there was an obvious protein expression in wild-type mice. The nonsense mutation in exon 7 (a mutation that changes the codon 347 from TAC to TAA) in the pde6b gene of KM/rd and B6/rd mice was identified by genomic DNA sequence analysis. All these findings revealed that the ocular phenotype and genotype of KM/rd and B6/rd mice were similar to those of rd1 mice, which indicates that KM/rd and B6/rd mice can be used as an RP mouse model. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

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.

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.

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

Strains of Sarcocystis neurona exhibit differences in their surface antigens, including the absence of the major surface antigen SnSAG1.

PubMed

Howe, Daniel K; Gaji, Rajshekhar Y; Marsh, Antoinette E; Patil, Bhagyashree A; Saville, William J; Lindsay, David S; Dubey, J P; Granstrom, David E

2008-05-01

A gene family of surface antigens is expressed by merozoites of Sarcocystis neurona, the primary cause of equine protozoal myeloencephalitis (EPM). These surface proteins, designated SnSAGs, are immunodominant and therefore excellent candidates for development of EPM diagnostics or vaccines. Prior work had identified an EPM isolate lacking the major surface antigen SnSAG1, thus suggesting there may be some diversity in the SnSAGs expressed by different S. neurona isolates. Therefore, a bioinformatic, molecular and immunological study was conducted to assess conservation of the SnSAGs. Examination of an expressed sequence tag (EST) database revealed several notable SnSAG polymorphisms. In particular, the EST information implied that the EPM strain SN4 lacked the major surface antigen SnSAG1. The absence of this surface antigen from the SN4 strain was confirmed by both Western blot and Southern blot. To evaluate SnSAG polymorphisms in the S. neurona population, 14 strains were examined by Western blots using monospecific polyclonal antibodies against the four described SnSAGs. The results of these analyses demonstrated that SnSAG2, SnSAG3, and SnSAG4 are present in all 14 S. neurona strains tested, although some variance in SnSAG4 was observed. Importantly, SnSAG1 was not detected in seven of the strains, which included isolates from four cases of EPM and a case of fatal meningoencephalitis in a sea otter. Genetic analyses by PCR using gene-specific primers confirmed the absence of the SnSAG1 locus in six of these seven strains. Collectively, the data indicated that there is heterogeneity in the surface antigen composition of different S. neurona isolates, which is an important consideration for development of serological tests and prospective vaccines for EPM. Furthermore, the diversity reported herein likely extends to other phenotypes, such as strain virulence, and may have implications for the phylogeny of the various Sarcocystis spp. that undergo sexual stages of their life cycle in opossums.

Proteomic identification of plant proteins probed by mammalian nitric oxide synthase antibodies.

PubMed

Butt, Yoki Kwok-Chu; Lum, John Hon-Kei; Lo, Samuel Chun-Lap

2003-03-01

Several studies suggest that a mammalian-like nitric oxide synthase (NOS) exists in plants. Researchers have attempted to verify its presence using two approaches: (i) determination of NOS functional activity and (ii) probing with mammalian NOS antibodies. However, up to now, neither a NOS-like gene nor a protein has been found in plants. While there is still some controversy over whether the NOS functional activity seen is due to nitrate reductase, using the mammalian NOS antibodies in western blot analysis, several groups have reported the presence of immunoreactive protein bands in plant homogenates. Based on these results, immunohistochemical studies using these antibodies have also been used to localize NOS in plant tissues. However, plant NOS has never been positively identified or characterized. Thus, we used a proteomic approach to verify the identities of plant proteins that cross-reacted with the mammalian NOS antibodies. Proteins extracted from maize (Zea mays L.) embryonic axes were separated by two-dimensional gel electrophoresis and subjected to western blot analysis with the mammalian neuronal NOS and inducible NOS antibodies. Twenty immunoreactive protein spots recognized on a corresponding Coomassie blue-stained two-dimensional gel were subjected to tryptic digestion, followed by identification using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Fifteen proteins were successfully identified and they have described functions that are unrelated to NO metabolism. The remaining five proteins could not be identified. The amino acid sequences of these identified proteins and those used to raise the antibodies were aligned. However, no homologous region could be found. Our results demonstrate that the mammalian NOS antibodies recognize many NOS-unrelated plant proteins. Therefore, it is inappropriate to infer the presence of plant NOS using this immunological technique.

Helicobacter pylori HopE and HopV porins present scarce expression among clinical isolates

PubMed Central

Lienlaf, Maritza; Morales, Juan Pablo; Díaz, María Inés; Díaz, Rodrigo; Bruce, Elsa; Siegel, Freddy; León, Gloria; Harris, Paul R; Venegas, Alejandro

2010-01-01

AIM: To evaluate how widely Helicobacter pylori (H. pylori) HopE and HopV porins are expressed among Chilean isolates and how seroprevalent they are among infected patients in Chile. METHODS: H. pylori hopE and hopV genes derived from strain CHCTX-1 were cloned by polymerase chain reaction (PCR), sequenced and expressed in Escherichia coli AD494 (DE3). Gel-purified porins were used to prepare polyclonal antibodies. The presence of both genes was tested by PCR in a collection of H. pylori clinical isolates and their expression was detected in lysates by immunoblotting. Immune responses against HopE, HopV and other H. pylori antigens in sera from infected and non-infected patients were tested by Western blotting using these sera as first antibody on recombinant H. pylori antigens. RESULTS: PCR and Western blotting assays revealed that 60 and 82 out of 130 Chilean isolates carried hopE and hopV genes, respectively, but only 16 and 9, respectively, expressed these porins. IgG serum immunoreactivity evaluation of 69 H. pylori-infected patients revealed that HopE and HopV were infrequently recognized (8.7% and 10.1% respectively) compared to H. pylori VacA (68.1%) and CagA (59.5%) antigens. Similar values were detected for IgA serum immunoreactivity against HopE (11.6%) and HopV (10.5%) although lower values for VacA (42%) and CagA (17.4%) were obtained when compared to the IgG response. CONCLUSION: A scarce expression of HopE and HopV among Chilean isolates was found, in agreement with the infrequent seroconversion against these antigens when tested in infected Chilean patients. PMID:20082477

Novel mutations in the Wiskott-Aldrich syndrome protein gene and their effects on transcriptional, translational, and clinical phenotypes.

PubMed

Lemahieu, V; Gastier, J M; Francke, U

1999-01-01

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive immunodeficiency characterized by thrombocytopenia, eczema, and recurrent infections, and caused by mutations in the WAS protein (WASP) gene. WASP contains several functional domains through which it interacts with proteins involved in intracellular signaling and regulation of the actin cytoskeleton. In this report, 17 WASP gene mutations were identified, 12 of which are novel. DNA of affected males and obligate carriers was PCR amplified and analyzed by SSCA, heteroduplex analysis, and direct sequencing. The effects of the mutations at the mRNA and protein level were ascertained by RT-PCR and Western blot analyses. All missense mutations were located in exons 1-4. Most of the nonsense, frameshift and splice site mutations were found in exons 6-11. Mutations that alter splice sites led to the synthesis of several types of mRNAs, a fraction of which represented the normally spliced product. The presence of normally spliced transcripts was correlated with a milder phenotype. When one such case was studied by Western blotting, reduced amounts of normal-size WASP were present. In other cases as well, a correlation was found between the amount of normal or mutant WASP present and the phenotypes of the affected individuals. No protein was detected in two individuals with severe WAS. Reduced levels of a normal-size WASP with a missense mutation were seen in two individuals with XLT. It is concluded that mutation analysis at the DNA level is not sufficient for predicting clinical course. Studies at the transcript and protein level are needed for a better assessment.

Identification, expression and phylogenetic analysis of EgG1Y162 from Echinococcus granulosus

PubMed Central

Zhang, Fengbo; Ma, Xiumin; Zhu, Yuejie; Wang, Hongying; Liu, Xianfei; Zhu, Min; Ma, Haimei; Wen, Hao; Fan, Haining; Ding, Jianbing

2014-01-01

Objective: This study was to clone, identify and analyze the characteristics of egG1Y162 gene from Echinococcus granulosus. Methods: 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. Results: 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%. Conclusion: 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. PMID:25337206

Characterization of the immune response of domestic fowl following immunization with proteins extracted from Dermanyssus gallinae.

PubMed

Harrington, David; Din, Hatem Mohi El; Guy, Jonathan; Robinson, Karen; Sparagano, Olivier

2009-03-23

Dermanyssus gallinae is the most significant ectoparasite of European poultry egg laying production systems due to high costs of control and associated production losses as well as adverse effects on bird welfare. In this study, soluble proteins were extracted from unfed D. gallinae (DGE) using a urea-based detergent and ultra-filtration, passed through a 0.22 microm filter and blended aseptically with adjuvant. One group of laying hens was immunized with DGE and adjuvant (Montanide ISA 50 V) whilst another group (Control) received physiological saline and adjuvant. All birds were immunized on two occasions, 21 days apart. Antibody response to immunization was determined by ELISA and western blotting using immunoglobulins (Igs) extracted from egg yolk. DGE immunization of hens resulted in a significant (P<0.05) IgY response compared to controls, although there was no significant difference in IgM response between treatments. A number of proteins were identified by western blotting using IgY antibodies from DGE immunized birds, most prominently at 40 and 230kDa. Analysis of proteins from approximately corresponding bands on SDS-PAGE confirmed the identity of tropomyosin, whilst other proteins showed high sequence homology with myosin and actin from other arachnid and insect species. Immunization of hens with DGE resulted in a 50.6% increase in mite mortality (P<0.001) 17h after feeding when tested by an in vitro mite feeding model. Data in this study demonstrate that somatic antigens from D. gallinae can be used to stimulate a protective immune response in laying hens. Further work is needed to identify other proteins of interest that could confer higher protection against D. gallinae, as well as optimization of the vaccination and in vitro testing protocol.

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.

[Expression and identification of eukaryotic expression vectors of Brucella melitensis lipoprotein OMP19].

PubMed

He, Zuoping; Luo, Peifang; Hu, Feihuan; Weng, Yunceng; Wang, Wenjing; Li, Chengyao

2016-04-01

To construct eukaryotic expression vectors carrying Brucella melitensis outer membrane protein 19 (OMP19), express them in transfected Huh7.5.1 and JEG-3 cells, and analyze their role in cell apoptosis. Brucella melitensis lipidated OMP19 (L-OMP19) gene and unlipidated OMP19 (U-OMP19) gene were amplified by PCR and inserted into the vector pZeroBack/blunt. The correct L-OMP19 and U-OMP19 genes verified by XbaI and BamHI double digestion and sequencing were cloned into the lentivirus expression vector pHAGE-CMV-MCS-IZsGreen to construct vectors pHAGE-L-OMP19 and pHAGE-U-OMP19, which were separately transfected into 293FT cells, Huh7.5.1 and JEG-3 cells. L-OMP19 and U-OMP19 in the cells were detected by Western blotting and immunofluorescence technique. Flow cytometry combined with annexin V-PE/7-AAD staining was used to detect the cell apoptosis. The lentiviral vectors pHAGE-L-OMP19 and pHAGE-U-OMP19 were constructed correctly and the recombinant lipoproteins L-OMP19 and U-OMP19 expressed in the above cells were well recognized by the specific antibodies against L-OMP19 in Western blotting and immunofluorescence technique. L-OMP19 and U-OMP19 induced JEG-3 cell death, but did not induce the apoptosis of Huh7.5.1 cells. The eukaryotic expression vectors of L-OMP19 and U-OMP19 have been constructed successfully. Recombinant lipoproteins L-OMP19 and U-OMP19 expressed in cells have a good antigenicity, which could be used as experimental materials for the research on the relationship between host cells and lipoproteins in Brucella infection.

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.

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.

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.

[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.

Characterisation of single domain ATP-binding cassette protien homologues of Theileria parva.

PubMed

Kibe, M K; Macklin, M; Gobright, E; Bishop, R; Urakawa, T; ole-MoiYoi, O K

2001-09-01

Two distinct genes encoding single domain, ATP-binding cassette transport protein homologues of Theileria parva were cloned and sequenced. Neither of the genes is tandemly duplicated. One gene, TpABC1, encodes a predicted protein of 593 amino acids with an N-terminal hydrophobic domain containing six potential membrane-spanning segments. A single discontinuous ATP-binding element was located in the C-terminal region of TpABC1. The second gene, TpABC2, also contains a single C-terminal ATP-binding motif. Copies of TpABC2 were present at four loci in the T. parva genome on three different chromosomes. TpABC1 exhibited allelic polymorphism between stocks of the parasite. Comparison of cDNA and genomic sequences revealed that TpABC1 contained seven short introns, between 29 and 84 bp in length. The full-length TpABC1 protein was expressed in insect cells using the baculovirus system. Application of antibodies raised against the recombinant antigen to western blots of T. parva piroplasm lysates detected an 85 kDa protein in this life-cycle stage.

The effect of growth medium salinity of Photobacterium damselae subsp. piscicida on the immune response of hybrid bass (Morone saxatilis x M. chrysops).

PubMed

Nitzan, S; Shwartsburd, B; Heller, E D

2004-02-01

Photobacterium damselae subsp. piscicida (P. damselae) was grown on various media and the effect of media salinity on certain immune responses of hybrid bass was studied. In Israel, pasteurellosis outbreaks have not been reported at water salinities below 1.38 per thousand. During vaccination experiments the salinity of the medium on which P. damselae is grown, was shown to affect stimulation of the immune system. No correlation was found between antibody response and protection. Bacterial envelopes separated by electrophoresis and subjected to western blot analysis revealed an antibody response against some protein bands. Band sequencing was performed to identify the protein stimulating the immune response. Sequence identity of 80% was seen in 10-amino-acid overlap of the 36-kDa band with a specific gene of alkalophilic Bacillus firmus. A preparation of P. damselae grown in a 2.5% NaCl medium at 25 degrees C is the most effective vaccine against pasteurellosis, providing hybrid bass with quite good protection.

The recombinant expression and activity detection of MAF-1 fusion protein.

PubMed

Fu, Ping; Wu, Jianwei; Gao, Song; Guo, Guo; Zhang, Yong; Liu, Jian

2015-10-01

This study establishes the recombinant expression system of MAF-1 (Musca domestica antifungal peptide-1) and demonstrates the antifungal activity of the expression product and shows the relationship between biological activity and structure. The gene segments on mature peptide part of MAF-1 were cloned, based on the primers designed according to the cDNA sequence of MAF-1. We constructed the recombinant prokaryotic expression plasmid using prokaryotic expression vector (pET-28a(+)) and converted it to the competent cell of BL21(DE3) to gain recombinant MAF-1 fusion protein with His tag sequence through purifying affinity chromatographic column of Ni-NTA. To conduct the Western Blotting test, recombinant MAF-1 fusion protein was used to produce the polyclonal antibody of rat. The antifungal activity of the expression product was detected using Candida albicans (ATCC10231) as the indicator. The MAF-1 recombinant fusion protein was purified to exhibit obvious antifungal activity, which lays the foundation for the further study of MAF-1 biological activity, the relationship between structure and function, as well as control of gene expression.

Complete nucleotide sequence and genome organization of a Chinese isolate of Tobacco vein distorting virus.

PubMed

Mo, Xiao-han; Chen, Zheng-bin; Chen, Jian-ping

2010-12-01

Tobacco bushy top disease is caused by tobacco bushy top virus (TBTV, a member of the genus Umbravirus) which is dependent on tobacco vein-distorting virus (TVDV) to act as a helper virus encapsidating TBTV and enabling its transmission by aphids. Isometric virions from diseased tobacco plants were purified and disease symptoms were reproduced after experimental aphid transmission. The complete genome of TVDV was determined from cloned RT-PCR products derived from viral RNA. It was 5,920 nucleotides (nts) long and had the six major open reading frames (ORFs) typical of a member of the genus Polerovirus. Sequence comparisons showed that it differed significantly from any of the other species in the genus and this was confirmed by phylogenetic analyses of the RdRp and coat protein. SDS-PAGE analysis of purified virions gave two protein bands of about 26 and 59 kDa both of which reacted strongly in Western blots with antiserum produced to prokaryotically expressed TVDV CP showing that the two forms of the TVDV CP were the only protein components of the capsid.

Analyses of expression and localization of two mammalian-type transglutaminases in Physarum polycephalum, an acellular slime mold.

PubMed

Wada, Fumitaka; Ogawa, Atsuko; Hanai, Yuko; Nakamura, Akio; Maki, Masatoshi; Hitomi, Kiyotaka

2004-11-01

Transglutaminase (TGase) is an enzyme that modifies proteins by crosslinking or polyamination. Physarum polycephalum, an acellular slime mold, is the evolutionally lowest organism that has a mammalian-type transglutaminase. We have cloned a cDNA for Physarum polycephalum TGase (PpTGB), homologous to a previously identified TGase (PpTGA), whose sequence is similar to that of mammalian TGases. PpTGB encodes a primary sequence identical to that of PpTGA except for 11 amino acid residues at the N-terminus. Reverse transcription-PCR and Western blotting analyses showed that both PpTGA and PpTGB are expressed in microplasmodia and macroplasmodia during their life cycle, except for in sporangia. For biochemical characterization, we carried out the ectopical expressions of PpTGA and PpTGB in Dictyostelium discoideum. Subcellular fractionation of these Dictyostelium cells showed that the expressed PpTGA, but not PpTGB, localizes to the membrane fraction. Furthermore, in Physarum, subcellular fractionation and immunostaining indicated specific localization at the plasma membrane in macroplasmodia, while the localization was entirely cytoplasmic in microplasmodia.

Properties of an equine herpesvirus 1 mutant devoid of the internal inverted repeat sequence of the genomic short region

PubMed Central

Ahn, ByungChul; Zhang, Yunfei; Osterrieder, Nikolaus; O'Callaghan, Dennis J.

2010-01-01

The 150 kbp genome of equine herpesvirus -1 (EHV-1) is composed of a unique long (UL) region and a unique short (Us) segment, which is flanked by identical internal and terminal repeat (IR and TR) sequences of 12.7kbp. We constructed an EHV-1 lacking the entire IR (vL11ΔIR) and showed that the IR is dispensable for EHV-1 replication but that the vL11ΔIR exhibits a smaller plaque size and delayed growth kinetics. Western blot analyses of cells infected with vL11ΔIR showed that the synthesis of viral proteins encoded by the immediate-early, early, and late genes was reduced at immediate-early and early times, but by late stages of replication reached wild type levels. Intranasal infection of CBA mice revealed that the vL11ΔIR was significantly attenuated as mice infected with the vL11ΔIR showed a reduced lung viral titer and greater ability to survive infection compared to mice infected with parental or revertant virus. PMID:21176938

Generation of the novel monoclonal antibody against TLS/EWS-CHOP chimeric oncoproteins that is applicable to one of the most sensitive assays for myxoid and round cell liposarcomas.

PubMed

Oikawa, Kosuke; Ishida, Tsuyoshi; Imamura, Tetsuo; Yoshida, Keiichi; Takanashi, Masakatsu; Hattori, Hiroyuki; Ishikawa, Akio; Fujita, Koji; Yamamoto, Kengo; Matsubayashi, Jun; Kuroda, Masahiko; Mukai, Kiyoshi

2006-03-01

The fusion oncoproteins, TLS-CHOP and EWS-CHOP, are characteristic markers for myxoid and round cell liposarcomas (MLS/RCLS). Especially, the peptide sequence of 26 amino acids corresponding to the normally untranslated CHOP exon 2 and parts of exon 3 (5'-UTR) is a unique structure for these chimeric proteins. In this report, we have generated monoclonal antibodies against the unique peptide sequence of TLS/EWS-CHOP oncoproteins. These antibodies reacted with TLS-CHOP fusion protein, but not reacted with normal TLS and CHOP proteins by Western blot analysis. In addition, one of the antibodies also recognized the chimeric oncoprotein in archival paraffin-embedded tissue samples of MLS/RCLS. The oncoprotein was detectable by the antibody even in the paraffin-embedded tissue samples whose mRNAs were too degraded to be detected by a nested reverse transcription-polymerase chain reaction-based assay. Thus, the molecular assay using the novel antibody is expected to be one of the most sensitive diagnostic assays for MLS/RCLS.

Detection of Sirtuin-1 protein expression in peripheral blood leukocytes in dogs.

PubMed

Yoshimura, Kuniko; Matsuu, Aya; Sasaki, Kai; Momoi, Yasuyuki

2018-05-11

Sirtuin-1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD + )-dependent histone deacetylase with a large number of protein substrates. It has attracted a lot of attention in association with extending lifespan. The objective of this study was to enable the evaluation of SIRT1 expression in peripheral blood mononuclear cells (PBMCs) from dogs by flow cytometry. Three transcript variants were amplified from PBMCs by reverse transcription PCR and the nucleotide sequences were analyzed. On the basis deduced amino acid sequence, a monoclonal antibody against human SIRT1, 1F3, was selected to detect canine SIRT1. Canine SIRT1 in peripheral blood mononuclear cells was successfully detected by western blotting using this antibody. Intracellular canine SIRT1 was also detected in permeabilized 293T cells transfected with a canine SIRT1 expression plasmid by flow cytometry using this antibody. SIRT1 was detected in all leukocyte subsets including lymphocytes, granulocytes and monocytes. The expression level was markedly different among individual dogs. These results indicated that the method applied in this study is useful for evaluating canine SIRT1 levels in PBMCs from dogs.

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

Comparison of Multispot EIA with Western blot for confirmatory serodiagnosis of HIV.

PubMed

Torian, Lucia V; Forgione, Lisa A; Punsalang, Amado E; Pirillo, Robert E; Oleszko, William R

2011-12-01

Recent improvements in the sensitivity of immunoassays (IA) used for HIV screening, coupled with increasing recognition of the importance of rapid point-of-care testing, have led to proposals to adjust the algorithm for serodiagnosis of HIV so that screening and confirmation can be performed using a dual or triple IA sequence that does not require Western blotting for confirmation. One IA that has been proposed as a second or confirmatory test is the Bio-Rad Multispot(®) Rapid HIV-1/HIV-2 Test. This test would have the added advantage of differentiating between HIV-1 and HIV-2 antibodies. To compare the sensitivity and type-specificity of an algorithm combining a 3rd generation enzyme immunoassay (EIA) followed by a confirmatory Multispot with the conventional algorithm that combines a 3rd generation EIA (Bio-Rad GS HIV-1/HIV-2 Plus O EIA) followed by confirmatory Western blot (Bio-Rad GS HIV-1 WB). 8760 serum specimens submitted for HIV testing to the New York City Public Health Laboratory between May 22, 2007, and April 30, 2010, tested repeatedly positive on 3rd generation HIV-1-2+O EIA screening and received parallel confirmatory testing by WB and Multispot (MS). 8678/8760 (99.1%) specimens tested WB-positive; 82 (0.9%) tested WB-negative or indeterminate (IND). 8690/8760 specimens (99.2%) tested MS-positive, of which 14 (17.1%) had been classified as negative or IND by WB. Among the HIV-1 WB-positive specimens, MS classified 26 (0.29%) as HIV-2. Among the HIV-1 WB negative and IND, MS detected 12 HIV-2. MS detected an additional 14 HIV-1 infections among WB negative or IND specimens, differentiated 26 HIV-1 WB positives as HIV-2, and detected 12 additional HIV-2 infections among WB negative/IND. A dual 3rd generation EIA algorithm incorporating MS had equivalent HIV-1 sensitivity to the 3rd generation EIA-WB algorithm and had the added advantage of detecting 12 HIV-2 specimens that were not HIV-1 WB cross-reactors. In this series an algorithm using EIA followed by MS would have resulted in the expedited referral of 38 specimens for HIV-2 testing and 40 specimens for nucleic acid confirmation. Further testing using a combined gold standard of nucleic acid detection and WB is needed to calculate specificity and validate the substitution of MS for WB in the diagnostic algorithm used by a large public health laboratory. Copyright © 2011. Published by Elsevier B.V.

Up-regulation of VEZT by small activating RNA inhibits the proliferation, invasion and migration of gastric cancer cells

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

Xie, Detian; Shang, Liang; Peng, Lipan

Objective: To identify an effective saRNA sequence that can specifically up-regulate VEZT expression and to determine the influence of saRNA had on gastric cancer cell growth, proliferation, invasion and migration. Methods: Three various saRNAs, that target the VEZT gene promoter at different locations relative to the transcription start site were synthesized. A dsControl saRNA was synthesized as a negative control, and a specific shRNA was synthesized to knockdown VEZT and eliminate any off-target effects of the saRNA. Both SGC-7901 and M-28 cells were either transfected with the different saRNAs, or treated with Lipofectamine2000 alone. To determine the most effective saRNA, real-timemore » PCR and Western blot were used to determine the VEZT mRNA and protein content, respectively, of each treatment group. After selection, both cell lines were treated with the chosen saRNA, dsControl or Lipofectamine2000. The saRNA treated cells were divided into two groups: the first group was used immediately in the experiments, and the second group was transfected with shRNA by using RNAi-Mate. The proliferation of cells transfected with saRNA, or saRNA and shRNA, as well as the other control cells, was detected by CCK-8. The invasive and migratory abilities were determined using the transwell chamber assay. Results: We identified the most effective saRNA via real-time PCR and Western blot. The selected saRNA inhibited the growth, invasion and migration of GC cells by specially reactivating VEZT. The real-time PCR and Western blot results showed that treatment with saRNA caused a significant up-regulation of VEZT, and an obvious decrease in the proliferative, invasive and migratory abilities; compared with the control groups (P < 0.01); furthermore, there were no significant differences among the control groups (P > 0.05). This phenomenon provides a theoretical basis for saRNA design and gene therapy for gastric cancer. - Highlights: • saRNA is a brand-new type of small non-coding RNA. It provides a brand-new therapeutic tool for gastric cancer. • In this manuscript, we designed the new saRNA that was effective for VEZT for the first time. • VEZT is a new TSG, and the reactivation through saRNA is never reported before. • It's the first time to report the RNAa in gastric cancer.« less

Isolation and characterization of a c-type lysozyme from the nurse shark.

PubMed

Hinds Vaughan, Nichole; Smith, Sylvia L

2013-12-01

Lysozyme is a ubiquitous antibacterial enzyme that occurs in numerous invertebrate and vertebrate species. Three forms have been described c-type, g-type and i-type which differ in primary structure. Shark lysozyme has not been characterized; here we report on the isolation and characterization of lysozyme from unstimulated shark (Ginglymostoma cirratum) leukocytes and provide amino acid sequence data across the highly conserved active site of the molecule identifying it to be a c-type lysozyme. A leukocyte lysate was applied either (a) to the first of two sequential DE-52 cellulose columns or alternatively, (b) to a DEAE-Sepharose column. Lysozyme activity in lysate and active fractions was identified by zones of lysis of Micrococcus lysodeikticus cell walls on lysoplates and zones of growth inhibition in agar diffusion assays using Planococcus citreus as the target organism. SDS-PAGE analysis revealed a 14 kDa protein which was identified as lysozyme by mass spectroscopic analysis of peptides, reactivity against anti-HEWL antibodies on a Western blot, hydrolysis of M. lysodeikticus cell walls, and inhibition of growth of P. citreus on AU-gel blots in which the area of growth inhibition correlated to a 14 kDa protein. Copyright © 2013 Elsevier Ltd. All rights reserved.

Stability of transgene integration and expression in subsequent generations of doubled haploid oilseed rape transformed with chitinase and beta-1,3-glucanase genes in a double-gene construct.

PubMed

Melander, Margareta; Kamnert, Iréne; Happstadius, Ingrid; Liljeroth, Erland; Bryngelsson, Tomas

2006-09-01

A double-gene construct with one chitinase and one beta-1,3-glucanase gene from barley, both driven by enhanced 35S promoters, was transformed into oilseed rape. From six primary transformants expressing both transgenes 10 doubled haploid lines were produced and studied for five generations. The number of inserted copies for both the genes was determined by Southern blotting and real-time PCR with full agreement between the two methods. When copy numbers were analysed in different generations, discrepancies were found, indicating that at least part of the inserted sequences were lost in one of the alleles of some doubled haploids. Chitinase and beta-1,3-glucanase expression was analysed by Western blotting in all five doubled haploid generations. Despite that both the genes were present on the same T-DNA and directed by the same promoter their expression pattern between generations was different. The beta-1,3-glucanase was expressed at high and stable levels in all generations, while the chitinase displayed lower expression that varied between generations. The transgenic plants did not show any major impact on fungal resistance when assayed in greenhouse, although purified beta-1,3-glucanase and chitinase caused retardment of fungal growth in vitro.

Identification of interleukin-8 converting enzyme as cathepsin L.

PubMed

Ohashi, Kensaku; Naruto, Masanobu; Nakaki, Toshio; Sano, Emiko

2003-06-26

IL-8 is produced by various cells, and the NH(2)-terminal amino acid sequence of IL-8 displays heterogeneity among cell types. The mature form of IL-8 has 72 amino acids (72IL-8), while a precursor form (77IL-8) of IL-8 has five additional amino acids to the 72IL-8 NH(2)-terminal. However, it has been unclear how IL-8 is processed to yield the mature form. In this study, converting enzyme was purified as a single 31-kDa band on silver-stained polyacrylamide gel from 160 l of cultured fibroblast supernatant by sequential chromatography. NH(2)-terminal amino acid sequence analysis revealed a sequence, EAPRSVDWRE, which was identified as a partial sequence of cathepsin L. Polyclonal antibodies raised against cathepsin L recognized the purified converting enzyme on Western blot. Moreover, human hepatic cathepsin L cleaved 77IL-8 between Arg(5) and Ser(6), which is the same cleavage site as the putative converting enzyme, resulting in 72IL-8 formation. These data indicate that the converting enzyme of the partially purified fraction of the human fibroblast culture supernatant was cathepsin L. Furthermore, 72IL-8 was sevenfold more potent than 77IL-8 in a neutrophil chemotaxis assay. These results show that cathepsin L is secreted from human fibroblasts in response to external stimuli and plays an important role in IL-8 processing in inflammatory sites.

Molecular cloning, characterization, and immunolocalization of two lactate dehydrogenase homologous genes from Taenia solium.

PubMed

Du, Wuying; Hu, Fengyu; Yang, Yabo; Hu, Dong; Hu, Xuchu; Yu, Xinbing; Xu, Jin; Dai, Jialin; Liao, Xinjiang; Huang, Jiang

2011-09-01

Two novel genes encoding lactate dehydrogenase A (LDHA) and B (LDHB) homologues, respectively, were identified from the cDNA libraries of adult Taenia solium (T. solium). The two deduced amino acid sequences both show more than 50% identity to the homologues for Danio rerio, Xenopus laevis, Schistosoma japonicum, Sus scrofa, Homo sapiens, et al. The identity of the amino acid sequence between TsLDHA and TsLDHB is 57.4%, and that of the nucleotide sequence is 61.5%. Recombinant TsLDHA homologue (rTsLDHA) and TsLDHB homologue (rTsLDHB) were expressed in Escherichia coli BL21/DE3 and purified. Though there were some differences in the sequence, the two LDH isozyme homologues show similarity in the conserved LDH domain, topological structure, primary immunological traits, localization on the tegument of T. solium adult, and partial physicochemical properties. The linear B-cell epitope analysis of TsLDHA and TsLDHB discovered a TsLDHA specific epitope. The purified rTsLDHA and rTsLDHB could be recognized by rat immuno-sera, serum from swine, or a patient infected with T. solium, respectively, but Western blot analysis showed cross-reactions, not only between these two LDH members but also with other common human tapeworms or helminths. The results suggested that the two LDH homologues are similar in the characteristics of LDH family, and they are not specific antigens for immunodiagnosis.

Exome Sequencing Identifies a Novel CEACAM16 Mutation Associated with Autosomal Dominant Nonsyndromic Hearing Loss DFNA4B in a Chinese Family

PubMed Central

He, Chufeng; Li, Haibo; Qing, Jie; Grati, Mhamed; Hu, Zhengmao; Li, Jiada; Hu, Yiqiao; Xia, Kun; Mei, Lingyun; Wang, Xingwei; Yu, Jianjun; Chen, Hongsheng; Jiang, Lu; Liu, Yalan; Men, Meichao; Zhang, Hailin; Guan, Liping; Xiao, Jingjing; Zhang, Jianguo; Liu, Xuezhong; Feng, Yong

2014-01-01

Autosomal dominant nonsyndromic hearing loss (ADNSHL/DFNA) is a highly genetically heterogeneous disorder. Hitherto only about 30 ADNSHL-causing genes have been identified and many unknown genes remain to be discovered. In this research, genome-wide linkage analysis mapped the disease locus to a 4.3 Mb region on chromosome 19q13 in SY-026, a five-generation nonconsanguineous Chinese family affected by late-onset and progressive ADNSHL. This linkage region showed partial overlap with the previously reported DFNA4. Simultaneously, probands were analyzed using exome capture followed by next generation sequencing. Encouragingly, a heterozygous missense mutation, c.505G>A (p.G169R) in exon 3 of the CEACAM16 gene (carcinoembryonic antigen-related cell adhesion molecule 16), was identified via this combined strategy. Sanger sequencing verified that the mutation co-segregated with hearing loss in the family and that it was not present in 200 unrelated control subjects with matched ancestry. This is the second report in the literature of a family with ADNSHL caused by CEACAM16 mutation. Immunofluorescence staining and Western blots also prove CEACAM16 to be a secreted protein. Furthermore, our studies in transfected HEK293T cells show that the secretion efficacy of the mutant CEACAM16 is much lower than that of the wild-type, suggesting a deleterious effect of the sequence variant. PMID:25589040

Exome sequencing identifies a novel CEACAM16 mutation associated with autosomal dominant nonsyndromic hearing loss DFNA4B in a Chinese family.

PubMed

Wang, Honghan; Wang, Xinwei; He, Chufeng; Li, Haibo; Qing, Jie; Grati, Mhamed; Hu, Zhengmao; Li, Jiada; Hu, Yiqiao; Xia, Kun; Mei, Lingyun; Wang, Xingwei; Yu, Jianjun; Chen, Hongsheng; Jiang, Lu; Liu, Yalan; Men, Meichao; Zhang, Hailin; Guan, Liping; Xiao, Jingjing; Zhang, Jianguo; Liu, Xuezhong; Feng, Yong

2015-03-01

Autosomal dominant nonsyndromic hearing loss (ADNSHL/DFNA) is a highly genetically heterogeneous disorder. Hitherto only about 30 ADNSHL-causing genes have been identified and many unknown genes remain to be discovered. In this research, genome-wide linkage analysis mapped the disease locus to a 4.3 Mb region on chromosome 19q13 in SY-026, a five-generation nonconsanguineous Chinese family affected by late-onset and progressive ADNSHL. This linkage region showed partial overlap with the previously reported DFNA4. Simultaneously, probands were analyzed using exome capture followed by next-generation sequencing. Encouragingly, a heterozygous missense mutation, c.505G>A (p.G169R) in exon 3 of the CEACAM16 gene (carcinoembryonic antigen-related cell adhesion molecule 16), was identified via this combined strategy. Sanger sequencing verified that the mutation co-segregated with hearing loss in the family and that it was not present in 200 unrelated control subjects with matched ancestry. This is the second report in the literature of a family with ADNSHL caused by CEACAM16 mutation. Immunofluorescence staining and western blots also prove CEACAM16 to be a secreted protein. Furthermore, our studies in transfected HEK293T cells show that the secretion efficacy of the mutant CEACAM16 is much lower than that of the wild type, suggesting a deleterious effect of the sequence variant.

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.

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.

Avoidance of truncated proteins from unintended ribosome binding sites within heterologous protein coding sequences.

PubMed

Whitaker, Weston R; Lee, Hanson; Arkin, Adam P; Dueber, John E

2015-03-20

Genetic sequences ported into non-native hosts for synthetic biology applications can gain unexpected properties. In this study, we explored sequences functioning as ribosome binding sites (RBSs) within protein coding DNA sequences (CDSs) that cause internal translation, resulting in truncated proteins. Genome-wide prediction of bacterial RBSs, based on biophysical calculations employed by the RBS calculator, suggests a selection against internal RBSs within CDSs in Escherichia coli, but not those in Saccharomyces cerevisiae. Based on these calculations, silent mutations aimed at removing internal RBSs can effectively reduce truncation products from internal translation. However, a solution for complete elimination of internal translation initiation is not always feasible due to constraints of available coding sequences. Fluorescence assays and Western blot analysis showed that in genes with internal RBSs, increasing the strength of the intended upstream RBS had little influence on the internal translation strength. Another strategy to minimize truncated products from an internal RBS is to increase the relative strength of the upstream RBS with a concomitant reduction in promoter strength to achieve the same protein expression level. Unfortunately, lower transcription levels result in increased noise at the single cell level due to stochasticity in gene expression. At the low expression regimes desired for many synthetic biology applications, this problem becomes particularly pronounced. We found that balancing promoter strengths and upstream RBS strengths to intermediate levels can achieve the target protein concentration while avoiding both excessive noise and truncated protein.

Characterization of capsaicin synthase and identification of its gene (csy1) for pungency factor capsaicin in pepper (Capsicum sp.)

PubMed Central

Prasad, B. C. Narasimha; Kumar, Vinod; Gururaj, H. B.; Parimalan, R.; Giridhar, P.; Ravishankar, G. A.

2006-01-01

Capsaicin is a unique alkaloid of the plant kingdom restricted to the genus Capsicum. Capsaicin is the pungency factor, a bioactive molecule of food and of medicinal importance. Capsaicin is useful as a counterirritant, antiarthritic, analgesic, antioxidant, and anticancer agent. Capsaicin biosynthesis involves condensation of vanillylamine and 8-methyl nonenoic acid, brought about by capsaicin synthase (CS). We found that CS activity correlated with genotype-specific capsaicin levels. We purified and characterized CS (≈35 kDa). Immunolocalization studies confirmed that CS is specifically localized to the placental tissues of Capsicum fruits. Western blot analysis revealed concomitant enhancement of CS levels and capsaicin accumulation during fruit development. We determined the N-terminal amino acid sequence of purified CS, cloned the CS gene (csy1) and sequenced full-length cDNA (981 bp). The deduced amino acid sequence of CS from full-length cDNA was 38 kDa. Functionality of csy1 through heterologous expression in recombinant Escherichia coli was also demonstrated. Here we report the gene responsible for capsaicin biosynthesis, which is unique to Capsicum spp. With this information on the CS gene, speculation on the gene for pungency is unequivocally resolved. Our findings have implications in the regulation of capsaicin levels in Capsicum genotypes. PMID:16938870

Exploring the Midgut Transcriptome and Brush Border Membrane Vesicle Proteome of the Rice Stem Borer, Chilo suppressalis (Walker)

PubMed Central

Peng, Chuanhua; Wang, Xiaoping; Li, Fei; Lin, Yongjun

2012-01-01

The rice stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), is one of the most detrimental pests affecting rice crops. The use of Bacillus thuringiensis (Bt) toxins has been explored as a means to control this pest, but the potential for C. suppressalis to develop resistance to Bt toxins makes this approach problematic. Few C. suppressalis gene sequences are known, which makes in-depth study of gene function difficult. Herein, we sequenced the midgut transcriptome of the rice stem borer. In total, 37,040 contigs were obtained, with a mean size of 497 bp. As expected, the transcripts of C. suppressalis shared high similarity with arthropod genes. Gene ontology and KEGG analysis were used to classify the gene functions in C. suppressalis. Using the midgut transcriptome data, we conducted a proteome analysis to identify proteins expressed abundantly in the brush border membrane vesicles (BBMV). Of the 100 top abundant proteins that were excised and subjected to mass spectrometry analysis, 74 share high similarity with known proteins. Among these proteins, Western blot analysis showed that Aminopeptidase N and EH domain-containing protein have the binding activities with Bt-toxin Cry1Ac. These data provide invaluable information about the gene sequences of C. suppressalis and the proteins that bind with Cry1Ac. PMID:22666467

Piroplasms in brown hyaenas (Parahyaena brunnea) and spotted hyaenas (Crocuta crocuta) in Namibia and South Africa are closely related to Babesia lengau.

PubMed

Burroughs, Richard E J; Penzhorn, Barend L; Wiesel, Ingrid; Barker, Nancy; Vorster, Ilse; Oosthuizen, Marinda C

2017-02-01

The objective of our study was identification and molecular characterization of piroplasms and rickettsias occurring in brown (Parahyaena brunnea) and spotted hyaenas (Crocuta crocuta) from various localities in Namibia and South Africa. Whole blood (n = 59) and skin (n = 3) specimens from brown (n = 15) and spotted hyaenas (n = 47) were screened for the presence of Babesia, Theileria, Ehrlichia and Anaplasma species using the reverse line blot (RLB) hybridization technique. PCR products of 52/62 (83.9%) of the specimens hybridized only with the Theileria/Babesia genus-specific probes and not with any of the species-specific probes, suggesting the presence of a novel species or variant of a species. No Ehrlichia and/or Anaplasma species DNA could be detected. A parasite 18S ribosomal RNA gene of brown (n = 3) and spotted hyaena (n = 6) specimens was subsequently amplified and cloned, and the recombinants were sequenced. Homologous sequence searches of databases indicated that the obtained sequences were most closely related to Babesia lengau, originally described from cheetahs (Acinonyx jubatus). Observed sequence similarities were subsequently confirmed by phylogenetic analyses which showed that the obtained hyaena sequences formed a monophyletic group with B. lengau, B abesia conradae and sequences previously isolated from humans and wildlife in the western USA. Within the B. lengau clade, the obtained sequences and the published B. lengau sequences were grouped into six distinct groups, of which groups I to V represented novel B. lengau genotypes and/or gene variants. We suggest that these genotypes cannot be classified as new Babesia species, but rather as variants of B. lengau. This is the first report of occurrence of piroplasms in brown hyaenas.

The hypervariable region 1 protein of hepatitis C virus broadly reactive with sera of patients with chronic hepatitis C has a similar amino acid sequence with the consensus sequence.

PubMed

Watanabe, K; Yoshioka, K; Ito, H; Ishigami, M; Takagi, K; Utsunomiya, S; Kobayashi, M; Kishimoto, H; Yano, M; Kakumu, S

1999-11-10

Hypervariable region 1 (HVR1) proteins of hepatitis C virus (HCV) have been reported to react broadly with sera of patients with HCV infection. However, the variability of the broad reactivity of individual HVR1 proteins has not been elucidated. We assessed the reactivity of 25 different HVR1 proteins (genotype 1b) with sera of 81 patients with HCV infection (genotype 1b) by Western blot. HVR1 proteins reacted with 2-60 sera. The number of sera reactive with each HVR1 protein significantly correlated with the number of amino acid residues identical to the consensus sequence defined by Puntoriero et al. (G. Puntoriero, A. Lahm, S. Zucchelli, B. B. Ercole, R. Tafi, M. Penzzanera, M. U. Mondelli, R. Cortese, A. Tramontano, G. Galfre', and A. Nicosia. 1998. EMBO J. 17, 3521-3533. ) (r = 0.561, P < 0.005). The most widely reactive HVR1 protein, 12-22, had a sequence similar to the consensus sequence. The peptide with C-terminal 13-amino-acids sequence of HVR1 protein 12-22 (NH2-CSFTSLFTPGPSQK) was injected into rabbits as an immunogen. The rabbit immune sera reacted with 9 of 25 HVR1 proteins of genotype 1b including HVR1 protein 12-22 and with 3 of 12 proteins of genotype 2a. These results indicate that the HVR1 protein broadly reactive with patients' sera has a sequence similar to the consensus sequence, can induce broadly reactive sera, and could be one of the candidate immunogens in a prophylactic vaccine against HCV. Copyright 1999 Academic Press.

Sequence analysis and expression of the M1 and M2 matrix protein genes of hirame rhabdovirus (HIRRV)

USGS Publications Warehouse

Nishizawa, T.; Kurath, G.; Winton, J.R.

1997-01-01

We have cloned and sequenced a 2318 nucleotide region of the genomic RNA of hirame rhabdovirus (HIRRV), an important viral pathogen of Japanese flounder Paralichthys olivaceus. This region comprises approximately two-thirds of the 3' end of the nucleocapsid protein (N) gene and the complete matrix protein (M1 and M2) genes with the associated intergenic regions. The partial N gene sequence was 812 nucleotides in length with an open reading frame (ORF) that encoded the carboxyl-terminal 250 amino acids of the N protein. The M1 and M2 genes were 771 and 700 nucleotides in length, respectively, with ORFs encoding proteins of 227 and 193 amino acids. The M1 gene sequence contained an additional small ORF that could encode a highly basic, arginine-rich protein of 25 amino acids. Comparisons of the N, M1, and M2 gene sequences of HIRRV with the corresponding sequences of the fish rhabdoviruses, infectious hematopoietic necrosis virus (IHNV) or viral hemorrhagic septicemia virus (VHSV) indicated that HIRRV was more closely related to IHNV than to VHSV, but was clearly distinct from either. The putative consensus gene termination sequence for IHNV and VHSV, AGAYAG(A)(7), was present in the N-M1, M1-M2, and M2-G intergenic regions of HIRRV as were the putative transcription initiation sequences YGGCAC and AACA. An Escherichia coli expression system was used to produce recombinant proteins from the M1 and M2 genes of HIRRV. These were the same size as the authentic M1 and M2 proteins and reacted with anti-HIRRV rabbit serum in western blots. These reagents can be used for further study of the fish immune response and to test novel control methods.

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.

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

Horizontal Transfer of Segments of the 16S rRNA Genes between Species of the Streptococcus anginosus Group

PubMed Central

Schouls, Leo M.; Schot, Corrie S.; Jacobs, Jan A.

2003-01-01

The nature in variation of the 16S rRNA gene of members of the Streptococcus anginosus group was investigated by hybridization and DNA sequencing. A collection of 708 strains was analyzed by reverse line blot hybridization. This revealed the presence of distinct reaction patterns representing 11 different hybridization groups. The 16S rRNA genes of two strains of each hybridization group were sequenced to near-completion, and the sequence data confirmed the reverse line blot hybridization results. Closer inspection of the sequences revealed mosaic-like structures, strongly suggesting horizontal transfer of segments of the 16S rRNA gene between different species belonging to the Streptococcus anginosus group. Southern blot hybridization further showed that within a single strain all copies of the 16S rRNA gene had the same composition, indicating that the apparent mosaic structures were not PCR-induced artifacts. These findings indicate that the highly conserved rRNA genes are also subject to recombination and that these events may be fixed in the population. Such recombination may lead to the construction of incorrect phylogenetic trees based on the 16S rRNA genes. PMID:14645285

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.

Identification of immunodominant antigens for the laboratory diagnosis of toxocariasis.

PubMed

Zhan, Bin; Ajmera, Ravi; Geiger, Stefan Michael; Gonçalves, Marco Túlio Porto; Liu, Zhuyun; Wei, Junfei; Wilkins, Patricia P; Fujiwara, Ricardo; Gazzinelli-Guimaraes, Pedro Henrique; Bottazzi, Maria Elena; Hotez, Peter

2015-12-01

To identify immunodominant antigens of Toxocara canis recognised by Toxocara-infected sera as recombinant reagents for immunodiagnosis of toxocariasis. Pooled sera from human cases of toxocariasis were used to identify immunodominant antigens by immunoscreening a T. canis larval expression cDNA library. The positive clones were sequenced to reveal the identity of the antigens. The recombinant proteins were expressed in E. coli and then used to confirm their immunoreaction with sera of humans with toxocariasis. Two chosen antigens were also used to differentiate Toxocara infection from other helminth infections in mice. Eleven antigens with immunodiagnostic potential were identified, including two C-type lectins (CTLs) that reacted strongly with the Toxocara-positive serum pool. The first CTL (Tc-CTL-1) is the same as TES-32, previously identified as a major immunodominant component of TES; the second CTL (Tc-CTL-2) is a novel C-type lectin sharing 83% amino acid sequence identity within the functional domain of Tc-CTL-1. The E. coli-expressed recombinant Tc-CTL-1 was strongly recognised by the Toxocara-positive serum pool or sera from animals experimentally infected with T. canis. Reactivity with recombinant Tc-CTL-1 was higher when the unreduced protein was used in an enzyme-linked immunosorbent assay (ELISA), dot-blot assay or Western blot test compared to the protein under reduced condition. Both recombinant Tc-CTL-1- and Tc-CTL-2-based ELISAs were able to differentiate T. canis infection from other helminth infections in experimentally infected mice. Both Tc-CTL-1 and Tc-CTL-2 were able to differentiate Toxocara infection from other helminth infections and could potentially be used as sensitive and specific immunodiagnostic antigens. © 2015 John Wiley & Sons Ltd.

Isolation and Characterization of Dromedary Camel Coronavirus UAE-HKU23 from Dromedaries of the Middle East: Minimal Serological Cross-Reactivity between MERS Coronavirus and Dromedary Camel Coronavirus UAE-HKU23

PubMed Central

Woo, Patrick C. Y.; Lau, Susanna K. P.; Fan, Rachel Y. Y.; Lau, Candy C. Y.; Wong, Emily Y. M.; Joseph, Sunitha; Tsang, Alan K. L.; Wernery, Renate; Yip, Cyril C. Y.; Tsang, Chi-Ching; Wernery, Ulrich; Yuen, Kwok-Yung

2016-01-01

Recently, we reported the discovery of a dromedary camel coronavirus UAE-HKU23 (DcCoV UAE-HKU23) from dromedaries in the Middle East. In this study, DcCoV UAE-HKU23 was successfully isolated in two of the 14 dromedary fecal samples using HRT-18G cells, with cytopathic effects observed five days after inoculation. Northern blot analysis revealed at least seven distinct RNA species, corresponding to predicted subgenomic mRNAs and confirming the core sequence of transcription regulatory sequence motifs as 5′-UCUAAAC-3′ as we predicted previously. Antibodies against DcCoV UAE-HKU23 were detected in 58 (98.3%) and 59 (100%) of the 59 dromedary sera by immunofluorescence and neutralization antibody tests, respectively. There was significant correlation between the antibody titers determined by immunofluorescence and neutralization assays (Pearson coefficient = 0.525, p < 0.0001). Immunization of mice using recombinant N proteins of DcCoV UAE-HKU23 and Middle East respiratory syndrome coronavirus (MERS-CoV), respectively, and heat-inactivated DcCoV UAE-HKU23 showed minimal cross-antigenicity between DcCoV UAE-HKU23 and MERS-CoV by Western blot and neutralization antibody assays. Codon usage and genetic distance analysis of RdRp, S and N genes showed that the 14 strains of DcCoV UAE-HKU23 formed a distinct cluster, separated from those of other closely related members of Betacoronavirus 1, including alpaca CoV, confirming that DcCoV UAE-HKU23 is a novel member of Betacoronavirus 1. PMID:27164099

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.

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

Immunological detection of phenylalanine hydroxylase protein in Drosophila melanogaster.

PubMed Central

Silva, F J; Bel, Y; Botella, L M; Cotton, R G; Ferré, J

1992-01-01

A monoclonal antibody raised against monkey liver phenylalanine hydroxylase (PAH) has been used to detect this protein in Drosophila melanogaster. A cross-reacting material (CRM) band of apparent molecular mass 50-52 kDa, equivalent to that deduced for the Drosophila melanogaster PAH protein based on the pah gene cDNA sequence, has been detected. This CRM was analysed throughout development and showed an equivalent pattern to that reported for PAH activity in this insect, with maxima at pupariation and at pharate adult formation. Distribution of this CRM in larval tissues, the haemolymph and the adult body is mainly restricted to the larval fat body and the adult head. Demonstration of this CRM as the PAH protein comes from the correlation between the decreased PAH enzyme activities of two mutant strains and their decreased amounts of CRM by Western blotting. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:1417795

A case of imported paracoccidioidomycosis in a German legionnaire.

PubMed

Horré, R; Schumacher, G; Alpers, K; Seitz, H M; Adler, S; Lemmer, K; De Hoog, G S; Schaal, K P; Tintelno, K

2002-04-01

We report on a case of the chronic form of paracoccidioidomycosis with swelling and ulcerations of the mouth in a German legionnaire who also suffered from a chronic bronchitis. The patient had worked for many years in Brazil, an area endemic for the disease. Infection due to Paracoccidioides brasiliensis was diagnosed in Germany, more than 10 years after the patient's return. Diagnosis was established by the presence of yeast cells with multipolar budding in the tissue of the oral lesion. Furthermore, the fungus was grown in a liquid Leishmania culture medium. Identification of the fungus was based on morphology and genetic sequencing. Furthermore, IgG antibodies against a 43-kDa antigen of P. brasiliensis were detected in a western blot. After itraconazole therapy (400 mg day(-1)) for 4 weeks, the lesions had disappeared almost completely, but the therapy was continued for further 5 months to avoid relapse of the infection.

Canine and ovine tick-borne pathogens in camels, Nigeria.

PubMed

Lorusso, Vincenzo; Wijnveld, Michiel; Latrofa, Maria S; Fajinmi, Akinyemi; Majekodunmi, Ayodele O; Dogo, Abraham G; Igweh, Augustine C; Otranto, Domenico; Jongejan, Frans; Welburn, Susan C; Picozzi, Kim

2016-09-15

In April 2008, whole blood samples were collected from 36 dromedary camels in Sokoto, North-western Nigeria. Following PCR and reverse line blotting, twenty-two samples (61%) resulted positive for Ehrlichia/Anaplasma spp. and three (8%) for Theileria/Babesia spp., with three (8%) cases of co-infections being found. Both sequence and BLAST analyses identified Ehrlichia/Anaplasma spp. and Theileria/Babesia spp. positive cases as Anaplasma platys and Theileria ovis, respectively. This is the first report of the detection of A. platys and T. ovis in camels from sub-Saharan Africa. The epidemiological relevance of this finding is enhanced by the close living of these animals with both dogs and small ruminants. The high prevalence detected for A. platys suggests a possible role of camels as carriers of this infection. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Expression of recombinant myostatin propeptide pPIC9K-Msp plasmid in Pichia pastoris.

PubMed

Du, W; Xia, J; Zhang, Y; Liu, M J; Li, H B; Yan, X M; Zhang, J S; Li, N; Zhou, Z Y; Xie, W Z

2015-12-28

Myostatin propeptide can inhibit the biological activity of myostatin protein and promote muscle growth. To express myostatin propeptide in vitro with a higher biological activity, we performed codon optimization on the sheep myostatin propeptide gene sequence, and mutated aspartic acid-76 to alanine based on the codon usage bias of Pichia pastoris and the enhanced biological activity of myostatin propeptide mutant. Modified myostatin propeptide gene was cloned into the pPIC9K plasmid to form the recombinant plasmid pPIC9K-Msp. Recombinant plasmid pPIC9K-Msp was transformed into Pichia pastoris GS115 by electrotransformation. Transformed cells were screened, and methanol was used to induce expression. SDS-PAGE and western blotting were used to verify the successful expression of myostatin propeptide with biological activity in Pichia pastoris, providing the basis for characterization of this protein.

Determining the Localization of Carbohydrate Active Enzymes Within Gram-Negative Bacteria.

PubMed

McLean, Richard; Inglis, G Douglas; Mosimann, Steven C; Uwiera, Richard R E; Abbott, D Wade

2017-01-01

Investigating the subcellular location of secreted proteins is valuable for illuminating their biological function. Although several bioinformatics programs currently exist to predict the destination of a trafficked protein using its signal peptide sequence, these programs have limited accuracy and often require experimental validation. Here, we present a systematic method to fractionate gram-negative cells and characterize the subcellular localization of secreted carbohydrate active enzymes (CAZymes). This method involves four parallel approaches that reveal the relative abundance of protein within the cytoplasm, periplasm, outer membrane, and extracellular environment. Cytoplasmic and periplasmic proteins are fractionated by lysis and osmotic shock, respectively. Outer membrane bound proteins are determined by comparing cells before and after exoproteolytic digestion. Extracellularly secreted proteins are collected from the media and concentrated. These four different fractionations can then be probed for the presence and quantity of target proteins using immunochemical methods such as Western blots and ELISAs, or enzyme activity assays.

Expression of the Bacillus anthracis protective antigen gene by baculovirus and vaccinia virus recombinants.

PubMed Central

Iacono-Connors, L C; Schmaljohn, C S; Dalrymple, J M

1990-01-01

The gene encoding Bacillus anthracis protective antigen (PA) was modified by site-directed mutagenesis, subcloned into baculovirus and vaccinia virus plasmid transfer vectors (pAcYM1 and pSC-11, respectively), and inserted via homologous recombinations into baculovirus Autographa californica nuclear polyhedrosis virus or vaccinia virus (strains WR and Connaught). Expression of PA was detected in both systems by immunofluorescence assays with antisera from rabbits immunized with B. anthracis PA. Western blot (immunoblot) analysis showed that the expressed product of both systems was slightly larger (86 kilodaltons) than B. anthracis-produced PA (83.5 kilodaltons). Analysis of trypsin digests of virus-expressed and authentic PA suggested that the size difference was due to the presence of a signal sequence remaining with the virus-expressed protein. Immunization of mice with either recombinant baculovirus-infected Spodoptera frugiperda cells or with vaccinia virus recombinants elicited a high-titer, anti-PA antibody response. Images PMID:2105271

Generation and Characterization of a New Monoclonal Antibody Against CXCL4.

PubMed

Gao, Jing; Wu, Mingyuan; Gao, Jin; Wang, Xia; Zhang, Yang; Zhu, Shunying; Yu, Yan; Han, Wei

2015-04-01

CXCL4 plays important roles in numerous disease processes, which makes the CXCL4 signaling pathway a potential therapeutic target. In this study, we aimed to develop a neutralizing antibody against both human and mouse CXCL4. Rats were immunized with recombinant human CXCL4 (rhCXCL4). Hybridoma clones were created by fusion of the immunized rat spleen cells with mouse myeloma SP2/0 cells and screened using recombinant mouse CXCL4 (rmCXCL4) and rhCXCL4. The CXCL4 monoclonal antibody (CXCL4 MAb) produced by the 16D6-3 hybridoma clone was sequenced and characterized by Western blot and Biacore assays. It recognized both human and mouse CXCL4 with high affinity and neutralized the effect of rhCXCL4 in vitro. Thus, the antibody may be used in the studies of CXCL4 in murine disease models and as a template in the antibody humanization for clinical developments.

Novel GATAD2B loss-of-function mutations cause intellectual disability in two unrelated cases.

PubMed

Luo, Xiaomei; Zou, Yongyi; Tan, Bo; Zhang, Yue; Guo, Jing; Zeng, Lanlan; Zhang, Rui; Tan, Hu; Wei, Xianda; Hu, Yiqiao; Zheng, Yu; Liang, Desheng; Wu, Lingqian

2017-04-01

GATA zinc finger domain-containing 2B (GATAD2B) is a subunit of the methyl-CpG-binding protein-1 complex (MECP1), which deacetylates methylated nucleosomes and regresses transcriptional activity. Recently, GATAD2B has been elucidated as a candidate gene in patients with intellectual disability (ID). In this study, we identified two novel heterozygous frameshift mutations of GATAD2B in two unrelated ID cases through next-generation sequencing (NGS). Both of the mutations c.80_81insGATGT and c.552_555delGAAA cause truncated proteins that might be detrimental to neurodevelopment. We performed western blotting and observed a reduction in the target protein compared with normal controls. This is the first report of GATAD2B in Chinese ID patients. Our findings will broaden the spectrum of GATAD2B mutations and facilitate genetic diagnosis and counseling.

Identification of molting fluid carboxypeptidase A (MF-CPA) in Bombyx mori.

PubMed

Ote, Manabu; Mita, Kazuei; Kawasaki, Hideki; Daimon, Takaaki; Kobayashi, Masahiko; Shimada, Toru

2005-07-01

Using microarray analyses, we identified carboxypeptidase A (MF-CPA), which was induced during pupal ecdysis in the wing discs of Bombyx mori. Here, we report the functional characterization of MF-CPA. MF-CPA has amino acid sequence similarities with the proteins in the carboxypeptidase A/B subfamily, from human to nematode. The MF-CPA gene is expressed during the molting periods in the epithelial tissues. MF-CPA is detected in the molting fluid, which fills the space between the old and new cuticle during molting. By Western blot analysis, we show that MF-CPA is secreted as a zymogen and processed in the molting fluid. Recombinant MF-CPA expressed in the insect cells has carboxypeptidase A activity. We propose that MF-CPA degrades the proteins from the old cuticle during the molting periods and contributes to recycling of the amino acids.

Live, attenuated Salmonella typhimurium vectoring Campylobacter antigens.

PubMed

Sizemore, Donata R; Warner, Beth; Lawrence, Julie; Jones, Amy; Killeen, Kevin P

2006-05-01

We describe the evaluation of three live, attenuated deltaphoP/Q Salmonella enteric serovar Typhimurium strains expressing PEB1 minus its signal sequence (PEB1-ss) from three different plasmids: a pBR-based asd plasmid, an arabinose-based runaway plasmid, which each expressed PEB1-ss in the bacterial cytosol, and a PEB1::HlyA fusion plasmid that directs secretion of PEB1-ss into the extracellular milieu. Serum IgG responses specific for PEB1-ss were induced by pBR-derived and runaway plasmids, with 100 and 90% seroconversion, respectively, at a 1:500 dilution of anti-sera as measured by Western blot analysis, while the PEB1-ss::HlyA fusion plasmid induced serum IgG in only 20% of the mice. Although significant levels of anti-PEB serum IgG were induced, no protection against oral Campylobacter jejuni challenge was observed.

MARCC (Matrix-Assisted Reader Chromatin Capture): an antibody-free method to enrich and analyze combinatorial nucleosome modifications

PubMed Central

Su, Zhangli

2016-01-01

Combinatorial patterns of histone modifications are key indicators of different chromatin states. Most of the current approaches rely on the usage of antibodies to analyze combinatorial histone modifications. Here we detail an antibody-free method named MARCC (Matrix-Assisted Reader Chromatin Capture) to enrich combinatorial histone modifications. The combinatorial patterns are enriched on native nucleosomes extracted from cultured mammalian cells and prepared by micrococcal nuclease digestion. Such enrichment is achieved by recombinant chromatin-interacting protein modules, or so-called reader domains, which can bind in a combinatorial modification-dependent manner. The enriched chromatin can be quantified by western blotting or mass spectrometry for the co-existence of histone modifications, while the associated DNA content can be analyzed by qPCR or next-generation sequencing. Altogether, MARCC provides a reproducible, efficient and customizable solution to enrich and analyze combinatorial histone modifications. PMID:26131849

Characterization of 4 New Mutations in the CYBB Gene in 10 Iranian Families With X-linked Chronic Granulomatous Disease.

PubMed

Teimourian, Shahram; Sazgara, Faezeh; de Boer, Martin; van Leeuwen, Karin; Roos, Dirk; Lashkary, Sharzad; Chavoshzadeh, Zahra; Nabavi, Mohammad; Bemanian, Mohammad Hassan; Isaian, Anna

2018-04-26

Chronic granulomatous disease (CGD) is an inherited disease of the innate immune system that results from defects in 1 of the 5 subunits of nicotinamide adenine dinucleotide phosphate oxidase complex and leads to life-threatening infections with granuloma formation. During 3 years of study, we recognized 10 male patients with X-linked CGD from a tertiary referral center for immune deficiencies in Iran. The CGD patients were diagnosed according to clinical features and biochemical tests, including nitroblue tetrazolium and dihydrorhodamine-1, 2, 3 tests, performed on patients and their mothers. In all patients, Western blot analysis showed a gp91 phenotype. Mutation screening by single strand conformation polymorphism and multiplex ligation-dependent probe amplification analysis of the CYBB gene encoding gp91, followed by sequencing, showed 9 different mutations, 4 of them novel as far as we know.

[Neurological adaptations to hypoxia in Tibetan antelope (Pantholops hodgsonii) with a view of molecular biology of respiratory globin-neuroglobin].

PubMed

Bai, Zhen-Zhong; Yang, Ying-Zhong; Jin, Guo-En; Ma, Lan; Ge, Ri-Li

2012-11-01

Neuroglobin (Ngb) is a respiratory protein that is preferentially expressed in brain of mouse and man. In this article, Tibetan antelope, living at altitude of 3 000-5 000 m for millions of years, was selected as the model of hypoxia-tolerant adaptation species. Using reverse transcription polymerase chain reaction (RT-PCR) and Western blot techniques, expression of Ngb gene was amplified and analyzed in antelope brain tissue. Our results showed that Ngb homology protein in Tibetan antelope was identified with more sequence similarity with cattle (96%), sheep (95%), and human (95%). We detected that there were some mutations occurred in the Open Reading Frame of Ngb in Tibetan antelope compared with sheep. Phylogenetic analysis of Ngb chain showed that it was closer to cattle than the others. This study suggests possible roles of central nervous system enriched Ngb in adaptation of Tibetan antelope to extremely high altitude.

76 FR 7568 - Findings of Research Misconduct

Federal Register 2010, 2011, 2012, 2013, 2014

2011-02-10

... images of Western- blot bands from unrelated experiments done in 2005 that were falsely labeled as if.... The 2007 Journal of Neuroimmunology article was retracted in J. Neuroimmunol. 197(1):197, 2008. Dr...

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

The majority of atypical cpb2 genes in Clostridium perfringens isolates of different domestic animal origin are expressed.

PubMed

Kircanski, Jasmina; Parreira, Valeria R; Whiteside, Samantha; Pei, Yanlong; Prescott, John F

2012-10-12

This study examined the prevalence and expression of the "consensus" and the "atypical"cpb2 genes in Clostridium perfringens isolates from cattle, chickens, dogs, goats, horses, pigs and sheep using polymerase chain reaction (PCR), sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting. Almost all porcine isolates (12/14) carried and expressed the consensus form of cpb2 but, when present in 108 non-porcine isolates, the gene was usually the atypical form (40 atypical versus 9 consensus). Western blotting showed expression in 30 of 40 (75%) atypical cpb2-positive isolates, considerably more frequently than reported previously. CPB2 was expressed by almost all (20/21) the consensus cpb2-positive isolates, regardless of source. Copyright © 2012 Elsevier B.V. All rights reserved.

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.

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.

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.

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.

Comparative evaluation of western blotting in hepatic and pulmonary cystic echinococcosis.

PubMed

Akisu, C; Delibas, S B; Bicmen, C; Ozkoc, S; Aksoy, U; Turgay, N

2006-12-01

Many serological tests are widely used in the diagnosis of cystic echinococcosis (CE), caused by the larval stages of Echinococcus granulosus. The present study was carried for differentiation between hepatic and pulmonary cystic echinococcosis by Western Blotting (WB). A total of 121 sera from patients with hepatic CE (37), pulmonary CE (31) and controls (53; consisting of six healthy, seven Hymenolepis nana infection, 20 hepatic and 20 pulmonary diseases other than CE) were examined. In all of the CE patients, E. gronulosus infection was confirmed by surgical intervention. Sera were previously tested using IHA and ELISA to detect the E. gronulosus specific antibodies. Sera from hepatic cases of CE reacted with 16 polypeptides of 6-116 kDa and sera from pulmonary cases of CE reacted with 14 polypeptides of 4-130 kDa by Western Blotting. The WB test enabled the detection of antibodies in the hepatic CE samples for proteins of 24, 32 34, 44-46 and 52-54 kDa in molecular weight in 78.4%, 75.7%, 78.4% and 89.2% of the patients, respectively. In the pulmonary CE samples sera WB test enabled the detection of antibodies 24, 44-46, 100, 110, 116 and 120 124 kDa in molecular weight in 81.3%, 75.0%, 87.5%, 71.9%, 84.4% and 65.6% of the patients, respectively. We indicated that the antigenic components of high molecular weight can be good candidates for differentiation of hepatic CE from pulmonary CE.

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.

Development and Evaluation of Recombinant Nucleocapsid Protein Based Diagnostic ELISA for Detection of Nipah Virus Infection in Pigs.

PubMed

Kulkarni, Diwakar D; Venkatesh, Govindarajalu; Tosh, Chakradhar; Patel, Priyanka; Mashoria, Anita; Gupta, Vandana; Gupta, Sourabh; D, Senthilkumar

2016-01-01

The recombinant viral protein-based indirect enzyme-linked immunosorbent assay (ELISA) is a cost-effective, safe, specific, and rapid tool to diagnose the viral infection. Nipah virus nucleocapsid (NiV-N) protein was expressed in Escherichia coli and purified by histidine tag-based affinity chromatography. The N protein was selected based on its immuno dominance and conservation among different NiV strains. An indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for swine sera was optimized using the recombinant NiV-N protein as an antigen along with negative and positive controls. The background reading was blocked using skim milk powder and chicken serum. A total number of 1709 swine serum samples from various states of India were tested with indirect ELISA and Western blot. The test was considered positive only when its total reactivity reading was higher than 0.2 cut-off value and the ratio of the total reactivity to the background reading was more than 2.0. Since specificity is high for Western blotting it was used as standard test for comparison of results of indirect ELISA. Sensitivity and specificity of indirect ELISA was 100% and 98.7%, respectively, in comparison with Western blotting. Recombinant N protein-based ELISA can be used in screening large number of serum samples for epidemiological investigations in developing countries where high containment laboratories are not available to handle this zoonotic virus.

Docosahexaenoic Acid (DHA) Provides Neuroprotection in Traumatic Brain Injury Models via Activating Nrf2-ARE Signaling.

PubMed

Zhu, Wei; Ding, Yuexia; Kong, Wei; Li, Tuo; Chen, Hongguang

2018-04-16

In this study, we explored the neuroprotective effects of docosahexaenoic acid (DHA) in traumatic brain injury (TBI) models. In this study, we first confirmed that DHA was neuroprotective against TBI via the NSS test and Morris water maze experiment. Western blot was conducted to identify the expression of Bax, caspase-3, and Bcl-2. And the cell apoptosis of the TBI models was validated by TUNEL staining. Relationships between nuclear factor erythroid 2-related factor 2-antioxidant response element (Nrf2-ARE) pathway-related genes and DHA were explored by RT-PCR and Western blot. Rats of the DHA group performed remarkably better than those of the TBI group in both NSS test and water maze experiment. DHA conspicuously promoted the expression of Bcl-2 and diminished that of cleaved caspase-3 and Bax, indicating the anti-apoptotic role of DHA. Superoxide dismutase (SOD) activity and cortical malondialdehyde content, glutathione peroxidase (GPx) activity were renovated in rats receiving DHA treatment, implying that the neuroprotective influence of DHA was derived from lightening the oxidative stress caused by TBI. Moreover, immunofluorescence and Western blot experiments revealed that DHA facilitated the translocation of Nrf2 to the nucleus. DHA administration also notably increased the expression of the downstream factors NAD(P)H:quinone oxidoreductase (NQO-1) and heme oxygenase 1(HO-1). DHA exerted neuroprotective influence on the TBI models, potentially through activating the Nrf2- ARE pathway.

Identification of allergens in the box jellyfish Chironex yamaguchii that cause sting dermatitis.

PubMed

Horiike, Takumi; Nagai, Hiroshi; Kitani, Seiichi

2015-01-01

Jellyfish stings cause painful, papular-urticarial eruptions due to the immediate allergic, acute toxic and persistent inflammatory responses. In spite of many marine accidents and their economic impact, modes of first-aid treatment remain conventional and specific allergen and medical treatment are not yet available. The purpose of this study was to define the specific allergen of the box jellyfish Chironex yamaguchii and to study the precise mechanism of the resulting dermatitis. We comprehensively studied the immunoglobulin-binding molecules from the box jellyfish C. yamaguchii with a purification procedure and Western blotting, using sera from 1 patient and from several controls. From the nematocyst wall and spine, we detected IgG-binding acidic glycoprotein (of 66 and 30 kDa) as determined by Western blot and ion-exchange chromatography. In addition, the 66-kDa protein was found to be an asparagine residue-coupled N-linked glycoprotein and the epitope resided in the protein fraction. We found that CqTX-A, the major toxic protein of the nematocyst, is also a heat-stable IgE-binding allergen. This was confirmed as a 45-kDa protein by Western blot from both nematocyst extracts and purified CqTX-A. The detection of these proteins may, in part, explain the combined immediate allergic-toxic and persistent allergic responses. Hopefully, our findings will lead to the development of specific venom immunotherapy for marine professional workers and tourists for jellyfish-sting dermatitis and anaphylaxis. © 2015 S. Karger AG, Basel.

[Lactobacillus rhamnosus GG conditioned medium prevents E. coli meningitis by inhibiting nuclear factor-κB pathway].

PubMed

Zeng, Qing; He, Xiao-Long; Xiao, Han-Sheng; DU, Lei; Li, Yu-Jing; Chen, Le-Cheng; Tian, Hui-Wen; Huang, Sheng-He; Cao, Hong

2017-01-20

To investigate whether Lactobacillus rhamnosus GG conditioned medium(LGG-CM)has preventive effect against E. coli K1-induced neuropathogenicity in vitro by inhibiting nuclear factor-κB (NF-κB) signaling pathway. An in vitro blood-brain barrier (BBB) model was constructed using human brain microvascular endothelial cells (HBMECs). The effect of LGG-CM on E. coli-actived NF-κB signaling pathway was assayed using Western blotting. Invasion assay and polymorphonuclear leukocyte (PMN) transmigration assay were performed to explore whether LGG-CM could inhibit E. coli invasion and PMN transmigration across the BBB in vitro. The expressions of ZO-1 and CD44 were detected using Western blotting and immunofluorescence. The changes of trans-epithelial electric resistance (TEER) and bacterial translocation were determined to evaluate the BBB permeability. Pre-treament with LGG-CM inhibited E. coli-activated NF-κB signaling pathway in HBMECs and decreased the invasion of E. coli K1 and transmigration of PMN. Western blotting showed that LGG-CM could alleviate E. coli-induced up-regulation of CD44 and down-regulation of ZO-1 expressions in HBMECs. In addition, pre-treatment with LGG-CM alleviated E. coli K1-induced reduction of TEER and suppressed bacterial translocation across the BBB in vitro. LGG-CM can block E. coli-induced activation of NF-κB signaling pathway and thereby prevents E. coli K1-induced neuropathogenicity by decreasing E. coli K1 invasion rates and PMN transmigration.

Methods to Assess the Direct Interaction of C. jejuni with Mucins.

PubMed

Clyne, Marguerite; Duggan, Gina; Naughton, Julie; Bourke, Billy

2017-01-01

Studies of the interaction of bacteria with mucus-secreting cells can be complemented at a more mechanistic level by exploring the interaction of bacteria with purified mucins. Here we describe a far Western blotting approach to show how C. jejuni proteins separated by SDS PAGE and transferred to a membrane or slot blotted directly onto a membrane can be probed using biotinylated mucin. In addition we describe the use of novel mucin microarrays to assess bacterial interactions with mucins in a high-throughput manner.

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

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

Targeted Elimination of PCDH-PC Expressing Prostate Cancer Cells for Control of Hormone-Resistant Prostate Cancer

DTIC Science & Technology

2007-11-01

SDS-PAGE gel . The Western blot made from this gel was probed with antibody that recognizes the myc-tag. When compared to the extracts from the...SDS-PAGE gel and blotted onto a filter. The filter was probed with an anti-myc antibody. The levels of myc-tagged PCDH-PC protein in cells co...Specific Aim 2. Design and test antisense oligonucleotides ( ASOs ) that suppress PCDH-PC expression in prostate cancer cells. Work Done: We used

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

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.

Identification of parathyroid hormone-related protein in canine apocrine adenocarcinoma of the anal sac.

PubMed

Rosol, T J; Capen, C C; Danks, J A; Suva, L J; Steinmeyer, C L; Hayman, J; Ebeling, P R; Martin, T J

1990-03-01

The presence of parathyroid hormone-related protein (PTHrP) in the apocrine adenocarcinoma tumor line (CAC-8) derived from a hypercalcemic dog was demonstrated by western and northern blot analyses. Western blots of CAC-8 tumor extracts revealed a major protein with a molecular weight of approximately 18,000 daltons that cross-reacted with antiserum to human PTHrP. Northern blots demonstrated multiple-sized messenger RNA transcripts in CAC-8 that hybridized to a full-length cDNA probe to human PTHrP. Adenocarcinomas derived from apocrine glands of the anal sac also were stained immunohistochemically for antigens that cross-react with antiserum to human PTHrP. The tumor line (CAC-8) maintained in nude mice stained positively for PTHrP in 13 of 24 tumors. Three of ten apocrine adenocarcinomas from dogs with hypercalcemia stained for PTHrP, whereas zero of ten tumors were positive from normocalcemic dogs. Normal canine epidermal keratinocytes and areas of squamous metaplasia in a perianal gland carcinoma also were positive for PTHrP. These data demonstrated that canine tissues contained a homologue to human PTHrP that likely is important in the pathogenesis of humoral hypercalcemia of malignancy.

Epitope mapping of the variable repetitive region with the MB antigen of Ureaplasma urealyticum.

PubMed Central

Zheng, X; Lau, K; Frazier, M; Cassell, G H; Watson, H L

1996-01-01

One of the major surface structures of Ureaplasma urealyticum recognized by antibodies of patients during infection is the MB antigen. Previously, we showed by Western blot (immunoblot) analysis that any one of the anti-MB monoclonal antibodies (MAbs) 3B1.5, 5B1.1, and 10C6.6 could block the binding of patient antibodies to MB. Subsequent DNA sequencing revealed that a unique six-amino-acid direct tandem repeat region composed the carboxy two-thirds of this antigen. In the present study, using antibody-reactive peptide scanning of this repeat region, we demonstrated that the amino acids defining the epitopes for MAbs 3B1.5 5B1.1 and 10C6.6 are EQP, GK, and KEQPA, respectively. Peptide scanning analysis of an infected patient's serum antibody response showed that the dominant epitope was defined by the sequence PAGK. Mapping of these continuous epitopes revealed overlap between all MAb and patient polyclonal antibody binding sites, thus explaining the ability of a single MAb to apparently block all polyclonal antibody binding sites. We also show that a single amino acid difference in the sequence of the repeats of serovars 3 and 14 accounts for the lack of reactivity with serovar 14 of two of the serovar 3-specific MAbs. Finally, the data demonstrate the need to obtain the sequences of the mba genes of all serovars before an effective serovar-specific antibody detection method can be developed. PMID:8914774

Babesia lengau associated with cerebral and haemolytic babesiosis in two domestic cats.

PubMed

Bosman, Anna-Mari; Oosthuizen, Marinda C; Venter, Estelle H; Steyl, Johan C A; Gous, Tertius A; Penzhorn, Barend L

2013-05-01

Although reported sporadically from various countries, feline babesiosis appears to be a significant clinical entity only in South Africa, where Babesia felis is usually incriminated as the causative agent. Babesia lengau, recently described from asymptomatic cheetahs, has now possibly been incriminated as the causative agent in two severe clinical cases in domestic cats. Both cats were euthanised in extremis. While typical feline babesiosis in South Africa is an afebrile disease with a chronic manifestation, there was acute onset of severe clinical signs in both cats and their body temperatures were above the normal range when they were presented for treatment. Haemolytic anaemia was confirmed in one case. To our knowledge, this is the first report of cerebral babesiosis in cats.On reverse line blot 18S rDNA PCR products obtained from both cats showed positive hybridization profiles with the B. lengau species-specific probe. The two partial parasite 18S rRNA gene sequences obtained, showed high sequence similarity (99.9%) to B. lengau. In a representative tree constructed by the neighbor-joining method using the two-parameter model of Kimura the two obtained partial 18S rDNA sequences and that of B. lengau formed a monophyletic group with B. conradae and sequences previously isolated from humans and wildlife in the western USA. All clinical cases of feline babesiosis in South Africa are not necessarily caused by B. felis. Other piroplasms, e.g. B. lengau, may be incriminated in clinical cases, especially those occurring outside the known endemic area.

Genome of turbot rhabdovirus exhibits unusual non-coding regions and an additional ORF that could be expressed in fish cell.

PubMed

Zhu, Ruo-Lin; Lei, Xiao-Ying; Ke, Fei; Yuan, Xiu-Ping; Zhang, Qi-Ya

2011-02-01

Genomic sequence of Scophthalmus maximus rhabdovirus (SMRV) isolated from diseased turbot has been characterized. The complete genome of SMRV comprises 11,492 nucleotides and encodes five typical rhabdovirus genes N, P, M, G and L. In addition, two open reading frames (ORF) are predicted overlapping with P gene, one upstream of P and smaller than P (temporarily called Ps), and another in P gene which may encodes a protein similar to the vesicular stomatitis virus C protein. The C ORF is contained within the P ORF. The five typical proteins share the highest sequence identities (48.9%) with the corresponding proteins of rhabdoviruses in genus Vesiculovirus. Phylogenetic analysis of partial L protein sequence indicates that SMRV is close to genus Vesiculovirus. The first 13 nucleotides at the ends of the SMRV genome are absolutely inverse complementarity. The gene junctions between the five genes show conserved polyadenylation signal (CATGA(7)) and intergenic dinucleotide (CT) followed by putative transcription initiation sequence A(A/G)(C/G)A(A/G/T), which are different from known rhabdoviruses. The entire Ps ORF was cloned and expressed, and used to generate polyclonal antibody in mice. One obvious band could be detected in SMRV-infected carp leucocyte cells (CLCs) by anti-Ps/C serum via Western blot, and the subcellular localization of Ps-GFP fusion protein exhibited cytoplasm distribution as multiple punctuate or doughnut shaped foci of uneven size. Copyright © 2010 Elsevier B.V. All rights reserved.

Genomic sequence of mandarin fish rhabdovirus with an unusual small non-transcriptional ORF.

PubMed

Tao, Jian-Jun; Zhou, Guang-Zhou; Gui, Jian-Fang; Zhang, Qi-Ya

2008-03-01

The complete genome of mandarin fish Siniperca chuatsi rhabdovirus (SCRV) was cloned and sequenced. It comprises 11,545 nucleotides and contains five genes encoding the nucleoprotein N, the phosphoprotein P, the matrix protein M, the glycoprotein G, and the RNA-dependent RNA polymerase protein L. At the 3' and 5' termini of SCRV genome, leader and trailer sequences show inverse complementarity. The N, P, M and G proteins share the highest sequence identities (ranging from 14.8 to 41.5%) with the respective proteins of rhabdovirus 903/87, the L protein has the highest identity with those of vesiculoviruses, especially with Chandipura virus (44.7%). Phylogenetic analysis of L proteins showed that SCRV clustered with spring vireamia of carp virus (SVCV) and was most closely related to viruses in the genus Vesiculovirus. In addition, an overlapping open reading frame (ORF) predicted to encode a protein similar to vesicular stomatitis virus C protein is present within the P gene of SCRV. Furthermore, an unoverlapping small ORF downstream of M ORF within M gene is predicted (tentatively called orf4). Therefore, the genomic organization of SCRV can be proposed as 3' leader-N-P/C-M-(orf4)-G-L-trailer 5'. Orf4 transcription or translation products could not be detected by northern or Western blot, respectively, though one similar mRNA band to M mRNA was found. This is the first report on one small unoverlapping ORF in M gene of a fish rhabdovirus.

Cloning and analysis of DnaJ family members in the silkworm, Bombyx mori.

PubMed

Li, Yinü; Bu, Cuiyu; Li, Tiantian; Wang, Shibao; Jiang, Feng; Yi, Yongzhu; Yang, Huipeng; Zhang, Zhifang

2016-01-15

Heat shock proteins (Hsps) are involved in a variety of critical biological functions, including protein folding, degradation, and translocation and macromolecule assembly, act as molecular chaperones during periods of stress by binding to other proteins. Using expressed sequence tag (EST) and silkworm (Bombyx mori) transcriptome databases, we identified 27 cDNA sequences encoding the conserved J domain, which is found in DnaJ-type Hsps. Of the 27 J domain-containing sequences, 25 were complete cDNA sequences. We divided them into three types according to the number and presence of conserved domains. By analyzing the gene structures, intron numbers, and conserved domains and constructing a phylogenetic tree, we found that the DnaJ family had undergone convergent evolution, obtaining new domains to expand the diversity of its family members. The acquisition of the new DnaJ domains most likely occurred prior to the evolutionary divergence of prokaryotes and eukaryotes. The expression of DnaJ genes in the silkworm was generally higher in the fat body. The tissue distribution of DnaJ1 proteins was detected by western blotting, demonstrating that in the fifth-instar larvae, the DnaJ1 proteins were expressed at their highest levels in hemocytes, followed by the fat body and head. We also found that the DnaJ1 transcripts were likely differentially translated in different tissues. Using immunofluorescence cytochemistry, we revealed that in the blood cells, DnaJ1 was mainly localized in the cytoplasm. Copyright © 2015 Elsevier B.V. All rights reserved.

Putative Monofunctional Type I Polyketide Synthase Units: A Dinoflagellate-Specific Feature?

PubMed Central

Eichholz, Karsten; Beszteri, Bánk; John, Uwe

2012-01-01

Marine dinoflagellates (alveolata) are microalgae of which some cause harmful algal blooms and produce a broad variety of most likely polyketide synthesis derived phycotoxins. Recently, novel polyketide synthesase (PKS) transcripts have been described from the Florida red tide dinoflagellate Karenia brevis (gymnodiniales) which are evolutionarily related to Type I PKS but were apparently expressed as monofunctional proteins, a feature typical of Type II PKS. Here, we investigated expression units of PKS I-like sequences in Alexandrium ostenfeldii (gonyaulacales) and Heterocapsa triquetra (peridiniales) at the transcript and protein level. The five full length transcripts we obtained were all characterized by polyadenylation, a 3′ UTR and the dinoflagellate specific spliced leader sequence at the 5′end. Each of the five transcripts encoded a single ketoacylsynthase (KS) domain showing high similarity to K. brevis KS sequences. The monofunctional structure was also confirmed using dinoflagellate specific KS antibodies in Western Blots. In a maximum likelihood phylogenetic analysis of KS domains from diverse PKSs, dinoflagellate KSs formed a clade placed well within the protist Type I PKS clade between apicomplexa, haptophytes and chlorophytes. These findings indicate that the atypical PKS I structure, i.e., expression as putative monofunctional units, might be a dinoflagellate specific feature. In addition, the sequenced transcripts harbored a previously unknown, apparently dinoflagellate specific conserved N-terminal domain. We discuss the implications of this novel region with regard to the putative monofunctional organization of Type I PKS in dinoflagellates. PMID:23139807

Detection of Proteins on Blot Membranes

PubMed Central

Goldman, Aaron; Harper, Sandra; Speicher, David W.

2017-01-01

Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics. Protocols are included for seven general protein stains (amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, India ink, and MemCode) and three fluorescent protein stains (fluorescamine, IAEDANS, and SYPRO Ruby). Also included is an in-depth discussion of the different blot membrane types and the compatibility of different protein stains with downstream applications, such as immunoblotting or N-terminal Edman sequencing. PMID:27801518

Detection of Proteins on Blot Membranes.

PubMed

Goldman, Aaron; Harper, Sandra; Speicher, David W

2016-11-01

Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics. Protocols are included for seven general protein stains (amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, India ink, and MemCode) and three fluorescent protein stains (fluorescamine, IAEDANS, and SYPRO Ruby). Also included is an in-depth discussion of the different blot membrane types and the compatibility of different protein stains with downstream applications, such as immunoblotting or N-terminal Edman sequencing. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Identification, immunolocalization, and characterization analyses of an exopeptidase of papain superfamily, (cathepsin C) from Clonorchis sinensis.

PubMed

Liang, Pei; He, Lei; Xu, Yanquan; Chen, Xueqing; Huang, Yan; Ren, Mengyu; Liang, Chi; Li, Xuerong; Xu, Jin; Lu, Gang; Yu, Xinbing

2014-10-01

Cathepsin C is an important exopeptidase of papain superfamily and plays a number of great important roles during the parasitic life cycle. The amino acid sequence of cathepsin C from Clonorchis sinensis (C. sinensis) showed 54, 53, and 49% identities to that of Schistosoma japonicum, Schistosoma mansoni, and Homo sapiens, respectively. Phylogenetic analysis utilizing the sequences of papain superfamily of C. sinensis demonstrated that cathepsin C and cathepsin Bs came from a common ancestry. Cathepsin C of C. sinensis (Cscathepsin C) was identified as an excretory/secretory product by Western blot analysis. The results of transcriptional level and translational level of Cscathepsin C at metacercaria stage were higher than that at adult worms. Immunolocalization analysis indicated that Cscathepsin C was specifically distributed in the suckers (oral sucker and ventral sucker), eggs, vitellarium, intestines, and testis of adult worms. In the metacercaria, it was mainly detected on the cyst wall and excretory bladder. Combining with the results mentioned above, it implies that Cscathepsin C may be an essential proteolytic enzyme for proteins digestion of hosts, nutrition assimilation, and immune invasion of C. sinensis. Furthermore, it may be a potential diagnostic antigen and drug target against C. sinensis infection.

General characterization of Tityus fasciolatus scorpion venom. Molecular identification of toxins and localization of linear B-cell epitopes.

PubMed

Mendes, T M; Guimarães-Okamoto, P T C; Machado-de-Avila, R A; Oliveira, D; Melo, M M; Lobato, Z I; Kalapothakis, E; Chávez-Olórtegui, C

2015-06-01

This communication describes the general characteristics of the venom from the Brazilian scorpion Tityus fasciolatus, which is an endemic species found in the central Brazil (States of Goiás and Minas Gerais), being responsible for sting accidents in this area. The soluble venom obtained from this scorpion is toxic to mice being the LD50 is 2.984 mg/kg (subcutaneally). SDS-PAGE of the soluble venom resulted in 10 fractions ranged in size from 6 to 10-80 kDa. Sheep were employed for anti-T. fasciolatus venom serum production. Western blotting analysis showed that most of these venom proteins are immunogenic. T. fasciolatus anti-venom revealed consistent cross-reactivity with venom antigens from Tityus serrulatus. Using known primers for T. serrulatus toxins, we have identified three toxins sequences from T. fasciolatus venom. Linear epitopes of these toxins were localized and fifty-five overlapping pentadecapeptides covering complete amino acid sequence of the three toxins were synthesized in cellulose membrane (spot-synthesis technique). The epitopes were located on the 3D structures and some important residues for structure/function were identified. Copyright © 2015 Elsevier Ltd. All rights reserved.

Whole-exome sequencing in a single proband reveals a mutation in the CHST8 gene in autosomal recessive peeling skin syndrome

PubMed Central

Cabral, Rita M.; Kurban, Mazen; Wajid, Muhammad; Shimomura, Yutaka; Petukhova, Lynn; Christiano, Angela M.

2015-01-01

Generalized peeling skin syndrome (PSS) is an autosomal recessive genodermatosis characterized by lifelong, continuous shedding of the upper epidermis. Using whole-genome homozygozity mapping and whole-exome sequencing, we identified a novel homozygous missense mutation (c.229C>T, R77W) within the CHST8 gene, in a large consanguineous family with non-inflammatory PSS type A. CHST8 encodes a Golgi transmembrane N-acetylgalactosamine-4-O-sulfotransferase (GalNAc4-ST1), which we show by immunofluorescence staining to be expressed throughout normal epidermis. A colorimetric assay for total sulfated glycosaminoglycan (GAG) quantification, comparing human keratinocytes (CCD1106 KERTr) expressing wild type and mutant recombinant GalNAc4-ST1, revealed decreased levels of total sulfated GAGs in cells expressing mutant GalNAc4-ST1, suggesting loss of function. Western blotting revealed lower expression levels of mutant recombinant GalNAc4-ST1 compared to wild type, suggesting that accelerated degradation may result in loss of function, leading to PSS type A. This is the first report describing a mutation as the cause of PSS type A. PMID:22289416

Whole-exome sequencing in a single proband reveals a mutation in the CHST8 gene in autosomal recessive peeling skin syndrome.

PubMed

Cabral, Rita M; Kurban, Mazen; Wajid, Muhammad; Shimomura, Yutaka; Petukhova, Lynn; Christiano, Angela M

2012-04-01

Generalized peeling skin syndrome (PSS) is an autosomal recessive genodermatosis characterized by lifelong, continuous shedding of the upper epidermis. Using whole-genome homozygozity mapping and whole-exome sequencing, we identified a novel homozygous missense mutation (c.229C>T, R77W) within the CHST8 gene, in a large consanguineous family with non-inflammatory PSS type A. CHST8 encodes a Golgi transmembrane N-acetylgalactosamine-4-O-sulfotransferase (GalNAc4-ST1), which we show by immunofluorescence staining to be expressed throughout normal epidermis. A colorimetric assay for total sulfated glycosaminoglycan (GAG) quantification, comparing human keratinocytes (CCD1106 KERTr) expressing wild type and mutant recombinant GalNAc4-ST1, revealed decreased levels of total sulfated GAGs in cells expressing mutant GalNAc4-ST1, suggesting loss of function. Western blotting revealed lower expression levels of mutant recombinant GalNAc4-ST1 compared to wild type, suggesting that accelerated degradation may result in loss of function, leading to PSS type A. This is the first report describing a mutation as the cause of PSS type A. Copyright © 2012 Elsevier Inc. All rights reserved.

Sequencing, bioinformatic characterization and expression pattern of a putative amino acid transporter from the parasitic cestode Echinococcus granulosus.

PubMed

Camicia, Federico; Paredes, Rodolfo; Chalar, Cora; Galanti, Norbel; Kamenetzky, Laura; Gutierrez, Ariana; Rosenzvit, Mara C

2008-03-31

We have sequenced and partially characterized an Echinococcus granulosus cDNA, termed egat1, from a protoscolex signal sequence trap (SST) cDNA library. The isolated 1627 bp long cDNA contains an ORF of 489 amino acids and shows an amino acid identity of 30% with neutral and excitatory amino acid transporters members of the Dicarboxylate/Amino Acid Na+ and/or H+ Cation Symporter family (DAACS) (TC 2.A.23). Additional bioinformatics analysis of EgAT1, confirmed the results obtained by similarity searches and showed the presence of 9 to 10 transmembrane domains, consensus sequences for N-glycosylation between the third and fourth transmembrane domain, a highly similar hydropathy profile with ASCT1 (a known member of DAACS family), high score with SDF (Sodium Dicarboxilate Family) and similar motifs with EDTRANSPORT, a fingerprint of excitatory amino acid transporters. The localization of the putative amino acid transporter was analyzed by in situ hybridization and immunofluorescence in protoscoleces and associated germinal layer. The in situ hybridization labelling indicates the distribution of egat1 mRNA throughout the tegument. EgAT1 protein, which showed in Western blots a molecular mass of approximately 60 kD, is localized in the subtegumental region of the metacestode, particularly around suckers and rostellum of protoscoleces and layers from brood capsules. The sequence and expression analyses of EgAT1 pave the way for functional analysis of amino acids transporters of E. granulosus and its evaluation as new drug targets against cystic echinococcosis.

Vesicular monoamine transporter-1 (VMAT-1) mRNA and immunoreactive proteins in mouse brain.

PubMed

Ashe, Karen M; Chiu, Wan-Ling; Khalifa, Ahmed M; Nicolas, Antoine N; Brown, Bonnie L; De Martino, Randall R; Alexander, Clayton P; Waggener, Christopher T; Fischer-Stenger, Krista; Stewart, Jennifer K

2011-01-01

Vesicular monoamine transporter 1 (VMAT-1) mRNA and protein were examined (1) to determine whether adult mouse brain expresses full-length VMAT-1 mRNA that can be translated to functional transporter protein and (2) to compare immunoreactive VMAT-1 proteins in brain and adrenal. VMAT-1 mRNA was detected in mouse brain with RT-PCR. The cDNA was sequenced, cloned into an expression vector, transfected into COS-1 cells, and cell protein was assayed for VMAT-1 activity. Immunoreactive proteins were examined on western blots probed with four different antibodies to VMAT-1. Sequencing confirmed identity of the entire coding sequences of VMAT-1 cDNA from mouse medulla oblongata/pons and adrenal to a Gen-Bank reference sequence. Transfection of the brain cDNA into COS-1 cells resulted in transporter activity that was blocked by the VMAT inhibitor reserpine and a proton ionophore, but not by tetrabenazine, which has a high affinity for VMAT-2. Antibodies to either the C- or N- terminus of VMAT-1 detected two proteins (73 and 55 kD) in transfected COS-1 cells. The C-terminal antibodies detected both proteins in extracts of mouse medulla/pons, cortex, hypothalamus, and cerebellum but only the 73 kD protein and higher molecular weight immunoreactive proteins in mouse adrenal and rat PC12 cells, which are positive controls for rodent VMAT-1. These findings demonstrate that a functional VMAT-1 mRNA coding sequence is expressed in mouse brain and suggest processing of VMAT-1 protein differs in mouse adrenal and brain.

Characterization and biological properties of a new staphylococcal exotoxin

PubMed Central

1994-01-01

Staphylococcus aureus strain D4508 is a toxic shock syndrome toxin 1- negative clinical isolate from a nonmenstrual case of toxic shock syndrome (TSS). In the present study, we have purified and characterized a new exotoxin from the extracellular products of this strain. This toxin was found to have a molecular mass of 25.14 kD by mass spectrometry and an isoelectric point of 5.65 by isoelectric focusing. We have also cloned and sequenced its corresponding genomic determinant. The DNA sequence encoding the mature protein was found to be 654 base pairs and is predicted to encode a polypeptide of 218 amino acids. The deduced protein contains an NH2-terminal sequence identical to that of the native protein. The calculated molecular weight (25.21 kD) of the recombinant mature protein is also consistent with that of the native molecules. When injected intravenously into rabbits, both the native and recombinant toxins induce an acute TSS-like illness characterized by high fever, hypotension, diarrhea, shock, and in some cases death, with classical histological findings of TSS. Furthermore, the activity of the toxin is specifically enhanced by low quantities of endotoxins. The toxicity can be blocked by rabbit immunoglobulin G antibody specific for the toxin. Western blotting and DNA sequencing data confirm that the protein is a unique staphylococcal exotoxin, yet shares significant sequence homology with known staphylococcal enterotoxins, especially the SEA, SED, and SEE toxins. We conclude therefore that this 25-kD protein belongs to the staphylococcal enterotoxin gene family that is capable of inducing a TSS-like illness in rabbits. PMID:7964453

Sequence, molecular properties, and chromosomal mapping of mouse lumican

NASA Technical Reports Server (NTRS)

Funderburgh, J. L.; Funderburgh, M. L.; Hevelone, N. D.; Stech, M. E.; Justice, M. J.; Liu, C. Y.; Kao, W. W.; Conrad, G. W.; Spooner, B. S. (Principal Investigator)

1995-01-01

PURPOSE. Lumican is a major proteoglycan of vertebrate cornea. This study characterizes mouse lumican, its molecular form, cDNA sequence, and chromosomal localization. METHODS. Lumican sequence was determined from cDNA clones selected from a mouse corneal cDNA expression library using a bovine lumican cDNA probe. Tissue expression and size of lumican mRNA were determined using Northern hybridization. Glycosidase digestion followed by Western blot analysis provided characterization of molecular properties of purified mouse corneal lumican. Chromosomal mapping of the lumican gene (Lcn) used Southern hybridization of a panel of genomic DNAs from an interspecific murine backcross. RESULTS. Mouse lumican is a 338-amino acid protein with high-sequence identity to bovine and chicken lumican proteins. The N-terminus of the lumican protein contains consensus sequences for tyrosine sulfation. A 1.9-kb lumican mRNA is present in cornea and several other tissues. Antibody against bovine lumican reacted with recombinant mouse lumican expressed in Escherichia coli and also detected high molecular weight proteoglycans in extracts of mouse cornea. Keratanase digestion of corneal proteoglycans released lumican protein, demonstrating the presence of sulfated keratan sulfate chains on mouse corneal lumican in vivo. The lumican gene (Lcn) was mapped to the distal region of mouse chromosome 10. The Lcn map site is in the region of a previously identified developmental mutant, eye blebs, affecting corneal morphology. CONCLUSIONS. This study demonstrates sulfated keratan sulfate proteoglycan in mouse cornea and describes the tools (antibodies and cDNA) necessary to investigate the functional role of this important corneal molecule using naturally occurring and induced mutants of the murine lumican gene.

Sperm flagella protein components: Human meichroacidin constructed by the membrane occupation and recognition nexus motif

PubMed Central

MATSUOKA, YASUHIRO; NISHIMURA, HIROMI; NUMAZAWA, KAHORI; TSUCHIDA, JUNJI; MIYAGAWA, YASUSHI; TSUJIMURA, AKIRA; MATSUMIYA, KIYOMI; OKUYAMA, AKIHIKO; NISHIMUNE, YOSHITAKE

2005-01-01

Background and Aims:   In a previous study, the authors of the present study cloned mouse meichroacidin (MCA), which is expressed in stages of spermatogenesis from pachytene spermatocytes through round spermatid germ cells. MCA protein contains the membrane occupation and recognition nexus (MORN) motif and localizes to a male meiotic metaphase chromosome. Recently, a MCA homolog of carp (Cyprinus carpio), MORN motif‐containing sperm‐specific axonemal protein (MSAP), was reportedly identified and localized in sperm flagella. Present knowledge of human spermiogenesis requires the identification of proteins in human sperm. The present study identified the human orthologue of MCA. Methods:   Colony hybridization using a human testis plasmid cDNA library was carried out to clone human MCA (h‐MCA) cDNA. Northern blot, Western blot, and immunohistochemical analyses were carried out. Results:   h‐MCA was found to be specifically expressed in the testes. The h‐MCA amino acid sequence shared 79.8% identity with mouse MCA and contained MORN motifs. h‐MCA localized in the sperm flagellum and basal body, as does MSAP in carp. Conclusion:   Expression and localization analyses showed that h‐MCA is a component of the sperm flagellum and basal body and might play an important role in the development of the sperm flagellum in humans. (Reprod Med Biol 2005; 4: 213–219) PMID:29699225

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.

Cloning of human prourokinase cDNA without the signal peptide and expression in Escherichia coli.

PubMed

Hu, B; Li, J; Yu, W; Fang, J

1993-01-01

Human prourokinase (pro-UK) cDNA without the signal peptide was obtained using synthetic oligonucleotide and DNA recombination techniques and was successfully expressed in E. coli. The plasmid pMMUK which contained pro-UK cDNA (including both the entire coding sequence and the sequence for signal peptide) was digested with Hind III and PstI, so that the N-terminal 371-bp fragment could be recovered. A 304-bp fragment was collected from the 371-bp fragment after partial digestion with Fnu4HI in order to remove the signal peptide sequence. An intermediate plasmid was formed after this 304-bp fragment and the synthetic oligonucleotide was ligated with pUC18. Correctness of the ligation was confirmed by enzyme digestion and sequencing. By joining the PstI-PstI fragment of pro-UK to the plasmid we obtained the final plasmid which contained the entire coding sequence of pro-UK without the signal peptide. The coding sequence with correct orientation was inserted into pBV220 under the control of the temperature-induced promoter PRPL, and mature pro-UK was expressed in E. coli at 42 degrees C. Both sonicated supernatant and inclusion bodies of the bacterial host JM101 showed positive results by ELISA and FAPA assays. After renaturation, the biological activity of the expressed product was increased from 500-1000IU/L to about 60,000IU/L. The bacterial pro-UK showed a molecular weight of about 47,000 daltons by Western blot analysis. It can be completely inhibited by UK antiserum but not by t-PA antiserum nor by normal rabbit serum.

Congenital hypothyroidism with goiter in Tenterfield terriers.

PubMed

Dodgson, S E; Day, R; Fyfe, J C

2012-01-01

A cluster of cases of congenital hypothyroidism with goiter (CHG) in Tenterfield Terriers was identified and hypothesized to be dyshormonogenesis of genetic etiology with autosomal recessive inheritance. To describe the phenotype, thyroid histopathology, biochemistry, mode of inheritance, and causal mutation of CHG in Tenterfield Terriers. Thyroid tissue from 1 CHG-affected Tenterfield Terriers, 2 affected Toy Fox Terriers, and 7 normal control dogs. Genomic DNA from blood or buccal brushings of 114 additional Tenterfield Terriers. Biochemical and genetic segregation analysis of functional gene candidates in a Tenterfield Terrier kindred. Thyroid peroxidase (TPO) iodide oxidation activity was measured, and TPO protein and SDS-resistant thyroglobulin aggregation were assessed on western blots. TPO cDNA was amplified from thyroid RNA and sequenced. Exons and flanking splice sites were amplified from genomic DNA and sequenced. Variant TPO allele segregation was assessed by restriction enzyme digestion of PCR products. Thyroid from an affected pup had lesions consistent with dyshormonogenesis. TPO activity was absent, but normal sized immunocrossreactive TPO protein was present. Affected dog cDNA and genomic sequences revealed a homozygous TPO missense mutation in exon 9 (R593W) that was heterozygous in all obligate carriers and in 31% of other clinically normal Tenterfield Terriers. The mutation underlying CHG in Tenterfield Terriers was identified, and a convenient carrier test made available for screening Tenterfield Terriers used for breeding. Copyright © 2012 by the American College of Veterinary Internal Medicine.

Exploration of G-quadruplex function in c-Myb gene and its transcriptional regulation by topotecan.

PubMed

Li, Fangyuan; Zhou, Jiang; Xu, Ming; Yuan, Gu

2018-02-01

Our bioinformatics research shows that there are four G-rich sequences (S1-S4) in the upstream region of the transcription start site of c-Myb gene, and we have proved that these sequences have the ability to form G-quadruplex structures. This work mainly focuses on G-quadruplex function, recognition and transcription regulation in c-Myb gene, revealing a novel regulatory element in c-Myb proximal promoter region, and its transcription regulation by G-quadruplex binder. The research has identified that the enhancer effect in c-Myb transcription was primarily affected by the G-quadruplex formed by S1 sequence, and the up-regulation effect may due to the removal of repressive progress of MZF-1 by stabilizing G-quadruplex. Attentions were being paid to the development of G-quadruplex binders for selective recognition, and topotecan was found to have high binding affinity in vitro and could effectively affect the c-Myb transcription activities in cells. The regulation of G-quadruplex with binders in transcriptional, translational levels by Q-RT-PCR and western blot was in expectation of providing a strategy for gene expression modulation. In conclusion, our study revealed a G-quadruplex structure in c-Myb proximal promoter region, which was of great importance in the regulation of c-Myb function. Copyright © 2017 Elsevier B.V. All rights reserved.

Translational initiation in Leishmania tarentolae and Phytomonas serpens (Kinetoplastida) is strongly influenced by pre-ATG triplet and its 5' sequence context.

PubMed

Lukes, Julius; Paris, Zdenek; Regmi, Sandesh; Breitling, Reinhard; Mureev, Sergey; Kushnir, Susanna; Pyatkov, Konstantin; Jirků, Milan; Alexandrov, Kirill A

2006-08-01

To investigate the influence of sequence context of translation initiation codon on translation efficiency in Kinetoplastida, we constructed a library of expression plasmids randomized in the three nucleotides prefacing ATG of a reporter gene encoding enhanced green fluorescent protein (EGFP). All 64 possible combinations of pre-ATG triplets were individually stably integrated into the rDNA locus of Leishmania tarentolae and the resulting cell lines were assessed for EGFP expression. The expression levels were quantified directly by measuring the fluorescence of EGFP protein in living cells and confirmed by Western blotting. We observed a strong influence of the pre-ATG triplet on the level of protein expression over a 20-fold range. To understand the degree of evolutionary conservation of the observed effect, we transformed Phytomonas serpens, a trypanosomatid parasite of plants, with a subset of the constructs. The pattern of translational efficiency mediated by individual pre-ATG triplets in this species was similar to that observed in L. tarentolae. However, the pattern of translational efficiency of two other proteins (red fluorescent protein and tetracycline repressor) containing selected pre-ATG triplets did not correlate with either EGFP or each other. Thus, we conclude that a conserved mechanism of translation initiation site selection exists in kinetoplastids that is strongly influenced not only by the pre-ATG sequences but also by the coding region of the gene.

Bph32, a novel gene encoding an unknown SCR domain-containing protein, confers resistance against the brown planthopper in rice.

PubMed

Ren, Juansheng; Gao, Fangyuan; Wu, Xianting; Lu, Xianjun; Zeng, Lihua; Lv, Jianqun; Su, Xiangwen; Luo, Hong; Ren, Guangjun

2016-11-23

An urgent need exists to identify more brown planthopper (Nilaparvata lugens Stål, BPH) resistance genes, which will allow the development of rice varieties with resistance to BPH to counteract the increased incidence of this pest species. Here, using bioinformatics and DNA sequencing approaches, we identified a novel BPH resistance gene, LOC_Os06g03240 (MSU LOCUS ID), from the rice variety Ptb33 in the interval between the markers RM19291 and RM8072 on the short arm of chromosome 6, where a gene for resistance to BPH was mapped by Jirapong Jairin et al. and renamed as "Bph32". This gene encodes a unique short consensus repeat (SCR) domain protein. Sequence comparison revealed that the Bph32 gene shares 100% sequence identity with its allele in Oryza latifolia. The transgenic introgression of Bph32 into a susceptible rice variety significantly improved resistance to BPH. Expression analysis revealed that Bph32 was highly expressed in the leaf sheaths, where BPH primarily settles and feeds, at 2 and 24 h after BPH infestation, suggesting that Bph32 may inhibit feeding in BPH. Western blotting revealed the presence of Pph (Ptb33) and Tph (TN1) proteins using a Penta-His antibody, and both proteins were insoluble. This study provides information regarding a valuable gene for rice defence against insect pests.

Cloning and characterization of an alternative splicing transcript of the gene coding for human cytidine deaminase.

PubMed

Lisboa, Bianca Cristina Garcia; Machado, Tamara da Rocha; Pimenta, Daniel Carvalho; Han, Sang Won

2007-02-01

Human cytidine deaminase (HCD) catalyzes the deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. The genomic sequence of HCD is formed by 31 kb with 4 exons and several alternative splicing signals, but an alternative form of HCD has yet to be reported. Here we describe the cloning and characterization of a small form of HCD, HSCD, and it is likely to be a product of alternative splicing of HCD. The alignment of DNA sequences shows that the HSCD matches HCD in 2 parts, except for a deletion of 170 bp. Based on the HCD genome organization, exons 1 and 4 should be joined and all sequences of introns and exons 2 and 3 should be deleted by splicing. This alternative splicing shifted the translation of the reading frame from the point of splicing. The estimated molecular mass is 9.8 kDa, and this value was confirmed by Western blot and mass spectroscopy after expressing the gene fused with glutathionine-S-transferase in the pGEX vector. The deletion and shift of the reading frame caused a loss of HCD activity, which was confirmed by enzyme assay and also with NIH3T3 cells modified to express HSCD and challenged against cytosine arabinoside. In this work we describe the identification and characterization of HSCD, which is the product of alternative splicing of the HCD gene.

Growth hormone-releasing hormone stimulates and somatostatin inhibits the release of a novel protein by cultured rat pituitary cells.

PubMed

Tachibana, K; Marquardt, H; Yokoya, S; Friesen, H G

1988-10-01

We have reported that the secretion of at least 17 distinct peptides [including rat (rGH)] GH by cultured rat pituitary cells was stimulated by GH-releasing hormone and inhibited by somatostatin, when analyzed by two-dimensional polyacrylamide gel electrophoresis. Three of these peptides (no. 23, 24, and 25) were not rGH immunoreactive. In order to determine whether these three peptides are fragments, degradation products or posttranscriptionally modified forms of rGH, rGH and peptide no. 23 were characterized structurally. From partial peptide maps of rGH and peptide no. 23 by V8 protease or chymotrypsin, it appeared that these peptides were not related to each other. By N-terminal microsequencing of two-dimensional polyacrylamide gel electrophoresis purified peptide, we have obtained the sequence of 24 N-terminal amino acid residues of peptide no. 23. This sequence has no significant homology with rGH or any other reported protein sequence. Antiserum was generated against a synthetic oligopeptide corresponding to amino acid residues 3-24 of peptide no. 23. The antiserum cross-reacted with peptides no. 23, 24, and 25 upon Western blot analysis. These results indicate that peptide no. 23 has a novel structure unrelated to other pituitary hormones. Since its secretion is influenced by GH-releasing hormone and somatostatin, peptide no. 23 may represent a previously unrecognized structurally unique growth factor.

Functional characterization of recombinant bromelain of Ananas comosus expressed in a prokaryotic system.

PubMed

George, Susan; Bhasker, Salini; Madhav, Harish; Nair, Archana; Chinnamma, Mohankumar

2014-02-01

Bromelain (BRM) is a defense protein present in the fruit and stem of pineapple (Ananas comosus) and it is grouped as a cysteine protease enzyme with diversified medicinal uses. Based on its therapeutic applications, bromelain has got sufficient attention in pharmaceutical industries. In the present study, the full coding gene of bromelain in pineapple stem (1,093 bp) was amplified by RT-PCR. The PCR product was cloned, sequenced, and characterized. The sequence analysis of the gene revealed the single nucleotide polymorphism and its phylogenetic relatedness. The peptide sequence deduced from the gene showed the amino acid variations, physicochemical properties and secondary and tertiary structural features of the protein. The full BRM gene was transformed to prokaryotic vector pET32b and expressed in Escherichia coli BL21 DE3pLysS host cells successfully. The identity of the recombinant bromelain (rBRM) protein was confirmed by Western blot analysis using anti-BRM-rabbit IgG antibody. The activity of recombinant bromelain compared with purified native bromelain was determined by protease assay. The inhibitory effect of rBRM compared with native BRM in the growth of Gram-positive and Gram-negative strains of Streptococcus agalactiae and Escherichia coli O111 was evident from the antibacterial sensitivity test. To the best of our knowledge, this is the first report showing the bactericidal property of rBRM expressed in a prokaryotic system.

Bph32, a novel gene encoding an unknown SCR domain-containing protein, confers resistance against the brown planthopper in rice

PubMed Central

Ren, Juansheng; Gao, Fangyuan; Wu, Xianting; Lu, Xianjun; Zeng, Lihua; Lv, Jianqun; Su, Xiangwen; Luo, Hong; Ren, Guangjun

2016-01-01

An urgent need exists to identify more brown planthopper (Nilaparvata lugens Stål, BPH) resistance genes, which will allow the development of rice varieties with resistance to BPH to counteract the increased incidence of this pest species. Here, using bioinformatics and DNA sequencing approaches, we identified a novel BPH resistance gene, LOC_Os06g03240 (MSU LOCUS ID), from the rice variety Ptb33 in the interval between the markers RM19291 and RM8072 on the short arm of chromosome 6, where a gene for resistance to BPH was mapped by Jirapong Jairin et al. and renamed as “Bph32”. This gene encodes a unique short consensus repeat (SCR) domain protein. Sequence comparison revealed that the Bph32 gene shares 100% sequence identity with its allele in Oryza latifolia. The transgenic introgression of Bph32 into a susceptible rice variety significantly improved resistance to BPH. Expression analysis revealed that Bph32 was highly expressed in the leaf sheaths, where BPH primarily settles and feeds, at 2 and 24 h after BPH infestation, suggesting that Bph32 may inhibit feeding in BPH. Western blotting revealed the presence of Pph (Ptb33) and Tph (TN1) proteins using a Penta-His antibody, and both proteins were insoluble. This study provides information regarding a valuable gene for rice defence against insect pests. PMID:27876888

Shine-Dalgarno sequence enhances the efficiency of lacZ repression by artificial anti-lac antisense RNAs in Escherichia coli.

PubMed

Stefan, Alessandra; Schwarz, Flavio; Bressanin, Daniela; Hochkoeppler, Alejandro

2010-11-01

Silencing of the lacZ gene in Escherichia coli was attempted by means of the expression of antisense RNAs (asRNAs) in vivo. A short fragment of lacZ was cloned into the pBAD expression vector, in reverse orientation, using the EcoRI and PstI restriction sites. This construct (pBAD-Zcal1) was used to transform E. coli cells, and the antisense transcription was induced simply by adding arabinose to the culture medium. We demonstrated that the Zcal1 asRNA effectively silenced lacZ using β-galactosidase activity determinations, SDS-PAGE, and Western blotting. Because the concentration of the lac mRNA was always high in cells that expressed Zcal1, we hypothesize that this antisense acts by inhibiting messenger translation. Similar analyses, performed with a series of site-specific Zcal1 mutants, showed that the Shine-Dalgarno sequence, which is conferred by the pBAD vector, is an essential requisite for silencing competence. Indeed, the presence of the intact Shine-Dalgarno sequence positively affects asRNA stability and, hence, silencing effectiveness. Our observations will contribute to the understanding of the main determinants of silencing as exerted by asRNAs as well as provide useful support for the design of robust and efficient prokaryotic gene silencers. Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Osthole inhibits proliferation and induces apoptosis in human osteosarcoma cells.

PubMed

Ding, Yong; Lu, Xiongwei; Hu, Xiaopeng; Ma, Jie; Ding, Huan

2014-02-01

The purpose of this study was to investigate the effect of osthole on osteosarcoma cell proliferation and apoptosis. Cell counting Kit-8 assay was performed to establish the effects of osthole on osteosarcoma MG-63 cell proliferation. Annexin V-FITC/PI was performed to analyze the apoptotic rate of the cells. The inhibitory effects of osthole on the expression of BCL-2, BAX, and caspase-3 were detected by Western blotting. Osthole inhibited the growth of human osteosarcoma MG-63 cells by inhibiting cell proliferation and induced cell apoptosis. Western blotting demonstrated that osthole downregulated the expressions of BCL-2 and caspase-3 and upregulated the expression of BAX in human osteosarcoma cells. Osthole can inhibit osteosarcoma cell proliferation and induced apoptosis effectively in a dose-dependent manner through downregulating the expression of BCL-2 and caspase-3 proteins levels and upregulating the expression of BAX proteins levels.

Exploration of Spinal Cord Aging-Related Proteins Using a Proteomics Approach.

PubMed

Kamiya, Koshiro; Furuya, Takeo; Hashimoto, Masayuki; Mannoji, Chikato; Inada, Taigo; Ota, Mitsutoshi; Maki, Satoshi; Ijima, Yasushi; Saito, Junya; Kitamura, Mitsuhiro; Ohtori, Seiji; Orita, Sumihisa; Inage, Kazuhide; Yamazaki, Masashi; Koda, Masao

2017-01-01

How aging affects the spinal cord at a molecular level is unclear. The aim of this study was to explore spinal cord aging-related proteins that may be involved in pathological mechanisms of age-related changes in the spinal cord. Spinal cords of 2-year-old and 8-week-old female Sprague-Dawley rats were dissected from the animals. Protein samples were subjected to 2-dimentional polyacrylamide gel electrophoresis followed by mass spectrometry. Screened proteins were further investigated with immunohistochemistry and Western blotting. Among the screened proteins, we selected α-crystallin B-subunit (αB-crystallin) and peripherin for further investigation because these proteins were previously reported to be related to central nervous system pathologies. Immunohistochemistry and Western blotting revealed significant upregulation of αB-crystallin and peripherin expression in aged rat spinal cord. Further exploration is needed to elucidate the precise mechanism and potential role of these upregulated proteins in spinal cord aging processes.

Quality of different in-clinic test systems for feline immunodeficiency virus and feline leukaemia virus infection.

PubMed

Hartmann, Katrin; Griessmayr, Pascale; Schulz, Bianka; Greene, Craig E; Vidyashankar, Anand N; Jarrett, Os; Egberink, Herman F

2007-12-01

Many new diagnostic in-house tests for identification of feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) infection have been licensed for use in veterinary practice, and the question of the relative merits of these kits has prompted comparative studies. This study was designed to define the strengths and weaknesses of seven FIV and eight FeLV tests that are commercially available. In this study, 536 serum samples from randomly selected cats were tested. Those samples reacting FIV-positive in at least one of the tests were confirmed by Western blot, and those reacting FeLV-positive were confirmed by virus isolation. In addition, a random selection of samples testing negative in all test systems was re-tested by Western blot (100 samples) and by virus isolation (81 samples). Specificity, sensitivity, positive and negative predictive values of each test and the quality of the results were compared.

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.

Hemin offers neuroprotection through inducing exogenous neuroglobin in focal cerebral hypoxic-ischemia in rats

PubMed Central

Song, Xue; Xu, Rui; Xie, Fei; Zhu, Haiyuan; Zhu, Ji; Wang, Xin

2014-01-01

Objective: To investigate the inducible effect of hemin on exogenous neuroglobin (Ngb) in focal cerebral hypoxic-ischemia in rats. Methods: 125 healthy SD rats were randomly divided into five groups: sham-operation control group, operation group, hemin treatment group, exogenous Ngb treatment group, and hemin and exogenous Ngb joint treatment group. Twenty-four hours after focal cerebral hypoxic-ischemia, Ngb expression was evaluated by immunocytochemistry, RT-PCR, and western blot analyses, while the brain water content and infarct volume were examined. Results: Immunocytochemistry, RT-PCR, and western blot analyses showed more pronounced Ngb expression in the hemin and exogenous Ngb joint operation group than in the hemin or exogenous Ngb individual treatment groups, thus producing significant differences in brain water content and infarct volume (p < 0.05). Conclusions: Hemin may be beneficial in protecting against focal cerebral hypoxic-ischemia through inducing the expression of exogenous Ngb. PMID:24966924

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.

Practical diagnostic testing for human immunodeficiency virus.

PubMed Central

Jackson, J B; Balfour, H H

1988-01-01

Since the discovery of human immunodeficiency virus (HIV) as the causative agent of acquired immunodeficiency syndrome in 1983, there has been a proliferation of diagnostic tests. These assays can be used to detect the presence of HIV antibody, HIV antigen, HIV ribonucleic and deoxyribonucleic acids, and HIV reverse transcriptase. Enzyme-linked immunosorbent assays, Western blot, radioimmunoprecipitation assays, indirect immunofluorescence assays, reverse transcriptase assays, and several molecular hybridization techniques are currently available. Enzyme-linked immunosorbent, Western blot, and indirect immunofluorescence assays for HIV antibody are very sensitive, specific, and adaptable to most laboratories. An enzyme-linked immunosorbent assay for HIV antigen is also readily adaptable to most laboratories and will be commercially available soon. While the other assays are more tedious, they are valuable confirmatory tests and are suitable for reference laboratories. The biohazards of performing HIV testing can be minimized with proper biosafety measures. Images PMID:3060241

Single-band upconversion nanoprobes for multiplexed simultaneous in situ molecular mapping of cancer biomarkers.

PubMed

Zhou, Lei; Wang, Rui; Yao, Chi; Li, Xiaomin; Wang, Chengli; Zhang, Xiaoyan; Xu, Congjian; Zeng, Aijun; Zhao, Dongyuan; Zhang, Fan

2015-04-24

The identification of potential diagnostic markers and target molecules among the plethora of tumour oncoproteins for cancer diagnosis requires facile technology that is capable of quantitatively analysing multiple biomarkers in tumour cells and tissues. Diagnostic and prognostic classifications of human tumours are currently based on the western blotting and single-colour immunohistochemical methods that are not suitable for multiplexed detection. Herein, we report a general and novel method to prepare single-band upconversion nanoparticles with different colours. The expression levels of three biomarkers in breast cancer cells were determined using single-band upconversion nanoparticles, western blotting and immunohistochemical technologies with excellent correlation. Significantly, the application of antibody-conjugated single-band upconversion nanoparticle molecular profiling technology can achieve the multiplexed simultaneous in situ biodetection of biomarkers in breast cancer cells and tissue specimens and produce more accurate results for the simultaneous quantification of proteins present at low levels compared with classical immunohistochemical technology.

Ethanol extracts of black pepper or turmeric down-regulated SIRT1 protein expression in Daudi culture cells.

PubMed

Nishimura, Yuri; Kitagishi, Yasuko; Yoshida, Hitomi; Okumura, Naoko; Matsuda, Satoru

2011-01-01

SIRT1 is a mammalian candidate molecule involved in longevity and diverse metabolic processes. The present study aimed to determine the effects of certain herbs and spices on SIRT1 expression. Human cell lines Daudi, Jurkat, U937 and K562 were cultured in RPMI-1640. Herb and spice powders were prepared and the supernatants were collected. RT-PCR was used to quantify the expression level of the gene. Protein samples were then analyzed by Western blotting. Western blotting revealed the down-regulation of SIRT1 protein expression in Daudi cells treated with extracts of black pepper or turmeric. On the other hand, the effect on the SIRT1 gene expression examined by reverse transcription polymerase chain reaction was unaltered. In conclusion, component(s) of certain herbs and spices may induce the down-regulation of SIRT1 protein.

Enhanced production and purification of recombinant surface array protein (Sap) for use in detection of Bacillus anthracis.

PubMed

Puranik, Nidhi; Tripathi, N K; Pal, V; Goel, Ajay Kumar

2018-05-01

Surface array protein (Sap) can be an important biomarker for specific detection of Bacillus anthracis , which is released by the bacterium during its growth in culture broth. In the present work, we have cloned and expressed Sap in Escherichia coli . The culture conditions and cultivation media were optimized and used in batch fermentation process for scale up of Sap in soluble form. The recombinant Sap was purified employing affinity chromatography followed by diafiltration. The final yield of purified protein was 20 and 46 mg/l of culture during shake flasks and batch fermentation, respectively. The protein purity and its reactivity were confirmed employing SDS-PAGE and Western blot, respectively. The antibodies raised against purified Sap were evaluated by Western blotting for detection of Sap released by B. anthracis . Our results showed that the Sap could be a novel marker for detection and confirmation of B. anthracis .

Antibody reactivity to Borrelia burgdorferi sensu stricto antigens in patients from the Brazilian Amazon region with skin diseases not related to Lyme disease.

PubMed

Santos, Mônica; Ribeiro-Rodrigues, Rodrigo; Lobo, Rogério; Talhari, Sinésio

2010-05-01

In the present study, we report the occurrence of borreliosis in patients from the Brazilian Amazonic region. Nineteen (7.2%) out of 270 dermatological patients with different skin diseases (no one with clinical Lyme disease), tested positive by ELISA for Borrelia burgdorferi. Serum samples from 15 out of the 19 ELISA-positive patients were further evaluated by Western blot. Presence of Borrelia burgdorferi specific IgG was confirmed in eight (53.3%) out of the 15 patients. All eight patients with ELISA and Western blot positive reactions were treated with doxycycline, according to the Centers for Disease Control and Prevention guidelines. One of them had clinical manifestations of colagenosis and was sent to the Department of Internal Medicine for further investigation. Data presented here suggested that borreliosis "lato sensu" is in the Brazilian Amazon region.

Natural history, clinicoradiologic correlates, and response to triclabendazole in acute massive fascioliasis.

PubMed

Marcos, Luis A; Tagle, Martin; Terashima, Angelica; Bussalleu, Alejandro; Ramirez, Cesar; Carrasco, Carlos; Valdez, Luis; Huerta-Mercado, Jorge; Freedman, David O; Vinetz, Joseph M; Gotuzzo, Eduardo

2008-02-01

Fascioliasis is highly endemic in the Andean region of South America. Newer serological assays have improved our ability to diagnose acute fascioliasis. The diagnosis was established by Fasciola hepatica serology (Fas2-ELISA or Western blot) in 10 patients. Identifiable exposure included ingestion of watercress (N = 8), alfalfa juice (N = 5), and lettuce (N = 1). Computed tomography of the abdomen showed hepatomegaly (N = 9), track-like hypodense lesions with subcapsular location (N = 8), and subcapsular hematoma (N = 2). Radiologic sequelae included cyst calcifications detectable at least 3 years after treatment. Stool examinations were negative for F. hepatica eggs; serology was positive (Arc II [N = 2], Fas2-ELISA [N = 6], Western blot [N = 2]). The syndrome of eosinophilia, fever, and right upper quadrant pain, elevated transaminases without jaundice, hypodense liver lesions on CT, and an appropriate exposure history suggests acute fascioliasis. Fascioliasis is specifically treatable with a single dose of triclabendazole.

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

Effects of hypoxia-inducible factor-1α silencing on the proliferation of CBRH-7919 hepatoma cells

PubMed Central

Xu, Lin-Feng; Ni, Jia-Yan; Sun, Hong-Liang; Chen, Yao-Ting; Wu, Yu-Dan

2013-01-01

AIM: To study the effects of hypoxia-inducible factor-1α (HIF-1α) silencing on the proliferation of hypoxic CBRH-7919 rat hepatoma cells. METHODS: The CBRH-7919 rat hepatoma cell line was used in this study and the hypoxic model was constructed using CoCl2. The HIF-1α-specific RNAi sequences were designed according to the gene coding sequence of rat HIF-1α obtained from GeneBank. The secondary structure of the HIF-1α gene sequence was analyzed using RNA draw software. The small interfering RNA (siRNA) transfection mixture was produced by mixing the siRNA and Lipofectamine2000TM, and transfected into the hypoxic hepatoma cells. Real time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting assay were used to detect the expression levels of mRNA and protein. HIF-1α and vascular endothelial growth factor (VEGF) mRNA was determined using real time RT-PCR; the protein expression levels of AKT, p-AKT, p21 and cyclinD1 were determined using Western blotting. The proliferation of hepatoma cells was observed using the methyl thiazolyl tetrazolium (MTT) assay and the bromodeoxyuridine (BrdU) incorporation cell proliferation assay. RESULTS: Under induced hypoxia, the viability of the hepatoma cells reached a minimum at 800 μmol/L CoCl2; the viability of the cells was relatively high at CoCl2 concentrations between 100 μmol/L and 200 μmol/L. Under hypoxia, the mRNA and protein expression levels of HIF-1α and VEGF were significantly higher than that of hepatoma cells that were cultured in normaxia. HIF-1α-specific RNAi sequences were successfully transfected into hepatoma cells. The transfection of specific siRNAs significantly inhibited the mRNA and protein expression levels of HIF-1α and VEGF, along with the protein expression levels of p-AKT and cyclinD1; the protein expression of p21 was significantly increased, and there was no significant difference in the expression of AKT. The MTT assay showed that the amount of hepatoma cells in S phase in the siRNA transfection group was obviously smaller than that in the control group; in the siRNA transfection group, the amount of hepatoma cells in G1 phase was more than that in the control group. The BrdU incorporation assay showed that the number of BrdU positive hepatoma cells in the siRNA transfection group was less than that in the control group. The data of the MTT assay and BrdU incorporation assay suggested that HIF-1α silencing using siRNAs significantly inhibited the proliferation of hepatoma cells. CONCLUSION: Hypoxia increases the expression of HIF-1α, and HIF-1α silencing significantly inhibits the proliferation of hypoxic CBRH-7919 rat hepatoma cells. PMID:23555163

IMPACT (Imaging and Molecular Markers for Patients with Lung Cancer: Approaches with Molecular Targets and Complementary, Innovative Treatments and Therapeutic Modalities)

DTIC Science & Technology

2010-02-01

in t heir o ptimal g rowth media a nd h arvested an d l ysed t hen analyzed by Western bl otting f or t he expression of H sp90, Hsp27 , Hsp60...analyzed by western blotting for the expression of Hsp27 , 70 and 90. The same membrane was probed with actin as a control for protein loading

Identification of candidates for interacting partners of the tail domain of DcNMCP1, a major component of the Daucus carota nuclear lamina-like structure.

PubMed

Mochizuki, Ryota; Tsugama, Daisuke; Yamazaki, Michihiro; Fujino, Kaien; Masuda, Kiyoshi

2017-05-04

NMCP/CRWN (NUCLEAR MATRIX CONSTITUENT PROTEIN/CROWDED NUCLEI) is a major component of a protein fibrous meshwork (lamina-like structure) on the plant inner nuclear membrane. NMCP/CRWN contributes to regulating nuclear shape and nuclear functions. An NMCP/CRWN protein in Daucus carota (DcNMCP1) is localized to the nuclear periphery in interphase cells, and surrounds chromosomes in cells in metaphase and anaphase. The N-terminal region and the C-terminal region of DcNMCP1 are both necessary for localizing DcNMCP1 to the nuclear periphery. Here candidate interacting partners of the amino acid position 975-1053 of DcNMCP1 (T975-1053), which is present in the C-terminal region and contains a conserved sequence that plays a role in localizing DcNMCP1 to the nuclear periphery, are screened for. Arabidopsis thaliana nuclear proteins were subjected to far-Western blotting with GST-fused T975-1053 as a probe, and signals were detected at the positions corresponding to ∼70, ∼40, and ∼18 kDa. These ∼70, ∼40, and ∼18 kDa nuclear proteins were identified by mass spectrometry, and subjected to a yeast 2-hybrid (Y2H) analysis with T975-1053 as bait. In this analysis, the ∼40 kDa protein ARP7, which is a nuclear actin-related protein possibly involved in regulating chromatin structures, was confirmed to interact with T975-1053. Independently of the far-Western blotting, a Y2H screen was performed using T975-1053 as bait. Targeted Y2H assays confirmed that 3 proteins identified in the screen, MYB3, SINAT1, and BIM1, interact with T975-1053. These proteins might have roles in NMCP/CRWN protein-mediated biologic processes.

Aberrant EPHB4 gene methylation and childhood acute lymphoblastic leukemia

PubMed Central

Li, Yuhua; Wang, Huihui; Chen, Xiaowen; Mai, Huirong; Li, Changgang; Wen, Feiqiu

2017-01-01

The present study aimed to investigate the association between aberrant DNA methylation of the promoter region of the ephrin type-B receptor 4 (EPHB4) gene and the development of childhood acute lymphoblastic leukemia (ALL). Bisulfite sequencing polymerase chain reaction (BSP) was performed to determine the methylation density of cytosine-guanine pair islands in the promoter region of EPHB4, in bone marrow samples from 40 children with ALL. The mRNA and protein expression levels of EPHB4 were detected using reverse transcription-quantitative polymerase chain reaction and western blot analysis. A total of 10 children with idiopathic thrombocytopenic purpura (ITP) were recruited as controls. The results revealed that the average methylation density of the bone marrow samples from the patients with ALL was significantly higher, compared with the patients with ITP (P=0.046). The relative mRNA expression levels of EPHB4 in the patients with ITP (25.08±4.03) and the patients with ALL without methylation (12.33±2.16) were significantly higher, compared with that observed in the patients with ALL with methylation (6.48±2.73; P<0.01). Pearson analysis revealed a significant negative linear correlation between EPHB4 gene methylation and its expression levels (r=−0.957; P<0.01). Western blot analysis indicated that EPHB4 protein expression levels were low in the methylated ALL samples. An evaluation of the two-year disease-free survival (DFS) of the patients with ALL was performed, which revealed that the patients with unmethylated ALL exhibited a significantly higher two-year DFS rate, as compared with patients with methylated ALL (P=0.036). These results suggest that the methylation of the EPHB4 gene is prevalent in childhood ALL and may result in expressional inactivation, which consequently promotes ALL pathogenesis and is associated with an unfavorable prognosis. Therefore, the EPHB4 gene may function as a potential tumor suppressor in childhood ALL. PMID:29085439

Molecular characterization and analysis of a novel protein disulfide isomerase-like protein of Eimeria tenella.

PubMed

Han, Hongyu; Dong, Hui; Zhu, Shunhai; Zhao, Qiping; Jiang, Lianlian; Wang, Yange; Li, Liujia; Wu, Youlin; Huang, Bing

2014-01-01

Protein disulfide isomerase (PDI) and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE) according to the expressed sequence tag (EST). The EtPDIL cDNA contained 1129 nucleotides encoding 216 amino acids. The deduced EtPDIL protein belonged to thioredoxin-like superfamily and had a single predicted thioredoxin domain with a non-classical thioredoxin-like motif (SXXC). BLAST analysis showed that the EtPDIL protein was 55-59% identical to PDI-like proteins of other apicomplexan parasites. The transcript and protein levels of EtPDIL at different development stages were investigated by real-time quantitative PCR and western blot. The messenger RNA and protein levels of EtPDIL were higher in sporulated oocysts than in unsporulated oocysts, sporozoites or merozoites. Protein expression was barely detectable in unsporulated oocysts. Western blots showed that rabbit antiserum against recombinant EtPDIL recognized only a native 24 kDa protein from parasites. Immunolocalization with EtPDIL antibody showed that EtPDIL had a disperse distribution in the cytoplasm of whole sporozoites and merozoites. After sporozoites were incubated in complete medium, EtPDIL protein concentrated at the anterior of the sporozoites and appeared on the surface of parasites. Specific staining was more intense and mainly located on the parasite surface after merozoites released from mature schizonts invaded DF-1 cells. After development of parasites in DF-1 cells, staining intensified in trophozoites, immature schizonts and mature schizonts. Antibody inhibition of EtPDIL function reduced the ability of E. tenella to invade DF-1 cells. These results suggested that EtPDIL might be involved in sporulation in external environments and in host cell adhesion, invasion and development of E. tenella.

Prospective Study of Serologic Tests for Lyme Disease

PubMed Central

Steere, Allen C.; McHugh, Gail; Damle, Nitin; Sikand, Vijay K.

2017-01-01

Background Tests to determine serum antibody levels—the 2-tier sonicate immunoglobulin M (IgM) and immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and Western blot method or the IgG of the variable major protein-like sequence-expressed (VlsE) sixth invariant region (C6) peptide ELISA method—are the major tests available for support of the diagnosis of Lyme disease. However, these tests have not been assessed prospectively. Methods We used these tests prospectively to determine serologic responses in 134 patients with various manifestations of Lyme disease, 89 patients with other illnesses (with or without a history of Lyme disease), and 136 healthy subjects from areas of endemicity and areas in which the infection was not endemic. Results With 2-tier tests and the C6 peptide ELISA, only approximately one-third of 76 patients with erythema migrans had results that were positive for IgM or IgG seroreactivity with Borrelia burgdorferi in acute-phase samples. During convalescence, 3–4 weeks later, almost two-thirds of patients had seroreactivity with the spirochete B. burgdorferi. The frequencies of seroreactivity were significantly greater among patients with spirochetal dissemination than they were among those who lacked evidence of disseminated disease. Of the 44 patients with Lyme disease who had neurologic, heart, or joint involvement, all had positive C6 peptide ELISA results, 42 had IgG responses with 2-tier tests, and 2 patients with facial palsy had only IgM responses. However, among the control groups, the IgG Western blot was slightly more specific than the C6 peptide ELISA. The differences between the 2 test systems (2-tier testing and C6 peptide ELISA) with respect to sensitivity and specificity were not statistically significant. Conclusions Except in patients with erythema migrans, both test systems were sensitive for support of the diagnosis of Lyme disease. However, with current methods, 2-tier testing was associated with slightly better specificity. PMID:18532885

MiR-320 inhibits the growth of glioma cells through downregulating PBX3.

PubMed

Pan, Cuicui; Gao, Hua; Zheng, Ni; Gao, Qi; Si, Yuanquan; Zhao, Yueran

2017-09-21

MiR-320 is downregulated in multiple cancers, including glioma and acts as tumor suppressor through inhibiting tumor cells proliferation and inducing apoptosis. PBX3 (Pre-B cell leukemia homeobox 3), a putative target gene of miR-320, has been reported to be upregulated in various tumors and promote tumor cell growth through regulating MAKP/ERK pathway. This study aimed to verify whether miR-320 influences glioma cells growth through regulating PBX3. Twenty-four human glioma and paired adjacent nontumorous tissues were collected for determination of miR-320 and PBX3 expression using RT-qPCR and western blot assays. Luciferase reporter assay was performed to verify the interaction between miR-320 and its targeting sequence in the 3' UTR of PBX3 in glioma cells U87 and U251. Increased miR-320 level in U87 and U251 cells was achieved through miR-320 mimic transfection and the effect of which on glioma cells growth, proliferation, cell cycle, apoptosis and activation of Raf-1/MAPK pathway was determined using MTT, colony formation, flow cytometry and western blot assays. PBX3 knockdown was performed using shPBX3 and the influence on MAPK pathway activation was evaluated. MiR-320 downregulation and PBX3 upregulation was found in glioma tissues. Luciferase reporter assays identified miR-320 directly blinds to the 3' UTR of PBX3 in glioma cells. MiR-320 mimic transfection suppressed glioma cells proliferation, and induced cell cycle arrest and apoptosis. Both miR-320 overexpression and PBX3 knockdown inhibited Raf-1/MAPK activation. MiR-320 may suppress glioma cells growth and induced apoptosis through the PBX3/Raf-1/MAPK axis, and miR-320 oligonucleotides may be a potential cancer therapeutic for glioma.

[Protein interaction site of Toxoplasma gondii microneme protein 6 and aldolase determined by site-directed mutagenesis].

PubMed

Zheng, Bin; Yin, Zhi-Kui; Zhan, Xi-Mei

2014-06-01

To identify the protein interaction site of Toxoplasma gondii microneme protein 6 (MIC6) and aldolase by using site-directed mutagenesis. Based on Toxoplasma gondii MIC6 gene sequence (GenBank Accession No. AF110270), the specific primers were designed. Tryptophan (W)-348 of MIC6 C terminus (MIC6C) was mutated to valine (V) via site-directed mutagenesis. MIC6C W/V gene was obtained from cDNA library by PCR amplification and subcloned into pGEX-4T-1. The mutant protein GST-MIC6C W/V was expressed in E. coli, induced by 0.8 mmol/L IPTG, and purified by affinity chromatography. Glutathione sepharose beads were incubated with GST-MIC6C W/V and GST-MIC6C, respectively, and then incubated with T. gondii tachyzoites lysate, and bound proteins were eluted using sample buffer. Bound products were resolved by SDS-PAGE and Western blotting. Glutathione sepharose beads were incubated with GST-MIC6C W/V and GST-MIC6C, respectively, and then incubated with aldolase-His6. After incubation, the resin was washed and subjected to SDS-PAGE. The MIC6C W/N gene was obtained, and the recombinant plasmid MIC6C W/V/pGEX-4T-1 was successfully constructed. The mutant protein GST-MIC6C W/V was expressed and purified in vitro. SDS-PAGE analysis indicated that GST-MIC6C was co-precipitated with aldolase from T. gondii tachyzoites lysate or aldolase-His6, whereas GST-MIC6C W/V failed to precipitate aldolase from T. gondii tachyzoites lysate or aldolase-His6. Western blotting analysis using anti-aldolase antibody indicated that GST-MIC6C could pull-down aldolase from T. gondii tachyzoites lysate. Tryptophan (W348) was the interaction site of MIC6 and aldolase in T. gondii.

An image cytometric technique is a concise method to detect adenoviruses and host cell proteins and to monitor the infection and cellular responses induced.

PubMed

Morinaga, Takao; Nguyễn, Thảo Thi Thanh; Zhong, Boya; Hanazono, Michiko; Shingyoji, Masato; Sekine, Ikuo; Tada, Yuji; Tatsumi, Koichiro; Shimada, Hideaki; Hiroshima, Kenzo; Tagawa, Masatoshi

2017-11-10

Genetically modified adenoviruses (Ad) with preferential replications in tumor cells have been examined for a possible clinical applicability as an anti-cancer agent. A simple method to detect viral and cellular proteins is valuable to monitor the viral infections and to predict the Ad-mediated cytotoxicity. We used type 5 Ad in which the expression of E1A gene was activated by 5'-regulatory sequences of genes that were augmented in the expression in human tumors. The Ad were further modified to have the fiber-knob region replaced with that derived from type 35 Ad. We infected human mesothelioma cells with the fiber-replaced Ad, and sequentially examined cytotoxic processes together with an expression level of the viral E1A, hexon, and cellular cleaved caspase-3 with image cytometric and Western blot analyses. The replication-competent Ad produced cytotoxicity on mesothelioma cells. The infected cells expressed E1A and hexon 24 h after the infection and then showed cleavage of caspase-3, all of which were detected with image cytometry and Western blot analysis. Image cytometry furthermore demonstrated that increased Ad doses did not enhance an expression level of E1A and hexon in an individual cell and that caspase-3-cleaved cells were found more frequently in hexon-positive cells than in E1A-positive cells. Image cytometry thus detected these molecular changes in a sensitive manner and at a single cell level. We also showed that an image cytometric technique detected expression changes of other host cell proteins, cyclin-E and phosphorylated histone H3 at a single cell level. Image cytometry is a concise procedure to detect expression changes of Ad and host cell proteins at a single cell level, and is useful to analyze molecular events after the infection.

The guinea-pig expresses functional CYP2C and P-glycoprotein: further validation of its usefulness in drug biotransformation/transport studies.

PubMed

Hasibu, Ibrahim; Patoine, Dany; Pilote, Sylvie; Drolet, Benoit; Simard, Chantale

2015-04-01

The guinea-pig is an excellent animal model for studying cardiopulmonary physiology/pharmacology. Interestingly, it also possesses a number of drug-metabolizing enzymes found in humans, such as CYP1A, CYP2D and CYP3A. To evaluate the hypothesis that the guinea-pig also expresses a functional CYP2C drug-metabolizing enzyme and the P-glycoprotein (P-gp) drug transporter in various tissues. cDNAs encoding CYP2C and P-gp were obtained from guinea-pig liver or small intestine and sequenced. Western blotting was performed to confirm the expression of CYP2C and P-gp. The functional enzymatic activity of guinea-pig CYP2C was evaluated with microsomal preparations using diclofenac and tolbutamide as specific drug substrates in HPLC analyses. To further study both P-gp and CYP2C functional activities, the guinea-pig ABCB1/MDR1 and CYP2C genes were cloned. The recombinant plasmids were then transfected in HEK293 (human embryonic kidney) cells and either calcein-acetoxymethyl ester (AM) accumulation assays or 14,15-EET/DHET formation experiments were performed to evaluate either P-gp transport activity or CYP2C epoxygenase activity, respectively. The guinea-pig tissue distribution of P-gp was studied by Western blotting. Functional expression of CYP2C was demonstrated in guinea-pig liver microsomal preparations. CYP2C-mediated biotransformation of diclofenac and tolbutamide were shown. Expression of P-gp protein was detected in guinea-pig liver and small intestine. Functional activity of guinea-pig P-gp was demonstrated in ABCB1/MDR1-transfected cells. GP-CYP2C-transfected cells also showed functional epoxygenase activity. The guinea-pig expresses functional CYP2C and P-gp, thus suggesting its usefulness for further validating data obtained with other animal models in drug biotransformation/transport studies. Copyright © 2015 John Wiley & Sons, Ltd.

Phage display and synthetic peptides as promising biotechnological tools for the serological diagnosis of leprosy.

PubMed

Alban, Silvana Maria; de Moura, Juliana Ferreira; Thomaz-Soccol, Vanete; Bührer Sékula, Samira; Alvarenga, Larissa Magalhães; Mira, Marcelo Távora; Olortegui, Carlos Chávez; Minozzo, João Carlos

2014-01-01

The diagnosis of leprosy is primarily based on clinical manifestations, and there is no widely available laboratory test for the early detection of this disease, which is caused by Mycobacterium leprae. In fact, early detection and treatment are the key elements to the successful control of leprosy. Peptide ligands for antibodies from leprosy patients were selected from phage-displayed peptide libraries. Three peptide sequences expressed by reactive phage clones were chemically synthesized. Serological assays that used synthetic peptides were evaluated using serum samples from leprosy patients, household contacts (HC) of leprosy patients, tuberculosis patients and endemic controls (EC). A pool of three peptides identified 73.9% (17/23) of multibacillary (MB) leprosy patients using an enzyme-linked immunosorbent assay (ELISA). These peptides also showed some seroreactivities to the HC and EC individuals. The peptides were not reactive to rabbit polyclonal antisera against the different environmental mycobacteria. The same peptides that were conjugated to the carrier protein bovine serum albumin (BSA) induced the production of antibodies in the mice. The anti-peptide antibodies that were used in the Western blotting analysis of M. leprae crude extracts revealed a single band of approximately 30 kDa in one-dimensional electrophoresis and four 30 kDa isoforms in the two-dimensional gel. The Western blotting data indicated that the three peptides are derived from the same bacterial protein. These new antigens may be useful in the diagnosis of MB leprosy patients. Their potentials as diagnostic reagents must be more extensively evaluated in future studies using a large panel of positive and negative sera. Furthermore, other test approaches using peptides should be assessed to increase their sensitivity and specificity in detecting leprosy patients. We have revealed evidence in support of phage-displayed peptides as promising biotechnological tools for the design of leprosy diagnostic serological assays.

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.

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.

Autoantibody detection in type 2 autoimmune hepatitis using a chimera recombinant protein.

PubMed

Vitozzi, Susana; Lapierre, Pascal; Djilali-Saiah, Idriss; Alvarez, Fernando

2002-04-01

Autoantibodies against cytochrome P450 2D6 (CYP2D6), known as anti-liver/kidney microsome type 1 (LKM1) and/or anti-human formiminotransferase cyclodeaminase, formally known as anti-liver cytosol type 1 (LC1) define type 2 autoimmune hepatitis (AIH). The aims of this work are to develop a sensitive and specific test to detect anti-LKM1 and/or anti-LC1 autoantibodies and to establish the prevalence of anti-LC1. Sera from children with type 2 AIH (n=48) and those from a control group (n=100) were evaluated for anti-LKM1 and anti-LC1 by Enzyme-Linked Immunosorbent Assay (ELISA) and Western blotting. Each serum sample was assayed for reactivity against formiminotransferase cyclodeaminase and CYP2D6 alone or as part of a recombinant chimera protein. By ELISA with recombinant chimera protein, 50 serum samples were positive, 48 from patients with type 2 AIH and 2 from patients with chronic hepatitis C. Twenty-five of 48 (52%) patients studied were positive for both CYP2D6 and LC1 autoantibodies. Anti-LC1, either as the only marker or associated with anti-LKM1, was positive in 34/48 (71%). By Western blotting, anti-LC1 was found in 27/48 (56%) patients. This ELISA technique has proven to be antigen-specific and more sensitive than Western blot for the detection of anti-LC1 and anti-LKM1 autoantibodies. The prevalence of anti-LC1 (71%) confirms it as an important immunomarker in type 2 AIH.

Demethoxycurcumin Suppresses Migration and Invasion of Human Cervical Cancer HeLa Cells via Inhibition of NF-κB Pathways.

PubMed

Lin, Chin-Chung; Kuo, Chao-Lin; Huang, Yi-Ping; Chen, Cheng-Yen; Hsu, Ming-Jie; Chu, Yung Lin; Chueh, Fu-Shin; Chung, Jing-Gung

2018-05-01

Demethoxycurcumin (DMC), one of the curcuminoids present in turmeric, has been shown to induce cell death in many human cancer cell lines, however, there has not been any investigation on whether DMC inhibits metastatic activity in human cervical cancer cells in vitro. In the present study, DMC at 2.5-15 μM decreased cell number, thus, we used IC 20 (7.5 μM) for further investigation of its anti-metastatic activity in human cervical cancer HeLa cells. The wound healing, migration, invasion, zymography, and western blotting assays were used to investigate the effects of DMC on HeLa cells. The wound healing assay was used to show that DMC suppressed cell movement of HeLa cells. Furthermore, the trans-well chamber assay was used to show that DMC suppressed HeLa cell migration and invasion. Gelatin zymography assay did not show any significant effects of DMC on the gelatinolytic activity (MMP-2 and -9) in conditioned media of HeLa cells treated by DMC. Western blotting showed that DMC significantly reduced protein levels of GRB2, MMP-2, ERK1/2, N-cadherin and Ras but increased the levels of E-cadherin and NF-κB in HeLa cells. Confocal laser microscopy indicated that DMC increased NF-κB in HeLa cells confirming the results from Western blotting. DMC may be used as a novel anti-metastatic agent for the treatment of human cervical cancer in the future. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

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

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.

Sumoylated α-skeletal muscle actin in the skeletal muscle of adult rats.

PubMed

Uda, Munehiro; Kawasaki, Hiroaki; Iizumi, Kyoichi; Shigenaga, Ayako; Baba, Takeshi; Naito, Hisashi; Yoshioka, Toshitada; Yamakura, Fumiyuki

2015-11-01

Skeletal muscles are composed of two major muscle fiber types: slow-twitch oxidative fibers and fast-twitch glycolytic fibers. The proteins in these muscle fibers are known to differ in their expression, relative abundance, and post-translational modifications. In this study, we report a previously unreported post-translational modification of α-skeletal muscle actin in the skeletal muscles of adult male F344 rats in vivo. Using two-dimensional electrophoresis (2D-PAGE), we first examined the differences in the protein expression profiles between the soleus and plantaris muscles. We found higher intensity protein spots at approximately 60 kDa and pH 9 on 2D-PAGE for the soleus muscle compared with the plantaris muscle. These spots were identified as α-skeletal muscle actin by liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry and western blot analyses. In addition, we found that the 60 kDa α-skeletal muscle actin is modified by small ubiquitin-like modifier (SUMO) 1, using 2D-PAGE and western blot analyses. Furthermore, we found that α-skeletal muscle actin with larger molecular weight was localized in the nuclear and cytosol of the skeletal muscle, but not in the myofibrillar fraction by the combination of subcellular fractionation and western blot analyses. These results suggest that α-skeletal muscle actin is modified by SUMO-1 in the skeletal muscles, localized in nuclear and cytosolic fractions, and the extent of this modification is much higher in the slow muscles than in the fast muscles. This is the first study to show the presence of SUMOylated actin in animal tissues.

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.

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.

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 oculocerebrorenal syndrome gene product is a 105-kD protein localized to the Golgi complex.

PubMed Central

Olivos-Glander, I M; Jänne, P A; Nussbaum, R L

1995-01-01

The oculocerebrorenal syndrome of Lowe (OCRL) is a multisystem disorder affecting the lens, kidney, and CNS. The predicted amino acid sequence of the OCRL gene, OCRL-1, was used to develop antibodies against the OCRL-1 protein. Western blot analysis using affinity-purified serum against the amino terminus of the OCRL-1 gene product (ocrl-1) demonstrates a single protein of 105 kD in fibroblasts of a normal individual that is absent in fibroblasts of an OCRL patient who lacks OCRL-1 transcript. A single protein with the same electrophoretic mobility is found by western analysis in various human cultured cell lines, and approximately the same size protein is also found in all mouse tissues tested. Northern analysis of various human and mouse tissues demonstrate that OCRL-1 transcript is expressed in nearly all tissues examined. By immunofluorescence, the ocrl-1 antibody stains a juxtanuclear region in normal fibroblast cells, while no specific staining is evident in the OCRL patient who produces no transcript. Colocalization of the ocrl-1 protein to the Golgi complex was demonstrated using a known monoclonal antibody against a Golgi-specific coat protein, beta-COP (beta coatomer protein). Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7573041

IRAK-M alters the polarity of macrophages to facilitate the survival of Mycobacterium tuberculosis.

PubMed

Shen, Pei; Li, Quan; Ma, Jilei; Tian, Maopeng; Hong, Fei; Zhai, Xinjie; Li, Jianrong; Huang, Hanju; Shi, Chunwei

2017-08-23

Intracellular bacterium, Mycobacterium tuberculosis (M. tb), infects specifically macrophages as host cells. IRAK-M, a member of IRAK family, is a negative regulator in TLR signaling and specifically expresses in monocytes and macrophages. The role of IRAK-M in intracellular growth of M. tb and macrophage polarization was explored, for deeply understanding the pathogenesis of M. tb, the significance of IRAK-M to innate immunity and pathogen-host interaction. IRAK-M expression was detected in M. tb infected macrophages and in human lung tissue of pulmonary tuberculosis with immunofluorescence staining, Western blot and immunohistochemistry. IRAK-M knock-down and over-expressing cell strains were constructed and intracellular survival of M. tb was investigated by acid-fast staining and colony forming units. Molecular markers of M1-type (pSTAT1 and iNOS) and M2-type (pSTAT6 and Arg-1) macrophages were detected using Western blot in IRAK-M knockdown U937 cells infected with M. tb H37Rv. U937 cells were stimulated with immunostimulant CpG7909 into M1 status and then infected with M. tb H37Rv. Expression of IRAK-M, IRAK-4 and iNOS was detected with immunofluorescence staining and Western blot, to evaluate the effect of IRAK-M to CpG directed M1-type polarization of macrophages during M. tb infection. Molecules related with macrophage's bactericidal ability such as Hif-1 and phosphorylated ERK1/2 were detected with immunohistochemistry and Western blot. IRAK-M increased in M. tb infected macrophage cells and also in human lung tissue of pulmonary tuberculosis. IRAK-M over-expression resulted in higher bacterial load, while IRAK-M interference resulted in lower bacterial load in M. tb infected cells. During M. tb infection, IRAK-M knockdown induced M1-type, while inhibited M2-type polarization of macrophage. M1-type polarization of U937 cells induced by CpG7909 was inhibited by M. tb infection, which was reversed by IRAK-M knockdown in U937 cells. IRAK-M affected Hif-1 and MAPK signaling cascade during M. tb infection. Conclusively, IRAK-M might alter the polarity of macrophages, to facilitate intracellular survival of M. tb and affect Th1-type immunity of the host, which is helpful to understanding the pathogenesis of M. tb.

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.

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

HOXBES2: a novel epididymal HOXB2 homeoprotein and its domain-specific association with spermatozoa.

PubMed

Prabagaran, E; Bandivdekar, A H; Dighe, V; Raghavan, V P

2007-02-01

The sperm from the testis acquires complete fertilizing ability and forward progressive motility following its transit through the epididymis. Acquisition of these characteristics results from the modification of the sperm proteome following interactions with epididymal secretions. In our attempts to identify epididymis-specific sperm plasma membrane proteins, a partial 2.83-kb clone was identified by immunoscreening a monkey epididymal cDNA library with an agglutinating monoclonal antibody raised against washed human spermatozoa. The sequence of the 2.83-kb clone exhibited homology to the region between 1 and 1097 bp of the homeobox gene, Hoxb2. This sequence was found to be species conserved, as revealed by RT-PCR analysis. To obtain a full-length clone of the sequence, 5' RACE-PCR (rapid amplification of cDNA ends PCR) was carried out using rat epididymal RNA as the template. It resulted in a full-length 1.657-kb cDNA encoding a 32.9-kDa putative protein. The protein designated HOXBES2 exhibited homology to the conserved 61-amino acid homeodomain region of the HOXB2 homeoprotein. However, characteristic differences were noted in its amino and carboxyl termini compared with HOXB2. A putative 30-kDa protein was detected in the tissue extracts from adult rat epididymis and caudal spermatozoa, and a 37-kDa protein was detected in the rat embryo when probed with a polyclonal antibody against HOXB2 protein. Multiple tissue Western blot and immunohistochemical analysis further indicated its expression in the cytoplasm of the principal and basal epithelial cells, with maximal expression in the distal epididymal segments. Northern blot analysis detected a single approximately 2.5-kb transcript from the adult epididymis. Indirect immunofluorescence localized the protein to the acrosome, midpiece, and equatorial segments of rat caudal and ejaculated human and monkey spermatozoa, respectively. In conclusion, we have identified and characterized a novel epididymal homeoprotein different from HOXB2 protein and hereafter referred to as HOXBES2, (HOXB2 homeodomain containing epididymis-specific sperm protein) with a probable role in fertilization.

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

Efficient and heritable transformation of Phalaenopsis orchids.

PubMed

Hsing, Hong-Xian; Lin, Yi-Jyun; Tong, Chii-Gong; Li, Min-Jeng; Chen, Yun-Jin; Ko, Swee-Suak

2016-12-01

Phalaenopsis orchid (Phal. orchid) is visually attractive and it is important economic floriculture species. Phal. orchids have many unique biological features. However, investigation of these features and validation on their biological functions are limited due to the lack of an efficient transformation method. We developed a heritable and efficient Agrobacterium- mediated transformation using protocorms derived from tetraploid or diploid Phal. orchids. A T-DNA vector construct containing eGFP driven by ubiquitin promoter was subjected to transformation. An approximate 1.2-5.2 % transformation rate was achieved. Genomic PCR confirmed that hygromycin selection marker, HptII gene and target gene eGFP were integrated into the orchid genome. Southern blotting indicated a low T-DNA insertion number in the orchid genome of the transformants. Western blot confirmed the expression of eGFP protein in the transgenic orchids. Furthermore, the GFP signal was detected in the transgenic orchids under microscopy. After backcrossing the pollinia of the transgenic plants to four different Phal. orchid varieties, the BC1 progenies showed hygromycin resistance and all surviving BC1 seedlings were HptII positive in PCR and expressed GFP protein as shown by western blot. This study demonstrated a stable transformation system was generated for Phal. orchids. This useful transformation protocol enables functional genomics studies and molecular breeding.

Ionophore and Biometal Modulation of P-glycoprotein Expression and Function in Human Brain Microvascular Endothelial Cells.

PubMed

McInerney, Mitchell P; Volitakis, Irene; Bush, Ashley I; Banks, William A; Short, Jennifer L; Nicolazzo, Joseph A

2018-03-05

Biometals such as zinc and copper have been shown to affect tight junction expression and subsequently blood-brain barrier (BBB) integrity. Whether these biometals also influence the expression and function of BBB transporters such as P-glycoprotein (P-gp) however is currently unknown. Using the immortalised human cerebral microvascular endothelial (hCMEC/D3) cell line, an in-cell western assay (alongside western blotting) assessed relative P-gp expression after treatment with the metal ionophore clioquinol and biometals zinc and copper. The fluorescent P-gp substrate rhodamine-123 was employed to observe functional modulation, and inductively coupled plasma mass spectrometry (ICP-MS) provided information on biometal trafficking. A 24-h treatment with clioquinol, zinc and copper (0.5, 0.5 and 0.1 μM) induced a significant upregulation of P-gp (1.7-fold) assessed by in-cell western and this was confirmed with western blotting (1.8-fold increase). This same treatment resulted in a 23% decrease in rhodamine-123 accumulation over a 1 h incubation. ICP-MS demonstrated that while t8his combination treatment had no effect on intracellular zinc concentrations, the treatment significantly enhanced bioavailable copper (4.6-fold). Enhanced delivery of copper to human brain microvascular endothelial cells is associated with enhanced expression and function of the important efflux pump P-gp, which may provide therapeutic opportunities for P-gp modulation.

MDC9, a widely expressed cellular disintegrin containing cytoplasmic SH3 ligand domains

PubMed Central

1996-01-01

Cellular disintegrins are a family of proteins that are related to snake venom integrin ligands and metalloproteases. We have cloned and sequenced the mouse and human homologue of a widely expressed cellular disintegrin, which we have termed MDC9 (for metalloprotease/disintegrin/cysteine-rich protein 9). The deduced mouse and human protein sequences are 82% identical. MDC9 contains several distinct protein domains: a signal sequence is followed by a prodomain and a domain with sequence similarity to snake venom metalloproteases, a disintegrin domain, a cysteine-rich region, an EGF repeat, a membrane anchor, and a cytoplasmic tail. The cytoplasmic tail of MDC9 has two proline-rich sequences which can bind the SH3 domain of Src, and may therefore function as SH3 ligand domains. Western blot analysis shows that MDC9 is an approximately 84-kD glycoprotein in all mouse tissues examined, and in NIH 3T3 fibroblast and C2C12 myoblast mouse cell lines. MDC9 can be both cell surface biotinylated and 125I-labeled in NIH 3T3 mouse fibroblasts, indicating that the protein is present on the plasma membrane. Expression of MDC9 in COS-7 cells yields an 84-kD protein, and immunofluorescence analysis of COS-7 cells expressing MDC9 shows a staining pattern that is consistent with a plasma membrane localization. The apparent molecular mass of 84 kD suggests that MDC9 contains a membrane-anchored metalloprotease and disintegrin domain. We propose that MDC9 might function as a membrane-anchored integrin ligand or metalloprotease, or that MDC9 may combine both activities in one protein. PMID:8647900

Bmcystatin, a cysteine proteinase inhibitor characterized from the tick Boophilus microplus

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

Lima, Cassia A.; Sasaki, Sergio D.; Tanaka, Aparecida S.

2006-08-18

The bovine tick Rhipicephalus (Boophilus) microplus is a blood-sucking animal, which is responsible for Babesia spp and Anaplasma marginale transmission for cattle. From a B. microplus fat body cDNA library, 465 selected clones were sequenced randomly and resulted in 60 Contigs. An open reading frame (ORF) contains 98 amino acids named Bmcystatin, due to 70% amino acid identity to a classical type 1 cystatin from Ixodes scapularis tick (GenBank Accession No. DQ066227). The Bmcystatin amino acid sequence analysis showed two cysteine residues, theoretical pI of 5.92 and M{sub r} of 11kDa. Bmcystatin gene was cloned in pET 26b vector andmore » the protein expressed using bacteria Escherichia coli BL21 SI. Recombinant Bmcystatin (rBmcystatin) purified by affinity chromatography on Ni-NTA-agarose column and ionic exchange chromatography on HiTrap Q column presented molecular mass of 11kDa, by SDS-PAGE and the N-terminal amino acid sequenced revealed unprocessed N-terminal containing part of pelB signal sequence. Purified rBmcystatin showed to be a C1 cysteine peptidase inhibitor with K{sub i} value of 0.1 and 0.6nM for human cathepsin L and VTDCE (vitellin degrading cysteine endopeptidase), respectively. The rBmcystatin expression analyzed by semi-quantitative RT-PCR confirmed the amplification of a specific DNA sequence (294bp) in the fat body and ovary cDNA preparation. On the other hand, a protein band was detected in the fat body, ovary, and the salivary gland extracts using anti-Bmcystatin antibody by Western blot. The present results suggest a possible role of Bmcystatin in the ovary, even though the gene was cloned from the fat body, which could be another site of this protein synthesis.« less

Bmcystatin, a cysteine proteinase inhibitor characterized from the tick Boophilus microplus.

PubMed

Lima, Cassia A; Sasaki, Sergio D; Tanaka, Aparecida S

2006-08-18

The bovine tick Rhipicephalus (Boophilus) microplus is a blood-sucking animal, which is responsible for Babesia spp and Anaplasma marginale transmission for cattle. From a B. microplus fat body cDNA library, 465 selected clones were sequenced randomly and resulted in 60 Contigs. An open reading frame (ORF) contains 98 amino acids named Bmcystatin, due to 70% amino acid identity to a classical type 1 cystatin from Ixodes scapularis tick (GenBank Accession No. ). The Bmcystatin amino acid sequence analysis showed two cysteine residues, theoretical pI of 5.92 and M(r) of 11 kDa. Bmcystatin gene was cloned in pET 26b vector and the protein expressed using bacteria Escherichia coli BL21 SI. Recombinant Bmcystatin (rBmcystatin) purified by affinity chromatography on Ni-NTA-agarose column and ionic exchange chromatography on HiTrap Q column presented molecular mass of 11 kDa, by SDS-PAGE and the N-terminal amino acid sequenced revealed unprocessed N-terminal containing part of pelB signal sequence. Purified rBmcystatin showed to be a C1 cysteine peptidase inhibitor with K(i) value of 0.1 and 0.6 nM for human cathepsin L and VTDCE (vitellin degrading cysteine endopeptidase), respectively. The rBmcystatin expression analyzed by semi-quantitative RT-PCR confirmed the amplification of a specific DNA sequence (294 bp) in the fat body and ovary cDNA preparation. On the other hand, a protein band was detected in the fat body, ovary, and the salivary gland extracts using anti-Bmcystatin antibody by Western blot. The present results suggest a possible role of Bmcystatin in the ovary, even though the gene was cloned from the fat body, which could be another site of this protein synthesis.

Computationally predicted IgE epitopes of walnut allergens contribute to cross-reactivity with peanuts

PubMed Central

Maleki, Soheila J.; Teuber, Suzanne S.; Cheng, Hsiaopo; Chen, Deliang; Comstock, Sarah S.; Ruan, Sanbao; Schein, Catherine H.

2011-01-01

Background Cross reactivity between peanuts and tree nuts implies that similar IgE epitopes are present in their proteins. Objective To determine whether walnut sequences similar to known peanut IgE binding sequences, according to the property distance (PD) scale implemented in the Structural Database of Allergenic Proteins (SDAP), react with IgE from sera of patients with allergy to walnut and/or peanut. Methods Patient sera were characterized by Western blotting for IgE-binding to nut protein extracts, and to peptides from walnut and peanut allergens, similar to known peanut epitopes as defined by low PD values, synthesized on membranes. Competitive ELISA was used to show that peanut and predicted walnut epitope sequences compete with purified Ara h 2 for binding to IgE in serum from a cross-reactive patient. Results Sequences from the vicilin walnut allergen Jug r 2 which had low PD values to epitopes of the peanut allergen Ara h 2, a 2s-albumin, bound IgE in sera from five patients who reacted to either walnut, peanut or both. A walnut epitope recognized by 6 patients mapped to a surface-exposed region on a model of the N-terminal pro-region of Jug r 2. A predicted walnut epitope competed for IgE binding to Ara h 2 in serum as well as the known IgE epitope from Ara h 2. Conclusions Sequences with low PD value (<8.5) to known IgE epitopes could contribute to cross-reactivity between allergens. This further validates the PD scoring method for predicting cross-reactive epitopes in allergens. PMID:21883278

Molecular characterization of hypoxia and hypoxia-inducible factor 1 alpha (HIF-1α) from Taiwan voles (Microtus kikuchii).

PubMed

Jiang, Yi-Fan; Chou, Chung-Hsi; Lin, En-Chung; Chiu, Chih-Hsien

2011-02-01

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that senses and adapts cells to hypoxic environmental conditions. HIF-1 is composed of an oxygen-regulated α subunit (HIF-1α) and a constitutively expressed β subunit (HIF-1β). Taiwan voles (Microtus kikuchii) are an endemic species in Taiwan, found only in mountainous areas greater than 2000m above sea level. In this study, the full-length HIF-1α cDNA was cloned and sequenced from liver tissues of Taiwan voles. We found that HIF-1α of Taiwan voles had high sequence similarity to HIF-1α of other species. Sequence alignment of HIF-1α functional domains indicated basic helix-loop-helix (bHLH), PER-ARNT-SIM (PAS) and C-terminal transactivation (TAD-C) domains were conserved among species, but sequence variations were found between the oxygen-dependent degradation domains (ODDD). To measure Taiwan vole HIF-1α responses to hypoxia, animals were challenged with cobalt chloride, and HIF-1α mRNA and protein expression in brain, lung, heart, liver, kidney, and muscle was assessed by quantitative RT-PCR and Western blot analysis. Upon induction of hypoxic stress with cobalt chloride, an increase in HIF-1α mRNA levels was detected in lung, heart, kidney, and muscle tissue. In contrast, protein expression levels showed greater variation between individual animals. These results suggest that the regulation of HIF-1α may be important to the Taiwan vole under cobalt chloride treatments. But more details regarding the evolutionary effect of environmental pressure on HIF-1α primary sequence, HIF-1α function and regulation in Taiwan voles remain to be identified. Copyright © 2010 Elsevier Inc. All rights reserved.

Human scFv antibody fragments specific for hepatocellular carcinoma selected from a phage display library.

PubMed

Yu, Bing; Ni, Ming; Li, Wen-Han; Lei, Ping; Xing, Wei; Xiao, Dai-Wen; Huang, Yu; Tang, Zhen-Jie; Zhu, Hui-Fen; Shen, Guan-Xin

2005-07-14

To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library. A large human naive scFv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positive-selecting and the normal liver cell line L02 for the counter-selecting. After three rounds of biopanning, individual scFv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scFv gene sequence. The specific scFv phages were selected by ELISA and flow cytometry. DNA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3 730. The expression proteins of the specific scFv antibody fragments in E.coli HB2151 were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation. Two different positive clones were obtained and the functional variable sequences were identified. Their DNA sequences of the scFv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scFv antibody fragments were successfully expressed in E.coli HB2151. The relative molecular mass of the expression products was about 36 ku, according to its predicted M(r) value. The two soluble scFv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells. The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.

Automated design of genomic Southern blot probes

PubMed Central

2010-01-01

Background Sothern blotting is a DNA analysis technique that has found widespread application in molecular biology. It has been used for gene discovery and mapping and has diagnostic and forensic applications, including mutation detection in patient samples and DNA fingerprinting in criminal investigations. Southern blotting has been employed as the definitive method for detecting transgene integration, and successful homologous recombination in gene targeting experiments. The technique employs a labeled DNA probe to detect a specific DNA sequence in a complex DNA sample that has been separated by restriction-digest and gel electrophoresis. Critically for the technique to succeed the probe must be unique to the target locus so as not to cross-hybridize to other endogenous DNA within the sample. Investigators routinely employ a manual approach to probe design. A genome browser is used to extract DNA sequence from the locus of interest, which is searched against the target genome using a BLAST-like tool. Ideally a single perfect match is obtained to the target, with little cross-reactivity caused by homologous DNA sequence present in the genome and/or repetitive and low-complexity elements in the candidate probe. This is a labor intensive process often requiring several attempts to find a suitable probe for laboratory testing. Results We have written an informatic pipeline to automatically design genomic Sothern blot probes that specifically attempts to optimize the resultant probe, employing a brute-force strategy of generating many candidate probes of acceptable length in the user-specified design window, searching all against the target genome, then scoring and ranking the candidates by uniqueness and repetitive DNA element content. Using these in silico measures we can automatically design probes that we predict to perform as well, or better, than our previous manual designs, while considerably reducing design time. We went on to experimentally validate a number of these automated designs by Southern blotting. The majority of probes we tested performed well confirming our in silico prediction methodology and the general usefulness of the software for automated genomic Southern probe design. Conclusions Software and supplementary information are freely available at: http://www.genes2cognition.org/software/southern_blot PMID:20113467

Molecular Characterization of Transgenic Events Using Next Generation Sequencing Approach.

PubMed

Guttikonda, Satish K; Marri, Pradeep; Mammadov, Jafar; Ye, Liang; Soe, Khaing; Richey, Kimberly; Cruse, James; Zhuang, Meibao; Gao, Zhifang; Evans, Clive; Rounsley, Steve; Kumpatla, Siva P

2016-01-01

Demand for the commercial use of genetically modified (GM) crops has been increasing in light of the projected growth of world population to nine billion by 2050. A prerequisite of paramount importance for regulatory submissions is the rigorous safety assessment of GM crops. One of the components of safety assessment is molecular characterization at DNA level which helps to determine the copy number, integrity and stability of a transgene; characterize the integration site within a host genome; and confirm the absence of vector DNA. Historically, molecular characterization has been carried out using Southern blot analysis coupled with Sanger sequencing. While this is a robust approach to characterize the transgenic crops, it is both time- and resource-consuming. The emergence of next-generation sequencing (NGS) technologies has provided highly sensitive and cost- and labor-effective alternative for molecular characterization compared to traditional Southern blot analysis. Herein, we have demonstrated the successful application of both whole genome sequencing and target capture sequencing approaches for the characterization of single and stacked transgenic events and compared the results and inferences with traditional method with respect to key criteria required for regulatory submissions.

Isolation of mini- and microsatellite loci from chromosome 19 library

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

Prosnyak, M.I.; Belajeva, O.V.; Polukarova, L.G.

Mini- and microsatellite sequences are abundant in the human genome and are very useful as genetic markers. We report the isolation of a panel of clones containing marker sequences from chromosome 19. We screened 10,000 clones from the chromosome 19 cosmid library for the presence of di-(CA)n, tri-(TCC)n, (CAC)n microsatellites and M13-like minisatellite sequences. For this we have used synthetic oligonucleotides and polynucleotides, including micro- (CA, TCC, CAC) and minisatellite (M13 core) sequences. Preliminary results indicated that the chromosome 19 cosmid library contained both human and hamster clones. In order to identify human sequences from this library we have developedmore » the technique of colony and blot hybridization with Alu-PCR, L1-PCR and B1-PCR probes. Dozens of clones have been selected, some of which were analyzed by conventional Southern blot analysis and non-radioactive in situ hybridization of chromosomes. Highly informative markers derived from these clones will be used for physical and genetic mapping of chromosome 19.« less

Three-dimensional cell organization leads to almost immediate HRE activity as demonstrated by molecular imaging of MG-63 spheroids using two-photon excitation microscopy.

PubMed

Indovina, Paola; Collini, Maddalena; Chirico, Giuseppe; Santini, Maria Teresa

2007-02-20

Hypoxia through HRE (hypoxia-responsive element) activity in MG-63 human osteosarcoma cells grown in monolayer and as very small, three-dimensional tumor spheroids was investigated using molecular imaging techniques. MG-63 cells were stably transfected with a vector constructed with multiple copies of the HRE sequence of the human vascular endothelial growth factor (VEGF) gene and with the enhanced green fluorescent protein (EGFP) coding sequence. During hypoxia when HIF-1alpha (hypoxia-inducible factor-1alpha) is stabilized, the binding of HIF-1 to the HRE sequences of the vector allows the transcription of EGFP and the appearance of fluorescence. Transfected monolayer cells were characterized by flow cytometric analysis in response to various hypoxic conditions and HIF-1alpha expression in these cells was assessed by Western blotting. Two-photon excitation (TPE) microscopy was then used to examine both MG-63-transfected monolayer cells and spheroids at 2 and 5 days of growth in normoxic conditions. Monolayer cells reveal almost no fluorescence, whereas even very small spheroids (<100 microm) after 2 days of growth contain regions of high fluorescence. For the first time in the literature, at least to our knowledge, it is demonstrated, using highly sensitive and non-perturbing molecular imaging techniques, that three-dimensional cell organization leads to almost immediate HRE activation. This activation of the HRE sequences, which control a wide variety of genes, suggests that monolayer cells and spheroids of the MG-63 cell line have different genes activated and thus diverse functional activities.

Simplifying complex sequence information: a PCP-consensus protein binds antibodies against all four Dengue serotypes.

PubMed

Bowen, David M; Lewis, Jessica A; Lu, Wenzhe; Schein, Catherine H

2012-09-14

Designing proteins that reflect the natural variability of a pathogen is essential for developing novel vaccines and drugs. Flaviviruses, including Dengue (DENV) and West Nile (WNV), evolve rapidly and can "escape" neutralizing monoclonal antibodies by mutation. Designing antigens that represent many distinct strains is important for DENV, where infection with a strain from one of the four serotypes may lead to severe hemorrhagic disease on subsequent infection with a strain from another serotype. Here, a DENV physicochemical property (PCP)-consensus sequence was derived from 671 unique sequences from the Flavitrack database. PCP-consensus proteins for domain 3 of the envelope protein (EdomIII) were expressed from synthetic genes in Escherichia coli. The ability of the purified consensus proteins to bind polyclonal antibodies generated in response to infection with strains from each of the four DENV serotypes was determined. The initial consensus protein bound antibodies from DENV-1-3 in ELISA and Western blot assays. This sequence was altered in 3 steps to incorporate regions of maximum variability, identified as significant changes in the PCPs, characteristic of DENV-4 strains. The final protein was recognized by antibodies against all four serotypes. Two amino acids essential for efficient binding to all DENV antibodies are part of a discontinuous epitope previously defined for a neutralizing monoclonal antibody. The PCP-consensus method can significantly reduce the number of experiments required to define a multivalent antigen, which is particularly important when dealing with pathogens that must be tested at higher biosafety levels. Copyright © 2012 Elsevier Ltd. All rights reserved.

Comparative oncology DNA sequencing of canine T cell lymphoma via human hotspot panel

PubMed Central

Beheshti, Afshin; Pilichowska, Monika; Burgess, Kristine; Ricks-Santi, Luisel; McNiel, Elizabeth; London, Cheryl B.; Ravi, Dashnamoorthy; Evens, Andrew M.

2018-01-01

T-cell lymphoma (TCL) is an uncommon and aggressive form of human cancer. Lymphoma is the most common hematopoietic tumor in canines (companion animals), with TCL representing approximately 30% of diagnoses. Collectively, the canine is an appealing model for cancer research given the spontaneous occurrence of cancer, intact immune system, and phytogenetic proximity to humans. We sought to establish mutational congruence of the canine with known human TCL mutations in order to identify potential actionable oncogenic pathways. Following pathologic confirmation, DNA was sequenced in 16 canine TCL (cTCL) cases using a custom Human Cancer Hotspot Panel of 68 genes commonly mutated in human TCL. Sequencing identified 4,527,638 total reads with average length of 229 bases containing 346 unique variants and 1,474 total variants; each sample had an average of 92 variants. Among these, there were 258 germline and 32 somatic variants. Among the 32 somatic variants there were 8 missense variants, 1 splice junction variant and the remaining were intron or synonymous variants. A frequency of 4 somatic mutations per sample were noted with >7 mutations detected in MET, KDR, STK11 and BRAF. Expression of these associated proteins were also detected via Western blot analyses. In addition, Sanger sequencing confirmed three variants of high quality (MYC, MET, and TP53 missense mutation). Taken together, the mutational spectrum and protein analyses showed mutations in signaling pathways similar to human TCL and also identified novel mutations that may serve as drug targets as well as potential biomarkers. PMID:29854308

HSP70 from the Antarctic sea urchin Sterechinus neumayeri: molecular characterization and expression in response to heat stress.

PubMed

González-Aravena, Marcelo; Calfio, Camila; Mercado, Luis; Morales-Lange, Byron; Bethke, Jorn; De Lorgeril, Julien; Cárdenas, César A

2018-03-27

Heat stress proteins are implicated in stabilizing and refolding denatured proteins in vertebrates and invertebrates. Members of the Hsp70 gene family comprise the cognate heat shock protein (Hsc70) and inducible heat shock protein (Hsp70). However, the cDNA sequence and the expression of Hsp70 in the Antarctic sea urchin are unknown. We amplified and cloned a transcript sequence of 1991 bp from the Antarctic sea urchin Sterechinus neumayeri, experimentally exposed to heat stress (5  and 10 °C for 1, 24 and 48 h). RACE-PCR and qPCR were employed to determine Hsp70 gene expression, while western blot and ELISA methods were used to determine protein expression. The sequence obtained from S. neumayeri showed high identity with Hsp70 members. Several Hsp70 family features were identified in the deduced amino acid sequence and they indicate that the isolated Hsp70 is related to the cognate heat shock protein type. The corresponding 70 kDa protein, called Sn-Hsp70, was immune detected in the coelomocytes and the digestive tract of S. neumayeri using a monospecific polyclonal antibody. We showed that S. neumayeri do not respond to acute heat stress by up-regulation of Sn-Hsp70 at transcript and protein level. Furthermore, the Sn-Hsp70 protein expression was not induced in the digestive tract. Our results provide the first molecular evidence that Sn-Hsp70 is expressed constitutively and is non-induced by heat stress in S. neumayeri.

Structural analysis of the RH-like blood group gene products in nonhuman primates

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

Salvignol, I.; Calvas, P.; Blancher, A.

1995-03-01

Rh-related transcripts present in bone marrow samples from several species of nonhuman primates (chimpanzee, gorilla, gibbon, crab-eating macaque) have been amplified by RT-polymerase chain reaction using primers deduced from the sequence of human RH genes. Nucleotide sequence analysis of the nonhuman transcripts revealed a high degree of similarity to human blood group Rh sequences, suggesting a great conservation of the RH genes throughout evolution. Full-length transcripts, potentially encoding 417 amino acid long proteins homologous to Rh polypeptides, were characterized, as well as mRNA isoforms which harbored nucleotide deletions or insertions and potentially encode truncated proteins. Proteins of 30-40,000 M{sub r},more » immunologically related to human Rh proteins, were detected by western blot analysis with antipeptide antibodies, indicating that Rh-like transcripts are translated into membrane proteins. Comparison of human and nonhuman protein sequences was pivotal in clarifying the molecular basis of the blood group C/c polymorphism, showing that only the Pro103Ser substitution was correlated with C/c polymorphism. In addition, it was shown that a proline residue at position 102 was critical in the expression of C and c epitopes, most likely by providing an appropriate conformation of Rh polypeptides. From these data a phylogenetic reconstruction of the RH locus evolution has been calculated from which an unrooted phylogenetic tree could be proposed, indicating that African ape Rh-like genes would be closer to the human RhD gene than to the human RhCE gene. 55 refs., 4 figs., 1 tab.« less

Middle Eocene, older sequences in rifts key to potential in western Indonesia

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

Courteney, S.

1996-05-27

In Part 1 of this article three areas, the Tiga Puluh arch (Sumatra), the Tanjung Raya area (Kalimantan), and the East Java basin in western Indonesia were discussed in terms of sequence stratigraphy. Such data have been interpreted for each basin in western Indonesia and two example correlations are discussed. A correlation chart of the major basins of Western Indonesia is shown in this paper. This chart was prepared on the basis of the regional correlations using sequence stratigraphy discussed.

ASBESTOS-INDUCED ACTIVATION OF CELL SIGNALING PATHWAYS IN HUMAN BRONCHIAL EPITHELIAL CELLS

EPA Science Inventory

Using respiratory epithelial cells transfected with either superoxide dismutase (SOD) or catalase, the authors tested the hypothesis that the activation of the epidermal growth factor (EGF) receptor signal pathway after asbestos exposure involves an oxidative stress. Western blot...

Generation and purification of highly-specific antibodies for detecting post-translationally modified proteins in vivo

PubMed Central

Arur, Swathi; Schedl, Tim

2014-01-01

Post-translational modifications alter protein structure, affecting activity, stability, localization and/or binding partners. Antibodies that specifically recognize post-translationally modified proteins have a number of uses including immuno-cytochemistry and immuno-precipitation of the modified protein to purify protein-protein and protein-nucleic acid complexes. However, antibodies directed at modified sites on individual proteins are often non-specific. Here we describe a protocol to purify polyclonal antibodies that specifically detect the modified protein of interest. The approach uses iterative rounds of subtraction and affinity purification, using stringent washes to remove antibodies that recognize the unmodified protein and low sequence complexity epitopes containing the modified amino acid. Dot and western blots assays are employed to assess antibody preparation specificity. The approach is designed to overcome the common occurrence that a single round of subtraction and affinity purification is not sufficient to obtain a modified protein specific antibody preparation. One full round of antibody purification and specificity testing takes 6 days of discontinuous time. PMID:24457330

Expression of fox-related genes in the skin follicles of Inner Mongolia cashmere goat.

PubMed

Han, Wenjing; Li, Xiaoyan; Wang, Lele; Wang, Honghao; Yang, Kun; Wang, Zhixin; Wang, Ruijun; Su, Rui; Liu, Zhihong; Zhao, Yanhong; Zhang, Yanjun; Li, Jinquan

2018-03-01

This study investigated the expression of genes in cashmere goats at different periods of their fetal development. Bioinformatics analysis was used to evaluate data obtained by transcriptome sequencing of fetus skin samples collected from Inner Mongolia cashmere goats on days 45, 55, and 65 of fetal age. We found that FoxN1 , FoxE1 , and FoxI3 genes of the Fox gene family were probably involved in the growth and development of the follicle and the formation of hair, which is consistent with previous findings. Real-time quantitative polymerase chain reaction detecting system and Western blot analysis were employed to study the relative differentially expressed genes FoxN1 , FoxE1 , and FoxI3 in the body skin of cashmere goat fetuses and adult individuals. This study provided new fundamental information for further investigation of the genes related to follicle development and exploration of their roles in hair follicle initiation, growth, and development.

[Construction of the eukaryotic recombinant vector and expression of the outer membrane protein LipL32 gene from Leptospira serovar Lai].

PubMed

Huang, Bi; Bao, Lang; Zhong, Qi; Shang, Zheng-ling; Zhang, Hui-dong; Zhang, Ying

2008-02-01

To construct the eukaryotic experssion vector of LipL32 gene from Leptospira serovar Lai and express the recombinant plasmid in COS-7 cell. The LipL32 gene was amplified from Leptospira strain 017 genomic DNA by PCR and cloned into pcDNA3.1, through restriction nuclease enzyme digestion. Then the recombinant plasmid was transformed into E.coli DH5alpha. After identified by nuclease digestion, PCR and sequencing analysis, the recombinant vector was transfected into COS-7 cell with lipsome. The expression of the target gene was detected by RT-PCR and Western blot. The eukaryotic experssion vector pcDNA3.1-LipL32 was successfully constructed and stably expressed in COS-7 cell. The eukaryotic recombinant vector of outer membrane protein LipL32 gene from Leptospira serovar Lai can be expressed in mammalian cell, which provides an experimental basis for the application of the Leptospira DNA vaccine.

Molecular diversity and hypoglycemic polypeptide-P content of Momordica charantia in different accessions and different seasons.

PubMed

Tian, Miao; Zeng, Xiang-Qing; Song, Huan-Lei; Hu, Shan-Xin; Wang, Fu-Jun; Zhao, Jian; Hu, Zhi-Bi

2015-04-01

Momordica charantia (MC) has been used for treating diabetes mellitus from ancient times in Asia, Africa and South America. There are many MC accessions in local markets. Polypeptide-P as a main hypoglycemic component in MC was first studied in this experiment to illustrate the different contents in MC of different accessions and different harvesting times. Nineteen MC accessions collected from different regions were clustered into three groups using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) molecular markers. Content of polypeptide-P in the tested MC accessions was detected by western blot (WB) method. The WB results revealed that polypeptide-P was detected in MC accessions harvested in June and July but not in September and October. Furthermore, Polypeptide-P content corresponded well with the MC accessions. Our results suggest that the MC accessions and the harvesting times or the weather during harvest play significant roles in high content of polypeptide-P. © 2014 Society of Chemical Industry.

Antigenic validation of recombinant hemagglutinin-neuraminidase protein of Newcastle disease virus expressed in Saccharomyces cerevisiae.

PubMed

Khulape, S A; Maity, H K; Pathak, D C; Mohan, C Madhan; Dey, S

2015-09-01

The outer membrane glycoprotein, hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) is important for virus infection and subsequent immune response by host, and offers target for development of recombinant antigen-based immunoassays and subunit vaccines. In this study, the expression of HN protein of NDV is attempted in yeast expression system. Yeast offers eukaryotic environment for protein processing and posttranslational modifications like glycosylation, in addition to higher growth rate and easy genetic manipulation. Saccharomyces cerevisiae was found to be better expression system for HN protein than Pichia pastoris as determined by codon usage analysis. The complete coding  sequence of HN gene was amplified with the histidine tag, cloned in pESC-URA under GAL10 promotor and transformed in Saccharomyces cerevisiae. The recombinant HN (rHN) protein was characterized by western blot, showing glycosylation heterogeneity as observed with other eukaryotic expression systems. The recombinant protein was purified by affinity column purification. The protein could be further used as subunit vaccine.

Fabrication of functional fibronectin patterns by nanosecond excimer laser direct write for tissue engineering applications.

PubMed

Grigorescu, S; Hindié, M; Axente, E; Carreiras, F; Anselme, K; Werckmann, J; Mihailescu, I N; Gallet, O

2013-07-01

Laser direct write techniques represent a prospective alternative for engineering a new generation of hybrid biomaterials via the creation of patterns consisting of biological proteins onto practically any type of substrate. In this paper we report on the characterization of fibronectin features obtained onto titanium substrates by UV nanosecond laser transfer. Fourier-transform infrared spectroscopy measurements evidenced no modification in the secondary structure of the post-transferred protein. The molecular weight of the transferred protein was identical to the initial fibronectin, no fragment bands being found in the transferred protein's Western blot migration profile. The presence of the cell-binding domain sequence and the mannose groups within the transferred molecules was revealed by anti-fibronectin monoclonal antibody immunolabelling and FITC-Concanavalin-A staining, respectively. The in vitro tests performed with MC3T3-E1 osteoblast-like cells and Swiss-3T3 fibroblasts showed that the cells' morphology and spreading were strongly influenced by the presence of the fibronectin spots.

Trichomonas vaginalis Metalloproteinase Induces mTOR Cleavage of SiHa Cells

PubMed Central

Quan, Juan-Hua; Choi, In-Wook; Yang, Jung-Bo; Zhou, Wei; Cha, Guang-Ho; Zhou, Yu; Ryu, Jae-Sook

2014-01-01

Trichomonas vaginalis secretes a number of proteases which are suspected to be the cause of pathogenesis; however, little is understood how they manipulate host cells. The mammalian target of rapamycin (mTOR) regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. We detected various types of metalloproteinases including GP63 protein from T. vaginalis trophozoites, and T. vaginalis GP63 metalloproteinase was confirmed by sequencing and western blot. When SiHa cells were stimulated with live T. vaginalis, T. vaginalis excretory-secretory products (ESP) or T. vaginalis lysate, live T. vaginalis and T. vaginalis ESP induced the mTOR cleavage in both time- and parasite load-dependent manner, but T. vaginalis lysate did not. Pretreatment of T. vaginalis with a metalloproteinase inhibitor, 1,10-phenanthroline, completely disappeared the mTOR cleavage in SiHa cells. Collectively, T. vaginalis metallopeptidase induces host cell mTOR cleavage, which may be related to survival of the parasite. PMID:25548410

Identification of a Novel GJA8 (Cx50) Point Mutation Causes Human Dominant Congenital Cataracts

NASA Astrophysics Data System (ADS)

Ge, Xiang-Lian; Zhang, Yilan; Wu, Yaming; Lv, Jineng; Zhang, Wei; Jin, Zi-Bing; Qu, Jia; Gu, Feng

2014-02-01

Hereditary cataracts are clinically and genetically heterogeneous lens diseases that cause a significant proportion of visual impairment and blindness in children. Human cataracts have been linked with mutations in two genes, GJA3 and GJA8, respectively. To identify the causative mutation in a family with hereditary cataracts, family members were screened for mutations by PCR for both genes. Sequencing the coding regions of GJA8, coding for connexin 50, revealed a C > A transversion at nucleotide 264, which caused p.P88T mutation. To dissect the molecular consequences of this mutation, plasmids carrying wild-type and mutant mouse ORFs of Gja8 were generated and ectopically expressed in HEK293 cells and human lens epithelial cells, respectively. The recombinant proteins were assessed by confocal microscopy and Western blotting. The results demonstrate that the molecular consequences of the p.P88T mutation in GJA8 include changes in connexin 50 protein localization patterns, accumulation of mutant protein, and increased cell growth.

Exosome-like vesicles with dipeptidyl peptidase IV in human saliva.

PubMed

Ogawa, Yuko; Kanai-Azuma, Masami; Akimoto, Yoshihiro; Kawakami, Hayato; Yanoshita, Ryohei

2008-06-01

Saliva contains a large number of proteins that participate in the protection of oral tissue. We found, for the first time, small vesicles (30-130 nm in diameter) in human whole saliva. Vesicles from saliva were identified by electron microscopy after isolation by gel-filtration on Sepharose CL-4B. They resemble exosomes, which are vesicles with an endosome-derived limiting membrane that are secreted by a diverse range of cell types. We performed a biochemical characterization of these vesicles by amino acid sequence analysis and Western blot analysis. We found that they contain dipeptidyl peptidase IV (DPP IV), galectin-3 and immunoglobulin A, which have potential to influence immune response. The DPP IV in the vesicles was metabolically active in cleaving substance P and glucose-dependent insulinotropic polypeptide to release N-terminal dipeptides. Our results demonstrate that human whole saliva contains exosome-like vesicles; they might participate in the catabolism of bioactive peptides and play a regulatory role in local immune defense in the oral cavity.

Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies

PubMed Central

Chervyakova, Olga V.; Zaitsev, Valentin L.; Iskakov, Bulat K.; Tailakova, Elmira T.; Strochkov, Vitaliy M.; Sultankulova, Kulyaisan T.; Sandybayev, Nurlan T.; Stanbekova, Gulshan E.; Beisenov, Daniyar K.; Abduraimov, Yergali O.; Mambetaliyev, Muratbay; Sansyzbay, Abylay R.; Kovalskaya, Natalia Y.; Nemchinov, Lev. G.; Hammond, Rosemarie W.

2016-01-01

The aim of this work was to evaluate the immunogenicity and neutralizing activity of sheep pox virus (SPPV; genus Capripoxvirus, family Poxviridae) structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins of vaccinia virus (VACV) strain Copenhagen. Four SPPV proteins (SPPV-ORF 060, SPPV-ORF 095, SPPV-ORF 117, and SPPV-ORF 122), orthologs of immunodominant L1, A4, A27, and A33 VACV proteins, respectively, were produced in Escherichia coli. Western blot analysis revealed the antigenic and immunogenic properties of SPPV-060, SPPV-095, SPPV-117 and SPPV-122 proteins when injected with adjuvant into experimental rabbits. Virus-neutralizing activity against SPPV in lamb kidney cell culture was detected for polyclonal antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. To our knowledge, this is the first report demonstrating the virus-neutralizing activities of antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. PMID:27338444

Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies.

PubMed

Chervyakova, Olga V; Zaitsev, Valentin L; Iskakov, Bulat K; Tailakova, Elmira T; Strochkov, Vitaliy M; Sultankulova, Kulyaisan T; Sandybayev, Nurlan T; Stanbekova, Gulshan E; Beisenov, Daniyar K; Abduraimov, Yergali O; Mambetaliyev, Muratbay; Sansyzbay, Abylay R; Kovalskaya, Natalia Y; Nemchinov, Lev G; Hammond, Rosemarie W

2016-06-07

The aim of this work was to evaluate the immunogenicity and neutralizing activity of sheep pox virus (SPPV; genus Capripoxvirus, family Poxviridae) structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins of vaccinia virus (VACV) strain Copenhagen. Four SPPV proteins (SPPV-ORF 060, SPPV-ORF 095, SPPV-ORF 117, and SPPV-ORF 122), orthologs of immunodominant L1, A4, A27, and A33 VACV proteins, respectively, were produced in Escherichia coli. Western blot analysis revealed the antigenic and immunogenic properties of SPPV-060, SPPV-095, SPPV-117 and SPPV-122 proteins when injected with adjuvant into experimental rabbits. Virus-neutralizing activity against SPPV in lamb kidney cell culture was detected for polyclonal antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. To our knowledge, this is the first report demonstrating the virus-neutralizing activities of antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins.

Generation and Characterization of a New Monoclonal Antibody Against CXCL4

PubMed Central

Gao, Jing; Wu, Mingyuan; Gao, Jin; Wang, Xia; Zhang, Yang; Zhu, Shunying; Yu, Yan

2015-01-01

CXCL4 plays important roles in numerous disease processes, which makes the CXCL4 signaling pathway a potential therapeutic target. In this study, we aimed to develop a neutralizing antibody against both human and mouse CXCL4. Rats were immunized with recombinant human CXCL4 (rhCXCL4). Hybridoma clones were created by fusion of the immunized rat spleen cells with mouse myeloma SP2/0 cells and screened using recombinant mouse CXCL4 (rmCXCL4) and rhCXCL4. The CXCL4 monoclonal antibody (CXCL4 MAb) produced by the 16D6-3 hybridoma clone was sequenced and characterized by Western blot and Biacore assays. It recognized both human and mouse CXCL4 with high affinity and neutralized the effect of rhCXCL4 in vitro. Thus, the antibody may be used in the studies of CXCL4 in murine disease models and as a template in the antibody humanization for clinical developments. PMID:25897609

Construction and heterologous expression of a truncated Haemagglutinin (HA) protein from the avian influenza virus H5N1 in Escherichia coli.

PubMed

Chee Wei, T; Nurul Wahida, A G; Shaharum, S

2014-12-01

Malaysia first reported H5N1 poultry case in 2004 and subsequently outbreak in poultry population in 2007. Here, a recombinant gene encoding of peptide epitopes, consisting fragments of HA1, HA2 and a polybasic cleavage site of H5N1 strain Malaysia, was amplified and cloned into pET-47b(+) bacterial expression vector. DNA sequencing and alignment analysis confirmed that the gene had no alteration and in-frame to the vector. Then, His-tagged truncated HA protein was expressed in Escherichia coli BL21 (DE3) under 1 mM IPTG induction. The protein expression was optimized under a time-course induction study and further purified using Ni-NTA agarose under reducing condition. Migration size of protein was detected at 15 kDa by Western blot using anti-His tag monoclonal antibody and demonstrated no discrepancy compared to its calculated molecular weight.

A novel MYH7 mutation links congenital fiber type disproportion and myosin storage myopathy.

PubMed

Ortolano, Saida; Tarrío, Rosa; Blanco-Arias, Patricia; Teijeira, Susana; Rodríguez-Trelles, Francisco; García-Murias, María; Delague, Valerie; Lévy, Nicolas; Fernández, José M; Quintáns, Beatriz; Millán, Beatriz San; Carracedo, Angel; Navarro, Carmen; Sobrido, María-Jesús

2011-04-01

This study aimed to identify the genetic defect in a multigenerational family presenting an autosomal dominant myopathy with histological features of congenital fiber type disproportion. Linkage analysis and genetic sequencing identified, in all affected members of the family, the c.5807A>G heterozygous mutation in MYH7, which encodes the slow/β-cardiac myosin heavy chain. This mutation causes skeletal but not cardiac involvement. Myosin heavy chain expression pattern was also characterized by immunohistochemistry, western blot and q-PCR in muscle biopsies from two patients aged 25 and 62, respectively. While only congenital fiber type disproportion was observed in the younger patient, older patient's biopsy presented aggregates of slow myosin heavy chains, in fiber sub-sarcolemmal region. These clinico-pathologic findings suggest a novel phenotype within the emerging group of hereditary myosin myopathies, which in this family presents typical characteristics of congenital fiber type disproportion in early stages and later evolves to myosin storage myopathy. Copyright © 2011 Elsevier B.V. All rights reserved.

Vitellogenin in black turtle (Chelonia mydas agassizii): purification, partial characterization, and validation of an enzyme-linked immunosorbent assay for its detection.

PubMed

Sifuentes-Romero, Itzel; Vázquez-Boucard, Celia; Sierra-Beltrán, Arturo P; Gardner, Susan C

2006-02-01

Black turtle plasmatic vitellogenin (VTG) was purified from 17beta-estradiol-induced males using ion-exchange chromatography. The isolated protein was identified as VTG by its glycolipoprotein nature and amino acid sequence homology with other vertebrate VTG. It was characterized as a 500-kDa dimer composed of two identical, 200- to 240-kDa monomers. Polyclonal antibodies raised against black turtle VTG showed high titer and specificity, as demonstrated by enzyme-linked immunosorbent assay and Western blot analysis, respectively. The range of the assay was estimated to be between 15 ng/ml and 2 microg/ml, and the inter- and intra-assay coefficients of variation were 9.4 and 7.3%, respectively. Black turtle antibody cross-reacted with VTG of two other sea turtle species, Caretta caretta (loggerhead) and Eretmochelys imbricata (hawksbill), extending the applicability of the assay as part of a sea turtle health assessment program.

Secretion of clostridium cellulase by E. coli

DOEpatents

Yu, Ida Kuo

1998-01-01

A gene, encoding an endocellulase from a newly isolated mesophilic Clostridium strain IY-2 which can digest bamboo fibers, cellulose, rice straw, and sawdust, was isolated by shotgun cloning in an E. coli expression plasmid pLC2833. E. coli positive clones were selected based on their ability to hydrolyze milled bamboo fibers and cellulose present in agar plates. One clone contained a 2.8 kb DNA fragment that was responsible for cellulase activity. Western blot analyses indicated that the positive clone produced a secreted cellulase with a mass of about 58,000 daltons that was identical in size to the subunit of one of the three major Clostridium cellulases. The products of cellulose digestion by this cloned cellulase were cellotetraose and soluble higher polymers. The cloned DNA contained signal sequences capable of directing the secretion of heterologous proteins from an E. coli host. The invention describes a bioprocess for the treatment of cellulosic plant materials to produce cellular growth substrates and fermentation end products suitable for production of liquid fuels, solvents, and acids.

Elucidation of the critical epitope of an anti-EGFR monoclonal antibody EMab-134.

PubMed

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

2018-07-01

The epidermal growth factor receptor (EGFR) is a type-1 transmembrane receptor tyrosine kinase, which activates the downstream signaling cascades in many tumors, such as oral and lung cancers. We previously developed EMab-134, a novel anti-EGFR monoclonal antibody (mAb), which reacts with endogenous EGFR-expressing cancer cell lines and normal cells independent of glycosylation in Western blotting, flow cytometry, and immunohistochemical analysis. EMab-134 showed very high sensitivity (94.7%) to oral squamous cell carcinomas in immunohistochemical analysis. In this study, we performed enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of EMab-134. A blocking peptide (375-394 amino acids of EGFR) neutralized the EMab-134 reaction against oral cancer cells in flow cytometry and immunohistochemistry. The minimum epitope of EMab-134 was found to be the 377- RGDSFTHTPP -386 sequence. Our findings can be applied for the production of more functional anti-EGFR mAbs that in turn can be used for antitumor treatments.

Curcumin reverses irinotecan resistance in colon cancer cell by regulation of epithelial-mesenchymal transition.

PubMed

Zhang, Chunhong; Xu, Yangjie; Wang, Haowen; Li, Gang; Yan, Han; Fei, Zhenghua; Xu, Yunsheng; Li, Wenfeng

2018-04-01

The objective of this study was to investigate the effect and the mechanism by which curcumin reverses irinotecan-induced chemotherapy resistance in colon cancer. Construction of irinotecan-resistant colon cancer model LoVo/CPT-11R cells was performed by increasing drug concentration. The Cell Counting Kit-8 assay was used to detect inhibition of proliferation; cell morphology was observed by an optical microscope. Quantitative RT-PCR and western blotting were performed to detect molecular marker expressions during epithelial-mesenchymal transition (EMT); drug-resistant cells were treated with curcumin at different concentrations and Cell Counting Kit-8 was reperformed to detect cell proliferation after treatments. Drug-resistant cells were then divided into four groups: control group, irinotecan group, curcumin group, and irinotecan+curcumin group; quantitative RT-PCR and western blotting were performed to detect molecular marker expressions during epithelial-mesenchymal transition. Flow cytometry was used to detect cell apoptosis after grouping, and apoptosis-related protein was detected by western blotting. LoVo/CPT-11R cells could survive in culture medium containing irinotecan at 60 μg/ml and the drug-resistance index was 5.69; the drug-resistant cells had a larger volume than normal cells and were poorly connected to each other. E-cadherin expression was downregulated, whereas vimentin and N-cadherin expressions were upregulated. After curcumin treatment, drug-resistant cell proliferation was significantly inhibited; in the curcumin+irinotecan treatment group, E-cadherin expression was upregulated, whereas vimentin and N-cadherin expressions were downregulated. Curcumin could significantly increase cell apoptosis. EMT is involved in the development of irinotecan resistance and curcumin can reverse this drug resistance through reversion of the EMT process.

Curcumin suppresses transforming growth factor-β1-induced cardiac fibroblast differentiation via inhibition of Smad-2 and p38 MAPK signaling pathways

PubMed Central

LIU, HUZI; LIU, AIJUN; SHI, CHUNLI; LI, BAO

2016-01-01

The differentiation of cardiac fibroblasts (CFs) into myofibroblasts and the subsequent deposition of the extracellular matrix is associated with myocardial fibrosis following various types of myocardial injury. In the present study, the effect of curcumin, which is a pharmacologically-safe natural compound from the Curcuma longa herb, on transforming growth factor (TGF)-β1-induced CFs was investigated, and the underlying molecular mechanisms were examined. The expression levels of α-smooth muscle actin (SMA) stress fibers were investigated using western blotting and immunofluorescence in cultured neonatal rat CFs. Protein and mRNA expression levels of α-SMA and collagen type I (ColI) were determined by western blotting and reverse transcription-quantitative polymerase chain reaction. In addition, the activation of Smad2 and p38 was examined using western blotting. Curcumin, SB431542 (a TGF-βR-Smad2 inhibitor) and SB203580 (a p38 inhibitor) were used to inhibit the stimulation by TGF-β1. The results demonstrated that the TGF-β1-induced expression of α-SMA and ColI was suppressed by curcumin at the mRNA and protein levels, while SB431542 and SB203580 induced similar effects. Furthermore, phosphorylated Smad-2 and p38 were upregulated in TGF-β1-induced CFs, and these effects were substantially inhibited by curcumin administration. In conclusion, the results of the present study demonstrated that treatment with curcumin effectively suppresses TGF-β1-induced CF differentiation via Smad-2 and p38 signaling pathways. Thus, curcumin may be a potential therapeutic agent for the treatment of cardiac fibrosis. PMID:26998027

Proportion of collagen type II in the extracellular matrix promotes the differentiation of human adipose-derived mesenchymal stem cells into nucleus pulposus cells.

PubMed

Tao, Yiqing; Zhou, Xiaopeng; Liu, Dongyu; Li, Hao; Liang, Chengzhen; Li, Fangcai; Chen, Qixin

2016-01-01

During degeneration process, the catabolism of collagen type II and anabolism of collagen type I in nucleus pulposus (NP) may influence the bioactivity of transplanted cells. Human adipose-derived mesenchymal stem cells (hADMSCs) were cultured as a micromass or in a series of gradual proportion hydrogels of a mix of collagen types I and II. Cell proliferation and cytotoxicity were detected using CCK-8 and LDH assays respectively. The expression of differentiation-related genes and proteins, including SOX9, aggrecan, collagen type I, and collagen type II, was examined using RT-qPCR and Western blotting. Novel phenotypic genes were also detected by RT-qPCR and western blotting. Alcian blue and dimethylmethylene blue assays were used to investigate sulfate proteoglycan expression, and PI3K/AKT, MAPK/ERK, and Smad signaling pathways were examined by Western blotting. The results showed collagen hydrogels have good biocompatibility, and cell proliferation increased after collagen type II treatment. Expressions of SOX9, aggrecan, and collagen type II were increased in a collagen type II dependent manner. Sulfate proteoglycan synthesis increased in proportion to collagen type II concentration. Only hADMSCs highly expressed NP cell marker KRT19 in collagen type II culture. Additionally, phosphorylated Smad3, which is associated with phosphorylated ERK, was increased after collagen type II-stimulation. The concentration and type of collagen affect hADMSC differentiation into NP cells. Collagen type II significantly ameliorates hADMSC differentiation into NP cells and promotes extracellular matrix synthesis. Therefore, anabolism of collagen type I and catabolism of type II may attenuate the differentiation and biosynthesis of transplanted stem cells. © 2016 International Union of Biochemistry and Molecular Biology.

Autophagy promotes metastasis and glycolysis by upregulating MCT1 expression and Wnt/β-catenin signaling pathway activation in hepatocellular carcinoma cells.

PubMed

Fan, Qing; Yang, Liang; Zhang, Xiaodong; Ma, Yingbo; Li, Yan; Dong, Lei; Zong, Zhihong; Hua, Xiangdong; Su, Dongming; Li, Hangyu; Liu, Jingang

2018-01-19

Autophagy is a dynamic physiological process that can generate energy and nutrients for cell survival during stress. Autophagy can regulate the migration and invasive ability in cancer cells. However, the connection between autophagy and metabolism is unclear. Monocarboxylate transporter 1 (MCT1) plays an important role in lactic acid transport and H + clearance in cancer cells, and Wnt/β-catenin signaling can increase cancer cell glycolysis. We investigated whether autophagy promotes glycolysis in hepatocellular carcinoma (HCC) cells by activating the Wnt/β-catenin signaling pathway, accompanied by MCT1 upregulation. Autophagic activity was evaluated using western blotting, immunoblotting, and transmission electron microscopy. The underlying mechanisms of autophagy activation on HCC cell glycolysis were studied via western blotting, and Transwell, lactate, and glucose assays. MCT1 expression was detected using quantitative reverse transcription-PCR (real-time PCR), western blotting, and immunostaining of HCC tissues and the paired adjacent tissues. Autophagy promoted HCC cell glycolysis accompanied by MCT1 upregulation. Wnt/β-catenin signaling pathway activation mediated the effect of autophagy on HCC cell glycolysis. β-Catenin downregulation inhibited the autophagy-induced glycolysis in HCC cells, and reduced MCT1 expression in the HCC cells. MCT1 was highly expressed in HCC tissues, and high MCT1 expression correlated positively with the expression of microtubule-associated protein light chain 3 (LC3). Activation of autophagy can promote metastasis and glycolysis in HCC cells, and autophagy induces MCT1 expression by activating Wnt/β-catenin signaling. Our study describes the connection between autophagy and glucose metabolism in HCC cells and may provide a potential therapeutic target for HCC treatment.

[Overexpression of liver kinase B1 inhibits the proliferation of lung cancer cells].

PubMed

Li, Yang; Zhang, Libin; Wang, Ping

2017-01-01

Objective To explore the effect of overexpressed liver kinase B1(LKB1) on the proliferation of lung cancer cell lines. Methods The expression levels of LKB1 and PTEN in A549, NCI-H23, NCI-H157, XWLC-05, NCI-H446 lung cancer cells were detected by immunocytochemistry (ICC) and Western blotting. Plasmid pcDNA3.1 + -LKB1 and empty vector pcDNA3.1 + -null were separately transfected into the above five cell lines, and then the expression of LKB1 mRNA and protein were determined by quantitative real-time PCR and Western blotting, respectively. Finally, CCK-8 assay was used to analyze the proliferation ability of the transfected cells. Results LKB1 and PTEN were positive in NCI-H23 cells; LKB1 was negative while PTEN was positive in A549 and NCI-H446 cells; both LKB1 and PTEN were negative in NCI-H157 and XWLC-05 cells. Quantitative real-time PCR and Western blotting showed that the expression level of LKB1 significantly increased in the above cell lines transfected with plasmid pcDNA3.1 + -LKB1 compared with the ones with empty vector pcDNA3.1 + -null. Besides, CCK-8 assay showed that the overexpression of LKB1 in the lung cancer cells transfected with pcDNA3.1 + -LKB1 had an obvious inhibitory effect on cell proliferation. Conclusion The expression of LKB1 is down-regulated in most of the lung cell lines to different extent and the over-expression of LKB1 can remarkably inhibit the proliferation ability of lung cancer cell lines.

[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.

The Major Antigenic Membrane Protein of “Candidatus Phytoplasma asteris” Selectively Interacts with ATP Synthase and Actin of Leafhopper Vectors

PubMed Central

Galetto, Luciana; Bosco, Domenico; Balestrini, Raffaella; Genre, Andrea; Fletcher, Jacqueline; Marzachì, Cristina

2011-01-01

Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of “Candidatus Phytoplasma asteris”, the chrysanthemum yellows phytoplasmas (CYP) strain, and three others as non-vectors. Interactions between a labelled (recombinant) CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity. PMID:21799902

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.

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.

Persistence of RSV promotes proliferation and epithelial-mesenchymal transition of bronchial epithelial cells through Nodal signaling.

PubMed

Xiang, Zhao; Liang, Zhang; Yanfeng, Huang; Leitao, Kang

2017-10-01

Nodal may play an important role in the development of cancers. The present study was designed to determine the effects of Nodal induced by respiratory syncytial virus (RSV) infection on the occurrence and development of lung cancer and the underlying mechanisms. After verification of RSV infection by observation of cytopathic effect and indirect immunofluorescence, real-time PCR, Western blot and methylation assays were used to verify the influence of RSV on Nodal expression. Then, a Nodal overexpressed vector was constructed and the effects of Nodal on the proliferation and apoptosis of bronchial epithelial cells (BECs) and epithelial-mesenchymal transition (EMT) were assayed by flow cytometry and Western blot, respectively. Moreover, Lefty and pSmad2/3 were assayed by Western blot and Cyclin D1, CDK4, c-myc and Bcl-2 induced by Nodal overepression or RSV infection were also assayed by real-time PCR. The results showed that Nodal over expression and demethylation of the promoter were observed in BECs after RSV infection. Activation of Nodal promoted proliferation, colony formation and EMT and inhibited apoptosis of BECs. Nodal also promoted malignant change by promoting expression of cyclin D1 and related-dependent kinase and inhibiting apoptosis. Besides, RSV infection inhibited Lefty expression and promoted the activation of pSmad2/3. RSV also promoted Cyclin D1, CDK4, c-myc and Bcl-2 expression through the activation of pSmad2/3. Our data showed that persistence of RSV promoted the proliferation, epithelial-mesenchymal transition and expression of oncogenes through Nodal signaling, which may be associated with the occurrence and development of lung cancers.