Goat Anti-rabbit Immunoglobulin Research Articles

Gold Nanoparticle-Functionalized Diatom Biosilica as Label-Free Biosensor for Biomolecule Detection.

Diatom biosilica (DBs) is the cell wall of natural diatom called frustule, which is made of porous hydrogenated amorphous silica possessing periodic micro- to nanoscale features. In this study, a simple, sensitive, and label-free photoluminescence (PL) immune-detection platform based on functionalized diatom frustules was developed. Gold nanoparticles (AuNPs) deposited on poly-dopamine-coated diatom frustules via in situ deposition which considerably decreased the intrinsic blue PL intensity of diatom biosilica. Then, goat anti-rabbit immunoglobulin G (IgG) was added to functionalize diatom biosilica-poly-dopamine-AuNPs (DBs-PDA-AuNPs). PL studies revealed that the specific binding with antigen rabbit IgG increased the peak intensity of PL in comparison with the non-complimentary antigen (human IgG). The enhancement in PL intensity of DBs-PDA had a linear correlation with antigen (rabbit IgG) concentration, whose limit of detection (LOD) reached 8 × 10-6 mg/ml. Furthermore, PL detection based on DBs-PDA-AuNPs showed a high detection sensitivity with the LOD as low as 8 × 10-9 mg/ml and spread over almost eight orders of magnitude, making it suitable for the sensitive quantitative analysis of immune complex compared with traditional fluorescence immunoassay. Hence, the study proves that the AuNP-functionalized diatom frustules can serve as an effective biosensor platform for label-free PL-based immunoassay.

Development of Immunochromatographic Assay for the Rapid Detection of Mycoplasma capricolum subsp. capripneumoniae Antibodies.

Mycoplasma capricolum subsp. capripneumoniae (Mccp) is the cause of contagious caprine pleuropneumonia (CCPP), which is a highly significant respiratory disease in goats leading to significant economic losses in Africa and Asia. Currently available procedures for the diagnosis of CCPP have some limitations in sensitivity, specificity, operation time, requirement of sophisticated equipment or skilled personnel, and cost. In this study, we developed a rapid, sensitive, and specific colloidal gold-based immunochromatographic assay (GICA) strip for the efficient on-site detection of antibodies against Mccp in the serum within 10 min. For the preparation of this colloidal GICA strip, recombinant P20 protein, the membrane protein of Mccp, was expressed by Escherichia coli prokaryotic expression system after purification was used as the binding antigen in the test. The rabbit anti-goat immunoglobulin G labeled with the colloidal gold was used as the detection probe, whereas the goat anti-rabbit immunoglobulin G was coated on the nitrocellulose membrane as the control line. The concentration of the coating antibody was optimized, and the effectiveness of this colloidal GICA strip was evaluated. Our results proved that the detection limit of the test strip was up to 1:64 dilutions for the Mccp antibody-positive serum samples with no cross-reactivity with other pathogens commonly infecting small ruminants,including goat pox virus, peste des petits ruminants virus, foot-and-mouth disease virus type A, or other mycoplasmas. Moreover, the colloidal GICA strip was more sensitive and specific than the indirect hemagglutination assay for the detection of Mccp antibodies. The 106 clinical serum samples were detected by the colloidal GICA strip compared with the complement fixation test, demonstrating an 87.74% concordance with the complement fixation test. This novel colloidal GICA strip would be an effective tool for the cost-effective and rapid diagnosis of CCPP in the field.

Trophoblast Stem-Cell-Derived Exosomes Improve Doxorubicin-Induced Dilated Cardiomyopathy by Modulating the let-7i/YAP Pathway

Trophoblast stem cells (TSCs) have been confirmed to play a cardioprotective role in heart failure. However, whether TSC-derived exosomes (TSC-exos) can protect against cardiac injury remains unclear. In the present study, TSC-exos were isolated from the supernatant of TSCs using the ultracentrifugation method and characterized by transmission electron microscopy and western blotting. Utilizing the public Gene Expression Omnibus (GEO) database, we found that let-7i and Yes-associated protein 1 (YAP) could participate in the development of heart failure. In vitro, AC16 cardiomyocytes subjected to doxorubicin (DOX) were treated with TSC-exos or let-7i mimic. Flow cytometry showed that TSC-exos and let-7i both decreased cardiomyocyte apoptosis. In vivo, mice that were intraperitoneally injected into DOX received either PBS, TSC-exos, or AAV9-let7iup for let-7i overexpression. Mice receiving TSC-exos and AAV9-let7iup showed improved cardiac function and decreased inflammatory responses, accompanied by downregulated YAP signaling. Mechanistically, TSC-exos could transfer let-7i to cardiomyocytes and silence the YAP signaling pathway. In conclusion, TSC-exos could alleviate DOX-induced cardiac injury via the let-7i/YAP pathway, which sheds new light on the application of TSC-exos as a potential therapeutic tool for heart failure.

Evaluation of Apparatus Used to Test Liquid through Protective Materials: Comparison of a Modified Dot-Blot Apparatus to the ASTM Penetration Cell.

Personal protective equipment (PPE), such as gowns used in the latest Ebola outbreak in Western Africa, are critical in preventing the spread of deadly diseases. Appropriate test systems and test soils are needed to adequately evaluate PPE. ASTM F903, Standard Test Method for Resistance of Materials Used in Protective Clothing to Penetration by Liquid, has been used for decades to test fabrics' resistance to liquid penetration. However, this test apparatus requires at least 60 mL of test solutions, is labor intensive, and has problems with leakage around the gaskets. We compared the F903 test apparatus to a modified dot-blot apparatus to evaluate the visual penetration of a blood test soil. A series of commercially available gowns and drapes were tested in each apparatus. Using blood test soil at 2 psi, there was no statistically significant difference between the two methods except for in one gown. By comparing this gown in the ASTM test apparatus with and without a screen, the particular screen selected did not account for the difference between the dot-blot and F903 apparatuses; however, it is conceivable that a particular screen/fabric combination could account for this difference. The modified dot-blot apparatus was evaluated using three different test solutions: blood, vomit, and a labeled protein (goat anti-rabbit immunoglobulin G-horseradish peroxidase [GaR IgG-HRP]) in a blood test soil solution. This testing revealed significant difference in penetration for some of the PPE garments. The modified dot-blot had several large advantages over the ASTM apparatus-over six times less specimen volume and no edge or gasket leakage. In addition, nitrocellulose can be easily incorporated into the modified dot-blot apparatus, enabling the trapping of viruses and proteins that penetrate PPE-thus permitting the use of antibodies to quickly and sensitively detect penetration.

Hydrophobin HGFI–based fibre-optic biosensor for detection of antigen–antibody interaction

Abstract A compact S-tapered fibre biosensor functionalised with class I hydrophobin HGFI found in Grifola frondosa has been proposed for the first time to accrue label-free detection of reaction between goat–anti-rabbit immunoglobulin G (IgG) (GAR, antibody) and rabbit–anti-hemagglutinin IgG (R, antigen). The HGFI nanolayer self-assembled on fibre surface provides a distinguished analytical platform to implement biocompatible binding owing to its prominent amphiphilicity and remarkable optical as well as biochemical properties. Water contact angle measurements, atomic force microscopy, and immunofluorescence assay are utilised to characterize the modification properties of siliconised substrate surface with HGFI. Functionalisation of the S-tapered fibre is achieved by exploiting self-assembly of HGFI and immobilisation of GAR for specific R detection. HGFI with a concentration of 300 μg/ml allows the formation of a self-assembled amphipathic film on fibre surface, which can adsorb antibody smoothly with such desirable merits as ease of operation, fast response, good stability, good repeatability, and no side effects. Immunoassay experiments are conducted based on dip wavelengths interrogation of the interferometric transmission spectrum. In this study, we propose a novel antigen detection scheme, which could be extended for the detection of other biomolecules owing to its high integration, good specificity, real-time detection, and simple detection scheme.

Rapid and Sensitive Detection of Campylobacter jejuni in Poultry Products Using a Nanoparticle-Based Piezoelectric Immunosensor Integrated with Magnetic Immunoseparation

Campylobacter jejuni is one of the leading causes of foodborne human gastrointestinal diseases. Poultry and poultry products have been identified as the major transmission routes to humans for this pathogenic bacterium. The objective of this research was to develop a rapid and sensitive immunosensor for detection of C. jejuni in poultry products on the basis of a quartz crystal microbalance (QCM) using magnetic nanobeads (MNBs) for separation of target pathogen and gold nanoparticles for amplification of the measurement. A QCM sensor in a flow cell was prepared by immobilizing the mouse anti- C. jejuni monoclonal antibody (mAb1) on the sensor surface to specifically capture C. jejuni. Rabbit anti- C. jejuni polyclonal antibody (pAb1) was conjugated with MNBs to capture and separate C. jejuni from food matrices. MNB-pAb1- C. jejuni complexes were injected into the flow cell to bind with the mAb1 immobilized on the QCM sensor surface. Goat anti-rabbit immunoglobulin G polyclonal antibody conjugated with gold nanoparticles was injected into the flow cell to bind with pAb1 on MNBs. Finally, resonant frequency was measured with a QCM analyzer, and the change in resonant frequency was correlated to the cell number of C. jejuni. The specificity of this immunosensor was confirmed with different strains of Campylobacter, Salmonella, and other foodborne pathogens commonly colonized in the broiler gastrointestinal tract. Samples of broiler carcass wash and ground turkey were spiked with C. jejuni at different concentrations for use in tests. Results showed that the QCM immunosensor could rapidly detect C. jejuni in poultry products, with a detection limit of 20 to 30 CFU/mL without preenrichment, and a total detection time of less than 30 min. Characteristics of C. jejuni captured by the antibody immobilized on the surface of the QCM sensor were visualized by using atomic force microscopy. This highly adaptive and flexible method could provide the poultry industry a more rapid, sensitive, and effective method for detection of major foodborne pathogens in poultry products.

Development of a two-step immunochromatographic assay for microcystin-LR based on fluorescent microspheres

Microcystins (MCs) are a class of toxins that are mainly produced by cyanobacteria. Among them, microcystin-leucine arginine (MC-LR) is one of the most toxic and harmful of the freshwater toxins, and it has caused many accidents and threats to human health. In the present study, we report a neoteric lateral flow fluorescent microsphere immunochromatographic assay (FM-ICA) combined with UV light detection to rapidly and quantitatively detect MC-LR in freshwater food, based on direct competitive immunoreactions on chromatographic strips. In the method, MC-LR artificial antigen, MC-LR-bovine serum albumin (MC-LR-BSA), and goat anti-rabbit immunoglobulin G (IgG) were labelled with europium (Eu) nanospheres that functioned as a luminescence tracer. Goat anti-mouse IgG and rabbit IgG were spread on a nitrocellulose (NC) membrane for the test (T) line and control (C) line, respectively. The optimal parameters were 1 mg/mL for the goat anti-mouse IgG and rabbit IgG, 20 μg of MC-LR-BSA conjugated with Eu nanospheres, 1:100 for the dilution of Eu-MC-LR-BSA, and a 1:30000 dilution of anti-MC-LR monoclonal antibody (mAb) with an assay time of 15 min. The working range of the MC-LR standard curve was 0.1–5.0 μg/L with a limit of detection (LOD) of 0.0542 μg/L and a 50% inhibitory concentration (IC50) of 0.5613 μg/L. The average recoveries of the FM-ICA for detecting MC-LR in real samples ranged from 90.1 to 109.3%, with coefficients of variation under 15.8%. The FM-ICA test strips for quantitative determination of MC-LR provide sensitive, simple, and rapid performance, and can be used to monitor freshwater samples.

Chain-length dependent interfacial immunoreaction kinetics on self-assembled monolayers revealed by surface-enhanced infrared absorption spectroscopy

Self-assembled monolayer (SAM) has been extensively applied as ideal interface layer for construction of biosensors. Its chain length and end functional groups determine the physical and chemical properties of the modified surfaces, which will affect the performance of constructed biosensors. Herein, we studied the influence of chain length of n-alkanethiols SAMs on the immunoreaction kinetics employing attenuated total reflection surface-enhanced infrared absorption spectroscopy (ATR-SEIRAS). Antibody (rabbit immunoglobulin) is assembled on carboxyl terminated SAMs of n-alkanethiols with different chain lengths (n = 3, 6, 11, 16). The whole fabrication steps of the immunoassay can be monitored in situ by the ATR-SEIRAS. From the time-dependent SEIRA spectra, the interfacial immunoreaction kinetics between the immobilized antibody and antigen (goat anti-rabbit immunoglobulin) can be evaluated. We found that the immunoreaction became faster with increasing the chain length of SAMs. This chain length dependent kinetics might be attributed to different orientations of the assembled antibody caused by different packing densities of SAMs. The present research offers a sensing platform to evaluate immunoassay kinetics and provides fundamentals for construction of immunoassay with high performance.

Label-Free Immunosensor Based on Optical Fiber Fabry–Perot Interferometer

A label-free immunosensor based on optical fiber Fabry–Perot interferometer (FPI) is proposed. The FPI is fabricated by splicing both ends of a short section of hollow-core photonic crystal fiber to single mode fibers (SMFs) and cleaving the SMF pigtail to proper length. Then, the end surface of sensor is silanization modified and immobilized with goat anti-rabbit immunoglobulin G (IgG) by covalent binding to detect the specific rabbit IgG. The antibody immobilization and antigen binding will change the thickness of sensing layer, leading to the interference spectrum change of the FPI sensor. The responses of the sensor to immune reaction are analyzed theoretically and demonstrated experimentally. Results show that after antibody immobilization, the wavelength increases by 60 pm and the fringe contrast decreases by about 1 dB. And for antigen binding, the wavelength shows an obvious increase by 190 pm and the fringe contrast decreases by 2.15 dB. The wavelength shift during the antibody-antigen binding process conforms to the immune reaction kinetics. With the advantages of compact structure, good performance, capacity for real-time and label-free detection, easy fabrication, and low cost, the proposed optical fiber FPI sensor shows wide application prospect in label-free immunosensing.

Application of Goldmag immune probe in timely detection of syphilis based on GIS platform

The purpose of this study was to apply goldmag immunoprobes into establishment of nanoparticles-based colorimetric assay as well as construction of immunochromatography quantitative and qualitative system by exploring point-of-care testing of syphilis with goldmag particles carrier-based immunoprobe and analysis of spatial data of Geographic Information System (GIS) platform. Goat anti-rabbit immunoglobulin G (IgG) was coupled on the surface of modified nanoparticles, taking N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide as the connector. Then the nanoparticles were used for colorimetric detection of goat-anti-rabbit IgG in liquid phase system. Based on the analysis of spatial data in GIS platform, we found the probe constructed based on MUA-Fe304/Au nanoparticles responded more sensitive to detection objects compared with the probe designed based on PAA-Fe3O4/Au nanoparticles, and its reaction rate constant was two times that of PAA-Fe3O4/Au nanoparticles based goldmag immunoprobe. Goldmag particles not only can be coupled with biomolecules such as antibody/antigen and glycoprotein but also possess superparamagnetism.

Identification of a peptide ligand for antibody immobilization on biosensor surfaces

The construction of biosensors with antibodies as the biological sensing element requires the integration of antibodies into the biosensor such that their activity is reproducibly retained. In this work, a peptide ligand was identified from phage-displayed heptameric peptide libraries for the purpose of antibody immobilization on biosensor surfaces. For biopanning of peptides specific to the Fc region of the antibody, rabbit anti-goat immunoglobulin G (IgG) antibody was immobilized on a culture dish coated with goat IgG, which enabled exposure of the Fc region of rabbit antibody on the surface with the Fab region bound to the surface-adsorbed goat IgG. The library phages were added to the dish and phages displaying antibody-binding peptides were competitively eluted using the rabbit antibody. After four rounds of biopanning, 22 phage plaques were randomly selected and their nucleotide sequences corresponding to the random peptide were determined. Three enriched sequences were selected and analyzed for their rabbit antibody-binding activity. One of these sequences (KHRFNKD), which was rich in positive charge and homologous to the IgG-binding domains of Staphylococcal protein A, exhibited a high and specific affinity to rabbit antibody. This peptide was immobilized on a quartz crystal microbalance (QCM) biosensor, and the resulting QCM was found to be capable of capturing antibody and detecting antigen, with potential applications in antibody purification and enzyme immunoassay.

A versatile microparticle-based immunoaggregation assay for macromolecular biomarker detection and quantification.

The rapid, sensitive and low-cost detection of macromolecular biomarkers is critical in clinical diagnostics, environmental monitoring, research, etc. Conventional assay methods usually require bulky, expensive and designated instruments and relative long assay time. For hospitals and laboratories that lack immediate access to analytical instruments, fast and low-cost assay methods for the detection of macromolecular biomarkers are urgently needed. In this work, we developed a versatile microparticle (MP)-based immunoaggregation method for the detection and quantification of macromolecular biomarkers. Antibodies (Abs) were firstly conjugated to MP through streptavidin-biotin interaction; the addition of macromolecular biomarkers caused the aggregation of Ab-MPs, which were subsequently detected by an optical microscope or optical particle sizer. The invisible nanometer-scale macromolecular biomarkers caused detectable change of micrometer-scale particle size distributions. Goat anti-rabbit immunoglobulin and human ferritin were used as model biomarkers to demonstrate MP-based immunoaggregation assay in PBS and 10% FBS to mimic real biomarker assay in the complex medium. It was found that both the number ratio and the volume ratio of Ab-MP aggregates caused by biomarker to all particles were directly correlated to the biomarker concentration. In addition, we found that the detection range could be tuned by adjusting the Ab-MP concentration. We envision that this novel MP-based immunoaggregation assay can be combined with multiple detection methods to detect and quantify macromolecular biomarkers at the nanogram per milliliter level.

Synthesis and Characterization of Artificial Antigens for Copper and Application for Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay

Copper was conjugated to two carrier proteins using penicillin G sodium salt to synthesize artificial antigens for copper. The antigens were identified by ultraviolet spectrometry, circular dichroism, and inductively coupled plasma–optical emission spectrometry. The results of ultraviolet spectrometry showed characteristic absorption peak shifts between haptens and carrier proteins. Circular dichroism showed that the secondary structure of the conjugates was an α-helix. These results suggested that the artificial antigens for copper were synthesized successfully. The polyclonal antibodies of copper were produced by immunizing New Zealand white rabbits with copper antigens. An indirect competitive enzyme-linked immunosorbent assay based on the polyclonal antibodies was developed. The concentration of copper ion was quantified based on the ability of its ethylenediaminetetraacetic acid complexes to inhibit the binding of the goat anti-rabbit immunoglobulin G conjugated with horseradish peroxidase and, subsequently, color formation in the assay. The assay showed an IC50 value of 0.475 microgram per milliliter with a detection limit of 0.007 microgram per milliliter. The antibody showed high affinity with copper and low cross-reactivity with other metals. The recovery of copper from fortified water was between 78.0 and 122 percent. The results indicate that the assay is a convenient supplemental analytical tool for monitoring copper ions in aquatic environments.

Characterisation of novel cytokines in human atherosclerotic plaque

Inflammation plays a critical role in all stages of atheroscleroticplaque development, particularly so in inducing an unstable plaquephenotype [1]. Cytokines modulate this inflammatory process, withemergingdatashowingthatbothpro-andanti-inflammatorycytokinesare present in atherosclerotic plaques [2]. The cytokines IL-31, IL-32,IL-33 and IL-34 have recently been characterised with regard tovarious inflammatory conditions, with IL-31, -32 and -34 having pro-inflammatory effects whereas IL-33 has been shown to be potentiallyathero-protective both in vitro and in animal studies of experimentalatherosclerosis [3–6]. Our recently published data described the distri-butionofthesecytokinesinpost-mortemcoronarysamples[7];howev-er their expression in ex-vivo human carotid plaques, particularly theircorrelationwithstableversusunstableplaquephenotypesandrelation-ship with patient characteristics, has not yet been explored.Forty-five consecutive patients with haemodynamically significantcarotid artery disease and in whomcarotid endarterectomy was deemedappropriate were selected to participate in the study [8]. Patients wereclassified as symptomatic (n = 23) or asymptomatic (n = 22) accordingto the presence or absence of cerebrovascular symptoms, respectively.Aspreviouslydescribed,themajorityofplaquesfromasymptomaticsub-jects were Type V lesions, whilst plaques from symptomatic subjectswere predominantly Type VI lesions, as defined by the American HeartAssociation Vascular Lesion Classi fication [9]. Cardiovascular risk factors,lipidprofileandmedicationusewererecordedforeachpatient.Informedconsent was obtained from each patient and the study was approvedby the South Western Sydney Area Health Ethics Committee, NSW,Australia.Excised plaques were immediately fixed in formalin and transverse5mmparaffin blocks were prepared from each plaque, from which5 μM serial sections were obtained. Sections were stained withhaematoxylin–eosin and Movat's pentachrome, and further adjacentsections were used for immunohistochemistry (IL-31–IL-34). Each ofthe polyclonal rabbit anti-human primary antibodies (Abcam, Sydney,Australia)was followed bypolymer HRP goat anti-rabbit immunoglob-ulin (Dako, Sydney, Australia). The positive staining was photographedandquantifiedusingImage-Pro®Plus7.Quantitativereal-timePCRwasperformed on cDNA prepared from total RNA isolated from snap frozencarotid plaques(10asymptomatic, 8symptomatic). Gene expression ofIL-31–IL-34wasquantifiedaccordingtothemanufacturer'sinstructions(Qiagen, Chadstone, Australia) using relevant primers. The statisticalanalyses were performed using GraphPad Prism® Version 5.0 by oneway ANOVA. p-Value b 0.05 was considered significant.Asymptomatic and symptomatic groups were well matched interms of baseline patient characteristics (Table 1). IL-31, -32, -33and -34 were detected in plaques from both asymptomatic andsymptomatic patients. Immunostaining for IL-31, -32 and -34 wassignificantly greater in plaques from symptomatic compared withasymptomatic subjects (2.4-, 3.5- and 2.3-fold higher, respectively,p b 0.05 for all). However, IL-33 immunostaining was 1.9-fold greaterin stable versus unstable plaques (p b 0.05) (Fig. 1). Similarly mRNAtranscriptlevelsofIL-31,-32and-34weresignificantlyhigherinunsta-ble v stable plaques (1.5-, 1.8-, 1.9-fold, respectively, p b 0.05 for all)whereasfor IL-33there wasa significant2.1-foldincreaseinmRNAex-pression in stableversus unstableplaques (Fig. 2).When statin-treatedand statin-naive patients were compared, IL-33 immunostaining washigher in treated versus untreated (1.6-fold, p b 0.05), with thesegroups otherwise being well matched in terms of the presence orabsence of symptoms or other cardiovascular risk factors. There was anon-significant increase in IL-33 mRNA levels in statin-treated com-paredwithstatinnaivepatients.Cardiovascularriskfactorswereother-wise not significantly associated with the presence of any of thesecytokines.IL-31, -32, and -34 are potent inflammatory cytokines, whosepresencehasbeendemonstratedininflammatorydiseasessuchascoli-tis and psoriasis. We have previously shown the presence of these

Rapid diagnosis of Naja atra snakebites

Background. The clinical diagnosis of snakebites is critical and necessary in many parts of the world, especially in Southeastern Asia, where venomous snakebites are a burden on public health. It is difficult to define or recognize the species of venomous snake because of the overlapping clinical manifestations of envenomations. A quick and reliable method for identifying the snake species is necessary. We designed and tested a strip of lateral flow system for the diagnosis of cobra snake bites in Taiwan. Methods. We developed a kit based on an immunochromatographic method for rapid detection of cobra (Naja atra) venom in human serum. The test and control lines composed of 1 mg/ml polyclonal duck antivenom and 0.5 mg/ml goat anti-rabbit immunoglobulin antibody solutions, respectively, were coated on nitrocellulose strips. Colloidal gold was conjugated with rabbit polyclonal anti-cobra venom antibodies. From July 2007 to December 2012, we used the kit to test serum from snakebite patients and to examine the agreement between our rapid test and the currently used sandwich enzyme-linked immunosorbent assay (ELISA). Results. Our kit was able to detect cobra venom in serum samples in 20 minutes with a detection limit of 5 ng/ml. An absence of cross-reactivity with other non-cobra venoms from Taiwan was noted in vitro. A total of 88 snakebite patients (34 cobra and 54 other non-cobra) were tested. The sensitivity of the strips based on the ELISA results was 83.3% and the specificity was 100%. There was a strong agreement between the results of the ELISA and immunochromatographic strips (κ = 0.868). Discussion and conclusions. This data indicates that an immunochromatographic strip might be suitable for cobra venom detection and could be used as a quick diagnostic tool in cases of N. atra snakebite.

A Role for Glutathione Transferase Omega 1 (GSTO1-1) in the Glutathionylation Cycle

The glutathionylation of intracellular protein thiols can protect against irreversible oxidation and can act as a redox switch regulating metabolic pathways. In this study we discovered that the Omega class glutathione transferase GSTO1-1 plays a significant role in the glutathionylation cycle. The catalytic activity of GSTO1-1 was determined in vitro by assaying the deglutathionylation of a synthetic peptide by tryptophan fluorescence quenching and in T47-D epithelial breast cancer cells by both immunoblotting and the direct determination of total glutathionylation. Mutating the active site cysteine residue (Cys-32) ablated the deglutathionylating activity of GSTO1-1. Furthermore, we demonstrate that the expression of GSTO1-1 in T47-D cells that are devoid of endogenous GSTO1-1 resulted in a 50% reduction in total glutathionylation levels. Mass spectrometry and immunoprecipitation identified β-actin as a protein that is specifically deglutathionylated by GSTO1-1 in T47-D cells. In contrast to the deglutathionylation activity, we also found that GSTO1-1 is associated with the rapid glutathionylation of cellular proteins when the cells are exposed to S-nitrosoglutathione. The common A140D genetic polymorphism in GSTO1 was found to have significant effects on the kinetics of both the deglutathionylation and glutathionylation reactions. Genetic variation in GSTO1-1 has been associated with a range of diseases, and the discovery that a frequent GSTO1-1 polymorphism affects glutathionylation cycle reactions reveals a common mechanism where it can act on multiple proteins and pathways.

Application of bispecific antibody against antigen and hapten for immunodetection and immunopurification

We present a bispecific antibody that recognizes an antigen and a hapten and can be applied to various biological assays, including immunoblotting and immunoprecipitation. In immunoblot analysis of serum, an anti-C5 × anti-cotinine bispecific tandem single-chain variable fragment (scFv)-Fc fusion protein and cotinine-conjugated horseradish peroxidase (HRP) generated a clean signal without the high background that was observed in a parallel experiment using HRP-conjugated goat anti-rabbit immunoglobulin G (Fc-specific) antibody. In immunoprecipitation analysis of serum, use of the bispecific tandem scFv-Fc fusion protein and cotinine-crosslinked magnetic beads significantly reduced the amount of protein contaminants compared with a parallel experiment done with protein A agarose beads. In subsequent immunoblot analysis, use of cotinine–HRP as the secondary probe instead of HRP-conjugated goat anti-rabbit IgG (Fc-specific) antibody successfully eliminated the band corresponding to the bispecific tandem scFv-Fc fusion protein.

Islet Transplantation in a Subarachnoid Cavity Surrounding Olfactory Bulb of Diabetic Rats

Islet Transplantation in a Subarachnoid Cavity Surrounding Olfactory Bulb of Diabetic Rats

Dual Effects of Isoflurane on Proliferation, Differentiation, and Survival in Human Neuroprogenitor Cells

Previous studies have demonstrated that isoflurane can provide both neuroprotection and neurotoxicity in various tissue culture models and in rodent developing brains. The cellular and molecular mechanisms mediating these dual effects are not clear, but the exposure level and duration of isoflurane appear to be determinant factors. Using the ReNcell CX (Millipore, Billerica, MA) human neural progenitor cell line, the authors investigated the impact of prolonged exposure to varying isoflurane concentrations on cell survival and neurogenesis. In addition, the authors assessed the impact of short isoflurane preconditioning on elevation of cytosolic Ca concentration and cytotoxic effects mediated by prolonged isoflurane exposures and the contribution of inositol-1,4,5-trisphosphate or ryanodine receptor activation to these processes. Short exposures to low isoflurane concentrations promote proliferation and differentiation of ReNcell CX cells, with no cell damage. However, prolonged exposures to high isoflurane concentrations induced significant ReNcell CX cell damage and inhibited cell proliferation. These prolonged exposures suppressed neuronal cell fate and promoted glial cell fate. Preconditioning of ReNcell CX cultures with short exposures to low concentrations of isoflurane ameliorated the effects of prolonged exposures to isoflurane. Pretreatment of ReNcell cultures with inositol-1,4,5-trisphosphate or ryanodine receptor antagonists mostly prevented isoflurane-mediated effects on survival, proliferation, and differentiation. Finally, isoflurane-preconditioned cultures showed significantly less isoflurane-evoked changes in calcium concentration. The commonly used general anesthetic isoflurane exerts dual effects on neuronal stem cell survival, proliferation, and differentiation, which may be attributed to differential regulation of calcium release through activation of endoplasmic reticulum localized inositol-1,4,5-trisphosphate and/or ryanodine receptors.

Vibrio cholerae O395 Outer Membrane Vesicles Modulate Intestinal Epithelial Cells in a NOD1 Protein-dependent Manner and Induce Dendritic Cell-mediated Th2/Th17 Cell Responses

Like other Gram-negative pathogens, Vibrio cholerae, the causative agent of the diarrheal disease cholera, secretes outer membrane vesicles (OMVs). OMVs are complex entities composed of a subset of envelope lipid and protein components and play a role in the delivery of effector molecules to host cells. We previously showed that V. cholerae O395 cells secrete OMVs that are internalized by host cells, but their role in pathogenesis has not been well elucidated. In the present study, we evaluated the interaction of OMVs with intestinal epithelial cells. These vesicles induced expression of proinflammatory cytokines such as IL-8 and GM-CSF and chemokines such as CCL2, CCL20, and thymic stromal lymphopoietin in epithelial cells through activation of MAPK and NF-κB pathways in NOD1-dependent manner. Epithelial cells stimulated with OMVs activated dendritic cells (DCs) in a direct co-culture system. Activated DCs expressed high levels of co-stimulatory molecules; released inflammatory cytokines IL-1β, IL-6, TNF-α, and IL-23 and chemokines CCL22 and CCL17; and subsequently primed CD4(+) T cells leading to IL-4, IL-13, and IL-17 expression. These results suggest that V. cholerae O395 OMVs modulate the epithelial proinflammatory response and activate DCs, which promote T cell polarization toward an inflammatory Th2/Th17 response.