Blocking Reagent Research Articles

ESTIMATION OF ANTIBODIES AGAINST PASTEURELLA MULTOCIDA B:2 IN SERUM EMPLOYING ELISA: COMPARISON OF DIFFERENT BLOCKING REAGENTS

Haemorrhagic Septicaemia (HS), an endemic bacterial disease, is caused by specific serotypes of Pasteurella multocida and is one of the major economic diseases of bovines in south-east Asia including India. Sero-monitoring of vaccinal antibodies and definitive diagnosis of the disease is of utmost importance to combat the economic losses and ELISA system has remained the test of preference due to its versatility. Skimmed milk powder (SMP) and bovine serum albumin (BSA) respectively in concentration of 2, 3, 4% and 0.5, 1.0% have been compared as blocking reagents in ELISA for detection of antibodies against P. multocida . SMP and BSA blocking produced almost equal sensitivity. Results of the ELISA indicate that being inexpensive and easily available, SMP is a suitable blocking reagent for detection of antibodies against P. multocida B:2 for herd screening as well as in laboratories.

Optimized Blocking Of Porous Nitrocellulose Films For Sensitive Protein Microarrays

Super G Blocking Buffer was developed to produce low backgrounds in assays using nitrocellulose film-slides. Using this blocking reagent in combination with ONCYTE® SuperNOVA Film-Slides, we achieved an order of magnitude greater sensitivity in a cytokine microarray assay than with hydrogel- or silane-based glass substrates. Limit of detection (LOD) ranged from 160 – 690 fM for IL1α, IL1β, IL6, TNFα, TNFβ, and IFNγ without use of enzymatic signal amplification. The dynamic range for these microarrays on Film-Slides was greater than that of hydrogels and functionalized glass substrates and the LOD was 50-fold lower. On Film-Slides, the linear dynamic range spanned 6 orders of magnitude and the LOD was pushed into the zmole range (62.5 zmol). The advantages of Film-Slides are discussed in the larger context of developing the most sensitive protein microarrays for multiplexed clinical assays and quantitative studies of the cellular proteome.

Regulation of integrin αV subunit expression by sulfatide in hepatocellular carcinoma cells

Integrin is important in migration and metastasis of tumor cells. Changes of integrin expression and distribution will cause an alteration of cellular adhesion and migration behaviors. In this study, we investigated sulfatide regulation of the integrin αV subunit expression in hepatoma cells and observed that either exogenous or endogenous sulfatide elicited a robust upregulation of integrin αV subunit mRNA and protein expression in hepatoma cells. This regulatory effect occurred with a corresponding phosphorylation (T739) of the transcription factor Sp1. Based on the electrophoretic mobility shift assay, sulfatide enhanced the integrin αV promoter activity and strengthened the Sp1 complex super-shift. The results of chromatin immunoprecipitation analysis also indicated that sulfatide enhanced Sp1 binding to the integrin αV promoter in vivo. Silence of Sp1 diminished the stimulation of integrin αV expression by sulfatide. In the early stage of sulfatide stimulation, phosphorylation of Erk as well as c-Src was noted, and inhibition of Erk activation with either U0126 or PD98059 significantly suppressed Sp1 phosphorylation and integrin αV expression. We demonstrated that sulfatide regulated integrin αV expression and cell adhesion, which was associated with Erk activation.

An electrochemical aptasensor for thrombin detection based on direct electrochemistry of glucose oxidase using a functionalized graphene hybrid for amplification

In this work, we reported a new label-free electrochemical aptasensor for highly sensitive detection of thrombin using direct electron transfer of glucose oxidase (GOD) as a redox probe and a gold nanoparticle-polyaniline-graphene (Au-PANI-Gra) hybrid for amplification. The Au-PANI-Gra hybrid with large surface area provided a biocompatible sensing platform for the immobilization of GOD. GOD was encapsulated into the three-dimensional netlike (3-mercaptopropyl)trimethoxysilane (MPTS) to form the MPTS-GOD biocomposite, which not only retained the native functions and properties, but also exhibited tunable porosity, high thermal stability, and chemical inertness. With abundant thiol tail groups on MPTS, MPTS-GOD was able to chemisorb onto the surface of the Au-PANI-Gra modified electrode through the strong affinity of the Au-S bond. The electrochemical signal originated from GOD, avoiding the addition or labeling of other redox mediators. After immobilizing the thiolated thrombin binding aptamer through gold nanoparticles (AuNPs), GOD as a blocking reagent was employed to block the remaining active sites of the AuNPs and avoid the nonspecific adsorption. The proposed method avoided the labeling process of redox probes and increased the amount of electroactive GOD. The concentration of thrombin was monitored based on the decrease of current response through cyclic voltammetry (CV) in 0.1 M PBS (pH 7.4). With the excellent direct electron transfer of double layer GOD membranes, the resulting aptasensor exhibited high sensitivity for detection of thrombin with a wide linear range from 1.0 × 10(-12) to 3.0 × 10(-8) M. The proposed aptasensor also showed good stability, satisfactory reproducibility and high specificity, which provided a promising strategy for electrochemical aptamer-based detection of other biomolecules.

Plasmon enhanced fluoro-immunoassay using egg yolk antibodies for ultra-sensitive detection of herbicide diuron

Plasmon enhanced fluorescence immunoassay (PEFI) format has been reported in developing a sensitive heterogeneous fluoroimmunoassay for monitoring the phenylurea herbicide diuron. Computer-assisted molecular modeling was carried out to study the conformational and electrostatic effects of synthesized hapten for producing highly specific egg yolk antibody against a phenyl urea herbicide diuron. The generated antibodies were labeled with fluorescein isothiocyanate at different molar ratios and used as tracer in the developed fluorescence based immunoassay. The sensitivity of the assay format was enhanced by using silver nanoparticles tagged with bovine serum albumin as a new blocking reagent in the developed PEFI format. Enhancer treatment on the developed immunoassay showed a significant improvement of fluorescence signal intensity with approximately 10 fold increase in assay sensitivity. The immunoassay has a detection limit of 0.01 ng mL(-1) with good signal precision (~2%) in the optimum working concentration range between 1 pg mL(-1) to 10 μg mL(-1) of diuron. These findings facilitate high throughput fluorescence-based processes that could be useful in biology, drug discovery and compound screening applications.

Novel cutinase from Pseudomonas cepacia NRRL B 2320: Purification, characterization and identification of cutinase encoding genes

An extracellular cutinase from Pseudomonas cepacia NRRL B 2320 was purified to apparent homogeneity. Upon biochemical characterization, the purified cutinase was found to be tolerant to organic solvents and surfactants under assay conditions. The molecular mass of cutinase was found to be 26.25 kDa by MALDI-TOF-MS analysis. The enzyme was able to show activity towards synthetic esters of chain length C4-C16. The activity of cutinase was enhanced by mono cations and various effectors, whereas it was moderately inhibited by various divalent cations and serine blocking reagent, phenyl methyl sulphonyl fluoride (PMSF). The optimal pH and temperature for highest activity were found to be 7.9 and 36.5°C, respectively. An overall 1.42-fold increase in activity was observed after optimization of both assay and process conditions. The exposure of hydrophobic amino acid to an aqueous environment and change in secondary structure of cutinase was observed from thermodynamic parameters (ΔH(*), ΔS(*)), fluorescence and circular dichorism spectra during the deactivation process. Two cutinase encoding genes were identified in P. cepacia, cloned and expressed in E. coli BL21 (DE3).

Immunosuppression in inflammatory melanoma: can it be resisted by adoptively transferred T cells?

Discovery of tumor antigen (TA) recognized by autologous T cells (TCs) in patients with melanoma has led to clinical protocols using either vaccination or adoptive transfer of TA-specific TCs. However, efficacy of these treatments has been hampered by inhibitory effects exerted on tumor-infiltrating TCs by tumor-intrinsic mediators or by recruitment of immunosuppressive cells. A mouse model of autochthonous melanoma recapitulates some aspects of inflammatory melanoma development in patients. These include a systemic Th2-/Th17-oriented chronic inflammation, recruitment of immunosuppressive myeloid cells and acquisition by tumor-infiltrating TCs of an 'exhausted' phenotype characterized by expression of multiple inhibitory receptors including programmed death-1, also expressed on patients' melanoma-infiltrating TCs. Rather than using extracellular blocking reagents to inhibitory surface molecules on TCs, we sought to dampen negative signaling exerted on them. Adoptively transferred TCs presenting increased cytokine receptor signaling due to expression of an active Stat5 transcription factor were efficient at inducing melanoma regression in the preclinical melanoma model. These transferred TCs thrived and retained expression of effector molecules in the melanoma microenvironment, defining a protocol endowing TCs with the ability to resist melanoma-induced immunosuppression.

A dual amplified electrochemical immunosensor for ofloxacin: Polypyrrole film-Au nanocluster as the matrix and multi-enzyme-antibody functionalized gold nanorod as the label

In this work, an electrochemical immunosensor, basing on a dual signal amplified strategy by employing a biocompatible polypyrrole film-Au nanocluster matrix as a sensor platform and multi-enzyme-antibody functionalized gold nanorod as an electrochemical detection label, is established for sensitive detection of ofloxacin (OFL). Firstly, polypyrrole film and Au nanoclusters were progressively fabricated onto the surface of a glassy carbon electrode via electropolymerization and electrochemical deposition, respectively. Such PPy-Au nanocomposite modified electrode was used to immobilize OFL-OVA, blocked with the blocking reagent, and then associated with the corresponding antibody. Secondly, gold nanorod (GNR) was synthesized to load horseradish peroxidase (HRP) and horseradish peroxidase-secondary antibody (HRP-Ab2), and the resulting nanostructure (multi-HRP-GNR-Ab2) was applied as the detection label. The fabrication process of the ordered multilayer structure and immunosensor were characterized by scanning electron microscopy (SEM) and electrochemical measurements, respectively. Finally, based on a competitive immunoassay, i.e., the association ability with the corresponding antibody between the captured antigen and free OFL in the solution, the fabricated immunosensor exhibited a sensitive response to OFL in the range from 0.08 to 410ng/mL with a detection limit of 0.03ng/mL. The current immunosensor exhibited good sensitivity, selectivity and long-term stability. This amplification strategy shows excellent promise for food safety monitoring of other antibiotics and a potential application in other immunosensors.

An aptamer-based biosensing platform for highly sensitive detection of platelet-derived growth factor via enzyme-mediated direct electrochemistry

In this work, a new label-free electrochemical aptamer-based sensor (aptasensor) was constructed for detection of platelet-derived growth factor (PDGF) based on the direct electrochemistry of glucose oxidase (GOD). For this proposed aptasensor, poly(diallyldimethylammonium chloride) (PDDA)-protected graphene-gold nanoparticles (P-Gra-GNPs) composite was firstly coated on electrode surface to form the interface with biocompatibility and huge surface area for the adsorption of GOD layer. Subsequently, gold nanoclusters (GNCs) were deposited on the surface of GOD to capture PDGF binding aptamer (PBA). Finally, GOD as a blocking reagent was employed to block the remaining active sites of the GNCs and avoid the nonspecific adsorption. With the direct electron transfer of double layer GOD membranes, the aptasensor showed excellent electrochemical response and the peak current decreased linearly with increasing logarithm of PDGF concentration from 0.005nM to 60nM with a relatively low limit of detection of 1.7pM. The proposed aptasensor exhibited high specificity, good reproducibility and long-term stability, which provided a new promising technique for aptamer-based protein detection.

Targeting the Cellular and Molecular Components of CD28 Mediated Survival Signaling in Multiple Myeloma

Abstract 722Very little is known about the cellular or molecular nature of pro-survival interactions between multiple myeloma (MM) and their bone marrow stromal hosts. CD28, the prototypical co-stimulatory molecule on T cells is overexpressed on MM relative to their normal counterparts. Clinical studies have independently shown that expression of CD28 or its ligand CD86 on myeloma cells portends poor survival in patients. We and others have shown that CD28 activation increases MM survival via NFkB accompanied by increased PI3K activity. Previously (ASH2010 and 2011), and since published, we have shown that myeloid dendritic cells (DC) form a vital part of the myeloma microenvironment, and myeloma cells interact with DC via a CD28-CD80/CD86 (B7) interaction. On the myeloma side, this interaction drives a CD28 mediated pro-survival signal, while on the DC side, it drives a B7 mediated induction of growth and immunosuppressive factors that modulate the MM survival niches in the bone marrow. In this work, we delineated key molecules downstream of CD28 that transduce the pro survival signal on the myeloma side as well as explored agents and strategies to target DC that help MM survive in the microenvironment. We also studied the role of DC IL-6 for MM survival in cocultures.We have previously shown that CD28 activation by anti-CD28 mAb involves activation of the multifunctional pro-survival transcription factor NFkB accompanied by increased PI3K activity. Through immunoassays and radioactivity assays, using myeloma cell lines that were transfected with wild type murine CD28 (mCD28 differs only by 3 amino acids from human hCD28) and mutants for PI3K binding site YMNM, we show that the CD28 ligation with anti-mCD28 antibodies induces immediate tyrosine phosphorylation of mCD28 (just as observed by others in T cells), followed by binding of p85, the catalytic subunit of PI3K and activation of VAV1 (Fig 1). Association of p85 and VAV1 activation depended on an intact PI3K binding site on CD28's cytoplasmic tail. Our studies on how this signal is transduced show no involvement of GRB2 or SLP76 more traditionally associated with CD28 signaling. Moreover, silencing SLP76 using siRNA did not affect antibody mediated protection of MM cells against serum starvation. Activation of human CD28 with anti-CD28 mAb also up-regulated the basal tyrosine phosphorylation of human CD28 further just as observed in murine CD28.Through EMSAs, we also show that similar to what we have previously seen with activating antibodies, increase in NFkB activity was observed in myeloma cells when they were cocultured with DCs while being exposed to melphalan, but this spike did not dissipate in the presence of B7 blocking reagents such as CTLA4Ig (Abatacept®) suggesting a low threshold of CD28 activation. Next we tried using agents that have been known to modulate microenvironment such as lenalidomide, a novel drug that is currently used in combination with other chemotherapeutic agents such as dexamethasone and Bortezomib (Velcade®). Survival assays using flow cytometry, show that lenalidomide treated DCs provided significantly lower protection against cell death compared to wild type DC. Lenalidomide DCs also show lower levels of B7 receptors which might be a contributing factor to this phenomenon. Blocking CD28-B7 interaction with CTLA4Ig or CD28(Fab) molecules reduced the pro-survival effect of DC on MM against dexamethasone (Fig 2) melphalan or bortezomib. In order to determine the role of IL-6 in an environment where myeloma cells derive pro-survival signals from their interaction with DC, we used neutralizing antibodies against human IL-6 in cocultures of MM cells with DC. Surprisingly, IL-6 neutralization did not affect the protective effect of DC on MM in cocultures in the presence of chemotherapeutic drugs like melphalan or dexamethasone (Fig 2).All these suggest that targeting DC might be a viable strategy towards augmenting chemotherapy, either by using agents that attenuate DC differentiation or agents that can block the CD28-B7 interaction between DC and MM. Moreover, MM cells do not seem to depend much on IL-6 as a survival factor when they are in contact with DC and its pro-survival effect is more visible when myeloma cells have to survive outside the microenvironment without DC (previous presentation at ASH 2011). [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Molecular Biology Techniques Q&A

Molecular Biology Techniques Q&A

Development of Polyclonal Antibody‐Based Indirect Enzyme‐Linked Immunosorbent Assay for the Detection of Alicyclobacillus Strains in Apple Juice

A sort of specific polyclonal anti-Alicyclobacillus antibody was generated by immunizing New Zealand white rabbits, and a sensitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for Alicyclobacillus detection in apple juice. A set of experimental parameters such as concentration of antigen, dilutions of the antibody and goat anti-rabbit IgG-horseradish peroxidase conjugate, selection of the blocking reagent, incubation time, and temperature was optimized. The cross-reactivity of the antibody was evaluated by ELISA and the result was consistent with Western blot analysis. The detection limit of the ELISA was about 10(5) colony forming units (CFU)/mL in apple juice samples. Samples were detected by ELISA and conventional culture method, and the ELISA results gave a good agreement with the results obtained by plating on Alicyclobacillus acidoterrestris medium agar. ELISA takes a total detection time of 6 to 7 h, which is less than the time of conventional techniques requiring more than 24 to 48 h. These results indicated that the established ELISA was a potential useful analytical method for detection of Alicyclobacillus in apple juice.

Chondroitin Sulfate Is a Crucial Determinant for Skeletal Muscle Development/Regeneration and Improvement of Muscular Dystrophies

Skeletal muscle formation and regeneration require myoblast fusion to form multinucleated myotubes or myofibers, yet their molecular regulation remains incompletely understood. We show here that the levels of extra- and/or pericellular chondroitin sulfate (CS) chains in differentiating C2C12 myoblast culture are dramatically diminished at the stage of extensive syncytial myotube formation. Forced down-regulation of CS, but not of hyaluronan, levels enhanced myogenic differentiation in vitro. This characteristic CS reduction seems to occur through a cell-autonomous mechanism that involves HYAL1, a known catabolic enzyme for hyaluronan and CS. In vivo injection of a bacterial CS-degrading enzyme boosted myofiber regeneration in a mouse cardiotoxin-induced injury model and ameliorated dystrophic pathology in mdx muscles. Our data suggest that the control of CS abundance is a promising new therapeutic approach for the treatment of skeletal muscle injury and progressive muscular dystrophies.

Flow Karyotyping of Wheat Addition Line “T240” with a Haynaldia villosa 6VS Telosome

To construct a chromosome-specific DNA library of chromosome 6VS from Haynaldia villosa, a wheat alien chromosome addition line T240 with a 6VS chromosome arm and its parental common wheat cv. CA921 were used to optimize protocols for preparing chromosome suspension amenable to flow sorting of the 6VS chromosome. Our results showed that root tips incubated sequentially in Hogland’s solution containing 1.25 mM hydroxyurea (DNA synthesis inhibitor) for 18 h, a hydroxyurea-free period for 2 h, and 1 μM trifluralin for 4 h (metaphase blocking reagent) increased the metaphase index (MI) by up to 62 % . Many metaphase chromosomes were released to form a chromosome suspension when root tips fixed in 2 % paraformaldehyde were treated in a homogenizer at 9,500 rpm for 10–15 s. Most of the released chromosomes had intact morphology. The background solution of chromosome suspension was clear and relatively few of cell debris and chromosome clumps. Univariate flow karyotypes were established with chromosome suspension flow through FACS Vantage 2000 SE flow cytometer. The flow karyotype of CA921 consisted of four chromosome peaks, whereas that for T240 had a fifth peak. This fifth peak in T240 contained the telosome, which was further confirmed to be 6VS through fluorescence in situ hybridization.

Structure of a β-glucan from Grifola frondosa and its antitumor effect by activating Dectin-1/Syk/NF-κB signaling

A soluble homogeneous β-glucan, GFPBW1, with a molecular mass of 300 kDa was purified from the fraction of the fruit bodies of Grifola frondosa extracted with 5% NaOH. Using various methods, such as infrared spectroscopy, NMR, methylation and monosaccharide composition analysis, its structure was determined to be a β-D-(1-3)-linked glucan backbone with a single β-D-(1-6)-linked glucopyranosyl residue branched at C-6 on every third residue. It induced TNF-α and IL-6 production and the activation of Syk and NF-κB signaling in resident peritoneal macrophages from ICR mice, which could be significantly inhibited by the blocking reagent laminarin. A competitive phagocytosis assay with FITC-zymosan indicated that GFPBW1 could bind to DC-associated C-type lectin 1 (Dectin-1). The TNF-α secretion and activation of Syk/NF-κB signaling triggered by GFPBW1 were enhanced in RAW264.7 cells overexpressing wild but not mutant (Δ38 and Y15S) Dectin-1. Furthermore, GFPBW1 potentiated the Concanavalin A-induced proliferative response of splenocytes and inhibited Sarcoma-180 growth allografted in ICR mice but not in immunodeficient BALB/c nu/nu mice. These results suggested that the antitumor activity of GFPBW1 was partially associated with the activation of macrophages via the Dectin-1/Syk/NF-κB signaling pathway. This molecule could be a promising biological response modifier with clear application for antitumor therapies.

Methylsulfonyl Benzothiazole (MSBT): A Selective Protein Thiol Blocking Reagent

A new thiol blocking reagent, methylsulfonyl benzothiazole, was discovered. This reagent showed good selectivity and high reactivity for protein thiols.

Covalent Protein Modification with ISG15 via a Conserved Cysteine in the Hinge Region

The ubiquitin-like protein ISG15 (interferon-stimulated gene of 15 kDa) is strongly induced by type I interferons and displays antiviral activity. As other ubiquitin-like proteins (Ubls), ISG15 is post-translationally conjugated to substrate proteins by an isopeptide bond between the C-terminal glycine of ISG15 and the side chains of lysine residues in the substrates (ISGylation). ISG15 consists of two ubiquitin-like domains that are separated by a hinge region. In many orthologs, this region contains a single highly reactive cysteine residue. Several hundred potential substrates for ISGylation have been identified but only a few of them have been rigorously verified. In order to investigate the modification of several ISG15 substrates, we have purified ISG15 conjugates from cell extracts by metal-chelate affinity purification and immunoprecipitations. We found that the levels of proteins modified by human ISG15 can be decreased by the addition of reducing agents. With the help of thiol blocking reagents, a mutational analysis and miRNA mediated knock-down of ISG15 expression, we revealed that this modification occurs in living cells via a disulphide bridge between the substrates and Cys78 in the hinge region of ISG15. While the ISG15 activating enzyme UBE1L is conjugated by ISG15 in the classical way, we show that the ubiquitin conjugating enzyme Ubc13 can either be classically conjugated by ISG15 or can form a disulphide bridge with ISG15 at the active site cysteine 87. The latter modification would interfere with its function as ubiquitin conjugating enzyme. However, we found no evidence for an ISG15 modification of the dynamin-like GTPases MxA and hGBP1. These findings indicate that the analysis of potential substrates for ISG15 conjugation must be performed with great care to distinguish between the two types of modification since many assays such as immunoprecipitation or metal-chelate affinity purification are performed with little or no reducing agent present.

Restoration of T cell tolerance in primary ITP

Primary immune thrombocytopenia (ITP) has been traditionally thought as an antibody-mediated autoimmune disease involving platelet destruction by macrophages in the reticuloendothelia system. More recently it has become obvious that ITP is a more complex disorder in which T cell mediated immunity plays important roles in platelet destruction. Antiplatelet autoantibody production is under the control of platelet-specific helper T-cells, and loss of tolerance to self antigen by T cells is the critical step of the immune dysregulation in ITP. Dendritic cells (DCs) from ITP patients showed enhanced capacity in stimulating autologous T-cell proliferation in the presence of autologous/allogeneic platelets [1], and ITP patients’ T cells had elevated IL-2 secretion ability compared with controls [2,3], suggesting increased antiplaltelet T-cell reactivity in ITP. The epitopes that recognize platelet glycoprotein (GP) IIIa on T helper (Th) cells has been determined and mapped by several groups [4,5], thus sheding new lights on the “therapeutic vaccination” approach to reinstate tolerance in ITP. Autoreactive T-cell reactivity against platelet antigen in active ITP patients has been observed at polyclonal as well as oligoclonal levels [6,7]. Our group has demonstrated that blocking the B7-CD28 interaction with CTLA4-Ig/CsA could induce platelet GP-specific T-cell anergy, which could exert suppressive effect on GP-reactive T cells via inducing tolerogenic dendritic cells (DCs) [8,9]. It has been well established that apoptotic genes, such as Fas, A20, Bax, Calpastatin, IL2RB, were expressed aberrantly in patients with active ITP [10,11], leading to autoreactive T cells resistant to activation induced cell death (AICD), which could in turn support the expansion of self-reactive T-cell clones. A loss of resistant to AICD might be an important mechanism for the achievement of remission in ITP. Previous studies have revealed that dexamethasone could suppress T-cell proliferation and induce apoptosis of T-cells in ITP [11,12]. In addition, our group has demonstrated that a novel BAFF blocking reagent, BR3-Fc, could restore the apoptosis of both B and T cells [13]. Th polarization in ITP has been attributed to increased Th1 [2,14], and Th17 cells [15] or reduced number or function of CD4+CD25+Foxp3+ T-regulatory cells (Tregs) [16,17]. A parallel body of aberrant cytokine patterns, such as the elevated ratio of interleukin (IL) -18/IL-18 binding protein (BP) [18,19], the increased expression of B cell activating factor (BAFF) has been reported in active ITP patients [20,21]. High-dose dexamthasone (HD-DXM) could not only restore Th1/Th2 [14] or IL-18/IL-18BP balance [19], but also increase the number of Tregs [16], and inhibit the expression of BAFF [12]. Besides HD-DXM, multiple agents, such as rituximab [22], intravenous immunoglobulin (IVIg) [23], romiplostim, eltrombopag [24] as well as indirubin [25], could increase the number or restore the function of Tregs in ITP. Our recently study showed that GP-specific induced Tregs could be successfully generated de novo from nonregulatory CD4+CD25-CD45RA+ cells and could mediate both antigen-specific and linked suppression of proliferating antiplatelet CD4+ Th cells in vitro, and further research revealed that the de novo expanded Tregs mediated their suppressive effects on T cells via actually modulating the T-cell stimulatory capacity of DCs [26], thus providing a clue to the potential of producing antigen-specific Tregs from the patients in vitro for the purpose of antigen-targeted cellular immunotherapy. In conclusion, induction of T-cell tolerance may provide a useful strategy for the management of ITP.

Biosynthetic sorting of the sodium pump: Visualization of the segregation of newly synthesized epithelial Na,K‐ATPase from apically directed proteins

Regulation of transport protein function involves many mechanisms, including endocytosis, recycling and biosynthetic sorting. The Na,K‐ATPase generates the ion gradients that drive most transepithelial transport. In almost every polarized epithelial cell type, the Na,K‐ATPase is restricted to the basolateral domain of the plasma membrane. To study the regulation of Na,K‐ATPase trafficking and its separation from apical cargo, we have created a stable MDCK cell line expressing a SNAP‐tagged version of Na,K‐ATPase and a CLIP‐tagged version of gp135, a glycoprotein that traffics to the apical membrane. The SNAP and CLIP tags are modified forms of a DNA repair protein that react with benzylguanine (SNAP) and benzylcytosine (CLIP) substrates, resulting in covalent linkage of the benzyl group to an active thiol residue within the tag. Using a block/pulse method and SNAP/CLIP‐specific blocking reagents, newly synthesized cohorts of SNAP‐ and CLIP‐tagged protein can be visualized by confocal and electron microscopy, or subjected to biochemical analysis to reveal time‐dependent protein‐protein interactions. Our data suggest that Na,K‐ATPase and gp135 pursue the same post‐synthetic trafficking pathway through the trans‐Golgi network, after which they appear to segregate from one another. Kinetic analysis reveals that Na,K‐ATPase traffics through the biosynthetic pathway more rapidly than the apical gp135.

On the role of Cys150 in the mechanism of maleamate amidohydrolase (NicF)

The oxidative degradation of nicotinate (vitamin B3) to fumarate is considered a model system for the bacterial degradation of N‐heterocyclic compounds, yet only recently has the complete set of genes corresponding to the enzymes involved in this pathway been identified. The identification of these six genes has enabled structural and functional analyses of the enzymes of this catabolic pathway to be undertaken. Maleamate amidohydrolase (NicF), catalyzes the penultimate reaction of the pathway ‐ the hydrolytic deamination of maleamate to maleate. The crystal structure of NicF, determined at 2.4 Å using molecular replacement, shows that the enzyme belongs to the cysteine hydrolase superfamily. The crystal structure offers insight into the roles of potential catalytically important residues, notably a conserved triad (Asp29, Lys117, Cys150) hypothesized to catalyze an addition‐elimination mechanism. In an effort to examine the functional importance of Cys150 in the mechanism iodoacetamide was discovered to be a potent inactivator. To test whether inhibition of NicF by sulfhydryl blocking reagents is reversible a study with methyl methanethiolsulfonate (MMTS) will be conducted. In addition, results of the kinetic analyses of NicF variants, Cys150Ser/Ala and Asp29Asn/Ala, are reported. This work was supported by a HHMI Undergraduate Science Education Program Award to the College of Wooster.