Structural flexibility at a major conserved antibody target on hepatitis C virus E2 antigen.

PubMed

Kong, Leopold; Lee, David E; Kadam, Rameshwar U; Liu, Tong; Giang, Erick; Nieusma, Travis; Garces, Fernando; Tzarum, Netanel; Woods, Virgil L; Ward, Andrew B; Li, Sheng; Wilson, Ian A; Law, Mansun

2016-10-24

Hepatitis C virus (HCV) is a major cause of liver disease, affecting over 2% of the world's population. The HCV envelope glycoproteins E1 and E2 mediate viral entry, with E2 being the main target of neutralizing antibody responses. Structural investigations of E2 have produced templates for vaccine design, including the conserved CD81 receptor-binding site (CD81bs) that is a key target of broadly neutralizing antibodies (bNAbs). Unfortunately, immunization with recombinant E2 and E1E2 rarely elicits sufficient levels of bNAbs for protection. To understand the challenges for eliciting bNAb responses against the CD81bs, we investigated the E2 CD81bs by electron microscopy (EM), hydrogen-deuterium exchange (HDX), molecular dynamics (MD), and calorimetry. By EM, we observed that HCV1, a bNAb recognizing the N-terminal region of the CD81bs, bound a soluble E2 core construct from multiple angles of approach, suggesting components of the CD81bs are flexible. HDX of multiple E2 constructs consistently indicated the entire CD81bs was flexible relative to the rest of the E2 protein, which was further confirmed by MD simulations. However, E2 has a high melting temperature of 84.8 °C, which is more akin to proteins from thermophilic organisms. Thus, recombinant E2 is a highly stable protein overall, but with an exceptionally flexible CD81bs. Such flexibility may promote induction of nonneutralizing antibodies over bNAbs to E2 CD81bs, underscoring the necessity of rigidifying this antigenic region as a target for rational vaccine design.

Antigenic and 3D structural characterization of soluble X4 and hybrid X4-R5 HIV-1 Env trimers

PubMed Central

2014-01-01

Background HIV-1 is decorated with trimeric glycoprotein spikes that enable infection by engaging CD4 and a chemokine coreceptor, either CCR5 or CXCR4. The variable loop 3 (V3) of the HIV-1 envelope protein (Env) is the main determinant for coreceptor usage. The predominant CCR5 using (R5) HIV-1 Env has been intensively studied in function and structure, whereas the trimeric architecture of the less frequent, but more cytopathic CXCR4 using (X4) HIV-1 Env is largely unknown, as are the consequences of sequence changes in and near V3 on antigenicity and trimeric Env structure. Results Soluble trimeric gp140 Env constructs were used as immunogenic mimics of the native spikes to analyze their antigenic properties in the context of their overall 3D structure. We generated soluble, uncleaved, gp140 trimers from a prototypic T-cell line-adapted (TCLA) X4 HIV-1 strain (NL4-3) and a hybrid (NL4-3/ADA), in which the V3 spanning region was substituted with that from the primary R5 isolate ADA. Compared to an ADA (R5) gp140, the NL4-3 (X4) construct revealed an overall higher antibody accessibility, which was most pronounced for the CD4 binding site (CD4bs), but also observed for mAbs against CD4 induced (CD4i) epitopes and gp41 mAbs. V3 mAbs showed significant binding differences to the three constructs, which were refined by SPR analysis. Of interest, the NL4-3/ADA construct with the hybrid NL4-3/ADA CD4bs showed impaired CD4 and CD4bs mAb reactivity despite the presence of the essential elements of the CD4bs epitope. We obtained 3D reconstructions of the NL4-3 and the NL4-3/ADA gp140 trimers via electron microscopy and single particle analysis, which indicates that both constructs inherit a propeller-like architecture. The first 3D reconstruction of an Env construct from an X4 TCLA HIV-1 strain reveals an open conformation, in contrast to recently published more closed structures from R5 Env. Exchanging the X4 V3 spanning region for that of R5 ADA did not alter the open Env architecture as deduced from its very similar 3D reconstruction. Conclusions 3D EM analysis showed an apparent open trimer configuration of X4 NL4-3 gp140 that is not modified by exchanging the V3 spanning region for R5 ADA. PMID:24884925

Site specific replacements of a single loop nucleoside with a dibenzyl linker may switch the activity of TBA from anticoagulant to antiproliferative.

PubMed

Scuotto, Maria; Rivieccio, Elisa; Varone, Alessia; Corda, Daniela; Bucci, Mariarosaria; Vellecco, Valentina; Cirino, Giuseppe; Virgilio, Antonella; Esposito, Veronica; Galeone, Aldo; Borbone, Nicola; Varra, Michela; Mayol, Luciano

2015-09-18

Many antiproliferative G-quadruplexes (G4s) arise from the folding of GT-rich strands. Among these, the Thrombin Binding Aptamer (TBA), as a rare example, adopts a monomolecular well-defined G4 structure. Nevertheless, the potential anticancer properties of TBA are severely hampered by its anticoagulant action and, consequently, no related studies have appeared so far in the literature. We wish to report here that suitable chemical modifications in the TBA sequence can preserve its antiproliferative over anticoagulant activity. Particularly, we replaced one residue of the TT or TGT loops with a dibenzyl linker to develop seven new quadruplex-forming TBA based sequences (TBA-bs), which were studied for their structural (CD, CD melting, 1D NMR) and biological (fibrinogen, PT and MTT assays) properties. The three-dimensional structures of the TBA-bs modified at T13 (TBA-bs13) or T12 (TBA-bs12), the former endowed with selective antiproliferative activity, and the latter acting as potently as TBA in both coagulation and MTT assays, were further studied by 2D NMR restrained molecular mechanics. The comparative structural analyses indicated that neither the stability, nor the topology of the G4s, but the different localization of the two benzene rings of the linker was responsible for the loss of the antithrombin activity for TBA-bs13. © Crown copyright 2015.

Site specific replacements of a single loop nucleoside with a dibenzyl linker may switch the activity of TBA from anticoagulant to antiproliferative

PubMed Central

Scuotto, Maria; Rivieccio, Elisa; Varone, Alessia; Corda, Daniela; Bucci, Mariarosaria; Vellecco, Valentina; Cirino, Giuseppe; Virgilio, Antonella; Esposito, Veronica; Galeone, Aldo; Borbone, Nicola; Varra, Michela; Mayol, Luciano

2015-01-01

Many antiproliferative G-quadruplexes (G4s) arise from the folding of GT-rich strands. Among these, the Thrombin Binding Aptamer (TBA), as a rare example, adopts a monomolecular well-defined G4 structure. Nevertheless, the potential anticancer properties of TBA are severely hampered by its anticoagulant action and, consequently, no related studies have appeared so far in the literature. We wish to report here that suitable chemical modifications in the TBA sequence can preserve its antiproliferative over anticoagulant activity. Particularly, we replaced one residue of the TT or TGT loops with a dibenzyl linker to develop seven new quadruplex-forming TBA based sequences (TBA-bs), which were studied for their structural (CD, CD melting, 1D NMR) and biological (fibrinogen, PT and MTT assays) properties. The three-dimensional structures of the TBA-bs modified at T13 (TBA-bs13) or T12 (TBA-bs12), the former endowed with selective antiproliferative activity, and the latter acting as potently as TBA in both coagulation and MTT assays, were further studied by 2D NMR restrained molecular mechanics. The comparative structural analyses indicated that neither the stability, nor the topology of the G4s, but the different localization of the two benzene rings of the linker was responsible for the loss of the antithrombin activity for TBA-bs13. PMID:26250112

Improved Therapy by Pretargeted Radioimmunotherapy of Non-Hodgkin Lymphoma with a New Recombinant, Trivalent, Anti-CD20, Bispecific Antibody

PubMed Central

Sharkey, Robert M.; Karacay, Habibe; Litwin, Samuel; Rossi, Edmund A.; McBride, William J.; Chang, Chein-Hsing; Goldenberg, David M.

2008-01-01

We examined whether a pretargeting method using a new, recombinant anti-CD20 bispecific antibody (bsMAb) followed by 90Y-DOTA-peptide could reduce hematological toxicity yet improve therapeutic responses, compared to conventional 90Y-anti-CD20 IgG and a chemically-conjugated bsMAb. TF4, a humanized, tri-Fab, bsMAb with 2 Fabs binding CD20 and 1 Fab binding HSG (histamine-succinyl-glycine), developed by the Dock-and-Lock (DNL) method, was tested in nude mice with Ramos B-cell lymphomas. Optimal pretargeting required a 29-h interval between TF4 and 90Y-DOTA-HSG, and 20-fold more moles of TF4. TF4 cleared more rapidly from the blood than anti-CD20 IgG, with early processing in the liver, spleen and kidneys. At 24 h, TF4 improved tumor uptake of 111In-HSG-peptide 2.6-fold (13% vs 5% injected-dose/g) and enhanced tumor/blood ratios more than 45-fold (770 vs 17), compared to an anti-CD20 Fab x anti-HSG Fab chemical conjugate, and by 1.6-fold (9.0% vs 5.6% injected-dose/g) and 1600-fold (522 vs 0.32), respectively, compared to radiolabeled anti-CD20 IgG. A severe (≥90%) and prolonged reduction of white blood cells was observed at the maximum dose of 90Y-anti-CD20 IgG, whereas pretargeting resulted in a ≤60% transient drop. TF4-pretargeting resulted in highly significant improvement in survival, curing 33-90% of the animals, even at relatively low doses, while most tumors progressed quickly without cures with 90Y-anti-CD20 IgG. These results indicate an improved therapeutic index with pretargeting radioimmunotherapy using a DNL-constructed tri-Fab, bsMAb, as compared to conventional therapy with directly-radiolabeled antibody or with a chemically-conjugated bsMAb. These encouraging results prompt testing of these constructs for pretargeting radioimmunotherapy in patients. PMID:18593929

Antibody specificities associated with neutralization breadth in plasma from human immunodeficiency virus type 1 subtype C-infected blood donors.

PubMed

Gray, Elin S; Taylor, Natasha; Wycuff, Diane; Moore, Penny L; Tomaras, Georgia D; Wibmer, Constantinos Kurt; Puren, Adrian; DeCamp, Allan; Gilbert, Peter B; Wood, Blake; Montefiori, David C; Binley, James M; Shaw, George M; Haynes, Barton F; Mascola, John R; Morris, Lynn

2009-09-01

Defining the specificities of the anti-human immunodeficiency virus type 1 (HIV-1) envelope antibodies able to mediate broad heterologous neutralization will assist in identifying targets for an HIV-1 vaccine. We screened 70 plasmas from chronically HIV-1-infected individuals for neutralization breadth. Of these, 16 (23%) were found to neutralize 80% or more of the viruses tested. Anti-CD4 binding site (CD4bs) antibodies were found in almost all plasmas independent of their neutralization breadth, but they mainly mediated neutralization of the laboratory strain HxB2 with little effect on the primary virus, Du151. Adsorption with Du151 monomeric gp120 reduced neutralizing activity to some extent in most plasma samples when tested against the matched virus, although these antibodies did not always confer cross-neutralization. For one plasma, this activity was mapped to a site overlapping the CD4-induced (CD4i) epitope and CD4bs. Anti-membrane-proximal external region (MPER) (r = 0.69; P < 0.001) and anti-CD4i (r = 0.49; P < 0.001) antibody titers were found to be correlated with the neutralization breadth. These anti-MPER antibodies were not 4E10- or 2F5-like but spanned the 4E10 epitope. Furthermore, we found that anti-cardiolipin antibodies were correlated with the neutralization breadth (r = 0.67; P < 0.001) and anti-MPER antibodies (r = 0.6; P < 0.001). Our study suggests that more than one epitope on the envelope glycoprotein is involved in the cross-reactive neutralization elicited during natural HIV-1 infection, many of which are yet to be determined, and that polyreactive antibodies are possibly involved in this phenomenon.

Antibody Specificities Associated with Neutralization Breadth in Plasma from Human Immunodeficiency Virus Type 1 Subtype C-Infected Blood Donors▿ †

PubMed Central

Gray, Elin S.; Taylor, Natasha; Wycuff, Diane; Moore, Penny L.; Tomaras, Georgia D.; Wibmer, Constantinos Kurt; Puren, Adrian; DeCamp, Allan; Gilbert, Peter B.; Wood, Blake; Montefiori, David C.; Binley, James M.; Shaw, George M.; Haynes, Barton F.; Mascola, John R.; Morris, Lynn

2009-01-01

Defining the specificities of the anti-human immunodeficiency virus type 1 (HIV-1) envelope antibodies able to mediate broad heterologous neutralization will assist in identifying targets for an HIV-1 vaccine. We screened 70 plasmas from chronically HIV-1-infected individuals for neutralization breadth. Of these, 16 (23%) were found to neutralize 80% or more of the viruses tested. Anti-CD4 binding site (CD4bs) antibodies were found in almost all plasmas independent of their neutralization breadth, but they mainly mediated neutralization of the laboratory strain HxB2 with little effect on the primary virus, Du151. Adsorption with Du151 monomeric gp120 reduced neutralizing activity to some extent in most plasma samples when tested against the matched virus, although these antibodies did not always confer cross-neutralization. For one plasma, this activity was mapped to a site overlapping the CD4-induced (CD4i) epitope and CD4bs. Anti-membrane-proximal external region (MPER) (r = 0.69; P < 0.001) and anti-CD4i (r = 0.49; P < 0.001) antibody titers were found to be correlated with the neutralization breadth. These anti-MPER antibodies were not 4E10- or 2F5-like but spanned the 4E10 epitope. Furthermore, we found that anti-cardiolipin antibodies were correlated with the neutralization breadth (r = 0.67; P < 0.001) and anti-MPER antibodies (r = 0.6; P < 0.001). Our study suggests that more than one epitope on the envelope glycoprotein is involved in the cross-reactive neutralization elicited during natural HIV-1 infection, many of which are yet to be determined, and that polyreactive antibodies are possibly involved in this phenomenon. PMID:19553335

Tetravalent anti-CD20/CD3 bispecific antibody for the treatment of B cell lymphoma

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

Lu, Chia-Yen; Chen, Gregory J.; Tai, Pei-Han

Bispecific antibodies (bsAbs) are second generation antibodies for therapeutic application in immunotherapy. One of the major strategies of the bsAb platform is the recruitment of immune effector T cells by incorporating an anti-CD3 domain. A bispecific T-cell engager (BiTE), with one end having an affinity for CD3 and the other end with affinity for CD19, has been approved in the US and Europe for the treatment of acute lymphoblastic leukemia. However, due to their small size and lack of Fc region, these single-chain variable fragment (scFv) bsAbs have short half-lives in vivo. Additionally, poor solubility, structural instability, and low production yieldsmore » have also become major challenges in the bulk production process. To overcome these challenges, we have engineered a tetravalent bsAb with bivalent binding specificity for the CD20 and CD3 antigen in an immunoglobulin G (IgG) format. The fusion of the anti-CD3 scFvs to the CD20 antibody via a linker-hinge domain (LHD) results in improved antibody stabilization and properties. Here we demonstrate this antibody's highly efficient cancer cell elimination in a dose-dependent manner in a CD20-expressing B lymphoblastoid cell line in vitro. Our data suggest the potential clinical application of this bsAb for the treatment of CD20-expressing B cell malignancies. - Highlights: • A bispecific antibody (bsAb) can increase immunotherapeutic efficacy. • A tetravalent bsAb with binding specificity for the CD20 and CD3 antigens is proposed. • A linker-hinge domain (LHD) within the bsAb results in improved antibody properties.« less

Bispecific antibodies and trispecific immunocytokines for targeting the immune system against cancer: preparing for the future.

PubMed

Fournier, Philippe; Schirrmacher, Volker

2013-02-01

Monoclonal anti-tumor antibodies (mAbs) that are clinically effective usually recruit, via their constant fragment (Fc) domain, Fc receptor (FcR)-positive accessory cells of the immune system and engage these additionally against the tumor. Since T cells are FcR negative, these important cells are not getting involved. In contrast to mAbs, bispecific antibodies (bsAbs) can be designed in such a way that they involve T cells. bsAbs are artificially designed molecules that bind simultaneously to two different antigens, one on the tumor cell, the other one on an immune effector cell such as CD3 on T cells. Such dual antibody constructs can cross-link tumor cells and T cells. Many such bsAb molecules at the surface of tumor cells can thus build a bridge to T cells and aggregate their CD3 molecules, thereby activating them for cytotoxic activity. BsAbs can also contain a third binding site, for instance a Fc domain or a cytokine that would bind to its respective cytokine receptor. The present review discusses the pros and cons for the use of the Fc fragment during the development of bsAbs using either cell-fusion or recombinant DNA technologies. The recombinant antibody technology allows the generation of very efficient bsAbs containing no Fc domain such as the bi-specific T-cell engager (BiTE). The strong antitumor activity of these molecules makes them very interesting new cancer therapeutics. Over the last decade, we have developed another concept, namely to combine bsAbs and multivalent immunocytokines with a tumor cell vaccine. The latter are patient-derived tumor cells modified by infection with a virus. The virus-Newcastle Disease Virus (NDV)-introduces, at the surface of the tumor cells, viral molecules that can serve as general anchors for the bsAbs. Our strategy aims at redirecting, in an Fc-independent fashion, activities of T cells and accessory cells against autologous tumor antigens. It creates very promising perspectives for a new generation of efficient and safe cancer therapeutics that should confer long-lasting anti-tumor immunity.

Antigen-Presenting Intratumoral B Cells Affect CD4+ TIL Phenotypes in Non-Small Cell Lung Cancer Patients.

PubMed

Bruno, Tullia C; Ebner, Peggy J; Moore, Brandon L; Squalls, Olivia G; Waugh, Katherine A; Eruslanov, Evgeniy B; Singhal, Sunil; Mitchell, John D; Franklin, Wilbur A; Merrick, Daniel T; McCarter, Martin D; Palmer, Brent E; Kern, Jeffrey A; Slansky, Jill E

2017-10-01

Effective immunotherapy options for patients with non-small cell lung cancer (NSCLC) are becoming increasingly available. The immunotherapy focus has been on tumor-infiltrating T cells (TILs); however, tumor-infiltrating B cells (TIL-Bs) have also been reported to correlate with NSCLC patient survival. The function of TIL-Bs in human cancer has been understudied, with little focus on their role as antigen-presenting cells and their influence on CD4 + TILs. Compared with other immune subsets detected in freshly isolated primary tumors from NSCLC patients, we observed increased numbers of intratumoral B cells relative to B cells from tumor-adjacent tissues. Furthermore, we demonstrated that TIL-Bs can efficiently present antigen to CD4 + TILs and alter the CD4 + TIL phenotype using an in vitro antigen-presentation assay. Specifically, we identified three CD4 + TIL responses to TIL-Bs, which we categorized as activated, antigen-associated, and nonresponsive. Within the activated and antigen-associated CD4 + TIL population, activated TIL-Bs (CD19 + CD20 + CD69 + CD27 + CD21 + ) were associated with an effector T-cell response (IFNγ + CD4 + TILs). Alternatively, exhausted TIL-Bs (CD19 + CD20 + CD69 + CD27 - CD21 - ) were associated with a regulatory T-cell phenotype (FoxP3 + CD4 + TILs). Our results demonstrate a new role for TIL-Bs in NSCLC tumors in their interplay with CD4 + TILs in the tumor microenvironment, establishing them as a potential therapeutic target in NSCLC immunotherapy. Cancer Immunol Res; 5(10); 898-907. ©2017 AACR . ©2017 American Association for Cancer Research.

Human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis C virus.

PubMed

Giang, Erick; Dorner, Marcus; Prentoe, Jannick C; Dreux, Marlène; Evans, Matthew J; Bukh, Jens; Rice, Charles M; Ploss, Alexander; Burton, Dennis R; Law, Mansun

2012-04-17

Hepatitis C virus (HCV) infects ∼2% of the world's population. It is estimated that there are more than 500,000 new infections annually in Egypt, the country with the highest HCV prevalence. An effective vaccine would help control this expanding global health burden. HCV is highly variable, and an effective vaccine should target conserved T- and B-cell epitopes of the virus. Conserved B-cell epitopes overlapping the CD81 receptor-binding site (CD81bs) on the E2 viral envelope glycoprotein have been reported previously and provide promising vaccine targets. In this study, we isolated 73 human mAbs recognizing five distinct antigenic regions on the virus envelope glycoprotein complex E1E2 from an HCV-immune phage-display antibody library by using an exhaustive-panning strategy. Many of these mAbs were broadly neutralizing. In particular, the mAb AR4A, recognizing a discontinuous epitope outside the CD81bs on the E1E2 complex, has an exceptionally broad neutralizing activity toward diverse HCV genotypes and protects against heterologous HCV challenge in a small animal model. The mAb panel will be useful for the design and development of vaccine candidates to elicit broadly neutralizing antibodies to HCV.

Using Tryptophan Mutants To Probe the Structural and Functional Status of BsSCO, a Copper Binding, Cytochrome c Oxidase Assembly Protein from Bacillus subtilis.

PubMed

Hussain, Shina; Andrews, Diann; Hill, Bruce C

2017-12-05

The synthesis of cytochrome c oxidase protein from Bacillus subtilis (i.e., BsSCO) binds copper with picomolar affinity, which increases the protein's melting temperature (i.e., T M ) by 20 °C. Here two native tryptophans (i.e., W36 and W101) are identified as major contributors to BsSCO's structural form, and their contributions to the stability, intrinsic fluorescence, and copper binding properties of BsSCO are explored. Single mutations of tryptophan to phenylalanine decrease the T M by 10 °C and the folding free energy by 3-4 kcal/mol. A more severe change to alanine (i.e., W36A BsSCO) decreases the T M by 20 °C and the stability by 9 kcal/mol. However, these mutants bind copper with high affinity and assemble cytochrome c oxidase in vivo. Replacing phenylalanine at a position near (∼5 Å) the copper binding site with tryptophan (i.e., F42W) increases the T M of apo-BsSCO by 3 °C but diminishes the effect of copper binding. When both native tryptophans are changed to alanine, apo-BsSCO is unfolded in vitro and is not functional in cytochrome c oxidase assembly in vivo. A double-mutant of BsSCO in which W36A is combined with F42W exhibits a form of metastability. Apo-W36A/F42W BsSCO melts at 37 °C, which upon binding of copper shifts to 65 °C. B. subtilis expressing W36A/F42W BsSCO and grown at 37 °C does not assemble cytochrome c oxidase. However, when these cells are cooled to 25 °C, cytochrome c oxidase activity is recovered. Our results illustrate the subtle relationship between the structural stability and functional properties of BsSCO in the assembly of cytochrome c oxidase.

CpG Methylation Analysis of HPV16 in Laser Capture Microdissected Archival Tissue and Whole Tissue Sections from High Grade Anal Squamous Intraepithelial Lesions: A Potential Disease Biomarker

PubMed Central

Molano, Monica; Tabrizi, Sepehr N.; Garland, Suzanne M.; Roberts, Jennifer M.; Machalek, Dorothy A.; Phillips, Samuel; Chandler, David; Hillman, Richard J.; Grulich, Andrew E.; Jin, Fengyi; Poynten, I. Mary; Templeton, David J.; Cornall, Alyssa M.

2016-01-01

Incidence and mortality rates of anal cancer are increasing globally. More than 90% of anal squamous cell carcinomas (ASCC) are associated with human papillomavirus (HPV). Studies on HPV-related anogenital lesions have shown that patterns of methylation of viral and cellular DNA targets could potentially be developed as disease biomarkers. Lesion-specific DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissues from existing or prospective patient cohorts may constitute a valuable resource for methylation analysis. However, low concentrations of DNA make these samples technically challenging to analyse using existing methods. We therefore set out to develop a sensitive and reproducible nested PCR-pyrosequencing based method to accurately quantify methylation at 10 CpG sites within the E2BS1, E2BS2,3,4 and Sp1 binding sites in the viral upstream regulatory region of HPV16 genome. Methylation analyses using primary and nested PCR-pyrosequencing on 52 FFPE tissue [26 paired whole tissue sections (WTS) and laser capture microdissected (LCM) tissues] from patients with anal squamous intraepithelial lesions was performed. Using nested PCR, methylation results were obtained for the E2BS1, E2BS2,3,4 and Sp1 binding sites in 86.4% of the WTS and 81.8% of the LCM samples. Methylation patterns were strongly correlated within median values of matched pairs of WTS and LCM sections, but overall methylation was higher in LCM samples at different CpG sites. High grade lesions showed low methylation levels in the E2BS1 and E2BS2 regions, with increased methylation detected in the E2BS,3,4/Sp1 regions, showing the highest methylation at CpG site 37. The method developed is highly sensitive in samples with low amounts of DNA and demonstrated to be suitable for archival samples. Our data shows a possible role of specific methylation in the HPV16 URR for detection of HSIL. PMID:27529629

CpG Methylation Analysis of HPV16 in Laser Capture Microdissected Archival Tissue and Whole Tissue Sections from High Grade Anal Squamous Intraepithelial Lesions: A Potential Disease Biomarker.

PubMed

Molano, Monica; Tabrizi, Sepehr N; Garland, Suzanne M; Roberts, Jennifer M; Machalek, Dorothy A; Phillips, Samuel; Chandler, David; Hillman, Richard J; Grulich, Andrew E; Jin, Fengyi; Poynten, I Mary; Templeton, David J; Cornall, Alyssa M

2016-01-01

Incidence and mortality rates of anal cancer are increasing globally. More than 90% of anal squamous cell carcinomas (ASCC) are associated with human papillomavirus (HPV). Studies on HPV-related anogenital lesions have shown that patterns of methylation of viral and cellular DNA targets could potentially be developed as disease biomarkers. Lesion-specific DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissues from existing or prospective patient cohorts may constitute a valuable resource for methylation analysis. However, low concentrations of DNA make these samples technically challenging to analyse using existing methods. We therefore set out to develop a sensitive and reproducible nested PCR-pyrosequencing based method to accurately quantify methylation at 10 CpG sites within the E2BS1, E2BS2,3,4 and Sp1 binding sites in the viral upstream regulatory region of HPV16 genome. Methylation analyses using primary and nested PCR-pyrosequencing on 52 FFPE tissue [26 paired whole tissue sections (WTS) and laser capture microdissected (LCM) tissues] from patients with anal squamous intraepithelial lesions was performed. Using nested PCR, methylation results were obtained for the E2BS1, E2BS2,3,4 and Sp1 binding sites in 86.4% of the WTS and 81.8% of the LCM samples. Methylation patterns were strongly correlated within median values of matched pairs of WTS and LCM sections, but overall methylation was higher in LCM samples at different CpG sites. High grade lesions showed low methylation levels in the E2BS1 and E2BS2 regions, with increased methylation detected in the E2BS,3,4/Sp1 regions, showing the highest methylation at CpG site 37. The method developed is highly sensitive in samples with low amounts of DNA and demonstrated to be suitable for archival samples. Our data shows a possible role of specific methylation in the HPV16 URR for detection of HSIL.

Spores of two probiotic Bacillus species enhance cellular immunity in BALB/C mice.

PubMed

Gong, Li; Huang, Qin; Fu, Aikun; Wu, YanPing; Li, Yali; Xu, Xiaogang; Huang, Yi; Yu, Dongyou; Li, Weifen

2018-01-01

Previous studies found that Bacillus subtilis BS02 and B. subtilis subsp. natto BS04 isolated in our laboratory could activate the immune response of murine macrophages in vitro. This study aims to investigate the effects of dietary supplementation with Bacillus species spores on the systemic cellular immune response in BALB/C mice. Results showed that both B. subtilis BS02 and B. subtilis natto BS04 enhanced the phagocytic function of the mononuclear phagocyte system (MPS) and the cytotoxicity of natural killer (NK) cells. In addition, B. subtilis BS02 could increase the respiratory burst activity of blood phagocytes. Furthermore, B. subtilis BS02 and B. subtilis natto BS04 increased the percentage of gamma-interferon-producing CD4 + cells and CD8 + T-cells, but only BS04 increased the percentage of CD3 + cells and CD3 +  CD4 + cells in splenocytes. However, there were no effects on other subsets of splenic lymphocytes and mitogen-induced splenic lymphocyte proliferation. All data suggested that oral administration of B. subtilis BS02 or B. subtilis natto BS04 could significantly enhance cellular immunity in BALB/C mice by increasing phagocytic activity of MPS and cytotoxic activity of NK cells in a strain-specific manner.

Abnormal B cell memory subsets dominate HIV-specific responses in infected individuals

PubMed Central

Kardava, Lela; Moir, Susan; Shah, Naisha; Wang, Wei; Wilson, Richard; Buckner, Clarisa M.; Santich, Brian H.; Kim, Leo J.Y.; Spurlin, Emily E.; Nelson, Amy K.; Wheatley, Adam K.; Harvey, Christopher J.; McDermott, Adrian B.; Wucherpfennig, Kai W.; Chun, Tae-Wook; Tsang, John S.; Li, Yuxing; Fauci, Anthony S.

2014-01-01

Recently, several neutralizing anti-HIV antibodies have been isolated from memory B cells of HIV-infected individuals. Despite extensive evidence of B cell dysfunction in HIV disease, little is known about the cells from which these rare HIV-specific antibodies originate. Accordingly, we used HIV envelope gp140 and CD4 or coreceptor (CoR) binding site (bs) mutant probes to evaluate HIV-specific responses in peripheral blood B cells of HIV-infected individuals at various stages of infection. In contrast to non-HIV responses, HIV-specific responses against gp140 were enriched within abnormal B cells, namely activated and exhausted memory subsets, which are largely absent in the blood of uninfected individuals. Responses against the CoRbs, which is a poorly neutralizing epitope, arose early, whereas those against the well-characterized neutralizing epitope CD4bs were delayed and infrequent. Enrichment of the HIV-specific response within resting memory B cells, the predominant subset in uninfected individuals, did occur in certain infected individuals who maintained low levels of plasma viremia and immune activation with or without antiretroviral therapy. The distribution of HIV-specific responses among memory B cell subsets was corroborated by transcriptional analyses. Taken together, our findings provide valuable insight into virus-specific B cell responses in HIV infection and demonstrate that memory B cell abnormalities may contribute to the ineffectiveness of the antibody response in infected individuals. PMID:24892810

B cell recognition of the conserved HIV-1 co-receptor binding site is altered by endogenous primate CD4.

PubMed

Forsell, Mattias N E; Dey, Barna; Mörner, Andreas; Svehla, Krisha; O'dell, Sijy; Högerkorp, Carl-Magnus; Voss, Gerald; Thorstensson, Rigmor; Shaw, George M; Mascola, John R; Karlsson Hedestam, Gunilla B; Wyatt, Richard T

2008-10-03

The surface HIV-1 exterior envelope glycoprotein, gp120, binds to CD4 on the target cell surface to induce the co-receptor binding site on gp120 as the initial step in the entry process. The binding site is comprised of a highly conserved region on the gp120 core, as well as elements of the third variable region (V3). Antibodies against the co-receptor binding site are abundantly elicited during natural infection of humans, but the mechanism of elicitation has remained undefined. In this study, we investigate the requirements for elicitation of co-receptor binding site antibodies by inoculating rabbits, monkeys and human-CD4 transgenic (huCD4) rabbits with envelope glycoprotein (Env) trimers possessing high affinity for primate CD4. A cross-species comparison of the antibody responses showed that similar HIV-1 neutralization breadth was elicited by Env trimers in monkeys relative to wild-type (WT) rabbits. In contrast, antibodies against the co-receptor site on gp120 were elicited only in monkeys and huCD4 rabbits, but not in the WT rabbits. This was supported by the detection of high-titer co-receptor antibodies in all sera from a set derived from human volunteers inoculated with recombinant gp120. These findings strongly suggest that complexes between Env and (high-affinity) primate CD4 formed in vivo are responsible for the elicitation of the co-receptor-site-directed antibodies. They also imply that the naïve B cell receptor repertoire does not recognize the gp120 co-receptor site in the absence of CD4 and illustrate that conformational stabilization, imparted by primary receptor interaction, can alter the immunogenicity of a type 1 viral membrane protein.

Hydration Differences Explain the Large Variations in the Complexation Thermodynamics of Modified γ-Cyclodextrins with Bile Salts.

PubMed

Køhler, Jonatan; Schönbeck, Christian; Westh, Peter; Holm, René

2016-01-28

The structure and thermodynamics of inclusion complexes of seven different γ-cyclodextrins (γCDs) and three biologically relevant bile salts (BS) were investigated in the present study. Natural γCD and six modified γCDs [two methyl-γCDs, one sulfobutyl ether-γCD (SBEγCD), and three 2-hydroxypropyl-γCDs (HPγCD)] and their complexes with BS were investigated by isothermal titration calorimetry, NMR, and molecular dynamics simulations. With the exception of the fully methylated γCD, which did not bind the BSs investigated, all of the γCDs formed 1:1 complexes with the BS, and the structures were similar to those with natural γCD; i.e., the modifications of the γCD had limited structural impact on the formation of complexes. Isothermal titration calorimetry was carried out over in the temperature interval 5-55 °C to enable the calculation of the stability constant (K) and the thermodynamic parameters enthalpy (ΔH°), entropy (ΔS°), and heat capacity (ΔCp°). The stability constants decreased with an increased degree of substitution (DS), with methyl substituents having a lower effect on the stability constant than the sulfobutyl ether and hydroxypropyl substituents on the stability constants. Enthalpy-entropy compensation was observed, since both enthalpy and entropy increased with the degree of substitution, which may reflect dehydration of the hydrophobic surface on both CD and BS. Calculations based on ΔCp° data suggested that each of the substituents dehydrated 10-20 (hydroxypropyl), 22-33 (sulfobutyl ether), and 10-15 Å(2) (methyl) of the BS surface area, in reasonable agreement with estimates from the molecular dynamics simulations. Combined with earlier investigations on modified βCDs, these results indicate general trends of the substituents on the thermodynamics of complex formation.

Specific ganglioside binding to receptor sites on T lymphocytes that couple to ganglioside-induced decrease of CD4 expression

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

Morrison, W.J.; Offner, H.; Vandenbark, A.A.

1989-01-01

The binding of different gangliosides to rat T-helper lymphocytes was characterized under conditions that decrease CD4 expression on different mammalian T-helper lymphoctyes. Saturation binding by monosialylated ({sub 3}H)-GM{sub 1} to rat T-lymphocytes was time- and temperature-dependent, had a dissociation constant (K{sub D}) of 2.2 {plus minus} 1.4 {mu}M and a binding capacity near 2 fmoles/cell. Competitive inhibition of ({sup 3}H)- GM{sub 1} binding demonstrated a structural-activity related to the number of unconstrained sialic acid moieties on GM{sub 1}-congeneric gangliosides. A comparison between the results of these binding studies and gangliosides-induced decrease of CD4 expression demonstrated that every aspect of ({supmore » 3}H)-GM{sub 1} binding concurs with ganglioside modulation of CD4 expression. It is concluded that the specific decrease of CD4 expression induced by pretreatment with gangliosides involves the initial process of gangliosides binding to specific sites on CD4{sup {double dagger}} T-helper lymphocytes.« less

Pretargeted Molecular Imaging and Radioimmunotherapy

PubMed Central

Goldenberg, David M.; Chang, Chien-Hsing; Rossi, Edmund A.; J, William; McBride; Sharkey, Robert M.

2012-01-01

Pretargeting is a multi-step process that first has an unlabeled bispecific antibody (bsMAb) localize within a tumor by virtue of its anti-tumor binding site(s) before administering a small, fast-clearing radiolabeled compound that then attaches to the other portion of the bsMAb. The compound's rapid clearance significantly reduces radiation exposure outside of the tumor and its small size permits speedy delivery to the tumor, creating excellent tumor/nontumor ratios in less than 1 hour. Haptens that bind to an anti-hapten antibody, biotin that binds to streptavidin, or an oligonucleotide binding to a complementary oligonucleotide sequence have all been radiolabeled for use by pretargeting. This review will focus on a highly flexible anti-hapten bsMAb platform that has been used to target a variety of radionuclides to image (SPECT and PET) as well as treat tumors. PMID:22737190

Structure of an N276-Dependent HIV-1 Neutralizing Antibody Targeting a Rare V5 Glycan Hole Adjacent to the CD4 Binding Site.

PubMed

Wibmer, Constantinos Kurt; Gorman, Jason; Anthony, Colin S; Mkhize, Nonhlanhla N; Druz, Aliaksandr; York, Talita; Schmidt, Stephen D; Labuschagne, Phillip; Louder, Mark K; Bailer, Robert T; Abdool Karim, Salim S; Mascola, John R; Williamson, Carolyn; Moore, Penny L; Kwong, Peter D; Morris, Lynn

2016-11-15

All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report the isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage. The conserved CD4 binding site on gp120 is a major target for HIV-1 vaccine design, but key events in the elicitation and maturation of different antibody lineages to this site remain elusive. Studies have shown that strain-specific antibodies can evolve into broadly neutralizing antibodies or in some cases act as helper lineages. Therefore, characterizing the epitopes of strain-specific antibodies may help to inform the design of HIV-1 immunogens to elicit broadly neutralizing antibodies. In this study, we isolate a narrowly neutralizing N276 glycan-dependent antibody and use X-ray crystallography and viral deep sequencing to describe how gp120 lacking glycans in V5 might have elicited these early glycan-dependent CD4 binding site antibodies. These data highlight how glycan holes can play a role in the elicitation of B-cell lineages targeting the CD4 binding site. Copyright © 2016 Wibmer et al.

Structure of an N276-Dependent HIV-1 Neutralizing Antibody Targeting a Rare V5 Glycan Hole Adjacent to the CD4 Binding Site

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

Wibmer, Constantinos Kurt; Gorman, Jason; Anthony, Colin S.

ABSTRACT All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report themore » isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage. IMPORTANCEThe conserved CD4 binding site on gp120 is a major target for HIV-1 vaccine design, but key events in the elicitation and maturation of different antibody lineages to this site remain elusive. Studies have shown that strain-specific antibodies can evolve into broadly neutralizing antibodies or in some cases act as helper lineages. Therefore, characterizing the epitopes of strain-specific antibodies may help to inform the design of HIV-1 immunogens to elicit broadly neutralizing antibodies. In this study, we isolate a narrowly neutralizing N276 glycan-dependent antibody and use X-ray crystallography and viral deep sequencing to describe how gp120 lacking glycans in V5 might have elicited these early glycan-dependent CD4 binding site antibodies. These data highlight how glycan holes can play a role in the elicitation of B-cell lineages targeting the CD4 binding site.« less

Structure of an N276-Dependent HIV-1 Neutralizing Antibody Targeting a Rare V5 Glycan Hole Adjacent to the CD4 Binding Site

PubMed Central

Wibmer, Constantinos Kurt; Gorman, Jason; Anthony, Colin S.; Mkhize, Nonhlanhla N.; Druz, Aliaksandr; York, Talita; Schmidt, Stephen D.; Labuschagne, Phillip; Louder, Mark K.; Bailer, Robert T.; Abdool Karim, Salim S.; Mascola, John R.; Williamson, Carolyn; Moore, Penny L.

2016-01-01

ABSTRACT All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report the isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage. IMPORTANCE The conserved CD4 binding site on gp120 is a major target for HIV-1 vaccine design, but key events in the elicitation and maturation of different antibody lineages to this site remain elusive. Studies have shown that strain-specific antibodies can evolve into broadly neutralizing antibodies or in some cases act as helper lineages. Therefore, characterizing the epitopes of strain-specific antibodies may help to inform the design of HIV-1 immunogens to elicit broadly neutralizing antibodies. In this study, we isolate a narrowly neutralizing N276 glycan-dependent antibody and use X-ray crystallography and viral deep sequencing to describe how gp120 lacking glycans in V5 might have elicited these early glycan-dependent CD4 binding site antibodies. These data highlight how glycan holes can play a role in the elicitation of B-cell lineages targeting the CD4 binding site. PMID:27581986

Plumbagin inhibits cytokinesis in Bacillus subtilis by inhibiting FtsZ assembly--a mechanistic study of its antibacterial activity.

PubMed

Bhattacharya, Anusri; Jindal, Bhavya; Singh, Parminder; Datta, Anindya; Panda, Dulal

2013-09-01

The assembly of FtsZ plays a central role in construction of the cytokinetic Z-ring that orchestrates bacterial cell division. A naturally occurring naphthoquinone, plumbagin, is known to exhibit antibacterial properties against several types of bacteria. In this study, plumbagin was found to perturb formation of the Z-ring in Bacillus subtilis 168 cells and to cause elongation of these cells without an apparent effect on nucleoid segregation, indicating that it may inhibit FtsZ assembly. Furthermore, it bound to purified B. subtilis FtsZ (BsFtsZ) with a dissociation constant of 20.7 ± 5.6 μM, and inhibited the assembly and GTPase activity of BsFtsZ in vitro. Interestingly, plumbagin did not inhibit either the assembly or GTPase activity of Escherichia coli FtsZ (EcFtsZ) in vitro. Using docking analysis, a putative plumbagin-binding site on BsFtsZ was identified, and the analysis indicated that hydrophobic interactions and hydrogen bonds predominate. Based on the in silico analysis, two variants of BsFtsZ, namely D199A and V307R, were constructed to explore the binding interaction of plumbagin and BsFtsZ. The effects of plumbagin on the assembly and GTPase activity of the variant BsFtsZ proteins in vitro indicated that the residues D199 and V307 may be involved in the binding of plumbagin to BsFtsZ. The results suggest that plumbagin inhibits bacterial proliferation by inhibiting the assembly of FtsZ, and provide insight into the binding site of plumbagin on BsFtsZ, which may help in the design of potent FtsZ-targeted antibacterial agents. © 2013 FEBS.

Revegetation of high zinc and lead tailings with municipal biosolids and lime: greenhouse study.

PubMed

Svendson, Alex; Henry, Chuck; Brown, Sally

2007-01-01

Acidic (pH 4.1) and high Cd, Pb, and Zn mine tailings (mean +/- SD: 17 +/- 0.4, 3800 +/- 100, and 3500 +/- 100 mg kg(-1), respectively) from an alluvial tailings deposit in Leadville, Colorado were amended with municipal biosolids (BS) (224 Mg ha(-1)) and different types of lime (calcium carbonate equivalent of 224 Mg ha(-1) CaCO3) in a greenhouse column study to test the ability of the amendments to neutralize surface and subsoil acidity and restore plant growth. The types of lime included coarse, agricultural, and fine-textured lime (CL, AL, and FL), sugar beet lime (SBL), and lime kiln dust (LK). The FL was also added alone. All treatments increased bulk pH in the amended horizon in comparison to the control, with the most significant increases observed in the FL, SBL+BS, and LK+BS treatments (7.33, 7.34, and 7.63, respectively). All treatments, excluding the FL, increased the pH in the horizon directly below the amended layer, with the most significant increases observed in the SBL+BS and LK+BS treatments (6.01 and 5.41, respectively). Significant decreases in 0.01 M Ca(NO3)2-extractable Zn and Cd were observed in the subsoil for all treatments that included BS, with the largest decrease in the SBL+BS treatment (344 and 3.9 versus 4 and 0.1 mg kg(-1) Zn and Cd, respectively). Plant growth of annual rye (Lolium multiflorum L.) was vigorous in all treatments that included BS with plant Zn, Cd, and Pb concentrations reduced over the control.

Trichloroethylene-induced alterations in DNA methylation were enriched in polycomb protein binding sites in effector/memory CD4+ T cells

PubMed Central

Gilbert, Kathleen M.; Blossom, Sarah J.; Reisfeld, Brad; Erickson, Stephen W.; Vyas, Kanan; Maher, Mary; Broadfoot, Brannon; West, Kirk; Bai, Shasha; Cooney, Craig A.; Bhattacharyya, Sudeepa

2017-01-01

Abstract Exposure to industrial solvent and water pollutant trichloroethylene (TCE) can promote autoimmunity, and expand effector/memory (CD62L) CD4+ T cells. In order to better understand etiology reduced representation bisulfite sequencing was used to study how a 40-week exposure to TCE in drinking water altered methylation of ∼337 770 CpG sites across the entire genome of effector/memory CD4+ T cells from MRL+/+ mice. Regardless of TCE exposure, 62% of CpG sites in autosomal chromosomes were hypomethylated (0–15% methylation), and 25% were hypermethylated (85–100% methylation). In contrast, only 6% of the CpGs on the X chromosome were hypomethylated, and 51% had mid-range methylation levels. In terms of TCE impact, TCE altered (≥ 10%) the methylation of 233 CpG sites in effector/memory CD4+ T cells. Approximately 31.7% of these differentially methylated sites occurred in regions known to bind one or more Polycomb group (PcG) proteins, namely Ezh2, Suz12, Mtf2 or Jarid2. In comparison, only 23.3% of CpG sites not differentially methylated by TCE were found in PcG protein binding regions. Transcriptomics revealed that TCE altered the expression of ∼560 genes in the same effector/memory CD4+ T cells. At least 80% of the immune genes altered by TCE had binding sites for PcG proteins flanking their transcription start site, or were regulated by other transcription factors that were in turn ordered by PcG proteins at their own transcription start site. Thus, PcG proteins, and the differential methylation of their binding sites, may represent a new mechanism by which TCE could alter the function of effector/memory CD4+ T cells. PMID:29129997

Ligand Binding Site Detection by Local Structure Alignment and Its Performance Complementarity

PubMed Central

Lee, Hui Sun; Im, Wonpil

2013-01-01

Accurate determination of potential ligand binding sites (BS) is a key step for protein function characterization and structure-based drug design. Despite promising results of template-based BS prediction methods using global structure alignment (GSA), there is a room to improve the performance by properly incorporating local structure alignment (LSA) because BS are local structures and often similar for proteins with dissimilar global folds. We present a template-based ligand BS prediction method using G-LoSA, our LSA tool. A large benchmark set validation shows that G-LoSA predicts drug-like ligands’ positions in single-chain protein targets more precisely than TM-align, a GSA-based method, while the overall success rate of TM-align is better. G-LoSA is particularly efficient for accurate detection of local structures conserved across proteins with diverse global topologies. Recognizing the performance complementarity of G-LoSA to TM-align and a non-template geometry-based method, fpocket, a robust consensus scoring method, CMCS-BSP (Complementary Methods and Consensus Scoring for ligand Binding Site Prediction), is developed and shows improvement on prediction accuracy. The G-LoSA source code is freely available at http://im.bioinformatics.ku.edu/GLoSA. PMID:23957286

AtSPX1 affects the AtPHR1-DNA-binding equilibrium by binding monomeric AtPHR1 in solution.

PubMed

Qi, Wanjun; Manfield, Iain W; Muench, Stephen P; Baker, Alison

2017-10-23

Phosphorus is an essential macronutrient for plant growth and is deficient in ∼50% of agricultural soils. The transcription factor phosphate starvation response 1 (PHR1) plays a central role in regulating the expression of a subset of phosphate starvation-induced (PSI) genes through binding to a cis -acting DNA element termed P1BS (PHR1-binding sequences). In Arabidopsis and rice, activity of AtPHR1/OsPHR2 is regulated in part by their downstream target SPX ( S yg1, P ho81, X pr1) proteins through protein-protein interaction. Here, we provide kinetic and affinity data for interaction between AtPHR1 and P1BS sites. Using surface plasmon resonance, a tandem P1BS sequence showed ∼50-fold higher affinity for MBPAtdPHR1 (a fusion protein comprising the DNA-binding domain and coiled-coil domain of AtPHR1 fused to maltose-binding protein) than a single site. The affinity difference was largely reflected in a much slower dissociation rate from the 2× P1BS-binding site, suggesting an important role for protein co-operativity. Injection of AtSPX1 in the presence of phosphate or inositol hexakisphosphate (InsP6) failed to alter the MBPAtdPHR1-P1BS dissociation rate, while pre-mixing of these two proteins in the presence of either 5 mM Pi or 500 µM InsP6 resulted in a much lower DNA-binding signal from MBPAtdPHR1. These data suggest that, in the Pi-restored condition, AtSPX1 can bind to monomeric AtPHR1 in solution and therefore regulate PSI gene expression by tuning the AtPHR1-DNA-binding equilibrium. This Pi-dependent regulation of AtPHR1-DNA-binding equilibrium also generates a negative feedback loop on the expression of AtSPX1 itself, providing a tight control of PSI gene expression. © 2017 The Author(s).

Immunotherapy in a human ovarian cancer xenograft model with two bispecific monoclonal antibodies: OV-TL 3/CD3 and OC/TR.

PubMed

van Ravenswaay Claasen, H H; Eggermont, A M; Nooyen, Y A; Warnaar, S O; Fieuren, G J

1994-02-01

The bispecific antibodies (bs-mAbs) OV-TL 3/CD3 and OC/TR (MOv18/CD3) efficiently mediate ovarian tumor cell lysis by cytotoxic T cells and activated peripheral blood lymphocytes (PBL) in vitro. OV-TL 3/CD3 and OC/TR are reactive with tumor-associated antigens on ovarian carcinoma cells (OA3 and CA-MOv18, respectively), and CD3 on activated PBL, bridging both cells and simultaneously inducing activation of the effector cells. In a comparative study we investigated the therapeutic efficacy of OV-TL 3/CD3 and OC/TR by targeting activated PBL with the bs-mAbs against intraperitoneally growing NIH:OVCAR-3 human ovarian carcinoma cells. As they have good tumor localization characteristics, HPLC-purified bispecific F(ab')2 fragments were used to target highly active PHA and IL-2-stimulated PBL effector cells. The efficacy of OV-TL 3/CD3 was compared to OC/TR with respect to tumor-associated antigen (TAA) binding on NIH:OVCAR-3 ascites cells and NIH:OVCAR-3 tumor cell lysis in vitro. In this report we show that ip ovarian cancer-bearing nude mice treated with IL-2 and activated PBL coated with bispecific F(ab')2 had a significantly longer survival than the untreated mice. No significant difference in survival was found between the OC/TR or OV-TL 3/CD3 bispecific antibody, although MOv18 expression was higher on NIH:OVCAR-3 ascites cells and PBL targeted with OC/TR induced slightly higher tumor cell lysis in vitro. Thus, the therapeutic efficacy of these bs-mAbs in vivo could not be predicted by TAA expression or bs-mAb-mediated tumor cell lysis in vitro.

Pretargeting of carcinoembryonic antigen-expressing cancers with a trivalent bispecific fusion protein produced in myeloma cells.

PubMed

Rossi, Edmund A; Chang, Chien-Hsing; Losman, Michele J; Sharkey, Robert M; Karacay, Habibe; McBride, William; Cardillo, Thomas M; Hansen, Hans J; Qu, Zhengxing; Horak, Ivan D; Goldenberg, David M

2005-10-01

To characterize a novel trivalent bispecific fusion protein and evaluate its potential utility for pretargeted delivery of radionuclides to tumors. hBS14, a recombinant fusion protein that binds bispecifically to carcinoembryonic antigen (CEA) and the hapten, histamine-succinyl-glycine (HSG), was produced by transgenic myeloma cells and purified to near homogeneity in a single step using a novel HSG-based affinity chromatography system. Biochemical characterization included size-exclusion high-performance liquid chromatography (SE-HPLC), SDS-PAGE, and isoelectric focusing. Functional characterization was provided by BIAcore and SE-HPLC. The efficacy of hBS14 for tumor pretargeting was evaluated in CEA-expressing GW-39 human colon tumor-bearing nude mice using a bivalent HSG hapten (IMP-241) labeled with (111)In. Biochemical analysis showed that single-step affinity chromatography provided highly purified material. SE-HPLC shows a single protein peak consistent with the predicted molecular size of hBS14. SDS-PAGE analysis shows only two polypeptide bands, which are consistent with the calculated molecular weights of the hBS14 polypeptides. BIAcore showed the bispecific binding properties and suggested that hBS14 possesses two functional CEA-binding sites. This was supported by SE-HPLC immunoreactivity experiments. All of the data suggest that the structure of hBS14 is an 80 kDa heterodimer with one HSG and two CEA binding sites. Pretargeting experiments in the mouse model showed high uptake of radiopeptide in the tumor, with favorable tumor-to-nontumor ratios as early as 3 hours postinjection. The results indicate that hBS14 is an attractive candidate for use in a variety of pretargeting applications, particularly tumor therapy with radionuclides and drugs.

Non-peptidic antagonists of the CGRP receptor, BIBN4096BS and MK-0974, interact with the calcitonin receptor-like receptor via methionine-42 and RAMP1 via tryptophan-74.

PubMed

Miller, Philip S; Barwell, James; Poyner, David R; Wigglesworth, Mark J; Garland, Stephen L; Donnelly, Dan

2010-01-01

The receptor for calcitonin gene-related peptide (CGRP) has been the target for the development of novel small molecule antagonists for the treatment of migraine. Two such antagonists, BIBN4096BS and MK-0974, have shown great promise in clinical trials and hence a deeper understanding of the mechanism of their interaction with the receptor is now required. The structure of the CGRP receptor is unusual since it is comprised of a hetero-oligomeric complex between the calcitonin receptor-like receptor (CRL) and an accessory protein (RAMP1). Both the CLR and RAMP1 components have extracellular domains which interact with each other and together form part of the peptide-binding site. It seems likely that the antagonist binding site will also be located on the extracellular domains and indeed Trp-74 of RAMP1 has been shown to form part of the binding site for BIBN4096BS. However, despite a chimeric study demonstrating the role of the N-terminal domain of CLR in antagonist binding, no specific residues have been identified. Here we carry out a mutagenic screen of the extreme N-terminal domain of CLR (residues 23-63) and identify a mutant, Met-42-Ala, which displays 48-fold lower affinity for BIBN4096BS and almost 900-fold lower affinity for MK-0974. In addition, we confirm that the Trp-74-Lys mutation at human RAMP1 reduces BIBN4096BS affinity by over 300-fold and show for the first time a similar effect for MK-0974 affinity. The data suggest that the non-peptide antagonists occupy a binding site close to the interface of the N-terminal domains of CLR and RAMP1. Copyright 2009 Elsevier Inc. All rights reserved.

High-and low-affinity binding sites for Cd on the bacterial cell walls of Bacillus subtilis and Shewanella oneidensis.

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

Mishra, B.; Boyanov, M.; Bunker, B. A.

2010-08-01

Bulk Cd adsorption isotherm experiments, thermodynamic equilibrium modeling, and Cd K edge EXAFS were used to constrain the mechanisms of proton and Cd adsorption to bacterial cells of the commonly occurring Gram-positive and Gram-negative bacteria, Bacillus subtilis and Shewanella oneidensis, respectively. Potentiometric titrations were used to characterize the functional group reactivity of the S. oneidensis cells, and we model the titration data using the same type of non-electrostatic surface complexation approach as was applied to titrations of B. subtilis suspensions by Fein et al. (2005). Similar to the results for B. subtilis, the S. oneidensis cells exhibit buffering behavior frommore » approximately pH 3-9 that requires the presence of four distinct sites, with pK{sub a} values of 3.3 {+-} 0.2, 4.8 {+-} 0.2, 6.7 {+-} 0.4, and 9.4 {+-} 0.5, and site concentrations of 8.9({+-}2.6) x 10{sup -5}, 1.3({+-}0.2) x 10{sup -4}, 5.9({+-}3.3) x 10{sup -5}, and 1.1({+-}0.6) x 10{sup -4} moles/g bacteria (wet mass), respectively. The bulk Cd isotherm adsorption data for both species, conducted at pH 5.9 as a function of Cd concentration at a fixed biomass concentration, were best modeled by reactions with a Cd:site stoichiometry of 1:1. EXAFS data were collected for both bacterial species as a function of Cd concentration at pH 5.9 and 10 g/L bacteria. The EXAFS results show that the same types of binding sites are responsible for Cd sorption to both bacterial species at all Cd loadings tested (1-200 ppm). Carboxyl sites are responsible for the binding at intermediate Cd loadings. Phosphoryl ligands are more important than carboxyl ligands for Cd binding at high Cd loadings. For the lowest Cd loadings studied here, a sulfhydryl site was found to dominate the bound Cd budgets for both species, in addition to the carboxyl and phosphoryl sites that dominate the higher loadings. The EXAFS results suggest that both Gram-positive and Gram-negative bacterial cell walls have a low concentration of very high-affinity sulfhydryl sites which become masked by the more abundant carboxyl and phosphoryl sites at higher metal:bacteria ratios. This study demonstrates that metal loading plays a vital role in determining the important metal-binding reactions that occur on bacterial cell walls, and that high affinity, low-density sites can be revealed by spectroscopy of biomass samples. Such sites may control the fate and transport of metals in realistic geologic settings, where metal concentrations are low.« less

Structure of Simian Immunodeficiency Virus Envelope Spikes Bound with CD4 and Monoclonal Antibody 36D5.

PubMed

Hu, Guiqing; Liu, Jun; Roux, Kenneth H; Taylor, Kenneth A

2017-08-15

The human immunodeficiency virus type 1 (HIV-1)/simian immunodeficiency virus (SIV) envelope spike (Env) mediates viral entry into host cells. The V3 loop of the gp120 component of the Env trimer contributes to the coreceptor binding site and is a target for neutralizing antibodies. We used cryo-electron tomography to visualize the binding of CD4 and the V3 loop monoclonal antibody (MAb) 36D5 to gp120 of the SIV Env trimer. Our results show that 36D5 binds gp120 at the base of the V3 loop and suggest that the antibody exerts its neutralization effect by blocking the coreceptor binding site. The antibody does this without altering the dynamics of the spike motion between closed and open states when CD4 is bound. The interaction between 36D5 and SIV gp120 is similar to the interaction between some broadly neutralizing anti-V3 loop antibodies and HIV-1 gp120. Two conformations of gp120 bound with CD4 are revealed, suggesting an intrinsic dynamic nature of the liganded Env trimer. CD4 binding substantially increases the binding of 36D5 to gp120 in the intact Env trimer, consistent with CD4-induced changes in the conformation of gp120 and the antibody binding site. Binding by MAb 36D5 does not substantially alter the proportions of the two CD4-bound conformations. The position of MAb 36D5 at the V3 base changes little between conformations, indicating that the V3 base serves as a pivot point during the transition between these two states. IMPORTANCE Glycoprotein spikes on the surfaces of SIV and HIV are the sole targets available to the immune system for antibody neutralization. Spikes evade the immune system by a combination of a thick layer of polysaccharide on the surface (the glycan shield) and movement between spike domains that masks the epitope conformation. Using SIV virions whose spikes were "decorated" with the primary cellular receptor (CD4) and an antibody (36D5) at part of the coreceptor binding site, we visualized multiple conformations trapped by the rapid freezing step, which were separated using statistical analysis. Our results show that the CD4-induced conformational dynamics of the spike enhances binding of the antibody. Copyright © 2017 American Society for Microbiology.

Insertion of scFv into the hinge domain of full-length IgG1 monoclonal antibody results in tetravalent bispecific molecule with robust properties.

PubMed

Bezabeh, Binyam; Fleming, Ryan; Fazenbaker, Christine; Zhong, Haihong; Coffman, Karen; Yu, Xiang-Qing; Leow, Ching Ching; Gibson, Nerea; Wilson, Susan; Stover, C Kendall; Wu, Herren; Gao, Changshou; Dimasi, Nazzareno

By simultaneous binding two disease mediators, bispecific antibodies offer the opportunity to broaden the utility of antibody-based therapies. Herein, we describe the design and characterization of Bs4Ab, an innovative and generic bispecific tetravalent antibody platform. The Bs4Ab format comprises a full-length IgG1 monoclonal antibody with a scFv inserted into the hinge domain. The Bs4Ab design demonstrates robust manufacturability as evidenced by MEDI3902, which is currently in clinical development. To further demonstrate the applicability of the Bs4Ab technology, we describe the molecular engineering, biochemical, biophysical, and in vivo characterization of a bispecific tetravalent Bs4Ab that, by simultaneously binding vascular endothelial growth factor and angiopoietin-2, inhibits their function. We also demonstrate that the Bs4Ab platform allows Fc-engineering similar to that achieved with IgG1 antibodies, such as mutations to extend half-life or modulate effector functions.

Ligand accessibility to the HIV-1 Env co-receptor binding site can occur prior to CD4 engagement and is independent of viral tier category.

PubMed

Boliar, Saikat; Patil, Shilpa; Shukla, Brihaspati N; Ghobbeh, Ali; Deshpande, Suprit; Chen, Weizao; Guenaga, Javier; Dimitrov, Dimiter S; Wyatt, Richard T; Chakrabarti, Bimal K

2018-06-01

HIV-1 virus entry into target cells requires the envelope glycoprotein (Env) to first bind the primary receptor, CD4 and subsequently the co-receptor. Antibody access to the co-receptor binding site (CoRbs) in the pre-receptor-engaged state, prior to cell attachment, remains poorly understood. Here, we have demonstrated that for tier-1 Envs, the CoRbs is directly accessible to full-length CD4-induced (CD4i) antibodies even before primary receptor engagement, indicating that on these Envs the CoRbs site is either preformed or can conformationally sample post-CD4-bound state. Tier-2 and tier-3 Envs, which are resistant to full-length CD4i antibody, are neutralized by m36.4, a lower molecular mass of CD4i-directed domain antibody. In some tier-2 and tier-3 Envs, CoRbs is accessible to m36.4 even prior to cellular attachment in an Env-specific manner independent of their tier category. These data suggest differential structural arrangements of CoRbs and varied masking of ligand access to the CoRbs in different Env isolates. Copyright © 2018 Elsevier Inc. All rights reserved.

Crystal Structure of the Neutralizing Llama VHH D7 and Its Mode of HIV-1 gp120 Interaction

PubMed Central

Hinz, Andreas; Lutje Hulsik, David; Forsman, Anna; Koh, Willie Wee-Lee; Belrhali, Hassan; Gorlani, Andrea; de Haard, Hans; Weiss, Robin A.; Verrips, Theo; Weissenhorn, Winfried

2010-01-01

HIV-1 entry into host cells is mediated by the sequential binding of the envelope glycoprotein gp120 to CD4 and a chemokine receptor. Antibodies binding to epitopes overlapping the CD4-binding site on gp120 are potent inhibitors of HIV entry, such as the llama heavy chain antibody fragment VHH D7, which has cross-clade neutralizing properties and competes with CD4 and mAb b12 for high affinity binding to gp120. We report the crystal structure of the D7 VHH at 1.5 Å resolution, which reveals the molecular details of the complementarity determining regions (CDR) and substantial flexibility of CDR3 that could facilitate an induced fit interaction with gp120. Structural comparison of CDRs from other CD4 binding site antibodies suggests diverse modes of interaction. Mutational analysis identified CDR3 as a key component of gp120 interaction as determined by surface plasmon resonance. A decrease in affinity is directly coupled to the neutralization efficiency since mutations that decrease gp120 interaction increase the IC50 required for HIV-1 IIIB neutralization. Thus the structural study identifies the long CDR3 of D7 as the key determinant of interaction and HIV-1 neutralization. Furthermore, our data confirm that the structural plasticity of gp120 can accommodate multiple modes of antibody binding within the CD4 binding site. PMID:20463957

Structural considerations for functional anti-EGFR × anti-CD3 bispecific diabodies in light of domain order and binding affinity.

PubMed

Asano, Ryutaro; Nagai, Keisuke; Makabe, Koki; Takahashi, Kento; Kumagai, Takashi; Kawaguchi, Hiroko; Ogata, Hiromi; Arai, Kyoko; Umetsu, Mitsuo; Kumagai, Izumi

2018-03-02

We previously reported a functional humanized bispecific diabody (bsDb) that targeted EGFR and CD3 (hEx3-Db) and enhancement of its cytotoxicity by rearranging the domain order in the V domain. Here, we further dissected the effect of domain order in bsDbs on their cross-linking ability and binding kinetics to elucidate general rules regarding the design of functional bsDbs. Using Ex3-Db as a model system, we first classified the four possible domain orders as anti-parallel (where both chimeric single-chain components are variable heavy domain (VH)-variable light domain (VL) or VL-VH order) and parallel types (both chimeric single-chain components are mixed with VH-VL and VL-VH order). Although anti-parallel Ex3-Dbs could cross-link the soluble target antigens, their cross-linking ability between soluble targets had no correlation with their growth inhibitory effects. In contrast, the binding affinity of one of the two constructs with a parallel-arrangement V domain was particularly low, and structural modeling supported this phenomenon. Similar results were observed with E2x3-Dbs, in which the V region of the anti-EGFR antibody clone in hEx3 was replaced with that of another anti-EGFR clone. Only anti-parallel types showed affinity-dependent cancer inhibitory effects in each molecule, and E2x3-LH (both components in VL-VH order) showed the most intense anti-tumor activity in vitro and in vivo . Our results showed that, in addition to rearranging the domain order of bsDbs, increasing their binding affinity may be an ideal strategy for enhancing the cytotoxicity of anti-parallel constructs and that E2x3-LH is particularly attractive as a candidate next-generation anti-cancer drug.

Diphtheria toxin can simultaneously bind to its receptor and adenylyl-(3',5')-uridine 3'-monophosphate

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

Barbieri, J.T.; Collins, C.M.; Collier, R.J.

1986-10-21

Diphtheria toxin (DT) that was bound to receptors on BS-C-1 cells was able to bind approximately 1 molar equiv of adenylyl-(3',5')-uridine 3'-monophosphate (ApUp). In contrast, receptor-bound CRM197, a mutant form of toxin with greatly diminished affinity for dinucleotides, did not bind ApUp. Affinity of the dinucleotide for receptor-bound toxin differed from that for free toxin by less than an order of magnitude. These results indicate that the receptor site and the ApUp site on the toxin do not significantly overlap. BS-C-1 cells were incubated with or without /sup 125/I-DT or CRM 197. They were then incubated with (/sup 32/P)ApUp, andmore » assayed.« less

In vitro and ex vivo distribution of [3H]harmane, an endogenous beta-carboline, in rat brain.

PubMed

Anderson, Neil J; Tyacke, Robin J; Husbands, Stephen M; Nutt, David J; Hudson, Alan L; Robinson, Emma S J

2006-03-01

The endogenous beta-carboline, harmane, has been shown to bind to monoamine oxidase A (MAO-A) and a separate, high affinity, non-MAO site. Research in our laboratory has shown that harmane is an active component of clonidine-displacing substance (CDS), the proposed endogenous ligand for imidazoline binding sites (IBS). In the present study we have investigated the distribution of [3H]harmane in rat brain, and related the binding profile to the distribution of the MAO-A selective ligand [3H]Ro41-1049 and the I2BS ligand [3H]2-BFI. The in vivo distribution of [3H]harmane following intravenous administration was also investigated. Receptor autoradiography revealed a highly significant correlation for the distribution of [3H]harmane and [3H]Ro41-1049, and a significant correlation for [3H]harmane and the I2BS ligand [3H]2-BFI. The in vivo distribution of [3H]harmane suggests that the ligand accumulates in the adrenal gland and throughout the brain with the primary route of excretion occurring via the duodenum. In conclusion, these studies have shown that [3H]harmane labels a population of binding sites that reflect the distribution of MAO-A. Further evidence for a non-MAO, IBS [3H]harmane population has not been shown but the high level of expression of the MAO-A site is likely to have masked the much smaller population of I2BS.

Loss of a single N-linked glycan allows CD4-independent human immunodeficiency virus type 1 infection by altering the position of the gp120 V1/V2 variable loops.

PubMed

Kolchinsky, P; Kiprilov, E; Bartley, P; Rubinstein, R; Sodroski, J

2001-04-01

The gp120 envelope glycoprotein of primary human immunodeficiency virus type 1 (HIV-1) promotes virus entry by sequentially binding CD4 and the CCR5 chemokine receptor on the target cell. Previously, we adapted a primary HIV-1 isolate, ADA, to replicate in CD4-negative canine cells expressing human CCR5. The gp120 changes responsible for CD4-independent replication were limited to the V2 loop-V1/V2 stem. Here we show that elimination of a single glycosylation site at asparagine 197 in the V1/V2 stem is sufficient for CD4-independent gp120 binding to CCR5 and for HIV-1 entry into CD4-negative cells expressing CCR5. Deletion of the V1/V2 loops also allowed CD4-independent viral entry and gp120 binding to CCR5. The binding of the wild-type ADA gp120 to CCR5 was less dependent upon CD4 at 4 degrees C than at 37 degrees C. In the absence of the V1/V2 loops, neither removal of the N-linked carbohydrate at asparagine 197 nor lowering of the temperature increased the CD4-independent phenotypes. A CCR5-binding conformation of gp120, achieved by CD4 interaction or by modification of temperature, glycosylation, or variable loops, was preferentially recognized by the monoclonal antibody 48d. These results suggest that the CCR5-binding region of gp120 is occluded by the V1/V2 variable loops, the position of which can be modulated by temperature, CD4 binding, or an N-linked glycan in the V1/V2 stem.

Time resolved native ion-mobility mass spectrometry to monitor dynamics of IgG4 Fab arm exchange and "bispecific" monoclonal antibody formation.

PubMed

Debaene, François; Wagner-Rousset, Elsa; Colas, Olivier; Ayoub, Daniel; Corvaïa, Nathalie; Van Dorsselaer, Alain; Beck, Alain; Cianférani, Sarah

2013-10-15

Monoclonal antibodies (mAbs) and derivatives such as antibody-drug conjugates (ADC) and bispecific antibodies (bsAb), are the fastest growing class of human therapeutics. Most of the therapeutic antibodies currently on the market and in clinical trials are chimeric, humanized, and human immunoglobulin G1 (IgG1). An increasing number of IgG2s and IgG4s that have distinct structural and functional properties are also investigated to develop products that lack or have diminished antibody effector functions compared to IgG1. Importantly, wild type IgG4 has been shown to form half molecules (one heavy chain and one light chain) that lack interheavy chain disulfide bonds and form intrachain disulfide bonds. Moreover, IgG4 undergoes a process of Fab-arm exchange (FAE) in which the heavy chains of antibodies of different specificities can dissociate and recombine in bispecific antibodies both in vitro and in vivo. Here, native mass spectrometry (MS) and time-resolved traveling wave ion mobility MS (TWIM-MS) were used for the first time for online monitoring of FAE and bsAb formation using Hz6F4-2v3 and natalizumab, two humanized IgG4s which bind to human Junctional Adhesion Molecule-A (JAM-A) and alpha4 integrin, respectively. In addition, native MS analysis of bsAb/JAM-A immune complexes revealed that bsAb can bind up to two antigen molecules, confirming that the Hz6F4 family preferentially binds dimeric JAM-A. Our results illustrate how IM-MS can rapidly assess bsAb structural heterogeneity and be easily implemented into MS workflows for bsAb production follow up and bsAb/antigen complex characterization. Altogether, these results provide new MS-based methodologies for in-depth FAE and bsAb formation monitoring. Native MS and IM-MS will play an increasing role in next generation biopharmaceutical product characterization like bsAbs, antibody mixtures, and antibody-drug conjugates (ADC) as well as for biosimilar and biobetter antibodies.

CpG methylation of HPV 16 LCR at E2 binding site proximal to P97 is associated with cervical cancer in presence of intact E2.

PubMed

Bhattacharjee, Bornali; Sengupta, Sharmila

2006-10-25

Human papillomavirus type 16 (HPV-16) E2 protein negatively regulates transcription of the E6 and E7 genes. This study was done to test the hypothesis that methylation of the HPV 16 long control region (LCR) is overrepresented among cervical cancer (CaCx) cases compared to cytologically normal controls harboring intact E2 gene. Methylation of the E2 binding site (E2BS-I), proximal to the P97 promoter, was assessed by HpaII/ MspI restriction digestion while McrBC digestion was used to assess LCR-E6 (7289-540) for 57 CaCx samples and 15 normal controls. E2BS-I methylation was found to be significantly higher (56.14%) in cases compared to (20%) controls [OR(age-adjusted) (95% CI): 4.53 (1.05-19.43) p=0.042]. The difference between cases (54.39%) and controls (40%) with respect to LCR-E6 methylation status [OR(age-adjusted) (95% CI): 1.77(0.5-6.3); p=0.38] was not significant. Sequencing of a randomly selected set of 13 methylated malignant samples revealed absence or rare presence, of methylation at CpGs 7579, 7535, 7683 and 7862 respectively. Methylation was found to be more at CpGs within E2 binding sites proximal to the P97 promoter. These results indicate the involvement of E2 binding site methylation in presence of intact E2, leading to loss of E2 repressor activity in CaCx.

Editing of misaligned 3'-termini by an intrinsic 3'-5' exonuclease activity residing in the PHP domain of a family X DNA polymerase.

PubMed

Baños, Benito; Lázaro, José M; Villar, Laurentino; Salas, Margarita; de Vega, Miguel

2008-10-01

Bacillus subtilis gene yshC encodes a family X DNA polymerase (PolX(Bs)), whose biochemical features suggest that it plays a role during DNA repair processes. Here, we show that, in addition to the polymerization activity, PolX(Bs) possesses an intrinsic 3'-5' exonuclease activity specialized in resecting unannealed 3'-termini in a gapped DNA substrate. Biochemical analysis of a PolX(Bs) deletion mutant lacking the C-terminal polymerase histidinol phosphatase (PHP) domain, present in most of the bacterial/archaeal PolXs, as well as of this separately expressed protein region, allow us to state that the 3'-5' exonuclease activity of PolX(Bs) resides in its PHP domain. Furthermore, site-directed mutagenesis of PolX(Bs) His339 and His341 residues, evolutionary conserved in the PHP superfamily members, demonstrated that the predicted metal binding site is directly involved in catalysis of the exonucleolytic reaction. The implications of the unannealed 3'-termini resection by the 3'-5' exonuclease activity of PolX(Bs) in the DNA repair context are discussed.

Species B adenovirus serotypes 3, 7, 11 and 35 share similar binding sites on the membrane cofactor protein CD46 receptor.

PubMed

Fleischli, Christoph; Sirena, Dominique; Lesage, Guillaume; Havenga, Menzo J E; Cattaneo, Roberto; Greber, Urs F; Hemmi, Silvio

2007-11-01

We recently characterized the domains of the human cofactor protein CD46 involved in binding species B2 adenovirus (Ad) serotype 35. Here, the CD46 binding determinants are mapped for the species B1 Ad serotypes 3 and 7 and for the species B2 Ad11. Ad3, 7 and 11 bound and transduced CD46-positive rodent BHK cells at levels similar to Ad35. By using antibody-blocking experiments, hybrid CD46-CD4 receptor constructs and CD46 single point mutants, it is shown that Ad3, 7 and 11 share many of the Ad35-binding features on CD46. Both CD46 short consensus repeat domains SCR I and SCR II were necessary and sufficient for optimal binding and transgene expression, provided that they were positioned at an appropriate distance from the cell membrane. Similar to Ad35, most of the putative binding residues of Ad3, 7 and 11 were located on the same glycan-free, solvent-exposed face of the SCR I or SCR II domains, largely overlapping with the binding surface of the recently solved fiber knob Ad11-SCR I-II three-dimensional structure. Differences between species B1 and B2 Ads were documented with competition experiments based on anti-CD46 antibodies directed against epitopes flanking the putative Ad-binding sites, and with competition experiments based on soluble CD46 protein. It is concluded that the B1 and B2 species of Ad engage CD46 through similar binding surfaces.

Interfacial metal and antibody recognition.

PubMed

Zhou, Tongqing; Hamer, Dean H; Hendrickson, Wayne A; Sattentau, Quentin J; Kwong, Peter D

2005-10-11

The unique ligation properties of metal ions are widely exploited by proteins, with approximately one-third of all proteins estimated to be metalloproteins. Although antibodies use various mechanisms for recognition, to our knowledge, none has ever been characterized that uses an interfacial metal. We previously described a family of CD4-reactive antibodies, the archetype being Q425. CD4:Q425 engagement does not interfere with CD4:HIV-1 gp120 envelope glycoprotein binding, but it blocks subsequent steps required for viral entry. Here, we use surface-plasmon resonance to show that Q425 requires calcium for recognition of CD4. Specifically, Q425 binding of calcium resulted in a 55,000-fold enhancement in affinity for CD4. X-ray crystallographic analyses of Q425 in the presence of Ca(2+), Ba(2+), or EDTA revealed an exposed metal-binding site, partially coordinated by five atoms contributed from four antibody complementarity-determining regions. The results suggest that Q425 recognition of CD4 involves direct ligation of antigen by the Q425-held calcium, with calcium binding each ligating atom of CD4 with approximately 1.5 kcal/mol of binding energy. This energetic contribution, which is greater than that from a typical protein atom, demonstrates how interfacial metal ligation can play a unique role in antigen recognition.

Interfacial metal and antibody recognition

PubMed Central

Zhou, Tongqing; Hamer, Dean H.; Hendrickson, Wayne A.; Sattentau, Quentin J.; Kwong, Peter D.

2005-01-01

The unique ligation properties of metal ions are widely exploited by proteins, with approximately one-third of all proteins estimated to be metalloproteins. Although antibodies use various mechanisms for recognition, to our knowledge, none has ever been characterized that uses an interfacial metal. We previously described a family of CD4-reactive antibodies, the archetype being Q425. CD4:Q425 engagement does not interfere with CD4:HIV-1 gp120 envelope glycoprotein binding, but it blocks subsequent steps required for viral entry. Here, we use surface-plasmon resonance to show that Q425 requires calcium for recognition of CD4. Specifically, Q425 binding of calcium resulted in a 55,000-fold enhancement in affinity for CD4. X-ray crystallographic analyses of Q425 in the presence of Ca2+, Ba2+, or EDTA revealed an exposed metal-binding site, partially coordinated by five atoms contributed from four antibody complementarity-determining regions. The results suggest that Q425 recognition of CD4 involves direct ligation of antigen by the Q425-held calcium, with calcium binding each ligating atom of CD4 with ≈1.5 kcal/mol of binding energy. This energetic contribution, which is greater than that from a typical protein atom, demonstrates how interfacial metal ligation can play a unique role in antigen recognition. PMID:16195378

Controlling and fine tuning the physical properties of two identical metal coordination sites in de novo designed three stranded coiled coil peptides.

PubMed

Iranzo, Olga; Chakraborty, Saumen; Hemmingsen, Lars; Pecoraro, Vincent L

2011-01-19

Herein we report how de novo designed peptides can be used to investigate whether the position of a metal site along a linear sequence that folds into a three-stranded α-helical coiled coil defines the physical properties of Cd(II) ions in either CdS(3) or CdS(3)O (O-being an exogenous water molecule) coordination environments. Peptides are presented that bind Cd(II) into two identical coordination sites that are located at different topological positions at the interior of these constructs. The peptide GRANDL16PenL19IL23PenL26I binds two Cd(II) as trigonal planar 3-coordinate CdS(3) structures whereas GRANDL12AL16CL26AL30C sequesters two Cd(II) as pseudotetrahedral 4-coordinate CdS(3)O structures. We demonstrate how for the first peptide, having a more rigid structure, the location of the identical binding sites along the linear sequence does not affect the physical properties of the two bound Cd(II). However, the sites are not completely independent as Cd(II) bound to one of the sites ((113)Cd NMR chemical shift of 681 ppm) is perturbed by the metalation state (apo or [Cd(pep)(Hpep)(2)](+) or [Cd(pep)(3)](-)) of the second center ((113)Cd NMR chemical shift of 686 ppm). GRANDL12AL16CL26AL30C shows a completely different behavior. The physical properties of the two bound Cd(II) ions indeed depend on the position of the metal center, having pK(a2) values for the equilibrium [Cd(pep)(Hpep)(2)](+) → [Cd(pep)(3)](-) + 2H(+) (corresponding to deprotonation and coordination of cysteine thiols) that range from 9.9 to 13.9. In addition, the L26AL30C site shows dynamic behavior, which is not observed for the L12AL16C site. These results indicate that for these systems one cannot simply assign a "4-coordinate structure" and assume certain physical properties for that site since important factors such as packing of the adjacent Leu, size of the intended cavity (endo vs exo) and location of the metal site play crucial roles in determining the final properties of the bound Cd(II).

Noninvasive brain cancer imaging with a bispecific antibody fragment, generated via click chemistry.

PubMed

Luo, Haiming; Hernandez, Reinier; Hong, Hao; Graves, Stephen A; Yang, Yunan; England, Christopher G; Theuer, Charles P; Nickles, Robert J; Cai, Weibo

2015-10-13

Early diagnosis remains a task of upmost importance for reducing cancer morbidity and mortality. Successful development of highly specific companion diagnostics targeting aberrant molecular pathways of cancer is needed for sensitive detection, accurate diagnosis, and opportune therapeutic intervention. Herein, we generated a bispecific immunoconjugate [denoted as Bs-F(ab)2] by linking two antibody Fab fragments, an anti-epidermal growth factor receptor (EGFR) Fab and an anti-CD105 Fab, via bioorthogonal "click" ligation of trans-cyclooctene and tetrazine. PET imaging of mice bearing U87MG (EGFR/CD105(+/+)) tumors with (64)Cu-labeled Bs-F(ab)2 revealed a significantly enhanced tumor uptake [42.9 ± 9.5 percentage injected dose per gram (%ID/g); n = 4] and tumor-to-background ratio (tumor/muscle ratio of 120.2 ± 44.4 at 36 h postinjection; n = 4) compared with each monospecific Fab tracer. Thus, we demonstrated that dual targeting of EGFR and CD105 provides a synergistic improvement on both affinity and specificity of (64)Cu-NOTA-Bs-F(ab)2. (64)Cu-NOTA-Bs-F(ab)2 was able to visualize small U87MG tumor nodules (<5 mm in diameter), owing to high tumor uptake (31.4 ± 10.8%ID/g at 36 h postinjection) and a tumor/muscle ratio of 76.4 ± 52.3, which provided excellent sensitivity for early detection. Finally, we successfully confirmed the feasibility of a ZW800-1-labeled Bs-F(ab)2 for near-infrared fluorescence imaging and image-guided surgical resection of U87MG tumors. More importantly, our rationale can be used in the construction of other disease-targeting bispecific antibody fragments for early detection and diagnosis of small malignant lesions.

Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody

PubMed Central

Kwong, Peter D.; Wyatt, Richard; Robinson, James; Sweet, Raymond W.; Sodroski, Joseph; Hendrickson, Wayne A.

2017-01-01

The entry of human immunodeficiency virus (HIV) into cells requires the sequential interaction of the viral exterior envelope glycoprotein, gp120, with the CD4 glycoprotein and a chemokine receptor on the cell surface. These interactions initiate a fusion of the viral and cellular membranes. Although gpl20 can elicit virus-neutralizing antibodies, HIV eludes the immune system. We have solved the X-ray crystal structure at 2.5 Å resolution of an HIV-1 gp120 core complexed with a two-domain fragment of human CD4 and an antigen-binding fragment of a neutralizing antibody that blocks chemokine-receptor binding. The structure reveals a cavity-laden CD4-gp120 interface, a conserved binding site for the chemokine receptor, evidence for a conformational change upon CD4 binding, the nature of a CD4-induced antibody epitope, and specific mechanisms for immune evasion. Our results provide a framework for understanding the complex biology of HIV entry into cells and should guide efforts to intervene. PMID:9641677

PTGER4 Expression-Modulating Polymorphisms in the 5p13.1 Region Predispose to Crohn's Disease and Affect NF-κB and XBP1 Binding Sites

PubMed Central

Czamara, Darina; Pasciuto, Giulia; Diegelmann, Julia; Wetzke, Martin; Olszak, Torsten; Wolf, Christiane; Müller-Myhsok, Bertram; Balschun, Tobias; Achkar, Jean-Paul; Kamboh, M. Ilyas; Franke, Andre; Duerr, Richard H.; Brand, Stephan

2012-01-01

Background Genome-wide association studies identified a PTGER4 expression-modulating region on chromosome 5p13.1 as Crohn's disease (CD) susceptibility region. The study aim was to test this association in a large cohort of patients with inflammatory bowel disease (IBD) and to elucidate genotypic and phenotypic interactions with other IBD genes. Methodology/Principal Findings A total of 7073 patients and controls were genotyped: 844 CD and 471 patients with ulcerative colitis and 1488 controls were analyzed for the single nucleotide polymorphisms (SNPs) rs4495224 and rs7720838 on chromosome 5p13.1. The study included two replication cohorts of North American (CD: n = 684; controls: n = 1440) and of German origin (CD: n = 1098; controls: n = 1048). Genotype-phenotype, epistasis and transcription factor binding analyses were performed. In the discovery cohort, an association of rs4495224 (p = 4.10×10−5; 0.76 [0.67–0.87]) and of rs7720838 (p = 6.91×10−4; 0.81 [0.71–0.91]) with susceptibility to CD was demonstrated. These associations were confirmed in both replication cohorts. In silico analysis predicted rs4495224 and rs7720838 as essential parts of binding sites for the transcription factors NF-κB and XBP1 with higher binding scores for carriers of the CD risk alleles, providing an explanation of how these SNPs might contribute to increased PTGER4 expression. There was no association of the PTGER4 SNPs with IBD phenotypes. Epistasis detected between 5p13.1 and ATG16L1 for CD susceptibility in the discovery cohort (p = 5.99×10−7 for rs7720838 and rs2241880) could not be replicated in both replication cohorts arguing against a major role of this gene-gene interaction in the susceptibility to CD. Conclusions/Significance We confirmed 5p13.1 as a major CD susceptibility locus and demonstrate by in silico analysis rs4495224 and rs7720838 as part of binding sites for NF-κB and XBP1. Further functional studies are necessary to confirm the results of our in silico analysis and to analyze if changes in PTGER4 expression modulate CD susceptibility. PMID:23300802

A novel RNA binding protein affects rbcL gene expression and is specific to bundle sheath chloroplasts in C4 plants

PubMed Central

2013-01-01

Background Plants that utilize the highly efficient C4 pathway of photosynthesis typically possess kranz-type leaf anatomy that consists of two morphologically and functionally distinct photosynthetic cell types, the bundle sheath (BS) and mesophyll (M) cells. These two cell types differentially express many genes that are required for C4 capability and function. In mature C4 leaves, the plastidic rbcL gene, encoding the large subunit of the primary CO2 fixation enzyme Rubisco, is expressed specifically within BS cells. Numerous studies have demonstrated that BS-specific rbcL gene expression is regulated predominantly at post-transcriptional levels, through the control of translation and mRNA stability. The identification of regulatory factors associated with C4 patterns of rbcL gene expression has been an elusive goal for many years. Results RLSB, encoded by the nuclear RLSB gene, is an S1-domain RNA binding protein purified from C4 chloroplasts based on its specific binding to plastid-encoded rbcL mRNA in vitro. Co-localized with LSU to chloroplasts, RLSB is highly conserved across many plant species. Most significantly, RLSB localizes specifically to leaf bundle sheath (BS) cells in C4 plants. Comparative analysis using maize (C4) and Arabidopsis (C3) reveals its tight association with rbcL gene expression in both plants. Reduced RLSB expression (through insertion mutation or RNA silencing, respectively) led to reductions in rbcL mRNA accumulation and LSU production. Additional developmental effects, such as virescent/yellow leaves, were likely associated with decreased photosynthetic function and disruption of associated signaling networks. Conclusions Reductions in RLSB expression, due to insertion mutation or gene silencing, are strictly correlated with reductions in rbcL gene expression in both maize and Arabidopsis. In both plants, accumulation of rbcL mRNA as well as synthesis of LSU protein were affected. These findings suggest that specific accumulation and binding of the RLSB binding protein to rbcL mRNA within BS chloroplasts may be one determinant leading to the characteristic cell type-specific localization of Rubisco in C4 plants. Evolutionary modification of RLSB expression, from a C3 “default” state to BS cell-specificity, could represent one mechanism by which rbcL expression has become restricted to only one cell type in C4 plants. PMID:24053212

Co-receptor Binding Site Antibodies Enable CD4-Mimetics to Expose Conserved Anti-cluster A ADCC Epitopes on HIV-1 Envelope Glycoproteins.

PubMed

Richard, Jonathan; Pacheco, Beatriz; Gohain, Neelakshi; Veillette, Maxime; Ding, Shilei; Alsahafi, Nirmin; Tolbert, William D; Prévost, Jérémie; Chapleau, Jean-Philippe; Coutu, Mathieu; Jia, Manxue; Brassard, Nathalie; Park, Jongwoo; Courter, Joel R; Melillo, Bruno; Martin, Loïc; Tremblay, Cécile; Hahn, Beatrice H; Kaufmann, Daniel E; Wu, Xueling; Smith, Amos B; Sodroski, Joseph; Pazgier, Marzena; Finzi, Andrés

2016-10-01

Human immunodeficiency virus type 1 (HIV-1) has evolved a sophisticated strategy to conceal conserved epitopes of its envelope glycoproteins (Env) recognized by antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies. These antibodies, which are present in the sera of most HIV-1-infected individuals, preferentially recognize Env in its CD4-bound conformation. Accordingly, recent studies showed that small CD4-mimetics (CD4mc) able to "push" Env into this conformation sensitize HIV-1-infected cells to ADCC mediated by HIV+ sera. Here we test whether CD4mc also expose epitopes recognized by anti-cluster A monoclonal antibodies such as A32, thought to be responsible for the majority of ADCC activity present in HIV+ sera and linked to decreased HIV-1 transmission in the RV144 trial. We made the surprising observation that CD4mc are unable to enhance recognition of HIV-1-infected cells by this family of antibodies in the absence of antibodies such as 17b, which binds a highly conserved CD4-induced epitope overlapping the co-receptor binding site (CoRBS). Our results indicate that CD4mc initially open the trimeric Env enough to allow the binding of CoRBS antibodies but not anti-cluster A antibodies. CoRBS antibody binding further opens the trimeric Env, allowing anti-cluster A antibody interaction and sensitization of infected cells to ADCC. Therefore, ADCC responses mediated by cluster A antibodies in HIV-positive sera involve a sequential opening of the Env trimer on the surface of HIV-1-infected cells. The understanding of the conformational changes required to expose these vulnerable Env epitopes might be important in the design of new strategies aimed at fighting HIV-1. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Boosting of HIV envelope CD4 binding site antibodies with long variable heavy third complementarity determining region in the randomized double blind RV305 HIV-1 vaccine trial

PubMed Central

Ackerman, Margaret; Saunders, Kevin O.; Pollara, Justin; Vandergrift, Nathan; Parks, Rob; Michael, Nelson L.; O’Connell, Robert J.; Vasan, Sandhya; Rerks-Ngarm, Supachai; Kaewkungwal, Jaranit; Pitisuttithum, Punnee; Nitayaphan, Sorachai; Sinangil, Faruk; Phogat, Sanjay; Alam, S. Munir; Liao, Hua-Xin; Ferrari, Guido; Seaman, Michael S.; Montefiori, David C.; Harrison, Stephen C.; Haynes, Barton F.

2017-01-01

The canary pox vector and gp120 vaccine (ALVAC-HIV and AIDSVAX B/E gp120) in the RV144 HIV-1 vaccine trial conferred an estimated 31% vaccine efficacy. Although the vaccine Env AE.A244 gp120 is antigenic for the unmutated common ancestor of V1V2 broadly neutralizing antibody (bnAbs), no plasma bnAb activity was induced. The RV305 (NCT01435135) HIV-1 clinical trial was a placebo-controlled randomized double-blinded study that assessed the safety and efficacy of vaccine boosting on B cell repertoires. HIV-1-uninfected RV144 vaccine recipients were reimmunized 6–8 years later with AIDSVAX B/E gp120 alone, ALVAC-HIV alone, or a combination of ALVAC-HIV and AIDSVAX B/E gp120 in the RV305 trial. Env-specific post-RV144 and RV305 boost memory B cell VH mutation frequencies increased from 2.9% post-RV144 to 6.7% post-RV305. The vaccine was well tolerated with no adverse events reports. While post-boost plasma did not have bnAb activity, the vaccine boosts expanded a pool of envelope CD4 binding site (bs)-reactive memory B cells with long third heavy chain complementarity determining regions (HCDR3) whose germline precursors and affinity matured B cell clonal lineage members neutralized the HIV-1 CRF01 AE tier 2 (difficult to neutralize) primary isolate, CNE8. Electron microscopy of two of these antibodies bound with near-native gp140 trimers showed that they recognized an open conformation of the Env trimer. Although late boosting of RV144 vaccinees expanded a novel pool of neutralizing B cell clonal lineages, we hypothesize that boosts with stably closed trimers would be necessary to elicit antibodies with greater breadth of tier 2 HIV-1 strains. Trial Registration: ClinicalTrials.gov NCT01435135 PMID:28235027

Boosting of HIV envelope CD4 binding site antibodies with long variable heavy third complementarity determining region in the randomized double blind RV305 HIV-1 vaccine trial.

PubMed

Easterhoff, David; Moody, M Anthony; Fera, Daniela; Cheng, Hao; Ackerman, Margaret; Wiehe, Kevin; Saunders, Kevin O; Pollara, Justin; Vandergrift, Nathan; Parks, Rob; Kim, Jerome; Michael, Nelson L; O'Connell, Robert J; Excler, Jean-Louis; Robb, Merlin L; Vasan, Sandhya; Rerks-Ngarm, Supachai; Kaewkungwal, Jaranit; Pitisuttithum, Punnee; Nitayaphan, Sorachai; Sinangil, Faruk; Tartaglia, James; Phogat, Sanjay; Kepler, Thomas B; Alam, S Munir; Liao, Hua-Xin; Ferrari, Guido; Seaman, Michael S; Montefiori, David C; Tomaras, Georgia D; Harrison, Stephen C; Haynes, Barton F

2017-02-01

The canary pox vector and gp120 vaccine (ALVAC-HIV and AIDSVAX B/E gp120) in the RV144 HIV-1 vaccine trial conferred an estimated 31% vaccine efficacy. Although the vaccine Env AE.A244 gp120 is antigenic for the unmutated common ancestor of V1V2 broadly neutralizing antibody (bnAbs), no plasma bnAb activity was induced. The RV305 (NCT01435135) HIV-1 clinical trial was a placebo-controlled randomized double-blinded study that assessed the safety and efficacy of vaccine boosting on B cell repertoires. HIV-1-uninfected RV144 vaccine recipients were reimmunized 6-8 years later with AIDSVAX B/E gp120 alone, ALVAC-HIV alone, or a combination of ALVAC-HIV and AIDSVAX B/E gp120 in the RV305 trial. Env-specific post-RV144 and RV305 boost memory B cell VH mutation frequencies increased from 2.9% post-RV144 to 6.7% post-RV305. The vaccine was well tolerated with no adverse events reports. While post-boost plasma did not have bnAb activity, the vaccine boosts expanded a pool of envelope CD4 binding site (bs)-reactive memory B cells with long third heavy chain complementarity determining regions (HCDR3) whose germline precursors and affinity matured B cell clonal lineage members neutralized the HIV-1 CRF01 AE tier 2 (difficult to neutralize) primary isolate, CNE8. Electron microscopy of two of these antibodies bound with near-native gp140 trimers showed that they recognized an open conformation of the Env trimer. Although late boosting of RV144 vaccinees expanded a novel pool of neutralizing B cell clonal lineages, we hypothesize that boosts with stably closed trimers would be necessary to elicit antibodies with greater breadth of tier 2 HIV-1 strains. ClinicalTrials.gov NCT01435135.

PTGER4 expression-modulating polymorphisms in the 5p13.1 region predispose to Crohn's disease and affect NF-κB and XBP1 binding sites.

PubMed

Glas, Jürgen; Seiderer, Julia; Czamara, Darina; Pasciuto, Giulia; Diegelmann, Julia; Wetzke, Martin; Olszak, Torsten; Wolf, Christiane; Müller-Myhsok, Bertram; Balschun, Tobias; Achkar, Jean-Paul; Kamboh, M Ilyas; Franke, Andre; Duerr, Richard H; Brand, Stephan

2012-01-01

Genome-wide association studies identified a PTGER4 expression-modulating region on chromosome 5p13.1 as Crohn's disease (CD) susceptibility region. The study aim was to test this association in a large cohort of patients with inflammatory bowel disease (IBD) and to elucidate genotypic and phenotypic interactions with other IBD genes. A total of 7073 patients and controls were genotyped: 844 CD and 471 patients with ulcerative colitis and 1488 controls were analyzed for the single nucleotide polymorphisms (SNPs) rs4495224 and rs7720838 on chromosome 5p13.1. The study included two replication cohorts of North American (CD: n = 684; controls: n = 1440) and of German origin (CD: n = 1098; controls: n = 1048). Genotype-phenotype, epistasis and transcription factor binding analyses were performed. In the discovery cohort, an association of rs4495224 (p = 4.10×10⁻⁵; 0.76 [0.67-0.87]) and of rs7720838 (p = 6.91×10⁻⁴; 0.81 [0.71-0.91]) with susceptibility to CD was demonstrated. These associations were confirmed in both replication cohorts. In silico analysis predicted rs4495224 and rs7720838 as essential parts of binding sites for the transcription factors NF-κB and XBP1 with higher binding scores for carriers of the CD risk alleles, providing an explanation of how these SNPs might contribute to increased PTGER4 expression. There was no association of the PTGER4 SNPs with IBD phenotypes. Epistasis detected between 5p13.1 and ATG16L1 for CD susceptibility in the discovery cohort (p = 5.99×10⁻⁷ for rs7720838 and rs2241880) could not be replicated in both replication cohorts arguing against a major role of this gene-gene interaction in the susceptibility to CD. We confirmed 5p13.1 as a major CD susceptibility locus and demonstrate by in silico analysis rs4495224 and rs7720838 as part of binding sites for NF-κB and XBP1. Further functional studies are necessary to confirm the results of our in silico analysis and to analyze if changes in PTGER4 expression modulate CD susceptibility.

Thermal Adaptation of Dihydrofolate Reductase from the Moderate Thermophile Geobacillus stearothermophilus

PubMed Central

2014-01-01

The thermal melting temperature of dihydrofolate reductase from Geobacillus stearothermophilus (BsDHFR) is ∼30 °C higher than that of its homologue from the psychrophile Moritella profunda. Additional proline residues in the loop regions of BsDHFR have been proposed to enhance the thermostability of BsDHFR, but site-directed mutagenesis studies reveal that these proline residues contribute only minimally. Instead, the high thermal stability of BsDHFR is partly due to removal of water-accessible thermolabile residues such as glutamine and methionine, which are prone to hydrolysis or oxidation at high temperatures. The extra thermostability of BsDHFR can be obtained by ligand binding, or in the presence of salts or cosolvents such as glycerol and sucrose. The sum of all these incremental factors allows BsDHFR to function efficiently in the natural habitat of G. stearothermophilus, which is characterized by temperatures that can reach 75 °C. PMID:24730604

Incomplete Neutralization and Deviation from Sigmoidal Neutralization Curves for HIV Broadly Neutralizing Monoclonal Antibodies

PubMed Central

McCoy, Laura E.; Falkowska, Emilia; Doores, Katie J.; Le, Khoa; Sok, Devin; van Gils, Marit J.; Euler, Zelda; Burger, Judith A.; Seaman, Michael S.; Sanders, Rogier W.; Schuitemaker, Hanneke; Poignard, Pascal; Wrin, Terri; Burton, Dennis R.

2015-01-01

The broadly neutralizing HIV monoclonal antibodies (bnMAbs) PG9, PG16, PGT151, and PGT152 have been shown earlier to occasionally display an unusual virus neutralization profile with a non-sigmoidal slope and a plateau at <100% neutralization. In the current study, we were interested in determining the extent of non-sigmoidal slopes and plateaus at <100% for HIV bnMAbs more generally. Using both a 278 panel of pseudoviruses in a CD4 T-cell (U87.CCR5.CXCR4) assay and a panel of 117 viruses in the TZM-bl assay, we found that bnMAbs targeting many neutralizing epitopes of the spike had neutralization profiles for at least one virus that plateaued at <90%. Across both panels the bnMAbs targeting the V2 apex of Env and gp41 were most likely to show neutralization curves that plateaued <100%. Conversely, bnMAbs targeting the high-mannose patch epitopes were less likely to show such behavior. Two CD4 binding site (CD4bs) Abs also showed this behavior relatively infrequently. The phenomenon of incomplete neutralization was also observed in a large peripheral blood mononuclear cells (PBMC)-grown molecular virus clone panel derived from patient viral swarms. In addition, five bnMAbs were compared against an 18-virus panel of molecular clones produced in 293T cells and PBMCs and assayed in TZM-bl cells. Examples of plateaus <90% were seen with both types of virus production with no consistent patterns observed. In conclusion, incomplete neutralization and non-sigmoidal neutralization curves are possible for all HIV bnMAbs against a wide range of viruses produced and assayed in both cell lines and primary cells with implications for the use of antibodies in therapy and as tools for vaccine design. PMID:26267277

Incomplete Neutralization and Deviation from Sigmoidal Neutralization Curves for HIV Broadly Neutralizing Monoclonal Antibodies.

PubMed

McCoy, Laura E; Falkowska, Emilia; Doores, Katie J; Le, Khoa; Sok, Devin; van Gils, Marit J; Euler, Zelda; Burger, Judith A; Seaman, Michael S; Sanders, Rogier W; Schuitemaker, Hanneke; Poignard, Pascal; Wrin, Terri; Burton, Dennis R

2015-08-01

The broadly neutralizing HIV monoclonal antibodies (bnMAbs) PG9, PG16, PGT151, and PGT152 have been shown earlier to occasionally display an unusual virus neutralization profile with a non-sigmoidal slope and a plateau at <100% neutralization. In the current study, we were interested in determining the extent of non-sigmoidal slopes and plateaus at <100% for HIV bnMAbs more generally. Using both a 278 panel of pseudoviruses in a CD4 T-cell (U87.CCR5.CXCR4) assay and a panel of 117 viruses in the TZM-bl assay, we found that bnMAbs targeting many neutralizing epitopes of the spike had neutralization profiles for at least one virus that plateaued at <90%. Across both panels the bnMAbs targeting the V2 apex of Env and gp41 were most likely to show neutralization curves that plateaued <100%. Conversely, bnMAbs targeting the high-mannose patch epitopes were less likely to show such behavior. Two CD4 binding site (CD4bs) Abs also showed this behavior relatively infrequently. The phenomenon of incomplete neutralization was also observed in a large peripheral blood mononuclear cells (PBMC)-grown molecular virus clone panel derived from patient viral swarms. In addition, five bnMAbs were compared against an 18-virus panel of molecular clones produced in 293T cells and PBMCs and assayed in TZM-bl cells. Examples of plateaus <90% were seen with both types of virus production with no consistent patterns observed. In conclusion, incomplete neutralization and non-sigmoidal neutralization curves are possible for all HIV bnMAbs against a wide range of viruses produced and assayed in both cell lines and primary cells with implications for the use of antibodies in therapy and as tools for vaccine design.

64Cu-Labeled Trastuzumab Fab-PEG24-EGF Radioimmunoconjugates Bispecific for HER2 and EGFR: Pharmacokinetics, Biodistribution, and Tumor Imaging by PET in Comparison to Monospecific Agents.

PubMed

Kwon, Luke Yongkyu; Scollard, Deborah A; Reilly, Raymond M

2017-02-06

Heterodimerization of EGFR with HER2 coexpressed in breast cancer (BC) promotes tumor growth, and increased EGFR expression is associated with trastuzumab resistance. Our aim was to construct 64 Cu-labeled bispecific radioimmunoconjugates (bsRIC) composed of trastuzumab Fab, which binds HER2 linked through a polyethylene glycol (PEG 24 ) spacer to EGF, and to compare their pharmacokinetic, biodistribution, and tumor imaging characteristics by positron-emission tomography (PET). bsRICs were generated by linking maleimide modified trastuzumab Fab with thiolated EGF through a thioether bond. HER2 and EGFR binding were assessed in vitro in MDA-MB-231 (EGFR mod /HER2 low ), MDA-MB-468 (EGFR high /HER2 neg ), MDA-MB-231-H2N (EGFR mod /HER2 mod ), and SKOV3 (EGFR low /HER2 high ) cells by competition and saturation cell binding assays to estimate the dissociation constant (K d ). The elimination of the 64 Cu-NOTA-trastuzumab Fab-PEG 24 -EGF bsRICs from the blood of Balb/c mice was compared to monospecific 64 Cu-NOTA-trastuzumab Fab and 64 Cu-NOTA-EGF. MicroPET/CT imaging was performed in NOD/SCID mice bearing subcutaneous MDA-MB-468, MDA-MB-231/H2N, or SKOV3 human BC xenografts at 24 and 48 h postinjection (p.i.) of bsRICs. Tumor and normal tissue uptake were quantified by biodistribution studies and compared to monospecific agents. The binding of bsRICs to MDA-MB-231 cells was decreased to 24.5 ± 5.2% by excess EGF, while the binding of bsRICs to SKOV3 cells was decreased to 38.6 ± 5.4% by excess trastuzumab Fab, demonstrating specific binding to both EGFR and HER2. 64 Cu-labeled bsRICs incorporating the PEG 24 spacer were eliminated more slowly from the blood than 64 Cu-bsRICs without the PEG spacer and were cleared much more slowly than 64 Cu-NOTA-Fab or 64 Cu-NOTA-EGF. All three tumor xenografts were visualized by microPET/CT at 24 and 48 h p.i. of bsRICs. Biodistribution studies at 48 h p.i. in NOD/SCID mice with MDA-MB-231/H2N tumors demonstrated significantly greater tumor uptake of 64 Cu-NOTA-Fab-PEG 24 -EGF (4.9 ± 0.4%ID/g) than 64 Cu-NOTA-Fab (1.9 ± 0.3%ID/g; P < 0.0001) and 64 Cu-NOTA-EGF (0.7 ± 0.2%ID/g; P < 0.0001). Furthermore, preadministration of an excess of trastuzumab Fab or trastuzumab Fab-PEG 24 -EGF significantly decreased the tumor uptake of 64 Cu-NOTA-Fab-PEG 24 -EGF in SK-OV-3 and MDA-MB-468 xenografts by 4.4-fold (P = 0.0012) and 1.8-fold (P = 0.0031), respectively. 64 Cu-labeled bsRICs bound HER2 or EGFR and were taken up specifically in vivo in tumor xenografts expressing one or both receptors. The PEG 24 linker prolonged the blood residence time contributing to the higher tumor uptake of the bsRICs than monospecific agents.

Expression of the Bs2 pepper gene confers resistance to bacterial spot disease in tomato.

PubMed

Tai, T H; Dahlbeck, D; Clark, E T; Gajiwala, P; Pasion, R; Whalen, M C; Stall, R E; Staskawicz, B J

1999-11-23

The Bs2 resistance gene of pepper specifically recognizes and confers resistance to strains of Xanthomonas campestris pv. vesicatoria that contain the corresponding bacterial avirulence gene, avrBs2. The involvement of avrBs2 in pathogen fitness and its prevalence in many X. campestris pathovars suggests that the Bs2 gene may be durable in the field and provide resistance when introduced into other plant species. Employing a positional cloning strategy, the Bs2 locus was isolated and the gene was identified by coexpression with avrBs2 in an Agrobacterium-mediated transient assay. A single candidate gene, predicted to encode motifs characteristic of the nucleotide binding site-leucine-rich repeat class of resistance genes, was identified. This gene specifically controlled the hypersensitive response when transiently expressed in susceptible pepper and tomato lines and in a nonhost species, Nicotiana benthamiana, and was designated as Bs2. Functional expression of Bs2 in stable transgenic tomatoes supports its use as a source of resistance in other Solanaceous plant species.

Crystal Structure of HIV-1 Primary Receptor CD4 i Complex with a Potent Antiviral Antibody

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

Freeman, M.M.; Hong, X.; Seaman, M.S.

2010-06-18

Ibalizumab is a humanized, anti-CD4 monoclonal antibody. It potently blocks HIV-1 infection and targets an epitope in the second domain of CD4 without interfering with immune functions mediated by interaction of CD4 with major histocompatibility complex (MHC) class II molecules. We report here the crystal structure of ibalizumab Fab fragment in complex with the first two domains (D1-D2) of CD4 at 2.2 {angstrom} resolution. Ibalizumab grips CD4 primarily by the BC-loop (residues 121125) of D2, sitting on the opposite side of gp120 and MHC-II binding sites. No major conformational change in CD4 accompanies binding to ibalizumab. Both monovalent and bivalentmore » forms of ibalizumab effectively block viral infection, suggesting that it does not need to crosslink CD4 to exert antiviral activity. While gp120-induced structural rearrangements in CD4 are probably minimal, CD4 structural rigidity is dispensable for ibalizumab inhibition. These results could guide CD4-based immunogen design and lead to a better understanding of HIV-1 entry.« less

The effect of Berberine on the secondary structure of human serum albumin

NASA Astrophysics Data System (ADS)

Li, Ying; He, WenYing; Tian, Jianniao; Tang, Jianghong; Hu, Zhide; Chen, Xingguo

2005-05-01

The presence of several high affinity binding sites on human serum albumin (HSA) makes it a possible target for many drugs. This study is designed to examine the effect of Berberine (an ancient Chinese drug used for antimicrobial, antiplasmodial, antidiarrheal and cardiovascular) on the solution structure of HSA using fluorescence, Fourier transform infrared (FT-IR), circular dichroism (CD) spectroscopic methods. The fluorescence spectroscopic results show that the fluorescence intensity of HSA was significantly decreased in the presence of Berberine. The Scatchard's plots indicated that the binding of Berberine to HSA at 296, 303, 318 K is characterized by one binding site with the binding constant is 4.071(±0.128)×10 4, 3.741(±0.089)×10 4, 3.454(±0.110)×10 4 M -1, respectively. The protein conformation is altered (FT-IR and CD data) with reductions of α-helices from 54 to 47% for free HSA to 45-32% and with increases of turn structure5% for free HSA to 18% in the presence of Berberine. The binding process was exothermic, enthalpy driven and spontaneous, as indicated by the thermodynamic analyses, Berberine bound to HSA was mainly based on hydrophobic interaction and electrostatic interaction cannot be excluded from the binding. Furthermore, the displace experiments indicate that Berberine can bind to the subdomain IIA, that is, high affinity site (site II).

Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs: Arg- and Gln-type Bacterial CDO Homologs

DOE PAGES

Driggers, Camden M.; Hartman, Steven J.; Karplus, P. Andrew

2015-01-01

In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ~15–30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue (“Arg-type” enzymes) and some having a Gln substituted for this Arg (“Gln-type” enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis “Arg-type” enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha “Gln-type” CDO homolog ofmore » uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among “Gln-type” CDO enzymes, we conclude that the “Gln-type” CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases.« less

Evolution of DNA-Binding Sites of a Floral Master Regulatory Transcription Factor

PubMed Central

Muiño, Jose M.; de Bruijn, Suzanne; Pajoro, Alice; Geuten, Koen; Vingron, Martin; Angenent, Gerco C.; Kaufmann, Kerstin

2016-01-01

Flower development is controlled by the action of key regulatory transcription factors of the MADS-domain family. The function of these factors appears to be highly conserved among species based on mutant phenotypes. However, the conservation of their downstream processes is much less well understood, mostly because the evolutionary turnover and variation of their DNA-binding sites (BSs) among plant species have not yet been experimentally determined. Here, we performed comparative ChIP (chromatin immunoprecipitation)-seq experiments of the MADS-domain transcription factor SEPALLATA3 (SEP3) in two closely related Arabidopsis species: Arabidopsis thaliana and A. lyrata which have very similar floral organ morphology. We found that BS conservation is associated with DNA sequence conservation, the presence of the CArG-box BS motif and on the relative position of the BS to its potential target gene. Differences in genome size and structure can explain that SEP3 BSs in A. lyrata can be located more distantly to their potential target genes than their counterparts in A. thaliana. In A. lyrata, we identified transposition as a mechanism to generate novel SEP3 binding locations in the genome. Comparative gene expression analysis shows that the loss/gain of BSs is associated with a change in gene expression. In summary, this study investigates the evolutionary dynamics of DNA BSs of a floral key-regulatory transcription factor and explores factors affecting this phenomenon. PMID:26429922

Nuclear factor I-A represses expression of the cell adhesion molecule L1

PubMed Central

2009-01-01

Background The neural cell adhesion molecule L1 plays a crucial role in development and plasticity of the nervous system. Neural cells thus require precise control of L1 expression. Results We identified a full binding site for nuclear factor I (NFI) transcription factors in the regulatory region of the mouse L1 gene. Electrophoretic mobility shift assay (EMSA) showed binding of nuclear factor I-A (NFI-A) to this site. Moreover, for a brain-specific isoform of NFI-A (NFI-A bs), we confirmed the interaction in vivo using chromatin immunoprecipitation (ChIP). Reporter gene assays showed that in neuroblastoma cells, overexpression of NFI-A bs repressed L1 expression threefold. Conclusion Our findings suggest that NFI-A, in particular its brain-specific isoform, represses L1 gene expression, and might act as a second silencer of L1 in addition to the neural restrictive silencer factor (NRSF). PMID:20003413

Diabody-based recombinant formats of humanized IgG-like bispecific antibody with effective retargeting of lymphocytes to tumor cells.

PubMed

Asano, Ryutaro; Kawaguchi, Hiroko; Watanabe, Yasuhiro; Nakanishi, Takeshi; Umetsu, Mitsuo; Hayashi, Hiroki; Katayose, Yu; Unno, Michiaki; Kudo, Toshio; Kumagai, Izumi

2008-10-01

Recently, recombinant antibodies have been dissected into antigen-binding regions and rebuilt into multivalent high-avidity formats. These new structural designs are expected to improve in vivo pharmacokinetics and efficacy in clinical use. Here, we designed effective recombinant bispecific antibody (BsAb) formats based on hEx3, a humanized bispecific diabody with epidermal growth factor receptor and CD3 retargeting. The bispecific and bivalent IgG-like antibodies engineered from hEx3 (or its single-chain form, hEx3-scDb) and the human Fc region showed stronger binding to each target cell than did monovalent diabody formats, and their affinity was identical to that of the corresponding parent IgG. The bivalent effect of the constructed IgG-like BsAbs resulted in cell cytotoxicity 10 times that of monovalent diabodies, and further, the fusion of Fc portion contributed intense cytotoxicity in peripheral blood mononuclear cells by the induction of the antibody-dependent cellular cytotoxicity. The growth-inhibition effects of IgG-like BsAbs were superior to those of the approved therapeutic antibody cetuximab, which recognizes the same epidermal growth factor receptor antigen, even when peripheral blood mononuclear cells were used as effector cells. We thus demonstrated a critical improvement in the effect of hEx3 by the bottom-up construction of IgG-like BsAbs; in adoptive immunotherapy, monotherapy without supplemental molecules may be able to induce antibody-dependent cellular cytotoxicity.

CD/MCD/VTVH-MCD Studies of Escherichia coli Bacterioferritin Support a Binuclear Iron Cofactor Site.

PubMed

Kwak, Yeonju; Schwartz, Jennifer K; Huang, Victor W; Boice, Emily; Kurtz, Donald M; Solomon, Edward I

2015-12-01

Ferritins and bacterioferritins (Bfrs) utilize a binuclear non-heme iron binding site to catalyze oxidation of Fe(II), leading to formation of an iron mineral core within a protein shell. Unlike ferritins, in which the diiron site binds Fe(II) as a substrate, which then autoxidizes and migrates to the mineral core, the diiron site in Bfr has a 2-His/4-carboxylate ligand set that is commonly found in diiron cofactor enzymes. Bfrs could, therefore, utilize the diiron site as a cofactor rather than for substrate iron binding. In this study, we applied circular dichroism (CD), magnetic CD (MCD), and variable-temperature, variable-field MCD (VTVH-MCD) spectroscopies to define the geometric and electronic structures of the biferrous active site in Escherichia coli Bfr. For these studies, we used an engineered M52L variant, which is known to eliminate binding of a heme cofactor but to have very minor effects on either iron oxidation or mineral core formation. We also examined an H46A/D50A/M52L Bfr variant, which additionally disrupts a previously observed mononuclear non-heme iron binding site inside the protein shell. The spectral analyses define a binuclear and an additional mononuclear ferrous site. The biferrous site shows two different five-coordinate centers. After O2 oxidation and re-reduction, only the mononuclear ferrous signal is eliminated. The retention of the biferrous but not the mononuclear ferrous site upon O2 cycling supports a mechanism in which the binuclear site acts as a cofactor for the O2 reaction, while the mononuclear site binds the substrate Fe(II) that, after its oxidation to Fe(III), migrates to the mineral core.

New multi-site observations of the delta Scuti stars BS and BT Cancri. Results of the STEPHI VII campaign on the Praesepe cluster

NASA Astrophysics Data System (ADS)

Hernandez, M. M.; Michel, E.; Belmonte, J. A.; Jiang, S. Y.; Alvarez, M.; Chevreton, M.; Paparo, M.; Kjeldsen, H.; Bauduin, D.; Fromage, J.; Goupil, M. J.; Li, Z. P.; Liu, Y. Y.; Mangeney, A.; Massacrier, G.; Ringot, O.; Cortes, T. Roca; Servan, B.; Vidal, I.

1998-09-01

New observations of BS and BT Cnc were performed during the STEPHI VII campaign in 1996 February. An overall run of 115 hours of data was collected. Different methods have been used to analyse the time series, bringing the detection of 2 frequencies for BT Cnc and 3 frequencies for BS Cnc above the 99% confidence level. A comparison with the literature reveals that the dominant mode of BT Cnc ( ~ 9.8 c/d) has kept excited over tens of years, while the secondary modes have not been the same all the time. However, during the very last years the two frequencies detected by us have been the only ones noticeably excited in the spectrum.

Identity of a peptide domain of human C9 that is bound by the cell-surface complement inhibitor, CD59.

PubMed

Chang, C P; Hüsler, T; Zhao, J; Wiedmer, T; Sims, P J

1994-10-21

The CD59 antigen is a plasma membrane glycoprotein that serves as an inhibitor of the C5b-9 complex of complement. This inhibitory activity appears related to the capacity of CD59 to bind with high affinity to sites that are nascently exposed in the alpha-chain subunit of human C8, as well as within the C9b domain (amino acid residues 245-538) of human C9, during assembly of the C5b-9 complex on the target membrane (Ninomiya, H., and Sims, P. J. (1992) J. Biol. Chem. 267, 13675-13680). The CD59 binding site in C9 was first investigated by N-terminal sequencing of CD59-binding peptides generated by limited digest of the isolated C9b domain. These experiments revealed a 17-kDa fragment (starting at C9 residue Thr-320) that retained affinity for CD59, suggesting the possibility for localizing the CD59 binding site by mapping with small C9-derived peptides. Peptides spanning the entire C9b sequence were expressed in Escherichia coli and then probed with CD59. CD59 bound specifically to all peptides starting N-terminal to C9 residue 359 with C termini extending beyond residue 411. Little to no CD59 binding was observed for various C9-derived peptides that started C-terminal to residue 359 or that were truncated N-terminal to residue 411. Affinity-purified antibody against C9 residues 320-411 inhibited CD59 binding to C9 by > 50% and completely inhibited its binding to the isolated C9b domain. Little to no specific binding of CD59 was detected for peptides restricted to the putative hinge domain within C9b (residues 245-271). These results indicate that a CD59 binding site is located between residues 320 and 411 of the C9 polypeptide and suggest that the affinity of this site is principally determined by residues 359-411.

A Novel Rice Cytochrome P450 Gene, CYP72A31, Confers Tolerance to Acetolactate Synthase-Inhibiting Herbicides in Rice and Arabidopsis1[C][W][OPEN

PubMed Central

Saika, Hiroaki; Horita, Junko; Taguchi-Shiobara, Fumio; Nonaka, Satoko; Nishizawa-Yokoi, Ayako; Iwakami, Satoshi; Hori, Kiyosumi; Matsumoto, Takashi; Tanaka, Tsuyoshi; Itoh, Takeshi; Yano, Masahiro; Kaku, Koichiro; Shimizu, Tsutomu; Toki, Seiichi

2014-01-01

Target-site and non-target-site herbicide tolerance are caused by the prevention of herbicide binding to the target enzyme and the reduction to a nonlethal dose of herbicide reaching the target enzyme, respectively. There is little information on the molecular mechanisms involved in non-target-site herbicide tolerance, although it poses the greater threat in the evolution of herbicide-resistant weeds and could potentially be useful for the production of herbicide-tolerant crops because it is often involved in tolerance to multiherbicides. Bispyribac sodium (BS) is an herbicide that inhibits the activity of acetolactate synthase. Rice (Oryza sativa) of the indica variety show BS tolerance, while japonica rice varieties are BS sensitive. Map-based cloning and complementation tests revealed that a novel cytochrome P450 monooxygenase, CYP72A31, is involved in BS tolerance. Interestingly, BS tolerance was correlated with CYP72A31 messenger RNA levels in transgenic plants of rice and Arabidopsis (Arabidopsis thaliana). Moreover, Arabidopsis overexpressing CYP72A31 showed tolerance to bensulfuron-methyl (BSM), which belongs to a different class of acetolactate synthase-inhibiting herbicides, suggesting that CYP72A31 can metabolize BS and BSM to a compound with reduced phytotoxicity. On the other hand, we showed that the cytochrome P450 monooxygenase CYP81A6, which has been reported to confer BSM tolerance, is barely involved, if at all, in BS tolerance, suggesting that the CYP72A31 enzyme has different herbicide specificities compared with CYP81A6. Thus, the CYP72A31 gene is a potentially useful genetic resource in the fields of weed control, herbicide development, and molecular breeding in a broad range of crop species. PMID:24406793

Design and Validation of a Novel Generic Platform for the Production of Tetravalent IgG1-like Bispecific Antibodies.

PubMed

Golay, Josée; Choblet, Sylvie; Iwaszkiewicz, Justyna; Cérutti, Pierre; Ozil, Annick; Loisel, Séverine; Pugnière, Martine; Ubiali, Greta; Zoete, Vincent; Michielin, Olivier; Berthou, Christian; Kadouche, Jean; Mach, Jean-Pierre; Duonor-Cérutti, Martine

2016-04-01

We have designed and validated a novel generic platform for production of tetravalent IgG1-like chimeric bispecific Abs. The VH-CH1-hinge domains of mAb2 are fused through a peptidic linker to the N terminus of mAb1 H chain, and paired mutations at the CH1-CL interface mAb1 are introduced that force the correct pairing of the two different free L chains. Two different sets of these CH1-CL interface mutations, called CR3 and MUT4, were designed and tested, and prototypic bispecific Abs directed against CD5 and HLA-DR were produced (CD5xDR). Two different hinge sequences between mAb1 and mAb2 were also tested in the CD5xDR-CR3 or -MUT4 background, leading to bispecific Ab (BsAbs) with a more rigid or flexible structure. All four Abs produced bound with good specificity and affinity to CD5 and HLA-DR present either on the same target or on different cells. Indeed, the BsAbs were able to efficiently redirect killing of HLA-DR(+) leukemic cells by human CD5(+) cytokine-induced killer T cells. Finally, all BsAbs had a functional Fc, as shown by their capacity to activate human complement and NK cells and to mediate phagocytosis. CD5xDR-CR3 was chosen as the best format because it had overall the highest functional activity and was very stable in vitro in both neutral buffer and in serum. In vivo, CD5xDR-CR3 was shown to have significant therapeutic activity in a xenograft model of human leukemia. Copyright © 2016 by The American Association of Immunologists, Inc.

Probiotic Lactobacillus johnsonii BS15 Improves Blood Parameters Related to Immunity in Broilers Experimentally Infected with Subclinical Necrotic Enteritis

PubMed Central

Wang, Hesong; Ni, Xueqin; Qing, Xiaodan; Liu, Lei; Xin, Jinge; Luo, Min; Khalique, Abdul; Dan, Yan; Pan, Kangcheng; Jing, Bo; Zeng, Dong

2018-01-01

The probiotic strain Lactobacillus johnsonii BS15 could exert beneficial effects on growth performance, lipid metabolism, and intestinal microflora in healthy broilers and those afflicted with subclinical necrotic enteritis (SNE). In particular, BS15 prevents SNE by enhancing intestinal immunity. To further understand the immune regulatory mechanism of BS15, we evaluated its effects on the overall immunity of broilers by determining blood parameters in healthy and SNE broilers. In this study, two experiments were conducted. Experiment 1 involved a 42-day experimental period and used 450 1-day-old male chicks. The chicks were randomly divided into three groups and fed with a basal diet with or without 1 × 105 or 106 colony-forming units (cfu) BS15/g as feed. Experiment 2 involved a 28-day experimental period and used 180 1-day-old male chicks. The chicks were randomly allotted into three groups and given with or without 1 × 106 cfu BS15/g BS15 as feed. SNE infection was treated in all broilers, except in those in the normal diet group. Antioxidant abilities, immunoglobulins, and cytokines in the serum were assessed. T-lymphocyte subsets in peripheral blood were also determined. The first experiment demonstrated that BS15 enhanced the antioxidant abilities; the serum levels of immunoglobulins, interleukin-2, and interferon-gamma; and CD3+CD4+ T-lymphocyte percentage in peripheral blood on day 21. However, limited significant changes were observed on day 42. The second experiment revealed that BS15 supplementation positively influenced the antioxidant abilities and increased the serum levels of immunoglobulins and cytokines that were affected by SNE. BS15 also positively affected T-lymphocyte subsets in peripheral blood during SNE infection. These findings suggest that BS15 supplementation may prevent SNE in broilers by improving blood parameters related to immunity and enhancing intestinal immunity. Furthermore, BS15 supplementation can improve blood parameters in healthy broilers, especially at the starter phase. PMID:29441047

Probiotic Lactobacillus johnsonii BS15 Improves Blood Parameters Related to Immunity in Broilers Experimentally Infected with Subclinical Necrotic Enteritis.

PubMed

Wang, Hesong; Ni, Xueqin; Qing, Xiaodan; Liu, Lei; Xin, Jinge; Luo, Min; Khalique, Abdul; Dan, Yan; Pan, Kangcheng; Jing, Bo; Zeng, Dong

2018-01-01

The probiotic strain Lactobacillus johnsonii BS15 could exert beneficial effects on growth performance, lipid metabolism, and intestinal microflora in healthy broilers and those afflicted with subclinical necrotic enteritis (SNE). In particular, BS15 prevents SNE by enhancing intestinal immunity. To further understand the immune regulatory mechanism of BS15, we evaluated its effects on the overall immunity of broilers by determining blood parameters in healthy and SNE broilers. In this study, two experiments were conducted. Experiment 1 involved a 42-day experimental period and used 450 1-day-old male chicks. The chicks were randomly divided into three groups and fed with a basal diet with or without 1 × 10 5 or 10 6 colony-forming units (cfu) BS15/g as feed. Experiment 2 involved a 28-day experimental period and used 180 1-day-old male chicks. The chicks were randomly allotted into three groups and given with or without 1 × 10 6 cfu BS15/g BS15 as feed. SNE infection was treated in all broilers, except in those in the normal diet group. Antioxidant abilities, immunoglobulins, and cytokines in the serum were assessed. T-lymphocyte subsets in peripheral blood were also determined. The first experiment demonstrated that BS15 enhanced the antioxidant abilities; the serum levels of immunoglobulins, interleukin-2, and interferon-gamma; and CD3 + CD4 + T-lymphocyte percentage in peripheral blood on day 21. However, limited significant changes were observed on day 42. The second experiment revealed that BS15 supplementation positively influenced the antioxidant abilities and increased the serum levels of immunoglobulins and cytokines that were affected by SNE. BS15 also positively affected T-lymphocyte subsets in peripheral blood during SNE infection. These findings suggest that BS15 supplementation may prevent SNE in broilers by improving blood parameters related to immunity and enhancing intestinal immunity. Furthermore, BS15 supplementation can improve blood parameters in healthy broilers, especially at the starter phase.

The PBX1 lupus susceptibility gene regulates CD44 expression

PubMed Central

Niu, Yuxin; Sengupta, Mayami; Titov, Anton A.; Choi, Seung-Chul; Morel, Laurence

2017-01-01

PBX1-d is novel splice isoform of pre-B-cell leukemia homeobox 1 (PBX1) that lacks its DNA-binding and Hox-binding domains, and functions as a dominant negative. We have shown that PBX1-d expression in CD4+ T cells is associated with systemic lupus erythematosus (SLE) in a mouse model as well as in human subjects. More specifically, PBX1-d expression leads to the production of autoreactive activated CD4+ T cells, a reduced frequency and function of Foxp3+ regulatory T (Treg) cells and an expansion of follicular helper T (Tfh) cells. Very little is known about the function of PBX1 in T cells, except that it directly regulates the expression of miRNAs associated with Treg and Tfh homeostasis. In the present study, we show that PBX1 directly regulated the expression of CD44, a marker of T cell activation. Two PBX1 binding sites in the promoter directly regulated CD44 expression, with PBX1-d driving a higher expression than the normal isoform PBX1-b. In addition, mutations in each of the two binding sites had different effects of PBX1-b and PBX1-d. Finally, we showed that an enhanced recruitment of co-factor MEIS by PBX1-d over PBX1-b, while there was no difference for co-factor PREP1 recruitment. Therefore, this study demonstrates that the lupus-associated PBX1-d isoform directly transactivates CD44, a marker of CD44 activation and memory, and that it has different DNA binding and co-factor recruitment relative to the normal isoform. Taken together, these results confirm that PBX1 directly regulates genes related to T cell activation and show that the lupus-associated isoform PBX1-d has unique molecular functions. PMID:28257976

Expression and purification of soluble porcine CTLA-4 in yeast Pichia pastoris

PubMed Central

Peraino, Jaclyn; Zhang, Huiping; Hermanrud, Christina E.; Li, Guoying; Sachs, David H.; Huang, Christene A.; Wang, Zhirui

2012-01-01

Co-stimulation blockade can be used to modulate the immune response for induction of organ transplantation tolerance, treatment of autoimmune disease as well as cancer treatment. Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4), also known as CD152, is an important co-stimulatory molecule which serves as a negative regulator for T cell proliferation and differentiation. CTLA-4/CD28-CD80/CD86 pathway is a critical co-stimulatory pathway for adaptive immune response. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for CD80 and CD86. MGH MHC-defined miniature swine provide a unique large animal model useful for preclinical studies of transplantation tolerance and immune regulation. In this study, we have expressed the codon-optimized soluble porcine CTLA-4 in the yeast Pichia pastoris system. The secreted porcine CTLA-4 was captured using Ni-Sepharose 6 fast flow resin and further purified using strong anion exchange resin Poros 50HQ. Glycosylation analysis using PNGase F demonstrated the N-linked glycosylation on Pichia pastoris expressed soluble porcine CTLA-4. To improve the expression level and facilitate the downstream purification we mutated the two potential N-linked glycosylation sites with non-polarized alanines by site-directed mutagenesis. Removal of the two N-glycosylation sites significantly improved the production level from ~2 mg/L to ~8 mg/L. Biotinylated glycosylated and non-N-glycosylated soluble porcine CTLA-4 both bind to a porcine CD80-expressing B-cell lymphoma cell line (KD = 13 nM) and competitively inhibit the binding of an anti-CD80 monoclonal antibody. The availability of soluble porcine CTLA-4, especially the non-N-glycosylated CTLA-4, will provide a very valuable tool for assessing co-stimulatory blockade treatment for translational studies in the clinically relevant porcine model. PMID:22326797

Hints for Metal-Preference Protein Sequence Determinants: Different Metal Binding Features of the Five Tetrahymena thermophila Metallothioneins

PubMed Central

Espart, Anna; Marín, Maribel; Gil-Moreno, Selene; Palacios, Òscar; Amaro, Francisco; Martín-González, Ana; Gutiérrez, Juan C.; Capdevila, Mercè; Atrian, Sílvia

2015-01-01

The metal binding preference of metallothioneins (MTs) groups them in two extreme subsets, the Zn/Cd- and the Cu-thioneins. Ciliates harbor the largest MT gene/protein family reported so far, including 5 paralogs that exhibit relatively low sequence similarity, excepting MTT2 and MTT4. In Tetrahymena thermophila, three MTs (MTT1, MTT3 and MTT5) were considered Cd-thioneins and two (MTT2 and MTT4) Cu-thioneins, according to gene expression inducibility and phylogenetic analysis. In this study, the metal-binding abilities of the five MTT proteins were characterized, to obtain information about the folding and stability of their cognate- and non-cognate metal complexes, and to characterize the T. thermophila MT system at protein level. Hence, the five MTTs were recombinantly synthesized as Zn2+-, Cd2+- or Cu+-complexes, which were analyzed by electrospray mass spectrometry (ESI-MS), circular dichroism (CD), and UV-vis spectrophotometry. Among the Cd-thioneins, MTT1 and MTT5 were optimal for Cd2+ coordination, yielding unique Cd17- and Cd8- complexes, respectively. When binding Zn2+, they rendered a mixture of Zn-species. Only MTT5 was capable to coordinate Cu+, although yielding heteronuclear Zn-, Cu-species or highly unstable Cu-homometallic species. MTT3 exhibited poor binding abilities both for Cd2+ and for Cu+, and although not optimally, it yielded the best result when coordinating Zn2+. The two Cu-thioneins, MTT2 and MTT4 isoforms formed homometallic Cu-complexes (major Cu20-MTT) upon synthesis in Cu-supplemented hosts. Contrarily, they were unable to fold into stable Cd-complexes, while Zn-MTT species were only recovered for MTT4 (major Zn10-MTT4). Thus, the metal binding preferences of the five T. thermophila MTs correlate well with their previous classification as Cd- and Cu-thioneins, and globally, they can be classified from Zn/Cd- to Cu-thioneins according to the gradation: MTT1>MTT5>MTT3>MTT4>MTT2. The main mechanisms underlying the evolution and specialization of the MTT metal binding preferences may have been internal tandem duplications, presence of doublet and triplet Cys patterns in Zn/Cd-thioneins, and optimization of site specific amino acid determinants (Lys for Zn/Cd- and Asn for Cu-coordination). PMID:25798065

A Sequence in the loop domain of hepatitis C virus E2 protein identified in silico as crucial for the selective binding to human CD81

PubMed Central

Chang, Chun-Chun; Hsu, Hao-Jen; Yen, Jui-Hung; Lo, Shih-Yen

2017-01-01

Hepatitis C virus (HCV) is a species-specific pathogenic virus that infects only humans and chimpanzees. Previous studies have indicated that interactions between the HCV E2 protein and CD81 on host cells are required for HCV infection. To determine the crucial factors for species-specific interactions at the molecular level, this study employed in silico molecular docking involving molecular dynamic simulations of the binding of HCV E2 onto human and rat CD81s. In vitro experiments including surface plasmon resonance measurements and cellular binding assays were applied for simple validations of the in silico results. The in silico studies identified two binding regions on the HCV E2 loop domain, namely E2-site1 and E2-site2, as being crucial for the interactions with CD81s, with the E2-site2 as the determinant factor for human-specific binding. Free energy calculations indicated that the E2/CD81 binding process might follow a two-step model involving (i) the electrostatic interaction-driven initial binding of human-specific E2-site2, followed by (ii) changes in the E2 orientation to facilitate the hydrophobic and van der Waals interaction-driven binding of E2-site1. The sequence of the human-specific, stronger-binding E2-site2 could serve as a candidate template for the future development of HCV-inhibiting peptide drugs. PMID:28481946

Molecular-Level Insight into the Differential Oxidase and Oxygenase Reactivities of de Novo Due Ferri Proteins

DOE PAGES

Snyder, Rae Ana; Butch, Susan E.; Reig, Amanda J.; ...

2015-06-19

Using the single-chain due ferri (DFsc) peptide scaffold, the differential oxidase and oxygenase reactivities of two 4A → 4G variants, one with two histidines at the diiron center (G4DFsc) and the other with three histidines (3His-G4DFsc(Mut3)), are explored. By controlling the reaction conditions, the active form responsible for 4-aminophenol (4-AP) oxidase activity in both G4DFsc and 3His-G4DFsc(Mut3) is determined to be the substrate-bound biferrous site. Using circular dichroism (CD), magnetic CD (MCD), and variable-temperature, variable-field (VTVH) MCD spectroscopies, 4-AP is found to bind directly to the biferrous sites of the DF proteins. In G4DFsc, 4-AP increases the coordination of themore » biferrous site, while in 3His-G4DFsc(Mut3), the coordination number remains the same and the substrate likely replaces the additional bound histidine. This substrate binding enables a two-electron process where 4-AP is oxidized to benzoquinone imine and O 2 is reduced to H 2O 2. In contrast, only the biferrous 3His variant is found to be active in the oxygenation of p-anisidine to 4-nitroso-methoxybenzene. From CD, MCD, and VTVH MCD, p-anisidine addition is found to minimally perturb the biferrous centers of both G4DFsc and 3His-G4DFsc(Mut3), indicating that this substrate binds near the biferrous site. Lastly, in 3His-G4DFsc(Mut3), the coordinative saturation of one iron leads to the two-electron reduction of O 2 at the second iron to generate an end-on hydroperoxo-Fe(III) active oxygenating species.« less

c-Abl-Mediated Tyrosine Phosphorylation of the T-bet DNA-Binding Domain Regulates CD4+ T-Cell Differentiation and Allergic Lung Inflammation ▿

PubMed Central

Chen, An; Lee, Sang-Myeong; Gao, Beixue; Shannon, Stephen; Zhu, Zhou; Fang, Deyu

2011-01-01

The tyrosine kinase c-Abl is required for full activation of T cells, while its role in T-cell differentiation has not been characterized. We report that c-Abl deficiency skews CD4+ T cells to type 2 helper T cell (Th2) differentiation, and c-Abl−/− mice are more susceptible to allergic lung inflammation. c-Abl interacts with and phosphorylates T-bet, a Th1 lineage transcription factor. c-Abl-mediated phosphorylation enhances the transcriptional activation of T-bet. Interestingly, three tyrosine residues within the T-bet DNA-binding domain are the predominant sites of phosphorylation by c-Abl. Mutation of these tyrosine residues inhibits the promoter DNA-binding activity of T-bet. c-Abl regulates Th cell differentiation in a T-bet-dependent manner because genetic deletion of T-bet in CD4+ T cells abolishes c-Abl-deficiency-mediated enhancement of Th2 differentiation. Reintroduction of T-bet-null CD4+ T cells with wild-type T-bet, but not its tyrosine mutant, rescues gamma interferon (IFN-γ) production and inhibits Th2 cytokine production. Therefore, c-Abl catalyzes tyrosine phosphorylation of the DNA-binding domain of T-bet to regulate CD4+ T cell differentiation. PMID:21690296

The acetylation of transcription factor HBP1 by p300/CBP enhances p16INK4A expression

PubMed Central

Wang, Weibin; Pan, Kewu; Chen, Yifan; Huang, Chunyin; Zhang, Xiaowei

2012-01-01

HBP1 is a sequence-specific DNA-binding transcription factor with many important biological roles. It activates or represses the expression of some specific genes during cell growth and differentiation. Previous studies have exhibited that HBP1 binds to p16INK4A promoter and activates p16INK4A expression. We found that trichostatin A (TSA), an inhibitor of HDAC (histone deacetylase), induces p16INK4A expression in an HBP1-dependent manner. This result was drawn from a transactivation experiment by measuring relative luciferase activities of p16INK4A promoter with HBP1-binding site in comparison with that of the wild-type p16INK4A promoter by transient cotransfection with HBP1 into HEK293T cells and 2BS cells. HBP1 acetylation after TSA treatment was confirmed by immunoprecipitation assay. Our data showed that HBP1 interacted with histone acetyltransferase p300 and CREB-binding protein (CBP) and also recruited p300/CBP to p16INK4A promoter. HBP1 was acetylated by p300/CBP in two regions: repression domain (K297/305/307) and P domain (K171/419). Acetylation of Repression domain was not required for HBP1 transactivation on p16INK4A. However, luciferase assay and western blotting results indicate that acetylation of P domain, especially K419 acetylation is essential for HBP1 transactivation on p16INK4A. As assayed by SA-beta-gal staining, the acetylation of HBP1 at K419 enhanced HBP1-induced premature senescence in 2BS cells. In addition, HDAC4 repressed HBP1-induced premature senescence through permanently deacetylating HBP1. We conclude that our data suggest that HBP1 acetylation at K419 plays an important role in HBP1-induced p16INK4A expression. PMID:21967847

Crystallographic analysis of CD40 recognition and signaling by human TRAF2

PubMed Central

McWhirter, Sarah M.; Pullen, Steven S.; Holton, James M.; Crute, James J.; Kehry, Marilyn R.; Alber, Tom

1999-01-01

Tumor necrosis factor receptor superfamily members convey signals that promote diverse cellular responses. Receptor trimerization by extracellular ligands initiates signaling by recruiting members of the tumor necrosis factor receptor-associated factor (TRAF) family of adapter proteins to the receptor cytoplasmic domains. We report the 2.4-Å crystal structure of a 22-kDa, receptor-binding fragment of TRAF2 complexed with a functionally defined peptide from the cytoplasmic domain of the CD40 receptor. TRAF2 forms a mushroom-shaped trimer consisting of a coiled coil and a unique β-sandwich domain. Both domains mediate trimerization. The CD40 peptide binds in an extended conformation with every side chain in contact with a complementary groove on the rim of each TRAF monomer. The spacing between the CD40 binding sites on TRAF2 supports an elegant signaling mechanism in which trimeric, extracellular ligands preorganize the receptors to simultaneously recognize three sites on the TRAF trimer. PMID:10411888

Crystal Structure of the Catalytic Domain of Drosophila [beta]1,4-Galactosyltransferase-7

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

Ramakrishnan, Boopathy; Qasba, Pradman K.

2010-11-03

The {beta}1,4-galactosyltransferase-7 ({beta}4Gal-T7) enzyme, one of seven members of the {beta}4Gal-T family, transfers in the presence of manganese Gal from UDP-Gal to an acceptor sugar (xylose) that is attached to a side chain hydroxyl group of Ser/Thr residues of proteoglycan proteins. It exhibits the least protein sequence similarity with the other family members, including the well studied family member {beta}4Gal-T1, which, in the presence of manganese, transfers Gal from UDP-Gal to GlcNAc. We report here the crystal structure of the catalytic domain of {beta}4Gal-T7 from Drosophila in the presence of manganese and UDP at 1.81 {angstrom} resolution. In the crystalmore » structure, a new manganese ion-binding motif (HXH) has been observed. Superposition of the crystal structures of {beta}4Gal-T7 and {beta}4Gal-T1 shows that the catalytic pocket and the substrate-binding sites in these proteins are similar. Compared with GlcNAc, xylose has a hydroxyl group (instead of an N-acetyl group) at C2 and lacks the CH{sub 2}OH group at C5; thus, these protein structures show significant differences in their acceptor-binding site. Modeling of xylose in the acceptor-binding site of the {beta}4Gal-T7 crystal structure shows that the aromatic side chain of Tyr{sup 177} interacts strongly with the C5 atom of xylose, causing steric hindrance to any additional group at C5. Because Drosophila Cd7 has a 73% protein sequence similarity to human Cd7, the present crystal structure offers a structure-based explanation for the mutations in human Cd7 that have been linked to Ehlers-Danlos syndrome.« less

The Binding Site of Human Adenosine Deaminase for Cd26/Dipeptidyl Peptidase IV

PubMed Central

Richard, Eva; Arredondo-Vega, Francisco X.; Santisteban, Ines; Kelly, Susan J.; Patel, Dhavalkumar D.; Hershfield, Michael S.

2000-01-01

Human, but not murine, adenosine deaminase (ADA) forms a complex with the cell membrane protein CD26/dipeptidyl peptidase IV. CD26-bound ADA has been postulated to regulate extracellular adenosine levels and to modulate the costimulatory function of CD26 on T lymphocytes. Absence of ADA–CD26 binding has been implicated in causing severe combined immunodeficiency due to ADA deficiency. Using human–mouse ADA hybrids and ADA point mutants, we have localized the amino acids critical for CD26 binding to the helical segment 126–143. Arg142 in human ADA and Gln142 in mouse ADA largely determine the capacity to bind CD26. Recombinant human ADA bearing the R142Q mutation had normal catalytic activity per molecule, but markedly impaired binding to a CD26+ ADA-deficient human T cell line. Reduced CD26 binding was also found with ADA from red cells and T cells of a healthy individual whose only expressed ADA has the R142Q mutation. Conversely, ADA with the E217K active site mutation, the only ADA expressed by a severely immunodeficient patient, showed normal CD26 binding. These findings argue that ADA binding to CD26 is not essential for immune function in humans. PMID:11067872

Energetics of dendrimer binding to HIV-1 gp120-CD4 complex and mechanismic aspects of its role as an entry-inhibitor

NASA Astrophysics Data System (ADS)

Saurabh, Suman; Sahoo, Anil Kumar; Maiti, Prabal K.

2016-10-01

Experiments and computational studies have established that de-protonated dendrimers (SPL7013 and PAMAM) act as entry-inhibitors of HIV. SPL7013 based Vivagel is currently under clinical development. The dendrimer binds to gp120 in the gp120-CD4 complex, destabilizes it by breaking key contacts between gp120 and CD4 and prevents viral entry into target cells. In this work, we provide molecular details and energetics of the formation of the SPL7013-gp120-CD4 ternary complex and decipher modes of action of the dendrimer in preventing viral entry. It is also known from experiments that the dendrimer binds weakly to gp120 that is not bound to CD4. It binds even more weakly to the CD4-binding region of gp120 and thus cannot directly block gp120-CD4 complexation. In this work, we examine the feasibility of dendrimer binding to the gp120-binding region of CD4 and directly blocking gp120-CD4 complex formation. We find that the process of the dendrimer binding to CD4 can compete with gp120-CD4 binding due to comparable free energy change for the two processes, thus creating a possibility for the dendrimer to directly block gp120-CD4 complexation by binding to the gp120-binding region of CD4.

Binding of human and rat CD59 to the terminal complement complexes.

PubMed Central

Lehto, T; Morgan, B P; Meri, S

1997-01-01

CD59-antigen (protectin) is a widely distributed glycolipid-anchored inhibitor of complement lysis. CD59 interacts with complement components C8 and C9 during assembly of the membrane attack complex (MAC). To evaluate species specificity of these interactions we have in the present study examined cross-species binding of isolated human and rat CD59 to the terminal complement components C8 and C9. By using primarily soluble CD59 isolated from urine (CD59U) potentially non-specific binding interactions of the phospholipid portion of the membrane forms of CD59 could be avoided. Sucrose density gradient ultracentrifugation analysis showed that human CD59U bound to both human and rat C8 in the SC5b-8 complexes. Similar binding occurred when rat CD59U was used. The degree of binding did not significantly differ between the heterologous and homologous CD59-C8 combinations. C9 from both species inhibited the binding of CD59 to soluble SC5b-8. In ligand blotting analysis human and rat CD59U bound to human and rat C8 alpha gamma-subunit and C9. Binding of human and rat CD59U was stronger to human than rat C9. In plate binding assays the erythrocyte form of CD59 (CD59E) bound to both human and rat C8. Binding of CD59E to heterologous C9 was considerably weaker than to homologous C9. Our results imply that the reciprocal binding sites between C8 and CD59 and to a lesser degree between CD59 and C9 are conserved between human and rat. Interactions of CD59 with the terminal C components are thus species selective but not 'homologously restricted'. Images Figure 4 Figure 5 PMID:9038722

Generation of Affibody ligands binding interleukin-2 receptor alpha/CD25.

PubMed

Grönwall, Caroline; Snelders, Eveline; Palm, Anna Jarelöv; Eriksson, Fredrik; Herne, Nina; Ståhl, Stefan

2008-06-01

Affibody molecules specific for human IL-2Ralpha, the IL-2 (interleukin-2) receptor alpha subunit, also known as CD25, were selected by phage-display technology from a combinatorial protein library based on the 58-residue Protein A-derived Z domain. The IL-2R system plays a major role in T-cell activation and the regulation of cellular immune responses. Moreover, CD25 has been found to be overexpressed in organ rejections, a number of autoimmune diseases and T-cell malignancies. The phage-display selection using Fc-fused target protein generated 16 unique Affibody molecules targeting CD25. The two most promising binders were characterized in more detail using biosensor analysis and demonstrated strong and selective binding to CD25. Kinetic biosensor analysis revealed that the two monomeric Affibody molecules bound to CD25 with apparent affinities of 130 and 240 nM respectively. The Affibody molecules were, on biosensor analysis, found to compete for the same binding site as the natural ligand IL-2 and the IL-2 blocking monoclonal antibody 2A3. Hence the Affibody molecules were assumed to have an overlapping binding site with IL-2 and antibodies targeting the IL-2 blocking Tac epitope (for example, the monoclonal antibodies Daclizumab and Basiliximab, both of which have been approved for therapeutic use). Furthermore, immunofluorescence microscopy and flow-cytometric analysis of CD25-expressing cells demonstrated that the selected Affibody molecules bound to CD4+ CD25+ PMBCs (peripheral-blood mononuclear cells), the IL-2-dependent cell line NK92 and phytohaemagglutinin-activated PMBCs. The potential use of the CD25-binding Affibody molecules as targeting agents for medical imaging and for therapeutic applications is discussed.

Binding of mitomycin C to blood proteins: A spectroscopic analysis and molecular docking

NASA Astrophysics Data System (ADS)

Jang, Jongchol; Liu, Hui; Chen, Wei; Zou, Guolin

2009-06-01

Mitomycin C (MMC) was the first recognized bioreductive alkylating agent, and has been widely used clinically for antitumor therapy. The binding of MMC to two human blood proteins, human serum albumin (HSA) and human hemoglobin (HHb), have been investigated by fluorescence quenching, synchronous fluorescence, circular dichroism (CD) spectroscopy and molecular docking methods. The fluorescence data showed that binding of MMC to proteins caused strong fluorescence quenching of proteins through a static quenching way, and each protein had only one binding site for the drug. The binding constants of MMC to HSA and HHb at 298 K were 2.71 × 10 4 and 2.56 × 10 4 L mol -1, respectively. Thermodynamic analysis suggested that both hydrophobic interaction and hydrogen bonding played major roles in the binding of MMC to HSA or HHb. The CD spectroscopy indicated that the secondary structures of the two proteins were not changed in the presence of MMC. The study of molecular docking showed that MMC was located in the entrance of site I of HSA, and in the central cavity of HHb.

Solution NMR studies provide structural basis for endotoxin pattern recognition by the innate immune receptor CD14

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

Albright, Seth; Chen Bin; Holbrook, Kristen

CD14 functions as a key pattern recognition receptor for a diverse array of Gram-negative and Gram-positive cell-wall components in the host innate immune response by binding to pathogen-associated molecular patterns (PAMPs) at partially overlapping binding site(s). To determine the potential contribution of CD14 residues in this pattern recognition, we have examined using solution NMR spectroscopy, the binding of three different endotoxin ligands, lipopolysaccharide, lipoteichoic acid, and a PGN-derived compound, muramyl dipeptide to a {sup 15}N isotopically labeled 152-residue N-terminal fragment of sCD14 expressed in Pichia pastoris. Mapping of NMR spectral changes upon addition of ligands revealed that the pattern ofmore » residues affected by binding of each ligand is partially similar and partially different. This first direct structural observation of the ability of specific residue combinations of CD14 to differentially affect endotoxin binding may help explain the broad specificity of CD14 in ligand recognition and provide a structural basis for pattern recognition. Another interesting finding from the observed spectral changes is that the mode of binding may be dynamically modulated and could provide a mechanism for binding endotoxins with structural diversity through a common binding site.« less

Envelope conformational changes induced by human immunodeficiency virus type 1 attachment inhibitors prevent CD4 binding and downstream entry events.

PubMed

Ho, Hsu-Tso; Fan, Li; Nowicka-Sans, Beata; McAuliffe, Brian; Li, Chang-Ben; Yamanaka, Gregory; Zhou, Nannan; Fang, Hua; Dicker, Ira; Dalterio, Richard; Gong, Yi-Fei; Wang, Tao; Yin, Zhiwei; Ueda, Yasutsugu; Matiskella, John; Kadow, John; Clapham, Paul; Robinson, James; Colonno, Richard; Lin, Pin-Fang

2006-04-01

BMS-488043 is a small-molecule human immunodeficiency virus type 1 (HIV-1) CD4 attachment inhibitor with demonstrated clinical efficacy. The compound inhibits soluble CD4 (sCD4) binding to the 11 distinct HIV envelope gp120 proteins surveyed. Binding of BMS-488043 and that of sCD4 to gp120 are mutually exclusive, since increased concentrations of one can completely block the binding of the other without affecting the maximal gp120 binding capacity. Similarly, BMS-488043 inhibited virion envelope trimers from binding to sCD4-immunoglobulin G (IgG), with decreasing inhibition as the sCD4-IgG concentration increased, and BMS-488043 blocked the sCD4-induced exposure of the gp41 groove in virions. In both virion binding assays, BMS-488043 was active only when added prior to sCD4. Collectively, these results indicate that obstruction of gp120-sCD4 interactions is the primary inhibition mechanism of this compound and that compound interaction with envelope must precede CD4 binding. By three independent approaches, BMS-488043 was further shown to induce conformational changes within gp120 in both the CD4 and CCR5 binding regions. These changes likely prevent gp120-CD4 interactions and downstream entry events. However, BMS-488043 could only partially inhibit CD4 binding to an HIV variant containing a specific envelope truncation and altered gp120 conformation, despite effectively inhibiting the pseudotyped virus infection. Taken together, BMS-488043 inhibits viral entry primarily through altering the envelope conformation and preventing CD4 binding, and other downstream entry events could also be inhibited as a result of these induced conformational changes.

Differential requirements for Runx proteins in CD4 repression and epigenetic silencing during T lymphocyte development.

PubMed

Taniuchi, Ichiro; Osato, Motomi; Egawa, Takeshi; Sunshine, Mary Jean; Bae, Suk Chul; Komori, Toshihisa; Ito, Yoshiaki; Littman, Dan R

2002-11-27

T lymphocytes differentiate in discrete stages within the thymus. Immature thymocytes lacking CD4 and CD8 coreceptors differentiate into double-positive cells (CD4(+)CD8(+)), which are selected to become either CD4(+)CD8(-)helper cells or CD4(-)CD8(+) cytotoxic cells. A stage-specific transcriptional silencer regulates expression of CD4 in both immature and CD4(-)CD8(+) thymocytes. We show here that binding sites for Runt domain transcription factors are essential for CD4 silencer function at both stages, and that different Runx family members are required to fulfill unique functions at each stage. Runx1 is required for active repression in CD4(-)CD8(-) thymocytes whereas Runx3 is required for establishing epigenetic silencing in cytotoxic lineage thymocytes. Runx3-deficient cytotoxic T cells, but not helper cells, have defective responses to antigen, suggesting that Runx proteins have critical functions in lineage specification and homeostasis of CD8-lineage T lymphocytes.

Anti-CD22–chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia

PubMed Central

Haso, Waleed; Lee, Daniel W.; Shah, Nirali N.; Stetler-Stevenson, Maryalice; Yuan, Constance M.; Pastan, Ira H.; Dimitrov, Dimiter S.; Morgan, Richard A.; FitzGerald, David J.; Barrett, David M.; Wayne, Alan S.; Mackall, Crystal L.

2013-01-01

Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains ± an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3ζ constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL. PMID:23243285

Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia.

PubMed

Haso, Waleed; Lee, Daniel W; Shah, Nirali N; Stetler-Stevenson, Maryalice; Yuan, Constance M; Pastan, Ira H; Dimitrov, Dimiter S; Morgan, Richard A; FitzGerald, David J; Barrett, David M; Wayne, Alan S; Mackall, Crystal L; Orentas, Rimas J

2013-02-14

Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains ± an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3 constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL.

Bispecific antibodies and CARs: generalized immunotherapeutics harnessing T cell redirection

PubMed Central

Zhukovsky, Eugene A.; Morse, Richard J.; Maus, Marcela V.

2016-01-01

To realize the full potential of cancer immunotherapy, the latest generation immunotherapeutics are designed to harness the potent tumor-killing capacity of T cells. Thus, to mobilize T cells, new optimized bispecific antibody (BsAb) designs, enabling efficient polyclonal redirection of cytotoxic activity through binding to CD3 and a Tumor Associated Antigen (TAA) and refined genetically-modified T cells have recently expanded the arsenal of available options for cancer treatment. This review presents the current understanding of the parameters crucial to the design of optimal T cell redirecting BsAb and chimeric antigen receptor (CAR)-modified T cells. However, there are additional questions that require thorough elucidation. Both modalities will benefit from design changes that may increase the therapeutic window. One such approach could employ the discrimination afforded by multiple TAA to significantly increase selectivity. PMID:26963133

Evolution of Metal(Loid) Binding Sites in Transcriptional Regulators

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

Ordonez, E.; Thiyagarajan, S.; Cook, J.D.

2009-05-22

Expression of the genes for resistance to heavy metals and metalloids is transcriptionally regulated by the toxic ions themselves. Members of the ArsR/SmtB family of small metalloregulatory proteins respond to transition metals, heavy metals, and metalloids, including As(III), Sb(III), Cd(II), Pb(II), Zn(II), Co(II), and Ni(II). These homodimeric repressors bind to DNA in the absence of inducing metal(loid) ion and dissociate from the DNA when inducer is bound. The regulatory sites are often three- or four-coordinate metal binding sites composed of cysteine thiolates. Surprisingly, in two different As(III)-responsive regulators, the metalloid binding sites were in different locations in the repressor, andmore » the Cd(II) binding sites were in two different locations in two Cd(II)-responsive regulators. We hypothesize that ArsR/SmtB repressors have a common backbone structure, that of a winged helix DNA-binding protein, but have considerable plasticity in the location of inducer binding sites. Here we show that an As(III)-responsive member of the family, CgArsR1 from Corynebacterium glutamicum, binds As(III) to a cysteine triad composed of Cys{sup 15}, Cys{sup 16}, and Cys{sup 55}. This binding site is clearly unrelated to the binding sites of other characterized ArsR/SmtB family members. This is consistent with our hypothesis that metal(loid) binding sites in DNA binding proteins evolve convergently in response to persistent environmental pressures.« less

Structural basis of Bloom syndrome (BS) causing mutations in the BLM helicase domain.

PubMed Central

Rong, S. B.; Väliaho, J.; Vihinen, M.

2000-01-01

BACKGROUND: Bloom syndrome (BS) is characterized by mutations within the BLM gene. The Bloom syndrome protein (BLM) has similarity to the RecQ subfamily of DNA helicases, which contain seven conserved helicase domains and share significant sequence and structural similarity with the Rep and PcrA DNA helicases. We modeled the three-dimensional structure of the BLM helicase domain to analyze the structural basis of BS-causing mutations. MATERIALS AND METHODS: The sequence alignment was performed for RecQ DNA helicases and Rep and PcrA helicases. The crystal structure of PcrA helicase (PDB entry 3PJR) was used as the template for modeling the BLM helicase domain. The model was used to infer the function of BLM and to analyze the effect of the mutations. RESULTS: The structural model with good stereochemistry of the BLM helicase domain contains two subdomains, 1A and 2A. The electrostatic potential of the model is highly negative over most of the surface, except for the cleft between subdomains 1A and 2A which is similar to the template protein. The ATP-binding site is located inside the model between subdomains 1A and 2A; whereas, the DNA-binding region is situated at the surface cleft, with positive potential between 1A and 2A. CONCLUSIONS: The three-dimensional structure of the BLM helicase domain was modeled and applied to interpret BS-causing mutations. The mutation I841T is likely to weaken DNA binding, while the mutations C891R, C901Y, and Q672R presumably disturb the ATP binding. In addition, other critical positions are discussed. PMID:10965492

Properties of the recombinant TNF-binding proteins from variola, monkeypox, and cowpox viruses are different.

PubMed

Gileva, Irina P; Nepomnyashchikh, Tatiana S; Antonets, Denis V; Lebedev, Leonid R; Kochneva, Galina V; Grazhdantseva, Antonina V; Shchelkunov, Sergei N

2006-11-01

Tumor necrosis factor (TNF), a potent proinflammatory and antiviral cytokine, is a critical extracellular immune regulator targeted by poxviruses through the activity of virus-encoded family of TNF-binding proteins (CrmB, CrmC, CrmD, and CrmE). The only TNF-binding protein from variola virus (VARV), the causative agent of smallpox, infecting exclusively humans, is CrmB. Here we have aligned the amino acid sequences of CrmB proteins from 10 VARV, 14 cowpox virus (CPXV), and 22 monkeypox virus (MPXV) strains. Sequence analyses demonstrated a high homology of these proteins. The regions homologous to cd00185 domain of the TNF receptor family, determining the specificity of ligand-receptor binding, were found in the sequences of CrmB proteins. In addition, a comparative analysis of the C-terminal SECRET domain sequences of CrmB proteins was performed. The differences in the amino acid sequences of these domains characteristic of each particular orthopoxvirus species were detected. It was assumed that the species-specific distinctions between the CrmB proteins might underlie the differences in these physicochemical and biological properties. The individual recombinant proteins VARV-CrmB, MPXV-CrmB, and CPXV-CrmB were synthesized in a baculovirus expression system in insect cells and isolated. Purified VARV-CrmB was detectable as a dimer with a molecular weight of 90 kDa, while MPXV- and CPXV-CrmBs, as monomers when fractioned by non-reducing SDS-PAGE. The CrmB proteins of VARV, MPXV, and CPXV differed in the efficiencies of inhibition of the cytotoxic effects of human, mouse, or rabbit TNFs in L929 mouse fibroblast cell line. Testing of CrmBs in the experimental model of LPS-induced shock using SPF BALB/c mice detected a pronounced protective effect of VARV-CrmB. Thus, our data demonstrated the difference in anti-TNF activities of VARV-, MPXV-, and CPXV-CrmBs and efficiency of VARV-CrmB rather than CPXV- or MPXV-CrmBs against LPS-induced mortality in mice.

Structural and Functional Analysis of the Human HDAC4 Catalytic Domain Reveals a Regulatory Structural Zinc-binding Domain*S⃞

PubMed Central

Bottomley, Matthew J.; Lo Surdo, Paola; Di Giovine, Paolo; Cirillo, Agostino; Scarpelli, Rita; Ferrigno, Federica; Jones, Philip; Neddermann, Petra; De Francesco, Raffaele; Steinkühler, Christian; Gallinari, Paola; Carfí, Andrea

2008-01-01

Histone deacetylases (HDACs) regulate chromatin status and gene expression, and their inhibition is of significant therapeutic interest. To date, no biological substrate for class IIa HDACs has been identified, and only low activity on acetylated lysines has been demonstrated. Here, we describe inhibitor-bound and inhibitor-free structures of the histone deacetylase-4 catalytic domain (HDAC4cd) and of an HDAC4cd active site mutant with enhanced enzymatic activity toward acetylated lysines. The structures presented, coupled with activity data, provide the molecular basis for the intrinsically low enzymatic activity of class IIa HDACs toward acetylated lysines and reveal active site features that may guide the design of class-specific inhibitors. In addition, these structures reveal a conformationally flexible structural zinc-binding domain conserved in all class IIa enzymes. Importantly, either the mutation of residues coordinating the structural zinc ion or the binding of a class IIa selective inhibitor prevented the association of HDAC4 with the N-CoR·HDAC3 repressor complex. Together, these data suggest a key role of the structural zinc-binding domain in the regulation of class IIa HDAC functions. PMID:18614528

Structural and functional analysis of the human HDAC4 catalytic domain reveals a regulatory structural zinc-binding domain.

PubMed

Bottomley, Matthew J; Lo Surdo, Paola; Di Giovine, Paolo; Cirillo, Agostino; Scarpelli, Rita; Ferrigno, Federica; Jones, Philip; Neddermann, Petra; De Francesco, Raffaele; Steinkühler, Christian; Gallinari, Paola; Carfí, Andrea

2008-09-26

Histone deacetylases (HDACs) regulate chromatin status and gene expression, and their inhibition is of significant therapeutic interest. To date, no biological substrate for class IIa HDACs has been identified, and only low activity on acetylated lysines has been demonstrated. Here, we describe inhibitor-bound and inhibitor-free structures of the histone deacetylase-4 catalytic domain (HDAC4cd) and of an HDAC4cd active site mutant with enhanced enzymatic activity toward acetylated lysines. The structures presented, coupled with activity data, provide the molecular basis for the intrinsically low enzymatic activity of class IIa HDACs toward acetylated lysines and reveal active site features that may guide the design of class-specific inhibitors. In addition, these structures reveal a conformationally flexible structural zinc-binding domain conserved in all class IIa enzymes. Importantly, either the mutation of residues coordinating the structural zinc ion or the binding of a class IIa selective inhibitor prevented the association of HDAC4 with the N-CoR.HDAC3 repressor complex. Together, these data suggest a key role of the structural zinc-binding domain in the regulation of class IIa HDAC functions.

A novel rice cytochrome P450 gene, CYP72A31, confers tolerance to acetolactate synthase-inhibiting herbicides in rice and Arabidopsis.

PubMed

Saika, Hiroaki; Horita, Junko; Taguchi-Shiobara, Fumio; Nonaka, Satoko; Nishizawa-Yokoi, Ayako; Iwakami, Satoshi; Hori, Kiyosumi; Matsumoto, Takashi; Tanaka, Tsuyoshi; Itoh, Takeshi; Yano, Masahiro; Kaku, Koichiro; Shimizu, Tsutomu; Toki, Seiichi

2014-11-01

Target-site and non-target-site herbicide tolerance are caused by the prevention of herbicide binding to the target enzyme and the reduction to a nonlethal dose of herbicide reaching the target enzyme, respectively. There is little information on the molecular mechanisms involved in non-target-site herbicide tolerance, although it poses the greater threat in the evolution of herbicide-resistant weeds and could potentially be useful for the production of herbicide-tolerant crops because it is often involved in tolerance to multiherbicides. Bispyribac sodium (BS) is an herbicide that inhibits the activity of acetolactate synthase. Rice (Oryza sativa) of the indica variety show BS tolerance, while japonica rice varieties are BS sensitive. Map-based cloning and complementation tests revealed that a novel cytochrome P450 monooxygenase, CYP72A31, is involved in BS tolerance. Interestingly, BS tolerance was correlated with CYP72A31 messenger RNA levels in transgenic plants of rice and Arabidopsis (Arabidopsis thaliana). Moreover, Arabidopsis overexpressing CYP72A31 showed tolerance to bensulfuron-methyl (BSM), which belongs to a different class of acetolactate synthase-inhibiting herbicides, suggesting that CYP72A31 can metabolize BS and BSM to a compound with reduced phytotoxicity. On the other hand, we showed that the cytochrome P450 monooxygenase CYP81A6, which has been reported to confer BSM tolerance, is barely involved, if at all, in BS tolerance, suggesting that the CYP72A31 enzyme has different herbicide specificities compared with CYP81A6. Thus, the CYP72A31 gene is a potentially useful genetic resource in the fields of weed control, herbicide development, and molecular breeding in a broad range of crop species. © 2014 American Society of Plant Biologists. All Rights Reserved.

Llama Antibody Fragments Recognizing Various Epitopes of the CD4bs Neutralize a Broad Range of HIV-1 Subtypes A, B and C

PubMed Central

Aasa-Chapman, Marlèn; Gorlani, Andrea; Forsman Quigley, Anna; Hulsik, David Lutje; Chen, Lei; Weiss, Robin; de Haard, Hans; Verrips, Theo

2012-01-01

Many of the neutralising antibodies, isolated to date, display limited activities against the globally most prevalent HIV-1 subtypes A and C. Therefore, those subtypes are considered to be an important target for antibody-based therapy. Variable domains of llama heavy chain antibodies (VHH) have some superior properties compared with classical antibodies. Therefore we describe the application of trimeric forms of envelope proteins (Env), derived from HIV-1 of subtype A and B/C, for a prolonged immunization of two llamas. A panel of VHH, which interfere with CD4 binding to HIV-1 Env were selected with use of panning. The results of binding and competition assays to various Env, including a variant with a stabilized CD4-binding state (gp120Ds2), cross-competition experiments, maturation analysis and neutralisation assays, enabled us to classify the selected VHH into three groups. The VHH of group I were efficient mainly against viruses of subtype A, C and B′/C. The VHH of group II resemble the broadly neutralising antibody (bnmAb) b12, neutralizing mainly subtype B and C viruses, however some had a broader neutralisation profile. A representative of the third group, 2E7, had an even higher neutralization breadth, neutralizing 21 out of the 26 tested strains belonging to the A, A/G, B, B/C and C subtypes. To evaluate the contribution of certain amino acids to the potency of the VHH a small set of the mutants were constructed. Surprisingly this yielded one mutant with slightly improved neutralisation potency against 92UG37.A9 (subtype A) and 96ZM651.02 (subtype C). These findings and the well-known stability of VHH indicate the potential application of these VHH as anti-HIV-1 microbicides. PMID:22438910

Thermodynamic and spectroscopic investigations of TMPyP4 association with guanine- and cytosine-rich DNA and RNA repeats of C9orf72.

PubMed

Alniss, Hasan; Zamiri, Bita; Khalaj, Melisa; Pearson, Christopher E; Macgregor, Robert B

2018-01-22

An expansion of the hexanucleotide repeat (GGGGCC)n·(GGCCCC)n in the C9orf72 promoter has been shown to be the cause of Amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). The C9orf72 repeat can form four-stranded structures; the cationic porphyrin (TMPyP4) binds and distorts these structures. Isothermal titration calorimetry (ITC), and circular dichroism (CD) were used to study the binding of TMPyP4 to the C-rich and G-rich DNA and RNA oligos containing the hexanucleotide repeat at pH 7.5 and 0.1 M K + . The CD spectra of G-rich DNA and RNA TMPyP4 complexes showed features of antiparallel and parallel G-quadruplexes, respectively. The shoulder at 260 nm in the CD spectrum becomes more intense upon formation of complexes between TMPyP4 and the C-rich DNA. The peak at 290 nm becomes more intense in the c-rich RNA molecules, suggesting induction of an i-motif structure. The ITC data showed that TMPyP4 binds at two independent sites for all DNA and RNA molecules. For DNA, the data are consistent with TMPyP4 stacking on the terminal tetrads and intercalation. For RNA, the thermodynamics of the two binding modes are consistent with groove binding and intercalation. In both cases, intercalation is the weaker binding mode. These findings are considered with respect to the structural differences of the folded DNA and RNA molecules and the energetics of the processes that drive site-specific recognition by TMPyP4; these data will be helpful in efforts to optimize the specificity and affinity of the binding of porphyrin-like molecules. Copyright © 2018 Elsevier Inc. All rights reserved.

The PBX1 lupus susceptibility gene regulates CD44 expression.

PubMed

Niu, Yuxin; Sengupta, Mayami; Titov, Anton A; Choi, Seung-Chul; Morel, Laurence

2017-05-01

PBX1-d is novel splice isoform of pre-B-cell leukemia homeobox 1 (PBX1) that lacks its DNA-binding and Hox-binding domains, and functions as a dominant negative. We have shown that PBX1-d expression in CD4 + T cells is associated with systemic lupus erythematosus (SLE) in a mouse model as well as in human subjects. More specifically, PBX1-d expression leads to the production of autoreactive activated CD4+ T cells, a reduced frequency and function of Foxp3+ regulatory T (Treg) cells and an expansion of follicular helper T (Tfh) cells. Very little is known about the function of PBX1 in T cells, except that it directly regulates the expression of miRNAs associated with Treg and Tfh homeostasis. In the present study, we show that PBX1 directly regulated the expression of CD44, a marker of T cell activation. Two PBX1 binding sites in the promoter directly regulated CD44 expression, with PBX1-d driving a higher expression than the normal isoform PBX1-b. In addition, mutations in each of the two binding sites had different effects of PBX1-b and PBX1-d. Finally, we showed that an enhanced recruitment of co-factor MEIS by PBX1-d over PBX1-b, while there was no difference for co-factor PREP1 recruitment. Therefore, this study demonstrates that the lupus-associated PBX1-d isoform directly transactivates CD44, a marker of CD44 activation and memory, and that it has different DNA binding and co-factor recruitment relative to the normal isoform. Taken together, these results confirm that PBX1 directly regulates genes related to T cell activation and shows that the lupus-associated isoform PBX1-d has unique molecular functions. Copyright © 2017 Elsevier Ltd. All rights reserved.

AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges

PubMed Central

Gardner, Matthew R.; Kattenhorn, Lisa M.; Kondur, Hema R.; von Schaewen, Markus; Dorfman, Tatyana; Chiang, Jessica J.; Haworth, Kevin G.; Decker, Julie M.; Alpert, Michael D.; Bailey, Charles C.; Neale, Ernest S.; Fellinger, Christoph H.; Joshi, Vinita R.; Fuchs, Sebastian P.; Martinez-Navio, Jose M.; Quinlan, Brian D.; Yao, Annie Y.; Mouquet, Hugo; Gorman, Jason; Zhang, Baoshan; Poignard, Pascal; Nussenzweig, Michel C.; Burton, Dennis R.; Kwong, Peter D.; Piatak, Michael; Lifson, Jeffrey D.; Gao, Guangping; Desrosiers, Ronald C.; Evans, David T.; Hahn, Beatrice H.; Ploss, Alexander; Cannon, Paula M.; Seaman, Michael S.; Farzan, Michael

2015-01-01

Long-term in vivo expression of a broad and potent entry inhibitor could circumvent the need for a conventional vaccine for HIV-1. Adeno-associated virus (AAV) vectors can stably express HIV-1 broadly neutralizing antibodies (bNAbs)1,2. However even the best bNAbs neutralize 10–50% of HIV-1 isolates inefficiently (IC80 > 5 μg/ml), suggesting that high concentrations of these antibodies would be necessary to achieve general protection3–6. Here we show that eCD4-Ig, a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, binds avidly and cooperatively to the HIV-1 envelope glycoprotein (Env) and is more potent than the best bNAbs (geometric mean IC50 < 0.05 μg/ml). Because eCD4-Ig binds only conserved regions of Env, it is also much broader than any bNAb. For example, eCD4-Ig efficiently neutralized 100% of a diverse panel of neutralization-resistant HIV-1, HIV-2, and SIV isolates, including a comprehensive set of isolates resistant to the CD4-binding site bNAbs VRC01, NIH45-46, and 3BNC117. Rhesus macaques inoculated with an AAV vector stably expressed 17 to 77 μg/ml of fully functional rhesus eCD4-Ig for 40 weeks, and these macaques were protected from multiple infectious challenges with SHIV-AD8. Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well characterized bNAbs. Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine. PMID:25707797

Investigation of the interaction between naringin and human serum albumin

NASA Astrophysics Data System (ADS)

Zhang, Yaheng; Li, Ying; Dong, Lijun; Li, Jiazhong; He, Wenying; Chen, Xingguo; Hu, Zhide

2008-03-01

The interaction between naringin and human serum albumin (HSA) has been thoroughly studied by fluorescence quenching technique in combination with UV absorption spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD) spectroscopy and molecular modeling method. Under the simulative physiological conditions, fluorescence data revealed the presence of the binding site on HSA and its binding constants ( K) are 1.62 × 10 4, 1.68 × 10 4, 1.72 × 10 4, and 1.79 × 10 4 M -1 at 289, 296, 303, and 310 K, respectively. The alterations of protein secondary structure in the presence of naringin aqueous solution were qualitative and quantitative calculated by the evidence from CD and FT-IR spectroscopes. In addition, according to the Van't Hoff equation, the thermodynamic functions standard enthalpy (Δ H0) and standard entropy (Δ S0) for the reaction were calculated to be 3.45 kJ mol -1 and 92.52 J mol -1 K -1. These results indicated that naringin binds to HSA mainly by a hydrophobic interaction. Furthermore, the displacement experiments confirmed that naringin could bind to the site I of HSA, which was also in agreement with the result of the molecular modeling study.

Allosteric modulation of semicarbazide-sensitive amine oxidase activities in vitro by imidazoline receptor ligands

PubMed Central

Holt, Andrew; Wieland, Barbara; Baker, Glen B

2004-01-01

Evidence indicates that imidazoline I2 binding sites (I2BSs) are present on monoamine oxidase (MAO) and on soluble (plasma) semicarbazide-sensitive amine oxidase enzymes. The binding site on MAO has been described as a modulatory site, although no effects on activity are thought to have been observed as a result of ligands binding to these sites. We examined the effects in vitro of several imidazoline binding site ligands on activities of bovine plasma amine oxidase (BPAO) and porcine kidney diamine oxidase (PKDAO) in a spectrophotometric protocol. While both enzymes were inhibited at high concentrations of all ligands, clonidine, cirazoline and oxymetazoline were seen, at lower concentrations, to increase activity of BPAO versus benzylamine, but not of PKDAO versus putrescine. This effect was substrate dependent, with mixed or biphasic inhibition of spermidine, methylamine, p-tyramine and β-phenylethylamine oxidation observed at cirazoline concentrations that increased benzylamine oxidation. With benzylamine as substrate, clonidine decreased KM (EC50 8.82 μM, Emax 75.1% of control) and increased Vmax (EC50 164.6 μM, Emax 154.1% of control). Cirazoline decreased Vmax (EC50 2.15 μM, Emax 91.4% of control), then decreased KM (EC50 5.63 μM, Emax 42.6% of control) and increased Vmax (EC50 49.0 μM, Emax 114.4% of decreased Vmax value). Data for clonidine fitted a mathematical model for two-site nonessential activation plus linear intersecting noncompetitive inhibition. Data for cirazoline were consistent with involvement of a fourth site. These results reveal an ability of imidazoline ligands to modulate BPAO kinetics allosterically. The derived mechanism may have functional significance with respect to modulation of MAO by I2BS ligands. PMID:15451775

Distribution of cyclophilin B-binding sites in the subsets of human peripheral blood lymphocytes.

PubMed

Denys, A; Allain, F; Foxwell, B; Spik, G

1997-08-01

Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway and released in biological fluids. We have recently demonstrated that both free CyPB and CyPB-CsA complex specifically bind to peripheral blood T lymphocytes and are internalized. These results suggest that CyPB might promote the targeting of the drug into sensitive cells. Peripheral blood lymphocytes are subdivided in several populations according to their biological functions and sensitivity to CsA. We have investigated the binding of CyPB to these different subsets using a CyPB derivatized by fluorescein through its single cysteine which retains its binding properties. We have confirmed that only T cells were involved in the interaction with CyPB. The ligand binding was found to be heterogeneously distributed on the different T-cell subsets and surface-bound CyPB was mainly associated with the CD4-positive cells. No significant difference was noted between the CD45RA and CD45RO subsets, demonstrating that CyPB-binding sites were equally distributed between native and memory T cells. CD3 stimulation of T lymphocytes led to a decrease in the CyPB-binding capacity, that may be explained by a down-regulation of the CyPB-receptor expression upon T-cell activation. Finally, we demonstrated that CyPB-receptor-positive cells, isolated on CyPB sulphydryl-coupled affinity matrices, are more sensitive to CyPB-complexed CsA than mixed peripheral blood lymphocytes, suggesting that CyPB potentiates CsA activity through the binding of the complex. Taken together, our results demonstrate that CyPB-binding sites are mainly associated with resting cells of the helper T lymphocyte, and that CyPB might modulate the distribution of CsA through the drug targeting to sensitive cells.

Inhibition of [11C]mirtazapine binding by alpha2-adrenoceptor antagonists studied by positron emission tomography in living porcine brain.

PubMed

Smith, Donald F; Dyve, Suzan; Minuzzi, Luciano; Jakobsen, Steen; Munk, Ole L; Marthi, Katalin; Cumming, Paul

2006-06-15

We have developed [(11)C]mirtazapine as a ligand for PET studies of antidepressant binding in living brain. However, previous studies have determined neither optimal methods for quantification of [(11)C]mirtazapine binding nor the pharmacological identity of this binding. To obtain that information, we have now mapped the distribution volume (V(d)) of [(11)C]mirtazapine relative to the arterial input in the brain of three pigs, in a baseline condition and after pretreatment with excess cold mirtazapine (3 mg/kg). Baseline V(d) ranged from 6 ml/ml in cerebellum to 18 ml/ml in frontal cortex, with some evidence for a small self-displaceable binding component in the cerebellum. Regional binding potentials (pBs) obtained by a constrained two-compartment model, using the V(d) observation in cerebellum, were consistently higher than pBs obtained by other arterial input or reference tissue methods. We found that adequate quantification of pB was obtained using the simplified reference tissue method. Concomitant PET studies with [(15)O]-water indicated that mirtazapine challenge increased CBF uniformly in cerebellum and other brain regions, supporting the use of this reference tissue for calculation of [(11)C]mirtazapine pB. Displacement by mirtazapine was complete in the cerebral cortex, but only 50% in diencephalon, suggesting the presence of multiple binding sites of differing affinities in that tissue. Competition studies with yohimbine and RX 821002 showed decreases in [(11)C]mirtazapine pB throughout the forebrain; use of the multireceptor version of the Michaelis-Menten equation indicated that 42% of [(11)C]mirtazapine binding in cortical regions is displaceable by yohimbine. Thus, PET studies confirm that [(11)C]mirtazapine affects alpha(2)-adrenoceptor binding sites in living brain. (c) 2006 Wiley-Liss, Inc.

Strength of Neisseria meningitidis binding to endothelial cells requires highly-ordered CD147/β2-adrenoceptor clusters assembled by alpha-actinin-4

PubMed Central

Maïssa, Nawal; Covarelli, Valentina; Janel, Sébastien; Durel, Beatrice; Simpson, Nandi; Bernard, Sandra C.; Pardo-Lopez, Liliana; Bouzinba-Ségard, Haniaa; Faure, Camille; Scott, Mark G.H.; Coureuil, Mathieu; Morand, Philippe C.; Lafont, Frank; Nassif, Xavier; Marullo, Stefano; Bourdoulous, Sandrine

2017-01-01

Neisseria meningitidis (meningococcus) is an invasive bacterial pathogen that colonizes human vessels, causing thrombotic lesions and meningitis. Establishment of tight interactions with endothelial cells is crucial for meningococci to resist haemodynamic forces. Two endothelial receptors, CD147 and the β2-adrenergic receptor (β2AR), are sequentially engaged by meningococci to adhere and promote signalling events leading to vascular colonization, but their spatiotemporal coordination is unknown. Here we report that CD147 and β2AR form constitutive hetero-oligomeric complexes. The scaffolding protein α-actinin-4 directly binds to the cytosolic tail of CD147 and governs the assembly of CD147–β2AR complexes in highly ordered clusters at bacterial adhesion sites. This multimolecular assembly process increases the binding strength of meningococci to endothelial cells under shear stress, and creates molecular platforms for the elongation of membrane protrusions surrounding adherent bacteria. Thus, the specific organization of cellular receptors has major impacts on host–pathogen interaction. PMID:28569760

Functional Mimetics of the HIV-1 CCR5 Co-Receptor Displayed on the Surface of Magnetic Liposomes.

PubMed

Kuzmina, Alona; Vaknin, Karin; Gdalevsky, Garik; Vyazmensky, Maria; Marks, Robert S; Taube, Ran; Engel, Stanislav

2015-01-01

Chemokine G protein coupled receptors, principally CCR5 or CXCR4, function as co-receptors for HIV-1 entry into CD4+ T cells. Initial binding of the viral envelope glycoprotein (Env) gp120 subunit to the host CD4 receptor induces a cascade of structural conformational changes that lead to the formation of a high-affinity co-receptor-binding site on gp120. Interaction between gp120 and the co-receptor leads to the exposure of epitopes on the viral gp41 that mediates fusion between viral and cell membranes. Soluble CD4 (sCD4) mimetics can act as an activation-based inhibitor of HIV-1 entry in vitro, as it induces similar structural changes in gp120, leading to increased virus infectivity in the short term but to virus Env inactivation in the long term. Despite promising clinical implications, sCD4 displays low efficiency in vivo, and in multiple HIV strains, it does not inhibit viral infection. This has been attributed to the slow kinetics of the sCD4-induced HIV Env inactivation and to the failure to obtain sufficient sCD4 mimetic levels in the serum. Here we present uniquely structured CCR5 co-receptor mimetics. We hypothesized that such mimetics will enhance sCD4-induced HIV Env inactivation and inhibition of HIV entry. Co-receptor mimetics were derived from CCR5 gp120-binding epitopes and functionalized with a palmitoyl group, which mediated their display on the surface of lipid-coated magnetic beads. CCR5-peptidoliposome mimetics bound to soluble gp120 and inhibited HIV-1 infectivity in a sCD4-dependent manner. We concluded that CCR5-peptidoliposomes increase the efficiency of sCD4 to inhibit HIV infection by acting as bait for sCD4-primed virus, catalyzing the premature discharge of its fusion potential.

Functional Mimetics of the HIV-1 CCR5 Co-Receptor Displayed on the Surface of Magnetic Liposomes

PubMed Central

Kuzmina, Alona; Vaknin, Karin; Gdalevsky, Garik; Vyazmensky, Maria; Marks, Robert S.; Taube, Ran

2015-01-01

Chemokine G protein coupled receptors, principally CCR5 or CXCR4, function as co-receptors for HIV-1 entry into CD4+ T cells. Initial binding of the viral envelope glycoprotein (Env) gp120 subunit to the host CD4 receptor induces a cascade of structural conformational changes that lead to the formation of a high-affinity co-receptor-binding site on gp120. Interaction between gp120 and the co-receptor leads to the exposure of epitopes on the viral gp41 that mediates fusion between viral and cell membranes. Soluble CD4 (sCD4) mimetics can act as an activation-based inhibitor of HIV-1 entry in vitro, as it induces similar structural changes in gp120, leading to increased virus infectivity in the short term but to virus Env inactivation in the long term. Despite promising clinical implications, sCD4 displays low efficiency in vivo, and in multiple HIV strains, it does not inhibit viral infection. This has been attributed to the slow kinetics of the sCD4-induced HIV Env inactivation and to the failure to obtain sufficient sCD4 mimetic levels in the serum. Here we present uniquely structured CCR5 co-receptor mimetics. We hypothesized that such mimetics will enhance sCD4-induced HIV Env inactivation and inhibition of HIV entry. Co-receptor mimetics were derived from CCR5 gp120-binding epitopes and functionalized with a palmitoyl group, which mediated their display on the surface of lipid-coated magnetic beads. CCR5-peptidoliposome mimetics bound to soluble gp120 and inhibited HIV-1 infectivity in a sCD4-dependent manner. We concluded that CCR5-peptidoliposomes increase the efficiency of sCD4 to inhibit HIV infection by acting as bait for sCD4-primed virus, catalyzing the premature discharge of its fusion potential. PMID:26629902

The distal short consensus repeats 1 and 2 of the membrane cofactor protein CD46 and their distance from the cell membrane determine productive entry of species B adenovirus serotype 35.

PubMed

Fleischli, Christoph; Verhaagh, Sandra; Havenga, Menzo; Sirena, Dominique; Schaffner, Walter; Cattaneo, Roberto; Greber, Urs F; Hemmi, Silvio

2005-08-01

The human regulator of complement activation membrane cofactor protein (CD46) has recently been identified as an attachment receptor for most species B adenoviruses (Ads), including Ad type 3 (Ad3), Ad11, and Ad35, as well as species D Ad37. To characterize the interaction between Ad35 and CD46, hybrid receptors composed of different CD46 short consensus repeat (SCR) domains fused to immunoglobulin-like domains of CD4 and a set of 36 CD46 mutants containing semiconservative changes of single amino acids within SCR domains I and II were tested in binding and in Ad35-mediated luciferase transduction assays. In addition, anti-CD46 antibodies and soluble polypeptides constituting various CD46 domains were used in binding inhibition studies. Our data indicate that (i) CD46 SCR I or SCR II alone confers low but significant Ad35 binding; (ii) the presence of SCR I and II is required for optimal binding and transgene expression; (iii) transduction efficiencies equivalent to that of full-length CD46 are obtained if SCR I and II are at an appropriate distance from the cell membrane; (iv) ablation of the N-glycan attached to SCR I has no influence on receptor function, whereas ablation of the SCR II N-glycan results in about a two- to threefold reduction of binding and transgene expression; (v) most putative Ad35 binding residues are located on the same solvent-exposed face of the SCR I or SCR II domain, which are twisted by about 90 degrees ; and (vi) the putative Ad35 binding sites partly overlap with the measles virus binding surface.

The Distal Short Consensus Repeats 1 and 2 of the Membrane Cofactor Protein CD46 and Their Distance from the Cell Membrane Determine Productive Entry of Species B Adenovirus Serotype 35

PubMed Central

Fleischli, Christoph; Verhaagh, Sandra; Havenga, Menzo; Sirena, Dominique; Schaffner, Walter; Cattaneo, Roberto; Greber, Urs F.; Hemmi, Silvio

2005-01-01

The human regulator of complement activation membrane cofactor protein (CD46) has recently been identified as an attachment receptor for most species B adenoviruses (Ads), including Ad type 3 (Ad3), Ad11, and Ad35, as well as species D Ad37. To characterize the interaction between Ad35 and CD46, hybrid receptors composed of different CD46 short consensus repeat (SCR) domains fused to immunoglobulin-like domains of CD4 and a set of 36 CD46 mutants containing semiconservative changes of single amino acids within SCR domains I and II were tested in binding and in Ad35-mediated luciferase transduction assays. In addition, anti-CD46 antibodies and soluble polypeptides constituting various CD46 domains were used in binding inhibition studies. Our data indicate that (i) CD46 SCR I or SCR II alone confers low but significant Ad35 binding; (ii) the presence of SCR I and II is required for optimal binding and transgene expression; (iii) transduction efficiencies equivalent to that of full-length CD46 are obtained if SCR I and II are at an appropriate distance from the cell membrane; (iv) ablation of the N-glycan attached to SCR I has no influence on receptor function, whereas ablation of the SCR II N-glycan results in about a two- to threefold reduction of binding and transgene expression; (v) most putative Ad35 binding residues are located on the same solvent-exposed face of the SCR I or SCR II domain, which are twisted by about 90°; and (vi) the putative Ad35 binding sites partly overlap with the measles virus binding surface. PMID:16014961

Combined Endoscopic/Sonographic-Based Risk Matrix Model for Predicting One-Year Risk of Surgery: A Prospective Observational Study of a Tertiary Center Severe/Refractory Crohn's Disease Cohort.

PubMed

Rispo, Antonio; Imperatore, Nicola; Testa, Anna; Bucci, Luigi; Luglio, Gaetano; De Palma, Giovanni Domenico; Rea, Matilde; Nardone, Olga Maria; Caporaso, Nicola; Castiglione, Fabiana

2018-03-08

In the management of Crohn's Disease (CD) patients, having a simple score combining clinical, endoscopic and imaging features to predict the risk of surgery could help to tailor treatment more effectively. AIMS: to prospectively evaluate the one-year risk factors for surgery in refractory/severe CD and to generate a risk matrix for predicting the probability of surgery at one year. CD patients needing a disease re-assessment at our tertiary IBD centre underwent clinical, laboratory, endoscopy and bowel sonography (BS) examinations within one week. The optimal cut-off values in predicting surgery were identified using ROC curves for Simple Endoscopic Score for CD (SES-CD), bowel wall thickness (BWT) at BS, and small bowel CD extension at BS. Binary logistic regression and Cox's regression were then carried out. Finally, the probabilities of surgery were calculated for selected baseline levels of covariates and results were arranged in a prediction matrix. Of 100 CD patients, 30 underwent surgery within one year. SES-CD©9 (OR 15.3; p<0.001), BWT©7 mm (OR 15.8; p<0.001), small bowel CD extension at BS©33 cm (OR 8.23; p<0.001) and stricturing/penetrating behavior (OR 4.3; p<0.001) were the only independent factors predictive of surgery at one-year based on binary logistic and Cox's regressions. Our matrix model combined these risk factors and the probability of surgery ranged from 0.48% to 87.5% (sixteen combinations). Our risk matrix combining clinical, endoscopic and ultrasonographic findings can accurately predict the one-year risk of surgery in patients with severe/refractory CD requiring a disease re-evaluation. This tool could be of value in clinical practice, serving as the basis for a tailored management of CD patients.

Structural and Physical Basis for Anti-IgE Therapy

NASA Astrophysics Data System (ADS)

Wright, Jon D.; Chu, Hsing-Mao; Huang, Chun-Hsiang; Ma, Che; Wen Chang, Tse; Lim, Carmay

2015-06-01

Omalizumab, an anti-IgE antibody, used to treat severe allergic asthma and chronic idiopathic urticaria, binds to IgE in blood or membrane-bound on B lymphocytes but not to IgE bound to its high (FcɛRI) or low (CD23) affinity receptor. Mutagenesis studies indicate overlapping FcɛRI and omalizumab-binding sites in the Cɛ3 domain, but crystallographic studies show FcɛRI and CD23-binding sites that are far apart, so how can omalizumab block IgE from binding both receptors? We report a 2.42-Å omalizumab-Fab structure, a docked IgE-Fc/omalizumab-Fab structure consistent with available experimental data, and the free energy contributions of IgE residues to binding omalizumab, CD23, and FcɛRI. These results provide a structural and physical basis as to why omalizumab cannot bind receptor-bound IgE and why omalizumab-bound IgE cannot bind to CD23/FcɛRI. They reveal the key IgE residues and their roles in binding omalizumab, CD23, and FcɛRI.

Differential binding of neutralizing and non-neutralizing antibodies to native-like soluble HIV-1 Env trimers, uncleaved Env proteins, and monomeric subunits

PubMed Central

2014-01-01

Background The trimeric envelope glycoproteins (Env) on the surface of HIV-1 virions are the targets for neutralizing antibodies (NAbs). No candidate HIV-1 immunogen has yet induced potent, broadly active NAbs (bNAbs). Part of the explanation may be that previously tested Env proteins inadequately mimic the functional, native Env complex. Trimerization and the proteolytic processing of Env precursors into gp120 and gp41 profoundly alter antigenicity, but soluble cleaved trimers are too unstable to serve as immunogens. By introducing stabilizing mutations (SOSIP), we constructed soluble, cleaved Env trimers derived from the HIV-1 subtype A isolate BG505 that resemble native Env spikes on virions both structurally and antigenically. Results We used surface plasmon resonance (SPR) to quantify antibody binding to different forms of BG505 Env: the proteolytically cleaved SOSIP.664 trimers, cleaved gp120-gp41ECTO protomers, and gp120 monomers. Non-NAbs to the CD4-binding site bound only marginally to the trimers but equally well to gp120-gp41ECTO protomers and gp120 monomers, whereas the bNAb VRC01, directed to the CD4bs, bound to all three forms. In contrast, bNAbs to V1V2 glycan-dependent epitopes bound preferentially (PG9 and PG16) or exclusively (PGT145) to trimers. We also explored the antigenic consequences of three different features of SOSIP.664 gp140 trimers: the engineered inter-subunit disulfide bond, the trimer-stabilizing I559P change in gp41ECTO, and proteolytic cleavage at the gp120-gp41ECTO junction. Each of these three features incrementally promoted native-like trimer antigenicity. We compared Fab and IgG versions of bNAbs and validated a bivalent model of IgG binding. The NAbs showed widely divergent binding kinetics and degrees of binding to native-like BG505 SOSIP.664. High off-rate constants and low stoichiometric estimates of NAb binding were associated with large amounts of residual infectivity after NAb neutralization of the corresponding BG505.T332N pseudovirus. Conclusions The antigenicity and structural integrity of cleaved BG505 SOSIP.664 trimers render these proteins good mimics of functional Env spikes on virions. In contrast, uncleaved gp140s antigenically resemble individual gp120-gp41ECTO protomers and gp120 monomers, but not native trimers. Although NAb binding to functional trimers may thus be both necessary and sufficient for neutralization, the kinetics and stoichiometry of the interaction influence the neutralizing efficacy of individual NAbs. PMID:24884783

Mechanistic and conformational studies on the interaction of food dye amaranth with human serum albumin by multispectroscopic methods.

PubMed

Zhang, Guowen; Ma, Yadi

2013-01-15

The mechanism of interaction between food dye amaranth and human serum albumin (HSA) in physiological buffer (pH 7.4) was investigated by fluorescence, UV-vis absorption, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy. Results obtained from analysis of fluorescence spectra indicated that amaranth had a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. The negative value of enthalpy change and positive value of entropy change elucidated that the binding of amaranth to HSA was driven mainly by hydrophobic and hydrogen bonding interactions. The surface hydrophobicity of HSA increased after binding with amaranth. The binding distance between HSA and amaranth was estimated to be 3.03 nm and subdomain IIA (Sudlow site I) was the primary binding site for amaranth on HSA. The results of CD and FT-IR spectra showed that binding of amaranth to HSA induced conformational changes of HSA. Copyright © 2012 Elsevier Ltd. All rights reserved.

CD14 and CD11b mediate serum-independent binding to human monocytes of an acylpolygalactoside isolated from Klebsiella pneumoniae.

PubMed Central

Hmama, Z; Mey, A; Normier, G; Binz, H; Revillard, J P

1994-01-01

A water-soluble acylpolygalactosyl (APG) of 34 kDa was obtained from the Klebsiella pneumoniae membrane by alkaline hydrolysis and delipidation. APG comprises a poly(1,3)galactose chain, a core, and a lipid moiety made of a glucosamine disaccharide with two N-linked beta OH-myristates. The monocyte binding sites for APG were investigated by flow cytometry. Biotin-labelled APG (Biot-APG) bound to monocytes at 4 degrees C in the absence of serum, calcium, and magnesium. The binding was dose dependent, saturable, and displaced by unlabelled APG. Neither the polysaccharide chain present in APG-related molecules nor the PPi group or additional ester-linked myristates and palmitates were required for APG binding. The role of CD11b and CD14 was demonstrated by competitive inhibition with monoclonal antibodies and by the uptake of APG by these solubilized proteins. APG was rapidly internalized into monocytes at 37 degrees C while CD14 and CD11b/CD18 molecules were partially down-modulated. Lipopolysaccharides (LPS) from the same K. pneumoniae strain and from Escherichia coli and Salmonella minnesota partially competed for Biot-APG binding in the absence but not in the presence of serum. When altered by alkaline hydrolysis, those LPS became strong competitors for APG binding. It was concluded that alkaline hydrolysis of the K. pneumoniae membrane yielded molecules structurally related to LPS which bind to LPS membrane receptors in the absence of serum. Images PMID:7513300

Elicitation of Neutralizing Antibodies Directed against CD4-Induced Epitope(s) Using a CD4 Mimetic Cross-Linked to a HIV-1 Envelope Glycoprotein

PubMed Central

Dey, Antu K.; Burke, Brian; Sun, Yide; Sirokman, Klara; Nandi, Avishek; Hartog, Karin; Lian, Ying; Geonnotti, Anthony R.; Montefiori, David; Franti, Michael; Martin, Grégoire; Carfi, Andrea; Kessler, Pascal; Martin, Loïc; Srivastava, Indresh K.; Barnett, Susan W.

2012-01-01

The identification of HIV-1 envelope glycoprotein (Env) structures that can generate broadly neutralizing antibodies (BNAbs) is pivotal to the development of a successful vaccine against HIV-1 aimed at eliciting effective humoral immune responses. To that end, the production of novel Env structure(s) that might induce BNAbs by presentation of conserved epitopes, which are otherwise occluded, is critical. Here, we focus on a structure that stabilizes Env in a conformation representative of its primary (CD4) receptor-bound state, thereby exposing highly conserved “CD4 induced” (CD4i) epitope(s) known to be important for co-receptor binding and subsequent virus infection. A CD4-mimetic miniprotein, miniCD4 (M64U1-SH), was produced and covalently complexed to recombinant, trimeric gp140 envelope glycoprotein (gp140) using site-specific disulfide linkages. The resulting gp140-miniCD4 (gp140-S-S-M64U1) complex was recognized by CD4i antibodies and the HIV-1 co-receptor, CCR5. The gp140-miniCD4 complex elicited the highest titers of CD4i binding antibodies as well as enhanced neutralizing antibodies against Tier 1 viruses as compared to gp140 protein alone following immunization of rabbits. Neutralization against HIV-27312/V434M and additional serum mapping confirm the specific elicitation of antibodies directed to the CD4i epitope(s). These results demonstrate the utility of structure-based approach in improving immunogenic response against specific region, such as the CD4i epitope(s) here, and its potential role in vaccine application. PMID:22291921

Cooperativity of CD44 and CD49d in leukemia cell homing, migration, and survival offers a means for therapeutic attack.

PubMed

Singh, Vibuthi; Erb, Ulrike; Zöller, Margot

2013-11-15

A CD44 blockade drives leukemic cells into differentiation and apoptosis by dislodging from the osteogenic niche. Because anti-CD49d also supports hematopoietic stem cell mobilization, we sought to determine the therapeutic efficacy of a joint CD49d/CD44 blockade. To unravel the underlying mechanism, the CD49d(-) EL4 lymphoma was transfected with CD49d or point-mutated CD49d, prohibiting phosphorylation and FAK binding; additionally, a CD44(-) Jurkat subline was transfected with murine CD44, CD44 with a point mutation in the ezrin binding site, or with cytoplasmic tail-truncated CD44. Parental and transfected EL4 and Jurkat cells were evaluated for adhesion, migration, and apoptosis susceptibility in vitro and in vivo. Ligand-binding and Ab-blocking studies revealed CD44-CD49d cooperation in vitro and in vivo in adhesion, migration, and apoptosis resistance. The cooperation depends on ligand-induced proximity such that both CD44 and CD49d get access to src, FAK, and paxillin and via lck to the MAPK pathway, with the latter also supporting antiapoptotic molecule liberation. Accordingly, synergisms were only seen in leukemia cells expressing wild-type CD44 and CD49d. Anti-CD44 together with anti-CD49d efficiently dislodged EL4-CD49d/Jurkat-CD44 in bone marrow and spleen. Dislodging was accompanied by increased apoptosis susceptibility that strengthened low-dose chemotherapy, the combined treatment most strongly interfering with metastatic settlement and being partly curative. Ab treatment also promoted NK and Ab-dependent cellular cytotoxicity activation, which affected leukemia cells independent of CD44/CD49d tail mutations. Thus, mostly owing to a blockade of joint signaling, anti-CD44 and anti-CD49d hamper leukemic cell settlement and break apoptosis resistance, which strongly supports low-dose chemotherapy.

An rbcL mRNA-binding protein is associated with C3 to C4 evolution and light-induced production of Rubisco in Flaveria.

PubMed

Yerramsetty, Pradeep; Agar, Erin M; Yim, Won C; Cushman, John C; Berry, James O

2017-07-20

Nuclear-encoded RLSB protein binds chloroplastic rbcL mRNA encoding the Rubisco large subunit. RLSB is highly conserved across all groups of land plants and is associated with positive post-transcriptional regulation of rbcL expression. In C3 leaves, RLSB and Rubisco occur in all chlorenchyma cell chloroplasts, while in C4 leaves these accumulate only within bundle sheath (BS) chloroplasts. RLSB's role in rbcL expression makes modification of its localization a likely prerequisite for the evolutionary restriction of Rubisco to BS cells. Taking advantage of evolutionarily conserved RLSB orthologs in several C3, C3-C4, C4-like, and C4 photosynthetic types within the genus Flaveria, we show that low level RLSB sequence divergence and modification to BS specificity coincided with ontogeny of Rubisco specificity and Kranz anatomy during C3 to C4 evolution. In both C3 and C4 species, Rubisco production reflected RLSB production in all cell types, tissues, and conditions examined. Co-localization occurred only in photosynthetic tissues, and both proteins were co-ordinately induced by light at post-transcriptional levels. RLSB is currently the only mRNA-binding protein to be associated with rbcL gene regulation in any plant, with variations in sequence and acquisition of cell type specificity reflecting the progression of C4 evolution within the genus Flaveria. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Insight into the binding mechanism of imipenem to human serum albumin by spectroscopic and computational approaches.

PubMed

Rehman, Md Tabish; Shamsi, Hira; Khan, Asad U

2014-06-02

The mechanism of interaction between imipenem and HSA was investigated by various techniques like fluorescence, UV.vis absorbance, FRET, circular dichroism, urea denaturation, enzyme kinetics, ITC, and molecular docking. We found that imipenem binds to HSA at a high affinity site located in subdomain IIIA (Sudlow's site I) and a low affinity site located in subdomain IIA.IIB. Electrostatic interactions played a vital role along with hydrogen bonding and hydrophobic interactions in stabilizing the imipenem.HSA complex at subdomain IIIA, while only electrostatic and hydrophobic interactions were present at subdomain IIA.IIB. The binding and thermodynamic parameters obtained by ITC showed that the binding of imipenem to HSA was a spontaneous process (ΔGD⁰(D)= -32.31 kJ mol(-1) for high affinity site and ΔGD⁰(D) = -23.02 kJ mol(-1) for low affinity site) with binding constants in the range of 10(4)-10(5) M(-1). Spectroscopic investigation revealed only one binding site of imipenem on HSA (Ka∼10(4) M(-1)). FRET analysis showed that the binding distance between imipenem and HSA (Trp-214) was optimal (r = 4.32 nm) for quenching to occur. Decrease in esterase-like activity of HSA in the presence of imipenem showed that Arg-410 and Tyr-411 of subdomain IIIA (Sudlow's site II) were directly involved in the binding process. CD spectral analysis showed altered conformation of HSA upon imipenem binding. Moreover, the binding of imipenem to subdomain IIIA (Sudlow's site II) of HSA also affected its folding pathway as clear from urea-induced denaturation studies.

In-Silico Analysis of Amotosalen Hydrochloride Binding to CD-61 of Platelets.

PubMed

Chaudhary, Hammad Tufail

2016-11-01

To determine the docking of Amotosalen hydrochloride (AH) at CD-61 of platelets, and to suggest the cause of bleeding in AH treated platelets transfusion. Descriptive study. Medical College, Taif University, Taif, Saudi Arabia, from October 2014 to May 2015. The study was carried out in-silico. PDB (protein data bank) code of Tirofiban bound to CD-61 was 2vdm. CD-61 was docked with Tirofiban using online docking tools, i.e. Patchdock and Firedock. Then, Amotosalen hydrochloride and CD-61 were also docked. Best docking poses to active sites of 2vdm were found. Ligplot of interactions of ligands and CD-61 were obtained. Then comparison of hydrogen bonds, hydrogen bond lengths, and hydrophobic bonds of 2vdm molecule and best poses of docking results were done. Patchdock and Firedock results of best poses were also analysed using SPSS version 16. More amino acids were involved in hydrogen and hydrophobic bonds in Patchdock and Firedock docking of Amotosalen hydrochloride with CD-61 than Patchdock and Firedock docking of CD-61 with Tirofiban. The binding energy was more in latter than former. Amotosalen hydrochloride binds to the active site of CD-61 with weaker binding force. Haemorrhage seen in Amotosalen hydrochloride-treated platelets might be due to binding of Amotosalen hydrochloride to CD-61.

Determining the Binding Sites of β-Cyclodextrin and Peptides by Electron-Capture Dissociation High Resolution Tandem Mass Spectrometry

NASA Astrophysics Data System (ADS)

Qi, Yulin; Geib, Timon; Volmer, Dietrich A.

2015-07-01

Cyclodextrins (CDs) are a group of cyclic oligosaccharides, which readily form inclusion complexes with hydrophobic compounds to increase bioavailability, thus making CDs ideal drug excipients. Recent studies have also shown that CDs exhibit a wide range of protective effects, preventing proteins from aggregation, degradation, and folding. These effects strongly depend on the binding sites on the protein surface. CDs only exhibit weak interactions with amino acids, however; conventional analytical techniques therefore usually fail to reveal the exact location of the binding sites. Moreover, some studies even suggest that CD inclusion complexes are merely electrostatic adducts. Here, electron capture dissociation (ECD) was applied in this proof-of-concept study to examine the exact nature of the CD/peptide complexes, and CD binding sites were unambiguously located for the first time via Fourier-transform ion cyclotron resonance (FTICR) tandem mass spectrometry.

Scavenger receptor function of mouse Fcγ receptor III contributes to progression of atherosclerosis in apolipoprotein E hyperlipidemic mice.

PubMed

Zhu, Xinmei; Ng, Hang Pong; Lai, Yen-Chun; Craigo, Jodi K; Nagilla, Pruthvi S; Raghani, Pooja; Nagarajan, Shanmugam

2014-09-01

Recent studies showed loss of CD36 or scavenger receptor-AI/II (SR-A) does not ameliorate atherosclerosis in a hyperlipidemic mouse model, suggesting receptors other than CD36 and SR-A may also contribute to atherosclerosis. In this report, we show that apolipoprotein E (apoE)-CD16 double knockout (DKO; apoE-CD16 DKO) mice have reduced atherosclerotic lesions compared with apoE knockout mice. In vivo and in vitro foam cell analyses showed apoE-CD16 DKO macrophages accumulated less neutral lipids. Reduced foam cell formation in apoE-CD16 DKO mice is not due to change in expression of CD36, SR-A, and LOX-1. This led to a hypothesis that CD16 may have scavenger receptor activity. We presented evidence that a soluble form of recombinant mouse CD16 (sCD16) bound to malondialdehyde-modified low-density lipoprotein (MDALDL), and this binding is blocked by molar excess of MDA- modified BSA and anti-MDA mAbs, suggesting CD16 specifically recognizes MDA epitopes. Interestingly, sCD16 inhibited MDALDL binding to macrophage cell line, as well as soluble forms of recombinant mouse CD36, SR-A, and LOX-1, indicating CD16 can cross-block MDALDL binding to other scavenger receptors. Anti-CD16 mAb inhibited immune complex binding to sCD16, whereas it partially inhibited MDALDL binding to sCD16, suggesting MDALDL binding site may be in close proximity to the immune complex binding site in CD16. Loss of CD16 expression resulted in reduced levels of MDALDL-induced proinflammatory cytokine expression. Finally, CD16-deficient macrophages showed reduced MDALDL-induced Syk phosphorylation. Collectively, our findings suggest scavenger receptor activity of CD16 may, in part, contribute to the progression of atherosclerosis. Copyright © 2014 by The American Association of Immunologists, Inc.

The CD44-initiated pathway of T-cell extravasation uses VLA-4 but not LFA-1 for firm adhesion

PubMed Central

Siegelman, Mark H.; Stanescu, Diana; Estess, Pila

2000-01-01

Leukocytes extravasate from the blood in response to physiologic or pathologic demands by means of complementary ligand interactions between leukocytes and endothelial cells. The multistep model of leukocyte extravasation involves an initial transient interaction (“rolling” adhesion), followed by secondary (firm) adhesion. We recently showed that binding of CD44 on activated T lymphocytes to endothelial hyaluronan (HA) mediates a primary adhesive interaction under shear stress, permitting extravasation at sites of inflammation. The mechanism for subsequent firm adhesion has not been elucidated. Here we demonstrate that the integrin VLA-4 is used in secondary adhesion after CD44-mediated primary adhesion of human and mouse T cells in vitro, and by mouse T cells in an in vivo model. We show that clonal cell lines and polyclonally activated normal T cells roll under physiologic shear forces on hyaluronate and require VCAM-1, but not ICAM-1, as ligand for subsequent firm adhesion. This firm adhesion is also VLA-4 dependent, as shown by antibody inhibition. Moreover, in vivo short-term homing experiments in a model dependent on CD44 and HA demonstrate that superantigen-activated T cells require VLA-4, but not LFA-1, for entry into an inflamed peritoneal site. Thus, extravasation of activated T cells initiated by CD44 binding to HA depends upon VLA-4–mediated firm adhesion, which may explain the frequent association of these adhesion receptors with diverse chronic inflammatory processes. PMID:10712440

The effect of metal loading on Cd adsorption onto Shewanella oneidensis bacterial cell envelopes: The role of sulfhydryl sites

NASA Astrophysics Data System (ADS)

Yu, Qiang; Fein, Jeremy B.

2015-10-01

The adsorption and desorption of Cd onto Shewanella oneidensis bacterial cells with and without blocking of sulfhydryl sites was measured in order to determine the effect of metal loading and to understand the role of sulfhydryl sites in the adsorption reactions. The observed adsorption/desorption behaviors display strong dependence on metal loading. Under a high loading of 40 μmol Cd/g bacterial cells, blocking the sulfhydryl sites within the cell envelope by exposure of the biomass to monobromo(trimethylammonio)bimane bromide (qBBr) does not significantly affect the extent of Cd adsorption, and we observed fully reversible adsorption under this condition. In contrast, under a low metal loading of 1.3 μmol Cd/g bacterial cells, the extent of Cd adsorption onto sulfhydryl-blocked S. oneidensis cells was significantly lower than that onto untreated cells, and only approximately 50-60% of the adsorbed Cd desorbed from the cells upon acidification. In conjunction with previous EXAFS results, our findings demonstrate that Cd adsorption onto S. oneidensis under low metal loading conditions is dominated by sulfhydryl binding, and thus is controlled by a distinct adsorption mechanism from the non-sulfhydryl site binding which controls Cd adsorption under high metal loading conditions. We use the data to develop a surface complexation model that constrains the values of the stability constants for individual Cd-sulfhydryl and Cd-non-sulfhydryl bacterial complexes, and we use this approach to account for the Cd adsorption behavior as a function of both pH and metal loading. This approach is crucial in order to predict metal adsorption onto bacteria under environmentally relevant metal loading conditions where sulfhydryl binding sites can dominate the adsorption reaction.

Interaction with glycosaminoglycans is required for cyclophilin B to trigger integrin-mediated adhesion of peripheral blood T lymphocytes to extracellular matrix

PubMed Central

Allain, Fabrice; Vanpouille, Christophe; Carpentier, Mathieu; Slomianny, Marie-Christine; Durieux, Sandrine; Spik, Geneviève

2002-01-01

Cyclophilins A and B (CyPA and CyPB) are cyclosporin A-binding proteins that are involved in inflammatory events. We have reported that CyPB interacts with two types of cell-surface-binding sites. The first site corresponds to a functional receptor and requires interaction with the central core of CyPB. This region is highly conserved in cyclophilins, suggesting that CyPA and CyPB might share biological activities mediated by interaction with this receptor. The second site is identified with glycosaminoglycans (GAGs), the binding region located in the N terminus of CyPB. The difference in the N-terminal extensions of CyPA and CyPB suggests that a unique interaction with GAGs might account for selective activity of CyPB. To explore this hypothesis, we analyzed the lymphocyte responses triggered by CyPA, CyPB, and CyPBKKK−, a mutant unable to interact with GAGs. The three ligands seemed capable enough to elicit calcium signal and chemotaxis by binding to the same signaling receptor. In contrast, only CyPB enhanced firm adhesion of T cells to the extracellular matrix. This activity depended on the interactions with GAGs and signaling receptor. CyPB-mediated adhesion required CD147 presumably because it was a costimulatory molecule and was related to an activation of α4β1 and α4β7 integrins. Finally, we showed that CyPB was capable mainly to enhance T cell adhesion of the CD4+CD45RO+ subset. The present data indicate that CyPB rather than CyPA is a proinflammatory factor for T lymphocytes and highlight the crucial role of CyPB–GAG interaction in the chemokine-like activity of this protein. PMID:11867726

Interaction with glycosaminoglycans is required for cyclophilin B to trigger integrin-mediated adhesion of peripheral blood T lymphocytes to extracellular matrix.

PubMed

Allain, Fabrice; Vanpouille, Christophe; Carpentier, Mathieu; Slomianny, Marie-Christine; Durieux, Sandrine; Spik, Geneviève

2002-03-05

Cyclophilins A and B (CyPA and CyPB) are cyclosporin A-binding proteins that are involved in inflammatory events. We have reported that CyPB interacts with two types of cell-surface-binding sites. The first site corresponds to a functional receptor and requires interaction with the central core of CyPB. This region is highly conserved in cyclophilins, suggesting that CyPA and CyPB might share biological activities mediated by interaction with this receptor. The second site is identified with glycosaminoglycans (GAGs), the binding region located in the N terminus of CyPB. The difference in the N-terminal extensions of CyPA and CyPB suggests that a unique interaction with GAGs might account for selective activity of CyPB. To explore this hypothesis, we analyzed the lymphocyte responses triggered by CyPA, CyPB, and CyPB(KKK-), a mutant unable to interact with GAGs. The three ligands seemed capable enough to elicit calcium signal and chemotaxis by binding to the same signaling receptor. In contrast, only CyPB enhanced firm adhesion of T cells to the extracellular matrix. This activity depended on the interactions with GAGs and signaling receptor. CyPB-mediated adhesion required CD147 presumably because it was a costimulatory molecule and was related to an activation of alpha4beta1 and alpha4beta7 integrins. Finally, we showed that CyPB was capable mainly to enhance T cell adhesion of the CD4+CD45RO+ subset. The present data indicate that CyPB rather than CyPA is a proinflammatory factor for T lymphocytes and highlight the crucial role of CyPB-GAG interaction in the chemokine-like activity of this protein.

Generation and Characterization of a Bispecific Antibody Targeting Both PD-1 and c-MET.

PubMed

Wu, Yi; Yu, Min; Sun, Zujun; Hou, Weihua; Wang, Yuxiong; Yuan, Qingyun; Mo, Wei

2018-02-08

Bispecific antibodies, BsAbs, are molecules with the ability to bind to two different epitopes on the same or different antigens. c-MET, cellular-mesenchymal to epithelial transition factor, is deregulated in many types of human malignancies. Abnormal c-MET activation in cancer correlates with poor prognosis. PD-1, programmed death-1, is an additional inhibitory receptor expressed by T cells. Blocking the interactions between PD-1 and PD-L1 has emerged as a promising immunotherapy for treating cancer. The goal of this study was to identify a novel bispecific antibody targeting both c-MET and PD-1 as an anti-cancer therapeutic candidate. The BsAb was produced using 293E expression system and purified by Protein A affinity chromatography. Then the binding specificity and affinity of the BsAb was examined by FACS and biolayer light interferometry. The ability of the BsAb to inhibit the proliferation of tuman cells was measured using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit; the potential signaling pathway involved was identified by Western Blot. Cytokine secreted by PHA-L stimulated PBMC was measured by ELISA. Effects of BsAb on PBMC-mediated lysis of MKN45 cells was measured by LDH cytotoxicity assay. Based on the original sequences of PD-1 and c-MET mAb, a BsAb gene was designed, cloned into pCEP4 vector for expression in 293E cells. The BsAb was obtained after purification of the cell culture supernatant. It can bind to PD-1 and c-MET simultaneously, the calculated affinity was 11.5 nM for PD-1 and 9.09 nM for c-MET. The BsAb enhanced IFN-γ production over control IgG by 2-3 folds. It also inhibit the c-MET pathway activation and the proliferation of tumor cells significantly, comparable to JnJ-38877605. The BsAb showed dose-dependent cytotoxic activity against MKN45 cells. Our results indicated that a novel BsAb recognizing PD-1 and c-MET was successfully generated. It could redirect T cells to kill tumor cells, while retaining its inherent ability to restore T cells and inhibit tumor cells. With this potential, this BsAb could be developed as a therapeutic candidate for the treatment of various solid tumors. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Simultaneous adsorption of Cd²⁺ and BPA on amphoteric surfactant activated montmorillonite.

PubMed

Liu, Chongmin; Wu, Pingxiao; Zhu, Yajie; Tran, Lytuong

2016-02-01

The study mainly investigated the simultaneous adsorption of bisphenol A (BPA) and Cd(2+) from aqueous solution on octadecane-betaine modified montmorillonite (BS-Mt). The characteristics of the obtained materials were analyzed by X-ray diffraction (XRD), Fourier-transform infrared (FTIR), Specific surface area (BET) and Scanning electron microscopy/Energy disperse spectroscopy (SEM/EDS), confirming that BS-18 was successfully introduced into Mt. Also, factors including initial solution pH, initial Cd(2+)/BPA concentration, contact time and adsorbent dosage on the adsorption processes were shown to be crucial for Cd(2+) adsorption, whereas had negligible effects on BPA adsorption. In this study, we found that pseudo-second-order model fitted well with the adsorption kinetic studies for both Cd(2+) and BPA with an equilibrium time of 24 h. The Cd(2+) and BPA adsorption isotherm could be well described by Freundlich model and Langmuir model, respectively. On the basis of kinetic models, the maximum adsorption capacity of Cd(2+) in aqueous solution was slightly enhanced after modification, indicating that Cd(2+) adsorption on BS-Mt was mainly attributed to direct electrostatic attraction and the chelate reaction, while the dramatic enhancement of maximum adsorption capacity for BPA was due to the hydrophobic interaction. Copyright © 2015 Elsevier Ltd. All rights reserved.

Neutralization Properties of Simian Immunodeficiency Viruses Infecting Chimpanzees and Gorillas

PubMed Central

Barbian, Hannah J.; Decker, Julie M.; Bibollet-Ruche, Frederic; Galimidi, Rachel P.; West, Anthony P.; Learn, Gerald H.; Parrish, Nicholas F.; Iyer, Shilpa S.; Li, Yingying; Pace, Craig S.; Song, Ruijiang; Huang, Yaoxing; Denny, Thomas N.; Mouquet, Hugo; Martin, Loic; Acharya, Priyamvada; Zhang, Baoshan; Kwong, Peter D.; Mascola, John R.; Verrips, C. Theo; Strokappe, Nika M.; Rutten, Lucy; McCoy, Laura E.; Weiss, Robin A.; Brown, Corrine S.; Jackson, Raven; Silvestri, Guido; Connors, Mark; Burton, Dennis R.; Shaw, George M.; Nussenzweig, Michel C.; Bjorkman, Pamela J.; Ho, David D.; Farzan, Michael

2015-01-01

ABSTRACT Broadly cross-reactive neutralizing antibodies (bNabs) represent powerful tools to combat human immunodeficiency virus type 1 (HIV-1) infection. Here, we examined whether HIV-1-specific bNabs are capable of cross-neutralizing distantly related simian immunodeficiency viruses (SIVs) infecting central (Pan troglodytes troglodytes) (SIVcpzPtt) and eastern (Pan troglodytes schweinfurthii) (SIVcpzPts) chimpanzees (n = 11) as well as western gorillas (Gorilla gorilla gorilla) (SIVgor) (n = 1). We found that bNabs directed against the CD4 binding site (n = 10), peptidoglycans at the base of variable loop 3 (V3) (n = 5), and epitopes at the interface of surface (gp120) and membrane-bound (gp41) envelope glycoproteins (n = 5) failed to neutralize SIVcpz and SIVgor strains. In addition, apex V2-directed bNabs (n = 3) as well as llama-derived (heavy chain only) antibodies (n = 6) recognizing both the CD4 binding site and gp41 epitopes were either completely inactive or neutralized only a fraction of SIVcpzPtt strains. In contrast, one antibody targeting the membrane-proximal external region (MPER) of gp41 (10E8), functional CD4 and CCR5 receptor mimetics (eCD4-Ig, eCD4-Igmim2, CD4-218.3-E51, and CD4-218.3-E51-mim2), as well as mono- and bispecific anti-human CD4 (iMab and LM52) and CCR5 (PRO140, PRO140-10E8) receptor antibodies neutralized >90% of SIVcpz and SIVgor strains with low-nanomolar (0.13 to 8.4 nM) potency. Importantly, the latter antibodies blocked virus entry not only in TZM-bl cells but also in Cf2Th cells expressing chimpanzee CD4 and CCR5 and neutralized SIVcpz in chimpanzee CD4+ T cells, with 50% inhibitory concentrations (IC50s) ranging from 3.6 to 40.5 nM. These findings provide new insight into the protective capacity of anti-HIV-1 bNabs and identify candidates for further development to combat SIVcpz infection. PMID:25900654

XAFS studies of metal-ligand interactions at organic surfaces and in solution

NASA Astrophysics Data System (ADS)

Boyanov, Maxim I.

X-ray absorption fine structure spectroscopy (XAFS) was used as a structural probe to determine the mechanism of metal adsorption to organic surfaces. Two specific systems were investigated, Pb adsorption to heneicosanoic acid Langmuir monolayers (CH3(CH2)19COOH), and Cd adsorption to isolated cell walls of the Bacillus subtilis bacterium. Although the study of these systems is important for quite different reasons, the goal in both is metal binding site speciation and structural characterization of the surface complex. The adsorption of aqueous Cd to B. subtilis was studied as a function of pH by fluorescence mode bulk XAFS. Samples were prepared at six pH values in the range 3.4 to 7.8, and the bacterial functional groups responsible for the adsorption were identified under each condition. Under the experimental Cd and bacterial concentrations, the spectroscopy results indicate that Cd binds predominantly to protonated phosphoryl ligands below pH 4.4, while at higher pH adsorption to carboxyl groups becomes increasingly important. At pH 7.8 we observe the activation of an additional binding site, which we tentatively ascribe to deprotonated phosphoryl ligands. A quantitative Cd speciation diagram for the pH range is presented. Grazing-incidence Pb L3 edge XAFS was used in situ to determine the adsorption complex structure in the Pb-Langmuir monolayer study. The results indicate covalent binding of the Pb cations to the carboxyl headgroups, and the observed Pb-Pb coordination suggests that the metal is adsorbed as a hydrolysis polymer, rather than as individual Pb 2+ ions. The data suggest a bidentate binding mechanism and a one Pb atom to one carboxyl headgroup binding stoichiometry. We discuss how this adsorption model can explain the peculiarities observed with Pb in previous metal-Langmuir monolayer studies. A systematic study of the metal local environment in aqueous solutions was conducted and used in the above analyses. Perchlorate and acetate salt solutions of Cd, Pb, Mn, Cr, and Cu were characterized as standards of hydrated ions and metal-carboxyl complexes. The utility of XAFS in differentiating between the ionic, monodentate, bridging-bidentate, and bidentate metal-carboxyl complexes through C-C multiple scattering effects and XANES features is demonstrated.

Localizing Carbohydrate Binding Sites in Proteins Using Hydrogen/Deuterium Exchange Mass Spectrometry

NASA Astrophysics Data System (ADS)

Zhang, Jingjing; Kitova, Elena N.; Li, Jun; Eugenio, Luiz; Ng, Kenneth; Klassen, John S.

2016-01-01

The application of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to localize ligand binding sites in carbohydrate-binding proteins is described. Proteins from three bacterial toxins, the B subunit homopentamers of Cholera toxin and Shiga toxin type 1 and a fragment of Clostridium difficile toxin A, and their interactions with native carbohydrate receptors, GM1 pentasaccharides (β-Gal-(1→3)-β-GalNAc-(1→4)[α-Neu5Ac-(2→3)]-β-Gal-(1→4)-Glc), Pk trisaccharide (α-Gal-(1→4)-β-Gal-(1→4)-Glc) and CD-grease (α-Gal-(1→3)-β-Gal-(1→4)-β-GlcNAcO(CH2)8CO2CH3), respectively, served as model systems for this study. Comparison of the differences in deuterium uptake for peptic peptides produced in the absence and presence of ligand revealed regions of the proteins that are protected against deuterium exchange upon ligand binding. Notably, protected regions generally coincide with the carbohydrate binding sites identified by X-ray crystallography. However, ligand binding can also result in increased deuterium exchange in other parts of the protein, presumably through allosteric effects. Overall, the results of this study suggest that HDX-MS can serve as a useful tool for localizing the ligand binding sites in carbohydrate-binding proteins. However, a detailed interpretation of the changes in deuterium exchange upon ligand binding can be challenging because of the presence of ligand-induced changes in protein structure and dynamics.

Characterization of the covalent binding of N-phenyl-N'-(2-chloroethyl)ureas to {beta}-tubulin: importance of Glu198 in microtubule stability.

PubMed

Fortin, Sébastien; Bouchon, Bernadette; Chambon, Christophe; Lacroix, Jacques; Moreau, Emmanuel; Chezal, Jean-Michel; Degoul, Françoise; C-Gaudreault, René

2011-02-01

N-Phenyl-N'-(2-chloroethyl)ureas (CEUs) are antimicrotubule agents interacting covalently with β-tubulin near the colchicine-binding site (C-BS). Glutamyl 198 residue in β-tubulin (Glu198), which is adjacent to the C-BS behind the two potent nucleophilic residues, Cys239 and Cys354, has been shown to covalently react with 1-(2-chloroethyl)-3-(4-iodophenyl)urea (ICEU). By use of mass spectrometry, we have now identified residues in β-tubulin that have become modified irreversibly by 1-(2-chloroethyl)-3-[3-(5-hydroxypentyl)phenyl]urea (HPCEU), 1-[4-(3-hydroxy-4-methoxystyryl)phenyl]-3-(2-chloroethyl)urea (4ZCombCEU), and N,N'-ethylenebis(iodoacetamide) (EBI). The binding of HPCEU and 4ZCombCEU to β-tubulin resulted in the acylation of Glu198, a protein modification of uncommon occurrence in living cells. Prototypical CEUs then were used as molecular probes to assess, in mouse B16F0 and human MDA-MB-231 cells, the role of Glu198 in microtubule stability. For that purpose, we studied the effect of Glu198 modification by ICEU, HPCEU, and 4ZCombCEU on the acetylation of Lys40 on α-tubulin, a key indicator of microtubule stability. We show that modification of Glu198 by prototypical CEUs correlates with a decrease in Lys40 acetylation, as observed also with other microtubule depolymerizing agents. Therefore, CEU affects the stability and the dynamics of microtubule, likewise a E198G mutation, which is unusual for xenobiotics. We demonstrate for the first time that EBI forms an intramolecular cross-link between Cys239 and Cys354 of β-tubulin in living cells. This work establishes a novel basis for the development of future chemotherapeutic agents and provides a framework for the design of molecules useful for studying the role of Asp and Glu residues in the structure/function and the biological activity of several cellular proteins under physiological conditions.

Conservation of CD44 exon v3 functional elements in mammals

PubMed Central

Vela, Elena; Hilari, Josep M; Delclaux, María; Fernández-Bellon, Hugo; Isamat, Marcos

2008-01-01

Background The human CD44 gene contains 10 variable exons (v1 to v10) that can be alternatively spliced to generate hundreds of different CD44 protein isoforms. Human CD44 variable exon v3 inclusion in the final mRNA depends on a multisite bipartite splicing enhancer located within the exon itself, which we have recently described, and provides the protein domain responsible for growth factor binding to CD44. Findings We have analyzed the sequence of CD44v3 in 95 mammalian species to report high conservation levels for both its splicing regulatory elements (the 3' splice site and the exonic splicing enhancer), and the functional glycosaminglycan binding site coded by v3. We also report the functional expression of CD44v3 isoforms in peripheral blood cells of different mammalian taxa with both consensus and variant v3 sequences. Conclusion CD44v3 mammalian sequences maintain all functional splicing regulatory elements as well as the GAG binding site with the same relative positions and sequence identity previously described during alternative splicing of human CD44. The sequence within the GAG attachment site, which in turn contains the Y motif of the exonic splicing enhancer, is more conserved relative to the rest of exon. Amplification of CD44v3 sequence from mammalian species but not from birds, fish or reptiles, may lead to classify CD44v3 as an exclusive mammalian gene trait. PMID:18710510

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

Wurzburg, Beth A.; Tarchevskaya, Svetlana S.; Jardetzky, Theodore S.

CD23, the low-affinity receptor for IgE (Fc{var_epsilon}RII), regulates IgE synthesis and also mediates IgE-dependent antigen transport and processing. CD23 is a unique Fc receptor belonging to the C-type lectin-like domain superfamily and binds IgE in an unusual, non-lectin-like manner, requiring calcium but not carbohydrate. We have solved the high-resolution crystal structures of the human CD23 lectin domain in the presence and absence of Ca{sup 2+}. The crystal structures differ significantly from a previously determined NMR structure and show that calcium binding occurs at the principal binding site, but not at an auxiliary site that appears to be absent in humanmore » CD23. Conformational differences between the apo and Ca{sup 2+} bound structures suggest how IgE-Fc binding can be both calcium-dependent and carbohydrate-independent.« less

Formononetin, a phyto-oestrogen, and its metabolites up-regulate interleukin-4 production in activated T cells via increased AP-1 DNA binding activity

PubMed Central

Park, Jin; Kim, Seung H; Cho, Daeho; Kim, Tae S

2005-01-01

Phyto-oestrogens are polyphenolic non-steroidal plant compounds with oestrogen-like biological activity. Phyto-oestrogens have many biological effects including oestrogen agonist/antagonist properties. However, the effect of phyto-oestrogens on allergic responses remains unclear. In this study we investigated whether formononetin, a phyto-oestrogen, and its metabolites, daidzein and equol, affect production of interleukin-4 (IL-4), a pro-inflammatory cytokine closely associated with allergic immune response, in primary CD4+ T cells and EL4 T lymphoma cells. Formononetin, daidzein and equol significantly enhanced IL-4 production from both CD4+ T cells and EL4 cells in a dose-dependent manner. Formononetin, daidzein and equol also enhanced IL-4 gene promoter activity in EL4 cells transiently transfected with IL-4 gene promoter constructs, but this effect was impaired in EL4 cells transfected with an IL-4 promoter construct deleted of P4 site carrying nuclear factor of activated T cells (NF-AT) and activator protein-1 (AP-1) binding sites. In addition, formononetin, daidzein and equol increased AP-1 DNA binding activities while did not affect NF-AT DNA binding activities. The enhancing effects on IL-4 production and AP-1 DNA binding activities were abrogated by specific inhibitors for phosphatidylinositol-3-kinase (PI3K), protein kinase C (PKC) and p38 mitogen-activated protein kinase (MAPK), indicating that formononetin, daidzein and equol might enhance IL-4 production by increased activation of AP-1 through the PI3-K/PKC/p38 MAPK signalling pathway. These results suggest that phyto-oestrogens and some of their metabolites may increase allergic responses via the enhancement of IL-4 production in T cells. PMID:16108819

Overexpression of a SNARE protein AtBS14b alters BR response in Arabidopsis.

PubMed

Zhu, Zhong Xin; Ye, Hong Bo; Xuan, Yuan Hu; Yao, Da Nian

2014-12-01

N-ethyl-maleimide sensitive factor adaptor protein receptor (SNAREs) domain-containing proteins were known as key players in vesicle-associated membrane fusion. Genetic screening has revealed the function of SNAREs in different aspects of plant biology, but the role of many SNAREs are still unknown. In this study, we have characterized the role of Arabidopsis Qc-SNARE protein AtBS14b in brassinosteroids (BRs) signaling pathway. AtBS14b overexpression (AtBS14b ox) plants exhibited short hypocotyl and petioles lengths as well as insensitivity to exogenously supplied BR, while AtBS14b mutants did not show any visible BR-dependent morphological differences. BR biosynthesis enzyme BR6OX2 expression was slightly lower in AtBS14b ox than in wild type plants. Further BR-mediated repression of BR6OX2, CPD and DWF4 was inhibited in AtBS14b ox plants. AtBS14b-mCherry fusion protein localized in vesicular compartments surrounding plasma membrane in N. benthamiana leaves. In addition, isolation of AtBS14b-interacting BR signaling protein, which localized in plasma membrane, showed that AtBS14b directly interacted with membrane steroid binding protein 1 (MSBP1), but did not interact with BAK1 or BRI1. These data suggested that Qc-SNARE protein AtBS14b is the first SNARE protein identified that interacts with MSBP1, and the overexpression of AtBS14b modulates BR response in Arabidopsis.

Saccharomyces boulardii and Bacillus subtilis B10 modulate TLRs mediated signaling to induce immunity by chicken BMDCs.

PubMed

Rajput, Imran Rashid; Hussain, Altaf; Li, Ya Li; Zhang, Xiaoping; Xu, Xin; Long, Mao Yu; You, Dong Yu; Li, Wei Fen

2014-01-01

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that play a critical role to activate immune response. They may be targeted for immunomodulation by microbes, including probiotics. In this study, chicken bone marrow dendrite cells (chi-BMDCs) were stimulated with lipopolysachride (LPS), Saccharomyces boulardii (Sb), Bacillus subtilis B10 (Bs), co-culture of Sb + Bs and phosphate buffer saline (PBS) as a control group (Ctr) at 3, 6, and 12 h intervals. Results revealed that treatment groups modulated the phenotype and biological functions of chi-BMDCs. Scan electron microscopy showed attachment of probiotics on the surface of chi-BMDCs. Additionally transmission electron microscopy (TEM) revealed efficiently engulfing and degradation of probiotics. Gene expression levels of MHC-II, CD40, CD80 and CD86 up-regulated in stimulated groups. Furthermore, toll-like receptors TLR1, TLR2, TLR4, and chicken specific TLR15 expressions were improved and downstream associated factors MyD88, TRAF6, TAB1, and NFκ-B mRNA levels increased in all treatment groups as compared to control. Surprisingly, NFκ-B response was noted significant higher in LPS treatment among all groups. Moreover, IL-1β, IL-17, IL-4, TGF-β, and IL-10 production levels were found higher, and lower concentration of INF-γ and IL-8 were observed in Sb, Bs, and Sb + Bs treatment groups. In contrast, LPS groups showed prominent increase in IL-12, INF-γ, and IL-8 concentration levels as compared to control group. Altogether, these results emphasize a potentially important role of Saccharomyces boulardii and Bacillus subtilis B10 in modulating immunological functions of chi-BMDCs by targeting specific toll like receptors (TLRs) and associated factors. The role of probiotics on chi-BMDCs functionality in a non-mammalian species have been presented for the first time. © 2013 Wiley Periodicals, Inc.

Emergence and Kinetics of Simian Immunodeficiency Virus-Specific CD8+ T Cells in the Intestines of Macaques during Primary Infection

PubMed Central

Veazey, Ronald S.; Gauduin, Marie-Claire; Mansfield, Keith G.; Tham, Irene C.; Altman, John D.; Lifson, Jeffrey D.; Lackner, Andrew A.; Johnson, R. Paul

2001-01-01

In this report, three Mamu-A*01+ rhesus macaques were examined to compare the emergence of simian immunodeficiency virus (SIV)-specific CD8+ T cells in the intestines and blood in early SIV infection using a major histocompatibility complex class I tetramer complexed with the Gag181–189 peptide. Fourteen days after intravenous inoculation with SIVmac251, large numbers of SIV Gag181–189-specific CD8+ T cells were detected in the intestinal mucosa (3.1 to 11.5% of CD3+ CD8+ lymphocytes) as well as in the blood (3.1 to 13.4%) of all three macaques. By 21 days postinoculation, levels of tetramer-binding cells had dropped in both the intestines and blood. At day 63, however, levels of SIV Gag181–189-specific CD8+ T cells in the intestines had rebounded in all three macaques to levels that were higher (8.6 to 18.7%) than those at day 21. In contrast, percentages of tetramer-binding cells in the peripheral blood remained comparatively stable (2.5 to 4.5%) at this time point. In summary, SIV Gag181–189-specific CD8+ T cells appeared in both the intestinal mucosa and peripheral blood at a comparable rate and magnitude in primary SIV infection. Given that the intestine is a major site of early viral replication as well as the site where most of the total body lymphocyte pool resides, these data indicate that it is also an early and important site of development of antiviral immune responses. PMID:11581423

Identification of contact sites between ankyrin and band 3 in the human erythrocyte membrane.

PubMed

Grey, Jesse L; Kodippili, Gayani C; Simon, Katya; Low, Philip S

2012-08-28

The red cell membrane is stabilized by a spectrin/actin-based cortical cytoskeleton connected to the phospholipid bilayer via multiple protein bridges. By virtue of its interaction with ankyrin and adducin, the anion transporter, band 3 (AE1), contributes prominently to these bridges. In a previous study, we demonstrated that an exposed loop comprising residues 175-185 of the cytoplasmic domain of band 3 (cdB3) constitutes a critical docking site for ankyrin on band 3. In this paper, we demonstrate that an adjacent loop, comprising residues 63-73 of cdB3, is also essential for ankyrin binding. Data that support this hypothesis include the following. (1) Deletion or mutation of residues within the latter loop abrogates ankyrin binding without affecting cdB3 structure or its other functions. (2) Association of cdB3 with ankyrin is inhibited by competition with the loop peptide. (3) Resealing of the loop peptide into erythrocyte ghosts alters membrane morphology and stability. To characterize cdB3-ankyrin interaction further, we identified their interfacial contact sites using molecular docking software and the crystal structures of D(3)D(4)-ankyrin and cdB3. The best fit for the interaction reveals multiple salt bridges and hydrophobic contacts between the two proteins. The most important ion pair interactions are (i) cdB3 K69-ankyrin E645, (ii) cdB3 E72-ankyrin K611, and (iii) cdB3 D183-ankyrin N601 and Q634. Mutation of these four residues on ankyrin yielded an ankyrin with a native CD spectrum but little or no affinity for cdB3. These data define the docking interface between cdB3 and ankyrin in greater detail.

Identification of contact sites between ankyrin and band 3 in the human erythrocyte membrane

PubMed Central

Grey, Jesse L.; Kodippili, Gayani C.; Simon, Katya; Low, Philip S.

2012-01-01

The red cell membrane is stabilized by a spectrin/actin-based cortical cytoskeleton connected to the phospholipid-bilayer via multiple protein bridges. By virtue of its interaction with ankyrin and adducin, the anion transporter, band 3 (AE1), contributes prominently to these bridges. In a previous study, we demonstrated that an exposed loop comprising residues 175–185 of the cytoplasmic domain of band 3 (cdB3) constitutes a critical docking site for ankyrin on band 3. In this paper, we demonstrate that an adjacent loop, comprising residues 63–73 of cdB3, is also essential for ankyrin binding. Data in support of this hypothesis include: 1) deletion or mutation of residues within the latter loop abrogates ankyrin binding without affecting cdB3 structure or its other functions, 2) association of cdB3 with ankyrin is inhibited by competition with the loop peptide, and 3) resealing of the loop peptide into erythrocyte ghosts alters membrane morphology and stability. To characterize cdB3-ankyrin interaction further, we identified their interfacial contact sites using molecular docking software and the crystal structures of D3D4-ankyrin and cdB3. The best fit for the interaction reveals multiple salt bridges and hydrophobic contacts between the two proteins. The most important ion pair interactions are: i) cdB3 K69 to ankyrin E645, ii) cdB3 E72 to ankyrin K611, and iii) cdB3 D183 to ankyrin N601 and Q634. Mutation of the above four residues on ankyrin yielded an ankyrin with native CD spectrum, but little or no affinity for cdB3. These data define the docking interface between cdB3 and ankyrin in greater detail. PMID:22861190

Differential cellular internalization of anti-CD19 and -CD22 immunotoxins results in different cytotoxic activity.

PubMed

Du, Xing; Beers, Richard; Fitzgerald, David J; Pastan, Ira

2008-08-01

B-cell malignancies routinely express surface antigens CD19 and CD22. Immunotoxins against both antigens have been evaluated, and the immunotoxins targeting CD22 are more active. To understand this disparity in cytotoxicity and guide the screening of therapeutic targets, we compared two immunotoxins, FMC63(Fv)-PE38-targeting CD19 and RFB4(Fv)-PE38 (BL22)-targeting CD22. Six lymphoma cell lines have 4- to 9-fold more binding sites per cell for CD19 than for CD22, but BL22 is 4- to 140-fold more active than FMC63(Fv)-PE38, although they have a similar cell binding affinity (Kd, approximately 7 nmol/L). In 1 hour, large amounts of BL22 are internalized (2- to 3-fold more than the number of CD22 molecules on the cell surface), whereas only 5.2% to 16.6% of surface-bound FMC63(Fv)-PE38 is internalized. The intracellular reservoir of CD22 decreases greatly after immunotoxin internalization, indicating that it contributes to the uptake of BL22. Treatment of cells with cycloheximide does not reduce the internalization of BL22. Both internalized immunotoxins are located in the same vesicles. Our results show that the rapid internalization of large amounts of BL22 bound to CD22 makes CD22 a better therapeutic target than CD19 for immunotoxins and probably for other immunoconjugates that act inside cells.

Kinetics of heavy metal adsorption and desorption in soil: Developing a unified model based on chemical speciation

NASA Astrophysics Data System (ADS)

Peng, Lanfang; Liu, Paiyu; Feng, Xionghan; Wang, Zimeng; Cheng, Tao; Liang, Yuzhen; Lin, Zhang; Shi, Zhenqing

2018-03-01

Predicting the kinetics of heavy metal adsorption and desorption in soil requires consideration of multiple heterogeneous soil binding sites and variations of reaction chemistry conditions. Although chemical speciation models have been developed for predicting the equilibrium of metal adsorption on soil organic matter (SOM) and important mineral phases (e.g. Fe and Al (hydr)oxides), there is still a lack of modeling tools for predicting the kinetics of metal adsorption and desorption reactions in soil. In this study, we developed a unified model for the kinetics of heavy metal adsorption and desorption in soil based on the equilibrium models WHAM 7 and CD-MUSIC, which specifically consider metal kinetic reactions with multiple binding sites of SOM and soil minerals simultaneously. For each specific binding site, metal adsorption and desorption rate coefficients were constrained by the local equilibrium partition coefficients predicted by WHAM 7 or CD-MUSIC, and, for each metal, the desorption rate coefficients of various binding sites were constrained by their metal binding constants with those sites. The model had only one fitting parameter for each soil binding phase, and all other parameters were derived from WHAM 7 and CD-MUSIC. A stirred-flow method was used to study the kinetics of Cd, Cu, Ni, Pb, and Zn adsorption and desorption in multiple soils under various pH and metal concentrations, and the model successfully reproduced most of the kinetic data. We quantitatively elucidated the significance of different soil components and important soil binding sites during the adsorption and desorption kinetic processes. Our model has provided a theoretical framework to predict metal adsorption and desorption kinetics, which can be further used to predict the dynamic behavior of heavy metals in soil under various natural conditions by coupling other important soil processes.

Differential affinity of BsSCO for Cu(II) and Cu(I) suggests a redox role in copper transfer to the Cu(A) center of cytochrome c oxidase.

PubMed

Hill, Bruce C; Andrews, Diann

2012-06-01

SCO (synthesis of cytochrome c oxidase) proteins are involved in the assembly of the respiratory chain enzyme cytochrome c oxidase acting to assist in the assembly of the Cu(A) center contained within subunit II of the oxidase complex. The Cu(A) center receives electrons from the reductive substrate ferrocytochrome c, and passes them on to the cytochrome a center. Cytochrome a feeds electrons to the oxygen reaction site composed of cytochrome a(3) and Cu(B). Cu(A) consists of two copper ions positioned within bonding distance and ligated by two histidine side chains, one methionine, a backbone carbonyl and two bridging cysteine residues. The complex structure and redox capacity of Cu(A) present a potential assembly challenge. SCO proteins are members of the thioredoxin family which led to the early suggestion of a disulfide exchange function for SCO in Cu(A) assembly, whereas the copper binding capacity of the Bacillus subtilis version of SCO (i.e., BsSCO) suggests a direct role for SCO proteins in copper transfer. We have characterized redox and copper exchange properties of apo- and metalated-BsSCO. The release of copper (II) from its complex with BsSCO is best achieved by reducing it to Cu(I). We propose a mechanism involving both disulfide and copper exchange between BsSCO and the apo-Cu(A) site. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes. Copyright © 2011 Elsevier B.V. All rights reserved.

Constants for mercury binding by organic matter isolates from the Florida Everglades

USGS Publications Warehouse

Benoit, J.M.; Mason, R.P.; Gilmour, C.C.; Aiken, G.R.

2001-01-01

Dissolved organic matter (DOM) has been implicated as an important complexing agent for Hg that can affect its mobility and bioavailability in aquatic ecosystems. However, binding constants for natural Hg-DOM complexes are not well known. We employed a competitive ligand approach to estimate conditional stability constants for Hg complexes with DOM isolates collected from Florida Everglades surface waters. The isolates examined were the hydrophobic fraction of DOM from a eutrophic, sulfidic site (F1-HPoA) and the hydrophilic fraction from an oligotrophic, low-sulfide site (2BS-HPiA). Our experimental determinations utilized overall octanol-water partitioning coefficients (Dow) for 203Hg at 0.01 M chloride and across pH and DOM concentration gradients. Use of this radioisotope allowed rapid determinations of Hg concentrations in both water and octanol phases without problems of matrix interference. Conditional stability constants (1 = 0.06, 23??C) were log K??? = 11.8 for F1-HPoA and log K' = 10.6 for 2BS-HPiA. These are similar to previously published stability constants for Hg binding to low-molecular-weight thiols. Further, F1-HPoA showed a pH-dependent decline in Dow that was consistent with models of Hg complexation with thiol groups as the dominant Hg binding sites in DOM. These experiments demonstrate that the DOM isolates are stronger ligands for Hg than chloride ion or ethylenediamine-tetraacetic acid. Speciation calculations indicate that at the DOM concentrations frequently measured in Everglades, 20 to 40 ??M, significant complexation of Hg by DOM would be expected in aerobic (sulfide-free) surface waters. Copyright ?? 2001 Elsevier Science Ltd.

Prostaglandin E2 induces expression of P-selectin (CD62P) on cultured human umbilical vein endothelial cells and enhances endothelial binding of CD4-T-cells.

PubMed

Hailer, N P; Oppermann, E; Leckel, K; Cinatl, J; Markus, B H; Blaheta, R A

2000-07-15

Interaction of endothelial P-selectin with sialyl Lewis(x)-glycoprotein or P-selectin glycoprotein ligand (PSGL)-1 on leukocytes represents an early step in leukocyte recruitment. Redistribution of P-selectin to the endothelial cell surface occurs rapidly after challenge with several proinflammatory agents, for example, histamine, leucopterins, or lipopolysaccharide. We present evidence that prostaglandin E2 (PGE2) is an efficient inductor of surface P-selectin on cultured human umbilical vein endothelial cells (HUVEC). The increase in P-selectin-immunoreactivity coincided with redistribution of cytoplasmic P-selectin-reactive granulae to the endothelial cell surface, as visualized by confocal laser microscopic examination. CD4-T-cell adhesion to PGE2-stimulated HUVEC was also enhanced by a factor of 4, and blocking mAb directed against the binding site of P-selectin almost completely abrogated this increase in CD4-T-cell adhesion. In summary, our findings show that liberation of PGE2 is an important inductor of P-selectin surface expression on endothelial cells, resulting in enhanced recruitment of inflammatory cells.

Sieve analysis of breakthrough HIV-1 sequences in HVTN 505 identifies vaccine pressure targeting the CD4 binding site of Env-gp120.

PubMed

deCamp, Allan C; Rolland, Morgane; Edlefsen, Paul T; Sanders-Buell, Eric; Hall, Breana; Magaret, Craig A; Fiore-Gartland, Andrew J; Juraska, Michal; Carpp, Lindsay N; Karuna, Shelly T; Bose, Meera; LePore, Steven; Miller, Shana; O'Sullivan, Annemarie; Poltavee, Kultida; Bai, Hongjun; Dommaraju, Kalpana; Zhao, Hong; Wong, Kim; Chen, Lennie; Ahmed, Hasan; Goodman, Derrick; Tay, Matthew Z; Gottardo, Raphael; Koup, Richard A; Bailer, Robert; Mascola, John R; Graham, Barney S; Roederer, Mario; O'Connell, Robert J; Michael, Nelson L; Robb, Merlin L; Adams, Elizabeth; D'Souza, Patricia; Kublin, James; Corey, Lawrence; Geraghty, Daniel E; Frahm, Nicole; Tomaras, Georgia D; McElrath, M Juliana; Frenkel, Lisa; Styrchak, Sheila; Tovanabutra, Sodsai; Sobieszczyk, Magdalena E; Hammer, Scott M; Kim, Jerome H; Mullins, James I; Gilbert, Peter B

2017-01-01

Although the HVTN 505 DNA/recombinant adenovirus type 5 vector HIV-1 vaccine trial showed no overall efficacy, analysis of breakthrough HIV-1 sequences in participants can help determine whether vaccine-induced immune responses impacted viruses that caused infection. We analyzed 480 HIV-1 genomes sampled from 27 vaccine and 20 placebo recipients and found that intra-host HIV-1 diversity was significantly lower in vaccine recipients (P ≤ 0.04, Q-values ≤ 0.09) in Gag, Pol, Vif and envelope glycoprotein gp120 (Env-gp120). Furthermore, Env-gp120 sequences from vaccine recipients were significantly more distant from the subtype B vaccine insert than sequences from placebo recipients (P = 0.01, Q-value = 0.12). These vaccine effects were associated with signatures mapping to CD4 binding site and CD4-induced monoclonal antibody footprints. These results suggest either (i) no vaccine efficacy to block acquisition of any viral genotype but vaccine-accelerated Env evolution post-acquisition; or (ii) vaccine efficacy against HIV-1s with Env sequences closest to the vaccine insert combined with increased acquisition due to other factors, potentially including the vaccine vector.

Sieve analysis of breakthrough HIV-1 sequences in HVTN 505 identifies vaccine pressure targeting the CD4 binding site of Env-gp120

PubMed Central

Edlefsen, Paul T.; Sanders-Buell, Eric; Hall, Breana; Magaret, Craig A.; Fiore-Gartland, Andrew J.; Juraska, Michal; Carpp, Lindsay N.; Karuna, Shelly T.; Bose, Meera; LePore, Steven; Miller, Shana; O'Sullivan, Annemarie; Poltavee, Kultida; Bai, Hongjun; Dommaraju, Kalpana; Zhao, Hong; Wong, Kim; Chen, Lennie; Ahmed, Hasan; Goodman, Derrick; Tay, Matthew Z.; Gottardo, Raphael; Koup, Richard A.; Bailer, Robert; Mascola, John R.; Graham, Barney S.; Roederer, Mario; O’Connell, Robert J.; Michael, Nelson L.; Robb, Merlin L.; Adams, Elizabeth; D’Souza, Patricia; Kublin, James; Corey, Lawrence; Geraghty, Daniel E.; Frahm, Nicole; Tomaras, Georgia D.; McElrath, M. Juliana; Frenkel, Lisa; Styrchak, Sheila; Tovanabutra, Sodsai; Sobieszczyk, Magdalena E.; Hammer, Scott M.; Kim, Jerome H.; Mullins, James I.; Gilbert, Peter B.

2017-01-01

Although the HVTN 505 DNA/recombinant adenovirus type 5 vector HIV-1 vaccine trial showed no overall efficacy, analysis of breakthrough HIV-1 sequences in participants can help determine whether vaccine-induced immune responses impacted viruses that caused infection. We analyzed 480 HIV-1 genomes sampled from 27 vaccine and 20 placebo recipients and found that intra-host HIV-1 diversity was significantly lower in vaccine recipients (P ≤ 0.04, Q-values ≤ 0.09) in Gag, Pol, Vif and envelope glycoprotein gp120 (Env-gp120). Furthermore, Env-gp120 sequences from vaccine recipients were significantly more distant from the subtype B vaccine insert than sequences from placebo recipients (P = 0.01, Q-value = 0.12). These vaccine effects were associated with signatures mapping to CD4 binding site and CD4-induced monoclonal antibody footprints. These results suggest either (i) no vaccine efficacy to block acquisition of any viral genotype but vaccine-accelerated Env evolution post-acquisition; or (ii) vaccine efficacy against HIV-1s with Env sequences closest to the vaccine insert combined with increased acquisition due to other factors, potentially including the vaccine vector. PMID:29149197

Interaction of flavonols with human serum albumin: a biophysical study showing structure-activity relationship and enhancement when coated on silver nanoparticles.

PubMed

Das, Pratyusa; Chaudhari, Sunil Kumar; Das, Asmita; Kundu, Somashree; Saha, Chabita

2018-04-24

Binding affinities of flavonols namely quercetin, myricetin, and kaempferol to human serum albumin (HSA) were determined fluorimetrically and the order was observed to be myricetin > quercetin > kaempferol demonstrating structure-activity relationship. Quercetin-coated silver nanoparticles (AgNPs) show higher binding affinity to HSA compared to free quercetin with binding constants 6.04 × 10 7  M -1 and 4.2 × 10 6  M -1 , respectively. Using site-specific markers it is concluded that free quercetin and that coated on AgNPs bind at different sites. Significant structural changes in circular dichroism (CD) spectra of HSA were recorded with quercetin-coated AgNPs compared to free quercetin. These results were further substantiated by time-resolved fluorescence spectroscopy where fluorescence life time of the tryptophan residue in HSA-quercetin-coated AgNPs complex decreased to 3.63 ns from 4.22 ns in HSA-quercetin complex. Isothermal calorimetric studies reveal two binding modes for quercetin-coated AgNPs and also higher binding constants compared to free quercetin. These higher binding affinities are attributed to altered properties of quercetin when coated on AgNPs enabling it to reach the binding sites other than site II where free quercetin mainly binds.

Syndecan-1/CD147 association is essential for cyclophilin B-induced activation of p44/42 mitogen-activated protein kinases and promotion of cell adhesion and chemotaxis.

PubMed

Pakula, Rachel; Melchior, Aurélie; Denys, Agnès; Vanpouille, Christophe; Mazurier, Joël; Allain, Fabrice

2007-05-01

Many of the biological functions attributed to cell surface proteoglycans are dependent on the interaction with extracellular mediators through their heparan sulphate (HS) moieties and the participation of their core proteins in signaling events. A class of recently identified inflammatory mediators is secreted cyclophilins, which are mostly known as cyclosporin A-binding proteins. We previously demonstrated that cyclophilin B (CyPB) triggers chemotaxis and integrin-mediated adhesion of T lymphocytes mainly of the CD4+/CD45RO+ phenotype. These activities are related to interactions with two types of binding sites, CD147 and cell surface HS. Here, we demonstrate that CyPB-mediated adhesion of CD4+/CD45RO+ T cells is related to p44/42 mitogen-activated protein kinase (MAPK) activation by a mechanism involving CD147 and HS proteoglycans (HSPG). Although HSPG core proteins are represented by syndecan-1, -2, -4, CD44v3 and betaglycan in CD4+/CD45RO+ T cells, we found that only syndecan-1 is physically associated with CD147. The intensity of the heterocomplex increased in response to CyPB, suggesting a transient enhancement and/or stabilization in the association of CD147 to syndecan-1. Pretreatment with anti-syndecan-1 antibodies or knockdown of syndecan-1 expression by RNA interference dramatically reduced CyPB-induced p44/p42 MAPK activation and consequent migration and adhesion, supporting the model in which syndecan-1 serves as a binding subunit to form the fully active receptor of CyPB. Altogether, our findings provide a novel example of a soluble mediator in which a member of the syndecan family plays a critical role in efficient interaction with signaling receptors and initiation of cellular responses.

A novel CD44-binding peptide from the pro-matrix metalloproteinase-9 hemopexin domain impairs adhesion and migration of chronic lymphocytic leukemia (CLL) cells.

PubMed

Ugarte-Berzal, Estefanía; Bailón, Elvira; Amigo-Jiménez, Irene; Albar, Juan Pablo; García-Marco, José A; García-Pardo, Angeles

2014-05-30

(pro)MMP-9 binds to CLL cells through the PEX9 domain and contributes to CLL progression. To biochemically characterize this interaction and identify potential therapeutic targets, we prepared GST-PEX9 forms containing structural blades B1B2 or B3B4. We recently described a sequence in blade B4 (P3 sequence) that bound α4β1 integrin and partially impaired cell adhesion and migration. We have now studied the possible contribution of the B1B2 region to cell interaction with PEX9. CLL cells bound to GST-B1B2 and CD44 was the primary receptor. GST-B1B2 inhibited CLL cell migration as effectively as GST-B3B4. Overlapping synthetic peptides spanning the B1B2 region identified the sequence FDAIAEIGNQLYLFKDGKYW, present in B1 and contained in peptide P6, as the most effective site. P6 inhibited cell adhesion to PEX9 in a dose-dependent manner and with an IC50 value of 90 μM. P6 also inhibited cell adhesion to hyaluronan but had no effect on adhesion to VCAM-1 (α4β1 integrin ligand), confirming its specific interaction with CD44. Spatial localization analyses mapped P6 to the central cavity of PEX9, in close proximity to the previously identified P3 sequence. Both P6 and P3 equally impaired cell adhesion to (pro)MMP-9. Moreover, P6 synergistically cooperated with P3, resulting in complete inhibition of CLL cell binding to PEX9, chemotaxis, and transendothelial migration. Thus, P6 is a novel sequence in PEX9 involved in cell-PEX9/(pro)MMP-9 binding by interacting with CD44. Targeting both sites, P6 and P3, should efficiently prevent (pro)MMP-9 binding to CLL cells and its pathological consequences. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Multiple oxygen entry pathways in globin proteins revealed by intrinsic pathway identification method

NASA Astrophysics Data System (ADS)

Takayanagi, Masayoshi; Kurisaki, Ikuo; Nagaoka, Masataka

2015-12-01

Each subunit of human hemoglobin (HbA) stores an oxygen molecule (O2) in the binding site (BS) cavity near the heme group. The BS is buried in the interior of the subunit so that there is a debate over the O2 entry pathways from solvent to the BS; histidine gate or multiple pathways. To elucidate the O2 entry pathways, we executed ensemble molecular dynamics (MD) simulations of T-state tetramer HbA in high concentration O2 solvent to simulate spontaneous O2 entry from solvent into the BS. By analyzing 128 independent 8 ns MD trajectories by intrinsic pathway identification by clustering (IPIC) method, we found 141 and 425 O2 entry events into the BS of the α and β subunits, respectively. In both subunits, we found that multiple O2 entry pathways through inside cavities play a significant role for O2 entry process of HbA. The rate constants of O2 entry estimated from the MD trajectories correspond to the experimentally observed values. In addition, by analyzing monomer myoglobin, we verified that the high O2 concentration condition can reproduce the ratios of each multiple pathway in the one-tenth lower O2 concentration condition. These indicate the validity of the multiple pathways obtained in our MD simulations.

Cd and proton adsorption onto bacterial consortia grown from industrial wastes and contaminated geologic settings.

PubMed

Borrok, David M; Fein, Jeremy B; Kulpa, Charles F

2004-11-01

To model the effects of bacterial metal adsorption in contaminated environments, results from metal adsorption experiments involving individual pure stains of bacteria must be extrapolated to systems in which potentially dozens of bacterial species are present. This extrapolation may be made easier because bacterial consortia from natural environments appear to exhibit similar metal binding properties. However, bacteria that thrive in highly perturbed contaminated environments may exhibit significantly different adsorptive behavior. Here we measure proton and Cd adsorption onto a range of bacterial consortia grown from heavily contaminated industrial wastes, groundwater, and soils. We model the results using a discrete site surface complexation approach to determine binding constants and site densities for each consortium. The results demonstrate that bacterial consortia from different contaminated environments exhibit a range of total site densities (approximately a 3-fold difference) and Cd-binding constants (approximately a 10-fold difference). These ranges for Cd binding constants may be small enough to suggest that bacteria-metal adsorption in contaminated environments can be described using relatively few "averaged" bacteria-metal binding constants (in conjunction with the necessary binding constants for competing surfaces and ligands). However, if additional precision is necessary, modeling parameters must be developed separately for each contaminated environment of interest.

Checkpoint Antibodies but not T Cell-Recruiting Diabodies Effectively Synergize with TIL-Inducing γ-Irradiation.

PubMed

Hettich, Michael; Lahoti, Jayashree; Prasad, Shruthi; Niedermann, Gabriele

2016-08-15

T cell-recruiting bispecific antibodies (bsAb) show promise in hematologic malignancies and are also being evaluated in solid tumors. In this study, we investigated whether T cell-recruiting bsAbs synergize with hypofractionated tumor radiotherapy (hRT) and/or blockade of the programmed death-1 (PD-1) immune checkpoint, both of which can increase tumor-infiltrating lymphocyte (TIL) numbers. Unexpectedly, large melanomas treated with hRT plus bsAb (AC133×CD3) relapsed faster than those treated with hRT alone, accompanied by massive TIL apoptosis. This fast relapse was delayed by the further addition of anti-PD-1. Mechanistic investigations revealed restimulation-induced cell death mediated by BIM and FAS as an additional cause of bsAb-mediated TIL depletion. In contrast, the double combination of hRT and anti-PD-1 strongly increased TIL numbers, and even very large tumors were completely eradicated. Our study reveals the risk that CD3-engaging bsAbs can induce apoptotic TIL depletion followed by rapid tumor regrowth, reminiscent of tolerance induction by CD3 mAb-mediated T-cell depletion, warranting caution in their use for the treatment of solid tumors. Our findings also argue that combining radiotherapy and anti-PD-1 can be quite potent, including against very large tumors. Cancer Res; 76(16); 4673-83. ©2016 AACR. ©2016 American Association for Cancer Research.

Restoration of high zinc and lead tailings with municipal biosolids and lime: a field study.

PubMed

Brown, Sally; Svendsen, Alex; Henry, Chuck

2009-01-01

A field study was conducted to test the ability of biosolids (BS) and different types of lime to increase soil pH, neutralize subsoil acidity, and restore a vegetative cover to alluvial mine tailings in Leadville, CO. The tailings had soil pH of 5.2 and total Cd, Pb, and Zn of 75+/-20, 2600+/-1100, and 6700+/-1900 mg kg(-1). Types of lime included agricultural lime (AL), sugar beet lime (SBL), and lime kiln dust (LKD) applied at 224 Mg ha(-1) calcium carbonate equivalent. Plots were established in 2000 and monitored intermittently through 2007. All amendments increased pH in surface and subsurface depths, with LKD, LKD+BS, and SBL+BS being the most effective. Amendments also reduced 0.01 mol L(-1) Ca(NO3)2 extractable Zn and Cd compared to the control. Plant growth was sparse on all treatments with limited yield for three of four harvests. Poor growth may have been related to elevated electrical conductivity (EC). All amendments except LKD alone (5.79 dS m(-1)) increased EC compared to the control treatment (5.28 dS m(-1)). Electrical conductivity was highest in 2002 which had the lowest summer rainfall. In 2005 EC in all treatments except the SBL+BS was similar in the surface soil. Aboveground plant tissue concentrations of Zn and Cd were also elevated. Limited precipitation and high electrical conductivity may be responsible for poor plant growth. Higher rainfall for the last sampling period resulted in significant growth in the LKD+BS, SBL+BS, and LKD alone treatments.

Characterization studies on cadmium-mycophosphatin from the mushroom Agaricus macrosporus.

PubMed Central

Meisch, H U; Schmitt, J A

1986-01-01

A low molecular weight Cd-binding phosphoglycoprotein, cadmium-mycophosphatin, has been isolated from the mushroom Agaricus macrosporus. This protein has a molecular weight of 12,000 dalton and contains no sulfur but a high amount of acid amino acids (Glu, Asp), and carbohydrates (glucose, galactose). Cadmium-mycophosphatin has an isoelectric point less than pH 2, binds cadmium with a dissociation constant of KD = 1.59 X 10 M (pKD = 6.8) and is saturated with 13.5 mole Cd/mole, all Cd-binding sites being equivalent. It is suggested that Cd is bound by phosphoserine groups, similar relations being known from calcium-binding proteins in animals. From A. macrosporus four other low-molecular weight glycoproteins have been isolated which contain sulfur and bind cadmium and copper. The biological significance of these Cd-binding proteins is discussed. PMID:3709455

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

Ngu, Thanh T.; Sturzenbaum, Stephen R.; Stillman, Martin J.

The earthworm Lumbricus rubellus has been found to inhabit cadmium-rich soils and accumulate cadmium within its tissues. Two metallothionein (MT) isoforms (1 and 2) have been identified and cloned from L. rubellus. In this study, we address the metalation status, metal coordination, and structure of recombinant MT-2 from L. rubellus using electrospray ionization mass spectrometry (ESI-MS), UV absorption, and circular dichroism (CD) spectroscopy. This is the first study to show the detailed mass and CD spectral properties for the important cadmium-containing earthworm MT. We report that the 20-cysteine L. rubellus MT-2 binds seven Cd{sup 2+} ions. UV absorption and CDmore » spectroscopy and ESI-MS pH titrations show a distinct biphasic demetalation reaction, which we propose results from the presence of two metal-thiolate binding domains. We propose stoichiometries of Cd{sub 3}Cys{sub 9} and Cd{sub 4}Cys{sub 11} based on the presence of 20 cysteines split into two isolated regions of the sequence with 11 cysteines in the N-terminal and 9 cysteines in the C-terminal. The CD spectrum reported is distinctly different from any other metallothionein known suggesting quite different binding site structure for the peptide.« less

Isolated receptor binding domains of HTLV-1 and HTLV-2 envelopes bind Glut-1 on activated CD4+ and CD8+ T cells

PubMed Central

Kinet, Sandrina; Swainson, Louise; Lavanya, Madakasira; Mongellaz, Cedric; Montel-Hagen, Amélie; Craveiro, Marco; Manel, Nicolas; Battini, Jean-Luc; Sitbon, Marc; Taylor, Naomi

2007-01-01

Background We previously identified the glucose transporter Glut-1, a member of the multimembrane-spanning facilitative nutrient transporter family, as a receptor for both HTLV-1 and HTLV-2. However, a recent report concluded that Glut-1 cannot serve as a receptor for HTLV-1 on CD4 T cells: This was based mainly on their inability to detect Glut-1 on this lymphocyte subset using the commercial antibody mAb1418. It was therefore of significant interest to thoroughly assess Glut-1 expression on CD4 and CD8 T cells, and its association with HTLV-1 and -2 envelope binding. Results As previously reported, ectopic expression of Glut-1 but not Glut-3 resulted in significantly augmented binding of tagged proteins harboring the receptor binding domains of either HTLV-1 or HTLV-2 envelope glycoproteins (H1RBD or H2RBD). Using antibodies raised against the carboxy-terminal peptide of Glut-1, we found that Glut-1 expression was significantly increased in both CD4 and CD8 cells following TCR stimulation. Corresponding increases in the binding of H1RBD as well as H2RBD, not detected on quiescent T cells, were observed following TCR engagement. Furthermore, increased Glut-1 expression was accompanied by a massive augmentation in glucose uptake in TCR-stimulated CD4 and CD8 lymphocytes. Finally, we determined that the apparent contradictory results obtained by Takenouchi et al were due to their monitoring of Glut-1 with a mAb that does not bind cells expressing endogenous Glut-1, including human erythrocytes that harbor 300,000 copies per cell. Conclusion Transfection of Glut-1 directly correlates with the capacities of HTLV-1 and HTLV-2 envelope-derived ligands to bind cells. Moreover, Glut-1 is induced by TCR engagement, resulting in massive increases in glucose uptake and binding of HTLV-1 and -2 envelopes to both CD4 and CD8 T lymphocytes. Therefore, Glut-1 is a primary binding receptor for HTLV-1 and HTLV-2 envelopes on activated CD4 as well as CD8 lymphocytes. PMID:17504522

Synthesis, Biological Evaluation and Molecular Docking of New Benzenesulfonylhydrazone as Potential anti-Trypanosoma cruzi Agents.

PubMed

Elizondo-Jimenez, Silvia; Moreno-Herrera, Antonio; Reyes-Olivares, Rogelio; Dorantes-Gonzalez, Edith; Nogueda-Torres, Benjamín; Oliveira, Eduardo A Gamosa de; Romeiro, Nelilma C; Lima, Lidia M; Palos, Isidro; Rivera, Gildardo

2017-01-01

Chagas disease is a public health problem caused by Trypanosoma cruzi. Cruzain is a pharmacological target for designing a new drug against this parasite. Hydrazone and Nacylhydrazone derivatives have been traditionally associated as potential Cruzain inhibitors. Additionally, benzenesulfonyl derivatives show trypanocidal activity. Therefore, in this study, the combination of both structures has been taken into account for drug design. Seven benzenesulfonylhydrazone (BS-H) and seven N-propionyl benzenesulfonylhydrazone (BS-NAH) derivatives were synthetized and elucidated by infrared spectroscopy, nuclear magnetic resonance, and elemental analysis. All compounds were evaluated biologically in vitro against two strains of Trypanosoma cruzi (NINOA and INC-5), which are endemic in Mexico, and compared with the reference drugs nifurtimox and benznidazole. In order to gain insight into the putative molecular origin of the trypanocidal properties of these derivatives, docking studies were carried out with Cruzain. Compounds 4 and 6 (BS-H) and 10, 12-14 (BS-NAH) showed the best biological activity against NINOA and INC-5 strains, respectively. Compound 13 was the most potent trypanocidal compound showing a LC50 of 0.06 µM against INC-5 strain. However, compound 4 showed the best activity against both strains (LC50 <30 µM). Theoretical binding modes obtained suggested covalent binding that could explain their biological activity. Benzenesulfonyl and N-propionyl benzenesulfonyl hydrazone derivatives are good options for developing new trypanocidal agents. Particularly, compound 4 could be considered a lead compound. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Structural changes at the metal ion binding site during the phosphoglucomutase reaction.

PubMed

Ray, W J; Post, C B; Liu, Y; Rhyu, G I

1993-01-12

An electron density map of the reactive, Cd2+ form of crystalline phosphoglucomutase from X-ray diffraction studies shows that the enzymic phosphate donates a nonbridging oxygen to the ligand sphere of the bound metal ion, which appears to be tetracoordinate. 31P and 113Cd NMR spectroscopy are used to assess changes in the properties of bound Cd2+ produced by substrate/product and by substrate/product analog inhibitors. The approximately 50 ppm downfield shift of the 113Cd resonance on formation of the complex of dephosphoenzyme and glucose 1,6-bisphosphate is associated with the initial sugar-phosphate binding step and likely involves a change in the geometry of the coordinating ligands. This interpretation is supported by spectral studies involving various complexes of the active Co2+ and Ni(2+)-enzyme. In addition, there is a loss of the 31P-113Cd J coupling that characterizes the monophosphate complexes of the Cd2+ enzyme either during or immediately after the PO3- transfer step that produces the bisphosphate complex, indicating a further change at the metal binding site. The implications of these observations with respect to the PO3- transfer process in the phosphoglucomutase reaction are considered. The apparent plasticity of the ligand sphere of the active site metal ion in this system may allow a single metal ion to act as a chaperone for a nonbridging oxygen during PO3- transfer or to allow a change in metal ion coordination during catalysis. A general NMR line shape/chemical-exchange analysis for evaluating binding in protein-ligand systems when exchange is intermediate to fast on the NMR time scale is described. Its application to the present system involves multiple exchange sites that depend on a single binding rate, thereby adding further constraints to the analysis.

The promiscuous protein binding ability of erythrosine B studied by metachromasy (metachromasia).

PubMed

Ganesan, Lakshmi; Buchwald, Peter

2013-04-01

The present study aims to elucidate aspects of the protein binding ability of erythrosine B (ErB), a poly-iodinated xanthene dye and an FDA-approved food colorant (FD&C Red No. 3), which we have identified recently as a promiscuous inhibitor of protein-protein interactions (PPIs) with a remarkably consistent median inhibitory concentration (IC50 ) in the 5- to 30-μM range. Because ErB exhibits metachromasy, that is, color change upon binding to several proteins, we exploited this property to quantify its binding to proteins such as bovine serum albumin (BSA) and CD40L (CD154) and to determine the corresponding binding constants (Kd ) and stoichiometry (nb ) using spectrophotometric methods. Binding was reversible, and the estimated affinities for both protein targets obtained here (Kd values of 14 and 20 μM for BSA and CD40L, respectively) were in good agreement with that expected from the PPI inhibitory activity of ErB. A stoichiometry greater than one was observed both for CD40L and BSA binding (nb of 5-6 and 8-9 for BSA and CD40L, respectively), indicating the possibility of nonspecific binding of the flat and rigid ErB molecule at multiple sites, which could explain the promiscuous PPI inhibitory activity if some of these overlap with the binding site of the protein partner and interfere with the binding. Copyright © 2013 John Wiley & Sons, Ltd.

The Promiscuous Protein Binding Ability of Erythrosine B Studied by Metachromasy (Metachromasia)

PubMed Central

Ganesan, Lakshmi; Buchwald, Peter

2013-01-01

The present study aims to elucidate aspects of the protein binding ability of erythrosine B (ErB), a poly-iodinated xanthene dye and an FDA-approved food colorant (FD&C Red No. 3), which we have identified recently as a promiscuous inhibitor of protein–protein interactions (PPI) with a remarkably consistent median inhibitory concentration (IC50) in the 5–30 µM range. Because ErB exhibits metachromasy, i.e., color change upon binding to several proteins, we exploited this property to quantify its binding to proteins such as bovine serum albumin (BSA) and CD40L (CD154) and to determine the corresponding binding constants (Kd) and stoichiometry (nb) using spectrophotometric methods. Binding was reversible and the estimated affinities for both protein targets obtained here (Kd values of 14 and 20 µM for BSA and CD40L, respectively) were in good agreement with that expected from the protein–protein interaction (PPI) inhibitory activity of ErB. A stoichiometry greater than one was observed both for CD40L and BSA binding (nb of 5–6 and 8–9 for BSA and CD40L, respectively) indicating the possibility of nonspecific binding of the flat an rigid ErB molecule at multiple sites, which could explain the promiscuous PPI inhibitory activity if some of these overlap with the binding site of the protein partner and interfere with the binding. PMID:23456742

Characterization of the dextran-binding domain in the glucan-binding protein C of Streptococcus mutans.

PubMed

Takashima, Y; Fujita, K; Ardin, A C; Nagayama, K; Nomura, R; Nakano, K; Matsumoto-Nakano, M

2015-10-01

Streptococcus mutans produces multiple glucan-binding proteins (Gbps), among which GbpC encoded by the gbpC gene is known to be a cell-surface-associated protein involved in dextran-induced aggregation. The purpose of the present study was to characterize the dextran-binding domain of GbpC using bioinformatics analysis and molecular techniques. Bioinformatics analysis specified five possible regions containing molecular binding sites termed GB1 through GB5. Next, truncated recombinant GbpC (rGbpC) encoding each region was produced using a protein expression vector and five deletion mutant strains were generated, termed CDGB1 through CDGB5 respectively. The dextran-binding rates of truncated rGbpC that included the GB1, GB3, GB4 and GB5 regions in the upstream sequences were higher than that of the construct containing GB2 in the downstream region. In addition, the rates of dextran-binding for strains CDGB4 and CD1, which was entire gbpC deletion mutant, were significantly lower than for the other strains, while those of all other deletion mutants were quite similar to that of the parental strain MT8148. Biofilm structures formed by CDGB4 and CD1 were not as pronounced as that of MT8148, while those formed by other strains had greater density as compared to that of CD1. Our results suggest that the dextran-binding domain may be located in the GB4 region in the interior of the gbpC gene. Bioinformatics analysis is useful for determination of functional domains in many bacterial species. © 2015 The Society for Applied Microbiology.

Crystal structure of thermostable p-nitrophenylphosphatase from Bacillus Stearothermophilus (Bs-TpNPPase).

PubMed

Guo, Zheng; Wang, Fengbin; Shen, Tiantian; Huang, Jing; Wang, Yuandong; Ji, Chaoneng

2014-05-01

Thermostable p-nitrophenylphosphatase from Bacillus Stearothermophilus (Bs-TpNPPase) is involved in the Mg(2+)-dependent hydrolysis of the phosphoenzyme at an optimum reaction temperature of 55°C. Bs-TpNPPase has been cloned and overexpressed in the E.coli M15 strain. Based on the conserved active sites, the protein was suggested to be a member of the haloalkanoate dehalogenase (HAD) superfamily. Two site-specific point mutants of Bs-TpNPPase were prepared by changing the catalytic Asp10 and Thr43 to Ala10 and Ala43, respectively. The activity of the two mutants further confirms Bs-TpNPPase as a member of the HAD superfamily. HAD superfamily can be divided into the four subfamilies and play several biochemical roles such as DNA repair, signal transduction and secondary metabolism. To understand the relationship between structure and thermostability in HAD superfamily, Bs-TpNPPase from Bacillus Stearothermophilus was selected. The X-ray crystal structure of Bs-TpNPPase was determined at 1.5A resolution using the molecular replacement phasing method. The structure of Bs-TpNPPase has been deposited and the PDB code is 4KN8. Compared with Bsp, a mesophilic prokaryotic putative p-nitrophenyl phosphatase from Bacillus Subtilis, Bs- TpNPPase showed highly homology but variations in the level of leucine content, aromatic clusters, cation-Pi and hydrophobic interaction. These differences may affect the thermal stability of the protein. The crystal structure of Bs-TpNPPase described herein may serve as a guide to better understand the mechanism of thermostability and provide insights for further mutation work.

Antibody-peptide-MHC fusion conjugates target non-cognate T cells to kill tumour cells.

PubMed

King, Ben C; Hamblin, Angela D; Savage, Philip M; Douglas, Leon R; Hansen, Ted H; French, Ruth R; Johnson, Peter W M; Glennie, Martin J

2013-06-01

Attempts to generate robust anti-tumour cytotoxic T lymphocyte (CTL) responses using immunotherapy are frequently thwarted by exhaustion and anergy of CTL recruited to tumour. One strategy to overcome this is to retarget a population of virus-specific CTL to kill tumour cells. Here, we describe a proof-of-principle study using a bispecific conjugate designed to retarget ovalbumin (OVA)-specific CTL to kill tumour cells via CD20. A single-chain trimer (SCT) consisting of MHCI H-2K(b)/SIINFEKL peptide/beta 2 microglobulin/BirA was expressed in bacteria, refolded and chemically conjugated to one (1:1; F2) or two (2:1; F3) anti-hCD20 Fab' fragments. In vitro, the [SCT × Fab'] (F2 and F3) redirected SIINFEKL-specific OT-I CTL to kill CD20(+) target cells, and in the presence of CD20(+) target cells to provide crosslinking, they were also able to induce proliferation of OT-I cells. In vivo, activated OT-I CTL could be retargeted to kill [SCT × Fab']-coated B cells from hCD20 transgenic (hCD20 Tg) mice and also EL4 and B16 mouse tumour cells expressing human CD20 (hCD20). Importantly, in a hCD20 Tg mouse model, [SCT × Fab'] administered systemically were able to retarget activated OT-I cells to deplete normal B cells, and their performance matched that of a bispecific antibody (BsAb) comprising anti-CD3 and anti-CD20. [SCT × Fab'] were also active therapeutically in an EL4 tumour model. Furthermore, measurement of serum cytokine levels suggests that [SCT × Fab'] are associated with a lower level of inflammatory cytokine release than the BsAb and so may be advantageous clinically in terms of reduced toxicity.

A computational ab initio study of surface diffusion of sulfur on the CdTe (111) surface

NASA Astrophysics Data System (ADS)

Naderi, Ebadollah; Ghaisas, S. V.

2016-08-01

In order to discern the formation of epitaxial growth of CdS shell over CdTe nanocrystals, kinetics related to the initial stages of the growth of CdS on CdTe is investigated using ab-initio methods. We report diffusion of sulfur adatom on the CdTe (111) A-type (Cd-terminated) and B-type (Te-terminated) surfaces within the density functional theory (DFT). The barriers are computed by applying the climbing Nudge Elastic Band (c-NEB) method. From the results surface hopping emerges as the major mode of diffusion. In addition, there is a distinct contribution from kick-out type diffusion in which a CdTe surface atom is kicked out from its position and is replaced by the diffusing sulfur atom. Also, surface vacancy substitution contributes to the concomitant dynamics. There are sites on the B- type surface that are competitively close in terms of the binding energy to the lowest energy site of epitaxy on the surface. The kick-out process is more likely for B-type surface where a Te atom of the surface is displaced by a sulfur adatom. Further, on the B-type surface, subsurface migration of sulfur is indicated. Furthermore, the binding energies of S on CdTe reveal that on the A-type surface, epitaxial sites provide relatively higher binding energies and barriers than on B-type.

A computational ab initio study of surface diffusion of sulfur on the CdTe (111) surface

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

Naderi, Ebadollah, E-mail: enaderi42@gmail.com; Ghaisas, S. V.

2016-08-15

In order to discern the formation of epitaxial growth of CdS shell over CdTe nanocrystals, kinetics related to the initial stages of the growth of CdS on CdTe is investigated using ab-initio methods. We report diffusion of sulfur adatom on the CdTe (111) A-type (Cd-terminated) and B-type (Te-terminated) surfaces within the density functional theory (DFT). The barriers are computed by applying the climbing Nudge Elastic Band (c-NEB) method. From the results surface hopping emerges as the major mode of diffusion. In addition, there is a distinct contribution from kick-out type diffusion in which a CdTe surface atom is kicked outmore » from its position and is replaced by the diffusing sulfur atom. Also, surface vacancy substitution contributes to the concomitant dynamics. There are sites on the B- type surface that are competitively close in terms of the binding energy to the lowest energy site of epitaxy on the surface. The kick-out process is more likely for B-type surface where a Te atom of the surface is displaced by a sulfur adatom. Further, on the B-type surface, subsurface migration of sulfur is indicated. Furthermore, the binding energies of S on CdTe reveal that on the A-type surface, epitaxial sites provide relatively higher binding energies and barriers than on B-type.« less

Genomic structure and chromosomal mapping of the human CD22 gene

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

Wilson, G.L.; Kozlow, E.; Kehrl, J.H.

1993-06-01

The human CD22 gene is expressed specifically in B lymphocytes and likely has an important function in cell-cell interactions. A nearly full length human CD22 cDNA clone was used to isolate genomic clones that span the CD22 gene. The CD22 gene is spread over 22 kb of DNA and is composed of 15 exons. The first exon contains the major transcriptional start sites. The translation initiation codon is located in exon 3, which also encodes a portion of the signal peptide. Exons 4 to 10 encode the seven Ig domains of CD22, exon 11 encodes the transmembrane domain, exons 12more » to 15 encode the intracytoplasmic domain of CD22, and exon 15 also contains the 3' untranslated region. A minor form of CD22 mRNA likely results from splicing of exon 5 to exon 8, skipping exons 6 and 7. A 4.6-kb Xbal fragment of the CD22 gene was used to map the chromosomal location of CD22 by fluorescence in situ hybridization. The hybridization locus was identified by combining fluorescent images of the probe with the chromosomal banding pattern generated by an Alu probe. The results demonstrate the CD22 is located within the band region q13.1 of chromosome 19. Two closely clustered major transcription start sites and several minor start sites were mapped by primer extension. Similarly to many other lymphoid-specific genes, the CD22 promoter lacks an obvious TATA box. Approximately 4 kb of DNA 5' of the transcription start sites were sequenced and found to contain multiple Alu elements. Potential binding sites for the transcriptional factors NF-kB, AP-1, and Oct-2 are located within 300 bp 5' of the major transcription start sites. A 400-bp fragment (bp -339 through +71) of the CD22 promoter region was subcloned into a pGEM-chloramphenicol acetyltransferase vector and after transfection into B and T cells was found to be active in both B and T cells. 45 refs., 7 figs., 2 tabs.« less

Elucidation of the Functional Metal Binding Profile of a CdII/PbII sensor CmtRSc from Streptomyces coelicolor

PubMed Central

Wang, Yun; Kendall, John; Cavet, Jennifer S.; Giedroc, David P.

2010-01-01

Metal homeostasis and resistance in bacteria is maintained by a panel of metal sensing transcriptional regulators that collectively control transition metal availability and mediate resistance to heavy metal xenobiotics, including AsIII, CdII, PbII and HgII. The ArsR family constitutes a superfamily of metal sensors that appear to conform to the same winged helical, homodimeric fold, that collectively “sense” a wide array of beneficial metal ions and heavy metal pollutants. The genomes of many actinomycetes, including the soil dwelling bacterium Streptomyces coelicolor and the human pathogen Mycobacterium tuberculosis, encode over ten ArsR family regulators, most of unknown function. Here, we present the characterization of a homolog of M. tuberculosis CmtR (CmtRMtb) from S. coelicolor, denoted CmtRSc. We show that CmtRSc, in contrast to CmtRMtb binds two monomer mol equivalents of PbII or CdII to form two pairs of trigonal S3 coordination complexes per dimer. Metal site 1 conforms exactly to the α4C site previously characterized in CmtRMtb while metal site 2 is coordinated by a C-terminal vicinal thiolate pair, Cys110 and Cys111. Biological assays reveal that only CdII and, to a lesser extent, PbII mediate transcriptional derepression in the heterologous host M. smegmatis in a way that requires metal site 1. In contrast, mutagenesis of metal site 2 ligands Cys110 or Cys111 significantly reduces CdII responsiveness, with no detectable effect on PbII sensing. The implications of these findings on the ability to predict metal specificity and function from metal-site “signatures” in the primary structure of ArsR family proteins are discussed. PMID:20586430

Mesophilic and hyperthermophilic adenylate kinases differ in their tolerance to random fragmentation.

PubMed

Segall-Shapiro, Thomas H; Nguyen, Peter Q; Dos Santos, Edgardo D; Subedi, Saurav; Judd, Justin; Suh, Junghae; Silberg, Jonathan J

2011-02-11

The extent to which thermostability influences the location of protein fragmentation sites that allow retention of function is not known. To evaluate this, we used a novel transposase-based approach to create libraries of vectors that express structurally-related fragments of Bacillus subtilis adenylate kinase (BsAK) and Thermotoga neapolitana adenylate kinase (TnAK) with identical modifications at their termini, and we selected for variants in each library that complement the growth of Escherichia coli with a temperature-sensitive adenylate kinase (AK). Mutants created using the hyperthermophilic TnAK were found to support growth with a higher frequency (44%) than those generated from the mesophilic BsAK (6%), and selected TnAK mutants complemented E. coli growth more strongly than homologous BsAK variants. Sequencing of functional clones from each library also identified a greater dispersion of fragmentation sites within TnAK. Nondisruptive fission sites were observed within the AMP binding and core domains of both AK homologs. However, only TnAK contained sites within the lid domain, which undergoes dynamic fluctuations that are critical for catalysis. These findings implicate the flexible lid domain as having an increased sensitivity to fission events at physiological temperatures. In addition, they provide evidence that comparisons of nondisruptive fission sites in homologous proteins could be useful for finding dynamic regions whose conformational fluctuations are important for function, and they show that the discovery of protein fragments that cooperatively function in mesophiles can be aided by the use of thermophilic enzymes as starting points for protein design. Copyright © 2010 Elsevier Ltd. All rights reserved.

Enhanced interleukin-4 production in CD4+ T cells and elevated immunoglobulin E levels in antigen-primed mice by bisphenol A and nonylphenol, endocrine disruptors: involvement of nuclear factor-AT and Ca2+

PubMed Central

Lee, Mee H; Chung, Su W; Kang, Bok Y; Park, Jin; Lee, Choon H; Hwang, Seung Y; Kim, Tae S

2003-01-01

Bisphenol A (BPA) and p-nonylphenol (NP) are representative endocrine disruptors (EDs) that may have adverse effects on human health. The influence of these compounds on allergic immune responses remains unclear. In this study, we have examined the effects of BPA and NP on production of interleukin-4 (IL-4), a pro-inflammatory cytokine closely associated with allergic immune responses. Both BPA and NP significantly enhanced IL-4 production in keyhole limpet haemocyanin (KLH)-primed CD4+ T cells in a concentration-dependent manner. Treatment with BPA or NP in vivo resulted in significant increase of IL-4 production in CD4+ T cells and of antigen-specific immunoglobulin E (IgE) levels in the sera of KLH-primed mice. Furthermore, BPA and NP enhanced the activation of IL-4 gene promoter in EL4 T cells transiently transfected with IL-4 promoter/reporter constructs, and the enhancing effect mapped to a region in the IL-4 promoter containing binding sites for nuclear factor (NF)-AT. Activation of T lymphocytes by phorbol 12-myristate 13-acetate/ionomycin resulted in markedly enhanced binding activities to the NF-AT site, which significantly increased upon addition of BPA or NP, as demonstrated by the electrophoretic mobility shift assay, indicating that the transcription factor NF-AT was involved in the enhancing effect of BPA and NP on IL-4 production. The enhancement of IL-4 production by BPA or NP was significantly reduced by nitrendipine, a blocker of Ca2+ influx, and by FK506, a calcineurin inhibitor. FK506 inhibited the NF-AT–DNA binding activity and IL-4 gene promoter activity enhanced by BPA or NP. These results represent the first report describing possible enhancement of allergic response by EDs through increasing IL-4 production in CD4+ T cells and antigen-specific IgE levels in the sera via the stimulation of Ca2+/calcineurin-dependent NF-AT activation. PMID:12709020

Characterization studies on cadmium-mycophosphatin from the mushroom Agaricus macrosporus

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

Meisch, H.U.; Schmitt, J.A.

A low molecular weight Cd-binding phosphoglycoprotein, cadmium-mycophosphatin, has been isolated from the mushroom Agaricus macrosporus. This protein has a molecular weight of 12,000 dalton and contains no sulfur but a high amount of acid amino acids (Glu, Asp), and carbohydrates (glucose, galactose). Cadmium-mycophosphatin has an isoelectric point less than pH 2, binds cadmium with a dissociation constant of K/sub D/ = 1.59 x 10 M (pK/sub D/ = 6.8) and is saturated with 13.5 mole Cd/mole, all Cd-binding sites being equivalent. It is suggested that Cd is bound by phosphoserine groups, similar relations being known from calcium-binding proteins in animals.more » From A. macrosporus four other low-molecular weight glycoproteins have been isolated which contain sulfur and bind cadmium and copper. The biological significance of these Cd-binding proteins is discussed.« less

Characteristics of the isomeric flavonoids apigenin and genistein binding to hemoglobin by spectroscopic methods

NASA Astrophysics Data System (ADS)

Yuan, Jiang-Lan; Liu, Hui; Kang, Xu; Lv, Zhong; Zou, Guo-Lin

2008-11-01

Apigenin (Ap) and genistein (Ge), a couple of isomeric flavonoids with extensive bioactivities, are the most common dietary ingredients. They have been widely investigated due to their potential therapeutic actions for some diseases. In our work, binding characteristics of Ap and Ge to hemoglobin (Hb) were analyzed with fluorescence spectroscopy, circular dichroism (CD) and UV-vis absorption spectroscopy. The results indicated that Ap and Ge caused strong fluorescence quenching of Hb by static quenching mechanism, but their quenching efficiency and mechanisms were different. The binding site n suggested that there was a single binding site in Hb for Ap and Ge. The results of synchronous fluorescence showed that the microenvironment around Tyr residues of Hb had a slight trend of polarity decreasing, but the polarity around Trp residues increased by adding Ap. Results of CD indicated that the Ap and Ge did not changed the secondary structure of Hb. According to the theory of Förster resonance energy transfer, the binding distance r between Trp 37 and Ap/Ge was predicted to be 3.4 nm and 3.32 nm, respectively. The affinity of Ge toward Hb was higher than that of Ap.

Synthesis and radiolabeling of a somatostatin analog for multimodal imaging

NASA Astrophysics Data System (ADS)

Edwards, W. Barry; Liang, Kexian; Xu, Baogang; Anderson, Carolyn J.; Achilefu, Samuel

2006-02-01

A new multimodal imaging agent for imaging the somatostatin receptor has been synthesized and evaluated in vitro and in vivo. A somatostatin analog, conjugated to both 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (DOTA) and cypate (BS-296), was synthesized entirely on the solid phase (Fmoc) and purified by RP-HPLC. DOTA was added as a ligand for radiometals such as 64Cu or 177Lu for either radio-imaging or radiotherapy respectively. Cytate, a cypatesomatostatin analog conjugate, has previously demonstrated the ability to visualize somatostatin receptor rich tumor xenografts and natural organs by optical imaging techniques. BS-296 exhibited low nanomolar inhibitory capacity toward the binding of radiolabeled somatostatin analogs in cell membranes enriched in the somatostatin receptor, demonstrating the high affinity of this multimodal imaging peptide and indicating its potential as a molecular imaging agent. 64Cu, an isotope for diagnostic imaging and radiotherapy, was selected as the isotope for radiolabeling BS-296. BS-296 was radiolabeled with 64Cu in high specific activity (200 μCi/μg) in 90% radiochemical yield. Addition of 2,5-dihydroxybenzoic acid (gentisic acid) prevented radiolysis of the sample, allowing for study of the 64Cu -BS-296 the day following radiolabeling. Furthermore, inclusion of DMSO at a level of 20% was found not to interfere with radiolabeling yields and prevented the adherence of 64Cu -BS-296 to the walls of the reaction vessel.

Rational design of a conformation-switchable Ca2+- and Tb3+-binding protein without the use of multiple coupled metal-binding sites.

PubMed

Li, Shunyi; Yang, Wei; Maniccia, Anna W; Barrow, Doyle; Tjong, Harianto; Zhou, Huan-Xiang; Yang, Jenny J

2008-10-01

Ca2+, as a messenger of signal transduction, regulates numerous target molecules via Ca2+-induced conformational changes. Investigation into the determinants for Ca2+-induced conformational change is often impeded by cooperativity between multiple metal-binding sites or protein oligomerization in naturally occurring proteins. To dissect the relative contributions of key determinants for Ca2+-dependent conformational changes, we report the design of a single-site Ca2+-binding protein (CD2.trigger) created by altering charged residues at an electrostatically sensitive location on the surface of the host protein rat Cluster of Differentiation 2 (CD2).CD2.trigger binds to Tb3+ and Ca2+ with dissociation constants of 0.3 +/- 0.1 and 90 +/- 25 microM, respectively. This protein is largely unfolded in the absence of metal ions at physiological pH, but Tb3+ or Ca2+ binding results in folding of the native-like conformation. Neutralization of the charged coordination residues, either by mutation or protonation, similarly induces folding of the protein. The control of a major conformational change by a single Ca2+ ion, achieved on a protein designed without reliance on sequence similarity to known Ca2+-dependent proteins and coupled metal-binding sites, represents an important step in the design of trigger proteins.

Atomistic Description of Thiostannate-Capped CdSe Nanocrystals: Retention of Four-Coordinate SnS4 Motif and Preservation of Cd-Rich Stoichiometry

PubMed Central

2016-01-01

Colloidal semiconductor nanocrystals (NCs) are widely studied as building blocks for novel solid-state materials. Inorganic surface functionalization, used to displace native organic capping ligands from NC surfaces, has been a major enabler of electronic solid-state devices based on colloidal NCs. At the same time, very little is known about the atomistic details of the organic-to-inorganic ligand exchange and binding motifs at the NC surface, severely limiting further progress in designing all-inorganic NCs and NC solids. Taking thiostannates (K4SnS4, K4Sn2S6, K6Sn2S7) as typical examples of chalcogenidometallate ligands and oleate-capped CdSe NCs as a model NC system, in this study we address these questions through the combined application of solution 1H NMR spectroscopy, solution and solid-state 119Sn NMR spectroscopy, far-infrared and X-ray absorption spectroscopies, elemental analysis, and by DFT modeling. We show that through the X-type oleate-to-thiostannate ligand exchange, CdSe NCs retain their Cd-rich stoichiometry, with a stoichiometric CdSe core and surface Cd adatoms serving as binding sites for terminal S atoms of the thiostannates ligands, leading to all-inorganic (CdSe)core[Cdm(Sn2S7)yK(6y-2m)]shell (taking Sn2S76– ligand as an example). Thiostannates SnS44– and Sn2S76– retain (distorted) tetrahedral SnS4 geometry upon binding to NC surface. At the same time, experiments and simulations point to lower stability of Sn2S64– (and SnS32–) in most solvents and its lower adaptability to the NC surface caused by rigid Sn2S2 rings. PMID:25597625

Characterizing regulatory and functional differentiation between maize mesophyll and bundle sheath cells by transcriptomic analysis.

PubMed

Chang, Yao-Ming; Liu, Wen-Yu; Shih, Arthur Chun-Chieh; Shen, Meng-Ni; Lu, Chen-Hua; Lu, Mei-Yeh Jade; Yang, Hui-Wen; Wang, Tzi-Yuan; Chen, Sean C-C; Chen, Stella Maris; Li, Wen-Hsiung; Ku, Maurice S B

2012-09-01

To study the regulatory and functional differentiation between the mesophyll (M) and bundle sheath (BS) cells of maize (Zea mays), we isolated large quantities of highly homogeneous M and BS cells from newly matured second leaves for transcriptome profiling by RNA sequencing. A total of 52,421 annotated genes with at least one read were found in the two transcriptomes. Defining a gene with more than one read per kilobase per million mapped reads as expressed, we identified 18,482 expressed genes; 14,972 were expressed in M cells, including 53 M-enriched transcription factor (TF) genes, whereas 17,269 were expressed in BS cells, including 214 BS-enriched TF genes. Interestingly, many TF gene families show a conspicuous BS preference in expression. Pathway analyses reveal differentiation between the two cell types in various functional categories, with the M cells playing more important roles in light reaction, protein synthesis and folding, tetrapyrrole synthesis, and RNA binding, while the BS cells specialize in transport, signaling, protein degradation and posttranslational modification, major carbon, hydrogen, and oxygen metabolism, cell division and organization, and development. Genes coding for several transporters involved in the shuttle of C(4) metabolites and BS cell wall development have been identified, to our knowledge, for the first time. This comprehensive data set will be useful for studying M/BS differentiation in regulation and function.

Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein epitopes exposed by CD4 binding

DOE PAGES

Guan, Yongjun; Pazgier, Marzena; Sajadi, Mohammad M.; ...

2012-12-13

The HIV-1 envelope glycoprotein (Env) undergoes conformational transitions consequent to CD4 binding and coreceptor engagement during viral entry. The physical steps in this process are becoming defined, but less is known about their significance as targets of antibodies potentially protective against HIV-1 infection. Here we probe the functional significance of transitional epitope exposure by characterizing 41 human mAbs specific for epitopes exposed on trimeric Env after CD4 engagement. These mAbs recognize three epitope clusters: cluster A, the gp120 face occluded by gp41 in trimeric Env; cluster B, a region proximal to the coreceptor-binding site (CoRBS) and involving the V1/V2 domain;more » and cluster C, the coreceptor-binding site. The mAbs were evaluated functionally by antibody-dependent, cell-mediated cytotoxicity (ADCC) and for neutralization of Tiers 1 and 2 pseudoviruses. All three clusters included mAbs mediating ADCC. However, there was a strong potency bias for cluster A, which harbors at least three potent ADCC epitopes whose cognate mAbs have electropositive paratopes. Cluster A epitopes are functional ADCC targets during viral entry in an assay format using virion-sensitized target cells. In contrast, only cluster C contained epitopes that were recognized by neutralizing mAbs. There was significant diversity in breadth and potency that correlated with epitope fine specificity. In contrast, ADCC potency had no relationship with neutralization potency or breadth for any epitope cluster. In conclusion, Fc-mediated effector function and neutralization coselect with specificity in anti-Env antibody responses, but the nature of selection is distinct for these two antiviral activities.« less

Fatty-binding protein and galectin of Baylisascaris schroederi: Prokaryotic expression and preliminary evaluation of serodiagnostic potential

PubMed Central

Sun, Ying; Li, Yu; Wu, Yiran; Xiong, Lang; Li, Caiwu; Wang, Chengdong; Li, Desheng; Lan, Jingchao; Zhang, Zhihe; Jing, Bo; Gu, Xiaobing; Xie, Yue; Lai, Weimin; Peng, Xuerong

2017-01-01

Baylisascaris schroederi is a common parasite of captive giant pandas. The diagnosis of this ascariasis is normally carried out by a sedimentation-floatation method or PCR to detect eggs in feces, but neither method is suitable for early diagnosis. Fatty acid-binding protein (FABP) and galectin (GAL) exist in various animals and participate in important biology of parasites. Because of their good immunogenicity, they are seen as potential antigens for the diagnosis of parasitic diseases. In this study, we cloned and expressed recombinant FABP and GAL from B. schroederi (rBs-FABP and rBs-GAL) and developed indirect enzyme-linked immunosorbent assays (ELISAs) to evaluate their potential for diagnosing ascariasis in giant pandas. Immunolocalization showed that Bs-FABP and Bs-GAL were widely distributed in adult worms. The ELISA based on rBs-FABP showed sensitivity of 95.8% (23/24) and specificity of 100% (12/12), and that based on rBs-GAL had sensitivity of 91.7% (22/24) and specificity of 100% (12/12). PMID:28750056

Cell–specific Variation in E-selectin Ligand Expression Among Human Peripheral Blood Mononuclear Cells: Implications for Immunosurveillance and Pathobiology

PubMed Central

Silva, Mariana; Fung, Ronald Kam Fai; Donnelly, Conor Brian; Videira, Paula Alexandra; Sackstein, Robert

2017-01-01

Both host defense and immunopathology are shaped by the ordered recruitment of circulating leukocytes to affected sites, a process initiated by binding of blood-borne cells to E-selectin displayed at target endothelial beds. Accordingly, knowledge of the expression and function of leukocyte E-selectin ligands is key to understanding the tempo and specificity of immunoreactivity. Here, we performed E-selectin adherence assays under hemodynamic flow conditions coupled with flow cytometry and western blot analysis to elucidate the function and structural biology of glycoprotein E-selectin ligands expressed on human peripheral blood mononuclear cells (PBMCs). Circulating monocytes uniformly express high levels of the canonical E-selectin binding determinant sLeX and display markedly greater adhesive interactions with E-selectin than do circulating lymphocytes, which exhibit variable E-selectin binding among CD4+ and CD8+ T-cells but no binding by B-cells. Monocytes prominently present sLeX decorations on an array of protein scaffolds including PSGL-1, CD43, and CD44 (rendering the E-selectin ligands CLA, CD43E, and HCELL, respectively), and B-cells altogether lack E-selectin ligands. Quantitative PCR gene expression studies of glycosyltransferases that regulate display of sLeX reveal high transcript levels among circulating monocytes and low levels among circulating B-cells, and, commensurately, cell surface α(1,3)-fucosylation reveals that acceptor sialyllactosaminyl glycans convertible into sLeX are abundantly expressed on human monocytes yet are relatively deficient on B-cells. Collectively, these findings unveil distinct cell-specific patterns of E-selectin ligand expression among human PBMCs, indicating that circulating monocytes are specialized to engage E-selectin and providing key insights into the molecular effectors mediating recruitment of these cells at inflammatory sites. PMID:28330896

Behçet's: A Disease or a Syndrome? Answer from an Expression Profiling Study.

PubMed

Oğuz, Ali Kemal; Yılmaz, Seda Taşır; Oygür, Çağdaş Şahap; Çandar, Tuba; Sayın, Irmak; Kılıçoğlu, Sibel Serin; Ergün, İhsan; Ateş, Aşkın; Özdağ, Hilal; Akar, Nejat

2016-01-01

Behçet's disease (BD) is a chronic, relapsing, multisystemic inflammatory disorder with unanswered questions regarding its etiology/pathogenesis and classification. Distinct manifestation based subsets, pronounced geographical variations in expression, and discrepant immunological abnormalities raised the question whether Behçet's is "a disease or a syndrome". To answer the preceding question we aimed to display and compare the molecular mechanisms underlying distinct subsets of BD. For this purpose, the expression data of the gene expression profiling and association study on BD by Xavier et al (2013) was retrieved from GEO database and reanalysed by gene expression data analysis/visualization and bioinformatics enrichment tools. There were 15 BD patients (B) and 14 controls (C). Three subsets of BD patients were generated: MB (isolated mucocutaneous manifestations, n = 7), OB (ocular involvement, n = 4), and VB (large vein thrombosis, n = 4). Class comparison analyses yielded the following numbers of differentially expressed genes (DEGs); B vs C: 4, MB vs C: 5, OB vs C: 151, VB vs C: 274, MB vs OB: 215, MB vs VB: 760, OB vs VB: 984. Venn diagram analysis showed that there were no common DEGs in the intersection "MB vs C" ∩ "OB vs C" ∩ "VB vs C". Cluster analyses successfully clustered distinct expressions of BD. During gene ontology term enrichment analyses, categories with relevance to IL-8 production (MB vs C) and immune response to microorganisms (OB vs C) were differentially enriched. Distinct subsets of BD display distinct expression profiles and different disease associated pathways. Based on these clear discrepancies, the designation as "Behçet's syndrome" (BS) should be encouraged and future research should take into consideration the immunogenetic heterogeneity of BS subsets. Four gene groups, namely, negative regulators of inflammation (CD69, CLEC12A, CLEC12B, TNFAIP3), neutrophil granule proteins (LTF, OLFM4, AZU1, MMP8, DEFA4, CAMP), antigen processing and presentation proteins (CTSS, ERAP1), and regulators of immune response (LGALS2, BCL10, ITCH, CEACAM8, CD36, IL8, CCL4, EREG, NFKBIZ, CCR2, CD180, KLRC4, NFAT5) appear to be instrumental in BS immunopathogenesis.

Attenuation of cancer-initiating cells stemness properties by abrogating S100A4 calcium binding ability in head and neck cancers.

PubMed

Cheng, Li-Hao; Hung, Kai-Feng; Huang, Tung-Fu; Hsieh, Hsin-Pei; Wang, Shu-Ying; Huang, Chih-Yang; Lo, Jeng-Fan

2016-11-29

S100A4 is a calcium-binding protein capable of promoting epithelial-mesenchymal transition. Previously, we have demonstrated that S100A4 is required to sustain the head and neck cancer-initiating cells (HN-CICs) subpopulation. In this study, to further investigate the molecular mechanism, we established the head and neck squamous cell carcinoma (HNSCC) cell lines stably expressing mutant S100A4 proteins with defective calcium-binding sites on either N-terminal (NM) or C-terminal (CM), or a deletion of the last 15 amino-acid residues (CD). We showed that the NM, CM and CD harboring sphere cells that were enriched with HN-CICs population exhibited impaired stemness and malignant properties in vitro, as well as reduced tumor growth ability in vivo. Mechanistically, we demonstrated that mutant S100A4 proteins decreased the promoter activity of Nanog, likely through inhibition of p53. Moreover, the biophysical analyses of purified recombinant mutant S100A4 proteins suggest that both NM and CM mutant S100A4 were very similar to the WT S100A4 with subtle difference on the secondary structure, and that the CD mutant protein displayed the unexpected monomeric form in the solution phase.Taken together, our results suggest that both the calcium-binding ability and the C-terminal region of S100A4 are important for HN-CICs to sustain its stemness property and malignancy, and that the mechanism could be mediated by repressing p53 and subsequently activating the Nanog expression.

Doxorubicin attached to HPMA copolymer via amide bond modifies the glycosylation pattern of EL4 cells.

PubMed

Kovar, Lubomir; Etrych, Tomas; Kabesova, Martina; Subr, Vladimir; Vetvicka, David; Hovorka, Ondrej; Strohalm, Jiri; Sklenar, Jan; Chytil, Petr; Ulbrich, Karel; Rihova, Blanka

2010-08-01

To avoid the side effects of the anti-cancer drug doxorubicin (Dox), we conjugated this drug to a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer backbone. Dox was conjugated via an amide bond (Dox-HPMA(AM), PK1) or a hydrazone pH-sensitive bond (Dox-HPMA(HYD)). In contrast to Dox and Dox-HPMA(HYD), Dox-HPMA(AM) accumulates within the cell's intracellular membranes, including those of the Golgi complex and endoplasmic reticulum, both involved in protein glycosylation. Flow cytometry was used to determine lectin binding and cell death, immunoblot to characterize the presence of CD7, CD43, CD44, and CD45, and high-performance anion exchange chromatography with pulsed amperometric detector analysis for characterization of plasma membrane saccharide composition. Incubation of EL4 cells with Dox-HPMA(AM) conjugate, in contrast to Dox or Dox-HPMA(HYD), increased the amounts of membrane surface-associated glycoproteins, as well as saccharide moieties recognized by peanut agglutinin, Erythrina cristagalli, or galectin-1 lectins. Only Dox-HPMA(AM) increased expression of the highly glycosylated membrane glycoprotein CD43, while expression of others (CD7, CD44, and CD45) was unaffected. The binding sites for galectin-1 are present on CD43 molecule. Furthermore, we present that EL4 treated with Dox-HPMA(AM) possesses increased sensitivity to galectin-1-induced apoptosis. In this study, we demonstrate that Dox-HPMA(AM) treatment changes glycosylation of the EL4 T cell lymphoma surface and sensitizes the cells to galectin-1-induced apoptosis.

Peptide P5 (residues 628–683), comprising the entire membrane proximal region of HIV-1 gp41 and its calcium-binding site, is a potent inhibitor of HIV-1 infection

PubMed Central

Yu, Huifeng; Tudor, Daniela; Alfsen, Annette; Labrosse, Beatrice; Clavel, François; Bomsel, Morgane

2008-01-01

The membrane proximal region (MPR) of the transmembrane subunit, gp41, of the HIV envelope glycoprotein plays a critical role in HIV-1 infection of CD4+ target cells and CD4-independent mucosal entry. It contains continuous epitopes recognized by neutralizing IgG antibodies 2F5, 4E10 and Z13, and is therefore considered to be a promising target for vaccine design. Moreover, some MPR-derived peptides, such as T20 (enfuvirtide), are in clinical use as HIV-1 inhibitors. We have shown that an extended MPR peptide, P5, harbouring the lectin-like domain of gp41 and a calcium-binding site, is implicated in the interaction of HIV with its mucosal receptor. We now investigate the potential antiviral activities of P5 and other such long MPR-derived peptides. Structural studies of gp41 MPR-derived peptides using circular dichroism showed that the peptides P5 (a.a.628–683), P1 (a.a.648–683), P5L (a.a.613–683) and P7 (a.a.613–746) displayed a well-defined α-helical structure. Peptides P5 inhibited HIV-1 envelope mediated cell-cell fusion and infection of peripheral blood mononuclear cells by both X4- and R5-tropic HIV-1 strains, whereas peptides P5 mutated in the calcium binding site or P1 lacked antiviral activity, when P5L blocked cell fusion in contrast to P7. Strikingly, P5 inhibited CD4-dependent infection by T20-resistant R5-tropic HIV-1 variants. Cell-cell fusion studies indicated that the anti-HIV-1 activity of P5, unlike T20, could not be abrogated in the presence of the N-terminal leucine zipper domain (LZ). These results suggested that P5 could serve as a potent fusion inhibitor. PMID:18925934

Lipopolysaccharide modulation of a CD14-like molecule on porcine alveolar macrophages

NASA Technical Reports Server (NTRS)

Kielian, T. L.; Ross, C. R.; McVey, D. S.; Chapes, S. K.; Blecha, F.; Spooner, B. S. (Principal Investigator)

1995-01-01

Cluster of differentiation antigen 14 (CD14) functions as a receptor for lipopolysaccharide (LPS) LPS-binding protein (LBP) complexes. Because LPS has varying effects on CD14 expression in vitro, we evaluated CD14 expression in response to LPS with a fully differentiated macrophage phenotype, the alveolar macrophage. By using flow microfluorometric analysis and a radioimmunoassay with an anti-human CD14 monoclonal antibody (My4) that cross-reacts with porcine CD14, we found that macrophages stimulated with LPS for 24 h exhibited a two- to fivefold increase in CD14-like antigen compared with unstimulated cells. At low concentrations of LPS, up-regulation of the CD14-like antigen was dependent on serum; at higher concentrations of LPS, serum was not required. In the absence of serum a 10-fold higher dose of LPS (10 ng/ml) was required to increase CD14-like expression. In addition, LPS-induced CD14-like up-regulation correlated with secretion of tumor necrosis factor-alpha, regardless of serum concentration. Blockade with My4 antibody significantly inhibited LPS-induced tumor necrosis factor-alpha secretion at 1 ng/ml of LPS. However, inhibition decreased as we increased the LPS concentration, suggesting the existence of CD14-independent pathways of macrophage activation in response to LPS. Alternatively, My4 may have a lower affinity for the porcine CD14 antigen than LPS, which may have only partially blocked the LPS-LBP binding site at high concentrations of LPS. Therefore, these data suggest that LPS activation of porcine alveolar macrophages for 24 h increased CD14-like receptor expression. The degree of CD14-like up-regulation was related to LPS concentration, however, activation did not require the presence of serum at high concentrations of LPS.

Comprehensive Analysis of Contributions from Protein Conformational Stability and Major Histocompatibility Complex Class II-Peptide Binding Affinity to CD4+ Epitope Immunogenicity in HIV-1 Envelope Glycoprotein

PubMed Central

Li, Tingfeng; Steede, N. Kalaya; Nguyen, Hong-Nam P.; Freytag, Lucy C.; McLachlan, James B.; Mettu, Ramgopal R.; Robinson, James E.

2014-01-01

ABSTRACT Helper T-cell epitope dominance in human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is not adequately explained by peptide binding to major histocompatibility complex (MHC) proteins. Antigen processing potentially influences epitope dominance, but few, if any, studies have attempted to reconcile the influences of antigen processing and MHC protein binding for all helper T-cell epitopes of an antigen. Epitopes of gp120 identified in both humans and mice occur on the C-terminal flanks of flexible segments that are likely to be proteolytic cleavage sites. In this study, the influence of gp120 conformation on the dominance pattern in gp120 from HIV strain 89.6 was examined in CBA mice, whose MHC class II protein has one of the most well defined peptide-binding preferences. Only one of six dominant epitopes contained the most conserved element of the I-Ak binding motif, an aspartic acid. Destabilization of the gp120 conformation by deletion of single disulfide bonds preferentially enhanced responses to the cryptic I-Ak motif-containing sequences, as reported by T-cell proliferation or cytokine secretion. Conversely, inclusion of CpG in the adjuvant with gp120 enhanced responses to the dominant CD4+ T-cell epitopes. The gp120 destabilization affected secretion of some cytokines more than others, suggesting that antigen conformation could modulate T-cell functions through mechanisms of antigen processing. IMPORTANCE CD4+ helper T cells play an essential role in protection against HIV and other pathogens. Thus, the sites of helper T-cell recognition, the dominant epitopes, are targets for vaccine design; and the corresponding T cells may provide markers for monitoring infection and immunity. However, T-cell epitopes are difficult to identify and predict. It is also unclear whether CD4+ T cells specific for one epitope are more protective than T cells specific for other epitopes. This work shows that the three-dimensional (3D) structure of an HIV protein partially determines which epitopes are dominant, most likely by controlling the breakdown of HIV into peptides. Moreover, some types of signals from CD4+ T cells are affected by the HIV protein 3D structure; and thus the protectiveness of a particular peptide vaccine could be related to its location in the 3D structure. PMID:24920818

Structure of the Apo Form of Bacillus stearothermophilus Phosphofructokinase

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

Mosser, Rockann; Reddy, Manchi C.M.; Bruning, John B.

2012-02-08

The crystal structure of the unliganded form of Bacillus stearothermophilus phosphofructokinase (BsPFK) was determined using molecular replacement to 2.8 {angstrom} resolution (Protein Data Bank entry 3U39). The apo BsPFK structure serves as the basis for the interpretation of any structural changes seen in the binary or ternary complexes. When the apo BsPFK structure is compared with the previously published liganded structures of BsPFK, the structural impact that the binding of the ligands produces is revealed. This comparison shows that the apo form of BsPFK resembles the substrate-bound form of BsPFK, a finding that differs from previous predictions.

Preferential Binding of Hot Spot Mutant p53 Proteins to Supercoiled DNA In Vitro and in Cells

PubMed Central

Brázdová, Marie; Navrátilová, Lucie; Tichý, Vlastimil; Němcová, Kateřina; Lexa, Matej; Hrstka, Roman; Pečinka, Petr; Adámik, Matej; Vojtesek, Borivoj; Paleček, Emil; Deppert, Wolfgang; Fojta, Miroslav

2013-01-01

Hot spot mutant p53 (mutp53) proteins exert oncogenic gain-of-function activities. Binding of mutp53 to DNA is assumed to be involved in mutp53-mediated repression or activation of several mutp53 target genes. To investigate the importance of DNA topology on mutp53-DNA recognition in vitro and in cells, we analyzed the interaction of seven hot spot mutp53 proteins with topologically different DNA substrates (supercoiled, linear and relaxed) containing and/or lacking mutp53 binding sites (mutp53BS) using a variety of electrophoresis and immunoprecipitation based techniques. All seven hot spot mutp53 proteins (R175H, G245S, R248W, R249S, R273C, R273H and R282W) were found to have retained the ability of wild-type p53 to preferentially bind circular DNA at native negative superhelix density, while linear or relaxed circular DNA was a poor substrate. The preference of mutp53 proteins for supercoiled DNA (supercoil-selective binding) was further substantiated by competition experiments with linear DNA or relaxed DNA in vitro and ex vivo. Using chromatin immunoprecipitation, the preferential binding of mutp53 to a sc mutp53BS was detected also in cells. Furthermore, we have shown by luciferase reporter assay that the DNA topology influences p53 regulation of BAX and MSP/MST1 promoters. Possible modes of mutp53 binding to topologically constrained DNA substrates and their biological consequences are discussed. PMID:23555710

Molecular docking guided structure based design of symmetrical N,N'-disubstituted urea/thiourea as HIV-1 gp120-CD4 binding inhibitors.

PubMed

Sivan, Sree Kanth; Vangala, Radhika; Manga, Vijjulatha

2013-08-01

Induced fit molecular docking studies were performed on BMS-806 derivatives reported as small molecule inhibitors of HIV-1 gp120-CD4 binding. Comprehensive study of protein-ligand interactions guided in identification and design of novel symmetrical N,N'-disubstituted urea and thiourea as HIV-1 gp120-CD4 binding inhibitors. These molecules were synthesized in aqueous medium using microwave irradiation. Synthesized molecules were screened for their inhibitory ability by HIV-1 gp120-CD4 capture enzyme-linked immunosorbent assay (ELISA). Designed compounds were found to inhibit HIV-1 gp120-CD4 binding in micromolar (0.013-0.247 μM) concentrations. Copyright © 2013 Elsevier Ltd. All rights reserved.

Characterization of biochemical properties of Bacillus subtilis RecQ helicase.

PubMed

Qin, Wei; Liu, Na-Nv; Wang, Lijun; Zhou, Min; Ren, Hua; Bugnard, Elisabeth; Liu, Jie-Lin; Zhang, Lin-Hu; Vendôme, Jeremie; Hu, Jin-Shan; Xi, Xu Guang

2014-12-01

RecQ family helicases function as safeguards of the genome. Unlike Escherichia coli, the Gram-positive Bacillus subtilis bacterium possesses two RecQ-like homologues, RecQ[Bs] and RecS, which are required for the repair of DNA double-strand breaks. RecQ[Bs] also binds to the forked DNA to ensure a smooth progression of the cell cycle. Here we present the first biochemical analysis of recombinant RecQ[Bs]. RecQ[Bs] binds weakly to single-stranded DNA (ssDNA) and blunt-ended double-stranded DNA (dsDNA) but strongly to forked dsDNA. The protein exhibits a DNA-stimulated ATPase activity and ATP- and Mg(2+)-dependent DNA helicase activity with a 3' → 5' polarity. Molecular modeling shows that RecQ[Bs] shares high sequence and structure similarity with E. coli RecQ. Surprisingly, RecQ[Bs] resembles the truncated Saccharomyces cerevisiae Sgs1 and human RecQ helicases more than RecQ[Ec] with regard to its enzymatic activities. Specifically, RecQ[Bs] unwinds forked dsDNA and DNA duplexes with a 3'-overhang but is inactive on blunt-ended dsDNA and 5'-overhung duplexes. Interestingly, RecQ[Bs] unwinds blunt-ended DNA with structural features, including nicks, gaps, 5'-flaps, Kappa joints, synthetic replication forks, and Holliday junctions. We discuss these findings in the context of RecQ[Bs]'s possible functions in preserving genomic stability. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Development of new multivalent-bispecific agents for pretargeting tumor localization and therapy.

PubMed

Rossi, Edmund A; Sharkey, Robert M; McBride, William; Karacay, Habibe; Zeng, Li; Hansen, Hans J; Goldenberg, David M; Chang, Chien-Hsing

2003-09-01

Two bispecific diabodies (BS1.5 and BS1.5H) and two bispecific trivalent proteins (BS6 and BS8) were produced and tested as potential agents for pretargeted delivery of radiolabeled bivalent haptens to tumors expressing carcinoembryonic antigen. Each of the four proteins was expressed in Escherichia coli and purified from the soluble fraction. BS1.5 and BS1.5H (a humanized version of BS1.5) were evaluated in the GW-39 human colonic tumor-nude mouse model using a di-HSG-1,4,7,10-tetra-azacyclododecane-N,N',N" N"'-tetraacetic acid peptide (IMP-241) radiolabeled with (111)In. The biodistribution and T/NT ratios were compared with those of hMN-14 x m679 (Fab' x Fab') prepared chemically. In animals, both BS1.5 and BS1.5H cleared more rapidly than hMN-14 x m679 and showed tumor to nontumor ratios far superior to those of hMN-14 x m679. For example, with BS1.5 injected 8 h before (111)In-IMP-241, the tumor uptake of (111)In was 10.3 +/- 2.7 and 6.3 +/- 2.2% ID/g at 3 and 24 h, respectively, with the tumor to blood ratios being 167 +/- 35 at 3 h and 631 +/- 231 at 24 h. In comparison, the tumor to blood ratios of (111)In observed for hMN-14 x m679 given 24 h earlier were 8 +/- 2 at 3 h and 16 +/- 3 at 24 h. These results indicate that BS1.5 and BS1.5H are promising candidates for use in a variety of pretargeting applications, including tumor therapy with radionuclides and drugs. BS6 and BS8 may be even more attractive because of their potential to achieve higher levels of tumor uptake because of divalent carcinoembryonic antigen binding.

Exploring the binding of 4-thiothymidine with human serum albumin by spectroscopy, atomic force microscopy, and molecular modeling methods.

PubMed

Zhang, Juling; Gu, Huaimin; Zhang, Xiaohui

2014-01-30

The interaction of 4-thiothymidine (S(4)TdR) with human serum albumin (HSA) was studied by equilibrium dialysis under normal physiological conditions. In this work, the mechanism of the interaction between S(4)TdR and human serum albumin (HSA) was exploited by fluorescence, UV, CD circular, and SERS spectroscopic. Fluorescence and UV spectroscopy suggest that HSA intensities are significantly decreased when adding S(4)TdR to HAS, and the quenching mechanism of the fluorescence is static. Also, the ΔG, ΔH, and ΔS values across temperature indicated that hydrophobic interaction was the predominant binding force. The CD circular results show that there is little change in the secondary structure of HSA except the environment of amino acid changes when adding S(4)TdR to HSA. The surface-enhanced Raman scattering (SERS) shows that the interaction between S(4)TdR and HSA can be achieved through different binding sites which are probably located in the II A and III A hydrophobic pockets of HSA which correspond to Sudlow's I and II binding sites. In addition, the molecular modeling displays that S(4)TdR-HSA complex is stabilized by hydrophobic forces, which result from amino acid residues. The atomic force microscopy results revealed that the single HSA molecular dimensions were larger after interaction of 4-thiothymidine. This work would be useful to understand the state of the transportation, distribution, and metabolism of the anticancer drugs in the human body, and it could provide a useful biochemistry parameter for the development of new anti-cancer drugs and research of pharmacology mechanisms. Copyright © 2013 Elsevier Ltd. All rights reserved.

Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus.

PubMed

Mutso, Margit; Morro, Ainhoa Moliner; Smedberg, Cecilia; Kasvandik, Sergo; Aquilimeba, Muriel; Teppor, Mona; Tarve, Liisi; Lulla, Aleksei; Lulla, Valeria; Saul, Sirle; Thaa, Bastian; McInerney, Gerald M; Merits, Andres; Varjak, Margus

2018-04-27

Infection by Chikungunya virus (CHIKV) of the Old World alphaviruses (family Togaviridae) in humans can cause arthritis and arthralgia. The virus encodes four non-structural proteins (nsP) (nsP1, nsp2, nsP3 and nsP4) that act as subunits of the virus replicase. These proteins also interact with numerous host proteins and some crucial interactions are mediated by the unstructured C-terminal hypervariable domain (HVD) of nsP3. In this study, a human cell line expressing EGFP tagged with CHIKV nsP3 HVD was established. Using quantitative proteomics, it was found that CHIKV nsP3 HVD can bind cytoskeletal proteins, including CD2AP, SH3KBP1, CAPZA1, CAPZA2 and CAPZB. The interaction with CD2AP was found to be most evident; its binding site was mapped to the second SH3 ligand-like element in nsP3 HVD. Further assessment indicated that CD2AP can bind to nsP3 HVDs of many different New and Old World alphaviruses. Mutation of the short binding element hampered the ability of the virus to establish infection. The mutation also abolished ability of CD2AP to co-localise with nsP3 and replication complexes of CHIKV; the same was observed for Semliki Forest virus (SFV) harbouring a similar mutation. Similar to CD2AP, its homolog SH3KBP1 also bound the identified motif in CHIKV and SFV nsP3.

Conjugation of Benzylvanillin and Benzimidazole Structure Improves DNA Binding with Enhanced Antileukemic Properties

PubMed Central

Al-Mudarris, Ban A.; Chen, Shih-Hsun; Liang, Po-Huang; Osman, Hasnah; Jamal Din, Shah Kamal Khan; Abdul Majid, Amin M. S.

2013-01-01

Benzyl-o-vanillin and benzimidazole nucleus serve as important pharmacophore in drug discovery. The benzyl vanillin (2-(benzyloxy)-3-methoxybenzaldehyde) compound shows anti-proliferative activity in HL60 leukemia cancer cells and can effect cell cycle progression at G2/M phase. Its apoptosis activity was due to disruption of mitochondrial functioning. In this study, we have studied a series of compounds consisting of benzyl vanillin and benzimidazole structures. We hypothesize that by fusing these two structures we can produce compounds that have better anticancer activity with improved specificity particularly towards the leukemia cell line. Here we explored the anticancer activity of three compounds namely 2-(2-benzyloxy-3-methoxyphenyl)-1H-benzimidazole, 2MP, N-1-(2-benzyloxy-3-methoxybenzyl)-2-(2-benzyloxy-3-methoxyphenyl)-1H-benzimidazole, 2XP, and (R) and (S)-1-(2-benzyloxy-3-methoxyphenyl)-2, 2, 2-trichloroethyl benzenesulfonate, 3BS and compared their activity to 2-benzyloxy-3-methoxybenzaldehyde, (Bn1), the parent compound. 2XP and 3BS induces cell death of U937 leukemic cell line through DNA fragmentation that lead to the intrinsic caspase 9 activation. DNA binding study primarily by the equilibrium binding titration assay followed by the Viscosity study reveal the DNA binding through groove region with intrinsic binding constant 7.39 µM/bp and 6.86 µM/bp for 3BS and 2XP respectively. 2XP and 3BS showed strong DNA binding activity by the UV titration method with the computational drug modeling showed that both 2XP and 3BS failed to form any electrostatic linkages except via hydrophobic interaction through the minor groove region of the nucleic acid. The benzylvanillin alone (Bn1) has weak anticancer activity even after it was combined with the benzimidazole (2MP), but after addition of another benzylvanillin structure (2XP), stronger activity was observed. Also, the combination of benzylvanillin with benzenesulfonate (3BS) significantly improved the anticancer activity of Bn1. The present study provides a new insight of benzyl vanillin derivatives as potential anti-leukemic agent. PMID:24260527

In silico Analysis of HIV-1 Env-gp120 Reveals Structural Bases for Viral Adaptation in Growth-Restrictive Cells

PubMed Central

Yokoyama, Masaru; Nomaguchi, Masako; Doi, Naoya; Kanda, Tadahito; Adachi, Akio; Sato, Hironori

2016-01-01

Variable V1/V2 and V3 loops on human immunodeficiency virus type 1 (HIV-1) envelope-gp120 core play key roles in modulating viral competence to recognize two infection receptors, CD4 and chemokine-receptors. However, molecular bases for the modulation largely remain unclear. To address these issues, we constructed structural models for a full-length gp120 in CD4-free and -bound states. The models showed topologies of gp120 surface loop that agree with those in reported structural data. Molecular dynamics simulation showed that in the unliganded state, V1/V2 loop settled into a thermodynamically stable arrangement near V3 loop for conformational masking of V3 tip, a potent neutralization epitope. In the CD4-bound state, however, V1/V2 loop was rearranged near the bound CD4 to support CD4 binding. In parallel, cell-based adaptation in the absence of anti-viral antibody pressures led to the identification of amino acid substitutions that individually enhance viral entry and growth efficiencies in association with reduced sensitivity to CCR5 antagonist TAK-779. Notably, all these substitutions were positioned on the receptors binding surfaces in V1/V2 or V3 loop. In silico structural studies predicted some physical changes of gp120 by substitutions with alterations in viral replication phenotypes. These data suggest that V1/V2 loop is critical for creating a gp120 structure that masks co-receptor binding site compatible with maintenance of viral infectivity, and for tuning a functional balance of gp120 between immune escape ability and infectivity to optimize HIV-1 replication fitness. PMID:26903989

Cd(II) Sorption on Montmorillonite-Humic acid-Bacteria Composites

PubMed Central

Du, Huihui; Chen, Wenli; Cai, Peng; Rong, Xingmin; Dai, Ke; Peacock, Caroline L.; Huang, Qiaoyun

2016-01-01

Soil components (e.g., clays, bacteria and humic substances) are known to produce mineral-organic composites in natural systems. Herein, batch sorption isotherms, isothermal titration calorimetry (ITC), and Cd K-edge EXAFS spectroscopy were applied to investigate the binding characteristics of Cd on montmorillonite(Mont)-humic acid(HA)-bacteria composites. Additive sorption and non-additive Cd(II) sorption behaviour is observed for the binary Mont-bacteria and ternary Mont-HA-bacteria composite, respectively. Specifically, in the ternary composite, the coexistence of HA and bacteria inhibits Cd adsorption, suggesting a “blocking effect” between humic acid and bacterial cells. Large positive entropies (68.1 ~ 114.4 J/mol/K), and linear combination fitting of the EXAFS spectra for Cd adsorbed onto Mont-bacteria and Mont-HA-bacteria composites, demonstrate that Cd is mostly bound to bacterial surface functional groups by forming inner-sphere complexes. All our results together support the assertion that there is a degree of site masking in the ternary clay mineral-humic acid-bacteria composite. Because of this, in the ternary composite, Cd preferentially binds to the higher affinity components-i.e., the bacteria. PMID:26792640

External protons destabilize the activated voltage sensor in hERG channels.

PubMed

Shi, Yu Patrick; Cheng, Yen May; Van Slyke, Aaron C; Claydon, Tom W

2014-03-01

Extracellular acidosis shifts hERG channel activation to more depolarized potentials and accelerates channel deactivation; however, the mechanisms underlying these effects are unclear. External divalent cations, e.g., Ca(2+) and Cd(2+), mimic these effects and coordinate within a metal ion binding pocket composed of three acidic residues in hERG: D456 and D460 in S2 and D509 in S3. A common mechanism may underlie divalent cation and proton effects on hERG gating. Using two-electrode voltage clamp, we show proton sensitivity of hERG channel activation (pKa = 5.6), but not deactivation, was greatly reduced in the presence of Cd(2+) (0.1 mM), suggesting a common binding site for the Cd(2+) and proton effect on activation and separable effects of protons on activation and deactivation. Mutational analysis confirmed that D509 plays a critical role in the pH dependence of activation, as shown previously, and that cooperative actions involving D456 and D460 are also required. Importantly, neutralization of all three acidic residues abolished the proton-induced shift of activation, suggesting that the metal ion binding pocket alone accounts for the effects of protons on hERG channel activation. Voltage-clamp fluorimetry measurements demonstrated that protons shifted the voltage dependence of S4 movement to more depolarized potentials. The data indicate a site and mechanism of action for protons on hERG activation gating; protonation of D456, D460 and D509 disrupts interactions between these residues and S4 gating charges to destabilize the activated configuration of S4.

Sequence of ligand binding and structure change in the diphtheria toxin repressor upon activation by divalent transition metals.

PubMed

Rangachari, Vijayaraghavan; Marin, Vedrana; Bienkiewicz, Ewa A; Semavina, Maria; Guerrero, Luis; Love, John F; Murphy, John R; Logan, Timothy M

2005-04-19

The diphtheria toxin repressor (DtxR) is an Fe(II)-activated transcriptional regulator of iron homeostatic and virulence genes in Corynebacterium diphtheriae. DtxR is a two-domain protein that contains two structurally and functionally distinct metal binding sites. Here, we investigate the molecular steps associated with activation by Ni(II)Cl(2) and Cd(II)Cl(2). Equilibrium binding energetics for Ni(II) were obtained from isothermal titration calorimetry, indicating apparent metal dissociation constants of 0.2 and 1.7 microM for two independent sites. The binding isotherms for Ni(II) and Cd(II) exhibited a characteristic exothermic-endothermic pattern that was used to infer the metal binding sequence by comparing the wild-type isotherm with those of several binding site mutants. These data were complemented by measuring the distance between specific backbone amide nitrogens and the first equivalent of metal through heteronuclear NMR relaxation measurements. Previous studies indicated that metal binding affects a disordered to ordered transition in the metal binding domain. The coupling between metal binding and structure change was investigated using near-UV circular dichroism spectroscopy. Together, the data show that the first equivalent of metal is bound by the primary metal binding site. This binding orients the DNA binding helices and begins to fold the N-terminal domain. Subsequent binding at the ancillary site completes the folding of this domain and formation of the dimer interface. This model is used to explain the behavior of several mutants.

Binding sites of resveratrol, genistein, and curcumin with milk α- and β-caseins.

PubMed

Bourassa, P; Bariyanga, J; Tajmir-Riahi, H A

2013-02-07

The binding sites of antioxidant polyphenols resveratrol, genistein, and curcumin are located with milk α- and β-caseins in aqueous solution. FTIR, CD, and fluorescence spectroscopic methods and molecular modeling were used to analyze polyphenol binding sites, the binding constant, and the effects of complexation on casein stability and conformation. Structural analysis showed that polyphenols bind casein via hydrophilic and hydrophobic interactions with the number of bound polyphenol molecules (n) 1.20 for resveratrol, 1.42 for genistein, and 1.43 for curcumin with α-casein and 1.14 for resveratrol, 1.27 for genistein, and 1.27 for curcumin with β-casein. The overall binding constants of the complexes formed are K(res-α-casein) = 1.9 (±0.6) × 10(4) M(-1), K(gen-α-casein) = 1.8 (±0.4) × 10(4) M(-1), and K(cur-α-casein) = 2.8 (±0.8) × 10(4) M(-1) with α-casein and K(res-β-casein) = 2.3 (±0.3) × 10(4) M(-1), K(gen-β-casein) = 3.0 (±0.5) × 10(4) M(-1), and K(cur-β-casein) = 3.1 (±0.5) × 10(4) M(-1) for β-casein. Molecular modeling showed the participation of several amino acids in polyphenol-protein complexes, which were stabilized by the hydrogen bonding network with the free binding energy of -11.56 (resveratrol-α-casein), -12.35 (resveratrol-β-casein), -9.68 (genistein-α-casein), -9.97 (genistein-β-casein), -8.89 (curcumin-α-casein), and -10.70 kcal/mol (curcumin-β-casein). The binding sites of polyphenols are different with α- and β-caseins. Polyphenol binding altered casein conformation with reduction of α-helix, indicating a partial protein destabilization. Caseins might act as carriers to transport polyphenol in vitro.

catena-Poly[[bis­(4-carboxy­cyclo­hexane­carboxyl­ato-κ2 O 1,O 1′)cadmium(II)]-μ-1,4-bis­(imidazol-1-ylmeth­yl)benzene-κ2 N 3:N 3′

PubMed Central

Li, Bing-Bing; Xiao, Bo

2009-01-01

In the title coordination polymer, [Cd(C8H11O4)2(C14H14N4)]n, the Cd atom (site symmetry 2) is six-coordin­ated by two O,O′-bidentate 4-carboxy­cyclo­hexa­necarboxyl­ate (Hchdc) ligands and two N atoms from two different 1,4-bis­(imidazol-1-ylmeth­yl)benzene (1,4-bix) mol­ecules in a very distorted cis-CdN2O4 octa­hedral environment. The 1,4-bix mol­ecules act as bridging ligands that bind two CdII atoms, thus forming an infinite chain propagating in [100], which is decorated by the Hchdc anions. The structure is completed by O—H⋯O hydrogen bonds, which link the chains together. PMID:21582692

Optimal combinations of broadly neutralizing antibodies for prevention and treatments of HIV-1 clade C infection

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

Wagh, Kshitij; Bhattacharya, Tanmoy; Williamson, Carolyn

In this study, the identification of a new generation of potent broadly neutralizing HIV-1 antibodies (bnAbs) has generated substantial interest in their potential use for the prevention and/or treatment of HIV-1 infection. While combinations of bnAbs targeting distinct epitopes on the viral envelope (Env) will likely be required to overcome the extraordinary diversity of HIV-1, a key outstanding question is which bnAbs, and how many, will be needed to achieve optimal clinical benefit. We assessed the neutralizing activity of 15 bnAbs targeting four distinct epitopes of Env, including the CD4-binding site (CD4bs), the V1/V2-glycan region, the V3-glycan region, and themore » gp41 membrane proximal external region (MPER), against a panel of 200 acute/early clade C HIV-1 Env pseudoviruses. A mathematical model was developed that predicted neutralization by a subset of experimentally evaluated bnAb combinations with high accuracy. Using this model, we performed a comprehensive and systematic comparison of the predicted neutralizing activity of over 1,600 possible double, triple, and quadruple bnAb combinations. The most promising bnAb combinations were identified based not only on breadth and potency of neutralization, but also other relevant measures, such as the extent of complete neutralization and instantaneous inhibitory potential (IIP). By this set of criteria, triple and quadruple combinations of bnAbs were identified that were significantly more effective than the best double combinations, and further improved the probability of having multiple bnAbs simultaneously active against a given virus, a requirement that may be critical for countering escape in vivo. These results provide a rationale for advancing bnAb combinations with the best in vitro predictors of success into clinical trials for both the prevention and treatment of HIV-1 infection.« less

Optimal combinations of broadly neutralizing antibodies for prevention and treatments of HIV-1 clade C infection

DOE PAGES

Wagh, Kshitij; Bhattacharya, Tanmoy; Williamson, Carolyn; ...

2016-03-30

In this study, the identification of a new generation of potent broadly neutralizing HIV-1 antibodies (bnAbs) has generated substantial interest in their potential use for the prevention and/or treatment of HIV-1 infection. While combinations of bnAbs targeting distinct epitopes on the viral envelope (Env) will likely be required to overcome the extraordinary diversity of HIV-1, a key outstanding question is which bnAbs, and how many, will be needed to achieve optimal clinical benefit. We assessed the neutralizing activity of 15 bnAbs targeting four distinct epitopes of Env, including the CD4-binding site (CD4bs), the V1/V2-glycan region, the V3-glycan region, and themore » gp41 membrane proximal external region (MPER), against a panel of 200 acute/early clade C HIV-1 Env pseudoviruses. A mathematical model was developed that predicted neutralization by a subset of experimentally evaluated bnAb combinations with high accuracy. Using this model, we performed a comprehensive and systematic comparison of the predicted neutralizing activity of over 1,600 possible double, triple, and quadruple bnAb combinations. The most promising bnAb combinations were identified based not only on breadth and potency of neutralization, but also other relevant measures, such as the extent of complete neutralization and instantaneous inhibitory potential (IIP). By this set of criteria, triple and quadruple combinations of bnAbs were identified that were significantly more effective than the best double combinations, and further improved the probability of having multiple bnAbs simultaneously active against a given virus, a requirement that may be critical for countering escape in vivo. These results provide a rationale for advancing bnAb combinations with the best in vitro predictors of success into clinical trials for both the prevention and treatment of HIV-1 infection.« less

Effect of Dioxygen on Copper(II) Binding to α-Synuclein

PubMed Central

Lucas, Heather R.; Lee, Jennifer C.

2010-01-01

Using the fluorescent amino acid tryptophan (Trp), we have characterized the copper(II) binding of F4W α-synuclein in the presence and absence of dioxygen at neutral pH. Variations in Trp fluorescence indicate that copper(II) binding is enhanced by the presence of dioxygen, with the apparent dissociation constant (Kd(app)) changing from 100 nM (anaerobic) to 10 nM (aerobic). To investigate the possible role of methionine oxidation, complementary work focused on synthetic peptide models of the N-terminal Cu(II)-α-syn site, MDV(F/W) and M*DV(F/W), where M*= methionine sulfoxide. Furthermore, we employed circular dichroism (CD) spectroscopy to demonstrate that the phenyl-to-indole (F→W) substitution does not alter copper(II) binding properties and to confirm the 1:1 metal-peptide binding stoichiometry. CD comparisons also revealed that Met1 oxidation does not affect the copper-peptide conformation and further suggested the possible existence of a CuII-Trp/Phe (cation-π) interaction. PMID:20064662

Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain

PubMed Central

Parrilla-Doblas, Jara Teresa; Ariza, Rafael R.; Roldán-Arjona, Teresa

2017-01-01

ABSTRACT DNA methylation is a crucial epigenetic mark associated to gene silencing, and its targeted removal is a major goal of epigenetic editing. In animal cells, DNA demethylation involves iterative 5mC oxidation by TET enzymes followed by replication-dependent dilution and/or replication-independent DNA repair of its oxidized derivatives. In contrast, plants use specific DNA glycosylases that directly excise 5mC and initiate its substitution for unmethylated C in a base excision repair process. In this work, we have fused the catalytic domain of Arabidopsis ROS1 5mC DNA glycosylase (ROS1_CD) to the DNA binding domain of yeast GAL4 (GBD). We show that the resultant GBD-ROS1_CD fusion protein binds specifically a GBD-targeted DNA sequence in vitro. We also found that transient in vivo expression of GBD-ROS1_CD in human cells specifically reactivates transcription of a methylation-silenced reporter gene, and that such reactivation requires both ROS1_CD catalytic activity and GBD binding capacity. Finally, we show that reactivation induced by GBD-ROS1_CD is accompanied by decreased methylation levels at several CpG sites of the targeted promoter. All together, these results show that plant 5mC DNA glycosylases can be used for targeted active DNA demethylation in human cells. PMID:28277978

Platelets Toll-like receptor-4 in Crohns disease.

PubMed

Schmid, Werner; Novacek, Gottfried; Vogelsang, Harald; Papay, Pavol; Primas, Christian; Eser, Alexander; Panzer, Simon

2017-02-01

Platelets are activated in Crohn's disease (CD) and interplay with leukocytes. Engagement of Toll-like receptor-4 (TLR-4), which is expressed in human platelets, may be involved in crosstalks between platelets and leukocytes leading to their mutual activation for host defense. Human neutrophil peptides (HNPs), lipoprotein binding peptides, and sCD14 were determined by enzyme-linked immunosorbent assays in 42 patients with active CD, in 43 patients with CD in remission, and in 30 healthy individuals. Neutrophil-platelet aggregates and binding of the TLR-4 monoclonal antibody to platelets were determined by flow cytometry. Levels of HNPs were higher in patients with CD than in controls (P = 0.0003 vs. active CD and P = 0.01 vs. CD in remission). Likewise, neutrophils with adhering platelets were higher in patients with active CD than in controls (P = 0.004). Binding of the TLR-4 antibody in patients with active CD was similar to that in controls, while patients in remission had significantly higher binding capacities (P = 0.59 and P = 0.003). Incubation of plasma from patients with active disease or patients in remission with platelets from healthy controls confirmed lower binding of the TLR-4 antibody in the presence of plasma from active diseased patients compared to controls (P = 0.039), possibly due to high levels of lipopolysaccharides, as suggested by high levels of sCD14 and lipoprotein binding protein. Our study indicates involvement of platelet TLR-4 in enhancing the secretion of antimicrobial peptides from neutrophils. While platelet aggregation can be due to a variety of mechanisms in inflammatory disease, the mutual activation of platelets and neutrophils may augment host defense. © 2016 Stichting European Society for Clinical Investigation Journal Foundation.

Multispectroscopic and molecular modeling studies on the interaction of copper-ibuprofenate complex with bovine serum albumin (BSA).

PubMed

Shiri, Farshad; Rahimi-Nasrabadi, Mehdi; Ahmadi, Farhad; Ehrlich, Hermann

2018-05-31

Bovine serum albumin (BSA) represents the well recognized model protein for investigations of diverse intermolecular reactions in studies on pharmacological activities of modern drugs. In the present work, the interaction between copper ibuprofenate ([Cu2(IBU)4]) and BSA under simulative physiological conditions was investigated by the using of diverse spectral methods including fluorescence, UV-vis absorption, CD spectroscopy and also molecular docking. The obtained results showed that there was a strong fluorescence quenching of BSA by [Cu2(IBU)4] (2.964E+4 M -1 at room temperature). Using the continuous variation method, a single class of binding sites, (1:1), for [Cu2(IBU)4] on BSA was put in evidence. The Stern-Volmer analysis of fluorescence quenching data shows the presence of the static quenching mechanism. The binding constants K b were calculated and the thermodynamic parameters ∆G°, ∆H° and ∆S° were given. The obtained thermodynamic values and the change observed in the alpha-helical content signature suggests that hydrogen bonding and hydrophobic forces play a major role in the [Cu2(IBU)4]-BSA binding interaction. Site marker competitive experiments indicated that the binding of [Cu2(IBU)4] to BSA primarily took place in sub-domain IIA that this observation were substantiated by molecular docking studies. The results of CD and UV-vis spectroscopy showed for the first time that the presence of [Cu2(IBU)4] increased the ɑ-helical content of BSA (from 48.56% to 55.71%) and conformational changes of BSA molecules. Copyright © 2017 Elsevier B.V. All rights reserved.

Photo-isomerization and oxidation of bilirubin in mammals is dependent on albumin binding.

PubMed

Goncharova, Iryna; Jašprová, Jana; Vítek, Libor; Urbanová, Marie

2015-12-01

The bilirubin (BR) photo-conversion in the human body is a protein-dependent process; an effective photo-isomerization of the potentially neurotoxic Z,Z-BR as well as its oxidation to biliverdin in the antioxidant redox cycle is possible only when BR is bound on serum albumin. We present a novel analytical concept in the study of linear tetrapyrroles metabolic processes based on an in-depth mapping of binding sites in the structure of human serum albumin (HSA). A combination of fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular modeling methods was used for recognition of the binding site for BR, its derivatives (mesobilirubin and bilirubin ditaurate), and the products of the photo-isomerization and oxidation (lumirubin, biliverdin, and xanthobilirubic acid) on HSA. The CD spectra and fluorescent quenching of the Trp-HSA were used to calculate the binding constants. The results of the CD displacement experiments performed with hemin were interpreted together with the findings of molecular docking performed on the pigment-HSA complexes. We estimated that Z,Z-BR and its metabolic products bind on two independent binding sites. Our findings support the existence of a reversible antioxidant redox cycle for BR and explain an additional pathway of the photo-isomerization process (increase of HSA binding capacity; the excess free [unbound] BR can be converted and also bound to HSA). Copyright © 2015 Elsevier Inc. All rights reserved.

Predicted structure of MIF/CD74 and RTL1000/CD74 complexes.

PubMed

Meza-Romero, Roberto; Benedek, Gil; Leng, Lin; Bucala, Richard; Vandenbark, Arthur A

2016-04-01

Macrophage migration inhibitory factor (MIF) is a key cytokine in autoimmune and inflammatory diseases that attracts and then retains activated immune cells from the periphery to the tissues. MIF exists as a homotrimer and its effects are mediated through its primary receptor, CD74 (the class II invariant chain that exhibits a highly structured trimerization domain), present on class II expressing cells. Although a number of binding residues have been identified between MIF and CD74 trimers, their spatial orientation has not been established. Using a docking program in silico, we have modeled binding interactions between CD74 and MIF as well as CD74 and a competitive MIF inhibitor, RTL1000, a partial MHC class II construct that is currently in clinical trials for multiple sclerosis. These analyses revealed 3 binding sites on the MIF trimer that each were predicted to bind one CD74 trimer through interactions with two distinct 5 amino acid determinants. Surprisingly, predicted binding of one CD74 trimer to a single RTL1000 antagonist utilized the same two 5 residue determinants, providing strong suggestive evidence in support of the MIF binding regions on CD74. Taken together, our structural modeling predicts a new MIF(CD74)3 dodecamer that may provide the basis for increased MIF potency and the requirement for ~3-fold excess RTL1000 to achieve full antagonism.

The retrovirus HTLV-1 inserts an ectopic CTCF-binding site into the human genome.

PubMed

Satou, Yorifumi; Miyazato, Paola; Ishihara, Ko; Yaguchi, Hiroko; Melamed, Anat; Miura, Michi; Fukuda, Asami; Nosaka, Kisato; Watanabe, Takehisa; Rowan, Aileen G; Nakao, Mitsuyoshi; Bangham, Charles R M

2016-03-15

Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes malignant and inflammatory diseases in ∼10% of infected people. A typical host has between 10(4) and 10(5) clones of HTLV-1-infected T lymphocytes, each clone distinguished by the genomic integration site of the single-copy HTLV-1 provirus. The HTLV-1 bZIP (HBZ) factor gene is constitutively expressed from the minus strand of the provirus, whereas plus-strand expression, required for viral propagation to uninfected cells, is suppressed or intermittent in vivo, allowing escape from host immune surveillance. It remains unknown what regulates this pattern of proviral transcription and latency. Here, we show that CTCF, a key regulator of chromatin structure and function, binds to the provirus at a sharp border in epigenetic modifications in the pX region of the HTLV-1 provirus in T cells naturally infected with HTLV-1. CTCF is a zinc-finger protein that binds to an insulator region in genomic DNA and plays a fundamental role in controlling higher order chromatin structure and gene expression in vertebrate cells. We show that CTCF bound to HTLV-1 acts as an enhancer blocker, regulates HTLV-1 mRNA splicing, and forms long-distance interactions with flanking host chromatin. CTCF-binding sites (CTCF-BSs) have been propagated throughout the genome by transposons in certain primate lineages, but CTCF binding has not previously been described in present-day exogenous retroviruses. The presence of an ectopic CTCF-BS introduced by the retrovirus in tens of thousands of genomic locations has the potential to cause widespread abnormalities in host cell chromatin structure and gene expression.

Lineage-Specific Differences between the gp120 Inner Domain Layer 3 of Human Immunodeficiency Virus and That of Simian Immunodeficiency Virus

PubMed Central

Ding, Shilei; Medjahed, Halima; Prévost, Jérémie; Coutu, Mathieu; Xiang, Shi-Hua

2016-01-01

ABSTRACT Binding of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) gp120 exterior envelope glycoprotein to CD4 triggers conformational changes in gp120 that promote its interaction with one of the chemokine receptors, usually CCR5, ultimately leading to gp41-mediated virus-cell membrane fusion and entry. We previously described that topological layers (layer 1, layer 2, and layer 3) in the gp120 inner domain contribute to gp120-trimer association in the unliganded state but also help secure CD4 binding. Relative to layer 1 of HIV-1 gp120, the SIVmac239 gp120 layer 1 plays a more prominent role in maintaining gp120-trimer association but is minimally involved in promoting CD4 binding, which could be explained by the existence of a well-conserved tryptophan at position 375 (Trp 375) in HIV-2/SIVsmm. In this study, we investigated the role of SIV layer 3 in viral entry, cell-to-cell fusion, and CD4 binding. We observed that a network of interactions involving some residues of the β8-α5 region in SIVmac239 layer 3 may contribute to CD4 binding by helping shape the nearby Phe 43 cavity, which directly contacts CD4. In summary, our results suggest that layer 3 in SIV has a greater impact on CD4 binding than in HIV-1. This work defines lineage-specific differences in layer 3 from HIV-1 and that from SIV. IMPORTANCE CD4-induced conformational changes in the gp120 inner domain involve rearrangements between three topological layers. While the role of layers 1 to 3 for HIV-1 and layers 1 and 2 for SIV on gp120 transition to the CD4-bound conformation has been reported, the role of SIV layer 3 remains unknown. Here we report that SIV layer 3 has a greater impact on CD4 binding than does layer 3 in HIV-1 gp120. This work defines lineage-specific differences in layer 3 from HIV-1 and SIV. PMID:27535053

The Antibacterial Cell Division Inhibitor PC190723 Is an FtsZ Polymer-stabilizing Agent That Induces Filament Assembly and Condensation*

PubMed Central

Andreu, José M.; Schaffner-Barbero, Claudia; Huecas, Sonia; Alonso, Dulce; Lopez-Rodriguez, María L.; Ruiz-Avila, Laura B.; Núñez-Ramírez, Rafael; Llorca, Oscar; Martín-Galiano, Antonio J.

2010-01-01

Cell division protein FtsZ can form single-stranded filaments with a cooperative behavior by self-switching assembly. Subsequent condensation and bending of FtsZ filaments are important for the formation and constriction of the cytokinetic ring. PC190723 is an effective bactericidal cell division inhibitor that targets FtsZ in the pathogen Staphylococcus aureus and Bacillus subtilis and does not affect Escherichia coli cells, which apparently binds to a zone equivalent to the binding site of the antitumor drug taxol in tubulin (Haydon, D. J., Stokes, N. R., Ure, R., Galbraith, G., Bennett, J. M., Brown, D. R., Baker, P. J., Barynin, V. V., Rice, D. W., Sedelnikova, S. E., Heal, J. R., Sheridan, J. M., Aiwale, S. T., Chauhan, P. K., Srivastava, A., Taneja, A., Collins, I., Errington, J., and Czaplewski, L. G. (2008) Science 312, 1673–1675). We have found that the benzamide derivative PC190723 is an FtsZ polymer-stabilizing agent. PC190723 induced nucleated assembly of Bs-FtsZ into single-stranded coiled protofilaments and polymorphic condensates, including bundles, coils, and toroids, whose formation could be modulated with different solution conditions. Under conditions for reversible assembly of Bs-FtsZ, PC190723 binding reduced the GTPase activity and induced the formation of straight bundles and ribbons, which was also observed with Sa-FtsZ but not with nonsusceptible Ec-FtsZ. The fragment 2,6-difluoro-3-methoxybenzamide also induced Bs-FtsZ bundling. We propose that polymer stabilization by PC190723 suppresses in vivo FtsZ polymer dynamics and bacterial division. The biochemical action of PC190723 on FtsZ parallels that of the microtubule-stabilizing agent taxol on the eukaryotic structural homologue tubulin. Both taxol and PC190723 stabilize polymers against disassembly by preferential binding to each assembled protein. It is yet to be investigated whether both ligands target structurally related assembly switches. PMID:20212044

Cryo-electron microscopy and single molecule fluorescent microscopy detect CD4 receptor induced HIV size expansion prior to cell entry

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

Pham, Son; CSIRO Australian Animal Health Laboratory, Victoria 3220; Tabarin, Thibault

Viruses are often thought to have static structure, and they only remodel after the viruses have entered target cells. Here, we detected a size expansion of virus particles prior to viral entry using cryo-electron microscopy (cryo-EM) and single molecule fluorescence imaging. HIV expanded both under cell-free conditions with soluble receptor CD4 (sCD4) targeting the CD4 binding site on the HIV-1 envelope protein (Env) and when HIV binds to receptor on cellular membrane. We have shown that the HIV Env is needed to facilitate receptor induced virus size expansions, showing that the ‘lynchpin’ for size expansion is highly specific. We demonstratemore » that the size expansion required maturation of HIV and an internal capsid core with wild type stability, suggesting that different HIV compartments are linked and are involved in remodelling. Our work reveals a previously unknown event in HIV entry, and we propose that this pre-entry priming process enables HIV particles to facilitate the subsequent steps in infection. - Highlights: • Cell free viruses are able to receive external trigger that leads to apparent size expansion. • Virus envelope and CD4 receptor engagement is the lynchpin of virus size expansion. • Internal capsid organisation can influence receptor mediated virus size expansion. • Pre-existing virus-associated lipid membrane in cell free virus can accommodate the receptor mediated virus size expansion.« less

Basic Fibroblast Growth Factor Fused with Tandem Collagen-Binding Domains from Clostridium histolyticum Collagenase ColG Increases Bone Formation.

PubMed

Sekiguchi, Hiroyuki; Uchida, Kentaro; Matsushita, Osamu; Inoue, Gen; Nishi, Nozomu; Masuda, Ryo; Hamamoto, Nana; Koide, Takaki; Shoji, Shintaro; Takaso, Masashi

2018-01-01

Basic fibroblast growth factor 2 (bFGF) accelerates bone formation during fracture healing. Because the efficacy of bFGF decreases rapidly following its diffusion from fracture sites, however, repeated dosing is required to ensure a sustained therapeutic effect. We previously developed a fusion protein comprising bFGF, a polycystic kidney disease domain (PKD; s2b), and collagen-binding domain (CBD; s3) sourced from the Clostridium histolyticum class II collagenase, ColH, and reported that the combination of this fusion protein with a collagen-like peptide, poly(Pro-Hyp-Gly) 10 , induced mesenchymal cell proliferation and callus formation at fracture sites. In addition, C. histolyticum produces class I collagenase (ColG) with tandem CBDs (s3a and s3b) at the C-terminus. We therefore hypothesized that a bFGF fusion protein containing ColG-derived tandem CBDs (s3a and s3b) would show enhanced collagen-binding activity, leading to improved bone formation. Here, we examined the binding affinity of four collagen anchors derived from the two clostridial collagenases to H-Gly-Pro-Arg-Gly-(Pro-Hyp-Gly) 12 -NH 2 , a collagenous peptide, by surface plasmon resonance and found that tandem CBDs (s3a-s3b) have the highest affinity for the collagenous peptide. We also constructed four fusion proteins consisting of bFGF and s3 (bFGF-s3), s2b-s3b (bFGF-s2b-s3), s3b (bFGF-s3b), and s3a-s3b (bFGF-s3a-s3b) and compared their biological activities to those of a previous fusion construct (bFGF-s2b-s3) using a cell proliferation assay in vitro and a mouse femoral fracture model in vivo. Among these CB-bFGFs, bFGF-s3a-s3b showed the highest capacity to induce mesenchymal cell proliferation and callus formation in the mice fracture model. The poly(Pro-Hyp-Gly) 10 /bFGF-s3a-s3b construct may therefore have the potential to promote bone formation in clinical settings.

Characterization of p-phenylenediamine-albumin binding sites and T-cell responses to hapten-modified protein.

PubMed

Jenkinson, Claire; Jenkins, Rosalind E; Aleksic, Maja; Pirmohamed, Munir; Naisbitt, Dean J; Park, B Kevin

2010-03-01

Exposure to p-phenylenediamine (PPD) is associated with the development of T-cell-mediated allergic contact dermatitis. The purpose of this study was to define the nature of the interaction of PPD with the protein and the antigenic determinant that stimulates T cells. Mass spectrometry was employed to show that PPD oxidation products bind irreversibly to cysteine (Cys, position 34) in human serum albumin (HSA). A modified tryptic peptide was characterized with an increase in mass of 106 Da, corresponding to the addition of PPD and not to the secondary products of self conjugation. Lymphocytes from 10 PPD-allergic patients, but not tolerant/naive individuals, were stimulated with PPD and PPD-modified HSA. A total of 70 PPD-specific and 10 PPD-HSA-specific CD4+, CD8+, and CD4+CD8+, Th2-secreting T-cell clones were generated from three allergic patients. In total, 40 clones were stimulated with both PPD and PPD-modified HSA. PPD-modified HSA triggered T-cell responses through a classical hapten mechanism involving processing. Presentation of PPD to several clones was dependent on protein complex formation (42 out of 48) and processing (32 out of 68); however, 12% of clones were triggered with PPD directly. These data identify Cys as the single target for PPD-HSA binding, and show that PPD protein adducts are antigenic determinants in patients with contact dermatitis.

Conformational Rearrangement Within the Soluble Domains of the CD4 Receptor is Ligand-Specific

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

Ashish,F.; Juncadella, I.; Garg, R.

2008-01-01

Ligand binding induces shape changes within the four modular ectodomains (D1-D4) of the CD4 receptor, an important receptor in immune signaling. Small angle x-ray scattering (SAXS) on both a two-domain and a four-domain construct of the soluble CD4 (sCD4) is consistent with known crystal structures demonstrating a bilobal and a semi-extended tetralobal Z conformation in solution, respectively. Detection of conformational changes within sCD4 as a result of ligand binding was followed by SAXS on sCD4 bound to two different glycoprotein ligands: the tick saliva immunosuppressor Salp15 and the HIV-1 envelope protein gp120. Ab initio modeling of these data showed thatmore » both Salp15 and gp120 bind to the D1 domain of sCD4 and yet induce drastically different structural rearrangements. Upon binding, Salp15 primarily distorts the characteristic lobal architecture of the sCD4 without significantly altering the semi-extended shape of the sCD4 receptor. In sharp contrast, the interaction of gp120 with sCD4 induces a shape change within sCD4 that can be described as a Z-to-U bi-fold closure of the four domains across its flexible D2-D3 linker. Placement of known crystal structures within the boundaries of the SAXS-derived models suggests that the ligand-induced shape changes could be a result of conformational changes within this D2-D3 linker. Functionally, the observed shape changes in CD4 receptor causes dissociation of lymphocyte kinase from the cytoplasmic domain of Salp15-bound CD4 and facilitates an interaction between the exposed V3 loops of CD4-bound gp120 molecule to the extracellular loops of its co-receptor, a step essential for HIV-1 viral entry.« less

An ultrasensitive lysozyme chemiluminescence biosensor based on surface molecular imprinting using ionic liquid modified magnetic graphene oxide/β-cyclodextrin as supporting material.

PubMed

Duan, Huimin; Wang, Xiaojiao; Wang, Yanhui; Sun, Yuanling; Li, Jianbo; Luo, Chuannan

2016-04-28

In this work, ionic liquid modified Fe3O4@dopamine/graphene oxide/β-cyclodextrin (ILs-Fe3O4@DA/GO/β-CD) was used as supporting material to synthesize surface molecularly imprinted polymer (SMIP) which then was introduced into chemiluminescence (CL) to achieve an ultrasensitive and selective biosensor for determination of lysozyme (Lys). ILs and β-CD was applied to provide multiple binding sites to prepare Lys SMIP and Fe3O4@DA was designed to make the product separate easily and prevent the aggregation of GO which could improve absorption capacity for its large specific surface area. The ILs-Fe3O4@DA/GO/β-CD-SMIP showed high adsorption capacity (Q = 101 mg/g) to Lys in the adsorption isotherm assays. The adsorption equilibrium was reached within 10 min for all the concentrations, attributing to the binding sites situated exclusively at the surface, and the adsorption model followed Langmuir isotherm. Under the suitable CL conditions, the proposed biosensor could response Lys linearly in the range of 1.0 × 10(-9)-8.0 × 10(-8) mg/mL with a detection limit of 3.0 × 10(-10) mg/mL. When used in practical samples in determination of Lys, the efficient biosensor exhibited excellent result with the recoveries ranging from 94% to 112%. Copyright © 2016 Elsevier B.V. All rights reserved.

Vaccine-Elicited Tier 2 HIV-1 Neutralizing Antibodies Bind to Quaternary Epitopes Involving Glycan-Deficient Patches Proximal to the CD4 Binding Site

PubMed Central

Crooks, Ema T.; Tong, Tommy; Chakrabarti, Bimal; Narayan, Kristin; Georgiev, Ivelin S.; Menis, Sergey; Huang, Xiaoxing; Kulp, Daniel; Osawa, Keiko; Muranaka, Janelle; Stewart-Jones, Guillaume; Destefano, Joanne; O’Dell, Sijy; LaBranche, Celia; Robinson, James E.; Montefiori, David C.; McKee, Krisha; Du, Sean X.; Doria-Rose, Nicole; Kwong, Peter D.; Mascola, John R.; Zhu, Ping; Schief, William R.; Wyatt, Richard T.; Whalen, Robert G.; Binley, James M.

2015-01-01

Eliciting broad tier 2 neutralizing antibodies (nAbs) is a major goal of HIV-1 vaccine research. Here we investigated the ability of native, membrane-expressed JR-FL Env trimers to elicit nAbs. Unusually potent nAb titers developed in 2 of 8 rabbits immunized with virus-like particles (VLPs) expressing trimers (trimer VLP sera) and in 1 of 20 rabbits immunized with DNA expressing native Env trimer, followed by a protein boost (DNA trimer sera). All 3 sera neutralized via quaternary epitopes and exploited natural gaps in the glycan defenses of the second conserved region of JR-FL gp120. Specifically, trimer VLP sera took advantage of the unusual absence of a glycan at residue 197 (present in 98.7% of Envs). Intriguingly, removing the N197 glycan (with no loss of tier 2 phenotype) rendered 50% or 16.7% (n = 18) of clade B tier 2 isolates sensitive to the two trimer VLP sera, showing broad neutralization via the surface masked by the N197 glycan. Neutralizing sera targeted epitopes that overlap with the CD4 binding site, consistent with the role of the N197 glycan in a putative “glycan fence” that limits access to this region. A bioinformatics analysis suggested shared features of one of the trimer VLP sera and monoclonal antibody PG9, consistent with its trimer-dependency. The neutralizing DNA trimer serum took advantage of the absence of a glycan at residue 230, also proximal to the CD4 binding site and suggesting an epitope similar to that of monoclonal antibody 8ANC195, albeit lacking tier 2 breadth. Taken together, our data show for the first time that strain-specific holes in the glycan fence can allow the development of tier 2 neutralizing antibodies to native spikes. Moreover, cross-neutralization can occur in the absence of protecting glycan. Overall, our observations provide new insights that may inform the future development of a neutralizing antibody vaccine. PMID:26023780

Vaccine-Elicited Tier 2 HIV-1 Neutralizing Antibodies Bind to Quaternary Epitopes Involving Glycan-Deficient Patches Proximal to the CD4 Binding Site.

PubMed

Crooks, Ema T; Tong, Tommy; Chakrabarti, Bimal; Narayan, Kristin; Georgiev, Ivelin S; Menis, Sergey; Huang, Xiaoxing; Kulp, Daniel; Osawa, Keiko; Muranaka, Janelle; Stewart-Jones, Guillaume; Destefano, Joanne; O'Dell, Sijy; LaBranche, Celia; Robinson, James E; Montefiori, David C; McKee, Krisha; Du, Sean X; Doria-Rose, Nicole; Kwong, Peter D; Mascola, John R; Zhu, Ping; Schief, William R; Wyatt, Richard T; Whalen, Robert G; Binley, James M

2015-05-01

Eliciting broad tier 2 neutralizing antibodies (nAbs) is a major goal of HIV-1 vaccine research. Here we investigated the ability of native, membrane-expressed JR-FL Env trimers to elicit nAbs. Unusually potent nAb titers developed in 2 of 8 rabbits immunized with virus-like particles (VLPs) expressing trimers (trimer VLP sera) and in 1 of 20 rabbits immunized with DNA expressing native Env trimer, followed by a protein boost (DNA trimer sera). All 3 sera neutralized via quaternary epitopes and exploited natural gaps in the glycan defenses of the second conserved region of JR-FL gp120. Specifically, trimer VLP sera took advantage of the unusual absence of a glycan at residue 197 (present in 98.7% of Envs). Intriguingly, removing the N197 glycan (with no loss of tier 2 phenotype) rendered 50% or 16.7% (n = 18) of clade B tier 2 isolates sensitive to the two trimer VLP sera, showing broad neutralization via the surface masked by the N197 glycan. Neutralizing sera targeted epitopes that overlap with the CD4 binding site, consistent with the role of the N197 glycan in a putative "glycan fence" that limits access to this region. A bioinformatics analysis suggested shared features of one of the trimer VLP sera and monoclonal antibody PG9, consistent with its trimer-dependency. The neutralizing DNA trimer serum took advantage of the absence of a glycan at residue 230, also proximal to the CD4 binding site and suggesting an epitope similar to that of monoclonal antibody 8ANC195, albeit lacking tier 2 breadth. Taken together, our data show for the first time that strain-specific holes in the glycan fence can allow the development of tier 2 neutralizing antibodies to native spikes. Moreover, cross-neutralization can occur in the absence of protecting glycan. Overall, our observations provide new insights that may inform the future development of a neutralizing antibody vaccine.

Probing the binding reaction of cytarabine to human serum albumin using multispectroscopic techniques with the aid of molecular docking.

PubMed

Xu, Liang; Hu, Yan-Xi; Li, Jin; Liu, Yu-Feng; Zhang, Li; Ai, Hai-Xin; Liu, Hong-Sheng

2017-08-01

Cytarabine is a kind of chemotherapy medication. In the present study, the molecular interaction between cytarabine and human serum albumin (HSA) was investigated via fluorescence, UV-vis absorption, circular dichroism (CD) spectroscopy and molecular docking method under simulative physiological conditions. It was found that cytarabine could effectively quench the intrinsic fluorescence of HSA through a static quenching process. The apparent binding constants between drug and HSA at 288, 293 and 298K were estimated to be in the order of 10 3 L·mol -1 . The thermodynamic parameters ΔH°, ΔG°and ΔS° were calculated, in which the negative ΔG°suggested that the binding of cytarabine to HSA was spontaneous, moreover the negative ΔS°and negative ΔH°revealed that van der Waals force and hydrogen bonds were the major forces to stabilize the protein-cytarabine (1:1) complex. The competitive binding experiments showed that the primary binding site of cytarabine was located in the site I (subdomain IIA) of HSA. In addition, the binding distance was calculated to be 3.4nm according to the Förster no-radiation energy transfer theory. The analysis of CD and three-dimensional (3D) fluorescence spectra demonstrated that the binding of drug to HSA induced some conformational changes in HSA. The molecular docking study also led to the same conclusion obtained from the spectral results. Copyright © 2017 Elsevier B.V. All rights reserved.

Design and Characterization of a Peptide Mimotope of the HIV-1 gp120 Bridging Sheet

PubMed Central

Schiavone, Marco; Fiume, Giuseppe; Caivano, Antonella; de Laurentiis, Annamaria; Falcone, Cristina; Masci, Francesca Fasanella; Iaccino, Enrico; Mimmi, Selena; Palmieri, Camillo; Pisano, Antonio; Pontoriero, Marilena; Rossi, Annalisa; Scialdone, Annarita; Vecchio, Eleonora; Andreozzi, Concetta; Trovato, Maria; Rafay, Jan; Ferko, Boris; Montefiori, David; Lombardi, Angela; Morsica, Giulia; Poli, Guido; Quinto, Ileana; Pavone, Vincenzo; de Berardinis, Piergiuseppe; Scala, Giuseppe

2012-01-01

The Bridging Sheet domain of HIV-1 gp120 is highly conserved among the HIV-1 strains and allows HIV-1 binding to host cells via the HIV-1 coreceptors. Further, the bridging sheet domain is a major target to neutralize HIV-1 infection. We rationally designed four linear peptide epitopes that mimic the three-dimensional structure of bridging sheet by using molecular modeling. Chemically synthesized peptides BS3 and BS4 showed a fair degree of antigenicity when tested in ELISA with IgG purified from HIV+ broadly neutralizing sera while the production of synthetic peptides BS1 and BS2 failed due to their high degree of hydrophobicity. To overcome this limitation, we linked all four BS peptides to the COOH-terminus of GST protein to test both their antigenicity and immunogenicity. Only the BS1 peptide showed good antigenicity; however, no envelope specific antibodies were elicited upon mice immunization. Therefore we performed further analyses by linking BS1 peptide to the NH2-terminus of the E2 scaffold from the Geobacillus Stearothermophylus PDH complex. The E2-BS1 fusion peptide showed good antigenic results, however only one immunized rabbit elicited good antibody titers towards both the monomeric and oligomeric viral envelope glycoprotein (Env). In addition, moderate neutralizing antibodies response was elicited against two HIV-1 clade B and one clade C primary isolates. These preliminary data validate the peptide mimotope approach as a promising tool to obtain an effective HIV-1 vaccine. PMID:22754323

Behçet's: A Disease or a Syndrome? Answer from an Expression Profiling Study

PubMed Central

Oğuz, Ali Kemal; Yılmaz, Seda Taşır; Oygür, Çağdaş Şahap; Çandar, Tuba; Sayın, Irmak; Kılıçoğlu, Sibel Serin; Ergün, İhsan; Ateş, Aşkın; Özdağ, Hilal; Akar, Nejat

2016-01-01

Behçet’s disease (BD) is a chronic, relapsing, multisystemic inflammatory disorder with unanswered questions regarding its etiology/pathogenesis and classification. Distinct manifestation based subsets, pronounced geographical variations in expression, and discrepant immunological abnormalities raised the question whether Behçet’s is “a disease or a syndrome”. To answer the preceding question we aimed to display and compare the molecular mechanisms underlying distinct subsets of BD. For this purpose, the expression data of the gene expression profiling and association study on BD by Xavier et al (2013) was retrieved from GEO database and reanalysed by gene expression data analysis/visualization and bioinformatics enrichment tools. There were 15 BD patients (B) and 14 controls (C). Three subsets of BD patients were generated: MB (isolated mucocutaneous manifestations, n = 7), OB (ocular involvement, n = 4), and VB (large vein thrombosis, n = 4). Class comparison analyses yielded the following numbers of differentially expressed genes (DEGs); B vs C: 4, MB vs C: 5, OB vs C: 151, VB vs C: 274, MB vs OB: 215, MB vs VB: 760, OB vs VB: 984. Venn diagram analysis showed that there were no common DEGs in the intersection “MB vs C” ∩ “OB vs C” ∩ “VB vs C”. Cluster analyses successfully clustered distinct expressions of BD. During gene ontology term enrichment analyses, categories with relevance to IL-8 production (MB vs C) and immune response to microorganisms (OB vs C) were differentially enriched. Distinct subsets of BD display distinct expression profiles and different disease associated pathways. Based on these clear discrepancies, the designation as “Behçet’s syndrome” (BS) should be encouraged and future research should take into consideration the immunogenetic heterogeneity of BS subsets. Four gene groups, namely, negative regulators of inflammation (CD69, CLEC12A, CLEC12B, TNFAIP3), neutrophil granule proteins (LTF, OLFM4, AZU1, MMP8, DEFA4, CAMP), antigen processing and presentation proteins (CTSS, ERAP1), and regulators of immune response (LGALS2, BCL10, ITCH, CEACAM8, CD36, IL8, CCL4, EREG, NFKBIZ, CCR2, CD180, KLRC4, NFAT5) appear to be instrumental in BS immunopathogenesis. PMID:26890122

Voltage- and ATP-dependent structural rearrangements of the P2X2 receptor associated with the gating of the pore

PubMed Central

Keceli, Batu; Kubo, Yoshihiro

2014-01-01

P2X2 is an extracellular ATP-gated cation channel which has a voltage-dependent gating property even though it lacks a canonical voltage sensor. It is a trimer in which each subunit has two transmembrane helices and a large extracellular domain. The three inter-subunit ATP binding sites are linked to the pore forming transmembrane (TM) domains by β-strands. We analysed structural rearrangements of the linker strands between the ATP binding site and TM domains upon ligand binding and voltage change, electrophysiologically in Xenopus oocytes, using mutants carrying engineered thiol-modifiable cysteine residues. (1) We demonstrated that the double mutant D315C&I67C (at β-14 and β-1, respectively) shows a 2- to 4-fold increase in current amplitude after treatment with a reducing reagent, dithiothreitol (DTT). Application of the thiol-reactive metal Cd2+ induced current decline due to bond formation between D315C and I67C. This effect was not observed in wild type (WT) or in single point mutants. (2) Cd2+-induced current decline was analysed in hyperpolarized and depolarized conditions with different pulse protocols, and also in the presence and absence of ATP. (3) Current decline induced by Cd2+ could be clearly observed in the presence of ATP, but was not clear in the absence of ATP, showing a state-dependent modification. (4) In the presence of ATP, Cd2+ modification was significantly faster in hyperpolarized than in depolarized conditions, showing voltage-dependent structural rearrangements of the linker strands. (5) Experiments using tandem trimeric constructs (TTCs) with controlled number and position of mutations in the trimer showed that the bridging by Cd2+ between 315 and 67 was not intra- but inter-subunit. (6) Finally, we performed similar analyses of a pore mutant T339S, which makes the channel activation voltage insensitive. Cd2+ modification rates of T339S were similar in hyperpolarized and depolarized conditions. Taking these results together, we demonstrated that structural rearrangements of the linker region of the P2X2 receptor channel are induced not only by ligand binding but also by membrane potential change. PMID:25172943

Biosurfactant production through Bacillus sp. MTCC 5877 and its multifarious applications in food industry.

PubMed

Anjum, Farhan; Gautam, Gunjan; Edgard, Gnansounou; Negi, Sangeeta

2016-08-01

In this study Bacillus sp. MTCC5877 was explored for the production of biosurfactant (BSs) and various carbon sources 1% (w/v), 0.5% (w/v) nitrogen sources were tested at different pH, and temperature. Yield was measured in terms of Emulsification index (EI), Oil Displacement Area (ODA) and Drop Collapse Area (DCA) and maximum emulsification activities of BSs were found (E24) 50%, 76% and 46%, respectively, and maximum ODA of 5.0, 6.2 and 4.7cm, were shown respectively. The BS was able to reduce the surface tension of water from 72 to 30mN/m and 72 to 32mN/m. Structural compositions of BS were confirmed by FTIR, GC-MS and NMR. Anti-adhesive property of BS was determined and found effective against biofilm formation. It could remove 73% Cd from vegetable which confirms its application in food industry. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cytoskeletal regulation of CD44 membrane organization and interactions with E-selectin.

PubMed

Wang, Ying; Yago, Tadayuki; Zhang, Nan; Abdisalaam, Salim; Alexandrakis, George; Rodgers, William; McEver, Rodger P

2014-12-19

Interactions of CD44 on neutrophils with E-selectin on activated endothelial cells mediate rolling under flow, a prerequisite for neutrophil arrest and migration into perivascular tissues. How CD44 functions as a rolling ligand despite its weak affinity for E-selectin is unknown. We examined the nanometer scale organization of CD44 on intact cells. CD44 on leukocytes and transfected K562 cells was cross-linked within a 1.14-nm spacer. Depolymerizing actin with latrunculin B reduced cross-linking. Fluorescence resonance energy transfer (FRET) revealed tight co-clustering between CD44 fused to yellow fluorescent protein (YFP) and CD44 fused to cyan fluorescent protein on K562 cells. Latrunculin B reduced FRET-reported co-clustering. Number and brightness analysis confirmed actin-dependent CD44-YFP clusters on living cells. CD44 lacking binding sites for ankyrin and for ezrin/radixin/moesin (ERM) proteins on its cytoplasmic domain (ΔANKΔERM) did not cluster. Unexpectedly, CD44 lacking only the ankyrin-binding site (ΔANK) formed larger but looser clusters. Fluorescence recovery after photobleaching demonstrated increased CD44 mobility by latrunculin B treatment or by deleting the cytoplasmic domain. ΔANKΔERM mobility increased only modestly, suggesting that the cytoplasmic domain engages the cytoskeleton by an additional mechanism. Ex vivo differentiated CD44-deficient neutrophils expressing exogenous CD44 rolled on E-selectin and activated Src kinases after binding anti-CD44 antibody. In contrast, differentiated neutrophils expressing ΔANK had impaired rolling and kinase activation. These data demonstrate that spectrin and actin networks regulate CD44 clustering and suggest that ankyrin enhances CD44-mediated neutrophil rolling and signaling. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Cytoskeletal Regulation of CD44 Membrane Organization and Interactions with E-selectin*

PubMed Central

Wang, Ying; Yago, Tadayuki; Zhang, Nan; Abdisalaam, Salim; Alexandrakis, George; Rodgers, William; McEver, Rodger P.

2014-01-01

Interactions of CD44 on neutrophils with E-selectin on activated endothelial cells mediate rolling under flow, a prerequisite for neutrophil arrest and migration into perivascular tissues. How CD44 functions as a rolling ligand despite its weak affinity for E-selectin is unknown. We examined the nanometer scale organization of CD44 on intact cells. CD44 on leukocytes and transfected K562 cells was cross-linked within a 1.14-nm spacer. Depolymerizing actin with latrunculin B reduced cross-linking. Fluorescence resonance energy transfer (FRET) revealed tight co-clustering between CD44 fused to yellow fluorescent protein (YFP) and CD44 fused to cyan fluorescent protein on K562 cells. Latrunculin B reduced FRET-reported co-clustering. Number and brightness analysis confirmed actin-dependent CD44-YFP clusters on living cells. CD44 lacking binding sites for ankyrin and for ezrin/radixin/moesin (ERM) proteins on its cytoplasmic domain (ΔANKΔERM) did not cluster. Unexpectedly, CD44 lacking only the ankyrin-binding site (ΔANK) formed larger but looser clusters. Fluorescence recovery after photobleaching demonstrated increased CD44 mobility by latrunculin B treatment or by deleting the cytoplasmic domain. ΔANKΔERM mobility increased only modestly, suggesting that the cytoplasmic domain engages the cytoskeleton by an additional mechanism. Ex vivo differentiated CD44-deficient neutrophils expressing exogenous CD44 rolled on E-selectin and activated Src kinases after binding anti-CD44 antibody. In contrast, differentiated neutrophils expressing ΔANK had impaired rolling and kinase activation. These data demonstrate that spectrin and actin networks regulate CD44 clustering and suggest that ankyrin enhances CD44-mediated neutrophil rolling and signaling. PMID:25359776

Trace metals and macroelements in mussels from Chinese coastal waters: National spatial patterns and normalization.

PubMed

Lu, Guang-Yuan; Wang, Wen-Xiong

2018-06-01

Metal contamination is one of the most ubiquitous and complex problems in the Chinese coastal environment. To explore the large-scale spatial patterns of bioavailable metals, we sampled three major mussels, including 784 blue mussels (Mytilus edulis Linnaeus, 1758) of 14 sites, 224 hard-shelled mussels (Mytilus unguiculatus Valenciennes, 1858) of 4 sites, and 392 green mussels (Perna viridis (Linnaeus, 1758)) of 7 sites, ranging from temperate to tropical coastlines of China, during August and September 2015. The concentrations of macroelements (Na, K, Ca, Mg, and P) and toxic trace metals (Ag, Cd, Cr, Cu, Ni, Pb, Ti, and Zn) in the mussel's whole soft tissues were determined. Among the four Chinese coastal basins, Cd, Ti and Cr in the mussel tissues were the highest at Bohai Sea (BS) and Yellow Sea (YS), and Cu, Ni, Pb and Ag in the mussel tissues were the highest at East China Sea (ECS) and South China Sea (SCS). Zinc concentrations in mussels from YS were significantly higher than those from the other regions. Given the variability of environmental conditions such as salinity and nutrients, we further normalized the measured tissue metal concentrations with tissue Na and P levels. After Na normalization as the salinity proxy, the variability of Cd, Cu, Zn, Ag, and Ni was reduced. Trace elements accumulation in the mussel tissues was significantly related to both macroelements (Na or P) and body dry weight. The present study demonstrated that nonlinear optimization of different elements was necessary in assessing metal bioaccumulation patterns in marine mussels at a large spatial scale. Copyright © 2018 Elsevier B.V. All rights reserved.

Dual-Affinity Re-Targeting proteins direct T cell–mediated cytolysis of latently HIV-infected cells

PubMed Central

Sung, Julia A.M.; Pickeral, Joy; Liu, Liqin; Stanfield-Oakley, Sherry A.; Lam, Chia-Ying Kao; Garrido, Carolina; Pollara, Justin; LaBranche, Celia; Bonsignori, Mattia; Moody, M. Anthony; Yang, Yinhua; Parks, Robert; Archin, Nancie; Allard, Brigitte; Kirchherr, Jennifer; Kuruc, JoAnn D.; Gay, Cynthia L.; Cohen, Myron S.; Ochsenbauer, Christina; Soderberg, Kelly; Liao, Hua-Xin; Montefiori, David; Moore, Paul; Johnson, Syd; Koenig, Scott; Haynes, Barton F.; Nordstrom, Jeffrey L.; Margolis, David M.; Ferrari, Guido

2015-01-01

Enhancement of HIV-specific immunity is likely required to eliminate latent HIV infection. Here, we have developed an immunotherapeutic modality aimed to improve T cell–mediated clearance of HIV-1–infected cells. Specifically, we employed Dual-Affinity Re-Targeting (DART) proteins, which are bispecific, antibody-based molecules that can bind 2 distinct cell-surface molecules simultaneously. We designed DARTs with a monovalent HIV-1 envelope-binding (Env-binding) arm that was derived from broadly binding, antibody-dependent cellular cytotoxicity–mediating antibodies known to bind to HIV-infected target cells coupled to a monovalent CD3 binding arm designed to engage cytolytic effector T cells (referred to as HIVxCD3 DARTs). Thus, these DARTs redirected polyclonal T cells to specifically engage with and kill Env-expressing cells, including CD4+ T cells infected with different HIV-1 subtypes, thereby obviating the requirement for HIV-specific immunity. Using lymphocytes from patients on suppressive antiretroviral therapy (ART), we demonstrated that DARTs mediate CD8+ T cell clearance of CD4+ T cells that are superinfected with the HIV-1 strain JR-CSF or infected with autologous reservoir viruses isolated from HIV-infected–patient resting CD4+ T cells. Moreover, DARTs mediated CD8+ T cell clearance of HIV from resting CD4+ T cell cultures following induction of latent virus expression. Combined with HIV latency reversing agents, HIVxCD3 DARTs have the potential to be effective immunotherapeutic agents to clear latent HIV-1 reservoirs in HIV-infected individuals. PMID:26413868

Natalizumab in the treatment of Crohn’s disease

PubMed Central

Guagnozzi, Danila; Caprilli, Renzo

2008-01-01

The pathogenesis of Crohn’s disease (CD) is multifactorial and the activation of specific pathways of immunological system is important. In particular, the adhesion molecules (integrins) mediate the selective binding between the leukocytes and the endothelial cells regulating the migration of leukocytes into the normal and inflamed intestine. Selective adhesion molecule inhibitors interfere with the migration of leukocytes to the sites of inflammation by targeting adhesion molecules (α4-integrin or α4β7-integrin). Natalizumab is a humanized IgG4 anti-α4-integrin monoclonal antibody that inhibits both α4β7-integrin/mucosal addressin-cell adhesion molecule-1 (MadCAM-1) interaction and α4β1/vascular-cell adhesion molecule-1 (VCAM-1) binding. Pooled data from the four studies, analyzed in a Cochrane review, suggest that natalizumab is effective for induction of clinical response and remission in patients with moderately to severely active CD. In particular, natalizumab may be beneficial for patients with active inflammation or chronically active disease despite the use of conventional therapies with high level of C-reactive protein values at baseline time. Nevertheless, many problems about the utilization of natalizumab in CD remain unsolved (such as the high placebo response, the final definition of dosage and timing schedule, the definition of outcomes and the development of adverse events). PMID:19707360

Bloom syndrome: a mendelian prototype of somatic mutational disease.

PubMed

German, J

1993-11-01

Spontaneous mutations in human somatic cells occur far more often than normal in individuals with Bloom syndrome. The basis for understanding these mutations and their developmental consequences emerges from examination of BS at the molecular, cellular, and clinical levels. The major clinical feature of BS, proportional dwarfism, as well as its major clinical complication, an exceptionally early emergence of neoplasia of the types and sites that affect the general population, are attributable to the excessive occurrence of mutations in somatic cells. Here, the following aspects of BS are discussed: (i) the BS phenotype; (ii) neoplasia in BS, including the means--the Bloom's Syndrome Registry--by which the significant risk for diverse sites and types of cancer in these patients was revealed; (iii) the biological basis for the cancer proneness of BS; and, finally, (iv) the significance for both basic human biology and clinical medicine of BS as the prototype of somatic mutational disease.

CD, MCD and VTVH MCD Studies of Biferrous and Mixed-Valent myo-Inositol Oxygenase: Insights into Substrate Activation of O2 Reactivity

PubMed Central

Snyder, Rae Ana; Bell, Caleb B.; Diao, Yinghui; Krebs, Carsten; Bollinger, J. Martin; Solomon, Edward I.

2013-01-01

Myo-inositol oxygenase (MIOX) catalyzes the 4e− oxidation of myo-inositol (MI) to D-glucuronate using a substrate activated Fe(II)Fe(III) site. The biferrous and Fe(II)Fe(III) forms of MIOX were studied with circular dichroism (CD), magnetic circular dichroism (MCD), and variable temperature variable field (VTVH) MCD spectroscopies. The MCD spectrum of biferrous MIOX shows two ligand field (LF) transitions near 10,000 cm−1, split by ~2,000 cm−1, characteristic of 6 coordinate (6C) Fe(II) sites, indicating that the modest reactivity of the biferrous form toward O2 can be attributed to the saturated coordination of both irons. Upon oxidation to the Fe(II)Fe(III) state, MIOX shows two LF transitions in the ~10,000 cm−1 region, again implying a coordinatively saturated Fe(II) site. Upon MI binding, these split in energy to 5,200 cm−1 and 11,200 cm−1, showing that MI binding causes the Fe(II) to become coordinately unsaturated. VTVH MCD magnetization curves of unbound and MI-bound Fe(II)Fe(III) forms show that upon substrate binding, the isotherms become more nested, requiring that the exchange coupling and ferrous zero field splitting (ZFS) both decrease in magnitude. These results imply that MI binds to the ferric site, weakening the Fe(III)-μ-OH bond and strengthening the Fe(II)-μ-OH bond. This perturbation results in the release of a coordinated water from the Fe(II) that enables its O2 activation. PMID:24066857

Immunoglobulin domain interface exchange as a platform technology for the generation of Fc heterodimers and bispecific antibodies.

PubMed

Skegro, Darko; Stutz, Cian; Ollier, Romain; Svensson, Emelie; Wassmann, Paul; Bourquin, Florence; Monney, Thierry; Gn, Sunitha; Blein, Stanislas

2017-06-09

Bispecific antibodies (bsAbs) are of significant importance to the development of novel antibody-based therapies, and heavy chain (Hc) heterodimers represent a major class of bispecific drug candidates. Current technologies for the generation of Hc heterodimers are suboptimal and often suffer from contamination by homodimers posing purification challenges. Here, we introduce a new technology based on biomimicry wherein the protein-protein interfaces of two different immunoglobulin (Ig) constant domain pairs are exchanged in part or fully to design new heterodimeric domains. The method can be applied across Igs to design Fc heterodimers and bsAbs. We investigated interfaces from human IgA CH3, IgD CH3, IgG1 CH3, IgM CH4, T-cell receptor (TCR) α/β, and TCR γ/δ constant domain pairs, and we found that they successfully drive human IgG1 CH3 or IgM CH4 heterodimerization to levels similar to or above those of reference methods. A comprehensive interface exchange between the TCR α/β constant domain pair and the IgG1 CH3 homodimer was evidenced by X-ray crystallography and used to engineer examples of bsAbs for cancer therapy. Parental antibody pairs were rapidly reformatted into scalable bsAbs that were free of homodimer traces by combining interface exchange, asymmetric Protein A binding, and the scFv × Fab format. In summary, we successfully built several new CH3- or CH4-based heterodimers that may prove useful for designing new bsAb-based therapeutics, and we anticipate that our approach could be broadly implemented across the Ig constant domain family. To our knowledge, CH4-based heterodimers have not been previously reported. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Analysis of tandem E-box motifs within human Complement receptor 2 (CR2/CD21) promoter reveals cell specific roles for RP58, E2A, USF and localized chromatin accessibility.

PubMed

Cruickshank, Mark N; Dods, James; Taylor, Rhonda L; Karimi, Mahdad; Fenwick, Emily J; Quail, Elizabeth A; Rea, Alexander J; Holers, V Michael; Abraham, Lawrence J; Ulgiati, Daniela

2015-07-01

Complement receptor 2 (CR2/CD21) plays an important role in the generation of normal B cell immune responses. As transcription appears to be the prime mechanism via which surface CR2/CD21 expression is controlled, understanding transcriptional regulation of this gene will have broader implications to B cell biology. Here we report opposing, cell-context specific control of CR2/CD21 promoter activity by tandem E-box elements, spaced 22 bp apart and within 70 bp of the transcription initiation site. We have identified E2A and USF transcription factors as binding to the distal and proximal E-box sites respectively in CR2-positive B-cells, at a site that is hypersensitive to restriction enzyme digestion compared to non-expressing K562 cells. However, additional unidentified proteins have also been found to bind these functionally important elements. By utilizing a proteomics approach we have identified a repressor protein, RP58, binding the distal E-box motif. Co-transfection experiments using RP58 overexpression constructs demonstrated a specific 10-fold repression of CR2/CD21 transcriptional activity mediated through the distal E-box repressor element. Taken together, our results indicate that repression of the CR2/CD21 promoter can occur through one of the E-box motifs via recruitment of RP58 and other factors to bring about a silenced chromatin context within CR2/CD21 non-expressing cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

Single-molecule Imaging Analysis of Binding, Processive Movement, and Dissociation of Cellobiohydrolase Trichoderma reesei Cel6A and Its Domains on Crystalline Cellulose*

PubMed Central

Nakamura, Akihiko; Tasaki, Tomoyuki; Ishiwata, Daiki; Yamamoto, Mayuko; Okuni, Yasuko; Visootsat, Akasit; Maximilien, Morice; Noji, Hiroyuki; Uchiyama, Taku; Samejima, Masahiro; Igarashi, Kiyohiko; Iino, Ryota

2016-01-01

Trichoderma reesei Cel6A (TrCel6A) is a cellobiohydrolase that hydrolyzes crystalline cellulose into cellobiose. Here we directly observed the reaction cycle (binding, surface movement, and dissociation) of single-molecule intact TrCel6A, isolated catalytic domain (CD), cellulose-binding module (CBM), and CBM and linker (CBM-linker) on crystalline cellulose Iα. The CBM-linker showed a binding rate constant almost half that of intact TrCel6A, whereas those of the CD and CBM were only one-tenth of intact TrCel6A. These results indicate that the glycosylated linker region largely contributes to initial binding on crystalline cellulose. After binding, all samples showed slow and fast dissociations, likely caused by the two different bound states due to the heterogeneity of cellulose surface. The CBM showed much higher specificity to the high affinity site than to the low affinity site, whereas the CD did not, suggesting that the CBM leads the CD to the hydrophobic surface of crystalline cellulose. On the cellulose surface, intact molecules showed slow processive movements (8.8 ± 5.5 nm/s) and fast diffusional movements (30–40 nm/s), whereas the CBM-Linker, CD, and a catalytically inactive full-length mutant showed only fast diffusional movements. These results suggest that both direct binding and surface diffusion contribute to searching of the hydrolysable point of cellulose chains. The duration time constant for the processive movement was 7.7 s, and processivity was estimated as 68 ± 42. Our results reveal the role of each domain in the elementary steps of the reaction cycle and provide the first direct evidence of the processive movement of TrCel6A on crystalline cellulose. PMID:27609516

Differential Recognition Preferences of the Three Src Homology 3 (SH3) Domains from the Adaptor CD2-associated Protein (CD2AP) and Direct Association with Ras and Rab Interactor 3 (RIN3)*

PubMed Central

Rouka, Evgenia; Simister, Philip C.; Janning, Melanie; Kumbrink, Joerg; Konstantinou, Tassos; Muniz, João R. C.; Joshi, Dhira; O'Reilly, Nicola; Volkmer, Rudolf; Ritter, Brigitte; Knapp, Stefan; von Delft, Frank; Kirsch, Kathrin H.; Feller, Stephan M.

2015-01-01

CD2AP is an adaptor protein involved in membrane trafficking, with essential roles in maintaining podocyte function within the kidney glomerulus. CD2AP contains three Src homology 3 (SH3) domains that mediate multiple protein-protein interactions. However, a detailed comparison of the molecular binding preferences of each SH3 remained unexplored, as well as the discovery of novel interactors. Thus, we studied the binding properties of each SH3 domain to the known interactor Casitas B-lineage lymphoma protein (c-CBL), conducted a peptide array screen based on the recognition motif PxPxPR and identified 40 known or novel candidate binding proteins, such as RIN3, a RAB5-activating guanine nucleotide exchange factor. CD2AP SH3 domains 1 and 2 generally bound with similar characteristics and specificities, whereas the SH3-3 domain bound more weakly to most peptide ligands tested yet recognized an unusually extended sequence in ALG-2-interacting protein X (ALIX). RIN3 peptide scanning arrays revealed two CD2AP binding sites, recognized by all three SH3 domains, but SH3-3 appeared non-functional in precipitation experiments. RIN3 recruited CD2AP to RAB5a-positive early endosomes via these interaction sites. Permutation arrays and isothermal titration calorimetry data showed that the preferred binding motif is Px(P/A)xPR. Two high-resolution crystal structures (1.65 and 1.11 Å) of CD2AP SH3-1 and SH3-2 solved in complex with RIN3 epitopes 1 and 2, respectively, indicated that another extended motif is relevant in epitope 2. In conclusion, we have discovered novel interaction candidates for CD2AP and characterized subtle yet significant differences in the recognition preferences of its three SH3 domains for c-CBL, ALIX, and RIN3. PMID:26296892

HIV-1 Env trimer opens through an asymmetric intermediate in which individual protomers adopt distinct conformations.

PubMed

Ma, Xiaochu; Lu, Maolin; Gorman, Jason; Terry, Daniel S; Hong, Xinyu; Zhou, Zhou; Zhao, Hong; Altman, Roger B; Arthos, James; Blanchard, Scott C; Kwong, Peter D; Munro, James B; Mothes, Walther

2018-03-21

HIV-1 entry into cells requires binding of the viral envelope glycoprotein (Env) to receptor CD4 and coreceptor. Imaging of individual Env molecules on native virions shows Env trimers to be dynamic, spontaneously transitioning between three distinct well-populated conformational states: a pre-triggered Env (State 1), a default intermediate (State 2) and a three-CD4-bound conformation (State 3), which can be stabilized by binding of CD4 and coreceptor-surrogate antibody 17b. Here, using single-molecule Fluorescence Resonance Energy Transfer (smFRET), we show the default intermediate configuration to be asymmetric, with individual protomers adopting distinct conformations. During entry, this asymmetric intermediate forms when a single CD4 molecule engages the trimer. The trimer can then transition to State 3 by binding additional CD4 molecules and coreceptor.

Spin-orbit effects on reflectance anisotropy spectroscopy of aclean CdTe(001) surface

NASA Astrophysics Data System (ADS)

Vázquez-Nava, Raül A.

2005-03-01

The spectroscopical reflectance anisotropy (RA) response of a clean (001) surface of CdTe, which exhibits a c(2 x2) surface reconstruction, is studied using a microscopic formulation based on a semi-empirical tight binding approach (SETB) which includes the spin-orbit (SO) interaction. Following Ref. 1, we apply an unitary transformation to the usual SETB sp^3s^* basis to describe the electronic states in terms of a set of atomic states which are eigenstates of the total angular momentum (TAM). These states are better suited to treat the SO interaction in this model, and their use in the computation of the RA signal is straightforward [1]. We show how the RA changes when SO is taken into account and compare our theoretical results with experimental measurements of Ref. 2. [1] R.A. V'azquez-Nava, B.S. Mendoza and C. Castillo, Phys. Rev. B 70, 165306 (2004). [2] J. R. Molina and R. Espinosa-Luna, J. Phys. D: Appl. Phys. (2004), accepted.

Molecular interaction of 2,4-diacetylphloroglucinol (DAPG) with human serum albumin (HSA): The spectroscopic, calorimetric and computational investigation

NASA Astrophysics Data System (ADS)

Pragna Lakshmi, T.; Mondal, Moumita; Ramadas, Krishna; Natarajan, Sakthivel

2017-08-01

Drug molecule interaction with human serum albumin (HSA) affects the distribution and elimination of the drug. The compound, 2,4-diacetylphloroglucinol (DAPG) has been known for its antimicrobial, antiviral, antihelminthic and anticancer properties. However, its interaction with HSA is not yet reported. In this study, the interaction between HSA and DAPG was investigated through steady-state fluorescence, time-resolved fluorescence (TRF), circular dichroism (CD), Fourier transform infrared (FT-IR) spectroscopy, isothermal titration calorimetry (ITC), molecular docking and molecular dynamics simulation (MDS). Fluorescence spectroscopy results showed the strong quenching of intrinsic fluorescence of HSA due to interaction with DAPG, through dynamic quenching mechanism. The compound bound to HSA with reversible and moderate affinity which explained its easy diffusion from circulatory system to target tissue. The thermodynamic parameters from fluorescence spectroscopic data clearly revealed the contribution of hydrophobic forces but, the role of hydrogen bonds was not negligible according to the ITC studies. The interaction was exothermic and spontaneous in nature. Binding with DAPG reduced the helical content of protein suggesting the unfolding of HSA. Site marker fluorescence experiments revealed the change in binding constant of DAPG in the presence of site I (warfarin) but not site II marker (ibuprofen) which confirmed that the DAPG bound to site I. ITC experiments also supported this as site I marker could not bind to HSA-DAPG complex while site II marker was accommodated in the complex. In silico studies further showed the lowest binding affinity and more stability of DAPG in site I than in site II. Thus the data presented in this study confirms the binding of DAPG to the site I of HSA which may help in further understanding of pharmacokinetic properties of DAPG.

Structure of a Potential Therapeutic Antibody Bound to Interleukin-16 (IL-16)

PubMed Central

Hall, Gareth; Cullen, Eilish; Sawmynaden, Kovilen; Arnold, Joanne; Fox, Simon; Cowan, Richard; Muskett, Frederick W.; Matthews, David; Merritt, Andrew; Kettleborough, Catherine; Cruikshank, William; Taylor, Debra; Bayliss, Richard; Carr, Mark D.

2016-01-01

Interleukin-16 (IL-16) is reported to be a chemoattractant cytokine and modulator of T-cell activation, and has been proposed as a ligand for the co-receptor CD4. The secreted active form of IL-16 has been detected at sites of TH1-mediated inflammation, such as those seen in autoimmune diseases, ischemic reperfusion injury (IRI), and tissue transplant rejection. Neutralization of IL-16 recruitment to its receptor, using an anti-IL16 antibody, has been shown to significantly attenuate inflammation and disease pathology in IRI, as well as in some autoimmune diseases. The 14.1 antibody is a monoclonal anti-IL-16 antibody, which when incubated with CD4+ cells is reported to cause a reduction in the TH1-type inflammatory response. Secreted IL-16 contains a characteristic PDZ domain. PDZ domains are typically characterized by a defined globular structure, along with a peptide-binding site located in a groove between the αB and βB structural elements and a highly conserved carboxylate-binding loop. In contrast to other reported PDZ domains, the solution structure previously reported for IL-16 reveals a tryptophan residue obscuring the recognition groove. We have solved the structure of the 14.1Fab fragment in complex with IL-16, revealing that binding of the antibody requires a conformational change in the IL-16 PDZ domain. This involves the rotation of the αB-helix, accompanied movement of the peptide groove obscuring tryptophan residue, and consequent opening up of the binding site for interaction. Our study reveals a surprising mechanism of action for the antibody and identifies new opportunities for the development of IL-16-targeted therapeutics, including small molecules that mimic the interaction of the antibody. PMID:27231345

Identification of thyroid hormone receptor binding sites and target genes using ChIP-on-chip in developing mouse cerebellum.

PubMed

Dong, Hongyan; Yauk, Carole L; Rowan-Carroll, Andrea; You, Seo-Hee; Zoeller, R Thomas; Lambert, Iain; Wade, Michael G

2009-01-01

Thyroid hormone (TH) is critical to normal brain development, but the mechanisms operating in this process are poorly understood. We used chromatin immunoprecipitation to enrich regions of DNA bound to thyroid receptor beta (TRbeta) of mouse cerebellum sampled on post natal day 15. Enriched target was hybridized to promoter microarrays (ChIP-on-chip) spanning -8 kb to +2 kb of the transcription start site (TSS) of 5000 genes. We identified 91 genes with TR binding sites. Roughly half of the sites were located in introns, while 30% were located within 1 kb upstream (5') of the TSS. Of these genes, 83 with known function included genes involved in apoptosis, neurodevelopment, metabolism and signal transduction. Two genes, MBP and CD44, are known to contain TREs, providing validation of the system. This is the first report of TR binding for 81 of these genes. ChIP-on-chip results were confirmed for 10 of the 13 binding fragments using ChIP-PCR. The expression of 4 novel TH target genes was found to be correlated with TH levels in hyper/hypothyroid animals providing further support for TR binding. A TRbeta binding site upstream of the coding region of myelin associated glycoprotein was demonstrated to be TH-responsive using a luciferase expression system. Motif searches did not identify any classic binding elements, indicating that not all TR binding sites conform to variations of the classic form. These findings provide mechanistic insight into impaired neurodevelopment resulting from TH deficiency and a rich bioinformatics resource for developing a better understanding of TR binding.

Functional Differentiation of Bundle Sheath and Mesophyll Maize Chloroplasts Determined by Comparative ProteomicsW⃞

PubMed Central

Majeran, Wojciech; Cai, Yang; Sun, Qi; van Wijk, Klaas J.

2005-01-01

Chloroplasts of maize (Zea mays) leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C4 photosynthesis. Consequences for other plastid functions are not well understood but are addressed here through a quantitative comparative proteome analysis of purified M and BS chloroplast stroma. Three independent techniques were used, including cleavable stable isotope coded affinity tags. Enzymes involved in lipid biosynthesis, nitrogen import, and tetrapyrrole and isoprenoid biosynthesis are preferentially located in the M chloroplasts. By contrast, enzymes involved in starch synthesis and sulfur import preferentially accumulate in BS chloroplasts. The different soluble antioxidative systems, in particular peroxiredoxins, accumulate at higher levels in M chloroplasts. We also observed differential accumulation of proteins involved in expression of plastid-encoded proteins (e.g., EF-Tu, EF-G, and mRNA binding proteins) and thylakoid formation (VIPP1), whereas others were equally distributed. Enzymes related to the C4 shuttle, the carboxylation and regeneration phase of the Calvin cycle, and several regulators (e.g., CP12) distributed as expected. However, enzymes involved in triose phosphate reduction and triose phosphate isomerase are primarily located in the M chloroplasts, indicating that the M-localized triose phosphate shuttle should be viewed as part of the BS-localized Calvin cycle, rather than a parallel pathway. PMID:16243905

The canonical Wnt pathway regulates the metastasis-promoting mucin MUC4 in pancreatic ductal adenocarcinoma.

PubMed

Pai, Priya; Rachagani, Satyanarayana; Lakshmanan, Imayavaramban; Macha, Muzafar A; Sheinin, Yuri; Smith, Lynette M; Ponnusamy, Moorthy P; Batra, Surinder K

2016-02-01

Aberrant Wnt signaling frequently occurs in pancreatic cancer (PC) and contributes to disease progression/metastases. Likewise, the transmembrane-mucin MUC4 is expressed de novo in early pancreatic intraepithelial neoplasia (PanINs) and incrementally increases with PC progression, contributing to metastasis. To determine the mechanism of MUC4 upregulation in PC, we examined factors deregulated in early PC progression, such as Wnt/β-catenin signaling. MUC4 promoter analysis revealed the presence of three putative TCF/LEF-binding sites, leading us to hypothesize that MUC4 can be regulated by β-catenin. Immunohistochemical (IHC) analysis of rapid autopsy PC tissues showed a correlation between MUC4 and cytosolic/nuclear β-catenin expression. Knock down (KD) of β-catenin in CD18/HPAF and T3M4 cell lines resulted in decreased MUC4 transcript and protein. Three MUC4 promoter luciferase constructs, p3778, p3000, and p2700, were generated. The construct p3778, encompassing the entire MUC4 promoter, elicited increased luciferase activity in the presence of stabilized β-catenin. Mutation of the TCF/LEF site closest to the transcription start site (i.e., -2629/-2612) and furthest from the start site (i.e., -3425/-3408) reduced MUC4 promoter luciferase activity. Transfection with dominant negative TCF4 decreased MUC4 transcript and protein levels. Chromatin immunoprecipitation confirmed enrichment of β-catenin on -2629/-2612 and -3425/-3408 of the MUC4 promoter in CD18/HPAF. Functionally, CD18/HPAF and T3M4 β-catenin KD cells showed decreased migration and decreased Vimentin, N-cadherin, and pERK1/2 expression. Tumorigenicity studies in athymic nude mice showed CD18/HPAF β-catenin KD cells significantly reduced primary tumor sizes and metastases compared to scrambled control cells. We show for the first time that β-catenin directly governs MUC4 in PC. Published by Elsevier B.V.

Sequence- and Interactome-Based Prediction of Viral Protein Hotspots Targeting Host Proteins: A Case Study for HIV Nef

PubMed Central

Sarmady, Mahdi; Dampier, William; Tozeren, Aydin

2011-01-01

Virus proteins alter protein pathways of the host toward the synthesis of viral particles by breaking and making edges via binding to host proteins. In this study, we developed a computational approach to predict viral sequence hotspots for binding to host proteins based on sequences of viral and host proteins and literature-curated virus-host protein interactome data. We use a motif discovery algorithm repeatedly on collections of sequences of viral proteins and immediate binding partners of their host targets and choose only those motifs that are conserved on viral sequences and highly statistically enriched among binding partners of virus protein targeted host proteins. Our results match experimental data on binding sites of Nef to host proteins such as MAPK1, VAV1, LCK, HCK, HLA-A, CD4, FYN, and GNB2L1 with high statistical significance but is a poor predictor of Nef binding sites on highly flexible, hoop-like regions. Predicted hotspots recapture CD8 cell epitopes of HIV Nef highlighting their importance in modulating virus-host interactions. Host proteins potentially targeted or outcompeted by Nef appear crowding the T cell receptor, natural killer cell mediated cytotoxicity, and neurotrophin signaling pathways. Scanning of HIV Nef motifs on multiple alignments of hepatitis C protein NS5A produces results consistent with literature, indicating the potential value of the hotspot discovery in advancing our understanding of virus-host crosstalk. PMID:21738584

Envelope residue 375 substitutions in simian–human immunodeficiency viruses enhance CD4 binding and replication in rhesus macaques

PubMed Central

Li, Hui; Wang, Shuyi; Kong, Rui; Ding, Wenge; Lee, Fang-Hua; Parker, Zahra; Kim, Eunlim; Learn, Gerald H.; Hahn, Paul; Policicchio, Ben; Brocca-Cofano, Egidio; Deleage, Claire; Hao, Xingpei; Chuang, Gwo-Yu; Gorman, Jason; Gardner, Matthew; Lewis, Mark G.; Hatziioannou, Theodora; Santra, Sampa; Apetrei, Cristian; Pandrea, Ivona; Alam, S. Munir; Liao, Hua-Xin; Shen, Xiaoying; Tomaras, Georgia D.; Farzan, Michael; Chertova, Elena; Keele, Brandon F.; Estes, Jacob D.; Lifson, Jeffrey D.; Doms, Robert W.; Montefiori, David C.; Haynes, Barton F.; Sodroski, Joseph G.; Kwong, Peter D.; Hahn, Beatrice H.; Shaw, George M.

2016-01-01

Most simian–human immunodeficiency viruses (SHIVs) bearing envelope (Env) glycoproteins from primary HIV-1 strains fail to infect rhesus macaques (RMs). We hypothesized that inefficient Env binding to rhesus CD4 (rhCD4) limits virus entry and replication and could be enhanced by substituting naturally occurring simian immunodeficiency virus Env residues at position 375, which resides at a critical location in the CD4-binding pocket and is under strong positive evolutionary pressure across the broad spectrum of primate lentiviruses. SHIVs containing primary or transmitted/founder HIV-1 subtype A, B, C, or D Envs with genotypic variants at residue 375 were constructed and analyzed in vitro and in vivo. Bulky hydrophobic or basic amino acids substituted for serine-375 enhanced Env affinity for rhCD4, virus entry into cells bearing rhCD4, and virus replication in primary rhCD4 T cells without appreciably affecting antigenicity or antibody-mediated neutralization sensitivity. Twenty-four RMs inoculated with subtype A, B, C, or D SHIVs all became productively infected with different Env375 variants—S, M, Y, H, W, or F—that were differentially selected in different Env backbones. Notably, SHIVs replicated persistently at titers comparable to HIV-1 in humans and elicited autologous neutralizing antibody responses typical of HIV-1. Seven animals succumbed to AIDS. These findings identify Env–rhCD4 binding as a critical determinant for productive SHIV infection in RMs and validate a novel and generalizable strategy for constructing SHIVs with Env glycoproteins of interest, including those that in humans elicit broadly neutralizing antibodies or bind particular Ig germ-line B-cell receptors. PMID:27247400

Local Conformational Stability of HIV-1 gp120 in Unliganded and CD4-Bound States as Defined by Amide Hydrogen/Deuterium Exchange▿ †

PubMed Central

Kong, Leopold; Huang, Chih-chin; Coales, Stephen J.; Molnar, Kathleen S.; Skinner, Jeff; Hamuro, Yoshitomo; Kwong, Peter D.

2010-01-01

The binding reaction of the HIV-1 gp120 envelope glycoprotein to the CD4 receptor involves exceptional changes in enthalpy and entropy. Crystal structures of gp120 in unliganded and various ligand-bound states, meanwhile, reveal an inner domain able to fold into diverse conformations, a structurally invariant outer domain, and, in the CD4-bound state, a bridging sheet minidomain. These studies, however, provide only hints as to the flexibility of each state. Here we use amide hydrogen/deuterium exchange coupled to mass spectrometry to provide quantifications of local conformational stability for HIV-1 gp120 in unliganded and CD4-bound states. On average, unliganded core gp120 displayed >10,000-fold slower exchange of backbone-amide hydrogens than a theoretically unstructured protein of the same composition, with binding by CD4 reducing the rate of gp120 amide exchange a further 10-fold. For the structurally constant CD4, alterations in exchange correlated well with alterations in binding surface (P value = 0.0004). For the structurally variable gp120, however, reductions in flexibility extended outside the binding surface, and regions of expected high structural diversity (inner domain/bridging sheet) displayed roughly 20-fold more rapid exchange in the unliganded state than regions of low diversity (outer domain). Thus, despite an extraordinary reduction in entropy, neither unliganded gp120 nor free CD4 was substantially unstructured, suggesting that most of the diverse conformations that make up the gp120 unliganded state are reasonably ordered. The results provide a framework for understanding how local conformational stability influences entropic change, conformational diversity, and structural rearrangements in the gp120-CD4 binding reaction. PMID:20660185

Human amniotic epithelial cells inhibit CD4+ T cell activation in acute kidney injury patients by influencing the miR-101-c-Rel-IL-2 pathway.

PubMed

Liu, Junfeng; Hua, Rong; Gong, Zhangbin; Shang, Bin; Huang, Yongyi; Guo, Lihe; Liu, Te; Xue, Jun

2017-01-01

In the pathogenesis of acute kidney injury (AKI), the release of multiple interleukins can lead to increased kidney damage. Human amniotic epithelial cells (HuAECs) can inhibit immune cell activation in vivo and in vitro. We hypothesized that HuAECs could weaken patient-derived peripheral blood CD4+ T-cell activation and decreasing the ability of these cells to express and release IL-2. -Cell proliferation assay revealed that under the same culture conditions, activated AKI patient-derived CD4+ T cells had a significantly reduced proliferation rate when were co-cultured with HuAECs. And the level of IL-2 released was also significantly reduced. Western blot and qRT-PCR assays showed that the expression of c-Rel in the CD4+ T cells was also significantly reduced. However, the expression level of endogenous miR-101 in the CD4+ T cells co-cultured with HuAECs was significantly increased. Luciferase reporter assay results suggested that miR-101 could bind to a specific site in the c-Rel 3' UTR and induce the post-transcriptional silencing of c-Rel. Subsequently, we over-expressed miR-101 in AKI patient-derived CD4+ T cells. The qRT-PCR and western blot assay results revealed that the expression of endogenous c-Rel was significantly reduced, while the ELISA results indicated that the level of IL-2 released was also significantly decreased. Finally, ChIP-PCR assay results showed that the miR-101-overexpressing CD4+ T-cell group and the HuAEC co-culture CD4+ T-cell group exhibited significantly decreased binding capacities between the 'c-Rel-NFκB' complex and the IL-2 gene promoter, and the transcriptional activity of IL-2 was also significantly decreased. Therefore, we confirmed that HuAECs can stimulate miR-101 expression in AKI patient-derived peripheral blood CD4+ T cells, thus inhibiting the expression of the miR-101 target gene c-Rel and leading to a reduction in IL-2 expression and release. Copyright © 2016 Elsevier Ltd. All rights reserved.

Dominant-negative effect of hetero-oligomerization on the function of the human immunodeficiency virus type 1 envelope glycoprotein complex

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

Herrera, Carolina; Klasse, Per Johan; Kibler, Christopher W.

2006-07-20

The human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein forms trimers that mediate interactions with the CD4 receptor and a co-receptor on the target cell surface, thereby triggering viral fusion with the cell membrane. Cleavage of Env into its surface, gp120, and transmembrane, gp41, moieties is necessary for activation of its fusogenicity. Here, we produced pseudoviruses with phenotypically mixed wild-type (Wt) and mutant, cleavage-incompetent Env in order to quantify the effects of incorporating uncleaved Env on virion infectivity, antigenicity and neutralization sensitivity. We modeled the relative infectivity of three such phenotypically mixed viral strains, JR-FL, HXBc2 and a derivativemore » of the latter, 3.2P, as a function of the relative amount of Wt Env. The data were fit very closely (R {sup 2} > 0.99) by models which assumed that only Wt homotrimers were functional, with different approximate thresholds of critical numbers of functional trimers per virion for the three strains. We also produced 3.2P pseudoviruses containing both a cleavage-competent Env that is defective for binding the neutralizing monoclonal antibody (NAb) 2G12, and a cleavage-incompetent Env that binds 2G12. The 2G12 NAb was not able to reduce the infectivity of these pseudoviruses detectably. Their neutralization by the CD4-binding site-directed agents CD4-IgG2 and NAb b12 was also unaffected by 2G12 binding to uncleaved Env. These results further strengthen the conclusion that only homotrimers consisting of cleaved Env are functional. They also imply that the function of a trimer is unaffected sterically by the binding of an antibody to an adjacent trimer.« less

Study on the binding of procaine hydrochloride to DNA/DNA bases and the effect of CdS nanoparticles on the binding behavior.

PubMed

Ping, Gang; Lv, Gang; Gutmann, Sebastian; Chen, Chen; Zhang, Renyun; Wang, Xuemei

2006-01-01

The interaction between procaine hydrochloride and DNA/DNA bases in the absence and presence of cadmium sulfide (CdS) nanoparticles has been explored in this study by using differential pulse voltammetry, atomic force microscopy (AFM) and so on, which illustrates the different binding behaviors of procaine hydrochloride with different DNA bases. The results clearly indicate that the binding of purines to procaine hydrochloride is stronger than that of pyrimidines and the binding affinity is in the order of G > A > T > C. In addition, it was observed that the presence of CdS nanoparticles could remarkably enhance the probing sensitivity for the interaction between procaine hydrochloride and DNA/DNA bases. Furthermore, AFM study illustrates that procaine hydrochloride can bind to some specific sites of DNA chains, which indicates that procaine hydrochloride may interact with some special sequences of DNA.

Expression of the Bs2 pepper gene confers resistance to bacterial spot disease in tomato

PubMed Central

Tai, Thomas H.; Dahlbeck, Douglas; Clark, Eszter T.; Gajiwala, Paresh; Pasion, Romela; Whalen, Maureen C.; Stall, Robert E.; Staskawicz, Brian J.

1999-01-01

The Bs2 resistance gene of pepper specifically recognizes and confers resistance to strains of Xanthomonas campestris pv. vesicatoria that contain the corresponding bacterial avirulence gene, avrBs2. The involvement of avrBs2 in pathogen fitness and its prevalence in many X. campestris pathovars suggests that the Bs2 gene may be durable in the field and provide resistance when introduced into other plant species. Employing a positional cloning strategy, the Bs2 locus was isolated and the gene was identified by coexpression with avrBs2 in an Agrobacterium-mediated transient assay. A single candidate gene, predicted to encode motifs characteristic of the nucleotide binding site–leucine-rich repeat class of resistance genes, was identified. This gene specifically controlled the hypersensitive response when transiently expressed in susceptible pepper and tomato lines and in a nonhost species, Nicotiana benthamiana, and was designated as Bs2. Functional expression of Bs2 in stable transgenic tomatoes supports its use as a source of resistance in other Solanaceous plant species. PMID:10570214

Separating the Role of Protein Restraints and Local Metal-Site Interaction Chemistry in the Thermodynamics of a Zinc Finger Protein

PubMed Central

Dixit, Purushottam D.; Asthagiri, D.

2011-01-01

We express the effective Hamiltonian of an ion-binding site in a protein as a combination of the Hamiltonian of the ion-bound site in vacuum and the restraints of the protein on the site. The protein restraints are described by the quadratic elastic network model. The Hamiltonian of the ion-bound site in vacuum is approximated as a generalized Hessian around the minimum energy configuration. The resultant of the two quadratic Hamiltonians is cast into a pure quadratic form. In the canonical ensemble, the quadratic nature of the resultant Hamiltonian allows us to express analytically the excess free energy, enthalpy, and entropy of ion binding to the protein. The analytical expressions allow us to separate the roles of the dynamic restraints imposed by the protein on the binding site and the temperature-independent chemical effects in metal-ligand coordination. For the consensus zinc-finger peptide, relative to the aqueous phase, the calculated free energy of exchanging Zn2+ with Fe2+, Co2+, Ni2+, and Cd2+ are in agreement with experiments. The predicted excess enthalpy of ion exchange between Zn2+ and Co2+ also agrees with the available experimental estimate. The free energy of applying the protein restraints reveals that relative to Zn2+, the Co2+, and Cd2+-site clusters are more destabilized by the protein restraints. This leads to an experimentally testable hypothesis that a tetrahedral metal binding site with minimal protein restraints will be less selective for Zn2+ over Co2+ and Cd2+ compared to a zinc finger peptide. No appreciable change is expected for Fe2+ and Ni2+. The framework presented here may prove useful in protein engineering to tune metal selectivity. PMID:21943427

Structural and functional characterization of the interaction between cyclophilin B and a heparin-derived oligosaccharide.

PubMed

Hanoulle, Xavier; Melchior, Aurélie; Sibille, Nathalie; Parent, Benjamin; Denys, Agnès; Wieruszeski, Jean-Michel; Horvath, Dragos; Allain, Fabrice; Lippens, Guy; Landrieu, Isabelle

2007-11-23

The chemotaxis and integrin-mediated adhesion of T lymphocytes triggered by secreted cyclophilin B (CypB) depend on interactions with both cell surface heparan sulfate proteoglycans (HSPG) and the extracellular domain of the CD147 membrane receptor. Here, we use NMR spectroscopy to characterize the interaction of CypB with heparin-derived oligosaccharides. Chemical shift perturbation experiments allowed the precise definition of the heparan sulfate (HS) binding site of CypB. The N-terminal extremity of CypB, which contains a consensus sequence for heparin-binding proteins was modeled on the basis of our experimental NMR data. Because the HS binding site extends toward the CypB catalytic pocket, we measured its peptidyl-prolyl cis-trans isomerase (PPIase) activity in the absence or presence of a HS oligosaccharide toward a CD147-derived peptide. We report the first direct evidence that CypB is enzymatically active on CD147, as it is able to accelerate the cis/trans isomerization of the Asp(179)-Pro(180) bond in a CD147-derived peptide. However, HS binding has no significant influence on this PPIase activity. We thus conclude that the glycanic moiety of HSPG serves as anchor for CypB at the cell surface, and that the signal could be transduced by CypB via its PPIase activity toward CD147.

Revealing interaction between sulfobutylether-β-cyclodextrin and reserpine by chemiluminescence and site-directed molecular docking.

PubMed

Xiong, Xunyu; Wu, Min; Zhao, Xinfeng; Song, Zhenghua

2014-09-01

The host-guest interaction between sulfobutylether-β-cyclodextrin (SBE-β-CD) and reserpine (RSP) is described using flow injection-chemiluminescence (FI-CL) and site-directed molecular docking methods. It was found that RSP could inhibit the CL intensity produced by a luminol/SBE-β-CD system. The decrease in CL intensity was logarithmic over an RSP concentration range of 0.03 to 700.0 nM, giving a regression equation of ∆I = 107.1lgCRES  + 186.1 with a detection limit of 10 pM (3σ). The CL assay was successfully applied in the determination of RSP in injection, saliva and urine samples with recoveries in the range 93.5-106.1%. Using the proposed CL model, the binding constant (KCD-R ) and the stoichiometric ratio of SBE-β-CD/RSP were calculated to be 7.4 × 10(6)  M(-1) and 1 : 1, respectively. Using molecular docking, it was confirmed that luminol binds to the small cavity of SBE-β-CD with a nonpolar interaction, while RSP targeted the larger cavity of SBE-β-CD and formed a 1 : 1 complex with hydrogen bonds. The proposed new CL method has the potential to become a powerful tool for revealing the host-guest interaction between CDs and drugs, as well as monitoring drugs with high sensitivity. Copyright © 2013 John Wiley & Sons, Ltd.

Engineering antigens for in situ erythrocyte binding induces T-cell deletion.

PubMed

Kontos, Stephan; Kourtis, Iraklis C; Dane, Karen Y; Hubbell, Jeffrey A

2013-01-02

Antigens derived from apoptotic cell debris can drive clonal T-cell deletion or anergy, and antigens chemically coupled ex vivo to apoptotic cell surfaces have been shown correspondingly to induce tolerance on infusion. Reasoning that a large number of erythrocytes become apoptotic (eryptotic) and are cleared each day, we engineered two different antigen constructs to target the antigen to erythrocyte cell surfaces after i.v. injection, one using a conjugate with an erythrocyte-binding peptide and another using a fusion with an antibody fragment, both targeting the erythrocyte-specific cell surface marker glycophorin A. Here, we show that erythrocyte-binding antigen is collected much more efficiently than free antigen by splenic and hepatic immune cell populations and hepatocytes, and that it induces antigen-specific deletional responses in CD4(+) and CD8(+) T cells. We further validated T-cell deletion driven by erythrocyte-binding antigens using a transgenic islet β cell-reactive CD4(+) T-cell adoptive transfer model of autoimmune type 1 diabetes: Treatment with the peptide antigen fused to an erythrocyte-binding antibody fragment completely prevented diabetes onset induced by the activated, autoreactive CD4(+) T cells. Thus, we report a translatable modular biomolecular approach with which to engineer antigens for targeted binding to erythrocyte cell surfaces to induce antigen-specific CD4(+) and CD8(+) T-cell deletion toward exogenous antigens and autoantigens.

Interactions of divalent cations with calcium binding sites of BK channels reveal independent motions within the gating ring.

PubMed

Miranda, Pablo; Giraldez, Teresa; Holmgren, Miguel

2016-12-06

Large-conductance voltage- and calcium-activated K + (BK) channels are key physiological players in muscle, nerve, and endocrine function by integrating intracellular Ca 2+ and membrane voltage signals. The open probability of BK channels is regulated by the intracellular concentration of divalent cations sensed by a large structure in the BK channel called the "gating ring," which is formed by four tandems of regulator of conductance for K + (RCK1 and RCK2) domains. In contrast to Ca 2+ that binds to both RCK domains, Mg 2+ , Cd 2+ , or Ba 2+ interact preferentially with either one or the other. Interaction of cations with their binding sites causes molecular rearrangements of the gating ring, but how these motions occur remains elusive. We have assessed the separate contributions of each RCK domain to the cation-induced gating-ring structural rearrangements, using patch-clamp fluorometry. Here we show that Mg 2+ and Ba 2+ selectively induce structural movement of the RCK2 domain, whereas Cd 2+ causes motions of RCK1, in all cases substantially smaller than those elicited by Ca 2+ By combining divalent species interacting with unique sites, we demonstrate that RCK1 and RCK2 domains move independently when their specific binding sites are occupied. Moreover, binding of chemically distinct cations to both RCK domains is additive, emulating the effect of fully occupied Ca 2+ binding sites.

Functional Interaction of CD154 Protein with α5β1 Integrin Is Totally Independent from Its Binding to αIIbβ3 Integrin and CD40 Molecules*

PubMed Central

El Fakhry, Youssef; Alturaihi, Haydar; Yacoub, Daniel; Liu, Lihui; Guo, Wenyan; Leveillé, Claire; Jung, Daniel; Khzam, Lara Bou; Merhi, Yahye; Wilkins, John A.; Li, Hongmin; Mourad, Walid

2012-01-01

In addition to its classical CD40 receptor, CD154 also binds to αIIbβ3, α5β1, and αMβ2 integrins. Binding of CD154 to these receptors seems to play a key role in the pathogenic processes of chronic inflammation. This investigation was aimed at analyzing the functional interaction of CD154 with CD40, αIIbβ3, and α5β1 receptors. We found that the binding affinity of CD154 for αIIbβ3 is ∼4-fold higher than for α5β1. We also describe the generation of sCD154 mutants that lost their ability to bind CD40 or αIIbβ3 and show that CD154 residues involved in its binding to CD40 or αIIbβ3 are distinct from those implicated in its interaction to α5β1, suggesting that sCD154 may bind simultaneously to different receptors. Indeed, sCD154 can bind simultaneously to CD40 and α5β1 and biologically activate human monocytic U937 cells expressing both receptors. The simultaneous engagement of CD40 and α5β1 activates the mitogen-activated protein kinases, p38, and extracellular signal-related kinases 1/2 and synergizes in the release of inflammatory mediators MMP-2 and -9, suggesting a cross-talk between these receptors. PMID:22461623

HIV envelope-mediated, CCR5/α4β7-dependent killing of CD4-negative γδ T cells which are lost during progression to AIDS.

PubMed

Li, Haishan; Pauza, C David

2011-11-24

HIV infects and replicates in CD4+ T cells but effects on host immunity and disease also involve depletion, hyper-activation, and modification of CD4-negative cell populations. In particular, the depletion of CD4-negative γδ T cells is common to all HIV+ individuals. We found that soluble or cell-associated envelope glycoproteins from CCR5-tropic strains of HIV could bind, activates the p38-caspase pathway, and induce the death of γδ cells. Envelope binding requires integrin α4β7 and chemokine receptor CCR5 which are at high levels and form a complex on the γδ T cell membrane. This receptor complex facilitated V3 loop binding to CCR5 in the absence of CD4-induced conformational changes. Cell death was increased by antigen stimulation after exposure to envelope glycoprotein. Direct signaling by envelope glycoprotein killed CD4-negative γδ T cells and reproduced a defect observed in all patients with HIV disease.

Lipopolysaccharide (LPS)-binding protein stimulates CD14-dependent Toll-like receptor 4 internalization and LPS-induced TBK1-IKKϵ-IRF3 axis activation.

PubMed

Tsukamoto, Hiroki; Takeuchi, Shino; Kubota, Kanae; Kobayashi, Yohei; Kozakai, Sao; Ukai, Ippo; Shichiku, Ayumi; Okubo, Misaki; Numasaki, Muneo; Kanemitsu, Yoshitomi; Matsumoto, Yotaro; Nochi, Tomonori; Watanabe, Kouichi; Aso, Hisashi; Tomioka, Yoshihisa

2018-05-14

Toll-like receptor 4 (TLR4) is an indispensable immune receptor for lipopolysaccharide (LPS), a major component of the Gram-negative bacterial cell wall. Following LPS stimulation, TLR4 transmits the signal from the cell surface and becomes internalized in an endosome. However, the spatial regulation of TLR4 signaling is not fully understood. Here, we investigated the mechanisms of LPS-induced TLR4 internalization and clarified the roles of the extracellular LPS-binding molecules, LPS-binding protein (LBP), and glycerophosphatidylinositol-anchored protein (CD14). LPS stimulation of CD14-expressing cells induced TLR4 internalization in the presence of serum, and an inhibitory anti-LBP mAb blocked its internalization. Addition of LBP to serum-free cultures restored LPS-induced TLR4 internalization to comparable levels of serum. The secretory form of the CD14 (sCD14) induced internalization but required a much higher concentration than LBP. An inhibitory anti-sCD14 mAb was ineffective for serum-mediated internalization. LBP lacking the domain for LPS transfer to CD14 and a CD14 mutant with reduced LPS binding both attenuated TLR4 internalization. Accordingly, LBP is an essential serum molecule for TLR4 internalization, and its LPS transfer to membrane-anchored CD14 (mCD14) is a prerequisite. LBP induced the LPS-stimulated phosphorylation of TBK1, IKKϵ, and IRF3, leading to IFN-β expression. However, LPS-stimulated late activation of NFκB or necroptosis were not affected. Collectively, our results indicate that LBP controls LPS-induced TLR4 internalization, which induces TLR adaptor molecule 1 (TRIF)-dependent activation of the TBK1-IKKϵ-IRF3-IFN-β pathway. In summary, we showed that LBP-mediated LPS transfer to mCD14 is required for serum-dependent TLR4 internalization and activation of the TRIF pathway. Copyright © 2018, The American Society for Biochemistry and Molecular Biology.

Characterization of the binding of metoprolol tartrate and guaifenesin drugs to human serum albumin and human hemoglobin proteins by fluorescence and circular dichroism spectroscopy.

PubMed

Duman, Osman; Tunç, Sibel; Kancı Bozoğlan, Bahar

2013-07-01

The interactions of metoprolol tartrate (MPT) and guaifenesin (GF) drugs with human serum albumin (HSA) and human hemoglobin (HMG) proteins at pH 7.4 were studied by fluorescence and circular dichroism (CD) spectroscopy. Drugs quenched the fluorescence spectra of HSA and HMG proteins through a static quenching mechanism. For each protein-drug system, the values of Stern-Volmer quenching constant, bimolecular quenching constant, binding constant and number of binding site on the protein molecules were determined at 288.15, 298.15, 310.15 and 318.15 K. It was found that the binding constants of HSA-MPT and HSA-GF systems were smaller than those of HMG-MPT and HMG-GF systems. For both drugs, the affinity of HMG was much higher than that of HSA. An increase in temperature caused a negative effect on the binding reactions. The number of binding site on blood proteins for MPT and GF drugs was approximately one. Thermodynamic parameters showed that MPT interacted with HSA through electrostatic attraction forces. However, hydrogen bonds and van der Waals forces were the main interaction forces in the formation of HSA-GF, HMG-MPT and HMG-GF complexes. The binding processes between protein and drug molecules were exothermic and spontaneous owing to negative ∆H and ∆G values, respectively. The values of binding distance between protein and drug molecules were calculated from Förster resonance energy transfer theory. It was found from CD analysis that the bindings of MPT and GF drugs to HSA and HMG proteins altered the secondary structure of HSA and HMG proteins.

An IFNG SNP with an estrogen-like response element selectively enhances promoter expression in peripheral but not lamina propria T cells.

PubMed

Gonsky, R; Deem, R L; Bream, J H; Young, H A; Targan, S R

2006-07-01

This study examines mucosa-specific regulatory pathways involved in modulation of interferon-gamma (IFN-gamma) in lamina propria T cells. Previous studies identified mucosa-specific CD2 cis-elements within the -204 to -108 bp IFNG promoter. Within this region, a single-site nucleotide polymorphism, -179G/T, imparts tumor necrosis factor-alpha stimulation of IFNG in peripheral blood lymphocytes, and is linked with accelerated AIDS progression. We discovered a putative estrogen response element (ERE) introduced by the -179T, which displays selective activation in peripheral blood mononuclear cells (PBMC) vs lamina propria mononuclear cells (LPMC). Transfection of PBMC with constructs containing the -179G or -179T site revealed CD2-mediated enhancement of the -179T compared to -179G allele, although, in LPMC, a similar level of expression was detected. Electrophoretic mobility shift assay (EMSA) analysis demonstrated CD2-mediated nucleoprotein binding to the -179T but not the -179G in PBMC. In LPMC, binding is constitutive to both -179G and -179T regions. Sequence and EMSA analysis suggests that the -179T allele creates an ERE-like binding site capable of binding recombinant estrogen receptor. Estrogen response element transactivation is enhanced by CD2 signaling, but inhibited by estrogen in PBMC but not in LPMC, although expression of estrogen receptor was similar. This is the first report to describe a potential molecular mechanism responsible for selectively controlling IFN-gamma production in LPMC.

In vitro DNA binding studies of Aspartame, an artificial sweetener.

PubMed

Kashanian, Soheila; Khodaei, Mohammad Mehdi; Kheirdoosh, Fahimeh

2013-03-05

A number of small molecules bind directly and selectively to DNA, by inhibiting replication, transcription or topoisomerase activity. In this work the interaction of native calf thymus DNA (CT-DNA) with Aspartame (APM), an artificial sweeteners was studied at physiological pH. DNA binding study of APM is useful to understand APM-DNA interaction mechanism and to provide guidance for the application and design of new and safer artificial sweeteners. The interaction was investigated using spectrophotometric, spectrofluorometric competition experiment and circular dichroism (CD). Hypochromism and red shift are shown in UV absorption band of APM. A strong fluorescence quenching reaction of DNA to APM was observed and the binding constants (Kf) of DNA with APM and corresponding number of binding sites (n) were calculated at different temperatures. Thermodynamic parameters, enthalpy changes (ΔH) and entropy changes (ΔS) were calculated to be +181kJmol(-1) and +681Jmol(-1)K(-1) according to Van't Hoff equation, which indicated that reaction is predominantly entropically driven. Moreover, spectrofluorometric competition experiment and circular dichroism (CD) results are indicative of non-intercalative DNA binding nature of APM. We suggest that APM interacts with calf thymus DNA via groove binding mode with an intrinsic binding constant of 5×10(+4)M(-1). Copyright © 2013 Elsevier B.V. All rights reserved.

Primary porcine Kupffer cell phagocytosis of human platelets involves the CD18 receptor.

PubMed

Chihara, Ray K; Paris, Leela L; Reyes, Luz M; Sidner, Richard A; Estrada, Jose L; Downey, Susan M; Wang, Zheng-Yu; Tector, A Joseph; Burlak, Christopher

2011-10-15

Hepatic failure has been treated successfully with clinical extracorporeal perfusions of porcine livers. However, dog-to-pig and pig-to-baboon liver xenotransplant models have resulted in severe bleeding secondary to liver xenograft-induced thrombocytopenia. Kupffer cells (KC) are abundant phagocytic cells in the liver. KC express the CD11b/CD18 receptor, which has been implicated in chilled platelet binding and phagocytosis through interaction with platelet surface proteins and carbohydrates. We sought to identify the role of KC CD18 in liver xenograft-induced thrombocytopenia. Primary pig KC were characterized by flow cytometry, immunoblots, and quantitative polymerase chain reaction. Pig KC were used in inhibition assays with fluorescently labeled human platelets. The CD18 receptor was targeted for siRNA knockdown. Domestic and α1,3-galactosyltransferase double knockout porcine KC cultures were approximately 92% positive for CD18 as detected by quantitative polymerase chain reaction and flow cytometry. Use of CD18 blocking antibodies resulted in reduction of human platelet binding and phagocytosis. Additionally, asialofetuin, not fetuin, inhibited platelet phagocytosis suggesting the involvement of an oligosaccharide-binding site. Furthermore, reduced CD18 expression by siRNA resulted in decreased human platelet binding. Our data suggest that primary pig KC bind and phagocytose human platelets with involvement of CD18. Further understanding and modification of CD18 expression in pigs may result in a liver xenograft with reduced thrombocytopenic effects, which could be used as a bridge to allogeneic liver transplantation.

Dissection of TALE-dependent gene activation reveals that they induce transcription cooperatively and in both orientations

PubMed Central

Streubel, Jana; Baum, Heidi; Grau, Jan; Stuttman, Johannes; Boch, Jens

2017-01-01

Plant-pathogenic Xanthomonas bacteria inject transcription activator-like effector proteins (TALEs) into host cells to specifically induce transcription of plant genes and enhance susceptibility. Although the DNA-binding mode is well-understood it is still ambiguous how TALEs initiate transcription and whether additional promoter elements are needed to support this. To systematically dissect prerequisites for transcriptional initiation the activity of one TALE was compared on different synthetic Bs4 promoter fragments. In addition, a large collection of artificial TALEs spanning the OsSWEET14 promoter was compared. We show that the presence of a TALE alone is not sufficient to initiate transcription suggesting the requirement of additional supporting promoter elements. At the OsSWEET14 promoter TALEs can initiate transcription from various positions, in a synergistic manner of multiple TALEs binding in parallel to the promoter, and even by binding in reverse orientation. TALEs are known to shift the transcriptional start site, but our data show that this shift depends on the individual position of a TALE within a promoter context. Our results implicate that TALEs function like classical enhancer-binding proteins and initiate transcription in both orientations which has consequences for in planta target gene prediction and design of artificial activators. PMID:28301511

Dissection of TALE-dependent gene activation reveals that they induce transcription cooperatively and in both orientations.

PubMed

Streubel, Jana; Baum, Heidi; Grau, Jan; Stuttman, Johannes; Boch, Jens

2017-01-01

Plant-pathogenic Xanthomonas bacteria inject transcription activator-like effector proteins (TALEs) into host cells to specifically induce transcription of plant genes and enhance susceptibility. Although the DNA-binding mode is well-understood it is still ambiguous how TALEs initiate transcription and whether additional promoter elements are needed to support this. To systematically dissect prerequisites for transcriptional initiation the activity of one TALE was compared on different synthetic Bs4 promoter fragments. In addition, a large collection of artificial TALEs spanning the OsSWEET14 promoter was compared. We show that the presence of a TALE alone is not sufficient to initiate transcription suggesting the requirement of additional supporting promoter elements. At the OsSWEET14 promoter TALEs can initiate transcription from various positions, in a synergistic manner of multiple TALEs binding in parallel to the promoter, and even by binding in reverse orientation. TALEs are known to shift the transcriptional start site, but our data show that this shift depends on the individual position of a TALE within a promoter context. Our results implicate that TALEs function like classical enhancer-binding proteins and initiate transcription in both orientations which has consequences for in planta target gene prediction and design of artificial activators.

The structure of cytomegalovirus immune modulator UL141 highlights structural Ig-fold versatility for receptor binding

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

Nemčovičová, Ivana; Slovak Academy of Sciences, Dúbravská cesta 9, SK 84505 Bratislava; Zajonc, Dirk M., E-mail: dzajonc@liai.org

2014-03-01

The crystal structure of Human cytomegalovirus immune modulator UL141 was solved at 3.25 Å resolution. Here, a detailed analysis of its intimate dimerization interface and the biophysical properties of its receptor (TRAIL-R2 and CD155) binding interactions are presented. Natural killer (NK) cells are critical components of the innate immune system as they rapidly detect and destroy infected cells. To avoid immune recognition and to allow long-term persistence in the host, Human cytomegalovirus (HCMV) has evolved a number of genes to evade or inhibit immune effector pathways. In particular, UL141 can inhibit cell-surface expression of both the NK cell-activating ligand CD155more » as well as the TRAIL death receptors (TRAIL-R1 and TRAIL-R2). The crystal structure of unliganded HCMV UL141 refined to 3.25 Å resolution allowed analysis of its head-to-tail dimerization interface. A ‘dimerization-deficient’ mutant of UL141 (ddUL141) was further designed, which retained the ability to bind to TRAIL-R2 or CD155 while losing the ability to cross-link two receptor monomers. Structural comparison of unliganded UL141 with UL141 bound to TRAIL-R2 further identified a mobile loop that makes intimate contacts with TRAIL-R2 upon receptor engagement. Superposition of the Ig-like domain of UL141 on the CD155 ligand T-cell immunoreceptor with Ig and ITIM domains (TIGIT) revealed that UL141 can potentially engage CD155 similar to TIGIT by using the C′C′′ and GF loops. Further mutations in the TIGIT binding site of CD155 (Q63R and F128R) abrogated UL141 binding, suggesting that the Ig-like domain of UL141 is a viral mimic of TIGIT, as it targets the same binding site on CD155 using similar ‘lock-and-key’ interactions. Sequence alignment of the UL141 gene and its orthologues also showed conservation in this highly hydrophobic (L/A)X{sub 6}G ‘lock’ motif for CD155 binding as well as conservation of the TRAIL-R2 binding patches, suggesting that these host–receptor interactions are evolutionary conserved.« less

Binding of CD40L to Mac-1’s I-domain involves the EQLKKSKTL motif and mediates leukocyte recruitment and atherosclerosis – but does not affect immunity and thrombosis in mice

PubMed Central

Wolf, Dennis; Hohmann, Jan-David; Wiedemann, Ansgar; Bledzka, Kamila; Blankenbach, Hermann; Marchini, Timoteo; Gutte, Katharina; Zeschky, Katharina; Bassler, Nicole; Hoppe, Natalie; Rodriguez, Alexandra Ortiz; Herr, Nadine; Hilgendorf, Ingo; Stachon, Peter; Willecke, Florian; Dürschmied, Daniel; von zur Mühlen, Constantin; Soloviev, Dmitry A.; Zhang, Li; Bode, Christoph; Plow, Edward F.; Libby, Peter; Peter, Karlheinz; Zirlik, Andreas

2012-01-01

Rationale CD40L figures prominently in chronic inflammatory diseases such as atherosclerosis. However, since CD40L potently regulates immune function and haemostasis by interaction with CD40 receptor and the platelet integrin GPIIb/IIIa, its global inhibition compromises host defense and generated thromboembolic complications in clinical trials. We recently reported that CD40L mediates atherogenesis independently of CD40 and proposed Mac-1 as an alternate receptor. Objective Here, we molecularly characterized the CD40L-Mac-1 interaction and tested whether its selective inhibition by a small peptide modulates inflammation and atherogenesis in vivo. Methods and Results CD40L concentration-dependently bound to Mac-1 I-domain in solid phase binding assays, and a high affinity interaction was revealed by surface-plasmon-resonance analysis. We identified the motif EQLKKSKTL, an exposed loop between the α1 helix and the β-sheet B, on Mac-1 as binding site for CD40L. A linear peptide mimicking this sequence, M7, specifically inhibited the interaction of CD40L and Mac-1. cM7, a cyclisized version optimized for in vivo use, decreased peritoneal inflammation and inflammatory cell recruitment in vivo. Finally, LDLr-/- mice treated with intraperitoneal injections of cM7 developed smaller, less inflamed atherosclerotic lesions featuring characteristics of stability. However, cM7 did not interfere with CD40L-CD40 binding in vitro and CD40L-GPIIb/IIIa-mediated thrombus formation in vivo. Conclusions We present the novel finding that CD40L binds to the EQLKKSKTL motif on Mac-1 mediating leukocyte recruitment and atherogenesis. Specific inhibition of CD40L-Mac-1 binding may represent an attractive anti-inflammatory treatment strategy for atherosclerosis and other inflammatory conditions, potentially avoiding the unwanted immunologic and thrombotic effects of global inhibition of CD40L. PMID:21998326

CD and MCD studies of the effects of component B variant binding on the biferrous active site of methane monooxygenase.

PubMed

Mitić, Natasa; Schwartz, Jennifer K; Brazeau, Brian J; Lipscomb, John D; Solomon, Edward I

2008-08-12

The multicomponent soluble form of methane monooxygenase (sMMO) catalyzes the oxidation of methane through the activation of O 2 at a nonheme biferrous center in the hydroxylase component, MMOH. Reactivity is limited without binding of the sMMO effector protein, MMOB. Past studies show that mutations of specific MMOB surface residues cause large changes in the rates of individual steps in the MMOH reaction cycle. To define the structural and mechanistic bases for these observations, CD, MCD, and VTVH MCD spectroscopies coupled with ligand-field (LF) calculations are used to elucidate changes occurring near and at the MMOH biferrous cluster upon binding of MMOB and the MMOB variants. Perturbations to both the CD and MCD are observed upon binding wild-type MMOB and the MMOB variant that similarly increases O 2 reactivity. MMOB variants that do not greatly increase O 2 reactivity fail to cause one or both of these changes. LF calculations indicate that reorientation of the terminal glutamate on Fe2 reproduces the spectral perturbations in MCD. Although this structural change allows O 2 to bridge the diiron site and shifts the redox active orbitals for good overlap, it is not sufficient for enhanced O 2 reactivity of the enzyme. Binding of the T111Y-MMOB variant to MMOH induces the MCD, but not CD changes, and causes only a small increase in reactivity. Thus, both the geometric rearrangement at Fe2 (observed in MCD) coupled with a more global conformational change that may control O 2 access (probed by CD), induced by MMOB binding, are critical factors in the reactivity of sMMO.

Inter-domain Synergism Is Required for Efficient Feeding of Cellulose Chain into Active Site of Cellobiohydrolase Cel7A.

PubMed

Kont, Riin; Kari, Jeppe; Borch, Kim; Westh, Peter; Väljamäe, Priit

2016-12-09

Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme systems. TrCel7A consists of a catalytic domain (CD) and a smaller carbohydrate-binding module (CBM) connected through the glycosylated linker peptide. A tunnel-shaped active site rests in the CD and contains 10 glucose unit binding sites. The active site of TrCel7A is lined with four Trp residues with two of them, Trp-40 and Trp-38, in the substrate binding sites near the tunnel entrance. Although addressed in numerous studies the elucidation of the role of CBM and active site aromatics has been obscured by a complex multistep mechanism of processive GHs. Here we studied the role of the CBM-linker and Trp-38 of TrCel7A with respect to binding affinity, on- and off-rates, processivity, and synergism with endoglucanase. The CBM-linker increased the on-rate and substrate affinity of the enzyme. The Trp-38 to Ala substitution resulted in increased off-rates and decreased processivity. The effect of the Trp-38 to Ala substitution on on-rates was strongly dependent on the presence of the CBM-linker. This compensation between CBM-linker and Trp-38 indicates synergism between CBM-linker and CD in feeding the cellulose chain into the active site. The inter-domain synergism was pre-requisite for the efficient degradation of cellulose in the presence of endoglucanase. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Inter-domain Synergism Is Required for Efficient Feeding of Cellulose Chain into Active Site of Cellobiohydrolase Cel7A*

PubMed Central

Kont, Riin; Kari, Jeppe; Borch, Kim; Westh, Peter; Väljamäe, Priit

2016-01-01

Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme systems. TrCel7A consists of a catalytic domain (CD) and a smaller carbohydrate-binding module (CBM) connected through the glycosylated linker peptide. A tunnel-shaped active site rests in the CD and contains 10 glucose unit binding sites. The active site of TrCel7A is lined with four Trp residues with two of them, Trp-40 and Trp-38, in the substrate binding sites near the tunnel entrance. Although addressed in numerous studies the elucidation of the role of CBM and active site aromatics has been obscured by a complex multistep mechanism of processive GHs. Here we studied the role of the CBM-linker and Trp-38 of TrCel7A with respect to binding affinity, on- and off-rates, processivity, and synergism with endoglucanase. The CBM-linker increased the on-rate and substrate affinity of the enzyme. The Trp-38 to Ala substitution resulted in increased off-rates and decreased processivity. The effect of the Trp-38 to Ala substitution on on-rates was strongly dependent on the presence of the CBM-linker. This compensation between CBM-linker and Trp-38 indicates synergism between CBM-linker and CD in feeding the cellulose chain into the active site. The inter-domain synergism was pre-requisite for the efficient degradation of cellulose in the presence of endoglucanase. PMID:27780868

Siglec-1 inhibits RSV-induced interferon gamma production by adult T cells in contrast to newborn T cells.

PubMed

Jans, Jop; Unger, Wendy W J; Vissers, Marloes; Ahout, Inge M L; Schreurs, Inge; Wickenhagen, Arthur; de Groot, Ronald; de Jonge, Marien I; Ferwerda, Gerben

2018-04-01

Interferon gamma (IFN-γ) plays an important role in the antiviral immune response during respiratory syncytial virus (RSV) infections. Monocytes and T cells are recruited to the site of RSV infection, but it is unclear whether cell-cell interactions between monocytes and T cells regulate IFN-γ production. In this study, micro-array data identified the upregulation of sialic acid-binding immunoglobulin-type lectin 1 (Siglec-1) in human RSV-infected infants. In vitro, RSV increased expression of Siglec-1 on healthy newborn and adult monocytes. RSV-induced Siglec-1 on monocytes inhibited IFN-γ production by adult CD4 + T cells. In contrast, IFN-γ production by RSV in newborns was not affected by Siglec-1. The ligand for Siglec-1, CD43, is highly expressed on adult CD4 + T cells compared to newborns. Our data show that Siglec-1 reduces IFN-γ release by adult T cells possibly by binding to the highly expressed CD43. The Siglec-1-dependent inhibition of IFN-γ in adults and the low expression of CD43 on newborn T cells provides a better understanding of the immune response against RSV in early life and adulthood. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cadmium is deposited in the gut content of larvae of the beetle Tenebrio molitor and involves a Cd-binding protein of the low cysteine type.

PubMed

Pedersen, S A; Kristiansen, E; Andersen, R A; Zachariassen, K E

2008-09-01

Binding of cadmium (Cd) to metallothionein (MT) and non-MT proteins with low contents of cysteine has been observed in terrestrial arthropods. We recently isolated a Cd-binding protein with no cysteine that was induced in Cd-exposed larvae of the beetle Tenebrio molitor. In this study we have examined the molecular distribution of Cd within extracts of different tissues and compartments of Cd-exposed T. molitor larvae. A Cd-peak consistent with the low cysteine Cd-binding protein was induced within the gut content where it could be detected after 4-8 days of exposure. Examination of gut wall tissue revealed no increase in Cd-binding capacity, indicating that no accumulation of MTs was taking place in this tissue. Incorporation of Cd in the gut wall tissue stabilized after 8 days of Cd-exposure at a rather low level compared to the other organs. There was a statistical trend towards Cd being incorporated in the gut content in a manner that was disproportionally high compared to the amount of Cd in the gut wall tissue. The possible role of the low cysteine Cd-binding protein in reducing the uptake of Cd in the tissues is discussed.

Towed Thermistor Chain Observations in JASIN,

DTIC Science & Technology

1980-07-01

Lij LiJ - S -Ky LJDC cYcc Ql I- 6? F-L T cy D- Hl-m L-L Lo i -,7W LOu CT KK _ _4 C7) 63 -) Ln CD Ln ;Lf FE - CS -JN V LO WLJCNK ua- -~ Lfl CO F (YD... DBD C _W2 + D2 Integrating (A5) yields: G8 bW -1 2cs + b z(s) = (- - 2c 3 / 2 [sinh (d - sinh- (-)] (a7) +- [(A + bs + cs) - /a]c where d = 4ac - b

Analysis of Qa-1bPeptide Binding Specificity and the Capacity of Cd94/Nkg2a to Discriminate between Qa-1–Peptide Complexes

PubMed Central

Kraft, Jennifer R.; Vance, Russell E.; Pohl, Jan; Martin, Amy M.; Raulet, David H.; Jensen, Peter E.

2000-01-01

The major histocompatibility complex class Ib protein, Qa-1b, serves as a ligand for murine CD94/NKG2A natural killer (NK) cell inhibitory receptors. The Qa-1b peptide-binding site is predominantly occupied by a single nonameric peptide, Qa-1 determinant modifier (Qdm), derived from the leader sequence of H-2D and L molecules. Five anchor residues were identified in this study by measuring the peptide-binding affinities of substituted Qdm peptides in experiments with purified recombinant Qa-1b. A candidate peptide-binding motif was determined by sequence analysis of peptides eluted from Qa-1 that had been folded in the presence of random peptide libraries or pools of Qdm derivatives randomized at specific anchor positions. The results indicate that Qa-1b can bind a diverse repertoire of peptides but that Qdm has an optimal primary structure for binding Qa-1b. Flow cytometry experiments with Qa-1b tetramers and NK target cell lysis assays demonstrated that CD94/NKG2A discriminates between Qa-1b complexes containing peptides with substitutions at nonanchor positions P4, P5, or P8. Our findings suggest that it may be difficult for viruses to generate decoy peptides that mimic Qdm and raise the possibility that competitive replacement of Qdm with other peptides may provide a novel mechanism for activation of NK cells. PMID:10974028

Human HDAC7 Harbors a Class IIa Histone Deacetylase-specific Zinc Binding Motif and Cryptic Deacetylase Activity*S⃞

PubMed Central

Schuetz, Anja; Min, Jinrong; Allali-Hassani, Abdellah; Schapira, Matthieu; Shuen, Michael; Loppnau, Peter; Mazitschek, Ralph; Kwiatkowski, Nick P.; Lewis, Timothy A.; Maglathin, Rebecca L.; McLean, Thomas H.; Bochkarev, Alexey; Plotnikov, Alexander N.; Vedadi, Masoud; Arrowsmith, Cheryl H.

2008-01-01

Histone deacetylases (HDACs) are protein deacetylases that play a role in repression of gene transcription and are emerging targets in cancer therapy. Here, we characterize the structure and enzymatic activity of the catalytic domain of human HDAC7 (cdHDAC7). Although HDAC7 normally exists as part of a multiprotein complex, we show that cdHDAC7 has a low level of deacetylase activity which can be inhibited by known HDAC inhibitors. The crystal structures of human cdHDAC7 and its complexes with two hydroxamate inhibitors are the first structures of the catalytic domain of class IIa HDACs and demonstrate significant differences with previously reported class I and class IIb-like HDAC structures. We show that cdHDAC7 has an additional class IIa HDAC-specific zinc binding motif adjacent to the active site which is likely to participate in substrate recognition and protein-protein interaction and may provide a site for modulation of activity. Furthermore, a different active site topology results in modified catalytic properties and in an enlarged active site pocket. Our studies provide mechanistic insights into class IIa HDACs and facilitate the design of specific modulators. PMID:18285338

Human HDAC7 harbors a class IIa histone deacetylase-specific zinc binding motif and cryptic deacetylase activity.

PubMed

Schuetz, Anja; Min, Jinrong; Allali-Hassani, Abdellah; Schapira, Matthieu; Shuen, Michael; Loppnau, Peter; Mazitschek, Ralph; Kwiatkowski, Nick P; Lewis, Timothy A; Maglathin, Rebecca L; McLean, Thomas H; Bochkarev, Alexey; Plotnikov, Alexander N; Vedadi, Masoud; Arrowsmith, Cheryl H

2008-04-25

Histone deacetylases (HDACs) are protein deacetylases that play a role in repression of gene transcription and are emerging targets in cancer therapy. Here, we characterize the structure and enzymatic activity of the catalytic domain of human HDAC7 (cdHDAC7). Although HDAC7 normally exists as part of a multiprotein complex, we show that cdHDAC7 has a low level of deacetylase activity which can be inhibited by known HDAC inhibitors. The crystal structures of human cdHDAC7 and its complexes with two hydroxamate inhibitors are the first structures of the catalytic domain of class IIa HDACs and demonstrate significant differences with previously reported class I and class IIb-like HDAC structures. We show that cdHDAC7 has an additional class IIa HDAC-specific zinc binding motif adjacent to the active site which is likely to participate in substrate recognition and protein-protein interaction and may provide a site for modulation of activity. Furthermore, a different active site topology results in modified catalytic properties and in an enlarged active site pocket. Our studies provide mechanistic insights into class IIa HDACs and facilitate the design of specific modulators.

An engineered promoter driving expression of a microbial avirulence gene confers recognition of TAL effectors and reduces growth of diverse Xanthomonas strains in citrus.

PubMed

Shantharaj, Deepak; Römer, Patrick; Figueiredo, Jose F L; Minsavage, Gerald V; Krönauer, Christina; Stall, Robert E; Moore, Gloria A; Fisher, Latanya C; Hu, Yang; Horvath, Diana M; Lahaye, Thomas; Jones, Jeffrey B

2017-09-01

Xanthomonas citri ssp. citri (X. citri), causal agent of citrus canker, uses transcription activator-like effectors (TALEs) as major pathogenicity factors. TALEs, which are delivered into plant cells through the type III secretion system (T3SS), interact with effector binding elements (EBEs) in host genomes to activate the expression of downstream susceptibility genes to promote disease. Predictably, TALEs bind EBEs in host promoters via known combinations of TALE amino acids to DNA bases, known as the TALE code. We introduced 14 EBEs, matching distinct X. citri TALEs, into the promoter of the pepper Bs3 gene (ProBs3 1EBE ), and fused this engineered promoter with multiple EBEs (ProBs3 14EBE ) to either the β-glucuronidase (GUS) reporter gene or the coding sequence (cds) of the pepper gene, Bs3. TALE-induced expression of the Bs3 cds in citrus leaves resulted in no visible hypersensitive response (HR). Therefore, we utilized a different approach in which ProBs3 1EBE and ProBs3 14EBE were fused to the Xanthomonas gene, avrGf1, which encodes a bacterial effector that elicits an HR in grapefruit and sweet orange. We demonstrated, in transient assays, that activation of ProBs3 14EBE by X. citri TALEs is T3SS dependent, and that the expression of AvrGf1 triggers HR and correlates with reduced bacterial growth. We further demonstrated that all tested virulent X. citri strains from diverse geographical locations activate ProBs3 14EBE . TALEs are essential for the virulence of X. citri strains and, because the engineered promoter traps are activated by multiple TALEs, this concept has the potential to confer broad-spectrum, durable resistance to citrus canker in stably transformed plants. © 2016 BSPP AND JOHN WILEY & SONS LTD.

Kinetics of Cd(ii) adsorption and desorption on ferrihydrite: experiments and modeling.

PubMed

Liang, Yuzhen; Tian, Lei; Lu, Yang; Peng, Lanfang; Wang, Pei; Lin, Jingyi; Cheng, Tao; Dang, Zhi; Shi, Zhenqing

2018-05-15

The kinetics of Cd(ii) adsorption/desorption on ferrihydrite is an important process affecting the fate, transport, and bioavailability of Cd(ii) in the environment, which was rarely systematically studied and understood at quantitative levels. In this work, a combination of stirred-flow kinetic experiments, batch adsorption equilibrium experiments, high-resolution transmission electron microscopy (HR-TEM), and mechanistic kinetic modeling were used to study the kinetic behaviors of Cd(ii) adsorption/desorption on ferrihydrite. HR-TEM images showed the open, loose, and sponge-like structure of ferrihydrite. The batch adsorption equilibrium experiments revealed that higher pH and initial metal concentration increased Cd(ii) adsorption on ferrihydrite. The stirred-flow kinetic results demonstrated the increased adsorption rate and capacity as a result of the increased pH, influent concentration, and ferrihydrite concentration. The mechanistic kinetic model successfully described the kinetic behaviors of Cd(ii) during the adsorption and desorption stages under various chemistry conditions. The model calculations showed that the adsorption rate coefficients varied as a function of solution chemistry, and the relative contributions of the weak and strong ferrihydrite sites for Cd(ii) binding varied with time at different pH and initial metal concentrations. Our model is able to quantitatively assess the contributions of each individual ferrihydrite binding site to the overall Cd(ii) adsorption/desorption kinetics. This study provided insights into the dynamic behavior of Cd(ii) and a predictive modeling tool for Cd(ii) adsorption/desorption kinetics when ferrihydrite is present, which may be helpful for the risk assessment and management of Cd contaminated sites.

Post-Synthetic Modification of Porphyrin-Encapsulating Metal-Organic Materials by Cooperative Addition of Inorganic Salts to Enhance CO 2/CH 4 Selectivity

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

Zhang, Zhenjie; Gao, Wen-Yang; Wojtas, Lukasz

Keeping MOM: Reaction of biphenyl-3,4',5-tricarboxylate and Cd(NO 3) 2 in the presence of meso-tetra(N-methyl-4-pyridyl)porphine tetratosylate afforded porph@MOM-11, a microporous metal–organic material (MOM) that encapsulates cationic porphyrins and solvent in alternating open channels. Porph@MOM-11 has cation and anion binding sites that facilitate cooperative addition of inorganic salts (such as M +Cl -) in a stoichiometric fashion.

Molecular modeling and spectroscopic studies on the interaction of the chiral drug venlafaxine hydrochloride with bovine serum albumin

NASA Astrophysics Data System (ADS)

Shahabadi, Nahid; Hadidi, Saba

2014-03-01

This study was designed to examine the interaction of racemic antidepressant drug "S,R-venlafaxine hydrochloride (VEN)" with bovine serum albumin (BSA) under physiological conditions. The mechanism of interaction was studied by spectroscopic techniques combination with molecular modeling. Stern-Volmer analysis of fluorescence quenching data shows the presence of the static quenching mechanism. The thermodynamic parameters indicated that the hydrogen bonding and weak van der Waals interactions are the predominant intermolecular forces stabilizing the complex. The number of binding sites (n) was calculated. Through the site marker competitive experiment, VEN was confirmed to be located in subdomain IIIA of BSA. The binding distance (r = 4.93 nm) between the donor BSA and acceptor VEN was obtained according to Förster's non-radiative energy transfer theory. According to UV-vis spectra and CD data binding of VEN leaded to conformational changes of BSA. Molecular docking simulations of S and R-VEN revealed that both isomers have similar interaction and the same binding sites, from this point of view S and R isomers are equal.

[Adsorption of heavy metals on the surface of birnessite relationship with its Mn average oxidation state and adsorption sites].

PubMed

Wang, Yan; Tan, Wen-Feng; Feng, Xiong-Han; Qiu, Guo-Hong; Liu, Fan

2011-10-01

Adsorption characteristics of mineral surface for heavy metal ions are largely determined by the type and amount of surface adsorption sites. However, the effects of substructure variance in manganese oxide on the adsorption sites and adsorption characteristics remain unclear. Adsorption experiments and powder X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) were combined to examine the adsorption characteristics of Pb2+, Cu2+, Zn2+ and Cd2+ sequestration by birnessites with different Mn average oxidation state (AOS), and the Mn AOS dependent adsorption sites and adsorption characteristics. The results show that the maximum adsorption capacity of Pb2+, Cu2+, Zn2+ and Cd2+ increased with increasing birnessite Mn AOS. The adsorption capacity followed the order of Pb2+ > Cu2+ > Zn2+ > Cd2+. The observations suggest that there exist two sites on the surface of birnessite, i. e., high-binding-energy site (HBE site) and low-binding-energy site (LBE site). With the increase of Mn AOS for birnessites, the amount of HBE sites for heavy metal ions adsorption remarkably increased. On the other hand, variation in the amount of LBE sites was insignificant. The amount of LBE sites is much more than those of HBE sites on the surface of birnessite with low Mn AOS. Nevertheless, both amounts on the surface of birnessite with high Mn AOS are very close to each other. Therefore, the heavy metal ions adsorption capacity on birnessite is largely determined by the amount of HBE sites. On birnessite surface, adsorption of Cu2+, Zn2+, and Cd2+ mostly occurred at HBE sites. In comparison with Zn2+ and Cd2+, more Cu2+ adsorbed on the LBW sites. Pb2+ adsorption maybe occupy at both LBE sites and HBE sites simultaneously.

An Aptamer That Neutralizes R5 Strains of Human Immunodeficiency Virus Type 1 Blocks gp120-CCR5 Interaction

PubMed Central

Dey, Antu K.; Khati, Makobetsa; Tang, Min; Wyatt, Richard; Lea, Susan M.; James, William

2005-01-01

We recently described the isolation and structural characterization of 2′-fluoropyrimidine-substituted RNA aptamers that bind to gp120 of R5 strains of human immunodeficiency virus type 1 and thereby potently neutralize the infectivity of phylogenetically diverse R5 strains. Here we investigate the physical basis of their antiviral action. We show that both N-linked oligosaccharides and the variable loops V1/V2 and V3 are not required for binding of one aptamer, B40, to gp120. Using surface plasmon resonance binding analyses, we show that the aptamer binds to the CCR5-binding site on gp120 in a relatively CD4-independent manner, providing a mechanistic explanation for its neutralizing potency. PMID:16227301

Human leucocyte antigen class I-redirected anti-tumour CD4+ T cells require a higher T cell receptor binding affinity for optimal activity than CD8+ T cells.

PubMed

Tan, M P; Dolton, G M; Gerry, A B; Brewer, J E; Bennett, A D; Pumphrey, N J; Jakobsen, B K; Sewell, A K

2017-01-01

CD4 + T helper cells are a valuable component of the immune response towards cancer. Unfortunately, natural tumour-specific CD4 + T cells occur in low frequency, express relatively low-affinity T cell receptors (TCRs) and show poor reactivity towards cognate antigen. In addition, the lack of human leucocyte antigen (HLA) class II expression on most cancers dictates that these cells are often unable to respond to tumour cells directly. These deficiencies can be overcome by transducing primary CD4 + T cells with tumour-specific HLA class I-restricted TCRs prior to adoptive transfer. The lack of help from the co-receptor CD8 glycoprotein in CD4 + cells might result in these cells requiring a different optimal TCR binding affinity. Here we compared primary CD4 + and CD8 + T cells expressing wild-type and a range of affinity-enhanced TCRs specific for the HLA A*0201-restricted NY-ESO-1- and gp100 tumour antigens. Our major findings are: (i) redirected primary CD4 + T cells expressing TCRs of sufficiently high affinity exhibit a wide range of effector functions, including cytotoxicity, in response to cognate peptide; and (ii) optimal TCR binding affinity is higher in CD4 + T cells than CD8 + T cells. These results indicate that the CD4 + T cell component of current adoptive therapies using TCRs optimized for CD8 + T cells is below par and that there is room for substantial improvement. © 2016 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.

Glutamine 89 is a key residue in the allosteric modulation of human serine racemase activity by ATP.

PubMed

Canosa, Andrea V; Faggiano, Serena; Marchetti, Marialaura; Armao, Stefano; Bettati, Stefano; Bruno, Stefano; Percudani, Riccardo; Campanini, Barbara; Mozzarelli, Andrea

2018-06-13

Serine racemase (SR) catalyses two reactions: the reversible racemisation of L-serine and the irreversible dehydration of L- and D-serine to pyruvate and ammonia. SRs are evolutionarily related to serine dehydratases (SDH) and degradative threonine deaminases (TdcB). Most SRs and TdcBs - but not SDHs - are regulated by nucleotides. SR binds ATP cooperatively and the nucleotide allosterically stimulates the serine dehydratase activity of the enzyme. A H-bond network comprising five residues (T52, N86, Q89, E283 and N316) and water molecules connects the active site with the ATP-binding site. Conservation analysis points to Q89 as a key residue for the allosteric communication, since its mutation to either Met or Ala is linked to the loss of control of activity by nucleotides. We verified this hypothesis by introducing the Q89M and Q89A point mutations in the human SR sequence. The allosteric communication between the active site and the allosteric site in both mutants is almost completely abolished. Indeed, the stimulation of the dehydratase activity by ATP is severely diminished and the binding of the nucleotide is no more cooperative. Ancestral state reconstruction suggests that the allosteric control by nucleotides established early in SR evolution and has been maintained in most eukaryotic lineages.

Blocking of HIV-1 Infectivity by a Soluble, Secreted Form of the CD4 Antigen

NASA Astrophysics Data System (ADS)

Smith, Douglas H.; Byrn, Randal A.; Marsters, Scot A.; Gregory, Timothy; Groopman, Jerome E.; Capon, Daniel J.

1987-12-01

The initial event in the infection of human T lymphocytes, macrophages, and other cells by human immunodeficiency virus (HIV-1) is the attachment of the HIV-1 envelope glycoprotein gp120 to its cellular receptor, CD4. As a step toward designing antagonists of this binding event, soluble, secreted forms of CD4 were produced by transfection of mammalian cells with vectors encoding versions of CD4 lacking its transmembrane and cytoplasmic domains. The soluble CD4 so produced binds gp120 with an affinity and specificity comparable to intact CD4 and is capable of neutralizing the infectivity of HIV-1. These studies reveal that the high-affinity CD4-gp120 interaction does not require other cell or viral components and may establish a novel basis for therapeutic intervention in the acquired immune deficiency syndrome (AIDS).

Convergent Use of Heptacoordination for Cation Selectivity by RNA and Protein Metalloregulators.

PubMed

Bachas, Sharrol T; Ferré-D'Amaré, Adrian R

2018-05-04

The large yybP-ykoY family of bacterial riboswitches is broadly distributed phylogenetically. Previously, these gene-regulatory RNAs were proposed to respond to Mn 2+ . X-ray crystallography revealed a binuclear cation-binding pocket. This comprises one hexacoordinate site, with six oxygen ligands, which preorganizes the second, with five oxygen and one nitrogen ligands. The relatively soft nitrogen ligand was proposed to confer affinity for Mn 2+ , but how this excludes other soft cations remained enigmatic. By subjecting representative yybP-ykoY riboswitches to diverse cations in vitro, we now find that these RNAs exhibit limited transition metal ion selectivity. Among the cations tested, Cd 2+ and Mn 2+ bind most tightly, and comparison of three new Cd 2+ -bound crystal structures suggests that these riboswitches achieve selectivity by enforcing heptacoordination (favored by high-spin Cd 2+ and Mn 2+ , but otherwise uncommon) in the softer site. Remarkably, the Cd 2+ - and Mn 2+ -selective bacterial transcription factor MntR also uses heptacoordination within a binuclear site to achieve selectivity. Published by Elsevier Ltd.

Combining transcription factor binding affinities with open-chromatin data for accurate gene expression prediction

PubMed Central

Schmidt, Florian; Gasparoni, Nina; Gasparoni, Gilles; Gianmoena, Kathrin; Cadenas, Cristina; Polansky, Julia K.; Ebert, Peter; Nordström, Karl; Barann, Matthias; Sinha, Anupam; Fröhler, Sebastian; Xiong, Jieyi; Dehghani Amirabad, Azim; Behjati Ardakani, Fatemeh; Hutter, Barbara; Zipprich, Gideon; Felder, Bärbel; Eils, Jürgen; Brors, Benedikt; Chen, Wei; Hengstler, Jan G.; Hamann, Alf; Lengauer, Thomas; Rosenstiel, Philip; Walter, Jörn; Schulz, Marcel H.

2017-01-01

The binding and contribution of transcription factors (TF) to cell specific gene expression is often deduced from open-chromatin measurements to avoid costly TF ChIP-seq assays. Thus, it is important to develop computational methods for accurate TF binding prediction in open-chromatin regions (OCRs). Here, we report a novel segmentation-based method, TEPIC, to predict TF binding by combining sets of OCRs with position weight matrices. TEPIC can be applied to various open-chromatin data, e.g. DNaseI-seq and NOMe-seq. Additionally, Histone-Marks (HMs) can be used to identify candidate TF binding sites. TEPIC computes TF affinities and uses open-chromatin/HM signal intensity as quantitative measures of TF binding strength. Using machine learning, we find low affinity binding sites to improve our ability to explain gene expression variability compared to the standard presence/absence classification of binding sites. Further, we show that both footprints and peaks capture essential TF binding events and lead to a good prediction performance. In our application, gene-based scores computed by TEPIC with one open-chromatin assay nearly reach the quality of several TF ChIP-seq data sets. Finally, these scores correctly predict known transcriptional regulators as illustrated by the application to novel DNaseI-seq and NOMe-seq data for primary human hepatocytes and CD4+ T-cells, respectively. PMID:27899623

Increased numbers of CD38 molecules on bright CD8+ T lymphocytes in infectious mononucleosis caused by Epstein–Barr virus infection

PubMed Central

ŽIDOVEC LEPEJ, S; VINCE, A; ÐAKOVIĆ RODE, O; REMENAR, A; JEREN, T

2003-01-01

The aim of this study was to quantify the expression of CD38 on CD8+ T lymphocytes of patients with infectious mononucleosis (IM) caused by Epstein–Barr virus (EBV) and cytomegalovirus (CMV). CD38 quantification technique chosen for this study was based on the enumeration of CD38 antibody binding sites in comparison to the quantification standards rather than determining relative fluorescence, which is difficult to standardize. The study enrolled 19 patients with typical clinical and laboratory parameters compatible with EBV-induced IM as well as 10 patients with atypical clinical presentation of this disease. Furthermore, CD38 expression was analysed in a group of 13 patients with IM caused by CMV infection. CD38 quantification was performed within 6 days of the presentation of symptoms. All three groups of IM patients showed a statistically significant increase in the number of anti-CD38 antibody binding sites (which correspond to the number of CD38 molecules) on bright CD8+ T lymphocytes compared to healthy controls. The numbers of CD38 molecules expressed on CD8+ T lymphocytes did not differ significantly between IM patients with typical and atypical clinical presentation of the disease. Patients with CMV-induced IM had significantly lower numbers of CD38 molecules expressed on CD8+ T lymphocytes. Therefore, we conclude that CD38 quantification could be helpful in differential diagnostics of IM cases with atypical clinical presentation. PMID:12930365

Retinoic acid prevents immunogenicity of milk lipocalin Bos d 5 through binding to its immunodominant T-cell epitope.

PubMed

Hufnagl, Karin; Ghosh, Debajyoti; Wagner, Stefanie; Fiocchi, Alessandro; Dahdah, Lamia; Bianchini, Rodolfo; Braun, Nina; Steinborn, Ralf; Hofer, Martin; Blaschitz, Marion; Roth, Georg A; Hofstetter, Gerlinde; Roth-Walter, Franziska; Pacios, Luis F; Jensen-Jarolim, Erika

2018-01-25

The major cow's milk allergen Bos d 5 belongs to the lipocalin protein family, with an intramolecular pocket for hydrophobic ligands. We investigated whether Bos d 5 when loaded with the active vitamin A metabolite retinoic acid (RA), would elicit differential immune responses compared to the unloaded state. By in silico docking an affinity energy of -7.8 kcal/mol was calculated for RA into Bos d 5. Loading of RA to Bos d 5 could be achieved in vitro, as demonstrated by ANS displacement assay, but had no effect on serum IgE binding in tolerant or challenge-positive milk allergic children. Bioinformatic analysis revealed that RA binds to the immunodominant T-cell epitope region of Bos d 5. In accordance, Bos d 5 significantly suppressed the CD3+ CD4+ cell numbers, proliferative response and IL-10, IL-13 and IFN-γ secretion from stimulated human PBMCs only when complexed with RA. This phenomenon was neither associated with apoptosis of T-cells nor with the activation of Foxp3+ T-cells, but correlated likely with enhanced stability to lysosomal digestion due to a predicted overlap of Cathepsin S cleavage sites with the RA binding site. Taken together, proper loading of Bos d 5 with RA may suppress its immunogenicity and prevent its allergenicity.

An anti-CD30 single-chain Fv selected by phage display and fused to Pseudomonas exotoxin A (Ki-4(scFv)-ETÁ) is a potent immunotoxin against a Hodgkin-derived cell line

PubMed Central

Klimka, A; Barth, S; Matthey, B; Roovers, R C; Lemke, H; Hansen, H; Arends, J-W; Diehl, V; Hoogenboom, H R; Engert, A

1999-01-01

The human CD30 receptor is highly overexpressed on the surface of Hodgkin Reed-Sternberg cells and has been shown to be an excellent target for selective immunotherapy using monoclonal antibody-based agents such as immunotoxins. To construct a new recombinant immunotoxin for possible clinical use in patients with Hodgkin's lymphoma, we have chosen the murine anti-CD30 hybridoma Ki-4 to generate a high-affinity Ki-4 single-chain variable fragment (scFv). Hybridoma V-genes were polymerase chain reaction-amplified, assembled, cloned and expressed as a mini-library for display on filamentous phage. Functional Ki-4 scFv were obtained by selection of binding phage on the Hodgkin lymphoma-derived, CD30-expressing cell line L540Cy. The selected recombinant Ki-4 scFv was shown to specifically bind to an overlapping epitope on the CD30 antigen with binding kinetics similar to those of the original antibody. The Ki-4 scFv was subsequently fused to a deletion mutant of Pseudomonas exotoxin A (ETÁ). The resulting immunotoxin Ki-4(scFv)-ETÁ specifically binds to CD30+ L540Cy cells and inhibits the protein synthesis by 50% at a concentration (IC50) of 43 pM. This recombinant immunotoxin is a promising candidate for further clinical evaluation in patients with Hodgkin's lymphoma or other CD30+ malignancies. © 1999 Cancer Research Campaign PMID:10376974

A novel mechanism of regulating breast cancer cell migration via palmitoylation-dependent alterations in the lipid raft affiliation of CD44.

PubMed

Babina, Irina S; McSherry, Elaine A; Donatello, Simona; Hill, Arnold D K; Hopkins, Ann M

2014-02-10

Most breast cancer-related deaths result from metastasis, a process involving dynamic regulation of tumour cell adhesion and migration. The adhesion protein CD44, a key regulator of cell migration, is enriched in cholesterol-enriched membrane microdomains termed lipid rafts. We recently reported that raft affiliation of CD44 negatively regulates interactions with its migratory binding partner ezrin. Since raft affiliation is regulated by post-translational modifications including palmitoylation, we sought to establish the contribution of CD44 palmitoylation and lipid raft affiliation to cell migration. Recovery of CD44 and its binding partners from raft versus non-raft membrane microdomains was profiled in non-migrating and migrating breast cancer cell lines. Site-directed mutagenesis was used to introduce single or double point mutations into both CD44 palmitoylation sites (Cys286 and Cys295), whereupon the implications for lipid raft recovery, phenotype, ezrin co-precipitation and migratory behaviour was assessed. Finally CD44 palmitoylation status and lipid raft affiliation was assessed in primary cultures from a small panel of breast cancer patients. CD44 raft affiliation was increased during migration of non-invasive breast cell lines, but decreased during migration of highly-invasive breast cells. The latter was paralleled by increased CD44 recovery in non-raft fractions, and exclusive non-raft recovery of its binding partners. Point mutation of CD44 palmitoylation sites reduced CD44 raft affiliation in invasive MDA-MB-231 cells, increased CD44-ezrin co-precipitation and accordingly enhanced cell migration. Expression of palmitoylation-impaired (raft-excluded) CD44 mutants in non-invasive MCF-10a cells was sufficient to reversibly induce the phenotypic appearance of epithelial-to-mesenchymal transition and to increase cell motility. Interestingly, cell migration was associated with temporal reductions in CD44 palmitoylation in wild-type breast cells. Finally, the relevance of these findings is underscored by the fact that levels of palmitoylated CD44 were lower in primary cultures from invasive ductal carcinomas relative to non-tumour tissue, while CD44 co-localisation with a lipid raft marker was less in invasive ductal carcinoma relative to ductal carcinoma in situ cultures. Our results support a novel mechanism whereby CD44 palmitoylation and consequent lipid raft affiliation inversely regulate breast cancer cell migration, and may act as a new therapeutic target in breast cancer metastasis.

Impact of ¹⁸F-fluoride PET-CT on implementing early treatment of painful bone metastases with Sm-153 EDTMP.

PubMed

Storto, Giovanni; Gallicchio, Rosj; Pellegrino, Teresa; Nardelli, Anna; De Luca, Serena; Capacchione, Daniela; Sirignano, Cesare; Pace, Leonardo

2013-05-01

This study evaluated the diagnostic impact of using skeletal (18)F-fluoride PET/CT on patients with painful bone metastases to schedule an early palliative radionuclide treatment. The skeletal involvement from prostate cancer metastases was assessed by both (99m)Tc-diphosphonate bone scan (BS) and (18)F-fluoride PET/CT within four weeks in 24 patients (67.7 ± 5.1 years) suffering from a borderline degree of bone pain for which radionuclide palliation was not shortly planned for administration. The BS and (18)F-fluoride PET/CT results were compared, assessing the number and extension of the skeletal sites involved. Afterward, the patients were randomly assigned either to the study group (N=12) receiving radionuclide therapy (Samarium-153 EDTMP) or to the control group (N=12) not receiving radionuclide therapy. The short-term results from the radionuclide palliation group (evaluated with a visual analogue scale) were compared with the controls. Overall, at BS, 7.6 ± 1.4 sites were considered metastatic, involving at least 5 ± 1 body regions. At (18)F-fluoride PET/CT, 116 ± 19 sites presented metastatic involvement with 12/12 body regions concerned. No differences were found in regards to either the number of metastatic sites or regions at both BS and (18)F-fluoride PET/CT between the study group and controls (p=ns). At CT, 88 blastic metastases were identified, whereas 110 were mainly lytic. Most of mainly lytic lesions were not detectable at BS. The reduction in total discomfort and bone pain in the study group was significantly greater than in the controls (p<0.0001). Sm-153 EDTMP therapy should be considered for patients with early bone pain from prostate cancer even if their BS only indicates a few metastases before the initiation of a severe pain syndrome. (18)F-fluoride PET/CT may be helpful in deciding if the implementation of bone pain palliation using bone-seeking radionuclides at pain onset is necessary. Copyright © 2013 Elsevier Inc. All rights reserved.

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Science.gov Websites

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Human HMG box transcription factor HBP1: a role in hCD2 LCR function.

PubMed Central

Zhuma, T; Tyrrell, R; Sekkali, B; Skavdis, G; Saveliev, A; Tolaini, M; Roderick, K; Norton, T; Smerdon, S; Sedgwick, S; Festenstein, R; Kioussis, D

1999-01-01

The locus control region (LCR) of the human CD2 gene (hCD2) confers T cell-specific, copy-dependent and position-independent gene expression in transgenic mice. This LCR consists of a strong T cell-specific enhancer and an element without enhancer activity (designated HSS3), which is required for prevention of position effect variegation (PEV) in transgenic mice. Here, we identified the HMG box containing protein-1 (HBP1) as a factor binding to HSS3 of the hCD2 LCR. Within the LCR, HBP1 binds to a novel TTCATTCATTCA sequence that is higher in affinity than other recently reported HBP1-binding sites. Mice transgenic for a hCD2 LCR construct carrying a deletion of the HBP1-binding sequences show a propensity for PEV if the transgene integrates in a heterochromatic region of the chromosome such as the centromere or telomere. We propose that HBP1 plays an important role in chromatin opening and remodelling activities by binding to and bending the DNA, thus allowing DNA-protein and/or protein-protein interactions, which increase the probability of establishing an active locus. PMID:10562551

An x-ray absorption spectroscopy study of Cd binding onto a halophilic archaeon

NASA Astrophysics Data System (ADS)

Showalter, Allison R.; Szymanowski, Jennifer E. S.; Fein, Jeremy B.; Bunker, Bruce A.

2016-05-01

X-ray absorption spectroscopy (XAS) and cadmium (Cd) isotherm experiments determine how Cd adsorbs to the surface of halophilic archaeon Halobacterium noricense. This archaeon, isolated from the Waste Isolation Pilot Plant (WIPP) near Carlsbad, New Mexico could be involved with the transport of toxic metals stored in the transuranic waste in the salt mine. The isotherm experiments show that adsorption is relatively constant across the tolerable pH range for H. noricense. The XAS results indicate that Cd adsorption occurs predominately via a sulfur site, most likely sulfhydryl, with the same site dominating all measured pH values.

Study on the interaction of the epilepsy drug, zonisamide with human serum albumin (HSA) by spectroscopic and molecular docking techniques

NASA Astrophysics Data System (ADS)

Shahabadi, Nahid; Khorshidi, Aref; Moghadam, Neda Hossinpour

2013-10-01

In the present investigation, an attempt has been made to study the interaction of zonisamide (ZNS) with the transport protein, human serum albumin (HSA) employing UV-Vis, fluorometric, circular dichroism (CD) and molecular docking techniques. The results indicated that binding of ZNS to HSA caused strong fluorescence quenching of HSA through static quenching mechanism, hydrogen bonds and van der Waals contacts are the major forces in the stability of protein ZNS complex and the process of the binding of ZNS with HSA was driven by enthalpy (ΔH = -193.442 kJ mol-1). The results of CD and UV-Vis spectroscopy showed that the binding of this drug to HSA induced conformational changes in HSA. Furthermore, the study of molecular docking also indicated that zonisamide could strongly bind to the site I (subdomain IIA) of HSA mainly by hydrophobic interaction and there were hydrogen bond interactions between this drug and HSA, also known as the warfarin binding site.

Structural changes in calcium-binding allergens: use of circular dichroism to study binding characteristics.

PubMed

Hebenstreit, D; Ferreira, F

2005-09-01

Several studies showed that calcium-binding proteins have a fixed place in the spectrum of allergenic substances. Often the binding of a calcium ion induces conformational changes and affects immunoglobulin E-binding to the allergen. Hence, the quantitative characterization of the binding to calcium is of importance to understand both the biologic and allergenic activity of these proteins. In the present study we describe a procedure for determining the stoichiometry and dissociation constant (K(D)) of calcium-binding allergens using circular dichroism (CD) techniques. For the experiments, we used recombinant Bet v 4, a two EF-hand allergen from birch pollen. Solutions of Bet v 4 were titrated with calcium and the change in molar ellipticity at 222 nm was monitored with a CD spectropolarimeter. The determination of the binding stoichiometry as well as of the K(D) for one EF-hand (4 microM) demonstrated the applicability of the method. CD-monitored calcium-titration of protein solutions represents a fast and easy method for determining the binding characteristics of calcium-binding allergens.

Binding of [3H]MSX-2 (3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine) to rat striatal membranes--a new, selective antagonist radioligand for A(2A) adenosine receptors.

PubMed

Müller, C E; Maurinsh, J; Sauer, R

2000-01-01

The present study describes the preparation and binding properties of a new, potent, and selective A(2A) adenosine receptor (AR) antagonist radioligand, [3H]3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargy lxanth ine ([3H]MSX-2). [3H]MSX-2 binding to rat striatal membranes was saturable and reversible. Saturation experiments showed that [3H]MSX-2 labeled a single class of binding sites with high affinity (K(d)=8.0 nM) and limited capacity (B(max)=1.16 fmol.mg(-1) of protein). The presence of 100 microM GTP, or 10 mM magnesium chloride, respectively, had no effect on [3H]MSX-2 binding. AR agonists competed with the binding of 1 nM [3H]MSX-2 with the following order of potency: 5'-N-ethylcarboxamidoadenosine (NECA)>2-[4-(carboxyethyl)phenylethylamino]-5'-N-ethylcarboxami doaden osine (CGS-21680)>2-chloroadenosine (2-CADO)>N(6)-cyclopentyladenosine (CPA). AR antagonists showed the following order of potency: 8-(m-bromostyryl)-3, 7-dimethyl-1-propargylxanthine (BS-DMPX)>1, 3-dipropyl-8-cyclopentylxanthine (DPCPX)>(R)-5, 6-dimethyl-7-(1-phenylethyl)-2-(4-pyridyl)-7H-pyrrolo[2, 3-d]pyrimidine-4-amine (SH-128)>3,7-dimethyl-1-propargylxanthine (DMPX)>caffeine. The K(i) values for antagonists were in accordance with data from binding studies with the agonist radioligand [3H]CGS21680, while agonist affinities were 3-7-fold lower. [3H]MSX-2 is a highly selective A(2A) AR antagonist radioligand exhibiting a selectivity of at least two orders of magnitude versus all other AR subtypes. The new radioligand shows high specific radioactivity (85 Ci/mmol, 3150 GBq/mmol) and acceptable nonspecific binding at rat striatal membranes of 20-30%, at 1 nM.

Immunodominant epitopes in herpes simplex virus type 2 glycoprotein D are recognized by CD4 lymphocytes from both HSV-1 and HSV-2 seropositive subjects.

PubMed

Kim, Min; Taylor, Janette; Sidney, John; Mikloska, Zorka; Bodsworth, Neil; Lagios, Katerina; Dunckley, Heather; Byth-Wilson, Karen; Denis, Martine; Finlayson, Robert; Khanna, Rajiv; Sette, Alessandro; Cunningham, Anthony L

2008-11-01

In human recurrent cutaneous herpes simplex, there is a sequential infiltrate of CD4 and then CD8 lymphocytes into lesions. CD4 lymphocytes are the major producers of the key cytokine IFN-gamma in lesions. They recognize mainly structural proteins and especially glycoproteins D and B (gD and gB) when restimulated in vitro. Recent human vaccine trials using recombinant gD showed partial protection of HSV seronegative women against genital herpes disease and also, in placebo recipients, showed protection by prior HSV1 infection. In this study, we have defined immunodominant peptide epitopes recognized by 8 HSV1(+) and/or 16 HSV2(+) patients using (51)Cr-release cytotoxicity and IFN-gamma ELISPOT assays. Using a set of 39 overlapping 20-mer peptides, more than six immunodominant epitopes were defined in gD2 (two to six peptide epitopes were recognized for each subject). Further fine mapping of these responses for 4 of the 20-mers, using a panel of 9 internal 12-mers for each 20-mers, combined with MHC II typing and also direct in vitro binding assay of these peptides to individual DR molecules, showed more than one epitope per 20-mers and promiscuous binding of individual 20-mers and 12-mers to multiple DR types. All four 20-mer peptides were cross-recognized by both HSV1(+)/HSV2(-) and HSV1(-)/HSV2(+) subjects, but the sites of recognition differed within the 20-mers where their sequences were divergent. This work provides a basis for CD4 lymphocyte cross-recognition of gD2 and possibly cross-protection observed in previous clinical studies and in vaccine trials.

Fluctuation Dynamics Analysis of gp120 Envelope Protein Reveals a Topologically Based Communication Network

PubMed Central

Shrivastava, Indira; LaLonde, Judith M.

2012-01-01

HIV infection is initiated by binding of the viral glycoprotein gp120, to the cellular receptor CD4. Upon CD4 binding, gp120 undergoes conformational change, permitting binding to the chemokine receptor. Crystal structures of gp120 ternary complex reveal the CD4 bound conformation of gp120. We report here the application of Gaussian Network Model (GNM) to the crystal structures of gp120 bound to CD4 or CD4 mimic and 17b, to study the collective motions of the gp120 core and determine the communication propensities of the residue network. The GNM fluctuation profiles identify residues in the inner domain and outer domain that may facilitate conformational change or stability, respectively. Communication propensities delineate a residue network that is topologically suited for signal propagation from the Phe43 cavity throughout the gp120 outer domain. . These results provide a new context for interpreting gp120 core envelope structure-function relationships. PMID:20718047

Bleeding score in type 1 von Willebrand disease patients using the condensed MCMDM-1 vWD validated questionnaire.

PubMed

Pathare, A; Al Hajri, F; Al Omrani, S; Al Obaidani, N; Al Balushi, B; Al Falahi, K

2018-05-13

Assessment of the severity of bleeding symptom has led to the evolution of bleeding assessment tools which are now validated. To administer the condensed molecular and clinical markers for the diagnosis and management of type 1 von Willebrand disease VWD (MCMDM-1 vWD) questionnaire to the Omani type 1 vWD patients and correlate it with the laboratory parameters. Patients and controls were personally interviewed and the condensed MCMDM-1 vWD questionnaire administered by a single investigator. Bleeding score (BS) was calculated, based on the presence or absence of the bleeding symptoms according to a standard validated questionnaire in both the patients and the controls. The median age of the patient cohort was 27 (range, 7-49) years with 60.87% of females. The median time to administer condensed MCMDM-1 BS questionnaire was 11 minutes (interquartile range-IQR;7,16). Overall, bleeding from the oral cavity was the most predominant symptom (63%). The median BS was 5 (IQR;1,8) although individual scores ranged between 0 and 29. However, there was no statistically significant difference in BS between genders (males: median 4; IQR 1,6 and females: median 5, IQR 1,10) (P > .05, Kruskal-Wallis test) The Spearman's correlation value of BS was weak with FVIII:C levels and von Willebrand Ristocetin co-factor activity; very weak with von Willebrand Antigen level, and moderate with vonWillebrand Collagen Binding activity being -0.29, -0.28, -0.14 and -0.43, respectively. The BS reflects the severity of bleeding among the vWD patients. Although the BS was abnormal, it did not correlate significantly with the surrogate laboratory parameters [P > .05]. © 2018 John Wiley & Sons Ltd.

The Use of Carbonaceous Particle Exposure Metrics in Health Impact Calculations

PubMed Central

Olstrup, Henrik; Johansson, Christer; Forsberg, Bertil

2016-01-01

Combustion-related carbonaceous particles seem to be a better indicator of adverse health effects compared to PM2.5 and PM10. Historical studies are based on black smoke (BS), but more recent studies use absorbance (Abs), black carbon (BC) or elemental carbon (EC) as exposure indicators. To estimate health risks based on BS, we review the literature regarding the relationship between Abs, BS, BC and EC. We also discuss the uncertainties associated with the comparison of relative risks (RRs) based on these conversions. EC is reported to represent a proportion between 5.2% and 27% of BS with a mean value of 12%. Correlations of different metrics at one particular site are higher than when different sites are compared. Comparing all traffic, urban and rural sites, there is no systematic site dependence, indicating that other properties of the particles or errors affect the measurements and obscure the results. It is shown that the estimated daily mortality associated with short-term levels of EC is in the same range as PM10, but this is highly dependent on the EC to BS relationship that is used. RRs for all-cause mortality associated with short-term exposure to PM10 seem to be higher at sites with higher EC concentrations, but more data are needed to verify this. PMID:26927139

Study binding of Al-curcumin complex to ds-DNA, monitoring by multispectroscopic and voltammetric techniques

NASA Astrophysics Data System (ADS)

Ahmadi, F.; Alizadeh, A. A.; Shahabadi, N.; Rahimi-Nasrabadi, M.

2011-09-01

In this work a complex of Al 3+ with curcumin ([Al(curcumin) (EtOH) 2](NO 3) 2) was synthesized and characterized by UV-vis, FT-IR, elemental analysis and spectrophotometric titration techniques. The mole ratio plot revealed a 1:1 complex between Al 3+ and curcumin in solution. For binding studies of this complex to calf thymus-DNA various methods such as: UV-vis, fluorescence, circular dichroism (CD), FT-IR spectroscopy and cyclic voltammetry were used. The intrinsic binding constant of ACC with DNA at 25 °C was calculated by UV-vis and cyclic voltammetry as 2.1 × 10 4 and 2.6 × 10 4, respectively. The thermodynamic studies showed that the reaction is enthalpy and entropy favored. The CD results showed that only the Δ-ACC interacts with DNA and the Δ-ACC form has not any tendency to interact with DNA, also the pure curcumin has not any stereoselective interaction with CT-DNA. Fluorimetric studies showed that fluorescence enhancement was initiated by a static process in the ground state. The cyclic voltammetry showed that ACC interact with DNA with a binding site size of 2. From the FT-IR we concluded that the Δ-ACC interacts with DNA via partial electrostatic and minor groove binding. In comparison with previous works it was concluded that curcumin significantly reduced the affinity of Al 3+ to the DNA.

Costimulatory receptors in a teleost fish: Typical CD28, elusive CTLA4

USGS Publications Warehouse

Bernard, D.; Riteau, B.; Hansen, J.D.; Phillips, R.B.; Michel, F.; Boudinot, P.; Benmansour, A.

2006-01-01

T cell activation requires both specific recognition of the peptide-MHC complex by the TCR and additional signals delivered by costimulatory receptors. We have identified rainbow trout sequences similar to CD28 (rbtCD28) and CTLA4 (rbtCTLA4). rbtCD28 and rbtCTLA4 are composed of an extracellular Ig-superfamily V domain, a transmembrane region, and a cytoplasmic tail. The presence of a conserved ligand binding site within the V domain of both molecules suggests that these receptors likely recognize the fish homologues of the B7 family. The mRNA expression pattern of rbtCD28 and rbtCTLA4 in naive trout is reminiscent to that reported in humans and mice, because rbtCTLA4 expression within trout leukocytes was quickly up-regulated following PHA stimulation and virus infection. The cytoplasmic tail of rbtCD28 possesses a typical motif that is conserved in mammalian costimulatory receptors for signaling purposes. A chimeric receptor made of the extracellular domain of human CD28 fused to the cytoplasmic tail of rbtCD28 promoted TCR-induced IL-2 production in a human T cell line, indicating that rbtCD28 is indeed a positive costimulator. The cytoplasmic tail of rtrtCTLA4 lacked obvious signaling motifs and accordingly failed to signal when fused to the huCD28 extracellular domain. Interestingly, rbtCTLA4 and rbtCD28 are not positioned on the same chromosome and thus do not belong to a unique costimulatory cluster as in mammals. Finally, oar results raise questions about the origin and evolution of positive and negative costimulation in vertebrate immune systems. Copyright ?? 2006 by The American Association of Immunologists, Inc.

Flow cytometric sex sorting affects CD4 membrane distribution and binding of exogenous DNA on bovine sperm cells.

PubMed

Domingues, William Borges; da Silveira, Tony Leandro Rezende; Komninou, Eliza Rossi; Monte, Leonardo Garcia; Remião, Mariana Härter; Dellagostin, Odir Antônio; Corcini, Carine Dahl; Varela Junior, Antônio Sergio; Seixas, Fabiana Kömmling; Collares, Tiago; Campos, Vinicius Farias

2017-08-01

Bovine sex-sorted sperm have been commercialized and successfully used for the production of transgenic embryos of the desired sex through the sperm-mediated gene transfer (SMGT) technique. However, sex-sorted sperm show a reduced ability to internalize exogenous DNA. The interaction between sperm cells and the exogenous DNA has been reported in other species to be a CD4-like molecule-dependent process. The flow cytometry-based sex-sorting process subjects the spermatozoa to different stresses causing changes in the cell membrane. The aim of this study was to elucidate the relationship between the redistribution of CD4-like molecules and binding of exogenous DNA to sex-sorted bovine sperm. In the first set of experiments, the membrane phospholipid disorder and the redistribution of the CD4 were evaluated. The second set of experiments was conducted to investigate the effect of CD4 redistribution on the mechanism of binding of exogenous DNA to sperm cells and the efficiency of lipofection in sex-sorted bovine sperm. Sex-sorting procedure increased the membrane phospholipid disorder and induced the redistribution of CD4-like molecules. Both X-sorted and Y-sorted sperm had decreased DNA bound to membrane in comparison with the unsorted sperm; however, the binding of the exogenous DNA was significantly increased with the addition of liposomes. Moreover, we demonstrated that the number of sperm-bound exogenous DNA was decreased when these cells were preincubated with anti-bovine CD4 monoclonal antibody, supporting our hypothesis that CD4-like molecules indeed play a crucial role in the process of exogenous DNA/bovine sperm cells interaction.

Vascular barrier protective effects of 3-N- or 3-O-cinnamoyl carbazole derivatives.

PubMed

Ku, Sae-Kwang; Lee, Jee-Hyun; O, Yuseok; Lee, Wonhwa; Song, Gyu-Yong; Bae, Jong-Sup

2015-10-01

In this Letter, we investigated the barrier protective effects of 3-N-(MeO)n-cinnamoyl carbazoles (BS 1; n=1, BS 2; n=2, BS 3; n=3) and 3-O-(MeO)3-cinnamoyl carbazole (BS 4) against high-mobility group box 1 (HMGB1)-mediated vascular disruptive responses in human umbilical vein endothelial cells (HUVECs) and in mice for the first time. Data showed that BS 2, BS 3, and BS 4, but not BS 1, inhibited HMGB1-mediated vascular disruptive responses and transendothelial migration of human neutrophils to HUVECs. BS 2, BS3, and BS 4 also suppressed HMGB1-induced hyperpermeability and leukocyte migration in mice. Interestingly, the barrier protective effects of BS 3 and BS 4 were better than those of BS 2. These results suggest that the number of methoxy groups substituted on the cinnamamide or cinnamate moiety of the 9H-3-carbazole derivative is an important pharmacophore for the barrier protective effects of these compounds. Copyright © 2015 Elsevier Ltd. All rights reserved.

Synthesis Characterization and DNA Interaction Studies of a New Zn(II) Complex Containing Different Dinitrogen Aromatic Ligands

PubMed Central

Shahabadi, Nahid; Mohammadi, Somaye

2012-01-01

A mononuclear complex of Zn(II), [Zn(DIP)2 (DMP)] (NO3)2 ·2H2O in which DIP is 4,7-diphenyl-1,10-phenanthroline and DMP is 4,4′-dimethyl-2,2′-bipyridine has been prepared and characterized by 1HNMR spectroscopy, FT-IR, UV-Vis and elemental analysis techniques. DNA-binding properties of the complex were studied using UV-vis spectra, circular dichroism (CD) spectra, fluorescence, cyclic voltammetry (CV), and viscosity measurements. The results indicate that this zinc(II) complex can intercalate into the stacked base pairs of DNA and compete with the strong intercalator ethidium bromide for the intercalative binding sites. PMID:22956919

Characterization of Cadmium Uptake by Plant Tissue 12

PubMed Central

Cutler, Jay M.; Rains, Donald W.

1974-01-01

The uptake of cadmium by excised root tissue of barley (Hordeum vulgare L. cv. Arivat) was investigated with respect to kinetics, concentration, and interactions with various cations. The role of metabolism in Cd absorption was examined using a range of temperatures, anaerobic treatments, and chemical inhibitors. The uptake and distribution of Cd in intact barley plants was also determined. A large fraction of the Cd taken up by excised barley roots was apparently the result of exchange adsorption and was displaced by subsequent desorption with unlabeled Cd, Zn, Cu, or Hg. Another fraction of Cd which could not be displaced by desorption in unlabeled Cd was thought to result from strong irreversible binding of Cd, perhaps on sites of the cell wall. The fraction of the Cd taken up beyond that by exchange adsorption by fresh roots was a linear function of temperature, and inhibited by conditions of low oxygen and by the presence of 2,4-dinitrophenol. It was concluded that this fraction of Cd entered excised barley roots by diffusion. Diffusion, when followed by sequestering, probably accounts for the accumulation of Cd observed in intact barley plants. PMID:16658840

Nanostructure and force spectroscopy analysis of human peripheral blood CD4+ T cells using atomic force microscopy.

PubMed

Hu, Mingqian; Wang, Jiongkun; Cai, Jiye; Wu, Yangzhe; Wang, Xiaoping

2008-09-12

To date, nanoscale imaging of the morphological changes and adhesion force of CD4(+) T cells during in vitro activation remains largely unreported. In this study, we used atomic force microscopy (AFM) to study the morphological changes and specific binding forces in resting and activated human peripheral blood CD4(+) T cells. The AFM images revealed that the volume of activated CD4(+) T cells increased and the ultrastructure of these cells also became complex. Using a functionalized AFM tip, the strength of the specific binding force of the CD4 antigen-antibody interaction was found to be approximately three times that of the unspecific force. The adhesion forces were not randomly distributed over the surface of a single activated CD4(+) T cell, indicated that the CD4 molecules concentrated into nanodomains. The magnitude of the adhesion force of the CD4 antigen-antibody interaction did not change markedly with the activation time. Multiple bonds involved in the CD4 antigen-antibody interaction were measured at different activation times. These results suggest that the adhesion force involved in the CD4 antigen-antibody interaction is highly selective and of high affinity.

CD4 molecules with a diversity of mutations encompassing the CDR3 region efficiently support human immunodeficiency virus type 1 envelope glycoprotein-mediated cell fusion.

PubMed Central

Broder, C C; Berger, E A

1993-01-01

The third complementarity-determining region (CDR3) within domain 1 of the human CD4 molecule has been suggested to play a critical role in membrane fusion mediated by the interaction of CD4 with the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein. To analyze in detail the role of CDR3 and adjacent regions in the fusion process, we used cassette mutagenesis to construct a panel of 30 site-directed mutations between residues 79 and 96 of the full-length CD4 molecule. The mutant proteins were transiently expressed by using recombinant vaccinia virus vectors and were analyzed for cell surface expression, recombinant gp120-binding activity, and overall structural integrity as assessed by reactivity with a battery of anti-CD4 monoclonal antibodies. Cells expressing the CD4 mutants were assayed for their ability to form syncytia when mixed with cells expressing the HIV-1 envelope glycoprotein. Surprisingly in view of published data from others, most of the mutations had little effect on syncytium-forming activity. Normal fusion was observed in 21 mutants, including substitution of human residues 85 to 95 with the corresponding sequences from either chimpanzee, rhesus, or mouse CD4; a panel of Ser-Arg double insertions after each residue from 86 to 91; and a number of other charge, hydrophobic, and proline substitutions and insertions within this region. The nine mutants that showed impaired fusion all displayed defective gp120 binding and disruption of overall structural integrity. In further contrast with results of other workers, we observed that transformant human cell lines expressing native chimpanzee or rhesus CD4 efficiently formed syncytia when mixed with cells expressing the HIV-1 envelope glycoprotein. These data refute the conclusion that certain mutations in the CDR3 region of CD4 abolish cell fusion activity, and they suggest that a wide variety of sequences can be functionally tolerated in this region, including those from highly divergent mammalian species. Syncytium formation mediated by several of the CDR3 mutants was partially or completely resistant to inhibition by the CDR3-directed monoclonal antibody L71, suggesting that the corresponding epitope is not directly involved in the fusion process.(ABSTRACT TRUNCATED AT 400 WORDS) Images PMID:8419649

CD4 molecules with a diversity of mutations encompassing the CDR3 region efficiently support human immunodeficiency virus type 1 envelope glycoprotein-mediated cell fusion.

PubMed

Broder, C C; Berger, E A

1993-02-01

The third complementarity-determining region (CDR3) within domain 1 of the human CD4 molecule has been suggested to play a critical role in membrane fusion mediated by the interaction of CD4 with the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein. To analyze in detail the role of CDR3 and adjacent regions in the fusion process, we used cassette mutagenesis to construct a panel of 30 site-directed mutations between residues 79 and 96 of the full-length CD4 molecule. The mutant proteins were transiently expressed by using recombinant vaccinia virus vectors and were analyzed for cell surface expression, recombinant gp120-binding activity, and overall structural integrity as assessed by reactivity with a battery of anti-CD4 monoclonal antibodies. Cells expressing the CD4 mutants were assayed for their ability to form syncytia when mixed with cells expressing the HIV-1 envelope glycoprotein. Surprisingly in view of published data from others, most of the mutations had little effect on syncytium-forming activity. Normal fusion was observed in 21 mutants, including substitution of human residues 85 to 95 with the corresponding sequences from either chimpanzee, rhesus, or mouse CD4; a panel of Ser-Arg double insertions after each residue from 86 to 91; and a number of other charge, hydrophobic, and proline substitutions and insertions within this region. The nine mutants that showed impaired fusion all displayed defective gp120 binding and disruption of overall structural integrity. In further contrast with results of other workers, we observed that transformant human cell lines expressing native chimpanzee or rhesus CD4 efficiently formed syncytia when mixed with cells expressing the HIV-1 envelope glycoprotein. These data refute the conclusion that certain mutations in the CDR3 region of CD4 abolish cell fusion activity, and they suggest that a wide variety of sequences can be functionally tolerated in this region, including those from highly divergent mammalian species. Syncytium formation mediated by several of the CDR3 mutants was partially or completely resistant to inhibition by the CDR3-directed monoclonal antibody L71, suggesting that the corresponding epitope is not directly involved in the fusion process.(ABSTRACT TRUNCATED AT 400 WORDS)

Sensing of hydrophobic cavity of serum albumin by an adenosine analogue: fluorescence correlation and ensemble spectroscopic studies.

PubMed

Nag, Moupriya; Bera, Kallol; Chakraborty, Sandipan; Basak, Soumen

2013-10-05

Adenosine is a naturally occurring purine nucleoside that plays important role in various biochemical processes. We have studied the binding of TNP-Ado (trinitrophenylated-adenosine), a fluorescent analogue of adenosine (which itself is a weak fluorophore), with a model transport protein, bovine serum albumin (BSA). The binding affinity was determined using Fluorescence correlation spectroscopy (FCS) and compared with its value obtained from macroscopic fluorescence spectroscopic studies. Fluorescence and circular dichroism (CD) spectroscopies were employed together with molecular docking study to locate the probable binding site of TNP-Ado on BSA and its effect on the conformation and stability of BSA. Fluorescence studies showed that TNP-Ado binds to BSA in 1:1 stoichiometry via an entropically favoured process. Induced CD spectra revealed that a chiro-optical switching of TNP-Ado occurs upon binding to BSA. Results on urea-induced denaturation of BSA and docking study suggested that the binding site for the ligand is in the hydrophobic subdomain IIA of BSA, consistent with the results of other measurements. This study establishes TNP-Ado as a sensor of hydrophobic regions in proteins like serum albumin, having the capability of detecting a minimum concentration of 140ng/ml protein. FCS measurement of binding interaction of rhodamine-labeled TNP-Ado (RTNP-Ado) with BSA yielded an association constant of KFCS=(1.03±0.06) × 10(4)M(-1). The association constants (Ka) obtained for binding of BSA with rhodamine-free (i.e. TNP-Ado) and rhodamine-labeled (RTNP-Ado) ligands, obtained using the ensemble spectroscopic technique, were (2.3±0.06) × 10(5)M(-1) and (3.4±0.03) × 10(4)M(-1), respectively. The difference between the values of Ka for the free and labeled ligands suggests that fluorescent labeling of small molecules perceptibly interferes with the binding process. On the other hand, the difference in Ka obtained by FCS and ensemble techniques is due to the fact that while the former measures the change in the diffusion constant (i.e. size) of RTNP-Ado upon binding to BSA, the latter focuses on the change of tryptophan emission properties of BSA due to the presence of bound RTNP-Ado. Copyright © 2013 Elsevier B.V. All rights reserved.

The investigation of the binding of 6-mercaptopurine to site I on human serum albumin.

PubMed

Sochacka, Jolanta; Baran, Wojciech

2012-12-01

6-Mercaptopurine (6-MP) is one of a large series of purine analogues which has been found active against human leukemias. The equilibrium dialysis, circular dichroism (CD) and molecular docking were employed to study the binding of 6-MP to human serum albumin (HSA). The binding of 6-MP to HSA in the equilibrium dialysis experiment was detected by measuring the displacement of 6-MP by specific markers for site I on HSA, warfarin (RWF), phenylbutazone (PhB) and n-butyl p-aminobenzoate (ABE). It was shown, according to CD data, that binding of 6-MP to HSA leads to alteration of HSA secondary structure. Based on the findings from displacement experiment and molecular docking simulation it was found that 6-MP was located within binding cavity of subdomain IIA and the space occupied by site markers overlapped with that of 6-MP. Displacement of 6-MP by the RWF or PhB was not up the level expected for a competitive mechanism, therefore displacement of 6-MP was rather by non-cooperative than that the direct competition. Instead, in case of the interaction between ABE and 6-MP, when the little enhancement of the binding of ABE by 6-MP was found, the interaction could be via a positively cooperative mechanism.

Competitive binding of (-)-epigallocatechin-3-gallate and 5-fluorouracil to human serum albumin: A fluorescence and circular dichroism study

NASA Astrophysics Data System (ADS)

Yuan, Lixia; Liu, Min; Liu, Guiqin; Li, Dacheng; Wang, Zhengping; Wang, Bingquan; Han, Jun; Zhang, Min

2017-02-01

Combination therapy with more than one therapeutic agent can improve therapeutic efficiency and decrease drug resistance. In this study, the interactions of human serum albumin (HSA) with individual or combined anticancer drugs, (-)-epigallocatechin-3-gallate (EGCG) and 5-fluorouracil (FU), were investigated by fluorescence and circular dichroism (CD) spectroscopy. The results demonstrated that the interaction of EGCG or FU with HSA is a process of static quenching and EGCG formed a more stable complex. The competitive experiments of site markers suggested that both anti-carcinogens mainly bound to site I (subdomain IIA). The interaction forces which play important roles in the binding process were discussed based on enthalpy and entropy changes. Moreover, the competition binding model for a ternary system was proposed so as to precisely calculate the binding parameters. The results demonstrated that one drug decreased the binding affinity of another drug with HSA, resulting in the increasing free drug concentration at the action sites. CD studies indicated that there was an alteration in HSA secondary structure due to the binding of EGCG and FU. It can be concluded that the combination of EGCG with FU may enhance anticancer efficacy. This finding may provide a theoretical basis for clinical treatments.

CD9 tetraspanin generates fusion competent sites on the egg membrane for mammalian fertilization

PubMed Central

Jégou, Antoine; Ziyyat, Ahmed; Barraud-Lange, Virginie; Perez, Eric; Wolf, Jean Philippe; Pincet, Frédéric; Gourier, Christine

2011-01-01

CD9 tetraspanin is the only egg membrane protein known to be essential for fertilization. To investigate its role, we have measured, on a unique acrosome reacted sperm brought in contact with an egg, the adhesion probability and strength with a sensitivity of a single molecule attachment. Probing the binding events at different locations of wild-type egg we described different modes of interaction. Here, we show that more gamete adhesion events occur on Cd9 null eggs but that the strongest interaction mode disappears. We propose that sperm–egg fusion is a direct consequence of CD9 controlled sperm–egg adhesion properties. CD9 generates adhesion sites responsible for the strongest of the observed gamete interaction. These strong adhesion sites impose, during the whole interaction lifetime, a tight proximity of the gamete membranes, which is a requirement for fusion to take place. The CD9-induced adhesion sites would be the actual location where fusion occurs. PMID:21690351

TMEM126A, a CD137 ligand binding protein, couples with the TLR4 signal transduction pathway in macrophages.

PubMed

Kim, Eun-Cheol; Moon, Ji-Hoi; Kang, Sang W; Kwon, Byungsuk; Lee, Hyeon-Woo

2015-04-01

We showed previously that a novel protein, transmembrane protein 126A (TMEM126A), binds to CD137 ligand (CD137L, 4-1BBL) and couples with its reverse signals in macrophages. Here, we present data showing that TMEM126A relays TLR4 signaling. Thus, up-regulation of CD54 (ICAM-1), MHC II, CD86 and CD40 expression in response to TLR4 activation was diminished in TMEM126A-deficient macrophages. Moreover in TMEM126A-deficient RAW264.7 cells, LPS/TLR4-induced late-phase JNK/SAPK and IRF-3 phosphorylation was abolished. These findings indicate that TMEM126A contributes to the TLR4 signal up-regulating the expression of genes whose products are involved in antigen presentation. Copyright © 2014 Elsevier Ltd. All rights reserved.

Differential Lectin Binding Patterns Identify Distinct Heart Regions in Giant Danio (Devario aequipinnatus) and Zebrafish (Danio rerio) Hearts

PubMed Central

Manalo, Trina; May, Adam; Quinn, Joshua; Lafontant, Dominique S.; Shifatu, Olubusola; He, Wei; Gonzalez-Rosa, Juan M.; Burns, Geoffrey C.; Burns, Caroline E.; Burns, Alan R.; Lafontant, Pascal J.

2016-01-01

Lectins are carbohydrate-binding proteins commonly used as biochemical and histochemical tools to study glycoconjugate (glycoproteins, glycolipids) expression patterns in cells, tissues, including mammalian hearts. However, lectins have received little attention in zebrafish (Danio rerio) and giant danio (Devario aequipinnatus) heart studies. Here, we sought to determine the binding patterns of six commonly used lectins—wheat germ agglutinin (WGA), Ulex europaeus agglutinin, Bandeiraea simplicifolia lectin (BS lectin), concanavalin A (Con A), Ricinus communis agglutinin I (RCA I), and Lycopersicon esculentum agglutinin (tomato lectin)—in these hearts. Con A showed broad staining in the myocardium. WGA stained cardiac myocyte borders, with binding markedly stronger in the compact heart and bulbus. BS lectin, which stained giant danio coronaries, was used to measure vascular reconstruction during regeneration. However, BS lectin reacted poorly in zebrafish. RCA I stained the compact heart of both fish. Tomato lectin stained the giant danio, and while low reactivity was seen in the zebrafish ventricle, staining was observed in their transitional cardiac myocytes. In addition, we observed unique staining patterns in the developing zebrafish heart. Lectins’ ability to reveal differential glycoconjugate expression in giant danio and zebrafish hearts suggests they can serve as simple but important tools in studies of developing, adult, and regenerating fish hearts. PMID:27680670

CD4/CD8/Dendritic cell complexes in the spleen: CD8+ T cells can directly bind CD4+ T cells and modulate their response

PubMed Central

Barinov, Aleksandr; Galgano, Alessia; Krenn, Gerald; Tanchot, Corinne; Vasseur, Florence

2017-01-01

CD4+ T cell help to CD8+ T cell responses requires that CD4+ and CD8+ T cells interact with the same antigen presenting dendritic cell (Ag+DC), but it remains controversial whether helper signals are delivered indirectly through a licensed DC and/or involve direct CD4+/CD8+ T cell contacts and/or the formation of ternary complexes. We here describe the first in vivo imaging of the intact spleen, aiming to evaluate the first interactions between antigen-specific CD4+, CD8+ T cells and Ag+DCs. We show that in contrast to CD4+ T cells which form transient contacts with Ag+DC, CD8+ T cells form immediate stable contacts and activate the Ag+DC, acquire fragments of the DC membranes by trogocytosis, leading to their acquisition of some of the DC properties. They express MHC class II, and become able to present the specific Marilyn peptide to naïve Marilyn CD4+ T cells, inducing their extensive division. In vivo, these CD8+ T cells form direct stable contacts with motile naïve CD4+ T cells, recruiting them to Ag+DC binding and to the formation of ternary complexes, where CD4+ and CD8+ T cells interact with the DC and with one another. The presence of CD8+ T cells during in vivo immune responses leads to the early activation and up-regulation of multiple functions by CD4+ T lymphocytes. Thus, while CD4+ T cell help is important to CD8+ T cell responses, CD8+ T cells can interact directly with naïve CD4+ T cells impacting their recruitment and differentiation. PMID:28686740

Novel highly luminescent amine-functionalized bridged silsesquioxanes

NASA Astrophysics Data System (ADS)

Pereira, Rui F. P.; Nunes, Sílvia C.; Toquer, Guillaume; Cardoso, Marita A.; Valente, Artur J. M.; Ferro, Marta C.; Silva, Maria M.; Carlos, Luís D.; Ferreira, Rute A. S.; de Zea Bermudez, Verónica

2017-12-01

Amine-functionalized bridged silsesquioxanes were synthesized from bis[(3-trimethoxysilyl)propyl] amine via a solvent-mediated route. BS-1 and BS-2 were obtained with neutral pH with sub- and stoichiometric amounts of water, respectively, and high tetrahydrofuran content. BS-3 was prepared with hyperstoichiometric water concentration, high tetrahydrofuran content and hydrochloric acid. BS-4 was synthesized with hyperstoichiometric water concentration, high ethanol content and sodium hydroxide. BS-1 and BS-2 were produced as transparent films, whereas BS-3 and BS-4 formed white powders. Face-to-face stacking of flat or folded lamellae yielded quasi-hydrophobic platelets with emission quantum yields of 0.05±0.01 (BS-1 and BS-2) or superhydrophilic onion-like nanoparticles with exciting emission quantum yields of 0.38±0.03 (BS-3) and 0.33±0.04 (BS-4), respectively. The latter two values are the largest ever reported for amine-functionalized siloxane-based hybrids lacking aromatic groups. Fast Grotthus proton hopping between =NH2+/=NH groups (BS-3) and =N-/=NH groups (BS-4), promoted by H+ and OH- ions, respectively, and aided by short amine-amine contacts provided by the onion-like morphology, account for this unique optical behavior.

Single N277A substitution in C2 of simian immunodeficiency virus envelope influences vaccine-elicited CD4i neutralizing and anti-V2 antibody responses.

PubMed

Tang, Xian; Guo, Jia; Cheng, Lin; Sun, Caijun; Liu, Li; Zuo, Teng; Wang, Hui; Chen, Ling; Zhang, Linqi; Chen, Zhiwei

2017-05-02

An effective HIV vaccine remains elusive, and immunogens capable of eliciting protective host humoral immunity have not yet been identified. Although HIV/SIV infections result in the abundant production of CD4-induced (CD4i) antibodies (Abs), these Abs are not protective due to steric restrictions following gp120 binding to CD4 on target cells. Here we report that both DNA- and vaccinia-based vaccines encoding SIV mac239 gp160 readily elicited high levels of CD4i Abs in experimental animals. We identified a highly conserved N-linked glycosylation site N277 in the C2 region which strongly affected the immunogenicity of the CD4i Ab domain. Moreover, a single N277A substitution significantly enhanced the immunogenicity of the V2 domain yielding higher titers and frequency of anti-V2 Ab responses as determined by ELISA and yeast antigen display mapping, respectively. Importantly, immune sera elicited by the N277A-mutated gp160 exhibited elevated antibody-dependent cellular cytotoxicity (ADCC) activity. ADCC activity correlated positively with the anti-V2 Ab titer yet, inversely with CD4i Ab titer. Thus, we identified a determinant of the CD4i domain that might affect vaccine-elicited anti-V2 Ab and ADCC responses to SIV mac239 . Our findings may have implications for design of immunogens to direct B cell recognition in the development of an Ab-based HIV vaccine. Copyright © 2017 Elsevier Ltd. All rights reserved.

The reaction mechanism of methyl-coenzyme M reductase: How an enzyme enforces strict binding order

DOE PAGES

Wongnate, Thanyaporn; Ragsdale, Stephen W.

2015-02-17

Methyl-coenzyme M reductase (MCR) is a nickel tetrahydrocorphinoid (coenzyme F430) containing enzyme involved in the biological synthesis and anaerobic oxidation of methane. MCR catalyzes the conversion of methyl-2-mercaptoethanesulfonate (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoB 7SH) to CH 4 and the mixed disulfide CoBS-SCoM. In this study, the reaction of MCR from Methanothermobacter marburgensis, with its native substrates was investigated using static binding, chemical quench, and stopped-flow techniques. Rate constants were measured for each step in this strictly ordered ternary complex catalytic mechanism. Surprisingly, in the absence of the other substrate, MCR can bind either substrate; however, only one binary complex (MCR·methyl-SCoM)more » is productive whereas the other (MCR·CoB 7SH) is inhibitory. Moreover, the kinetic data demonstrate that binding of methyl-SCoM to the inhibitory MCR·CoB 7SH complex is highly disfavored ( Kd = 56 mM). However, binding of CoB 7SH to the productive MCR·methyl-SCoM complex to form the active ternary complex (CoB 7SH·MCR(Ni I)·CH 3SCoM) is highly favored ( Kd = 79 μM). Only then can the chemical reaction occur ( kobs = 20 s -1 at 25 °C), leading to rapid formation and dissociation of CH 4 leaving the binary product complex (MCR(Ni II)·CoB 7S -·SCoM), which undergoes electron transfer to regenerate Ni(I) and the final product CoBS-SCoM. In conclusion, this first rapid kinetics study of MCR with its natural substrates describes how an enzyme can enforce a strictly ordered ternary complex mechanism and serves as a template for identification of the reaction intermediates.« less

Immune complexes formed following the binding of anti-platelet factor 4 (CXCL4) antibodies to CXCL4 stimulate human neutrophil activation and cell adhesion.

PubMed

Xiao, Zhihua; Visentin, Gian P; Dayananda, Kannayakanahalli M; Neelamegham, Sriram

2008-08-15

We tested the possibility that immune complexes formed following platelet factor 4 (PF4/CXCL4) binding to anti-PF4 antibody can stimulate neutrophil activation, similar to previous reports with platelets. Monoclonal Abs against PF4 and IgG from a heparin-induced thrombocytopenia (HIT) patient were applied. We observed that although PF4 or anti-PF4 antibody alone did not alter neutrophil function, costimulation with both reagents resulted in approximately 3-fold increase in cell surface Mac-1 expression, enhanced cell adhesion via L-selectin and CD18 integrins, and degranulation of secondary and tertiary granules. The level of Mac-1 up-regulation peaked at an intermediate PF4 dose, suggesting that functional response varies with antigen-antibody stoichiometry. PF4 binding to neutrophils was blocked by chondroitinase ABC. Cell activation was inhibited by both chondroitinase ABC and anti-CD32/FcgammaRII blocking mAb, IV.3. Confocal microscopy demonstrated that immune complexes colocalize with CD32a. Studies with HIT IgG demonstrated that neutrophils could be activated in the absence of exogenous heparin. These data, together, show that leukocyte surface chondroitin sulfates promote neutrophil activation by enhancing immune-complex binding to CD32a. Studies with recombinant PF4 suggest a role for arginine 49 in stabilizing PF4-chondroitin binding. Neutrophils activated via this mechanism may contribute to thrombosis and inflammation in patients mounting an immune response to PF4-heparin.

The increased binding affinity of curcumin with human serum albumin in the presence of rutin and baicalin: A potential for drug delivery system

NASA Astrophysics Data System (ADS)

Liu, Bing-Mi; Zhang, Jun; Hao, Ai-Jun; Xu, Liang; Wang, Dan; Ji, Hui; Sun, Shi-Jie; Chen, Bo-Qi; Liu, Bin

2016-02-01

The impacts of rutin and baicalin on the interaction of curcumin (CU) with human serum albumin (HSA) were investigated by fluorescence and circular dichroism (CD) spectroscopies under imitated physiological conditions. The results showed that the fluorescence quenching of HSA by CU was a simultaneous static and dynamic quenching process, irrespective of the presence or absence of flavonoids. The binding constants between CU and HSA in the absence and presence of rutin and baicalin were 2.268 × 105 M- 1, 3.062 × 105 M- 1, and 3.271 × 105 M- 1, indicating that the binding affinity was increased in the case of two flavonoids. Furthermore, the binding distance determined according to Förster's theory was decreased in the presence of flavonoids. Combined with the fact that flavonoids and CU have the same binding site (site I), it can be concluded that they may simultaneously bind in different regions in site I, and formed a ternary complex of flavonoid-HSA-CU. Meanwhile, the results of fluorescence quenching, CD and three-dimensional fluorescence spectra revealed that flavonoids further strengthened the microenvironmental and conformational changes of HSA induced by CU binding. Therefore, it is possible to develop a novel complex involving CU, flavonoid and HSA for CU delivery. The work may provide some valuable information in terms of improving the poor bioavailabiliy of CU.

In vivo emergence of HIV-1 highly sensitive to neutralizing antibodies.

PubMed

Aasa-Chapman, Marlén M I; Cheney, Kelly M; Hué, Stéphane; Forsman, Anna; O'Farrell, Stephen; Pellegrino, Pierre; Williams, Ian; McKnight, Áine

2011-01-01

The rapid and continual viral escape from neutralizing antibodies is well documented in HIV-1 infection. Here we report in vivo emergence of viruses with heightened sensitivity to neutralizing antibodies, sometimes paralleling the development of neutralization escape. Sequential viral envs were amplified from seven HIV-1 infected men monitored from seroconversion up to 5 years after infection. Env-recombinant infectious molecular clones were generated and tested for coreceptor use, macrophage tropism and neutralization sensitivity to homologous and heterologous serum, soluble CD4 and monoclonal antibodies IgG1b12, 2G12 and 17b. We found that HIV-1 evolves sensitivity to contemporaneous neutralizing antibodies during infection. Neutralization sensitive viruses grow out even when potent autologous neutralizing antibodies are present in patient serum. Increased sensitivity to neutralization was associated with susceptibility of the CD4 binding site or epitopes induced after CD4 binding, and mediated by complex envelope determinants including V3 and V4 residues. The development of neutralization sensitive viruses occurred without clinical progression, coreceptor switch or change in tropism for primary macrophages. We propose that an interplay of selective forces for greater virus replication efficiency without the need to resist neutralizing antibodies in a compartment protected from immune surveillance may explain the temporal course described here for the in vivo emergence of HIV-1 isolates with high sensitivity to neutralizing antibodies.

Polymeric Cd(II), trinuclear and mononuclear Ni(II) complexes of 5-methyl-4-phenyl-1,2,4-triazole-3-thione: Synthesis, structural characterization, thermal behaviour, fluorescence properties and antibacterial activity

NASA Astrophysics Data System (ADS)

Bharty, M. K.; Paswan, S.; Dani, R. K.; Singh, N. K.; Sharma, V. K.; Kharwar, R. N.; Butcher, R. J.

2017-02-01

Syntheses of a polymeric Cd(II) complex, [Cd(mptt)2]n (1), a trinuclear Ni(II) complex, [Ni3(μ-mptt)4(μ-H2O)2(H2O)2(ttfa)2]·3H2O (2) and a mononuclear Ni(II) complex [Ni(mptt)2(en)2] (3) have been performed using the ligand 5-methyl-4-phenyl-1,2,4-triazole-3-thione (Hmptt) and nickel(II)/cadmium(II) salts {ttfa = thenoyltrifluroacetonate). The ligand and the complexes have been characterized by various physicochemical methods in addition to their single crystal X-ray structure. The Cd centre in complex 1 adopts a distorted tetrahedral geometry with one sulfur atom and two mptt ligands provide three nitrogen atoms from three triazole units. The sulfur atom of the ligand binds covalently and overall the ligand acts as uninigative N,S/N,N bidentate moiety. The polymeric structure of complex 1 results from the N atoms of the neighboring triazole units coordinating with the Cd(II) centre. The three Ni(II) centres in the trinuclear Ni(II) complex 2 form a linear arrangement and all have six coordinated arrangements. The middle Ni(II) binds with four deprotonated triazole ring nitrogens and two water molecules form two bridges. The terminal Ni(II) centres bind through two thenoyl oxygens, two triazole nitrogens and water molecules that formed bridges with the middle Ni centre. In complex 3, the nickel(II) centre is covalently bonded through two deprotonated triazole ring nitrogens from two ligand moieties and other four sites are occupied by four nitrogens from two bidentate en ligands. Thermogravimetric analyses (TGA) of the complexes indicated for NiO as the final residue. The bioefficacy of the ligand and complexes 2 and 3 have been examined against the growth of bacteria to evaluate their anti-microbial potential. Complex 2 showed high antibacterial activity as compared to the ligand and complex 3. Complexes 1, 2 and 3 are fluorescent materials with maximum emissions at 425, 421 and 396 nm at an excitation wavelength of 323, 348 and 322 nm, respectively.

Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

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

Costantini, Lindsey M.; Irvin, Susan C.; Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461

The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFPmore » enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. - Highlights: • Development of fluorescent protein labeled HIV-1 envelope gp120. • Imaging of gp120 dynamics and trafficking in live cells. • Quantitative visual assay of antibody-mediated inhibition of gp120 binding to CD4 on live cells.« less

Identification of residues of CXCR4 critical for human immunodeficiency virus coreceptor and chemokine receptor activities.

PubMed

Brelot, A; Heveker, N; Montes, M; Alizon, M

2000-08-04

CXCR4 is a G-coupled receptor for the stromal cell-derived factor (SDF-1) chemokine, and a CD4-associated human immunodeficiency virus type 1 (HIV-1) coreceptor. These functions were studied in a panel of CXCR4 mutants bearing deletions in the NH(2)-terminal extracellular domain (NT) or substitutions in the NT, the extracellular loops (ECL), or the transmembrane domains (TMs). The coreceptor activity of CXCR4 was markedly impaired by mutations of two Tyr residues in NT (Y7A/Y12A) or at a single Asp residue in ECL2 (D193A), ECL3 (D262A), or TMII (D97N). These acidic residues could engage electrostatical interactions with basic residues of the HIV-1 envelope protein gp120, known to contribute to the selectivity for CXCR4. The ability of CXCR4 mutants to bind SDF-1 and mediate cell signal was consistent with the two-site model of chemokine-receptor interaction. Site I involved in SDF-1 binding but not signaling was located in NT with particular importance of Glu(14) and/or Glu(15) and Tyr(21). Residues required for both SDF-1 binding and signaling, and thus probably part of site II, were identified in ECL2 (Asp(187)), TMII (Asp(97)), and TMVII (Glu(288)). The first residues () of NT also seem required for SDF-1 binding and signaling. A deletion in the third intracellular loop abolished signaling, probably by disrupting the coupling with G proteins. The identification of CXCR4 residues involved in the interaction with both SDF-1 and HIV-1 may account for the signaling activity of gp120 and has implications for the development of antiviral compounds.

Spectroscopic studies of the interaction mechanisms between mono-caffeoylquinic acids and transferrin

NASA Astrophysics Data System (ADS)

Guan, Yanqing; Dong, Jing; Chen, Shizhong; Liu, Meixian; Wang, Daidong; Zhang, Xiaotian; Wang, Hong; Lin, Zongtao

2017-06-01

Transferrin (Tf) is an important protein responsible for circulating and transporting iron into cytoplasm. Tf can be taken into cells through endocytosis mediated by Tf receptor, which usually overexpresses in cancer cells. The Tf-Tf receptor pathway opens a possible avenue for novel targeted cancer therapy by utilizing Tf-binding active compounds. Among which, anti-cancer active caffeoylquinic acids (CQAs) were recently found to be promising Tf-binders by our group. For better understanding the anti-cancer activities of CQAs, it is important to unveil the binding mechanisms between CQAs and Tf. In this study, the fluorescence quenching, surface plasmon resonance (SPR), circular dichroism (CD) and molecular docking were used to investigate the interactions between CQA and Tf. The results showed that the calculated apparent association constants of interactions between 1-, 3-, 4- and 5-CQA and Tf at 298 K were 7.97 × 105 M- 1, 4.36 × 107 M- 1, 6.58 × 105 M- 1 and 4.42 × 106 M- 1, respectively. The thermodynamic parameters indicated that the interaction between 1-, 3-, 5-CQA and Tf is due to H-bonding, and electrostatic interactions were likely involved in the binding of 4-CQA and Tf. The CD results indicated that bindings of 1-CQA, 4-CQA and 5-CQA with Tf resulted in more stretched β-turn and random coil translated from β-sheet. In contrast, 3-CQA led to more stable a-helix conformation. Molecular docking studies of CQAs with Tf further displayed that CQAs were able to interact with residues near Fe3 + binding site. The spectroscopic studies revealed the action mechanisms, thermodynamics and interacting forces between CQAs and Tf, and thus are helpful for future design and discovery of Tf-binders for targeted cancer therapy applying Tf-Tf receptor pathway.

The pepper Bs4C proteins are localized to the endoplasmic reticulum (ER) membrane and confer disease resistance to bacterial blight in transgenic rice.

PubMed

Wang, Jun; Zeng, Xuan; Tian, Dongsheng; Yang, Xiaobei; Wang, Lanlan; Yin, Zhongchao

2018-03-30

Transcription activator-like effector (TALE)-dependent dominant disease resistance (R) genes in plants, also referred to as executor R genes, are induced on infection by phytopathogenic bacteria of the genus Xanthomonas harbouring the corresponding TALE genes. Unlike the traditional R proteins, the executor R proteins do not determine the resistance specificity and may function broadly in different plant species. The executor R gene Bs4C-R in the resistant genotype PI 235047 of the pepper species Capsicum pubescens (CpBs4C-R) confers disease resistance to Xanthomonas campestris pv. vesicatoria (Xcv) harbouring the TALE genes avrBsP/avrBs4. In this study, the synthetic genes of CpBs4C-R and two other Bs4C-like genes, the susceptible allele in the genotype PI585270 of C. pubescens (CpBs4C-S) and the CaBs4C-R homologue gene in the cultivar 'CM334' of Capsicum annum (CaBs4C), were characterized in tobacco (Nicotiana benthamiana) and rice (Oryza sativa). The Bs4C genes induced cell death in N. benthamiana. The functional Bs4C-eCFP fusion proteins were localized to the endoplasmic reticulum (ER) membrane in the leaf epidermal cells of N. benthamiana. The Xa10 promoter-Bs4C fusion genes in transgenic rice conferred strain-specific disease resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight in rice, and were specifically induced by the Xa10-incompatible Xoo strain PXO99 A (pHM1avrXa10). The results indicate that the Bs4C proteins from pepper species function broadly in rice and the Bs4C protein-mediated cell death from the ER is conserved between dicotyledonous and monocotyledonous plants, which can be utilized to engineer novel and enhanced disease resistance in heterologous plants. © 2018 TEMASEK LIFE SCIENCES LABORATORY. MOLECULAR PLANT PATHOLOGY © 2018 JOHN WILEY & SONS LTD.

Comparison and analysis on the serum-binding characteristics of aspirin-zinc complex and aspirin.

PubMed

Zhang, Hua-Xin; Zhang, Qun; Wang, Hong-Lin; Li, Li-Wei

2017-09-01

This study was designed to compare the protein-binding characteristics of aspirin-zinc complex (AZN) with those of aspirin itself. AZN was synthesized and interacted with a model transport protein, human serum albumin (HSA). Three-dimensional fluorescence, ultraviolet-visible and circular dichroism (CD) spectra were used to characterize the interaction of AZN with HSA under physiological conditions. The interaction mechanism was explored using a fluorescence quenching method and thermodynamic calculation. The binding site and binding locality of AZN on HSA were demonstrated using a fluorescence probe technique and Förster non-radiation energy transfer theory. Synchronous fluorescence and CD spectra were employed to reveal the effect of AZN on the native conformation of the protein. The HSA-binding results for AZN were compared with those for aspirin under consistent experimental conditions, and indicated that aspirin acts as a guide in AZN when binding to Sudlow's site I, in subdomain IIA of the HSA molecule. Moreover, compared with aspirin, AZN showed greater observed binding constants with, but smaller changes in the α-helicity of, HSA, which proved that AZN might be easier to transport and have less toxicity in vivo. Copyright © 2017 John Wiley & Sons, Ltd.

Visualization of novel virulence activities of the Xanthomonas type III effectors AvrBs1, AvrBs3 and AvrBs4.

PubMed

Gürlebeck, Doreen; Jahn, Simone; Gürlebeck, Norman; Szczesny, Robert; Szurek, Boris; Hahn, Simone; Hause, Gerd; Bonas, Ulla

2009-03-01

Xanthomonas campestris pv. vesicatoria secretes at least 20 effector proteins through the type III secretion system directly into plant cells. In this study, we uncovered virulence activities of the effector proteins AvrBs1, AvrBs3 and AvrBs4 using Agrobacterium-mediated transient expression of the corresponding genes in Nicotiana benthamiana, followed by microscopic analyses. We showed that, in addition to the nuclear-localized AvrBs3, the effector AvrBs1, which localizes to the plant cell cytoplasm, also induces a morphological change in mesophyll cells. Comparative analyses revealed that avrBs3-expressing plant cells contain highly active nuclei. Furthermore, plant cells expressing avrBs3 or avrBs1 show a decrease in the starch content in chloroplasts and an increased number of vesicles, indicating an enlargement of the central vacuole and the cell wall. Both AvrBs1 and AvrBs3 cause an increased ion efflux when expressed in N. benthamiana. By contrast, expression of the avrBs3 homologue avrBs4 leads to large catalase crystals in peroxisomes, suggesting a possible virulence function of AvrBs4 in the suppression of the plant defence responses. Taken together, our data show that microscopic inspection can uncover subtle and novel virulence activities of type III effector proteins.

Histidine pairing at the metal transport site of mammalian ZnT transporters controls Zn2+ over Cd2+ selectivity.

PubMed

Hoch, Eitan; Lin, Wei; Chai, Jin; Hershfinkel, Michal; Fu, Dax; Sekler, Israel

2012-05-08

Zinc and cadmium are similar metal ions, but though Zn(2+) is an essential nutrient, Cd(2+) is a toxic and common pollutant linked to multiple disorders. Faster body turnover and ubiquitous distribution of Zn(2+) vs. Cd(2+) suggest that a mammalian metal transporter distinguishes between these metal ions. We show that the mammalian metal transporters, ZnTs, mediate cytosolic and vesicular Zn(2+) transport, but reject Cd(2+), thus constituting the first mammalian metal transporter with a refined selectivity against Cd(2+). Remarkably, the bacterial ZnT ortholog, YiiP, does not discriminate between Zn(2+) and Cd(2+). A phylogenetic comparison between the tetrahedral metal transport motif of YiiP and ZnTs identifies a histidine at the mammalian site that is critical for metal selectivity. Residue swapping at this position abolished metal selectivity of ZnTs, and fully reconstituted selective Zn(2+) transport of YiiP. Finally, we show that metal selectivity evolves through a reduction in binding but not the translocation of Cd(2+) by the transporter. Thus, our results identify a unique class of mammalian transporters and the structural motif required to discriminate between Zn(2+) and Cd(2+), and show that metal selectivity is tuned by a coordination-based mechanism that raises the thermodynamic barrier to Cd(2+) binding.

Histidine pairing at the metal transport site of mammalian ZnT transporters controls Zn2+ over Cd2+ selectivity

PubMed Central

Hoch, Eitan; Lin, Wei; Chai, Jin; Hershfinkel, Michal; Fu, Dax; Sekler, Israel

2012-01-01

Zinc and cadmium are similar metal ions, but though Zn2+ is an essential nutrient, Cd2+ is a toxic and common pollutant linked to multiple disorders. Faster body turnover and ubiquitous distribution of Zn2+ vs. Cd2+ suggest that a mammalian metal transporter distinguishes between these metal ions. We show that the mammalian metal transporters, ZnTs, mediate cytosolic and vesicular Zn2+ transport, but reject Cd2+, thus constituting the first mammalian metal transporter with a refined selectivity against Cd2+. Remarkably, the bacterial ZnT ortholog, YiiP, does not discriminate between Zn2+ and Cd2+. A phylogenetic comparison between the tetrahedral metal transport motif of YiiP and ZnTs identifies a histidine at the mammalian site that is critical for metal selectivity. Residue swapping at this position abolished metal selectivity of ZnTs, and fully reconstituted selective Zn2+ transport of YiiP. Finally, we show that metal selectivity evolves through a reduction in binding but not the translocation of Cd2+ by the transporter. Thus, our results identify a unique class of mammalian transporters and the structural motif required to discriminate between Zn2+ and Cd2+, and show that metal selectivity is tuned by a coordination-based mechanism that raises the thermodynamic barrier to Cd2+ binding. PMID:22529353

Combining transcription factor binding affinities with open-chromatin data for accurate gene expression prediction.

PubMed

Schmidt, Florian; Gasparoni, Nina; Gasparoni, Gilles; Gianmoena, Kathrin; Cadenas, Cristina; Polansky, Julia K; Ebert, Peter; Nordström, Karl; Barann, Matthias; Sinha, Anupam; Fröhler, Sebastian; Xiong, Jieyi; Dehghani Amirabad, Azim; Behjati Ardakani, Fatemeh; Hutter, Barbara; Zipprich, Gideon; Felder, Bärbel; Eils, Jürgen; Brors, Benedikt; Chen, Wei; Hengstler, Jan G; Hamann, Alf; Lengauer, Thomas; Rosenstiel, Philip; Walter, Jörn; Schulz, Marcel H

2017-01-09

The binding and contribution of transcription factors (TF) to cell specific gene expression is often deduced from open-chromatin measurements to avoid costly TF ChIP-seq assays. Thus, it is important to develop computational methods for accurate TF binding prediction in open-chromatin regions (OCRs). Here, we report a novel segmentation-based method, TEPIC, to predict TF binding by combining sets of OCRs with position weight matrices. TEPIC can be applied to various open-chromatin data, e.g. DNaseI-seq and NOMe-seq. Additionally, Histone-Marks (HMs) can be used to identify candidate TF binding sites. TEPIC computes TF affinities and uses open-chromatin/HM signal intensity as quantitative measures of TF binding strength. Using machine learning, we find low affinity binding sites to improve our ability to explain gene expression variability compared to the standard presence/absence classification of binding sites. Further, we show that both footprints and peaks capture essential TF binding events and lead to a good prediction performance. In our application, gene-based scores computed by TEPIC with one open-chromatin assay nearly reach the quality of several TF ChIP-seq data sets. Finally, these scores correctly predict known transcriptional regulators as illustrated by the application to novel DNaseI-seq and NOMe-seq data for primary human hepatocytes and CD4+ T-cells, respectively. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Predominant nonproductive substrate binding by fungal cellobiohydrolase I and implications for activity improvement.

PubMed

Rabinovich, Mikhail L; Melnik, Maria S; Herner, Mikhail L; Voznyi, Yakov V; Vasilchenko, Lilia G

2018-05-21

Enzymatic conversion of the most abundant renewable source of organic compounds, cellulose to fermentable sugars is attractive for production of green fuels and chemicals. The major component of industrial enzyme systems, cellobiohydrolase I from Hypocrea jecorina (Trichoderma reesei) (HjCel7A) processively splits disaccharide units from the reducing ends of tightly packed cellulose chains. HjCel7A consists of a catalytic domain (CD) and a carbohydrate-binding module (CBM) separated by a linker peptide. A tunnel-shaped substrate-binding site in the CD includes 9 subsites for β-D-glucose units, 7 of which (-7 to -1) precede the catalytic center. Low catalytic activity of Cel7A is the bottleneck and the primary target for improvement. Here it is shown for the first time that, in spite of much lower apparent k cat of HjCel7A at the hydrolysis of β-1,4-glucosidic linkages in the fluorogenic cellotetra- and -pentaose compared to the structurally related endoglucanase I (HjCel7B), the specificity constants (catalytic efficiency) k cat /K m for both enzymes are almost equal in these reactions. The observed activity difference appears from strong nonproductive substrate binding by HjCel7A, particularly significant for MU-β-cellotetraose (MUG 4 ). Interaction of substrates with the subsites -6 and -5 proximal to the non-conserved Gln101 residue in HjCel7A decreases K m,ap by >1500 times. HjCel7A can be nonproductively bound onto cellulose surface with K d ∼2-9 nM via CBM and CD that captures 6 terminal glucose units of cellulose chain. Decomposition of this nonproductive complex can determine the rate of cellulose conversion. MUG 4 is a promising substrate to select active cellobiohydrolase I variants with reduced nonproductive substrate binding. This article is protected by copyright. All rights reserved.

The Human Membrane Cofactor CD46 Is a Receptor for Species B Adenovirus Serotype 3

PubMed Central

Sirena, Dominique; Lilienfeld, Benjamin; Eisenhut, Markus; Kälin, Stefan; Boucke, Karin; Beerli, Roger R.; Vogt, Lorenz; Ruedl, Christiane; Bachmann, Martin F.; Greber, Urs F.; Hemmi, Silvio

2004-01-01

Many human adenovirus (Ad) serotypes use the coxsackie B virus-Ad receptor (CAR). Recently, CD46 was suggested to be a receptor of species B Ad serotype 11 (Ad11), Ad14, Ad16, Ad21, Ad35, and Ad50. Using Sindbis virus-mediated cDNA library expression, we identify here the membrane cofactor protein CD46 as a surface receptor of species B Ad3. All four major CD46 transcripts and one minor CD46 transcript expressed in nucleated human cells were isolated. Rodent BHK cells stably expressing the BC1 form of CD46 bound radiolabeled Ad3 with a dissociation constant of 0.3 nM, identical to that of CD46-positive HeLa cells expressing twice as many Ad3 binding sites. Pull-down experiments with recombinant Ad3 fibers and a soluble form of the CD46 extracellular domain linked to the Fc portion of human immunoglobulin G (CD46ex-Fc) indicated direct interactions of the Ad3 fiber knob with CD46ex-Fc but not CARex-Fc (Fc-linked extracellular domain of CAR). Ad3 colocalized with cell surface CD46 in both rodent and human cells at the light and electron microscopy levels. Anti-CD46 antibodies and CD46ex-Fc inhibited Ad3 binding to CD46-expressing BHK cells more than 10-fold and to human cells 2-fold. In CD46-expressing BHK cells, wild-type Ad3 and a chimeric Ad consisting of the Ad5 capsid and the Ad3 fiber elicited dose-dependent cytopathic effects and transgene expression, albeit less efficiently than in human cells. Together, our results show that all of the major splice forms of CD46 are predominant and functional binding sites of Ad3 on CD46-expressing rodent and human cells but may not be the sole receptor of species B Ads on human cells. These results have implications for understanding viral pathogenesis and therapeutic gene delivery. PMID:15078926

The human membrane cofactor CD46 is a receptor for species B adenovirus serotype 3.

PubMed

Sirena, Dominique; Lilienfeld, Benjamin; Eisenhut, Markus; Kälin, Stefan; Boucke, Karin; Beerli, Roger R; Vogt, Lorenz; Ruedl, Christiane; Bachmann, Martin F; Greber, Urs F; Hemmi, Silvio

2004-05-01

Many human adenovirus (Ad) serotypes use the coxsackie B virus-Ad receptor (CAR). Recently, CD46 was suggested to be a receptor of species B Ad serotype 11 (Ad11), Ad14, Ad16, Ad21, Ad35, and Ad50. Using Sindbis virus-mediated cDNA library expression, we identify here the membrane cofactor protein CD46 as a surface receptor of species B Ad3. All four major CD46 transcripts and one minor CD46 transcript expressed in nucleated human cells were isolated. Rodent BHK cells stably expressing the BC1 form of CD46 bound radiolabeled Ad3 with a dissociation constant of 0.3 nM, identical to that of CD46-positive HeLa cells expressing twice as many Ad3 binding sites. Pull-down experiments with recombinant Ad3 fibers and a soluble form of the CD46 extracellular domain linked to the Fc portion of human immunoglobulin G (CD46ex-Fc) indicated direct interactions of the Ad3 fiber knob with CD46ex-Fc but not CARex-Fc (Fc-linked extracellular domain of CAR). Ad3 colocalized with cell surface CD46 in both rodent and human cells at the light and electron microscopy levels. Anti-CD46 antibodies and CD46ex-Fc inhibited Ad3 binding to CD46-expressing BHK cells more than 10-fold and to human cells 2-fold. In CD46-expressing BHK cells, wild-type Ad3 and a chimeric Ad consisting of the Ad5 capsid and the Ad3 fiber elicited dose-dependent cytopathic effects and transgene expression, albeit less efficiently than in human cells. Together, our results show that all of the major splice forms of CD46 are predominant and functional binding sites of Ad3 on CD46-expressing rodent and human cells but may not be the sole receptor of species B Ads on human cells. These results have implications for understanding viral pathogenesis and therapeutic gene delivery.

Activity of the HIV-1 Attachment Inhibitor BMS-626529, the Active Component of the Prodrug BMS-663068, against CD4-Independent Viruses and HIV-1 Envelopes Resistant to Other Entry Inhibitors

PubMed Central

Li, Zhufang; Zhou, Nannan; Sun, Yongnian; Ray, Neelanjana; Lataillade, Max; Hanna, George J.

2013-01-01

BMS-626529 is a novel small-molecule HIV-1 attachment inhibitor active against both CCR5- and CXCR4-tropic viruses. BMS-626529 functions by preventing gp120 from binding to CD4. A prodrug of this compound, BMS-663068, is currently in clinical development. As a theoretical resistance pathway to BMS-663068 could be the development of a CD4-independent phenotype, we examined the activity of BMS-626529 against CD4-independent viruses and investigated whether resistance to BMS-626529 could be associated with a CD4-independent phenotype. Finally, we evaluated whether cross-resistance exists between BMS-626529 and other HIV-1 entry inhibitors. Two laboratory-derived envelopes with a CD4-independent phenotype (one CXCR4 tropic and one CCR5 tropic), five envelopes from clinical isolates with preexisting BMS-626529 resistance, and several site-specific mutant BMS-626529-resistant envelopes were examined for their dependence on CD4 for infectivity or susceptibility to BMS-626529. Viruses resistant to other entry inhibitors (enfuvirtide, maraviroc, and ibalizumab) were also examined for susceptibility to BMS-626529. Both CD4-independent laboratory isolates retained sensitivity to BMS-626529 in CD4− cells, while HIV-1 envelopes from viruses resistant to BMS-626529 exhibited no evidence of a CD4-independent phenotype. BMS-626529 also exhibited inhibitory activity against ibalizumab- and enfuvirtide-resistant envelopes. While there appeared to be some association between maraviroc resistance and reduced susceptibility to BMS-626529, an absolute correlation cannot be presumed, since some CCR5-tropic maraviroc-resistant envelopes remained sensitive to BMS-626529. Clinical use of the prodrug BMS-663068 is unlikely to promote resistance via generation of CD4-independent virus. No cross-resistance between BMS-626529 and other HIV entry inhibitors was observed, which could allow for sequential or concurrent use with different classes of entry inhibitors. PMID:23774428

A novel mechanism of regulating breast cancer cell migration via palmitoylation-dependent alterations in the lipid raft affiliation of CD44

PubMed Central

2014-01-01

Introduction Most breast cancer-related deaths result from metastasis, a process involving dynamic regulation of tumour cell adhesion and migration. The adhesion protein CD44, a key regulator of cell migration, is enriched in cholesterol-enriched membrane microdomains termed lipid rafts. We recently reported that raft affiliation of CD44 negatively regulates interactions with its migratory binding partner ezrin. Since raft affiliation is regulated by post-translational modifications including palmitoylation, we sought to establish the contribution of CD44 palmitoylation and lipid raft affiliation to cell migration. Methods Recovery of CD44 and its binding partners from raft versus non-raft membrane microdomains was profiled in non-migrating and migrating breast cancer cell lines. Site-directed mutagenesis was used to introduce single or double point mutations into both CD44 palmitoylation sites (Cys286 and Cys295), whereupon the implications for lipid raft recovery, phenotype, ezrin co-precipitation and migratory behaviour was assessed. Finally CD44 palmitoylation status and lipid raft affiliation was assessed in primary cultures from a small panel of breast cancer patients. Results CD44 raft affiliation was increased during migration of non-invasive breast cell lines, but decreased during migration of highly-invasive breast cells. The latter was paralleled by increased CD44 recovery in non-raft fractions, and exclusive non-raft recovery of its binding partners. Point mutation of CD44 palmitoylation sites reduced CD44 raft affiliation in invasive MDA-MB-231 cells, increased CD44-ezrin co-precipitation and accordingly enhanced cell migration. Expression of palmitoylation-impaired (raft-excluded) CD44 mutants in non-invasive MCF-10a cells was sufficient to reversibly induce the phenotypic appearance of epithelial-to-mesenchymal transition and to increase cell motility. Interestingly, cell migration was associated with temporal reductions in CD44 palmitoylation in wild-type breast cells. Finally, the relevance of these findings is underscored by the fact that levels of palmitoylated CD44 were lower in primary cultures from invasive ductal carcinomas relative to non-tumour tissue, while CD44 co-localisation with a lipid raft marker was less in invasive ductal carcinoma relative to ductal carcinoma in situ cultures. Conclusion Our results support a novel mechanism whereby CD44 palmitoylation and consequent lipid raft affiliation inversely regulate breast cancer cell migration, and may act as a new therapeutic target in breast cancer metastasis. PMID:24512624

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

Pasek, Marta; Boeggeman, Elizabeth; Ramakrishnan, Boopathy

The expression of recombinant proteins in Escherichia coli often leads to inactive aggregated proteins known as the inclusion bodies. To date, the best available tool has been the use of fusion tags, including the carbohydrate-binding protein; e.g., the maltose-binding protein (MBP) that enhances the solubility of recombinant proteins. However, none of these fusion tags work universally with every partner protein. We hypothesized that galectins, which are also carbohydrate-binding proteins, may help as fusion partners in folding the mammalian proteins in E. coli. Here we show for the first time that a small soluble lectin, human galectin-1, one member of amore » large galectin family, can function as a fusion partner to produce soluble folded recombinant human glycosyltransferase, {beta}-1,4-galactosyltransferase-7 ({beta}4Gal-T7), in E. coli. The enzyme {beta}4Gal-T7 transfers galactose to xylose during the synthesis of the tetrasaccharide linker sequence attached to a Ser residue of proteoglycans. Without a fusion partner, {beta}4Gal-T7 is expressed in E. coli as inclusion bodies. We have designed a new vector construct, pLgals1, from pET-23a that includes the sequence for human galectin-1, followed by the Tev protease cleavage site, a 6x His-coding sequence, and a multi-cloning site where a cloned gene is inserted. After lactose affinity column purification of galectin-1-{beta}4Gal-T7 fusion protein, the unique protease cleavage site allows the protein {beta}4Gal-T7 to be cleaved from galectin-1 that binds and elutes from UDP-agarose column. The eluted protein is enzymatically active, and shows CD spectra comparable to the folded {beta}4Gal-T1. The engineered galectin-1 vector could prove to be a valuable tool for expressing other proteins in E. coli.« less

Nanostructure and force spectroscopy analysis of human peripheral blood CD4{sup +} T cells using atomic force microscopy

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

Hu Mingqian; Wang Jiongkun; Cai Jiye

2008-09-12

To date, nanoscale imaging of the morphological changes and adhesion force of CD4{sup +} T cells during in vitro activation remains largely unreported. In this study, we used atomic force microscopy (AFM) to study the morphological changes and specific binding forces in resting and activated human peripheral blood CD4{sup +} T cells. The AFM images revealed that the volume of activated CD4{sup +} T cells increased and the ultrastructure of these cells also became complex. Using a functionalized AFM tip, the strength of the specific binding force of the CD4 antigen-antibody interaction was found to be approximately three times thatmore » of the unspecific force. The adhesion forces were not randomly distributed over the surface of a single activated CD4{sup +} T cell, indicated that the CD4 molecules concentrated into nanodomains. The magnitude of the adhesion force of the CD4 antigen-antibody interaction did not change markedly with the activation time. Multiple bonds involved in the CD4 antigen-antibody interaction were measured at different activation times. These results suggest that the adhesion force involved in the CD4 antigen-antibody interaction is highly selective and of high affinity.« less

A view of the E2-CD81 interface at the binding site of a neutralizing antibody against hepatitis C virus.

PubMed

Harman, Christine; Zhong, Lilin; Ma, Li; Liu, Peter; Deng, Lu; Zhao, Zhong; Yan, Hailing; Struble, Evi; Virata-Theimer, Maria Luisa; Zhang, Pei

2015-01-01

Hepatitis C virus (HCV) glycoprotein E2 is considered a major target for generating neutralizing antibodies against HCV, primarily due to its role of engaging host entry factors, such as CD81, a key cell surface protein associated with HCV entry. Based on a series of biochemical analyses in combination with molecular docking, we present a description of a potential binding interface formed between the E2 protein and CD81. The virus side of this interface includes a hydrophobic helix motif comprised of residues W(437)LAGLF(442), which encompasses the binding site of a neutralizing monoclonal antibody, mAb41. The helical conformation of this motif provides a structural framework for the positioning of residues F442 and Y443, serving as contact points for the interaction with CD81. The cell side of this interface likewise involves a surface-exposed hydrophobic helix, namely, the D-helix of CD81, which coincides with the binding site of 1D6, a monoclonal anti-CD81 antibody known to block HCV entry. Our illustration of this virus-host interface suggests an important role played by the W(437)LAGLF(442) helix of the E2 protein in the hydrophobic interaction with the D-helix of CD81, thereby facilitating our understanding of the mechanism for antibody-mediated neutralization of HCV. Characterization of the interface established between a virus and host cells can provide important information that may be used for the control of virus infections. The interface that enables hepatitis C virus (HCV) to infect human liver cells has not been well understood because of the number of cell surface proteins, factors, and conditions found to be associated with the infection process. Based on a series of biochemical analyses in combination with molecular docking, we present such an interface, consisting of two hydrophobic helical structures, from the HCV E2 surface glycoprotein and the CD81 protein, a major host cell receptor recognized by all HCV strains. Our study reveals the critical role played by hydrophobic interactions in the formation of this virus-host interface, thereby contributing to our understanding of the mechanism for antibody-mediated neutralization of HCV. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The monomeric form of Neisseria DNA mimic protein DMP19 prevents DNA from binding to the histone-like HU protein

PubMed Central

Ko, Tzu-Ping; Liao, Yi-Ting; Hsu, Kai-Cheng

2017-01-01

DNA mimicry is a direct and effective strategy by which the mimic competes with DNA for the DNA binding sites on other proteins. Until now, only about a dozen proteins have been shown to function via this strategy, including the DNA mimic protein DMP19 from Neisseria meningitides. We have shown previously that DMP19 dimer prevents the operator DNA from binding to the transcription factor NHTF. Here, we provide new evidence that DMP19 monomer can also interact with the Neisseria nucleoid-associated protein HU. Using BS3 crosslinking, gel filtration and isothermal titration calorimetry assays, we found that DMP19 uses its monomeric form to interact with the Neisseria HU dimer. Crosslinking conjugated mass spectrometry was used to investigate the binding mode of DMP19 monomer and HU dimer. Finally, an electrophoretic mobility shift assay (EMSA) confirmed that the DNA binding affinity of HU is affected by DMP19. These results showed that DMP19 is bifunctional in the gene regulation of Neisseria through its variable oligomeric forms. PMID:29220372

Broadly Neutralizing Antibody Responses in a Large Longitudinal Sub-Saharan HIV Primary Infection Cohort

PubMed Central

Landais, Elise; Huang, Xiayu; Havenar-Daughton, Colin; Murrell, Ben; Price, Matt A.; Wickramasinghe, Lalinda; Ramos, Alejandra; Bian, Charoan B.; Simek, Melissa; Allen, Susan; Karita, Etienne; Kilembe, William; Lakhi, Shabir; Inambao, Mubiana; Kamali, Anatoli; Sanders, Eduard J.; Anzala, Omu; Edward, Vinodh; Bekker, Linda-Gail; Tang, Jianming; Gilmour, Jill; Kosakovsky-Pond, Sergei L.; Phung, Pham; Wrin, Terri; Crotty, Shane; Godzik, Adam; Poignard, Pascal

2016-01-01

Broadly neutralizing antibodies (bnAbs) are thought to be a critical component of a protective HIV vaccine. However, designing vaccines immunogens able to elicit bnAbs has proven unsuccessful to date. Understanding the correlates and immunological mechanisms leading to the development of bnAb responses during natural HIV infection is thus critical to the design of a protective vaccine. The IAVI Protocol C program investigates a large longitudinal cohort of primary HIV-1 infection in Eastern and South Africa. Development of neutralization was evaluated in 439 donors using a 6 cross-clade pseudo-virus panel predictive of neutralization breadth on larger panels. About 15% of individuals developed bnAb responses, essentially between year 2 and year 4 of infection. Statistical analyses revealed no influence of gender, age or geographical origin on the development of neutralization breadth. However, cross-clade neutralization strongly correlated with high viral load as well as with low CD4 T cell counts, subtype-C infection and HLA-A*03(-) genotype. A correlation with high overall plasma IgG levels and anti-Env IgG binding titers was also found. The latter appeared not associated with higher affinity, suggesting a greater diversity of the anti-Env responses in broad neutralizers. Broadly neutralizing activity targeting glycan-dependent epitopes, largely the N332-glycan epitope region, was detected in nearly half of the broad neutralizers while CD4bs and gp41-MPER bnAb responses were only detected in very few individuals. Together the findings suggest that both viral and host factors are critical for the development of bnAbs and that the HIV Env N332-glycan supersite may be a favorable target for vaccine design. PMID:26766578

Broadly Neutralizing Antibody Responses in a Large Longitudinal Sub-Saharan HIV Primary Infection Cohort.

PubMed

Landais, Elise; Huang, Xiayu; Havenar-Daughton, Colin; Murrell, Ben; Price, Matt A; Wickramasinghe, Lalinda; Ramos, Alejandra; Bian, Charoan B; Simek, Melissa; Allen, Susan; Karita, Etienne; Kilembe, William; Lakhi, Shabir; Inambao, Mubiana; Kamali, Anatoli; Sanders, Eduard J; Anzala, Omu; Edward, Vinodh; Bekker, Linda-Gail; Tang, Jianming; Gilmour, Jill; Kosakovsky-Pond, Sergei L; Phung, Pham; Wrin, Terri; Crotty, Shane; Godzik, Adam; Poignard, Pascal

2016-01-01

Broadly neutralizing antibodies (bnAbs) are thought to be a critical component of a protective HIV vaccine. However, designing vaccines immunogens able to elicit bnAbs has proven unsuccessful to date. Understanding the correlates and immunological mechanisms leading to the development of bnAb responses during natural HIV infection is thus critical to the design of a protective vaccine. The IAVI Protocol C program investigates a large longitudinal cohort of primary HIV-1 infection in Eastern and South Africa. Development of neutralization was evaluated in 439 donors using a 6 cross-clade pseudo-virus panel predictive of neutralization breadth on larger panels. About 15% of individuals developed bnAb responses, essentially between year 2 and year 4 of infection. Statistical analyses revealed no influence of gender, age or geographical origin on the development of neutralization breadth. However, cross-clade neutralization strongly correlated with high viral load as well as with low CD4 T cell counts, subtype-C infection and HLA-A*03(-) genotype. A correlation with high overall plasma IgG levels and anti-Env IgG binding titers was also found. The latter appeared not associated with higher affinity, suggesting a greater diversity of the anti-Env responses in broad neutralizers. Broadly neutralizing activity targeting glycan-dependent epitopes, largely the N332-glycan epitope region, was detected in nearly half of the broad neutralizers while CD4bs and gp41-MPER bnAb responses were only detected in very few individuals. Together the findings suggest that both viral and host factors are critical for the development of bnAbs and that the HIV Env N332-glycan supersite may be a favorable target for vaccine design.

Extracellular Membrane-proximal Domain of HAb18G/CD147 Binds to Metal Ion-dependent Adhesion Site (MIDAS) Motif of Integrin β1 to Modulate Malignant Properties of Hepatoma Cells*

PubMed Central

Li, Yong; Wu, Jiao; Song, Fei; Tang, Juan; Wang, Shi-Jie; Yu, Xiao-Ling; Chen, Zhi-Nan; Jiang, Jian-Li

2012-01-01

Several lines of evidence suggest that HAb18G/CD147 interacts with the integrin variants α3β1 and α6β1. However, the mechanism of the interaction remains largely unknown. In this study, mammalian protein-protein interaction trap (MAPPIT), a mammalian two-hybrid method, was used to study the CD147-integrin β1 subunit interaction. CD147 in human hepatocellular carcinoma (HCC) cells was interfered with by small hairpin RNA. Nude mouse xenograft model and metastatic model of HCC were used to detect the role of CD147 in carcinogenesis and metastasis. We found that the extracellular membrane-proximal domain of HAb18G/CD147 (I-type domain) binds at the metal ion-dependent adhesion site in the βA domain of the integrin β1 subunit, and Asp179 in the I-type domain of HAb18G/CD147 plays an important role in the interaction. The levels of the proteins that act downstream of integrin, including focal adhesion kinase (FAK) and phospho-FAK, were decreased, and the cytoskeletal structures of HCC cells were rearranged bearing the HAb18G/CD147 deletion. Simultaneously, the migration and invasion capacities, secretion of matrix metalloproteinases, colony formation rate in vitro, and tumor growth and metastatic potential in vivo were decreased. These results indicate that the interaction of HAb18G/CD147 extracellular I-type domain with the integrin β1 metal ion-dependent adhesion site motif activates the downstream FAK signaling pathway, subsequently enhancing the malignant properties of HCC cells. PMID:22130661

The alpha-fetoprotein (AFP) third domain: a search for AFP interaction sites of cell cycle proteins.

PubMed

Mizejewski, G J

2016-09-01

The carboxy-terminal third domain of alpha-fetoprotein (AFP-3D) is known to harbor binding and/or interaction sites for hydrophobic ligands, receptors, and binding proteins. Such reports have established that AFP-3D consists of amino acid (AA) sequence stretches on the AFP polypeptide that engages in protein-to-protein interactions with various ligands and receptors. Using a computer software program specifically designed for such interactions, the present report identified AA sequence fragments on AFP-3D that could potentially interact with a variety of cell cycle proteins. The cell cycle proteins identified were (1) cyclins, (2) cyclin-dependent kinases, (3) cell cycle-associated proteins (inhibitors, checkpoints, initiators), and (4) ubiquitin ligases. Following detection of the AFP-3D to cell cycle protein interaction sites, the computer-derived AFP localization AA sequences were compared and aligned with previously reported hydrophobic ligand and receptor interaction sites on AFP-3D. A literature survey of the association of cell cycle proteins with AFP showed both positive relationships and correlations. Previous reports of experimental AFP-derived peptides effects on various cell cycle proteins served to confirm and verify the present computer cell cycle protein identifications. Cell cycle protein interactions with AFP-CD peptides have been reported in cultured MCF-7 breast cancer cells subjected to mRNA microarray analysis. After 7 days in culture with MCF-7 cells, the AFP-derived peptides were shown to downregulate cyclin E, SKP2, checkpoint suppressors, cyclin-dependent kinases, and ubiquitin ligases that modulate cyclin E/CdK2 transition from the G1 to the S-phase of the cell cycle. Thus, the experimental data on AFP-CD interaction with cell cycle proteins were consistent with the "in silico" findings.

Reduced Human α-defensin 6 in Noninflamed Jejunal Tissue of Patients with Crohn's Disease.

PubMed

Hayashi, Ryohei; Tsuchiya, Kiichiro; Fukushima, Keita; Horita, Nobukatsu; Hibiya, Shuji; Kitagaki, Keisuke; Negi, Mariko; Itoh, Eisaku; Akashi, Takumi; Eishi, Yoshinobu; Okada, Eriko; Araki, Akihiro; Ohtsuka, Kazuo; Fukuda, Shinji; Ohno, Hiroshi; Okamoto, Ryuichi; Nakamura, Tetsuya; Tanaka, Shinji; Chayama, Kazuaki; Watanabe, Mamoru

2016-05-01

Mucosal barrier dysfunction is considered a critical component of Crohn's disease (CD) pathogenesis after the identification of susceptibility genes. However, the precise mechanism underlying mucosal barrier dysfunction has not yet been elucidated. We therefore aimed to elucidate the molecular mechanism underlying the expression of human α-defensin 6 (HD6) in patients with CD. HD6 expression was induced by the transfection of an atonal homolog 1 (Atoh1) transgene and was assessed by reverse transcription polymerase chain reaction. The HD6 promoter region targeted by Atoh1 and β-catenin was determined by reporter analysis and chromatin immunoprecipitation assay. HD5/HD6/Atoh1/β-catenin expression in noninflamed jejunal samples collected by balloon endoscopy from 15 patients with CD and 9 non-inflammatory bowel disease patients were assessed by immunofluorescence. Both promoter activity and gene expression of HD6 was significantly upregulated by the Atoh1 transgene in human colonic cancer cell line. We identified a TCF4 binding site and an E-box site, critical for the regulation of HD6 transcriptional activity by directly binding of Atoh1 in the 200-bp HD6 promoter region. The treatment with β-catenin inhibitor also decreases HD6 promoter activity and gene expression. Moreover, HD6 expression, but not HD5 expression, was found to be decreased in noninflamed jejunal regions from patients with CD. In HD6-negative crypts, nuclear accumulation of β-catenin was impaired. HD6 expression was found to be regulated by cooperation between Atoh1 and β-catenin within the HD6 promoter region. Downregulation of HD6 in noninflamed mucosa may contribute to mucosal barrier dysfunction of patients with CD.

Investigation of the interaction of 2,4-dimethoxy-6,7-dihydroxyphenanthrene with α-glucosidase using inhibition kinetics, CD, FT-IR and molecular docking methods.

PubMed

Zhang, Songsong; Qiu, Beibei; Zhu, Jinhua; Khan, M Z H; Liu, Xiuhua

2018-05-25

Applying enzyme kinetics, spectroscopic, and molecular docking methods, the interaction properties of 2,4-dimethoxy-6,7-dihydroxyphenanthrene with α-glucosidase were systematically investigated. The α-glucosidase inhibitory activities (IC 50  = 0.40 mM) were significantly higher than that of acarbose (as control) and the spectrometric results revealed that 2,4-dimethoxy-6,7-dihydroxyphenanthrene inhibited α-glucosidase in a reversible and noncompetitive manner, which is that the inhibitor bind to the inactive region of α-glucosidase and could be separated from the bind sites. Hydrogen bond was the key interaction force obtained from the results of the molecular docking study, and the binding energy was -27.754 kJ/mol. The CD studies showed that the content of α-helix in α-glucosidase increased from 17.2% to 17.8% with the concentration varying of 2,4-dimethoxy-6,7-dihydroxyphenanthrene. The α-helix increasing trend (19.70% - 21.43%) of α-glucosidase secondary structure was further proved by Fourier transform infrared spectra (FT-IR) results and the FT-IR spectra of α-glucosidase resulted in obvious red shift with the addition of 2,4-dimethoxy-6,7-dihydroxyphenanthrene. All the measurements proved the interaction of 2,4-dimethoxy-6,7-dihydroxyphenanthrene with α-glucosidase and revealed the conformational change of α-glucosidase secondary structure. Copyright © 2018 Elsevier B.V. All rights reserved.

The investigation of the interaction between piracetam and bovine serum albumin by spectroscopic methods

NASA Astrophysics Data System (ADS)

Guo, Xingjia; Han, Xiaowei; Tong, Jian; Guo, Chuang; Yang, Wenfeng; Zhu, Jifen; Fu, Bing

2010-03-01

The interaction between piracetam (OPA) with bovine serum albumin (BSA) has been thoroughly studied by fluorescence quenching technique in combination with UV-vis absorption, Fourier transform infrared (FT-IR), and circular dichroism (CD) spectroscopies under the simulative physiological conditions. The quenching of BSA fluorescence by OPA was found to be a static quenching process. The binding constants ( K a) are 3.014, 2.926, and 2.503 × 10 3 M -1 at 292, 298, and 309 K, respectively. According to the van't Hoff equation, the thermodynamic functions standard enthalpy (Δ H) and standard entropy (Δ S) for the reaction were calculated to be -74.560 kJ mol -1 and -159.380 J mol -1 K -1, which indicated that OPA binds to BSA mainly by hydrogen bonds and van der Waals interactions. The binding distance between BSA and OPA was calculated to be 4.10 nm according to the theory of FÖrster's non-radiation energy transfer. The displacement experiments confirmed that OPA could bind to the site I of BSA. Furthermore, the effects of pH and some common ions on the binding constant were also examined. And the alterations of protein secondary structure in the presence of OPA were observed by the CD and FT-IR spectra.

Single, binary and multi-component adsorption of some anions and heavy metals on environmentally friendly Carpobrotus edulis plant.

PubMed

Chiban, Mohamed; Soudani, Amina; Sinan, Fouad; Persin, Michel

2011-02-01

A low-cost adsorbent and environmentally friendly adsorbent from Carpobrotus edulis plant was used for the removal of NO(3)(-), H(2)PO(4)(-), Pb(2+) and Cd(2+) ions from single, binary and multi-component systems. The efficiency of the adsorbent was studied using batch adsorption technique under different experimental conditions by varying parameters such as pH, initial concentration and contact time. In single component systems, the dried C. edulis has the highest affinity for Pb(2+), followed by NO(3)(-), Cd(2+) and H(2)PO(4)(-), with adsorption capacities of 175mg/g, 125mg/g, 28mg/g and 26mg/g, respectively. These results showed that the adsorption of NO(3)(-) and H(2)PO(4)(-) ions from single and binary component systems can be successfully described by Langmuir and Freundlich isotherms. Freundlich adsorption model, showed the best fit to the single and binary experimental adsorption data. These results also indicated that the adsorption yield of Pb(2+) ion was reduced by the presence of Cd(2+) ion in binary metal mixture. The competitive adsorption of NO(3)(-), H(2)PO(4)(-), Pb(2+) and Cd(2+) ions on dried C. edulis plant shows that NO(3)(-) and H(2)PO(4)(-) anions are able to adsorb on different free binding sites and Pb(2+) and Cd(2+) cations are able to adsorb on the same active sites of C. edulis particles. The dried C. edulis was found to be efficient in removing nitrate, phosphate, cadmium and lead from aqueous solution as compared to other adsorbents already used for the removal of these ions. Copyright © 2010 Elsevier B.V. All rights reserved.

The exaggerated inflammatory response in Behçet's syndrome: identification of dysfunctional post-transcriptional regulation of the IFN-γ/CXCL10 IP-10 pathway

PubMed Central

Ambrose, N; Khan, E; Ravindran, R; Lightstone, L; Abraham, S; Botto, M; Johns, M; Haskard, D O

2015-01-01

The mechanisms underlying the exaggerated inflammatory response in Behçet's syndrome (BS) remain poorly understood. We investigated the response of CD14+ blood monocytes to interferon (IFN)-γ, focusing on the chemokine CXCL10. Chemokine synthesis and release were analysed at a protein and mRNA level following stimulation with IFN-γ. Findings in BS patients were compared with 25 healthy controls (HC), 15 rheumatoid arthritis (RA) and 15 systemic lupus erythematosus (SLE) disease control patients. BS monocytes produced significantly more CXCL10 protein than HC monocytes from 2 h following IFN-γ stimulation, despite equivalent quantities of mRNA, suggesting more efficient translation. This was significantly more pronounced in BS with high disease activity and in those with ocular and neurological clinical manifestations. The imbalance between CXCL10 protein and mRNA expression was not observed in either RA or SLE patients, and was not seen with other chemokines studied (CXCL9, CXCL11 and CCL2). Furthermore, BS monocytes treated with an alternative stimulant (LPS) did not show abnormal tumour necrosis factor (TNF)-α release. Sucrose density gradients to segregate monocyte CXCL10 mRNA into free RNA or polysome-associated RNA showed equal proportions in BS and HC samples, suggesting that the difference between BS and HC may be due to reduced negative control of CXCL10 translation in BS at a post-initiation level. We conclude that BS monocytes have dysfunctional post-transcriptional regulation of CXCL10 mRNA, resulting in over-expression of CXCL10 protein upon IFN-γ stimulation. As CXCL10 is a chemokine that recruits mononuclear cells, this abnormality may contribute to the exaggerated inflammatory responses that characterizes BS. PMID:25982097

The exaggerated inflammatory response in Behçet's syndrome: identification of dysfunctional post-transcriptional regulation of the IFN-γ/CXCL10 IP-10 pathway.

PubMed

Ambrose, N; Khan, E; Ravindran, R; Lightstone, L; Abraham, S; Botto, M; Johns, M; Haskard, D O

2015-09-01

The mechanisms underlying the exaggerated inflammatory response in Behçet's syndrome (BS) remain poorly understood. We investigated the response of CD14(+) blood monocytes to interferon (IFN)-γ, focusing on the chemokine CXCL10. Chemokine synthesis and release were analysed at a protein and mRNA level following stimulation with IFN-γ. Findings in BS patients were compared with 25 healthy controls (HC), 15 rheumatoid arthritis (RA) and 15 systemic lupus erythematosus (SLE) disease control patients. BS monocytes produced significantly more CXCL10 protein than HC monocytes from 2 h following IFN-γ stimulation, despite equivalent quantities of mRNA, suggesting more efficient translation. This was significantly more pronounced in BS with high disease activity and in those with ocular and neurological clinical manifestations. The imbalance between CXCL10 protein and mRNA expression was not observed in either RA or SLE patients, and was not seen with other chemokines studied (CXCL9, CXCL11 and CCL2). Furthermore, BS monocytes treated with an alternative stimulant (LPS) did not show abnormal tumour necrosis factor (TNF)-α release. Sucrose density gradients to segregate monocyte CXCL10 mRNA into free RNA or polysome-associated RNA showed equal proportions in BS and HC samples, suggesting that the difference between BS and HC may be due to reduced negative control of CXCL10 translation in BS at a post-initiation level. We conclude that BS monocytes have dysfunctional post-transcriptional regulation of CXCL10 mRNA, resulting in over-expression of CXCL10 protein upon IFN-γ stimulation. As CXCL10 is a chemokine that recruits mononuclear cells, this abnormality may contribute to the exaggerated inflammatory responses that characterizes BS. © 2015 British Society for Immunology.

Kinetic barriers for Cd and Te adatoms on Cd and Te terminated CdTe (111) surface using ab initio simulations

NASA Astrophysics Data System (ADS)

Naderi, Ebadollah; Nanavati, Sachin P.; Majumder, Chiranjib; Ghaisas, S. V.

2014-03-01

In the present work we have calculated using density functional theory (DFT), diffusion barrier potentials on both the CdTe (111) surfaces, Cd terminated (A-type) & Te terminated (B-type). We employ nudge elastic band method (NEB) for obtaining the barrier potentials. The barrier is computed for Cd and for Te adatoms on both A-type and B-type surfaces. We report two energetically favourable positions along the normal to the surface, one above and other below the surface. The one above the surface has binding energy slightly more the one below. According to the results of this work, binding energy (in all cases) for adatoms are reasonable and close to experimental data. The barrier potential for hopping adatoms (Cd and Te) on both the surfaces is less than 0.35 eV. Apart from these most probable sites, there are other at least two sites on both the types of surfaces which are meta stable. We have also computed barriers for hopping to and from these meta stable positions. The present results can shade light on the defect formation mechanism in CdTe thin films during growth. The authors would like to thank C-DAC for the computing time on its PARAM series of supercomputers and DST Govt. of India, for partial funding.

CD14 and TLR4 are expressed early in tammar (Macropus eugenii) neonate development.

PubMed

Daly, Kerry A; Lefévre, Christophe; Nicholas, Kevin; Deane, Elizabeth; Williamson, Peter

2008-04-01

Marsupials are born in a relatively underdeveloped state and develop during a period of intensive maturation in the postnatal period. During this period, the young marsupial lacks a competent immune system, but manages to survive despite the potential of exposure to environmental pathogens. Passive immune transfer via the milk is one well-recognised strategy to compensate the neonate, but there also may be innate immune mechanisms in place. In this study, CD14 and Toll-like receptor 4 (TLR4), integral molecular components of pathogen recognition, were identified and characterised for the first time in a marsupial, the tammar wallaby (Macropus eugenii). Functional motifs of tammar CD14 and the toll/interleukin receptor (TIR) domain of TLR4 were highly conserved. The lipopolysaccharide (LPS) binding residues and the TLR4 interaction site of CD14 were conserved in all marsupials. The TIR signalling domain had 84% identity within marsupials and 77% with eutherians. Stimulation of adult tammar leukocytes resulted in the induction of a biphasic pattern of CD14 and TLR4 expression, and coincided with increased production of the pro-inflammatory cytokine TNF-alpha. Differential patterns of expression of CD14 and TLR4 were observed in tammar pouch young early in development, suggesting that early maturation of the innate immune system in these animals may have developed as an immune survival strategy to protect the marsupial neonate from exposure to microbial pathogens.

Immune complexes formed following the binding of anti–platelet factor 4 (CXCL4) antibodies to CXCL4 stimulate human neutrophil activation and cell adhesion

PubMed Central

Xiao, Zhihua; Visentin, Gian P.; Dayananda, Kannayakanahalli M.

2008-01-01

We tested the possibility that immune complexes formed following platelet factor 4 (PF4/CXCL4) binding to anti-PF4 antibody can stimulate neutrophil activation, similar to previous reports with platelets. Monoclonal Abs against PF4 and IgG from a heparin-induced thrombocytopenia (HIT) patient were applied. We observed that although PF4 or anti-PF4 antibody alone did not alter neutrophil function, costimulation with both reagents resulted in approximately 3-fold increase in cell surface Mac-1 expression, enhanced cell adhesion via L-selectin and CD18 integrins, and degranulation of secondary and tertiary granules. The level of Mac-1 up-regulation peaked at an intermediate PF4 dose, suggesting that functional response varies with antigen-antibody stoichiometry. PF4 binding to neutrophils was blocked by chondroitinase ABC. Cell activation was inhibited by both chondroitinase ABC and anti-CD32/FcγRII blocking mAb, IV.3. Confocal microscopy demonstrated that immune complexes colocalize with CD32a. Studies with HIT IgG demonstrated that neutrophils could be activated in the absence of exogenous heparin. These data, together, show that leukocyte surface chondroitin sulfates promote neutrophil activation by enhancing immune-complex binding to CD32a. Studies with recombinant PF4 suggest a role for arginine 49 in stabilizing PF4-chondroitin binding. Neutrophils activated via this mechanism may contribute to thrombosis and inflammation in patients mounting an immune response to PF4-heparin. PMID:18539895

NeutrAvidin Functionalization of CdSe/CdS Quantum Nanorods and Quantification of Biotin Binding Sites using Biotin-4-Fluorescein Fluorescence Quenching.

PubMed

Lippert, Lisa G; Hallock, Jeffrey T; Dadosh, Tali; Diroll, Benjamin T; Murray, Christopher B; Goldman, Yale E

2016-03-16

We developed methods to solubilize, coat, and functionalize with NeutrAvidin elongated semiconductor nanocrystals (quantum nanorods, QRs) for use in single molecule polarized fluorescence microscopy. Three different ligands were compared with regard to efficacy for attaching NeutrAvidin using the "zero-length cross-linker" 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). Biotin-4-fluorescene (B4F), a fluorophore that is quenched when bound to avidin proteins, was used to quantify biotin binding activity of the NeutrAvidin coated QRs and biotin binding activity of commercially available streptavidin coated quantum dots (QDs). All three coating methods produced QRs with NeutrAvidin coating density comparable to the streptavidin coating density of the commercially available quantum dots (QDs) in the B4F assay. One type of QD available from the supplier (ITK QDs) exhibited ∼5-fold higher streptavidin surface density compared to our QRs, whereas the other type of QD (PEG QDs) had 5-fold lower density. The number of streptavidins per QD increased from ∼7 streptavidin tetramers for the smallest QDs emitting fluorescence at 525 nm (QD525) to ∼20 tetramers for larger, longer wavelength QDs (QD655, QD705, and QD800). QRs coated with NeutrAvidin using mercaptoundecanoicacid (MUA) and QDs coated with streptavidin bound to biotinylated cytoplasmic dynein in single molecule TIRF microscopy assays, whereas Poly(maleic anhydride-alt-1-ocatdecene) (PMAOD) or glutathione (GSH) QRs did not bind cytoplasmic dynein. The coating methods require optimization of conditions and concentrations to balance between substantial NeutrAvidin binding vs tendency of QRs to aggregate and degrade over time.

Besides an ITIM/SHP-1-dependent pathway, CD22 collaborates with Grb2 and plasma membrane calcium-ATPase in an ITIM/SHP-1-independent pathway of attenuation of Ca2+i signal in B cells

PubMed Central

Chen, Jie; Wang, Hong; Xu, Wei-Ping; Wei, Si-Si; Li, Hui Joyce; Mei, Yun-Qing; Li, Yi-Gang; Wang, Yue-Peng

2016-01-01

CD22 is a surface immunoglobulin implicated in negative regulation of B cell receptor (BCR) signaling; particularly inhibiting intracellular Ca2+ (Ca2+i)signals. Its cytoplasmic tail contains six tyrosine residues (Y773/Y783/Y817/Y828/Y843/Y863, designated Y1~Y6 respectively), including three (Y2/5/6) lying within immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that serve to recruit the protein tyrosine phosphatase SHP-1 after BCR activation-induced phosphorylation. The mechanism of inhibiting Ca2+i by CD22 has been poorly understood. Previous study demonstrated that CD22 associated with plasma membrane calcium-ATPase (PMCA) and enhanced its activity (Chen, J. et al. Nat Immunol 2004;5:651-7). The association is dependent on BCR activation-induced cytoplasmic tyrosine phosphorylation, because CD22 with either all six tyrosines mutated to phenylalanines or cytoplasmic tail truncated loses its ability to associate with PMCA. However, which individual or a group of tyrosine residues determine the association and how CD22 and PMCA interacts, are still unclear. In this study, by using a series of CD22 tyrosine mutants, we found that ITIM Y2/5/6 accounts for 34.3~37.1% Ca2+i inhibition but is irrelevant for CD22/PMCA association. Non-ITIM Y4 and its YEND motif contribute to the remaining 69.4~71.7% Ca2+i inhibition and is the binding site for PMCA-associated Grb2. Grb2, independently of BCR cross-linking, is constitutively associated with and directly binds to PMCA in both chicken and human B cells. Knockout of Grb2 by CRISPR/Cas9 completely disrupted the CD22/PMCA association. Thus, our results demonstrate for the first time that in addition to previously-identified ITIM/SHP-1-dependent pathway, CD22 holds a major pathway of negative regulation of Ca2+i signal, which is ITIM/SHP-1-independent, but Y4/Grb2/PMCA-dependent. PMID:27276708

Besides an ITIM/SHP-1-dependent pathway, CD22 collaborates with Grb2 and plasma membrane calcium-ATPase in an ITIM/SHP-1-independent pathway of attenuation of Ca2+i signal in B cells.

PubMed

Chen, Jie; Wang, Hong; Xu, Wei-Ping; Wei, Si-Si; Li, Hui Joyce; Mei, Yun-Qing; Li, Yi-Gang; Wang, Yue-Peng

2016-08-30

CD22 is a surface immunoglobulin implicated in negative regulation of B cell receptor (BCR) signaling; particularly inhibiting intracellular Ca2+ (Ca2+i)signals. Its cytoplasmic tail contains six tyrosine residues (Y773/Y783/Y817/Y828/Y843/Y863, designated Y1~Y6 respectively), including three (Y2/5/6) lying within immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that serve to recruit the protein tyrosine phosphatase SHP-1 after BCR activation-induced phosphorylation. The mechanism of inhibiting Ca2+i by CD22 has been poorly understood. Previous study demonstrated that CD22 associated with plasma membrane calcium-ATPase (PMCA) and enhanced its activity (Chen, J. et al. Nat Immunol 2004;5:651-7). The association is dependent on BCR activation-induced cytoplasmic tyrosine phosphorylation, because CD22 with either all six tyrosines mutated to phenylalanines or cytoplasmic tail truncated loses its ability to associate with PMCA. However, which individual or a group of tyrosine residues determine the association and how CD22 and PMCA interacts, are still unclear. In this study, by using a series of CD22 tyrosine mutants, we found that ITIM Y2/5/6 accounts for 34.3~37.1% Ca2+i inhibition but is irrelevant for CD22/PMCA association. Non-ITIM Y4 and its YEND motif contribute to the remaining 69.4~71.7% Ca2+i inhibition and is the binding site for PMCA-associated Grb2. Grb2, independently of BCR cross-linking, is constitutively associated with and directly binds to PMCA in both chicken and human B cells. Knockout of Grb2 by CRISPR/Cas9 completely disrupted the CD22/PMCA association. Thus, our results demonstrate for the first time that in addition to previously-identified ITIM/SHP-1-dependent pathway, CD22 holds a major pathway of negative regulation of Ca2+i signal, which is ITIM/SHP-1-independent, but Y4/Grb2/PMCA-dependent.

Induction of CD69 expression by cagPAI-positive Helicobacter pylori infection

PubMed Central

Mori, Naoki; Ishikawa, Chie; Senba, Masachika

2011-01-01

AIM: To investigate and elucidate the molecular mechanism that regulates inducible expression of CD69 by Helicobacter pylori (H. pylori) infection. METHODS: The expression levels of CD69 in a T-cell line, Jurkat, primary human peripheral blood mononuclear cells (PBMCs), and CD4+ T cells, were assessed by immunohistochemistry, reverse transcription polymerase chain reaction, and flow cytometry. Activation of CD69 promoter was detected by reporter gene. Nuclear factor (NF)-κB activation in Jurkat cells infected with H. pylori was evaluated by electrophoretic mobility shift assay. The role of NF-κB signaling in H. pylori-induced CD69 expression was analyzed using inhibitors of NF-κB and dominant-negative mutants. The isogenic mutants with disrupted cag pathogenicity island (cagPAI) and virD4 were used to elucidate the role of cagPAI-encoding type IV secretion system and CagA in CD69 expression. RESULTS: CD69 staining was detected in mucosal lymphocytes and macrophages in specimens of patients with H. pylori-positive gastritis. Although cagPAI-positive H. pylori and an isogenic mutant of virD4 induced CD69 expression, an isogenic mutant of cagPAI failed to induce this in Jurkat cells. H. pylori also induced CD69 expression in PBMCs and CD4+ T cells. The activation of the CD69 promoter by H. pylori was mediated through NF-κB. Transfection of dominant-negative mutants of IκBs, IκB kinases, and NF-κB-inducing kinase inhibited H. pylori-induced CD69 activation. Inhibitors of NF-κB suppressed H. pylori-induced CD69 mRNA expression. CONCLUSION: The results suggest that H. pylori induces CD69 expression through the activation of NF-κB. cagPAI might be relevant in the induction of CD69 expression in T cells. CD69 in T cells may play a role in H. pylori-induced gastritis. PMID:21990950

Characterization of the slow calcium channel binding sites for ( sup 3 H)SR 33557 in rat heart sarcolemmal membranes

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

Chatelain, P.; Beaufort, P.; Meysmans, L.

1991-01-01

SR 33557 represents a new class of compounds (indolizine sulfone) that inhibit L-type Ca2+ channels. ({sup 3}H)SR 33557 has been shown to bind with high affinity (Kd congruent to 0.36 nM, calculated from saturation isotherms and association/dissociation kinetics) to a single class of sites in a purified preparation of rat cardiac sarcolemmal membranes. The binding was found to be saturable and reversible. The maximal binding capacity was in approximately 1:1 stoichiometry with that of other Ca2+ channel antagonists. Various divalent cations (Mg2+, Mn2+, Ca2+, Ba2+, and Cd2+) were shown to inhibit specific ({sup 3}H)SR 33557 binding, with Cd2+ being themore » most potent. Among several receptor or channel ligands (including omega-conotoxin and Na+ and K+ channel modulators), only the L-type Ca2+ channel antagonists were found to displace ({sup 3}H)SR 33557. However, dihydropyridines, phenylalkylamines, benzothiazepines, and diphenylbutylpiperidines were found to inhibit ({sup 3}H)SR 33557 in a noncompetitive manner as demonstrated by displacement and saturation experiments in addition to dissociation kinetics. From these results, we suggest that SR 33557 binds with high affinity to a unique site on the L-type Ca2+ channel found in rat cardiac sarcolemmal membranes.« less

Cell wall reactivity of acidophilic and alkaliphilic bacteria determined by potentiometric titrations and Cd adsorption experiments.

PubMed

Kenney, Janice P L; Fein, Jeremy B

2011-05-15

In this study, we used potentiometric titrations and Cd adsorption experiments to determine the binding capacities of two acidophilic (A. cryptum and A. acidophilum) and two alkaliphilic (B. pseudofirmus and B. circulans) bacterial species in order to determine if any consistent trends could be observed relating bacterial growth environment to proton and Cd binding properties and to compare those binding behaviors to those of neutrophilic bacteria. All of the bacterial species studied exhibited significant proton buffering over the pH range in this study, with the alkaliphiles exhibiting significantly higher acidity constants than the acidophiles as well as the neutrophilic bacterial consortia. The calculated average site concentrations for each of the bacteria in this study are within 2σ experimental error of each other, with the exception of A. cryptum, which has a significantly higher Site 2 concentration than the other species. Despite differing acidity constants between the acidophiles and alkaliphiles, all bacteria except A. cryptum exhibited remarkably similar Cd adsorption behavior to each other, and the observed extent of adsorption was also similar to that predicted from a generalized model derived using neutrophilic bacterial consortia. This study demonstrates that bacteria that grow under extreme conditions exhibit similar proton and metal adsorption behavior to that of previously studied neutrophilic species and that a single set of proton and metal binding constants can be used to model the behavior of bacterial adsorption under a wide range of environmental conditions.

The Glycosylphosphatidylinositol-Anchored Variable Region of Llama Heavy Chain-Only Antibody JM4 Efficiently Blocks both Cell-Free and T Cell-T Cell Transmission of Human Immunodeficiency Virus Type 1

PubMed Central

Liu, Lihong; Wang, Weiming; Matz, Julie; Ye, Chaobaihui; Bracq, Lucie; Delon, Jerome; Kimata, Jason T.; Chen, Zhiwei

2016-01-01

ABSTRACT The variable regions (VHHs) of two heavy chain-only antibodies, JM2 and JM4, from llamas that have been immunized with a trimeric gp140 bound to a CD4 mimic have been recently isolated (here referred to as VHH JM2 and VHH JM4, respectively). JM2 binds the CD4-binding site of gp120 and neutralizes HIV-1 strains from subtypes B, C, and G. JM4 binds gp120 and neutralizes HIV-1 strains from subtypes A, B, C, A/E, and G in a CD4-dependent manner. In the present study, we constructed glycosylphosphatidylinositol (GPI)-anchored VHH JM2 and JM4 along with an E4 control and transduced them into human CD4+ cell lines and primary CD4 T cells. We report that by genetically linking the VHHs with a GPI attachment signal, VHHs are targeted to the lipid rafts of the plasma membranes. Expression of GPI-VHH JM4, but not GPI-VHH E4 and JM2, on the surface of transduced TZM.bl cells potently neutralizes multiple subtypes of HIV-1 isolates, including tier 2 or 3 strains, transmitted founders, quasispecies, and soluble single domain antibody (sdAb) JM4-resistant viruses. Moreover, transduction of CEMss-CCR5 cells with GPI-VHH JM4, but not with GPI-VHH E4, confers resistance to both cell-free and T cell-T cell transmission of HIV-1 and HIV-1 envelope-mediated fusion. Finally, GPI-VHH JM4-transduced human primary CD4 T cells efficiently resist both cell-free and T cell-T cell transmission of HIV-1. Thus, we conclude that VHH JM4, when targeted to the lipid rafts of the plasma membrane, efficiently neutralizes HIV-1 infection via both cell-free and T cell-T cell transmission. Our findings should have important implications for GPI-anchored antibody-based therapy against HIV-1. IMPORTANCE Lipid rafts are specialized dynamic microdomains of the plasma membrane and have been shown to be gateways for HIV-1 budding as well as entry into T cells and macrophages. In nature, many glycosylphosphatidylinositol (GPI)-anchored proteins localize in the lipid rafts. In the present study, we developed GPI-anchored variable regions (VHHs) of two heavy chain-only antibodies, JM2 and JM4, from immunized llamas. We show that by genetically linking the VHHs with a GPI attachment signal, VHHs are targeted to the lipid rafts of the plasma membranes. GPI-VHH JM4, but not GPI-VHH JM2, in transduced CD4+ cell lines and human primary CD4 T cells not only efficiently blocks diverse HIV-1 strains, including tier 2 or 3 strains, transmitted founders, quasispecies, and soluble sdAb JM4-resistant strains, but also efficiently interferes T cell-T cell transmissions of HIV-1 and HIV-1 envelope-mediated fusion. Our findings should have important implications in GPI-anchored antibody-based therapy against HIV-1. PMID:27654286

Tolerance exists towards resident intestinal flora but is broken in active inflammatory bowel disease (IBD)

PubMed

Duchmann, R; Kaiser, I; Hermann, E; Mayet, W; Ewe, K; Meyer zum Büschenfelde, K H

1995-12-01

Hyporesponsiveness to a universe of bacterial and dietary antigens from the gut lumen is a hallmark of the intestinal immune system. Since hyperresponsiveness against these antigens might be associated with inflammation, we studied the immune response to the indigenous intestinal microflora in peripheral blood, inflamed and non-inflamed human intestine. Lamina propria monocuclear cells (LPMC) isolated from inflamed intestine but not peripheral blood mononuclear cells (PBMC) of IBD patients with active inflammatory disease strongly proliferated after co-culture with sonicates of bacteria from autologous intestine (BsA). Proliferation was inhibitable by anti-MHC class II MoAb, suggesting that it was driven by antigen. LPMC from adjacent non-inflamed intestinal areas of the same IBD patients and PBMC or LPMC isolated from non-inflamed intestine of controls and patients with IBD in remission, in contrast, did not proliferate. PBMC or LPMC which had been tolerant to bacteria from autologous intestine, however, strongly proliferated after co-culture with bacterial sonicates from heterologous intestine (BsH). This proliferation was associated with an expansion of CD8+ T cells, increased expression of activation markers on both CD4+ and CD8+ lymphocyte subsets, and production of IL-12, interferon-gamma (IFN-gamma), and IL-10 protein. These results show that tolerance selectively exists to intestinal flora from autologous but not heterologous intestine, and that tolerance is broken in intestinal inflammation. This may be an important mechanism for the perpetuation of chronic IBD.

Tolerance exists towards resident intestinal flora but is broken in active inflammatory bowel disease (IBD)

PubMed Central

Duchmann, R; Kaiser, I; Hermann, E; Mayet, W; Ewe, K; Meyer zum Büschenfelde, K H

1995-01-01

Hyporesponsiveness to a universe of bacterial and dietary antigens from the gut lumen is a hallmark of the intestinal immune system. Since hyperresponsiveness against these antigens might be associated with inflammation, we studied the immune response to the indigenous intestinal microflora in peripheral blood, inflamed and non-inflamed human intestine. Lamina propria monocuclear cells (LPMC) isolated from inflamed intestine but not peripheral blood mononuclear cells (PBMC) of IBD patients with active inflammatory disease strongly proliferated after co-culture with sonicates of bacteria from autologous intestine (BsA). Proliferation was inhibitable by anti-MHC class II MoAb, suggesting that it was driven by antigen. LPMC from adjacent non-inflamed intestinal areas of the same IBD patients and PBMC or LPMC isolated from non-inflamed intestine of controls and patients with IBD in remission, in contrast, did not proliferate. PBMC or LPMC which had been tolerant to bacteria from autologous intestine, however, strongly proliferated after co-culture with bacterial sonicates from heterologous intestine (BsH). This proliferation was associated with an expansion of CD8+ T cells, increased expression of activation markers on both CD4+ and CD8+ lymphocyte subsets, and production of IL-12, interferon-gamma (IFN-gamma), and IL-10 protein. These results show that tolerance selectively exists to intestinal flora from autologous but not heterologous intestine, and that tolerance is broken in intestinal inflammation. This may be an important mechanism for the perpetuation of chronic IBD. PMID:8536356

Immunogenicity and safety of different injection routes and schedules of IC41, a Hepatitis C virus (HCV) peptide vaccine.

PubMed

Firbas, Christa; Boehm, Thomas; Buerger, Vera; Schuller, Elisabeth; Sabarth, Nicolas; Jilma, Bernd; Klade, Christoph S

2010-03-11

An effective vaccine would be a significant progress in the management of chronic HCV infections. This study was designed to examine whether different application schedules and injection routes may enhance the immunogenicity of the HCV peptide vaccine IC41. In this randomized trial 54 healthy subjects received either subcutaneous (s.c.) or intradermal (i.d.) vaccinations weekly (16 injections) or every other week (8 injections). One group additionally received imiquimod, an activator of the toll-like receptor (TLR) 7. The T cell epitope-specific immune response to IC41 was assessed using [(3)H]-thymidine CD4+ T cell proliferation, interferon-gamma (IFN-gamma) CD8+ and CD4+ ELIspot and HLA-A*0201 fluorescence-activated cell sorting (FACS) tetramer-binding assays. More than 60% of vaccinees responded in the CD4+ T cell proliferation assay in all groups. An HLA-A*0201 FACS tetramer-binding assay and IFN-gamma CD8+ ELIspot class I response of more than 70% was induced in four and three groups, respectively. IC41 induced significant immunological responses in all groups with responder rates of up to 100%. Interestingly, topical imiquimod was not able to enhance immunogenicity but was associated with a lower immune response. Local injection site reactions were mostly transient. Intradermal injections caused more pronounced reactions compared to s.c., especially erythema and edema. Compared to a previous study intensified dosing and/or i.d. injections enhanced the response rates to the vaccine IC41 in three assays measuring T cell function. Immunization with IC41 was generally safe in this study. These results justify testing IC41 in further clinical trials with HCV-infected individuals.

Macrophage Migration Inhibitory Factor-CXCR4 Receptor Interactions

PubMed Central

Rajasekaran, Deepa; Gröning, Sabine; Schmitz, Corinna; Zierow, Swen; Drucker, Natalie; Bakou, Maria; Kohl, Kristian; Mertens, André; Lue, Hongqi; Weber, Christian; Xiao, Annie; Luker, Gary; Kapurniotu, Aphrodite; Lolis, Elias; Bernhagen, Jürgen

2016-01-01

An emerging number of non-chemokine mediators are found to bind to classical chemokine receptors and to elicit critical biological responses. Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that exhibits chemokine-like activities through non-cognate interactions with the chemokine receptors CXCR2 and CXCR4, in addition to activating the type II receptor CD74. Activation of the MIF-CXCR2 and -CXCR4 axes promotes leukocyte recruitment, mediating the exacerbating role of MIF in atherosclerosis and contributing to the wealth of other MIF biological activities. Although the structural basis of the MIF-CXCR2 interaction has been well studied and was found to engage a pseudo-ELR and an N-like loop motif, nothing is known about the regions of CXCR4 and MIF that are involved in binding to each other. Using a genetic strain of Saccharomyces cerevisiae that expresses a functional CXCR4 receptor, site-specific mutagenesis, hybrid CXCR3/CXCR4 receptors, pharmacological reagents, peptide array analysis, chemotaxis, fluorescence spectroscopy, and circular dichroism, we provide novel molecular information about the structural elements that govern the interaction between MIF and CXCR4. The data identify similarities with classical chemokine-receptor interactions but also provide evidence for a partial allosteric agonist compared with CXCL12 that is possible due to the two binding sites of CXCR4. PMID:27226569

Interaction of milk whey protein with common phenolic acids

NASA Astrophysics Data System (ADS)

Zhang, Hao; Yu, Dandan; Sun, Jing; Guo, Huiyuan; Ding, Qingbo; Liu, Ruihai; Ren, Fazheng

2014-01-01

Phenolics-rich foods such as fruit juices and coffee are often consumed with milk. In this study, the interactions of α-lactalbumin and β-lactoglobulin with the phenolic acids (chlorogenic acid, caffeic acid, ferulic acid, and coumalic acid) were examined. Fluorescence, CD, and FTIR spectroscopies were used to analyze the binding modes, binding constants, and the effects of complexation on the conformation of whey protein. The results showed that binding constants of each whey protein-phenolic acid interaction ranged from 4 × 105 to 7 × 106 M-n and the number of binding sites n ranged from 1.28 ± 0.13 to 1.54 ± 0.34. Because of these interactions, the conformation of whey protein was altered, with a significant reduction in the amount of α-helix and an increase in the amounts of β-sheet and turn structures.

Site- and strand-specific nicking of DNA by fusion proteins derived from MutH and I-SceI or TALE repeats.

PubMed

Gabsalilow, Lilia; Schierling, Benno; Friedhoff, Peter; Pingoud, Alfred; Wende, Wolfgang

2013-04-01

Targeted genome engineering requires nucleases that introduce a highly specific double-strand break in the genome that is either processed by homology-directed repair in the presence of a homologous repair template or by non-homologous end-joining (NHEJ) that usually results in insertions or deletions. The error-prone NHEJ can be efficiently suppressed by 'nickases' that produce a single-strand break rather than a double-strand break. Highly specific nickases have been produced by engineering of homing endonucleases and more recently by modifying zinc finger nucleases (ZFNs) composed of a zinc finger array and the catalytic domain of the restriction endonuclease FokI. These ZF-nickases work as heterodimers in which one subunit has a catalytically inactive FokI domain. We present two different approaches to engineer highly specific nickases; both rely on the sequence-specific nicking activity of the DNA mismatch repair endonuclease MutH which we fused to a DNA-binding module, either a catalytically inactive variant of the homing endonuclease I-SceI or the DNA-binding domain of the TALE protein AvrBs4. The fusion proteins nick strand specifically a bipartite recognition sequence consisting of the MutH and the I-SceI or TALE recognition sequences, respectively, with a more than 1000-fold preference over a stand-alone MutH site. TALE-MutH is a programmable nickase.

Identification of a human erythroid progenitor cell population which expresses the CD34 antigen and binds the plant lectin Ulex europaeus I.

PubMed

Unverzagt, K L; Martinson, J; Lee, W; Stiff, P J; Williams, S; Bender, J G

1996-01-01

Two and three color flow cytometry of normal human bone marrow was used to identify CD34+ progenitor cells and examine their binding to the plant lectin Ulex europaeus I (Ulex). In normal bone marrow, 48.48 +/- 17.4% of the CD34+ cells bind to Ulex. Two color flow cytometry was used to sort CD34 + cells, and subsets of CD34+ cells, CD34+ Ulex+ and CD34+ Ulex-. These populations were sorted into colony assays to assess myeloid (CFU-GM) and erythroid (BFU-E) progenitors. The CD34+ Ulex+ subset was 84 +/- 14% BFU-E colonies (mean +/- S.D.) and had the highest cloning efficiency of 28 +/- 13%. Three color analysis of CD34+ Ulex+ cells showed staining with other erythroid (CD71, GlyA) antibodies and lack of stain. ing with myeloid (CD13, CD45RA) antibodies. These studies confirmed the erythroid characteristics of this subpopulation.

Important role of N108 residue in binding of bovine foamy virus transactivator Tas to viral promoters.

PubMed

Bing, Tiejun; Zhang, Suzhen; Liu, Xiaojuan; Liang, Zhibin; Shao, Peng; Zhang, Song; Qiao, Wentao; Tan, Juan

2016-06-30

Bovine foamy virus (BFV) encodes the transactivator BTas, which enhances viral gene transcription by binding to the long terminal repeat promoter and the internal promoter. In this study, we investigated the different replication capacities of two similar BFV full-length DNA clones, pBS-BFV-Y and pBS-BFV-B. Here, functional analysis of several chimeric clones revealed a major role for the C-terminal region of the viral genome in causing this difference. Furthermore, BTas-B, which is located in this C-terminal region, exhibited a 20-fold higher transactivation activity than BTas-Y. Sequence alignment showed that these two sequences differ only at amino acid 108, with BTas-B containing N108 and BTas-Y containing D108 at this position. Results of mutagenesis studies demonstrated that residue N108 is important for BTas binding to viral promoters. In addition, the N108D mutation in pBS-BFV-B reduced the viral replication capacity by about 1.5-fold. Our results suggest that residue N108 is important for BTas binding to BFV promoters and has a major role in BFV replication. These findings not only advances our understanding of the transactivation mechanism of BTas, but they also highlight the importance of certain sequence polymorphisms in modulating the replication capacity of isolated BFV clones.

Sorption selectivity of birnessite particle edges: a d-PDF analysis of Cd(ii) and Pb(ii) sorption by δ-MnO2 and ferrihydrite.

PubMed

van Genuchten, Case M; Peña, Jasquelin

2016-08-10

Birnessite minerals (layer-type MnO2), which bear both internal (cation vacancies) and external (particle edges) metal sorption sites, are important sinks of contaminants in soils and sediments. Although the particle edges of birnessite minerals often dominate the total reactive surface area, especially in the case of nanoscale crystallites, the metal sorption reactivity of birnessite particle edges remains elusive. In this study, we investigated the sorption selectivity of birnessite particle edges by combining Cd(ii) and Pb(ii) adsorption isotherms at pH 5.5 with surface structural characterization by differential pair distribution function (d-PDF) analysis. We compared the sorption reactivity of δ-MnO2 to that of the nanomineral, 2-line ferrihydrite, which exhibits only external surface sites. Our results show that, whereas Cd(ii) and Pb(ii) both bind to birnessite layer vacancies, only Pb(ii) binds extensively to birnessite particle edges. For ferrihydrite, significant Pb(ii) adsorption to external sites was observed (roughly 20 mol%), whereas Cd(ii) sorption was negligible. These results are supported by bond valence calculations that show comparable degrees of saturation of oxygen atoms on birnessite and ferrihydrite particle edges. Therefore, we propose that the sorption selectivity of birnessite edges follows the same order of that reported previously for ferrihydrite: Ca(ii) < Cd(ii) < Ni(ii) < Zn(ii) < Cu(ii) < Pb(ii).

Mobilization of Cd from human serum albumin by small molecular weight thiols.

PubMed

Morris, Thomas T; Keir, Jennifer L A; Boshart, Steven J; Lobanov, Victor P; Ruhland, Anthony M A; Bahl, Nishita; Gailer, Jürgen

2014-05-01

Although the toxic metal Cd is an established human nephrotoxin, little is known about the role that interactions with plasma constitutents play in determining its mammalian target organs. To gain insight, a Cd-human serum albumin (HSA) complex was analyzed on a system consisting of size exclusion chromatography (SEC) coupled on-line to a flame atomic absorption spectrometer (FAAS). Using phosphate buffered saline (pH 7.4) as the mobile phase, we investigated the effect of 1-10mM oxidized glutathione (GSSG), l-cysteine (Cys), l-glutathione (GSH), or N-acetyl-l-cysteine (NAC) on the elution of Cd. As expected, GSSG did not mobilize Cd from the Cd-HSA complex up to a concentration of 4mM. With 1.0mM NAC, ∼30% of the injected Cd-HSA complex eluted as such, while the mobilized Cd was lost on the column. With 1.0mM of Cys or GSH, no parent Cd-HSA complex was detected and 88% and 82% of the protein bound Cd eluted close to the elution volume, likely in form of Cd(Cys)2 and a Cd-GSH 1:1 complex. Interestingly, with GSH and NAC concentrations >4.0mM, a Cd double peak was detected, which was rationalized in terms of the elution of a polynuclear Cd complex baseline-separated from a mononuclear Cd complex. In contrast, mobile phases which contained Cys concentrations ≥2mM resulted in the detection of only a single Cd peak, probably Cd(Cys)4. Our results establish SEC-FAAS as a viable tool to probe the mobilization of Cd from binding sites on plasma proteins at near physiological conditions. The detected complexes between Cd and Cys or GSH may be involved in the translocation of Cd to mammalian target organs. Copyright © 2014 Elsevier B.V. All rights reserved.

The ASPP interaction network: electrostatic differentiation between pro- and anti-apoptotic proteins.

PubMed

Benyamini, Hadar; Friedler, Assaf

2011-01-01

The ASPP proteins are apoptosis regulators: ASPP1 and ASPP2 promote, while iASPP inhibits, apoptosis. The mechanism by which these different outcomes are achieved is still unknown. The C-terminal ankyrin repeats and SH3 domain (ANK-SH3) mediate the interactions of the ASPP proteins with major apoptosis regulators such as p53, Bcl-2, and NFκB. The structure of the complex between ASPP2(ANK-SH3) and the core domain of p53 (p53CD) was previously determined. We have recently characterized the individual interactions of ASPP2(ANK-SH3) with Bcl-2 and NFκB, as well as a regulatory intramolecular interaction with the proline rich domain of ASPP2. Here we compared the ASPP interactions at two levels: ASPP2(ANK-SH3) with different proteins, and different ASPP family members with each protein partner. We found that the binding sites of ASPP2 to p53CD, Bcl-2, and NFκB are different, yet lie on the same face of ASPP2(ANK-SH3) . The intramolecular binding site to the proline rich domain overlaps the three intermolecular binding sites. To reveal the basis of functional diversity in the ASPP family, we compared their protein-binding domains. A subset of surface-exposed residues differentiates ASPP1 and ASPP2 from iASPP: ASPP1/2 are more negatively charged in specific residues that contact positively charged residues of p53CD, Bcl-2, and NFκB. We also found a gain of positive charge at the non-protein binding face of ASPP1/2, suggesting a role in electrostatic direction towards the negatively charged protein binding face. The electrostatic differences in binding interfaces between the ASPP proteins may be one of the causes for their different function. Copyright © 2010 John Wiley & Sons, Ltd.

Surface conformations of an anti-ricin aptamer and its affinity for ricin determined by atomic force microscopy and surface plasmon resonance.

PubMed

Wang, B; Lou, Z; Park, B; Kwon, Y; Zhang, H; Xu, B

2015-01-07

We used atomic force microscopy (AFM) and surface plasmon resonance (SPR) to study the surface conformations of an anti-ricin aptamer and its specific binding affinity for ricin molecules. The effect of surface modification of the Au(111) substrate on the aptamer affinity was also estimated. The AFM topography images had a resolution high enough to distinguish different aptamer conformations. The specific binding site on the aptamer molecule was clearly located by the AFM recognition images. The aptamer on a Au(111) surface modified with carboxymethylated-dextran (CD) showed both similarities to and differences from the one without CD modification. The influence of CD modification was evaluated using AFM images of various aptamer conformations on the Au(111) surface. The affinity between ricin and the anti-ricin aptamer was estimated using the off-rate values measured using AFM and SPR. The SPR measurements of the ricin sample were conducted in the range from 83.3 pM to 8.33 nM, and the limit of detection was estimated as 25 pM (1.5 ng mL(-1)). The off-rate values of the ricin-aptamer interactions were estimated using both single-molecule dynamic force spectroscopy (DFS) and SPR as (7.3 ± 0.4) × 10(-4) s(-1) and (1.82 ± 0.067) × 10(-2) s(-1), respectively. The results show that single-molecule measurements can obtain different reaction parameters from bulk solution measurements. In AFM single-molecule measurements, the various conformations of the aptamer immobilized on the gold surface determined the availability of each specific binding site to the ricin molecules. The SPR bulk solution measurements averaged the signals from specific and non-specific interactions. AFM images and DFS measurements provide more specific information on the interactions of individual aptamer and ricin molecules.

Toward Effective HIV Vaccination INDUCTION OF BINARY EPITOPE REACTIVE ANTIBODIES WITH BROAD HIV NEUTRALIZING ACTIVITY

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

Nishiyama, Yasuhiro; Planque, Stephanie; Mitsuda, Yukie

2009-11-23

We describe murine monoclonal antibodies (mAbs) raised by immunization with an electrophilic gp120 analog (E-gp120) expressing the rare ability to neutralize genetically heterologous human immunodeficiency virus (HIV) strains. Unlike gp120, E-gp120 formed covalent oligomers. The reactivity of gp120 and E-gp120 with mAbs to reference neutralizing epitopes was markedly different, indicating their divergent structures. Epitope mapping with synthetic peptides and electrophilic peptide analogs indicated binary recognition of two distinct gp120 regions by anti-E-gp120 mAbs, the 421-433 and 288-306 peptide regions. Univalent Fab and single chain Fv fragments expressed the ability to recognize both peptides. X-ray crystallography of an anti-E-gp120 Fab fragmentmore » revealed two neighboring cavities, the typical antigen-binding cavity formed by the complementarity determining regions (CDRs) and another cavity dominated by antibody heavy chain variable (VH) domain framework (FR) residues. Substitution of the FR cavity VH Lys-19 residue by an Ala residue resulted in attenuated binding of the 421-433 region peptide probe. The CDRs and VH FR replacement/silent mutation ratios exceeded the ratio for a random mutation process, suggesting adaptive development of both putative binding sites. All mAbs studied were derived from VH1 family genes, suggesting biased recruitment of the V gene germ line repertoire by E-gp120. The conserved 421-433 region of gp120 is essential for HIV binding to host CD4 receptors. This region is recognized weakly by the FR of antibodies produced without exposure to HIV, but it usually fails to induce adaptive synthesis of neutralizing antibodies. We present models accounting for improved CD4-binding site recognition and broad HIV neutralizing activity of the mAbs, long sought goals in HIV vaccine development.« less

T Regulatory Cell Induced Foxp3 Binds the IL2, IFNγ, and TNFα Promoters in Virus-Specific CD8+ T Cells from Feline Immunodeficiency Virus Infected Cats.

PubMed

Wang, Yan; Nag, Mukta; Tuohy, Joanne L; De Paris, Kristina; Fogle, Jonathan E

2018-03-01

Polyfunctional CD8 + T cells play a critical role in controlling viremia during AIDS lentiviral infections. However, for most HIV-infected individuals, virus-specific CD8 + T cells exhibit loss of polyfunctionality, including loss of IL2, TNFα, and IFNγ. Using the feline immunodeficiency virus (FIV) model for AIDS lentiviral persistence, our laboratory has demonstrated that FIV-activated Treg cells target CD8 + T cells, leading to a reduction in IL2 and IFNγ production. Furthermore, we have demonstrated that Treg cells induce expression of the repressive transcription factor, Foxp3, in CD8 + T cells. Based upon these findings, we asked if Treg-induced Foxp3 could bind to the IL2, TNFα, and IFNγ promoter regions in virus-specific CD8 + T cells. Following coculture with autologous Treg cells, we demonstrated decreased mRNA levels of IL2 and IFNγ at weeks 4 and 8 postinfection and decreased TNFα at week 4 postinfection in virus-specific CD8 + T cells. We also clearly demonstrated Treg cell-induced Foxp3 expression in virus-specific CD8 + T cells at weeks 1, 4, and 8 postinfection. Finally, we documented Foxp3 binding to the IL2, TNFα, and IFNγ promoters at 8 weeks and 6 months postinfection in virus-specific CD8 + T cells following Treg cell coculture. In summary, the results here clearly demonstrate that Foxp3 inhibits IL2, TNFα, and IFNγ transcription by binding to their promoter regions in lentivirus-specific CD8 + T cells. We believe this is the first description of this process during the course of AIDS lentiviral infection.

Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities

PubMed Central

Yang, Li-yun; Liu, Xiao-fang; Yang, Yang; Yang, Lin-lin; Liu, Kai-wen; Tang, Yu-bo; Zhang, Min; Tan, Min-jia; Cheng, Shan-mei; Xu, Ye-chun; Yang, Huai-yu; Liu, Zhi-jie; Song, Gao-jie; Huang, Wei

2017-01-01

CD97 belongs to the adhesion GPCR family characterized by a long ECD linked to the 7TM via a GPCR proteolytic site (GPS) and plays important roles in modulating cell migration and invasion. CD97 (EGF1-5) is a splicing variant of CD97 that recognizes a specific ligand chondroitin sulfate on cell membranes and the extracellular matrix. The aim of this study was to elucidate the extracellular molecular basis of the CD97 EGF1-5 isoform in protein expression, auto-proteolysis and cell adhesion, including epidermal growth factor (EGF)-like domain, GPCR autoproteolysis-inducing (GAIN) domain, as well as GPS mutagenesis and N-glycosylation. Both wild-type (WT) CD97-ECD and its truncated, GPS mutated, PNGase F-deglycosylated, and N-glycosylation site mutated forms were expressed and purified. The auto-proteolysis of the proteins was analyzed with Western blotting and SDS-PAGE. Small angle X-ray scattering (SAXS) and molecular modeling were used to determine a structural profile of the properly expressed receptor. Potential N-glycosylation sites were identified using MS and were modulated with PNGase F digestion and glyco-site mutations. A flow cytometry-based HeLa cell attachment assay was used for all aforementioned CD97 variants to elucidate the molecular basis of CD97-HeLa interactions. A unique concentration-dependent GPS auto-proteolysis was observed in CD97 EGF1-5 isoform with the highest concentration (4 mg/mL) per sample was self-cleaved much faster than the lower concentration (0.1 mg/mL), supporting an intermolecular mechanism of auto-proteolysis that is distinct to the reported intramolecular mechanism for other CD97 isoforms. N-glycosylation affected the auto-proteolysis of CD97 EGF1-5 isoform in a similar way as the other previously reported CD97 isoforms. SAXS data for WT and deglycosylated CD97ECD revealed a spatula-like shape with GAIN and EGF domains constituting the body and handle, respectively. Structural modeling indicated a potential interaction between the GAIN and EGF5 domains accounting for the absence of expression of the GAIN domain itself, although EGF5-GAIN was expressed similarly in the wild-type protein. For HeLa cell adhesion, the GAIN-truncated forms showed dramatically reduced binding affinity. The PNGase F-deglycosylated and GPS mutated forms also exhibited reduced HeLa attachment compared with WT CD97. However, neither N-glycosylation mutagenesis nor auto-proteolysis inhibition caused by N-glycosylation mutagenesis affected CD97-HeLa cell interactions. A comparison of the HeLa binding affinities of PNGase F-digested, GPS-mutated and N-glycosylation-mutated CD97 samples revealed diverse findings, suggesting that the functions of CD97 ECD were complex, and various technologies for function validation should be utilized to avoid single-approach bias when investigating N-glycosylation and auto-proteolysis of CD97. A unique mechanism of concentration-dependent auto-proteolysis of the CD97 EGF1-5 isoform was characterized, suggesting an intermolecular mechanism that is distinct from that of other previously reported CD97 isoforms. The EGF5 and GAIN domains are likely associated with each other as CD97 expression and SAXS data revealed a potential interaction between the two domains. Finally, the GAIN and EGF domains are also important for CD97-HeLa adhesion, whereas N-glycosylation of the CD97 GAIN domain and GPS auto-proteolysis are not required for HeLa cell attachment. PMID:27641734

Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities.

PubMed

Yang, Li-Yun; Liu, Xiao-Fang; Yang, Yang; Yang, Lin-Lin; Liu, Kai-Wen; Tang, Yu-Bo; Zhang, Min; Tan, Min-Jia; Cheng, Shan-Mei; Xu, Ye-Chun; Yang, Huai-Yu; Liu, Zhi-Jie; Song, Gao-Jie; Huang, Wei

2017-01-01

CD97 belongs to the adhesion GPCR family characterized by a long ECD linked to the 7TM via a GPCR proteolytic site (GPS) and plays important roles in modulating cell migration and invasion. CD97 (EGF1-5) is a splicing variant of CD97 that recognizes a specific ligand chondroitin sulfate on cell membranes and the extracellular matrix. The aim of this study was to elucidate the extracellular molecular basis of the CD97 EGF1-5 isoform in protein expression, auto-proteolysis and cell adhesion, including epidermal growth factor (EGF)-like domain, GPCR autoproteolysis-inducing (GAIN) domain, as well as GPS mutagenesis and N-glycosylation. Both wild-type (WT) CD97-ECD and its truncated, GPS mutated, PNGase F-deglycosylated, and N-glycosylation site mutated forms were expressed and purified. The auto-proteolysis of the proteins was analyzed with Western blotting and SDS-PAGE. Small angle X-ray scattering (SAXS) and molecular modeling were used to determine a structural profile of the properly expressed receptor. Potential N-glycosylation sites were identified using MS and were modulated with PNGase F digestion and glyco-site mutations. A flow cytometry-based HeLa cell attachment assay was used for all aforementioned CD97 variants to elucidate the molecular basis of CD97-HeLa interactions. A unique concentration-dependent GPS auto-proteolysis was observed in CD97 EGF1-5 isoform with the highest concentration (4 mg/mL) per sample was self-cleaved much faster than the lower concentration (0.1 mg/mL), supporting an intermolecular mechanism of auto-proteolysis that is distinct to the reported intramolecular mechanism for other CD97 isoforms. N-glycosylation affected the auto-proteolysis of CD97 EGF1-5 isoform in a similar way as the other previously reported CD97 isoforms. SAXS data for WT and deglycosylated CD97ECD revealed a spatula-like shape with GAIN and EGF domains constituting the body and handle, respectively. Structural modeling indicated a potential interaction between the GAIN and EGF5 domains accounting for the absence of expression of the GAIN domain itself, although EGF5-GAIN was expressed similarly in the wild-type protein. For HeLa cell adhesion, the GAIN-truncated forms showed dramatically reduced binding affinity. The PNGase F-deglycosylated and GPS mutated forms also exhibited reduced HeLa attachment compared with WT CD97. However, neither N-glycosylation mutagenesis nor auto-proteolysis inhibition caused by N-glycosylation mutagenesis affected CD97-HeLa cell interactions. A comparison of the HeLa binding affinities of PNGase F-digested, GPS-mutated and N-glycosylation-mutated CD97 samples revealed diverse findings, suggesting that the functions of CD97 ECD were complex, and various technologies for function validation should be utilized to avoid single-approach bias when investigating N-glycosylation and auto-proteolysis of CD97. A unique mechanism of concentration-dependent auto-proteolysis of the CD97 EGF1-5 isoform was characterized, suggesting an intermolecular mechanism that is distinct from that of other previously reported CD97 isoforms. The EGF5 and GAIN domains are likely associated with each other as CD97 expression and SAXS data revealed a potential interaction between the two domains. Finally, the GAIN and EGF domains are also important for CD97-HeLa adhesion, whereas N-glycosylation of the CD97 GAIN domain and GPS auto-proteolysis are not required for HeLa cell attachment.

Analysis of hepatic transcriptome demonstrates altered lipid metabolism following Lactobacillus johnsonii BS15 prevention in chickens with subclinical necrotic enteritis.

PubMed

Qing, Xiaodan; Zeng, Dong; Wang, Hesong; Ni, Xueqin; Lai, Jing; Liu, Lei; Khalique, Abdul; Pan, Kangcheng; Jing, Bo

2018-04-20

Subclinical necrotic enteritis (SNE) widely outbreaks in chickens which inflicted growth-slowing, causing enormous social and economic burdens. To better understand the molecular underpinnings of SNE on lipid metabolism and explore novel preventative strategies against SNE, we studied the regulatory mechanism of a potential probiotic, Lactobacillus johnsonii BS15 on the lipid metabolism pathways involved in chickens with SNE. One hundred eighty one-day-old chickens were randomly divided into three groups and arranged with basal diet (control and SNE group). Added with BS15 (1 × 10 6  cfu/g) or Man Rogosa Sharpe (MRS) liquid medium for 28 days. The hepatic gene expression of each group was then measured using high-throughput analysis methods (RNA-Seq). Quantitative real-time PCR (qRT-PCR) was used to detect the expression changes of the related genes. The results showed that there are eleven lipid metabolic pathways were found during the prevention of BS15 treatment in SNE chickens by RNA-Seq, including the peroxisome proliferator-activated receptor (PPAR) signaling pathway and arachidonic acid metabolism. BS15 notably facilitated the expressions of fatty acid binding protein 2 (FABP2), acyl-CoA synthetase bubblegum family member 1 (ACSBG1), perilipin 1 (PLIN1) and perilipin 2 (PLIN2), which were involved in PPAR signaling pathway of SNE chickens. Besides, suppression of phospholipase A2 group IVA (PLA2G4A) in arachidonic acid metabolism was observed in SNE chickens after BS15 prevention. The expression patterns of FABP2, ACSBG1, PLIN1, PLIN2 and PLA24G in qRT-PCR validation were consistent with RNA-Seq results. These findings indicate that SNE may affect the hepatic lipid metabolism of chickens. Meanwhile, BS15 pretreatment may provide a prospective natural prophylaxis strategy against SNE through improving the PPAR signaling pathway and arachidonic acid metabolism.

Antibody-mediated immunotherapy of macaques chronically infected with SHIV suppresses viraemia

NASA Astrophysics Data System (ADS)

Shingai, Masashi; Nishimura, Yoshiaki; Klein, Florian; Mouquet, Hugo; Donau, Olivia K.; Plishka, Ronald; Buckler-White, Alicia; Seaman, Michael; Piatak, Michael; Lifson, Jeffrey D.; Dimitrov, Dimiter; Nussenzweig, Michel C.; Martin, Malcolm A.

2013-11-01

Neutralizing antibodies can confer immunity to primate lentiviruses by blocking infection in macaque models of AIDS. However, earlier studies of anti-human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies administered to infected individuals or humanized mice reported poor control of virus replication and the rapid emergence of resistant variants. A new generation of anti-HIV-1 monoclonal antibodies, possessing extraordinary potency and breadth of neutralizing activity, has recently been isolated from infected individuals. These neutralizing antibodies target different regions of the HIV-1 envelope glycoprotein including the CD4-binding site, glycans located in the V1/V2, V3 and V4 regions, and the membrane proximal external region of gp41 (refs 9, 10, 11, 12, 13, 14). Here we have examined two of the new antibodies, directed to the CD4-binding site and the V3 region (3BNC117 and 10-1074, respectively), for their ability to block infection and suppress viraemia in macaques infected with the R5 tropic simian-human immunodeficiency virus (SHIV)-AD8, which emulates many of the pathogenic and immunogenic properties of HIV-1 during infections of rhesus macaques. Either antibody alone can potently block virus acquisition. When administered individually to recently infected macaques, the 10-1074 antibody caused a rapid decline in virus load to undetectable levels for 4-7days, followed by virus rebound during which neutralization-resistant variants became detectable. When administered together, a single treatment rapidly suppressed plasma viraemia for 3-5weeks in some long-term chronically SHIV-infected animals with low CD4+ T-cell levels. A second cycle of anti-HIV-1 monoclonal antibody therapy, administered to two previously treated animals, successfully controlled virus rebound. These results indicate that immunotherapy or a combination of immunotherapy plus conventional antiretroviral drugs might be useful as a treatment for chronically HIV-1-infected individuals experiencing immune dysfunction.

Protein dynamics and motions in relation to their functions: several case studies and the underlying mechanisms

PubMed Central

Yang, Li-Quan; Sang, Peng; Tao, Yan; Fu, Yun-Xin; Zhang, Ke-Qin; Xie, Yue-Hui; Liu, Shu-Qun

2013-01-01

Proteins are dynamic entities in cellular solution with functions governed essentially by their dynamic personalities. We review several dynamics studies on serine protease proteinase K and HIV-1 gp120 envelope glycoprotein to demonstrate the importance of investigating the dynamic behaviors and molecular motions for a complete understanding of their structure–function relationships. Using computer simulations and essential dynamic (ED) analysis approaches, the dynamics data obtained revealed that: (i) proteinase K has highly flexible substrate-binding site, thus supporting the induced-fit or conformational selection mechanism of substrate binding; (ii) Ca2+ removal from proteinase K increases the global conformational flexibility, decreases the local flexibility of substrate-binding region, and does not influence the thermal motion of catalytic triad, thus explaining the experimentally determined decreased thermal stability, reduced substrate affinity, and almost unchanged catalytic activity upon Ca2+ removal; (iii) substrate binding affects the large concerted motions of proteinase K, and the resulting dynamic pocket can be connected to substrate binding, orientation, and product release; (iv) amino acid mutations 375 S/W and 423 I/P of HIV-1 gp120 have distinct effects on molecular motions of gp120, facilitating 375 S/W mutant to assume the CD4-bound conformation, while 423 I/P mutant to prefer for CD4-unliganded state. The mechanisms underlying protein dynamics and protein–ligand binding, including the concept of the free energy landscape (FEL) of the protein–solvent system, how the ruggedness and variability of FEL determine protein's dynamics, and how the three ligand-binding models, the lock-and-key, induced-fit, and conformational selection are rationalized based on the FEL theory are discussed in depth. PMID:23527883

Protein dynamics and motions in relation to their functions: several case studies and the underlying mechanisms.

PubMed

Yang, Li-Quan; Sang, Peng; Tao, Yan; Fu, Yun-Xin; Zhang, Ke-Qin; Xie, Yue-Hui; Liu, Shu-Qun

2014-01-01

Proteins are dynamic entities in cellular solution with functions governed essentially by their dynamic personalities. We review several dynamics studies on serine protease proteinase K and HIV-1 gp120 envelope glycoprotein to demonstrate the importance of investigating the dynamic behaviors and molecular motions for a complete understanding of their structure-function relationships. Using computer simulations and essential dynamic (ED) analysis approaches, the dynamics data obtained revealed that: (i) proteinase K has highly flexible substrate-binding site, thus supporting the induced-fit or conformational selection mechanism of substrate binding; (ii) Ca(2+) removal from proteinase K increases the global conformational flexibility, decreases the local flexibility of substrate-binding region, and does not influence the thermal motion of catalytic triad, thus explaining the experimentally determined decreased thermal stability, reduced substrate affinity, and almost unchanged catalytic activity upon Ca(2+) removal; (iii) substrate binding affects the large concerted motions of proteinase K, and the resulting dynamic pocket can be connected to substrate binding, orientation, and product release; (iv) amino acid mutations 375 S/W and 423 I/P of HIV-1 gp120 have distinct effects on molecular motions of gp120, facilitating 375 S/W mutant to assume the CD4-bound conformation, while 423 I/P mutant to prefer for CD4-unliganded state. The mechanisms underlying protein dynamics and protein-ligand binding, including the concept of the free energy landscape (FEL) of the protein-solvent system, how the ruggedness and variability of FEL determine protein's dynamics, and how the three ligand-binding models, the lock-and-key, induced-fit, and conformational selection are rationalized based on the FEL theory are discussed in depth.

Crystal Structure of Poliovirus 3CD Protein: Virally Encoded Protease and Precursor to the RNA-Dependent RNA Polymerase

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

Marcotte,L.; Wass, A.; Gohara, D.

2007-01-01

Poliovirus 3CD is a multifunctional protein that serves as a precursor to the protease 3Cpro and the viral polymerase 3Dpol and also plays a role in the control of viral replication. Although 3CD is a fully functional protease, it lacks polymerase activity. We have solved the crystal structures of 3CD at a 3.4- Angstroms resolution and the G64S fidelity mutant of 3Dpol at a 3.0- Angstroms resolution. In the 3CD structure, the 3C and 3D domains are joined by a poorly ordered polypeptide linker, possibly to facilitate its cleavage, in an arrangement that precludes intramolecular proteolysis. The polymerase active sitemore » is intact in both the 3CD and the 3Dpol G64S structures, despite the disruption of a network proposed to position key residues in the active site. Therefore, changes in molecular flexibility may be responsible for the differences in fidelity and polymerase activities. Extensive packing contacts between symmetry-related 3CD molecules and the approach of the 3C domain's N terminus to the VPg binding site suggest how 3Dpol makes biologically relevant interactions with the 3C, 3CD, and 3BCD proteins that control the uridylylation of VPg during the initiation of viral replication. Indeed, mutations designed to disrupt these interfaces have pronounced effects on the uridylylation reaction in vitro.« less

Progressive CD4+ central–memory T cell decline results in CD4+ effector–memory insufficiency and overt disease in chronic SIV infection

PubMed Central

Okoye, Afam; Meier-Schellersheim, Martin; Brenchley, Jason M.; Hagen, Shoko I.; Walker, Joshua M.; Rohankhedkar, Mukta; Lum, Richard; Edgar, John B.; Planer, Shannon L.; Legasse, Alfred; Sylwester, Andrew W.; Piatak, Michael; Lifson, Jeffrey D.; Maino, Vernon C.; Sodora, Donald L.; Douek, Daniel C.; Axthelm, Michael K.; Grossman, Zvi; Picker, Louis J.

2007-01-01

Primary simian immunodeficiency virus (SIV) infections of rhesus macaques result in the dramatic depletion of CD4+ CCR5+ effector–memory T (TEM) cells from extra-lymphoid effector sites, but in most infections, an increased rate of CD4+ memory T cell proliferation appears to prevent collapse of effector site CD4+ TEM cell populations and acute-phase AIDS. Eventually, persistent SIV replication results in chronic-phase AIDS, but the responsible mechanisms remain controversial. Here, we demonstrate that in the chronic phase of progressive SIV infection, effector site CD4+ TEM cell populations manifest a slow, continuous decline, and that the degree of this depletion remains a highly significant correlate of late-onset AIDS. We further show that due to persistent immune activation, effector site CD4+ TEM cells are predominantly short-lived, and that their homeostasis is strikingly dependent on the production of new CD4+ TEM cells from central–memory T (TCM) cell precursors. The instability of effector site CD4+ TEM cell populations over time was not explained by increasing destruction of these cells, but rather was attributable to progressive reduction in their production, secondary to decreasing numbers of CCR5− CD4+ TCM cells. These data suggest that although CD4+ TEM cell depletion is a proximate mechanism of immunodeficiency, the tempo of this depletion and the timing of disease onset are largely determined by destruction, failing production, and gradual decline of CD4+ TCM cells. PMID:17724130

Progressive CD4+ central memory T cell decline results in CD4+ effector memory insufficiency and overt disease in chronic SIV infection.

PubMed

Okoye, Afam; Meier-Schellersheim, Martin; Brenchley, Jason M; Hagen, Shoko I; Walker, Joshua M; Rohankhedkar, Mukta; Lum, Richard; Edgar, John B; Planer, Shannon L; Legasse, Alfred; Sylwester, Andrew W; Piatak, Michael; Lifson, Jeffrey D; Maino, Vernon C; Sodora, Donald L; Douek, Daniel C; Axthelm, Michael K; Grossman, Zvi; Picker, Louis J

2007-09-03

Primary simian immunodeficiency virus (SIV) infections of rhesus macaques result in the dramatic depletion of CD4(+) CCR5(+) effector-memory T (T(EM)) cells from extra-lymphoid effector sites, but in most infections, an increased rate of CD4(+) memory T cell proliferation appears to prevent collapse of effector site CD4(+) T(EM) cell populations and acute-phase AIDS. Eventually, persistent SIV replication results in chronic-phase AIDS, but the responsible mechanisms remain controversial. Here, we demonstrate that in the chronic phase of progressive SIV infection, effector site CD4(+) T(EM) cell populations manifest a slow, continuous decline, and that the degree of this depletion remains a highly significant correlate of late-onset AIDS. We further show that due to persistent immune activation, effector site CD4(+) T(EM) cells are predominantly short-lived, and that their homeostasis is strikingly dependent on the production of new CD4(+) T(EM) cells from central-memory T (T(CM)) cell precursors. The instability of effector site CD4(+) T(EM) cell populations over time was not explained by increasing destruction of these cells, but rather was attributable to progressive reduction in their production, secondary to decreasing numbers of CCR5(-) CD4(+) T(CM) cells. These data suggest that although CD4(+) T(EM) cell depletion is a proximate mechanism of immunodeficiency, the tempo of this depletion and the timing of disease onset are largely determined by destruction, failing production, and gradual decline of CD4(+) T(CM) cells.

Functional analysis of the Arabidopsis PLDZ2 promoter reveals an evolutionarily conserved low-Pi-responsive transcriptional enhancer element

PubMed Central

Oropeza-Aburto, Araceli; Cruz-Ramírez, Alfredo; Acevedo-Hernández, Gustavo J.; Pérez-Torres, Claudia-Anahí; Caballero-Pérez, Juan; Herrera-Estrella, Luis

2012-01-01

Plants have evolved a plethora of responses to cope with phosphate (Pi) deficiency, including the transcriptional activation of a large set of genes. Among Pi-responsive genes, the expression of the Arabidopsis phospholipase DZ2 (PLDZ2) is activated to participate in the degradation of phospholipids in roots in order to release Pi to support other cellular activities. A deletion analysis was performed to identify the regions determining the strength, tissue-specific expression, and Pi responsiveness of this regulatory region. This study also reports the identification and characterization of a transcriptional enhancer element that is present in the PLDZ2 promoter and able to confer Pi responsiveness to a minimal, inactive 35S promoter. This enhancer also shares the cytokinin and sucrose responsive properties observed for the intact PLDZ2 promoter. The EZ2 element contains two P1BS motifs, each of which is the DNA binding site of transcription factor PHR1. Mutation analysis showed that the P1BS motifs present in EZ2 are necessary but not sufficient for the enhancer function, revealing the importance of adjacent sequences. The structural organization of EZ2 is conserved in the orthologous genes of at least eight families of rosids, suggesting that architectural features such as the distance between the two P1BS motifs are also important for the regulatory properties of this enhancer element. PMID:22210906

Lipopolysaccharide-induced inhibition of transcription of tlr4 in vitro is reversed by dexamethasone and correlates with presence of conserved NFκB binding sites

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

Bonin, Camila P., E-mail: mila_bonin@yahoo.com.br; Baccarin, Raquel Y.A., E-mail: baccarin@usp.br; Nostell, Katarina, E-mail: katarina.nostell@slu.se

2013-03-08

Highlights: ► Chimpanzees, horses and humans have regions of similarity on TLR4 and MD2 promoters. ► Rodents have few regions of similarity on TLR4 promoter when compared to primates. ► Conserved NFkB binding sites were found in the promoters of TLR4 and MD2. ► LPS-induced inhibition of TLR4 transcription is reversed by dexamethasone. ► LPS-induced transcription of MD2 is inhibited by dexamethasone. -- Abstract: Engagement of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS) is a master trigger of the deleterious effects of septic shock. Horses and humans are considered the most sensitive species to septic shock, but the mechanisms explainingmore » these phenomena remain elusive. Analysis of tlr4 promoters revealed high similarity among LPS-sensitive species (human, chimpanzee, and horse) and low similarity with LPS-resistant species (mouse and rat). Four conserved nuclear factor kappa B (NFκB) binding sites were found in the tlr4 promoter and two in the md2 promoter sequences that are likely to be targets for dexamethasone regulation. In vitro treatment of equine peripheral blood mononuclear cells (eqPBMC) with LPS decreased transcripts of tlr4 and increased transcription of md2 (myeloid differentiation factor 2) and cd14 (cluster of differentiation 14). Treatment with dexamethasone rescued transcription of tlr4 after LPS inhibition. LPS-induced transcription of md2 was inhibited in the presence of dexamethasone. Dexamethasone alone did not affect transcription of tlr4 and md2.« less

SLP-76-ADAP adaptor module regulates LFA-1 mediated costimulation and T cell motility.

PubMed

Wang, Hongyan; Wei, Bin; Bismuth, Georges; Rudd, Christopher E

2009-07-28

Although adaptor ADAP (FYB) and its binding to SLP-76 has been implicated in TcR-induced "inside-out" signaling for LFA-1 activation in T cells, little is known regarding its role in LFA-1-mediated "outside-in" signaling. In this study, we demonstrate that ADAP and SLP-76-ADAP binding are coupled to LFA-1 costimulation of IL-2 production, F-actin clustering, cell polarization, and T cell motility. LFA-1 enhancement of anti-CD3-induced IL-2 production was completely dependent on SLP-76-ADAP binding. Further, anti-CD3 was found to require CD11a ligation by antibody or ICAM1 to cause T cell polarization. ADAP augmented this polarization induced by anti-CD3/CD11a, but not by anti-CD3 alone. ADAP expression with LFA-1 ligation alone was sufficient to polarize T cells directly and to increase T cell motility whereas the loss of ADAP in ADAP-/- primary T cells reduced motility. A mutant lacking SLP-76-binding sites (M12) blocked LFA-1 costimulation of IL-2 production, polarization, and motility. LFA-1-ADAP polarization was also dependent on src kinases, Rho GTPases, phospholipase C, and phosphoinositol 3-kinase. Our findings provide evidence of an obligatory role for the SLP-76-ADAP module in LFA-1-mediated costimulation in T cells.

Some novel insights on HPV16 related cervical cancer pathogenesis based on analyses of LCR methylation, viral load, E7 and E2/E4 expressions.

PubMed

Das Ghosh, Damayanti; Bhattacharjee, Bornali; Sen, Shrinka; Premi, Laikangbam; Mukhopadhyay, Indranil; Chowdhury, Rahul Roy; Roy, Sudipta; Sengupta, Sharmila

2012-01-01

This study was undertaken to decipher the interdependent roles of (i) methylation within E2 binding site I and II (E2BS-I/II) and replication origin (nt 7862) in the long control region (LCR), (ii) expression of viral oncogene E7, (iii) expression of the transcript (E7-E1/E4) that encodes E2 repressor protein and (iv) viral load, in human papillomavirus 16 (HPV16) related cervical cancer (CaCx) pathogenesis. The results revealed over-representation (p<0.001) of methylation at nucleotide 58 of E2BS-I among E2-intact CaCx cases compared to E2-disrupted cases. Bisulphite sequencing of LCR revealed overrepresentation of methylation at nucleotide 58 or other CpGs in E2BS-I/II, among E2-intact cases than E2-disrupted cases and lack of methylation at replication origin in case of both. The viral transcript (E7-E1/E4) that produces the repressor E2 was analyzed by APOT (amplification of papillomavirus oncogenic transcript)-coupled-quantitative-RT-PCR (of E7 and E4 genes) to distinguish episomal (pure or concomitant with integrated) from purely integrated viral genomes based on the ratio, E7 C(T)/E4 C(T). Relative quantification based on comparative C(T) (threshold cycle) method revealed 75.087 folds higher E7 mRNA expression in episomal cases over purely integrated cases. Viral load and E2 gene copy numbers were negatively correlated with E7 C(T) (p = 0.007) and E2 C(T) (p<0.0001), respectively, each normalized with ACTB C(T), among episomal cases only. The k-means clustering analysis considering E7 C(T) from APOT-coupled-quantitative-RT-PCR assay, in conjunction with viral load, revealed immense heterogeneity among the HPV16 positive CaCx cases portraying integrated viral genomes. The findings provide novel insights into HPV16 related CaCx pathogenesis and highlight that CaCx cases that harbour episomal HPV16 genomes with intact E2 are likely to be distinct biologically, from the purely integrated viral genomes in terms of host genes and/or pathways involved in cervical carcinogenesis.

Unusual interaction of human apurinic/apyrimidinic endonuclease 1 (APE1) with abasic sites via the Schiff-base-dependent mechanism.

PubMed

Ilina, Ekaterina S; Khodyreva, Svetlana N; Lavrik, Olga I

2018-05-03

Clustered apurinic/apyrimidinic (AP) sites are more cytotoxic than isolated AP lesions because double strand breaks (DSB) can be formed during repair of closely positioned bistranded AP sites. Formation of DSB due to simultaneous cleavage of bistranded AP sites may be regulated by proteins specifically interacting with this complex lesion. A set of AP DNA duplexes containing AP sites in both strands in different mutual orientation (BS-AP DNAs) was used for search in the extracts of human cells proteins specifically recognizing clustered AP sites. A protein, which formed the Schiff-base-dependent covalent products having an apparent molecular mass of 50 kDa with the subset of BS-AP DNAs, was identified by mass spectrometry as apurinic/apyrimidinic endonuclease 1 (APE1). The identity of trapped protein was confirmed by Western blot analysis with anti-APE1 antibodies. Purified recombinant human APE1 is also capable of forming the 50 kDa-adducts with efficiency of BS-AP DNAs cross-linking to APE1 being dependent on the mutual orientation of AP sites. In spite of formation of the Schiff-base-dependent intermediate, which is prerequisite for the β-elimination mechanism, APE1 is unable to cleave AP sites. APE1 lacking the first 34 amino acids at the N-terminus, unlike wild type enzyme, is unable to form cross-links with BS-AP DNAs that testifies to the involvement of disordered N-terminal extension, which is enriched in lysine residues, in the interaction with AP sites. The yield of APE1-AP DNA cross-links was found to correlate with the enzyme amount in the extracts estimated by the immunochemical approach; therefore the BS-AP DNA-probes can be useful for comparative analysis of APE1 content in cell extracts. Copyright © 2018. Published by Elsevier B.V.

Processing, fusogenicity, virion incorporation and CXCR4-binding activity of a feline immunodeficiency virus envelope glycoprotein lacking the two conserved N-glycosylation sites at the C-terminus of the V3 domain.

PubMed

González, Silvia A; Affranchino, José L

2016-07-01

The process of feline immunodeficiency virus (FIV) entry into its target cells is initiated by the association of the surface (SU) subunit of the viral envelope glycoprotein (Env) with the cellular receptors CD134 and CXCR4. This event is followed by the fusion of the viral and cellular membranes, which is mediated by the transmembrane (TM) subunit of Env. We and others have previously demonstrated that the V3 domain of the SU subunit of Env is essential for CXCR4 binding. Of note, there are two contiguous and highly conserved potential N-glycosylation sites ((418)NST(420) and (422)NLT(424)) located at the C-terminal side of the V3 domain. We therefore decided to study the relevance for Env functions of these N-glycosylation motifs and found that disruption of both of them by introducing the N418Q/N422Q double amino acid substitution drastically impairs Env processing into the SU and TM subunits. Moreover, the simultaneous mutation of these N-glycosylation sites prevents Env incorporation into virions and Env-mediated cell-to-cell fusion. Notably, a recombinant soluble version of the SU glycoprotein carrying the double amino acid replacement N418Q/N422Q at the V3 C-terminal side binds to CXCR4 with an efficiency similar to that of wild-type SU.

The IgV domain of human B7-2 (CD86) is sufficient to co-stimulate T lymphocytes and induce cytokine secretion.

PubMed

Rennert, P; Furlong, K; Jellis, C; Greenfield, E; Freeman, G J; Ueda, Y; Levine, B; June, C H; Gray, G S

1997-06-01

B7-1 (CD80) and B7-2 (CD86) are genetically and structurally related molecules expressed on antigen-presenting cells. Both bind CD28 to co-stimulate T lymphocytes, resulting in proliferation and cytokine production. The extracellular portions of B7-1 and B7-2 which bind to CD28 and CTLA-4 are related to Ig variable (V) and Ig constant (C) domain sequences. Recent reports have described splice variant forms of B7 proteins which occur in vivo and are of unknown function. Here we describe soluble recombinant forms of B7-1 and B7-2 containing either both of the Ig-like extracellular domains or the individual IgV or IgC domains coupled to an Ig Fc tail. Soluble B7-1 and B7-2 bind to CD28 and CTLA-4, and effectively co-stimulate T lymphocytes resulting in their proliferation and the secretion of cytokines. Furthermore, the IgV domain of B7-2 binds CD28 and CTLA-4, competes with B7-1 and B7-2 for binding to these receptors, and co-stimulates T lymphocytes. Cross-linked soluble B7-2v was the most potent co-stimulatory molecule tested and was active at a concentration approximately 100-fold lower than cross-linked soluble B7-1 or B7-2 proteins. When bound to tosyl-activated beads, B7-2v was capable of sustaining multiple rounds of T cell expansion. These data complement the description of naturally occurring variants to suggest that T cell co-stimulation in vivo may be regulated by soluble or truncated forms of B7 proteins.

Fusobacterium nucleatum binding to complement regulatory protein CD46 modulates the expression and secretion of cytokines and matrix metalloproteinases by oral epithelial cells.

PubMed

Mahtout, Hayette; Chandad, Fatiha; Rojo, Jose M; Grenier, Daniel

2011-02-01

Periodontitis is a chronic inflammatory disease that results in the destruction of the supporting tissues of the teeth. Gingival epithelial cells are an important mechanical barrier and participate in the host inflammatory response to periodontopathogens. The aim of the present study is to investigate the capacity of Fusobacterium nucleatum to bind to the complement regulatory protein CD46 expressed by oral epithelial cells and to determine the impact of the binding on the gene expression and protein secretion of interleukin (IL)-6, IL-8, and matrix metalloproteinase (MMP)-9 by oral epithelial cells. Binding of recombinant human CD46 to the surface of F. nucleatum was demonstrated by immunologic assays. After stimulation of oral epithelial cells with F. nucleatum, gene expression was determined by real-time polymerase chain reaction analysis while protein secretion was monitored by enzyme-linked immunosorbent assays. Heat and protease treatments of bacterial cells reduced CD46 binding. F. nucleatum-bound CD46 mediated the cleavage of C3b in the presence of factor I. Stimulating oral epithelial cells with F. nucleatum at a multiplicity of infection of 50 resulted in a significant upregulation of the gene expression and protein secretion of IL-6, IL-8, and MMP-9 by oral epithelial cells. However, pretreating the epithelial cells with an anti-CD46 polyclonal antibody attenuated the production of IL-6, IL-8, and MMP-9 in response to F. nucleatum. Such an inhibitory effect was not observed with non-specific antibodies. The present study demonstrates that F. nucleatum can bind the complement regulatory protein CD46. The interaction of F. nucleatum with epithelial cell surface CD46 may contribute to increasing the levels of proinflammatory mediators and MMPs in periodontal sites and consequently modulate tissue destruction.

Studies on the interaction of a synthetic nitro-flavone derivative with DNA: A multi-spectroscopic and molecular docking approach.

PubMed

Mitra, A; Saikh, F; Das, J; Ghosh, S; Ghosh, R

2018-05-22

Interaction of a ligand with DNA is often the basis of drug action of many molecules. Flavones are important in this regard as their structural features confer them the ability to bind to DNA. 2-(4-Nitrophenyl)-4H-chromen-4-one (4NCO) is an important biologically active synthetic flavone derivative. We are therefore interested in studying its interaction with DNA. Absorption spectroscopy studies included standard and reverse titration, effect of ionic strength on titration, determination of stoichiometry of binding and thermal denaturation. Spectrofluorimetry techniques included fluorimetric titration, quenching studies and fluorescence displacement assay. Assessment of relative viscosity and estimation of thermodynamic parameters from CD spectral studies were also undertaken. Furthermore, molecular docking analyses were also done with different short DNA sequences. The fluorescent flavone 4NCO reversibly interacted with DNA through partial intercalation as well as minor-groove binding. The binding constant and the number of binding sites were of the order 10 4  M -1 and 1 respectively. The binding stoichiometry with DNA was found to be 1:1. The nature of the interaction of 4NCO with DNA was hydrophobic in nature and the process of binding was spontaneous, endothermic and entropy-driven. The flavone also showed a preference for binding to GC rich sequences. The study presents a profile for structural and thermodynamic parameters, for the binding of 4NCO with DNA. DNA is an important target for ligands that are effective against cell proliferative disorders. In this regard, the molecule 4NCO is important since it can exert its biological activity through its DNA binding ability and can be a potential drug candidate. Copyright © 2018 Elsevier B.V. All rights reserved.

Directing stem cell trafficking via GPS.

PubMed

Sackstein, Robert

2010-01-01

The success of stem-cell-based regenerative therapeutics critically hinges on delivering relevant stem/progenitor cells to sites of tissue injury. To achieve adequate parenchymal infiltration following intravascular administration, it is first necessary that circulating cells bind to target tissue endothelium with sufficient strength to overcome the prevailing forces of hemodynamic shear. The principal mediators of these shear-resistant binding interactions consist of a family of C-type lectins known as "selectins" that bind discrete sialofucosylated glycans on their respective ligands. One member of this family, E-selectin, is an endothelial molecule that is inducibly expressed on postcapillary venules at all sites of tissue injury, but is also constitutively expressed on the luminal surface of bone marrow and dermal microvascular endothelium. Most stem/progenitor cells express high levels of CD44, and, in particular, human hematopoietic stem cells express a specialized sialofucosylated glycoform of CD44 known as "hematopoietic cell E-/L-selectin ligand" (HCELL) that functions as a potent E-selectin ligand. This chapter describes a method called "glycosyltransferase-programmed stereosubstitution" (GPS) for custom-modifying CD44 glycans to create HCELL on the surface of living cells that natively lack HCELL. Ex vivo glycan engineering of HCELL via GPS licenses trafficking of infused cells to endothelial beds that express E-selectin, thereby enabling efficient vascular delivery of stem/progenitor cells to sites where they are needed. Copyright (c) 2010 Elsevier Inc. All rights reserved.

MCT4 surpasses the prognostic relevance of the ancillary protein CD147 in clear cell renal cell carcinoma.

PubMed

Fisel, Pascale; Stühler, Viktoria; Bedke, Jens; Winter, Stefan; Rausch, Steffen; Hennenlotter, Jörg; Nies, Anne T; Stenzl, Arnulf; Scharpf, Marcus; Fend, Falko; Kruck, Stephan; Schwab, Matthias; Schaeffeler, Elke

2015-10-13

Cluster of differentiation 147 (CD147/BSG) is a transmembrane glycoprotein mediating oncogenic processes partly through its role as binding partner for monocarboxylate transporter MCT4/SLC16A3. As demonstrated for MCT4, CD147 is proposed to be associated with progression in clear cell renal cell carcinoma (ccRCC). In this study, we evaluated the prognostic relevance of CD147 in comparison to MCT4/SLC16A3 expression and DNA methylation. CD147 protein expression was assessed in two independent ccRCC-cohorts (n = 186, n = 59) by immunohistochemical staining of tissue microarrays and subsequent manual as well as automated software-supported scoring (Tissue Studio, Definien sAG). Epigenetic regulation of CD147 was investigated using RNAseq and DNA methylation data of The Cancer Genome Atlas. These results were validated in our cohort. Relevance of prognostic models for cancer-specific survival, comprising CD147 and MCT4 expression or SLC16A3 DNA methylation, was compared using chi-square statistics. CD147 protein expression generated with Tissue Studio correlated significantly with those from manual scoring (P < 0.0001, rS = 0.85), indicating feasibility of software-based evaluation exemplarily for the membrane protein CD147 in ccRCC. Association of CD147 expression with patient outcome differed between cohorts. DNA methylation in the CD147/BSG promoter was not associated with expression. Comparison of prognostic relevance of CD147/BSG and MCT4/SLC16A3, showed higher significance for MCT4 expression and superior prognostic power for DNA methylation at specific CpG-sites in the SLC16A3 promoter (e.g. CD147 protein: P = 0.7780,Harrell's c-index = 53.7% vs. DNA methylation: P = 0.0076, Harrell's c-index = 80.0%). Prognostic significance of CD147 protein expression could not surpass that of MCT4, especially of SLC16A3 DNA methylation, corroborating the role of MCT4 as prognostic biomarker for ccRCC.

MCT4 surpasses the prognostic relevance of the ancillary protein CD147 in clear cell renal cell carcinoma

PubMed Central

Winter, Stefan; Rausch, Steffen; Hennenlotter, Jörg; Nies, Anne T.; Stenzl, Arnulf; Scharpf, Marcus; Fend, Falko; Kruck, Stephan; Schwab, Matthias; Schaeffeler, Elke

2015-01-01

Cluster of differentiation 147 (CD147/BSG) is a transmembrane glycoprotein mediating oncogenic processes partly through its role as binding partner for monocarboxylate transporter MCT4/SLC16A3. As demonstrated for MCT4, CD147 is proposed to be associated with progression in clear cell renal cell carcinoma (ccRCC). In this study, we evaluated the prognostic relevance of CD147 in comparison to MCT4/SLC16A3 expression and DNA methylation. Methods CD147 protein expression was assessed in two independent ccRCC-cohorts (n = 186, n = 59) by immunohistochemical staining of tissue microarrays and subsequent manual as well as automated software-supported scoring (Tissue Studio, Definien sAG). Epigenetic regulation of CD147 was investigated using RNAseq and DNA methylation data of The Cancer Genome Atlas. These results were validated in our cohort. Relevance of prognostic models for cancer-specific survival, comprising CD147 and MCT4 expression or SLC16A3 DNA methylation, was compared using chi-square statistics. Results CD147 protein expression generated with Tissue Studio correlated significantly with those from manual scoring (P < 0.0001, rS = 0.85), indicating feasibility of software-based evaluation exemplarily for the membrane protein CD147 in ccRCC. Association of CD147 expression with patient outcome differed between cohorts. DNA methylation in the CD147/BSG promoter was not associated with expression. Comparison of prognostic relevance of CD147/BSG and MCT4/SLC16A3, showed higher significance for MCT4 expression and superior prognostic power for DNA methylation at specific CpG-sites in the SLC16A3 promoter (e.g. CD147 protein: P = 0.7780, Harrell's c-index = 53.7% vs. DNA methylation: P = 0.0076, Harrell's c-index = 80.0%). Conclusions Prognostic significance of CD147 protein expression could not surpass that of MCT4, especially of SLC16A3 DNA methylation, corroborating the role of MCT4 as prognostic biomarker for ccRCC. PMID:26384346

Molecular functions of Xanthomonas type III effector AvrBsT and its plant interactors in cell death and defense signaling.

PubMed

Han, Sang Wook; Hwang, Byung Kook

2017-02-01

Xanthomonas effector AvrBsT interacts with plant defense proteins and triggers cell death and defense response. This review highlights our current understanding of the molecular functions of AvrBsT and its host interactor proteins. The AvrBsT protein is a member of a growing family of effector proteins in both plant and animal pathogens. Xanthomonas type III effector AvrBsT, a member of the YopJ/AvrRxv family, suppresses plant defense responses in susceptible hosts, but triggers cell death signaling leading to hypersensitive response (HR) and defense responses in resistant plants. AvrBsT interacts with host defense-related proteins to trigger the HR cell death and defense responses in plants. Here, we review and discuss recent progress in understanding the molecular functions of AvrBsT and its host interactor proteins in pepper (Capsicum annuum). Pepper arginine decarboxylase1 (CaADC1), pepper aldehyde dehydrogenase1 (CaALDH1), pepper heat shock protein 70a (CaHSP70a), pepper suppressor of the G2 allele of skp1 (CaSGT1), pepper SNF1-related kinase1 (SnRK1), and Arabidopsis acetylated interacting protein1 (ACIP1) have been identified as AvrBsT interactors in pepper and Arabidopsis. Gene expression profiling, virus-induced gene silencing, and transient transgenic overexpression approaches have advanced the functional characterization of AvrBsT-interacting proteins in plants. AvrBsT is localized in the cytoplasm and forms protein-protein complexes with host interactors. All identified AvrBsT interactors regulate HR cell death and defense responses in plants. Notably, CaSGT1 physically binds to both AvrBsT and pepper receptor-like cytoplasmic kinase1 (CaPIK1) in the cytoplasm. During infection with Xanthomonas campestris pv. vesicatoria strain Ds1 (avrBsT), AvrBsT is phosphorylated by CaPIK1 and forms the active AvrBsT-CaSGT1-CaPIK1 complex, which ultimately triggers HR cell death and defense responses. Collectively, the AvrBsT interactor proteins are involved in plant cell death and immunity signaling.

Fab-based bispecific antibody formats with robust biophysical properties and biological activity.

PubMed

Wu, Xiufeng; Sereno, Arlene J; Huang, Flora; Lewis, Steven M; Lieu, Ricky L; Weldon, Caroline; Torres, Carina; Fine, Cody; Batt, Micheal A; Fitchett, Jonathan R; Glasebrook, Andrew L; Kuhlman, Brian; Demarest, Stephen J

2015-01-01

A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with CH1/CL domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore 2 novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when 2 Fabs are co-expressed simultaneously, thus allowing the use of 2 different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2×HER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFR × CD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a bispecific T cell engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity.

Fab-based bispecific antibody formats with robust biophysical properties and biological activity

PubMed Central

Wu, Xiufeng; Sereno, Arlene J; Huang, Flora; Lewis, Steven M; Lieu, Ricky L; Weldon, Caroline; Torres, Carina; Fine, Cody; Batt, Micheal A; Fitchett, Jonathan R; Glasebrook, Andrew L; Kuhlman, Brian; Demarest, Stephen J

2015-01-01

A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with CH1/CL domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore 2 novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when 2 Fabs are co-expressed simultaneously, thus allowing the use of 2 different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2×HER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFR × CD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a bispecific T cell engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity. PMID:25774965

Soil-solution chemistry in a low-elevation spruce-fir ecosystem, Howland, Maine

USGS Publications Warehouse

Fernandez, Ivan J.; Lawrence, Gregory B.; Son, Yowhan

1995-01-01

Soil solutions were collected monthly by tension and zero-tension lysimeters in a low-elevation red spruce stand in east-central Maine from May 1987 through December 1992. Soil solutions collected by Oa tension lysimeters had higher concentrations of most constituents than the Oa zero-tension lysimeters. In Oa horizon soil solutions growing season concentrations for SO4, Ca, and Mg averaged 57, 43, and 30 μmol L−1 in tension lysimeters, and 43, 28, and 19 μmol L−1 in zero-tension lysimeters, respectively. Because tension lysimeters remove water held by the soil at tensions up to 10 kPa, solutions are assumed to have more time to react with the soil compared to freely draining solutions collected by zero-tension lysimeters. Solutions collected in the Bs horizon by both types of collectors were similar which was attributed to the frequency of time periods when the water table was above the Bs lysimeters. Concentrations of SO4 and NO3 at this site were lower than concentrations reported for most other eastern U.S. spruce-fir sites, but base cation concentrations fell in the same range. Aluminum concentrations in this study were also lower than reported for other sites in the eastern U.S. and Ca/Al ratios did not suggest inhibition of Ca uptake by roots. Concentrations of SO4, Ca, K, and Cl decreased significantly in both the Oa and Bs horizons over the 56-month sampling period, which could reflect decreasing deposition rates for sulfur and base cations, climatic influences, or natural variation. A longer record of measured fluxes will be needed to adequately define temporal trends in solution chemistry and their causes.

CD studies on ribonuclease A - oligonucleotides interactions.

PubMed Central

White, M D; Keren-Zur, M; Lapidot, Y

1977-01-01

The interaction of ApU, Aps4U, Aps4Up, ApAps4Up and Gps4U with RNase A was studied by CD difference spectroscopy. The use of 4-thiouridine (s4U) containing oligonucleotides enables to distinguish between the interaction of the different components of the ligand with the enzyme. The mode of binding of the oligonucleotides to the enzyme is described. From this mode of binding it is explained why Aps4U, for example, inhibits RNase A, while s4UpA serves as a substrate. PMID:866194

CD80 and CD86 IgC domains are important for quaternary structure, receptor binding and co-signaling function.

PubMed

Girard, Tanya; Gaucher, Denis; El-Far, Mohamed; Breton, Gaëlle; Sékaly, Rafick-Pierre

2014-09-01

CD86 and CD80, the ligands for the co-stimulatory molecules CD28 and CTLA-4, are members of the Ig superfamily. Their structure includes Ig variable-like (IgV) domains, Ig constant-like (IgC) domains and intracellular domains. Although crystallographic studies have clearly identified the IgV domain to be responsible for receptor interactions, earlier studies suggested that both Ig domains are required for full co-signaling function. Herein, we have used deletion and chimeric human CD80 and CD86 molecules in co-stimulation assays to study the impact of the multimeric state of IgV and IgC domains on receptor binding properties and on co-stimulatory function in a peptide-specific T cell activation model. We report for the first time the presence of CD80 dimers and CD86 monomers in living cells. Moreover, we show that the IgC domain of both molecules inhibits multimer formation and greatly affects binding to the co-receptors CD28 and CTLA-4. Finally, both IgC and intracellular domains are required for full co-signaling function. These findings reveal the distinct but complementary roles of CD80 and CD86 IgV and IgC domains in T cell activation. Copyright © 2014 Elsevier B.V. All rights reserved.

Characterizing multiple metal ion binding sites within a ribozyme by cadmium-induced EPR silencing

PubMed Central

Kisseleva, Natalia; Kraut, Stefanie; Jäschke, Andres; Schiemann, Olav

2007-01-01

In ribozyme catalysis, metal ions are generally known to make structural and∕or mechanistic contributions. The catalytic activity of a previously described Diels-Alderase ribozyme was found to depend on the concentration of divalent metal ions, and crystallographic data revealed multiple binding sites. Here, we elucidate the interactions of this ribozyme with divalent metal ions in solution using electron paramagnetic resonance (EPR) spectroscopy. Manganese ion titrations revealed five high-affinity Mn2+ binding sites with an upper Kd of 0.6±0.2 μM. In order to characterize each binding site individually, EPR-silent Cd2+ ions were used to saturate the other binding sites. This cadmium-induced EPR silencing showed that the Mn2+ binding sites possess different affinities. In addition, these binding sites could be assigned to three different types, including innersphere, outersphere, and a Mn2+ dimer. Based on simulations, the Mn2+-Mn2+ distance within the dimer was found to be ∼6 Å, which is in good agreement with crystallographic data. The EPR-spectroscopic characterization reveals no structural changes upon addition of a Diels-Alder product, supporting the concept of a preorganized catalytic pocket in the Diels-Alder ribozyme and the structural role of these ions. PMID:19404418

Interaction of lafutidine in binding to human serum albumin in gastric ulcer therapy: STD-NMR, WaterLOGSY-NMR, NMR relaxation times, Tr-NOESY, molecule docking, and spectroscopic studies.

PubMed

Yang, Hongqin; Huang, Yanmei; He, Jiawei; Li, Shanshan; Tang, Bin; Li, Hui

2016-09-15

In this study, lafutidine (LAF) was used as a model compound to investigate the binding mechanism between antiulcer drugs and human serum albumin (HSA) through various techniques, including STD-NMR, WaterLOGSY-NMR, (1)H NMR relaxation times, tr-NOESY, molecule docking calculation, FT-IR spectroscopy, and CD spectroscopy. The analyses of STD-NMR, which derived relative STD (%) intensities, and WaterLOGSY-NMR, determined that LAF bound to HSA. In particular, the pyridyl group of LAF was in close contact with HSA binding pocket, whereas furyl group had a secondary binding. Competitive STD-NMR and WaterLOGSY-NMR experiments, with warifarin and ibuprofen as site-selective probes, indicated that LAF preferentially bound to site II in the hydrophobic subdomains IIIA of HSA. The bound conformation of LAF at the HSA binding site was further elucidated by transferred NOE effect (tr-NOESY) experiment. Relaxation experiments provided quantitative information about the relationship between the affinity and structure of LAF. The molecule docking simulations conducted with AutoDock and the restraints derived from STD results led to three-dimensional models that were consistent with the NMR spectroscopic data. The presence of hydrophobic forces and hydrogen interactions was also determined. Additionally, FT-IR and CD spectroscopies showed that LAF induced secondary structure changes of HSA. Copyright © 2016 Elsevier Inc. All rights reserved.

Amount of cadmium associated with Cd-binding protein in roots of young plants. [Agrostis gigantea

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

Rauser, W.E.

1986-04-01

The partitioning of Cd between roots and shoots was determined for young plants exposed to Cd in nutrient solution. The intentionally high concentration of 3 ..mu..m Cd was used to assess the role of root Cd-binding protein (Cd-BP) in Cd detoxification. The roots of tomatoes exposed to Cd retained 60-84% of the plant Cd from day 2 through day 9 without toxicity symptoms evident. Cd-BP did not contribute to Cd retention over the initial 4 days, only 1-4% of the root Cd was in this protein fraction. Maize roots retained 59-66% of the plant Cd from day 1 through daymore » 7. The Cd-BP fraction bound 8-19% of the root Cd on day 1 and 31-55% by day 7. Cd toxicity symptoms occurred in leaves by 4 days. In the grass Agrostis gigantea the roots retained 73-85% of the seedling Cd after 1 day and for another 6 days. A high proportion of the root Cd(34-68%) was in the Cd-BP fraction after one day and continued to be so to day 7 (46-64%). No Cd toxicity symptoms were evident. Only the specific pattern of rapid, early and sustained production of Cd-BP observed in Agrostis was consistent with the putative detoxification role for Cd-BP.« less

Sequential CD4-Coreceptor Interactions in Human Immunodeficiency Virus Type 1 Env Function: Soluble CD4 Activates Env for Coreceptor-Dependent Fusion and Reveals Blocking Activities of Antibodies against Cryptic Conserved Epitopes on gp120

PubMed Central

Salzwedel, Karl; Smith, Erica D.; Dey, Barna; Berger, Edward A.

2000-01-01

We devised an experimental system to examine sequential events by which the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) interacts with CD4 and coreceptor to induce membrane fusion. Recombinant soluble CD4 (sCD4) activated fusion between effector cells expressing Env and target cells expressing coreceptor (CCR5 or CXCR4) but lacking CD4. sCD4-activated fusion was dose dependent, occurred comparably with two- and four-domain proteins, and demonstrated Env-coreceptor specificities parallel to those reported in conventional fusion and infectivity systems. Fusion activation occurred upon sCD4 preincubation and washing of the Env-expressing effector cells but not the coreceptor-bearing target cells, thereby demonstrating that sCD4 exerts its effects by acting on Env. These findings provide direct functional evidence for a sequential two-step model of Env-receptor interactions, whereby gp120 binds first to CD4 and becomes activated for subsequent functional interaction with coreceptor, leading to membrane fusion. We used the sCD4-activated system to explore neutralization by the anti-gp120 human monoclonal antibodies 17b and 48d. These antibodies reportedly bind conserved CD4-induced epitopes involved in coreceptor interactions but neutralize HIV-1 infection only weakly. We found that 17b and 48d had minimal effects in the standard cell fusion system using target cells expressing both CD4 and coreceptor but potently blocked sCD4-activated fusion with target cells expressing coreceptor alone. Both antibodies strongly inhibited sCD4-activated fusion by Envs from genetically diverse HIV-1 isolates. Thus, the sCD4-activated system reveals conserved Env-blocking epitopes that are masked in native Env and hence not readily detected by conventional systems. PMID:10590121

Mg2+ ions: do they bind to nucleobase nitrogens?

PubMed Central

Leonarski, Filip; D'Ascenzo, Luigi; Auffinger, Pascal

2017-01-01

Given the many roles proposed for Mg2+ in nucleic acids, it is essential to accurately determine their binding modes. Here, we surveyed the PDB to classify Mg2+ inner-sphere binding patterns to nucleobase imine N1/N3/N7 atoms. Among those, purine N7 atoms are considered to be the best nucleobase binding sites for divalent metals. Further, Mg2+ coordination to N7 has been implied in several ribozyme catalytic mechanisms. We report that Mg2+ assigned near imine nitrogens derive mostly from poor interpretations of electron density patterns and are most often misidentified Na+, K+, NH4+ ions, water molecules or spurious density peaks. Consequently, apart from few documented exceptions, Mg2+ ions do not bind to N7 atoms. Without much of a surprise, Mn2+, Zn2+ and Cd2+, which have a higher affinity for nitrogens, may contact N7 atoms when present in crystallization buffers. In this respect, we describe for the first time a potential Zn2+ ribosomal binding site involving two purine N7 atoms. Further, we provide a set of guidelines to help in the assignment of Mg2+ in crystallographic, cryo-EM, NMR and model building practices and discuss implications of our findings related to ion substitution experiments. PMID:27923930

CdTe/CdSe quantum dots improve the binding affinities between α-amylase and polyphenols.

PubMed

Ni, Xiaoling

2012-03-01

People exposed to engineered nanomaterials have potential health risks associated. Human α-amylase is one of the key enzymes in the digestive system. There are few reports about the influence of quantum dots (QDs) on the digestive enzymes and their inhibition system. This work focused on the toxic effect of CdTe/CdSe QDs on the interactions between α-amylase and its natural inhibitors. Thirty-six dietary polyphenols, natural α-amylase inhibitors from food, were studied for their affinities for α-amylase in the absence and presence of CdTe/CdSe QDs by a fluorescence quenching method. The magnitudes of apparent binding constants of polyphenols for α-amylase were almost in the range of 10(5)-10(7) L mol(-1) in the presence of CdTe/CdSe QDs, which were higher than the magnitudes of apparent binding constants in the absence of CdTe/CdSe QDs (10(4)-10(6) L mol(-1)). CdTe/CdSe QDs obviously improved the affinities of dietary polyphenols for α-amylase up to 389.04 times. It is possible that the binding interaction between polyphenols and α-amylase in the presence of CdTe/CdSe QDs was mainly caused by electrostatic interactions. QDs significantly influence the digestive enzymes and their inhibition system. This journal is © The Royal Society of Chemistry 2012

Successful construction and stable expression of an anti-CD45RA scFv-EGFP fusion protein in Chinese hamster ovary cells.

PubMed

Wang, Zhujun; Chen, Yuanyuan; Li, Sisi; Cheng, Yuping; Zhao, Haizhao; Jia, Ming; Luo, Zebin; Tang, Yongmin

2014-02-01

CD45RA has been found highly expressed on leukemia cells and may be a potential target of the disease. In this study, an anti-CD45RA single-chain antibody fragment (scFv3A4) was genetically linked to the N terminus of the enhanced green fluorescent protein (EGFP) to generate a scFv3A4-EGFP fusion protein. The scFv3A4-EGFP with a molecular weight of 57kDa was stably expressed and secreted from the transfected CHO cells through the ER/Golgi-dependent pathway. The fusion protein was soluble in the culture supernatant and the yield was 1350μg/L. Flow cytometry analysis showed that the scFv3A4-EGFP had the same binding site and a very similar reactivity pattern with its parental murine monoclonal antibody (mAb) 3A4. Furthermore, comparing to conventional labeled 3A4-FITC antibody, the scFv3A4-EGFP was more resistant to illumination and more suitable for immunofluorescence histology (IFH) detection. Therefore, the scFv3A4-EGFP fusion protein can be a powerful tool to investigate the targeting of CD45RA on leukemia cells, biological activity of the target and possibly for the genetic manipulation of the antibody. Copyright © 2013 Elsevier Inc. All rights reserved.

Lactose binding to galectin-1 modulates structural dynamics, increases conformational entropy, and occurs with apparent negative cooperativity.

PubMed

Nesmelova, Irina V; Ermakova, Elena; Daragan, Vladimir A; Pang, Mabel; Menéndez, Margarita; Lagartera, Laura; Solís, Dolores; Baum, Linda G; Mayo, Kevin H

2010-04-16

Galectins are a family of lectins with a conserved carbohydrate recognition domain that interacts with beta-galactosides. By binding cell surface glycoconjugates, galectin-1 (gal-1) is involved in cell adhesion and migration processes and is an important regulator of tumor angiogenesis. Here, we used heteronuclear NMR spectroscopy and molecular modeling to investigate lactose binding to gal-1 and to derive solution NMR structures of gal-1 in the lactose-bound and unbound states. Structure analysis shows that the beta-strands and loops around the lactose binding site, which are more open and dynamic in the unbound state, fold in around the bound lactose molecule, dampening internal motions at that site and increasing motions elsewhere throughout the protein to contribute entropically to the binding free energy. CD data support the view of an overall more open structure in the lactose-bound state. Analysis of heteronuclear single quantum coherence titration binding data indicates that lactose binds the two carbohydrate recognition domains of the gal-1 dimer with negative cooperativity, in that the first lactose molecule binds more strongly (K(1)=21+/-6 x 10(3) M(-1)) than the second (K(2)=4+/-2 x 10(3) M(-1)). Isothermal calorimetry data fit using a sequential binding model present a similar picture, yielding K(1)=20+/-10 x 10(3) M(-1) and K(2)=1.67+/-0.07 x 10(3) M(-1). Molecular dynamics simulations provide insight into structural dynamics of the half-loaded lactose state and, together with NMR data, suggest that lactose binding at one site transmits a signal through the beta-sandwich and loops to the second binding site. Overall, our results provide new insight into gal-1 structure-function relationships and to protein-carbohydrate interactions in general. Copyright (c) 2010. Published by Elsevier Ltd.

Impairment of Fas-ligand-caveolin-1 interaction inhibits Fas-ligand translocation to rafts and Fas-ligand-induced cell death.

PubMed

Glukhova, Xenia A; Trizna, Julia A; Proussakova, Olga V; Gogvadze, Vladimir; Beletsky, Igor P

2018-01-22

Fas-ligand/CD178 belongs to the TNF family proteins and can induce apoptosis through death receptor Fas/CD95. The important requirement for Fas-ligand-dependent cell death induction is its localization to rafts, cholesterol- and sphingolipid-enriched micro-domains of membrane, involved in regulation of different signaling complexes. Here, we demonstrate that Fas-ligand physically associates with caveolin-1, the main protein component of rafts. Experiments with cells overexpressing Fas-ligand revealed a FasL N-terminal pre-prolin-rich region, which is essential for the association with caveolin-1. We found that the N-terminal domain of Fas-ligand bears two caveolin-binding sites. The first caveolin-binding site binds the N-terminal domain of caveolin-1, whereas the second one appears to interact with the C-terminal domain of caveolin-1. The deletion of both caveolin-binding sites in Fas-ligand impairs its distribution between cellular membranes, and attenuates a Fas-ligand-induced cytotoxicity. These results demonstrate that the interaction of Fas-ligand and caveolin-1 represents a molecular basis for Fas-ligand translocation to rafts, and the subsequent induction of Fas-ligand-dependent cell death. A possibility of a similar association between other TNF family members and caveolin-1 is discussed.

Cadmium exposure and the epigenome

PubMed Central

Sanders, Alison P; Smeester, Lisa; Rojas, Daniel; DeBussycher, Tristan; Wu, Michael C; Wright, Fred A; Zhou, Yi-Hui; Laine, Jessica E; Rager, Julia E; Swamy, Geeta K; Ashley-Koch, Allison; Lynn Miranda, Marie; Fry, Rebecca C

2014-01-01

Cadmium (Cd) is prevalent in the environment yet understudied as a developmental toxicant. Cd partially crosses the placental barrier from mother to fetus and is linked to detrimental effects in newborns. Here we examine the relationship between levels of Cd during pregnancy and 5-methylcytosine (5mC) levels in leukocyte DNA collected from 17 mother-newborn pairs. The methylation of cytosines is an epigenetic mechanism known to impact transcriptional signaling and influence health endpoints. A methylated cytosine-guanine (CpG) island recovery assay was used to assess over 4.6 million sites spanning 16,421 CpG islands. Exposure to Cd was classified for each mother-newborn pair according to maternal blood levels and compared with levels of cotinine. Subsets of genes were identified that showed altered DNA methylation levels in their promoter regions in fetal DNA associated with levels of Cd (n = 61), cotinine (n = 366), or both (n = 30). Likewise, in maternal DNA, differentially methylated genes were identified that were associated with Cd (n = 92) or cotinine (n = 134) levels. While the gene sets were largely distinct between maternal and fetal DNA, functional similarities at the biological pathway level were identified including an enrichment of genes that encode for proteins that control transcriptional regulation and apoptosis. Furthermore, conserved DNA motifs with sequence similarity to specific transcription factor binding sites were identified within the CpG islands of the gene sets. This study provides evidence for distinct patterns of DNA methylation or “footprints” in fetal and maternal DNA associated with exposure to Cd. PMID:24169490

Synergistic effect of ATP for RuvA-RuvB-Holliday junction DNA complex formation.

PubMed

Iwasa, Takuma; Han, Yong-Woon; Hiramatsu, Ryo; Yokota, Hiroaki; Nakao, Kimiko; Yokokawa, Ryuji; Ono, Teruo; Harada, Yoshie

2015-12-14

The Escherichia coli RuvB hexameric ring motor proteins, together with RuvAs, promote branch migration of Holliday junction DNA. Zero mode waveguides (ZMWs) constitute of nanosized holes and enable the visualization of a single fluorescent molecule under micromolar order of the molecules, which is applicable to characterize the formation of RuvA-RuvB-Holliday junction DNA complex. In this study, we used ZMWs and counted the number of RuvBs binding to RuvA-Holliday junction DNA complex. Our data demonstrated that different nucleotide analogs increased the amount of Cy5-RuvBs binding to RuvA-Holliday junction DNA complex in the following order: no nucleotide, ADP, ATPγS, and mixture of ADP and ATPγS. These results suggest that not only ATP binding to RuvB but also ATP hydrolysis by RuvB facilitates a stable RuvA-RuvB-Holliday junction DNA complex formation.

SH2-catalytic domain linker heterogeneity influences allosteric coupling across the SFK family.

PubMed

Register, A C; Leonard, Stephen E; Maly, Dustin J

2014-11-11

Src-family kinases (SFKs) make up a family of nine homologous multidomain tyrosine kinases whose misregulation is responsible for human disease (cancer, diabetes, inflammation, etc.). Despite overall sequence homology and identical domain architecture, differences in SH3 and SH2 regulatory domain accessibility and ability to allosterically autoinhibit the ATP-binding site have been observed for the prototypical SFKs Src and Hck. Biochemical and structural studies indicate that the SH2-catalytic domain (SH2-CD) linker, the intramolecular binding epitope for SFK SH3 domains, is responsible for allosterically coupling SH3 domain engagement to autoinhibition of the ATP-binding site through the conformation of the αC helix. As a relatively unconserved region between SFK family members, SH2-CD linker sequence variability across the SFK family is likely a source of nonredundant cellular functions between individual SFKs via its effect on the availability of SH3 and SH2 domains for intermolecular interactions and post-translational modification. Using a combination of SFKs engineered with enhanced or weakened regulatory domain intramolecular interactions and conformation-selective inhibitors that report αC helix conformation, this study explores how SH2-CD sequence heterogeneity affects allosteric coupling across the SFK family by examining Lyn, Fyn1, and Fyn2. Analyses of Fyn1 and Fyn2, isoforms that are identical but for a 50-residue sequence spanning the SH2-CD linker, demonstrate that SH2-CD linker sequence differences can have profound effects on allosteric coupling between otherwise identical kinases. Most notably, a dampened allosteric connection between the SH3 domain and αC helix leads to greater autoinhibitory phosphorylation by Csk, illustrating the complex effects of SH2-CD linker sequence on cellular function.

Sorption selectivity of birnessite particle edges: a d-PDF analysis of Cd( ii ) and Pb( ii ) sorption by δ-MnO 2 and ferrihydrite

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

van Genuchten, Case M.; Peña, Jasquelin

2016-01-01

Birnessite minerals (layer-type MnO2), which bear both internal (cation vacancies) and external (particle edges) metal sorption sites, are important sinks of contaminants in soils and sediments. Although the particle edges of birnessite minerals often dominate the total reactive surface area, especially in the case of nanoscale crystallites, the metal sorption reactivity of birnessite particle edges remains elusive. In this study, we investigated the sorption selectivity of birnessite particle edges by combining Cd(II) and Pb(II) adsorption isotherms at pH 5.5 with surface structural characterization by differential pair distribution function (d-PDF) analysis. We compared the sorption reactivity of δ-MnO2 to that ofmore » the nanomineral, 2-line ferrihydrite, which exhibits only external surface sites. Our results show that, whereas Cd(II) and Pb(II) both bind to birnessite layer vacancies, only Pb(II) binds extensively to birnessite particle edges. For ferrihydrite, significant Pb(II) adsorption to external sites was observed (roughly 20 mol%), whereas Cd(II) sorption was negligible. These results are supported by bond valence calculations that show comparable degrees of saturation of oxygen atoms on birnessite and ferrihydrite particle edges. Therefore, we propose that the sorption selectivity of birnessite edges follows the same order of that reported previously for ferrihydrite: Ca(II) < Cd(II) < Ni(II) < Zn(II) < Cu(II) < Pb(II).« less

CD and MCD spectroscopic studies of the two Dps miniferritin proteins from Bacillus anthracis: role of O2 and H2O2 substrates in reactivity of the diiron catalytic centers.

PubMed

Schwartz, Jennifer K; Liu, Xiaofeng S; Tosha, Takehiko; Diebold, Adrienne; Theil, Elizabeth C; Solomon, Edward I

2010-12-14

DNA protection during starvation (Dps) proteins are miniferritins found in bacteria and archaea that provide protection from uncontrolled Fe(II)/O radical chemistry; thus the catalytic sites are targets for antibiotics against pathogens, such as anthrax. Ferritin protein cages synthesize ferric oxymineral from Fe(II) and O(2)/H(2)O(2), which accumulates in the large central cavity; for Dps, H(2)O(2) is the more common Fe(II) oxidant contrasting with eukaryotic maxiferritins that often prefer dioxygen. To better understand the differences in the catalytic sites of maxi- versus miniferritins, we used a combination of NIR circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field MCD (VTVH MCD) to study Fe(II) binding to the catalytic sites of the two Bacillus anthracis miniferritins: one in which two Fe(II) react with O(2) exclusively (Dps1) and a second in which both O(2) or H(2)O(2) can react with two Fe(II) (Dps2). Both result in the formation of iron oxybiomineral. The data show a single 5- or 6-coordinate Fe(II) in the absence of oxidant; Fe(II) binding to Dps2 is 30× more stable than Dps1; and the lower limit of K(D) for binding a second Fe(II), in the absence of oxidant, is 2-3 orders of magnitude weaker than for the binding of the single Fe(II). The data fit an equilibrium model where binding of oxidant facilitates formation of the catalytic site, in sharp contrast to eukaryotic M-ferritins where the binuclear Fe(II) centers are preformed before binding of O(2). The two different binding sequences illustrate the mechanistic range possible for catalytic sites of the family of ferritins.

Complementary Phosphorylation Sites in the Adaptor Protein SLP-76 Promote Synergistic Activation of Natural Killer Cells

PubMed Central

Kim, Hun Sik; Long, Eric O.

2013-01-01

The cytotoxic effects of natural killer (NK) cells and their ability to secrete cytokines require the induction of synergistic signals from co-activation receptors, such as CD314 (NKG2D) and CD244 (2B4), which bind to ligands expressed on target cells. Synergy is required to overcome inhibition of the guanine nucleotide exchange factor (GEF) Vav1, a central regulator of NK cell activation, by the E3 ubiquitin ligase c-Cbl. However, the molecular basis for this synergy is unknown. Here, we showed that the adaptor protein Src homology 2 (SH2) domain–containing leukocyte phosphoprotein of 76 kD (SLP-76) was required for this synergy, and that distinct tyrosine residues in SLP-76 were phosphorylated by each receptor of a synergistic pair. Selective phosphorylation of tyrosine 113 or tyrosine 128 in SLP-76, each of which enables binding of SLP-76 to Vav1, was unique to receptors that stimulate ligand-dependent target cell killing, because antibody-dependent stimulation by Fc receptor CD16 promoted phosphorylation at both sites. Knockdown and reconstitution experiments with SLP-76 showed the distinct role of each tyrosine in the synergistic mobilization of Ca2+, revealing an unexpected degree of selectivity in the phosphorylation of SLP-76 by NK cell co-activation receptors. Together, these data suggest that complementation of separate phospho-tyrosine targets in SLP-76 forms the basis of synergistic NK cell activation. PMID:22786724

Complementary phosphorylation sites in the adaptor protein SLP-76 promote synergistic activation of natural killer cells.

PubMed

Kim, Hun Sik; Long, Eric O

2012-07-10

The cytotoxic effects of natural killer (NK) cells and their ability to secrete cytokines require synergistic signals from specific pairs of co-activation receptors, such as CD314 (also known as NKG2D) and CD244 (2B4), which bind to distinct ligands present on target cells. These signals are required to overcome inhibition mediated by the E3 ubiquitin ligase c-Cbl of the guanine nucleotide exchange factor Vav1, which promotes activation of NK cells. Here, we showed that the adaptor protein SLP-76 (Src homology 2 domain-containing leukocyte phosphoprotein of 76 kilodaltons) was required for this synergy and that distinct tyrosine residues in SLP-76 were phosphorylated by each member of a pair of synergistic receptors. Selective phosphorylation of tyrosine 113 or tyrosine 128 in SLP-76 enabled binding of SLP-76 to Vav1. Selective phosphorylation of SLP-76 at these residues was restricted to receptors that stimulated ligand-dependent target cell killing; antibody-dependent stimulation of the Fc receptor CD16 promoted phosphorylation at both sites. Knockdown and reconstitution experiments with SLP-76 mutant proteins showed the distinct role of each tyrosine in the synergistic mobilization of Ca2+, revealing an unexpected degree of selectivity in the phosphorylation of SLP-76 by NK cell co-activation receptors. Together, these data suggest that combined phosphorylation of separate tyrosine residues in SLP-76 forms the basis of synergistic NK cell activation.

Multispectroscopic methods reveal different modes of interaction of anti cancer drug mitoxantrone with Poly(dG-dC).Poly(dG-dC) and Poly(dA-dT).Poly(dA-dT).

PubMed

Awasthi, Pamita; Dogra, Shilpa; Barthwal, Ritu

2013-10-05

The interaction of mitoxantrone with alternating Poly(dG-dC).Poly(dG-dC) and Poly(dA-dT).Poly(dA-dT) duplex has been studied by absorption, fluorescence and Circular Dichroism (CD) spectroscopy at Drug to Phosphate base pair ratios D/P=20.0-0.04. Binding to GC polymer occurs in two distinct modes: partial stacking characterized by red shifts of 18-23nm at D/P=0.2-0.8 and external binding at D/P=1.0-20.0 whereas that to AT polymer occurs externally in the entire range of D/P. The binding constant and number of binding sites is 3.7×10(5)M(-1), 0.3 and 1.3× 10(4)M(-1), 1.5 in GC and AT polymers, respectively at low D/P ratios. CD binding isotherms show breakpoints at D/P=0.1, 0.5 and 0.25, 0.5 in GC and AT polymers, respectively. The intrinsic CD bands indicate that the distortions in GC polymer are significantly higher than that in AT polymer. Docking studies show partial insertion of mitoxantrone rings between to GC base pairs in alternating GC polymer. Side chains of mitoxantrone interact specifically with base pairs and DNA backbone. The studies are relevant to the understanding of suppression or inhibition of DNA cleavage on formation of ternary complex with topoisomerase-II enzyme and hence the anti cancer action. Copyright © 2013 Elsevier B.V. All rights reserved.

pH-Assisted control over the binding and relocation of an acridine guest between a macrocyclic nanocarrier and natural DNA.

PubMed

Sayed, Mhejabeen; Pal, Haridas

2015-04-14

The differential binding affinity of the hydroxypropyl-β-cyclodextrin (HPβCD) macrocycle, a drug delivery vehicle, towards the protonated and deprotonated forms of the well-known DNA binder and model anticancer drug acridine has been exploited as a strategy for dye-drug transportation and pH-responsive delivery to a natural DNA target. From pH-sensitive changes in the ground state absorption and steady-state fluorescence characteristics of the studied acridine dye-HPβCD-DNA ternary system and strongly supported by fluorescence lifetime, fluorescence anisotropy, Job's plots, (1)H NMR and circular dichroism results, it is revealed that in a moderately alkaline solution (pH ∼ 8.5), the dye can be predominantly bound to the HPβCD macrocycle and when the pH is lowered to a moderately acidic region (pH ∼ 4), the dye efficiently detaches from the HPβCD cavity and almost exclusively binds to DNA. In the present study we are thus able to construct a pH-sensitive supramolecular assembly where pH acts as a simple stimulus for controlled uptake and targeted release of the dye-drug. As pH is an essential and sensitive factor in various biological processes, a simple yet reliable pH-sensitive model such as is demonstrated here can have promising applications in the host-assisted delivery of prodrug to the target sites, such as cancer or tumour microenvironments, with an enhanced stability, bioavailability and activity, and also in the design of new fluorescent probes, sensors and smart materials for applications in nano-science.

Unravelling the binding mechanism and protein stability of human serum albumin while interacting with nefopam analogues: a biophysical and insilico approach.

PubMed

Gokara, Mahesh; Narayana, Vidadala V; Sadarangani, Vineet; Chowdhury, Shatabdi Roy; Varkala, Sreelaxmi; Ramachary, Dhevalapally B; Subramanyam, Rajagopal

2017-08-01

In this study, molecular binding affinity was investigated for Nefopam analogues (NFs), a functionalized benzoxazocine, with human serum albumin (HSA), a major transport protein in the blood. Its binding affinity and concomitant changes in its conformation, binding site and simulations were also studied. Fluorescence data revealed that the fluorescence quenching of HSA upon binding of NFs analogues is based on a static mechanism. The three analogues of NFs binding constants (K A ) are in the order of NF3 > NF2 > NF1 with values of 1.53 ± .057 × 10 4 , 2.16 ± .071 × 10 4 and 3.6 ± .102 × 10 5  M -1 , respectively. Concurrently, thermodynamic parameters indicate that the binding process was spontaneous, and the complexes were stabilized mostly by hydrophobic interactions, except for NF2 has one hydrogen bond stabilizes it along with hydrophobic interactions. Circular dichroism (CD) studies revealed that there is a decrease in α-helix with an increase in β-sheets and random coils signifying partial unfolding of the protein upon binding of NFs, which might be due to the formation of NFs-HSA complexes. Further, molecular docking studies showed that NF1, NF2 and NF3 bound to subdomains IIIA, IB and IIA through hydrophobic interactions. However, NF1 have additionally formed a single hydrogen bond with LYS 413. Furthermore, molecular simulations unveiled that NFs binding was in support with the structural perturbation observed in CD, which is evident from the root mean square deviation and R g fluctuations. We hope our insights will provide ample scope for engineering new drugs based on the resemblances with NFs for enhanced efficacy with HSA.

Interaction mechanisms between organic UV filters and bovine serum albumin as determined by comprehensive spectroscopy exploration and molecular docking.

PubMed

Ao, Junjie; Gao, Li; Yuan, Tao; Jiang, Gaofeng

2015-01-01

Organic UV filters are a group of emerging PPCP (pharmaceuticals and personal care products) contaminants. Current information is insufficient to understand the in vivo processes and health risks of organic UV filters in humans. The interaction mechanism of UV filters with serum albumin provides critical information for the health risk assessment of these active ingredients in sunscreen products. This study investigates the interaction mechanisms of five commonly used UV filters (2-hydroxy-4-methoxybenzophenone, BP-3; 2-ethylhexyl 4-methoxycinnamate, EHMC; 4-methylbenzylidene camphor, 4-MBC; methoxydibenzoylmethane, BDM; homosalate, HMS) with bovine serum albumin (BSA) by spectroscopic measurements of fluorescence, circular dichroism (CD), competitive binding experiments and molecular docking. Our results indicated that the fluorescence of BSA was quenched by these UV filters through a static quenching mechanism. The values of the binding constant (Ka) ranged from (0.78±0.02)×10(3) to (1.29±0.01)×10(5) L mol(-1). Further exploration by synchronous fluorescence and CD showed that the conformation of BSA was demonstrably changed in the presence of these organic UV filters. It was confirmed that the UV filters can disrupt the α-helical stability of BSA. Moreover, the results of molecular docking revealed that the UV filter molecule is located in site II (sub-domain IIIA) of BSA, which was further confirmed by the results of competitive binding experiments. In addition, binding occurred mainly through hydrogen bonding and hydrophobic interaction. This study raises critical concerns regarding the transportation, distribution and toxicity effects of organic UV filters in human body. Copyright © 2014 Elsevier Ltd. All rights reserved.

Selection of a novel CD19 aptamer for targeted delivery of doxorubicin to lymphoma cells.

PubMed

Hu, Yan; Li, Xiaoou; An, Yacong; Duan, Jinhong; Yang, Xian-Da

2018-06-01

CD19 is overexpressed in most human B cell malignancies and considered an important tumor marker for diagnosis and treatment. Aptamers are oligonucleotides that may potentially serve as tumor-homing ligand for targeted cancer therapy with excellent affinity and specificity. In this study, we selected a novel CD19 aptamer (LC1) that was a 59-nucleotide single strand DNA. The aptamer could bind to recombinant CD19 protein with a K d of 85.4 nM, and had minimal cross reactivity to bovine serum albumin (BSA) or ovalbumin (OVA). Moreover, the aptamer was found capable of binding with the CD19-positive lymphoma cells (Ramos and Raji), but not the CD19-negative cell lines (Jurkat and NB4). An aptamer-doxorubicin complex (Apt-Dox) was also formulated, and selectively delivered doxorubicin to CD19-positive lymphoma cells in vitro . The results indicate that aptamer LC1 can recognize CD19-positive tumor cells and may potentially function as a CD19-targeting ligand.

The alpha-fetoprotein third domain receptor binding fragment: in search of scavenger and associated receptor targets.

PubMed

Mizejewski, G J

2015-01-01

Recent studies have demonstrated that the carboxyterminal third domain of alpha-fetoprotein (AFP-CD) binds with various ligands and receptors. Reports within the last decade have established that AFP-CD contains a large fragment of amino acids that interact with several different receptor types. Using computer software specifically designed to identify protein-to-protein interaction at amino acid sequence docking sites, the computer searches identified several types of scavenger-associated receptors and their amino acid sequence locations on the AFP-CD polypeptide chain. The scavenger receptors (SRs) identified were CD36, CD163, Stabilin, SSC5D, SRB1 and SREC; the SR-associated receptors included the mannose, low-density lipoprotein receptors, the asialoglycoprotein receptor, and the receptor for advanced glycation endproducts (RAGE). Interestingly, some SR interaction sites were localized on the AFP-derived Growth Inhibitory Peptide (GIP) segment at amino acids #480-500. Following the detection studies, a structural subdomain analysis of both the receptor and the AFP-CD revealed the presence of epidermal growth factor (EGF) repeats, extracellular matrix-like protein regions, amino acid-rich motifs and dimerization subdomains. For the first time, it was reported that EGF-like sequence repeats were identified on each of the three domains of AFP. Thereafter, the localization of receptors on specific cell types were reviewed and their functions were discussed.

Binding of human immunodeficiency virus type 1 gp120 to CXCR4 induces mitochondrial transmembrane depolarization and cytochrome c-mediated apoptosis independently of Fas signaling.

PubMed

Roggero, R; Robert-Hebmann, V; Harrington, S; Roland, J; Vergne, L; Jaleco, S; Devaux, C; Biard-Piechaczyk, M

2001-08-01

Apoptosis of CD4(+) T lymphocytes, induced by contact between human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp120) and its receptors, could contribute to the cell depletion observed in HIV-infected individuals. CXCR4 appears to play an important role in gp120-induced cell death, but the mechanisms involved in this apoptotic process remain poorly understood. To get insight into the signal transduction pathways connecting CXCR4 to apoptosis following gp120 binding, we used different cell lines expressing wild-type CXCR4 and a truncated form of CD4 that binds gp120 but lacks the ability to transduce signals. The present study demonstrates that (i) the interaction of cell-associated gp120 with CXCR4-expressing target cells triggers a rapid dissipation of the mitochondrial transmembrane potential resulting in the cytosolic release of cytochrome c from the mitochondria to cytosol, concurrent with activation of caspase-9 and -3; (ii) this apoptotic process is independent of Fas signaling; and (iii) cooperation with a CD4 signal is not required. In addition, following coculture with cells expressing gp120, a Fas-independent apoptosis involving mitochondria and caspase activation is also observed in primary umbilical cord blood CD4(+) T lymphocytes expressing high levels of CXCR4. Thus, this gp120-mediated apoptotic pathway may contribute to CD4(+) T-cell depletion in AIDS.

Binding of Human Immunodeficiency Virus Type 1 gp120 to CXCR4 Induces Mitochondrial Transmembrane Depolarization and Cytochrome c-Mediated Apoptosis Independently of Fas Signaling

PubMed Central

Roggero, Rodolphe; Robert-Hebmann, Véronique; Harrington, Steve; Roland, Joachim; Vergne, Laurence; Jaleco, Sara; Devaux, Christian; Biard-Piechaczyk, Martine

2001-01-01

Apoptosis of CD4+ T lymphocytes, induced by contact between human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp120) and its receptors, could contribute to the cell depletion observed in HIV-infected individuals. CXCR4 appears to play an important role in gp120-induced cell death, but the mechanisms involved in this apoptotic process remain poorly understood. To get insight into the signal transduction pathways connecting CXCR4 to apoptosis following gp120 binding, we used different cell lines expressing wild-type CXCR4 and a truncated form of CD4 that binds gp120 but lacks the ability to transduce signals. The present study demonstrates that (i) the interaction of cell-associated gp120 with CXCR4-expressing target cells triggers a rapid dissipation of the mitochondrial transmembrane potential resulting in the cytosolic release of cytochrome c from the mitochondria to cytosol, concurrent with activation of caspase-9 and -3; (ii) this apoptotic process is independent of Fas signaling; and (iii) cooperation with a CD4 signal is not required. In addition, following coculture with cells expressing gp120, a Fas-independent apoptosis involving mitochondria and caspase activation is also observed in primary umbilical cord blood CD4+ T lymphocytes expressing high levels of CXCR4. Thus, this gp120-mediated apoptotic pathway may contribute to CD4+ T-cell depletion in AIDS. PMID:11462036

Isolation and N-terminal sequencing of a novel cadmium-binding protein from Boletus edulis

NASA Astrophysics Data System (ADS)

Collin-Hansen, C.; Andersen, R. A.; Steinnes, E.

2003-05-01

A Cd-binding protein was isolated from the popular edible mushroom Boletus edulis, which is a hyperaccumulator of both Cd and Hg. Wild-growing samples of B. edulis were collected from soils rich in Cd. Cd radiotracer was added to the crude protein preparation obtained from ethanol precipitation of heat-treated cytosol. Proteins were then further separated in two consecutive steps; gel filtration and anion exchange chromatography. In both steps the Cd radiotracer profile showed only one distinct peak, which corresponded well with the profiles of endogenous Cd obtained by atomic absorption spectrophotometry (AAS). Concentrations of the essential elements Cu and Zn were low in the protein fractions high in Cd. N-terminal sequencing performed on the Cd-binding protein fractions revealed a protein with a novel amino acid sequence, which contained aromatic amino acids as well as proline. Both the N-terminal sequencing and spectrofluorimetric analysis with EDTA and ABD-F (4-aminosulfonyl-7-fluoro-2, 1, 3-benzoxadiazole) failed to detect cysteine in the Cd-binding fractions. These findings conclude that the novel protein does not belong to the metallothionein family. The results suggest a role for the protein in Cd transport and storage, and they are of importance in view of toxicology and food chemistry, but also for environmental protection.

Biometal binding-site mimicry with modular, hetero-bifunctionally modified architecture encompassing a Trp/His motif: insights into spatiotemporal noncovalent interactions from a comparative spectroscopic study.

PubMed

Yang, Chi Ming

2011-03-28

Metal-site Trp/His interactions are crucial to diverse metalloprotein functions. This paper presents a study using metal-motif mimicry to capture and dissect the static and transient components of physicochemical properties underlying the Trp/His aromatic side-chain noncovalent interactions across the first- and second-coordination spheres of biometal ions. Modular biomimetic constructs, EDTA-(L-Trp, L-His) or EWH and DTPA-(L-Trp, L-His) or DWH, featuring a function-significant Trp/His pair, enabled extracting the putative hydrophobic/hydrophilic aromatic interactions surrounding metal centers. Fluorescence, circular dichroism (CD) spectroscopic titrations and ESI mass spectrometry demonstrated that both the constructs stoichiometrically bind to Ca(2+), Co(2+), Cu(2+), Ni(2+), Mn(2+), Zn(2+), Cd(2+), and Fe(2+), and such binding was strongly coupled to stereospecific side-chain structure reorientations of the Trp indole and His imidazole rings. A mechanistic dichotomy corresponding to the participation of the indole unit in the binding event was revealed by a scaffold-platform correlation of steady-state fluorescence-response landscape, illuminating that secondary-coordination-sphere ligand cation-π interactions were immediately followed by subsequent transient physicochemical processes including through-space energy transfer, charge transfer and/or electron transfer, depending on the type of metals. The fluorescence quenching of Trp side chain by 3d metal ions can be ascribed to through-space d-π interactions. While the fluorescence titration was capable of illuminating a two-component energetic model, clean isosbestic/isodichroic points in the CD titration spectra indicated that the metallo-constructs, such as Cu(2+)-EWH complex, fold thermodynamically by means of a two-state equilibrium. Further, the metal-ion dependence of Trp conformational variation in the modular architecture of metal-bound scaffolds was evidenced unambiguously by the CD spectra and supported by MMFF calculations; both were capable of distinguishing between the coordination geometry and the preference for metal binding mode. The study thus helps understand how aromatic rings around metal-sites have unique capabilities through the control of the spatiotemporal distribution of noncovalent interaction elements to achieve diverse chemical functionality.

Fatty acid modulated human serum albumin binding of the β-carboline alkaloids norharmane and harmane.

PubMed

Domonkos, Celesztina; Fitos, Ilona; Visy, Júlia; Zsila, Ferenc

2013-12-02

Harmane and norharmane are representative members of the large group of natural β-carboline alkaloids featured with diverse pharmacological activities. In blood, these agents are transported by human serum albumin (HSA) which has a profound impact on the pharmacokinetic and pharmacodynamic properties of many therapeutic drugs and xenobiotics. By combination of various spectroscopic methods, the present contribution is aimed to elucidate how nonesterified fatty acids (FAs), the primary endogenous ligands of HSA, affect the binding properties of harmane and norharmane. Analysis of induced circular dichroism (CD) and fluorescence spectroscopic data indicates the inclusion of the neutral form of both molecules into the binding pocket of subdomain IIIA, which hosts two FA binding sites, too. The induced CD and UV absorption spectra of harmane and norharmane exhibit peculiar changes upon addition of FAs, suggesting the formation of ternary complexes in which the lipid ligands significantly alter the binding mode of the alkaloids via cooperative allosteric mechanism. To our knowledge, it is the first instance of the demonstration of drug-FA cobinding at site IIIA. In line with these results, molecular docking calculations showed two distinct binding positions of norharmane within subdomain IIIA. The profound increase in the affinity constants of β-carbolines estimated in the presence of FAs predicts that the unbound, pharmacologically active serum fraction of these compounds strongly depends on the actual lipid binding profile of HSA.

Targeting BRCAness in Gastric Cancer

DTIC Science & Technology

2017-10-01

inhibitors. We also generated a modified CRISPR system using dCas9-KRAB expressing variants of these cells, and validated them for CRISPRi screening...Figure 2. Validation of CRISPR activity following transduction with sgRNAs targeting CD55 and FACS staining with the anti-CD55 antibody. Data shown...interpretation of CRISPR experiments Morgan Diolaiti Specialist UCSF PH.D. Experimental planning and reporting Jefferson Woods SRA UCSF B.S. Perform drug

Ligand reprogramming in dinuclear helicate complexes: a consequence of allosteric or electrostatic effects?

PubMed

Jeffery, John C; Rice, Craig R; Harding, Lindsay P; Baylies, Christian J; Riis-Johannessen, Thomas

2007-01-01

The ditopic ligand 6,6'-bis(4-methylthiazol-2-yl)-3,3'-([18]crown-6)-2,2'-bipyridine (L(1)) contains both a potentially tetradentate pyridyl-thiazole (py-tz) N-donor chain and an additional "external" crown ether binding site which spans the central 2,2'-bipyridine unit. In polar solvents (MeCN, MeNO(2)) this ligand forms complexes with Zn(II), Cd(II), Hg(II) and Cu(I) ions via coordination of the N donors to the metal ion. Reaction with both Hg(II) and Cu(I) ions results in the self-assembly of dinuclear double-stranded helicate complexes. The ligands are partitioned by rotation about the central py--py bond, such that each can coordinate to both metals as a bis-bidentate donor ligand. With Zn(II) ions a single-stranded mononuclear species is formed in which one ligand coordinates the metal ion in a planar tetradentate fashion. Reaction with Cd(II) ions gives rise to an equilibrium between both the dinuclear double-stranded helicate and the mononuclear species. These complexes can further coordinate s-block metal cations via the remote crown ether O-donor domains; a consequence of which are some remarkable changes in the binding modes of the N-donor domains. Reaction of the Hg(II)- or Cd(II)-containing helicate with either Ba(2+) or Sr(2+) ions effectively reprogrammes the ligand to form only the single-stranded heterobinuclear complexes [MM'(L(1))](4+) (M=Hg(II), Cd(II); M'=Ba(2+), Sr(2+)), where the transition and s-block cations reside in the N- and O-donor sites, respectively. In contrast, the same ions have only a minor structural impact on the Zn(II) species, which already exists as a single-stranded mononuclear complex. Similar reactions with the Cd(II) system result in a shift in equilibrium towards the single-stranded species, the extent of which depends on the size and charge of the s-block cation in question. Reaction of the dicopper(I) double-stranded helicate with Ba(2+) shows that the dinuclear structure still remains intact but the pitch length is significantly increased.

Chemotaxis and adherence to fungal surfaces are key components of the behavioral response of Burkholderia terrae BS001 to two selected soil fungi.

PubMed

Haq, Irshad Ul; Calixto, Renata Oliveira da Rocha; Yang, Pu; Dos Santos, Giulia Maria Pires; Barreto-Bergter, Eliana; van Elsas, Jan Dirk

2016-11-01

Burkholderia terrae BS001 has previously been proposed to be a 'generalist' associate of soil fungi, but its strategies of interaction have been largely ignored. Here, we studied the chemotactic behavior of B. terrae BS001 towards Lyophyllum sp. strain Karsten and Trichoderma asperellum 302 and the role of fungal surface molecules in their physical interaction with the bacteria. To assess the involvement of the type 3 secretion system (T3SS), wild-type strain BS001 and T3SS mutant strain BS001-ΔsctD were used in the experiments. First, the two fungi showed divergent behavior when confronted with B. terrae BS001 on soil extract agar medium. Lyophyllum sp. strain Karsten revealed slow growth towards the bacterium, whereas T. asperellum 302 grew avidly over it. Both on soil extract and M9 agar, B. terrae BS001 and BS001-ΔsctD moved chemotactically towards the hyphae of both fungi, with a stronger response to Lyophyllum sp. strain Karsten than to T. asperellum 302. The presence of a progressively increasing glycerol level in the M9 agar enhanced the level of movement. Different oxalic acid concentrations exerted varied effects, with a significantly raised chemotactic response at lower, and a subdued response at higher concentrations. Testing of the adherence of B. terrae BS001 and BS001-ΔsctD to Lyophyllum sp. strain Karsten and to cell envelope-extracted ceramide monohexosides (CMHs) revealed that CMHs in both conidia and hyphae could bind strain BS001 cells. As BS001-ΔsctD adhered significantly less to the CMHs than BS001, the T3SS was presumed to have a role in the interaction. In contrast, such avid adherence was not detected with T. asperellum 302. Thus, B. terrae BS001 shows a behavior characterized by swimming towards Lyophyllum sp. strain Karsten and T. asperellum 302 and attachment to the CMH moiety in the cell envelope, in particular of the former. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Low molecular weight (LMW) heparin inhibits injury-induced femoral artery remodeling in mouse via upregulating CD44 expression.

PubMed

Zhao, Gaofeng; Shaik, Rahamthulla S; Zhao, Hang; Beagle, John; Kuo, Shuennwen; Hales, Charles A

2011-05-01

The mechanism of postangioplasty restenosis remains poorly understood. Low molecular weight (LMW) heparin has been shown to inhibit the proliferation of vascular smooth muscle cells (VSMCs), which is the principal characteristic of restenosis. Studies have shown that LMW heparin could bind to CD44. We hypothesized that LMW heparin might modulate CD44 expression thereby decreasing vascular remodeling. Vascular remodeling was induced in CD44(+/+) and CD44(-/-) mice and treated with LMW heparin. The arteries were harvested for histologic assessment and determination of CD44 expression. Bone marrow transplantation was introduced to further explore the role and functional sites of CD44. Effects of LMW heparin on growth capacity, CD44 expression were further studied using the cultured mouse VSMCs. Transluminal injury induced remarkable remodeling in mouse femoral artery (sham wall thickness percentage [WT%]: 3.4 ± 1.2% vs injury WT%: 31.8 ± 4.7%; P < .001). LMW heparin reduced the remodeling significantly (WT%: 17.8 ± 3.5%, P < .005). CD44(-/-) mice demonstrated considerably thicker arterial wall remodeling (WT%: 46.2 ± 7.6%, P = .0035), and CD44-chimeric mice exhibited equal contributions of the local and circulating CD44 signal to the neointima formation. LMW heparin markedly upregulated CD44 expression in the injured femoral arteries. In vitro, LMW heparin decreased mouse VSMC growth capacity and upregulated its CD44 expression simultaneously in a dose-dependent and time-dependent manner, which could be partially blocked by CD44 inhibitor. LMW heparin inhibits injury-induced femoral artery remodeling, at least partially, by upregulating CD44 expression. Copyright © 2011. Published by Mosby, Inc.

A calix[4]arene strapped calix[4]pyrrole: an ion-pair receptor displaying three different cesium cation recognition modes.

PubMed

Kim, Sung Kuk; Sessler, Jonathan L; Gross, Dustin E; Lee, Chang-Hee; Kim, Jong Seung; Lynch, Vincent M; Delmau, Laetitia H; Hay, Benjamin P

2010-04-28

An ion-pair receptor, the calix[4]pyrrole-calix[4]arene pseudodimer 2, bearing a strong anion-recognition site but not a weak cation-recognition site, has been synthesized and characterized by standard spectroscopic means and via single-crystal X-ray diffraction analysis. In 10% CD(3)OD in CDCl(3) (v/v), this new receptor binds neither the Cs(+) cation nor the F(-) anion when exposed to these species in the presence of other counterions; however, it forms a stable 1:1 solvent-separated CsF complex when exposed to these two ions in concert with one another in this same solvent mixture. In contrast to what is seen in the case of a previously reported crown ether "strapped" calixarene-calixpyrrole ion-pair receptor 1 (J. Am. Chem. Soc. 2008, 130, 13162-13166), where Cs(+) cation recognition takes place within the crown, in 2.CsF cation recognition takes place within the receptor cavity itself, as inferred from both single-crystal X-ray diffraction analyses and (1)H NMR spectroscopic studies. This binding mode is supported by calculations carried out using the MMFF94 force field model. In 10% CD(3)OD in CDCl(3) (v/v), receptor 2 shows selectivity for CsF over the Cs(+) salts of Cl(-), Br(-), and NO(3)(-) but will bind these other cesium salts in the absence of fluoride, both in solution and in the solid state. In the case of CsCl, an unprecedented 2:2 complex is observed in the solid state that is characterized by two different ion-pair binding modes. One of these consists of a contact ion pair with the cesium cation and chloride anion both being bound within the central binding pocket and in direct contact with one another. The other mode involves a chloride anion bound to the pyrrole NH protons of a calixpyrrole subunit and a cesium cation sandwiched between two cone shaped calix[4]pyrroles originating from separate receptor units. In contrast to what is seen for CsF and CsCl, single-crystal X-ray structural analyses and (1)H NMR spectroscopic studies reveal that receptor 2 forms a 1:1 complex with CsNO(3), with the ions bound in the form of a contact ion pair. Thus, depending on the counteranion, receptor 2 is able to stabilize three different ion-pair binding modes with Cs(+), namely solvent-bridged, contact, and host-separated.

HLA Class I Binding 9mer Peptides from Influenza A Virus Induce CD4+ T Cell Responses

PubMed Central

Wang, Mingjun; Larsen, Mette V.; Nielsen, Morten; Harndahl, Mikkel; Justesen, Sune; Dziegiel, Morten H.; Buus, Søren; Tang, Sheila T.; Lund, Ole; Claesson, Mogens H.

2010-01-01

Background Identification of human leukocyte antigen class I (HLA-I) restricted cytotoxic T cell (CTL) epitopes from influenza virus is of importance for the development of new effective peptide-based vaccines. Methodology/Principal Findings In the present work, bioinformatics was used to predict 9mer peptides derived from available influenza A viral proteins with binding affinity for at least one of the 12 HLA-I supertypes. The predicted peptides were then selected in a way that ensured maximal coverage of the available influenza A strains. One hundred and thirty one peptides were synthesized and their binding affinities for the HLA-I supertypes were measured in a biochemical assay. Influenza-specific T cell responses towards the peptides were quantified using IFNγ ELISPOT assays with peripheral blood mononuclear cells (PBMC) from adult healthy HLA-I typed donors as responder cells. Of the 131 peptides, 21 were found to induce T cell responses in 19 donors. In the ELISPOT assay, five peptides induced responses that could be totally blocked by the pan-specific anti-HLA-I antibody W6/32, whereas 15 peptides induced responses that could be completely blocked in the presence of the pan-specific anti-HLA class II (HLA-II) antibody IVA12. Blocking of HLA-II subtype reactivity revealed that 8 and 6 peptide responses were blocked by anti-HLA-DR and -DP antibodies, respectively. Peptide reactivity of PBMC depleted of CD4+ or CD8+ T cells prior to the ELISPOT culture revealed that effectors are either CD4+ (the majority of reactivities) or CD8+ T cells, never a mixture of these subsets. Three of the peptides, recognized by CD4+ T cells showed binding to recombinant DRA1*0101/DRB1*0401 or DRA1*0101/DRB5*0101 molecules in a recently developed biochemical assay. Conclusions/Significance HLA-I binding 9mer influenza virus-derived peptides induce in many cases CD4+ T cell responses restricted by HLA-II molecules. PMID:20479886

A new metal binding domain involved in cadmium, cobalt and zinc transport

DOE PAGES

Smith, Aaron T.; Barupala, Dulmini; Stemmler, Timothy L.; ...

2015-07-20

The P 1B-ATPases, which couple cation transport across membranes to ATP hydrolysis, are central to metal homeostasis in all organisms. An important feature of P 1B-ATPases is the presence of soluble metal binding domains (MBDs) that regulate transport activity. Only one type of MBD has been characterized extensively, but bioinformatics analyses indicate that a diversity of MBDs may exist in nature. In this paper, we report the biochemical, structural and functional characterization of a new MBD from the Cupriavidus metallidurans P 1B-4-ATPase CzcP (CzcP MBD). The CzcP MBD binds two Cd 2+, Co 2+ or Zn 2+ ions in distinctmore » and unique sites and adopts an unexpected fold consisting of two fused ferredoxin-like domains. Both in vitro and in vivo activity assays using full-length CzcP, truncated CzcP and several variants indicate a regulatory role for the MBD and distinct functions for the two metal binding sites. Finally, taken together, these findings elucidate a previously unknown MBD and suggest new regulatory mechanisms for metal transport by P 1B-ATPases.« less

On the contributions of photorespiration and compartmentation to the contrasting intramolecular 2H profiles of C3 and C4 plant sugars

Treesearch

Youping Zhou; Benli Zhang; Hilary Stuart-Williams; Kliti Grice; Charles H. Hocart; Arthur Gessler; Zachary E. Kayler; Graham D. Farquhar

2018-01-01

Compartmentation of C4 photosynthetic biochemistry into bundle sheath (BS) and mesophyll (M) cells, and photorespiration in C3 plants is predicted to have hydrogen isotopic consequences for metabolites at both molecular and site-specific levels. Molecular-level evidence was recently reported (Zhou et al., 2016), but...

Safe-Site Effects on Rhizosphere Bacterial Communities in a High-Altitude Alpine Environment

PubMed Central

Zerbe, Stefan

2014-01-01

The rhizosphere effect on bacterial communities associated with three floristic communities (RW, FI, and M sites) which differed for the developmental stages was studied in a high-altitude alpine ecosystem. RW site was an early developmental stage, FI was an intermediate stage, M was a later more matured stage. The N and C contents in the soils confirmed a different developmental stage with a kind of gradient from the unvegetated bare soil (BS) site through RW, FI up to M site. The floristic communities were composed of 21 pioneer plants belonging to 14 species. Automated ribosomal intergenic spacer analysis showed different bacterial genetic structures per each floristic consortium which differed also from the BS site. When plants of the same species occurred within the same site, almost all their bacterial communities clustered together exhibiting a plant species effect. Unifrac significance value (P < 0.05) on 16S rRNA gene diversity revealed significant differences (P < 0.05) between BS site and the vegetated sites with a weak similarity to the RW site. The intermediate plant colonization stage FI did not differ significantly from the RW and the M vegetated sites. These results pointed out the effect of different floristic communities rhizospheres on their soil bacterial communities. PMID:24995302

Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry.

PubMed

Wang, Meiyao; Misakian, Martin; He, Hua-Jun; Bajcsy, Peter; Abbasi, Fatima; Davis, Jeffrey M; Cole, Kenneth D; Turko, Illarion V; Wang, Lili

2014-01-01

In our previous study that characterized different human CD4+ lymphocyte preparations, it was found that both commercially available cryopreserved peripheral blood mononuclear cells (PBMC) and a commercially available lyophilized PBMC (Cyto-Trol™) preparation fulfilled a set of criteria for serving as biological calibrators for quantitative flow cytometry. However, the biomarker CD4 protein expression level measured for T helper cells from Cyto-Trol was about 16% lower than those for cryopreserved PBMC and fresh whole blood using flow cytometry and mass cytometry. A primary reason was hypothesized to be due to steric interference in anti- CD4 antibody binding to the smaller sized lyophilized control cells. Targeted multiple reaction monitoring (MRM) mass spectrometry (MS) is used to quantify the copy number of CD4 receptor protein per CD4+ lymphocyte. Scanning electron microscopy (SEM) is utilized to assist searching the underlying reasons for the observed difference in CD4 receptor copy number per cell determined by MRM MS and CD4 expression measured previously by flow cytometry. The copy number of CD4 receptor proteins on the surface of the CD4+ lymphocyte in cryopreserved PBMCs and in lyophilized control cells is determined to be (1.45 ± 0.09) × 10(5) and (0.85 ± 0.11) × 10(5), respectively, averaged over four signature peptides using MRM MS. In comparison with cryopreserved PBMCs, there are more variations in the CD4 copy number in lyophilized control cells determined based on each signature peptide. SEM images of CD4+ lymphocytes from lyophilized control cells are very different when compared to the CD4+ T cells from whole blood and cryopreserved PBMC. Because of the lyophilization process applied to Cyto-Trol control cells, a lower CD4 density value, defined as the copy number of CD4 receptors per CD4+ lymphocyte, averaged over three different production lots is most likely explained by the loss of the CD4 receptors on damaged and/or broken microvilli where CD4 receptors reside. Steric hindrance of antibody binding and the association of CD4 receptors with other biomolecules likely contribute significantly to the nearly 50% lower CD4 receptor density value for cryopreserved PBMC determined from flow cytometry compared to the value obtained from MRM MS.

The antigenic complex in HIT binds to B cells via complement and complement receptor 2 (CD21)

PubMed Central

Khandelwal, Sanjay; Lee, Grace M.; Hester, C. Garren; Poncz, Mortimer; McKenzie, Steven E.; Sachais, Bruce S.; Rauova, Lubica; Kelsoe, Garnett; Cines, Douglas B.; Frank, Michael

2016-01-01

Heparin-induced thrombocytopenia is a prothrombotic disorder caused by antibodies to platelet factor 4 (PF4)/heparin complexes. The mechanism that incites such prevalent anti-PF4/heparin antibody production in more than 50% of patients exposed to heparin in some clinical settings is poorly understood. To investigate early events associated with antigen exposure, we first examined the interaction of PF4/heparin complexes with cells circulating in whole blood. In healthy donors, PF4/heparin complexes bind preferentially to B cells (>90% of B cells bind to PF4/heparin in vitro) relative to neutrophils, monocytes, or T cells. Binding of PF4 to B cells is heparin dependent, and PF4/heparin complexes are found on circulating B cells from some, but not all, patients receiving heparin. Given the high proportion of B cells that bind PF4/heparin, we investigated complement as a mechanism for noncognate antigen recognition. Complement is activated by PF4/heparin complexes, co-localizes with antigen on B cells from healthy donors, and is present on antigen-positive B cells in patients receiving heparin. Binding of PF4/heparin complexes to B cells is mediated through the interaction between complement and complement receptor 2 (CR2 [CD21]). To the best of our knowledge, these are the first studies to demonstrate complement activation by PF4/heparin complexes, opsonization of PF4/heparin to B cells via CD21, and the presence of complement activation fragments on circulating B cells in some patients receiving heparin. Given the critical contribution of complement to humoral immunity, our observations provide new mechanistic insights into the immunogenicity of PF4/heparin complexes. PMID:27412887

p56Lck and p59Fyn Regulate CD28 Binding to Phosphatidylinositol 3-Kinase, Growth Factor Receptor-Bound Protein GRB-2, and T Cell-Specific Protein-Tyrosine Kinase ITK: Implications for T-Cell Costimulation

NASA Astrophysics Data System (ADS)

Raab, Monika; Cai, Yun-Cai; Bunnell, Stephen C.; Heyeck, Stephanie D.; Berg, Leslie J.; Rudd, Christopher E.

1995-09-01

T-cell activation requires cooperative signals generated by the T-cell antigen receptor ξ-chain complex (TCRξ-CD3) and the costimulatory antigen CD28. CD28 interacts with three intracellular proteins-phosphatidylinositol 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine kinase ITK (formerly TSK or EMT), and the complex between growth factor receptor-bound protein 2 and son of sevenless guanine nucleotide exchange protein (GRB-2-SOS). PI 3-kinase and GRB-2 bind to the CD28 phosphotyrosine-based Tyr-Met-Asn-Met motif by means of intrinsic Src-homology 2 (SH2) domains. The requirement for tyrosine phosphorylation of the Tyr-Met-Asn-Met motif for SH2 domain binding implicates an intervening protein-tyrosine kinase in the recruitment of PI 3-kinase and GRB-2 by CD28. Candidate kinases include p56Lck, p59Fyn, ξ-chain-associated 70-kDa protein (ZAP-70), and ITK. In this study, we demonstrate in coexpression studies that p56Lck and p59Fyn phosphorylate CD28 primarily at Tyr-191 of the Tyr-Met-Asn-Met motif, inducing a 3- to 8-fold increase in p85 (subunit of PI 3-kinase) and GRB-2 SH2 binding to CD28. Phosphatase digestion of CD28 eliminated binding. In contrast to Src kinases, ZAP-70 and ITK failed to induce these events. Further, ITK binding to CD28 was dependent on the presence of p56Lck and is thus likely to act downstream of p56Lck/p59Fyn in a signaling cascade. p56Lck is therefore likely to be a central switch in T-cell activation, with the dual function of regulating CD28-mediated costimulation as well as TCR-CD3-CD4 signaling.

Hyaluronan as an Immune Regulator in Human Diseases

PubMed Central

NOBLE, PAUL W.; LIANG, JIURONG; JIANG, DIANHUA

2010-01-01

Accumulation and turnover of extracellular matrix components are the hallmarks of tissue injury. Fragmented hyaluronan stimulates the expression of inflammatory genes by a variety of immune cells at the injury site. Hyaluronan binds to a number of cell surface proteins on a variety of cell types. Hyaluronan fragments signal through both Toll-like receptor (TLR) 4 and TLR2 as well as CD44 to stimulate inflammatory genes in inflammatory cells. Hyaluronan is also present on the cell surface of epithelial cells and provides protection against tissue damage by interacting with TLR2 and TLR4 on these parenchymal cells. Hyaluronan and hyaluronan-binding proteins regulate inflammation, tissue injury and repair through regulating inflammatory cell recruitment, release of inflammatory cytokines, and stem cell migration. This review focuses on the role of hyaluronan as an immune regulator in human diseases. PMID:21248167

STAT3-activated CD36 facilitates fatty acid uptake in chronic lymphocytic leukemia cells

PubMed Central

Rozovski, Uri; Harris, David M.; Li, Ping; Liu, Zhiming; Jain, Preetesh; Ferrajoli, Alessandra; Burger, Jan; Thompson, Phillip; Jain, Nitin; Wierda, William; Keating, Michael J.; Estrov, Zeev

2018-01-01

Although several studies established that unlike normal B cells chronic lymphocytic leukemia (CLL) cells metabolize fatty acids (FA), how CLL cells internalize FA is poorly understood. Because in various cell types CD36 facilitates FA uptake, we wondered whether a similar mechanism is operative CLL. We found that CD36 levels are higher in CLL cells than in normal B cells, and that small interfering RNA, CD36 neutralizing antibodies or sulfosuccinimidyl oleate (SSO) that inhibits CD36 significantly reduced the oxygen consumption of CLL cells incubated with FA. Because CD36 is oeverexpressed and STAT3 is constitutively activated in CLL cells, we wondered whether STAT3 induces CD36 expression. Sequence analysis identified putative STAT3 binding sites in the CD36 gene promoter. Chromatin immunoprecipitation and an electrophoretic mobility shift assay revealed that STAT3 binds to the CD36 gene promoter. A luciferase assay and STAT3-small hairpin RNA, that significantly decreased the levels of CD36 in CLL cells, established that STAT3 activates the transcription of the CD36 gene. Furthermore, SSO induced a dose-dependent apoptosis of CLL cells. Taken together, our data suggest that STAT3 activates CD36 and that CD36 facilitates FA uptake in CLL cells. Whether CD36 inhibition would provide clinical benefits in CLL remains to be determined. PMID:29765537

Human milk galectin-3 binding protein and breast-feeding-associated HIV transmission.

PubMed

Chan, Christina S; Kim, Hae-Young; Autran, Chloe; Kim, Jae H; Sinkala, Moses; Kankasa, Chipepo; Mwiya, Mwiya; Thea, Donald M; Aldrovandi, Grace M; Kuhn, Louise; Bode, Lars

2013-12-01

Analysis of milk from 247 HIV-infected Zambian mothers showed that galectin-3 binding protein concentrations were significantly higher among HIV-infected mothers who transmitted HIV through breast-feeding (6.51 ± 2.12 μg/mL) than among nontransmitters but were also correlated with higher milk and plasma HIV RNA copies/mL and lower CD4+ cell counts. The association between galectin-3 binding protein and postnatal transmission was attenuated after adjustment for milk and plasma HIV load and CD4+ cell counts. This suggests that although milk galectin-3 binding protein is a marker of advanced maternal disease, it does not independently modify transmission risk.

Labeling by ( sup 3 H)1,3-di(2-tolyl)guanidine of two high affinity binding sites in guinea pig brain: Evidence for allosteric regulation by calcium channel antagonists and pseudoallosteric modulation by sigma ligands

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

Rothman, R.B.; Reid, A.; Mahboubi, A.

1991-02-01

Equilibrium binding studies with the sigma receptor ligand ({sup 3}H)1,3-di(2-tolyl)guanidine (({sup 3}H)DTG) demonstrated two high affinity binding sites in membranes prepared from guinea pig brain. The apparent Kd values of DTG for sites 1 and 2 were 11.9 and 37.6 nM, respectively. The corresponding Bmax values were 1045 and 1423 fmol/mg of protein. Site 1 had high affinity for (+)-pentazocine, haloperidol, (R)-(+)-PPP, carbepentane, and other sigma ligands, suggesting a similarity with the dextromethorphan/sigma 1 binding site described by Musacchio et al. (Life Sci. 45:1721-1732 (1989)). Site 2 had high affinity for DTG and haloperidol (Ki = 36.1 nM) and lowmore » affinity for most other sigma ligands. Kinetic experiments demonstrated that ({sup 3}H)DTG dissociated in a biphasic manner from both site 1 and site 2. DTG and haloperidol increased the dissociation rate of ({sup 3}H)DTG from site 1 and site 2, demonstrating the presence of pseudoallosteric interactions. Inorganic calcium channel blockers such as Cd2+ selectively increased the dissociation rate of ({sup 3}H)DTG from site 2, suggesting an association of this binding site with calcium channels.« less

The Nucleotide-Binding Oligomerization Domain-Like Receptor Family Pyrin Domain-Containing 3 Inflammasome Regulates Bronchial Epithelial Cell Injury and Proapoptosis after Exposure to Biomass Fuel Smoke.

PubMed

Li, Chen; Zhihong, Huang; Wenlong, Li; Xiaoyan, Liu; Qing, Chen; Wenzhi, Luo; Siming, Xie; Shengming, Liu

2016-12-01

The number of individuals in the population exposed to biomass fuel smoke (BS) is far greater than the number of cigarette smokers. About 20% of cigarette smokers develop chronic obstructive pulmonary disease (COPD) due to smoke-induced irreversible damage and sustained inflammation of the airway epithelium. However, the role of BS in COPD pathogenesis remains to be elucidated. In this study, we investigated the expression of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing (NLRP) 3 and caspase-1 in the bronchial epithelium from patients with COPD, and further determined the specific role of the NLRP3 inflammasome in bronchial epithelium injury using two in vitro models (BS and cigarette smoke [CS]) in the human bronchial epithelial (HBE) cell line (16HBE). After exposure to BS and CS, the release of damage-associated molecular patterns, the transcriptional and translational up-regulation of NLRP3, and the activation of caspase-1 were observed in cells at different time points. Because IL-1β secretion was dependent on the NLRP3 inflammasome, we assessed CXCL-8 production in response to smoke. Using a transwell migration assay in which 16HBE cells and human alveolar macrophages were cocultured, we showed that smoke-induced NLRP3 activation in 16HBE cells increased the migration of human alveolar macrophages. When the NLRP3 expression was silenced, the average migration distance of 16HBE was increased in scratch assay, because the activation of NLRP3 induced apoptosis by the p53-Bax mitochondrial pathway in the smoke-induced response. These results demonstrate the importance of the NLRP3 inflammasome in mediating BS- and CS-induced HBE cell damage and proapoptosis.

Acute effects of heavy-load squats on consecutive squat jump performance.

PubMed

Weber, Kurt R; Brown, Lee E; Coburn, Jared W; Zinder, Steven M

2008-05-01

Postactivation potentiation (PAP) and complex training have generated interest within the strength and conditioning community in recent years, but much of the research to date has produced confounding results. The purpose of this study was to observe the acute effects of a heavy-load back squat [85% 1 repetition maximum (1RM)] condition on consecutive squat jump performance. Twelve in-season Division I male track-and-field athletes participated in two randomized testing conditions: a five-repetition back squat at 85% 1RM (BS) and a five-repetition squat jump (SJ). The BS condition consisted of seven consecutive squat jumps (BS-PRE), followed by five repetitions of the BS at 85% 1RM, followed by another set of seven consecutive squat jumps (BS-POST). The SJ condition was exactly the same as the BS condition except that five consecutive SJs replaced the five BSs, with 3 minutes' rest between each set. BS-PRE, BS-POST, SJ-PRE, and SJ-POST were analyzed and compared for mean and peak jump height, as well as mean and peak ground reaction force (GRF). The BS condition's mean and peak jump height and peak GRF increased 5.8% +/- 4.8%, 4.7% +/- 4.8%, and 4.6% +/- 7.4%, respectively, whereas the SJ condition's mean and peak jump height and peak GRF decreased 2.7% +/- 5.0%, 4.0% +/- 4.9%, and 1.3% +/- 7.5%, respectively. The results indicate that performing a heavy-load back squat before a set of consecutive SJs may enhance acute performance in average and peak jump height, as well as peak GRF.

Thymic pathogenicity of an HIV-1 envelope is associated with increased CXCR4 binding efficiency and V5-gp41-dependent activity, but not V1/V2-associated CD4 binding efficiency and viral entry

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

Meissner, Eric G.; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599; Coffield, Vernon M.

2005-06-05

We previously described a thymus-tropic HIV-1 envelope (R3A Env) from a rapid progressor obtained at the time of transmission. An HIV-1 molecular recombinant with the R3A Env supported extensive replication and pathogenesis in the thymus and did not require Nef. Another Env from the same patient did not display the same thymus-tropic pathogenesis (R3B Env). Here, we show that relative to R3B Env, R3A Env enhances viral entry of T cells, increases fusion-induced cytopathicity, and shows elevated binding efficiency for both CD4 and CXCR4, but not CCR5, in vitro. We created chimeric envelopes to determine the region(s) responsible for eachmore » in vitro phenotype and for thymic pathogenesis. Surprisingly, while V1/V2 contributed to enhanced viral entry, CD4 binding efficiency, and cytopathicity in vitro, it made no contribution to thymic pathogenesis. Rather, CXCR4 binding efficiency and V5-gp41-associated activity appear to independently contribute to thymic pathogenesis of the R3A Env. These data highlight the contribution of unique HIV pathogenic factors in the thymic microenvironment and suggest that novel mechanisms may be involved in Env pathogenic activity in vivo.« less

β1,4-galactosyltransferase 1 is a novel receptor for IgA in human mesangial cells.

PubMed

Molyneux, Karen; Wimbury, David; Pawluczyk, Izabella; Muto, Masahiro; Bhachu, Jasraj; Mertens, Peter R; Feehally, John; Barratt, Jonathan

2017-12-01

IgA nephropathy is characterized by mesangial deposition of IgA, mesangial cell proliferation, and extracellular matrix production. Mesangial cells bind IgA, but the identity of all potential receptors involved remains incomplete. The transferrin receptor (CD71) acts as a mesangial cell IgA receptor and its expression is upregulated in many forms of glomerulonephritis, including IgA nephropathy. CD71 is not expressed in healthy glomeruli and blocking CD71 does not completely abrogate mesangial cell IgA binding. Previously we showed that mesangial cells express a receptor that binds the Fc portion of IgA and now report that this receptor is an isoform of β-1,4-galactosyltransferase. A human mesangial cell cDNA library was screened for IgA binding proteins and β-1,4-galactosyltransferase identified. Cell surface expression of the long isoform of β-1,4-galactosyltransferase was shown by flow cytometry and confocal microscopy and confirmed by immunoblotting. Glomerular β-1,4-galactosyltransferase expression was increased in IgA nephropathy. IgA binding and IgA-induced mesangial cell phosphorylation of spleen tyrosine kinase and IL-6 synthesis were inhibited by a panel of β-1,4-galactosyltransferase-specific antibodies, suggesting IgA binds to the catalytic domain of β-1,4-galactosyltransferase. Thus, β-1,4-galactosyltransferase is a constitutively expressed mesangial cell IgA receptor with an important role in both mesangial IgA clearance and the initial response to IgA deposition. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Models of metal binding structures in fulvic acid from the Suwannee River, Georgia

USGS Publications Warehouse

Leenheer, J.A.; Brown, G.K.; MacCarthy, P.; Cabaniss, S.E.

1998-01-01

Fulvic acid, isolated from the Suwannee River, Georgia, was assessed for its ability to bind Ca2+, Cd2+, Cu2+, Ni2+, and Zn2+ ions at pH 6 before and after extensive fractionation that was designed to reveal the nature of metal binding functional groups. The binding constant for Ca2+ ion had the greatest increase of all the ions in a metal binding fraction that was selected for intensive characterization for the purpose of building quantitative average model structures. The 'metal binding' fraction was characterized by quantitative 13C NMR, 1H NMR, and FT-1R spectrometry and elemental, titrimetric, and molecular weight determinations. The characterization data revealed that carboxyl groups were clustered in short- chain aliphatic dibasic acid structures. The Ca2+ binding data suggested that ether-substituted oxysuccinic acid structures are good models for the metal binding sites at pH 6. Structural models were derived based upon oxidation and photolytic rearrangements of cutin, lignin, and tannin precursors. These structural models rich in substituted dibasic acid structures revealed polydentate binding sites with the potential for both inner-sphere and outer-sphere type binding. The majority of the fulvic acid molecule was involved with metal binding rather than a small substructural unit.Fulvic acid, isolated from the Suwannee River, Georgia, was assessed for its ability to bind Ca2+, Cd2+, Cu2+, Ni2+, and Zn2+ ions at pH 6 before and after extensive fractionation that was designed to reveal the nature of metal binding functional groups. The binding constant for Ca2+ ion had the greatest increase of all the ions in a metal binding fraction that was selected for intensive characterization for the purpose of building quantitative average model structures. The `metal binding' fraction was characterized by quantitative 13C NMR, 1H NMR, and FT-IR spectrometry and elemental, titrimetric, and molecular weight determinations. The characterization data revealed that carboxyl groups were clustered in short-chain aliphatic dibasic acid structures. The Ca2+ binding data suggested that ether-substituted oxysuccinic acid structures are good models for the metal binding sites at pH 6. Structural models were derived based upon oxidation and photolytic rearrangements of cutin, lignin, and tannin precursors. These structural models rich in substituted dibasic acid structures revealed polydentate binding sites with the potential for both inner-sphere and outer-sphere type binding. The majority of the fulvic acid molecule was involved with metal binding rather than a small substructural unit.

Crystal Structures and Thermodynamic Analysis Reveal Distinct Mechanisms of CD28 Phosphopeptide Binding to the Src Homology 2 (SH2) Domains of Three Adaptor Proteins*

PubMed Central

Inaba, Satomi; Numoto, Nobutaka; Ogawa, Shuhei; Morii, Hisayuki; Ikura, Teikichi; Abe, Ryo; Ito, Nobutoshi; Oda, Masayuki

2017-01-01

Full activation of T cells and differentiation into effector T cells are essential for many immune responses and require co-stimulatory signaling via the CD28 receptor. Extracellular ligand binding to CD28 recruits protein-tyrosine kinases to its cytoplasmic tail, which contains a YMNM motif. Following phosphorylation of the tyrosine, the proteins growth factor receptor-bound protein 2 (Grb2), Grb2-related adaptor downstream of Shc (Gads), and p85 subunit of phosphoinositide 3-kinase may bind to pYMNM (where pY is phosphotyrosine) via their Src homology 2 (SH2) domains, leading to downstream signaling to distinct immune pathways. These three adaptor proteins bind to the same site on CD28 with variable affinity, and all are important for CD28-mediated co-stimulatory function. However, the mechanism of how these proteins recognize and compete for CD28 is unclear. To visualize their interactions with CD28, we have determined the crystal structures of Gads SH2 and two p85 SH2 domains in complex with a CD28-derived phosphopeptide. The high resolution structures obtained revealed that, whereas the CD28 phosphopeptide bound to Gads SH2 is in a bent conformation similar to that when bound to Grb2 SH2, it adopts a more extended conformation when bound to the N- and C-terminal SH2 domains of p85. These differences observed in the peptide-protein interactions correlated well with the affinity and other thermodynamic parameters for each interaction determined by isothermal titration calorimetry. The detailed insight into these interactions reported here may inform the development of compounds that specifically inhibit the association of CD28 with these adaptor proteins to suppress excessive T cell responses, such as in allergies and autoimmune diseases. PMID:27927989

Crystal Structures and Thermodynamic Analysis Reveal Distinct Mechanisms of CD28 Phosphopeptide Binding to the Src Homology 2 (SH2) Domains of Three Adaptor Proteins.

PubMed

Inaba, Satomi; Numoto, Nobutaka; Ogawa, Shuhei; Morii, Hisayuki; Ikura, Teikichi; Abe, Ryo; Ito, Nobutoshi; Oda, Masayuki

2017-01-20

Full activation of T cells and differentiation into effector T cells are essential for many immune responses and require co-stimulatory signaling via the CD28 receptor. Extracellular ligand binding to CD28 recruits protein-tyrosine kinases to its cytoplasmic tail, which contains a YMNM motif. Following phosphorylation of the tyrosine, the proteins growth factor receptor-bound protein 2 (Grb2), Grb2-related adaptor downstream of Shc (Gads), and p85 subunit of phosphoinositide 3-kinase may bind to pYMNM (where pY is phosphotyrosine) via their Src homology 2 (SH2) domains, leading to downstream signaling to distinct immune pathways. These three adaptor proteins bind to the same site on CD28 with variable affinity, and all are important for CD28-mediated co-stimulatory function. However, the mechanism of how these proteins recognize and compete for CD28 is unclear. To visualize their interactions with CD28, we have determined the crystal structures of Gads SH2 and two p85 SH2 domains in complex with a CD28-derived phosphopeptide. The high resolution structures obtained revealed that, whereas the CD28 phosphopeptide bound to Gads SH2 is in a bent conformation similar to that when bound to Grb2 SH2, it adopts a more extended conformation when bound to the N- and C-terminal SH2 domains of p85. These differences observed in the peptide-protein interactions correlated well with the affinity and other thermodynamic parameters for each interaction determined by isothermal titration calorimetry. The detailed insight into these interactions reported here may inform the development of compounds that specifically inhibit the association of CD28 with these adaptor proteins to suppress excessive T cell responses, such as in allergies and autoimmune diseases. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of a conformationally sensitive murine monoclonal antibody directed to the metal ion-dependent adhesion site face of integrin CD11b.

PubMed

Li, Rui; Haruta, Ikuko; Rieu, Philippe; Sugimori, Takashi; Xiong, Jian-Ping; Arnaout, M Amin

2002-02-01

Integrin binding to physiologic ligands requires divalent cations and an inside-out-driven switch of the integrin to a high-affinity state. Divalent cations at the metal ion-dependent adhesion site (MIDAS) face of the alpha subunit-derived A domain provide a direct bridge between ligands and the integrin, and it has been proposed that activation dependency is caused by reorientation of the surrounding residues relative to the metal ion, forming an optimal binding interface. To gain more insight into the functional significance of the protein movements on the MIDAS face, we raised and characterized a murine mAb 107 directed against the MIDAS face of the A domain from integrin CD11b. We find that mAb 107 behaves as a ligand mimic. It binds in a divalent-cation-dependent manner to solvent-exposed residues on the MIDAS face of CD11b, blocks interaction of 11bA or the holoreceptor with ligands, and inhibits spreading and phagocytosis by human neutrophils. However, in contrast to physiologic ligands, mAb 107 preferentially binds to the inactive low-affinity form of the integrin, suggesting that its antagonistic effects are exerted in part by stabilizing the receptor in the low-affinity state. These data support a functional relevance of the protein movements on the MIDAS face and suggest that stabilizing the A domain in the low-affinity state may have therapeutic benefit.

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

Gavva, S.R.; Harris, B.G.; Cook, P.F.

A thiol group at the malate-binding site of the NAD-malic enzyme from Ascaris suum has been modified to thiocyanate. The modified enzyme generally exhibits slight increases in K{sub NAD} and K{sub i metal} and decreases in V{sub max} as the metal size increases from Mg{sup 2+} to Mn{sup 2+} to Cd{sup 2+}, indicative of crowding in the site. The K{sub malate} value increases 10- to 30-fold, suggesting that malate does not bind optimally to the modified enzyme. Deuterium isotope effects on V and V/K{sub malate} increase with all three metal ions compared to the native enzyme concomitant with a decreasemore » in the {sup 13}C isotope effect, suggesting a switch in the rate limitation of the hydride transfer and decarboxylation steps with hydride transfer becoming more rate limiting. The {sup 13}C effect decreases only slightly when obtained with deuterated malate, suggestive of the presence of a secondary {sup 13}C effect in the hydride transfer step, similar to data obtained with non-nicotinamide-containing dinucleotide substrates for the native enzyme (see the preceding paper in this issue). The native enzyme is inactivated in a time-dependent manner by Cd{sup 2+}. This inactivation occurs whether the enzyme alone is present or whether the enzyme is turning over with Cd{sup 2+} as the divalent metal activator. Upon inactivation, only Cd{sup 2+} ions are bound at high stoichiometry to the enzyme, which eventually becomes denatured. Conversion of the active-site thiol to thiocyanate makes it more difficult to inactivate the enzyme by treatment with Cd{sup 2+}.« less

[Changes of monocyte and monocyte-platelet aggregates in different subgroups of thrombotic events in patients with acute myocardial infarction during PCI].

PubMed

Wang, Sheng; Sun, Cuifang; Liao, Wang; Wu, Zhongwei; Wang, Yudai; Huang, Xiuxian; Lu, Sijia; Dong, Xiaoli; Shuai, Fujie; Li, Bin

2017-07-01

Objective To investigate the impact of thrombotic events on the alterations of monocyte and monocyte-platelet aggregates (MPAs) in patients with acute myocardial infarction (AMI) during percutaneous coronary intervention (PCI). Methods Blood was collected before PCI for flow cytometry. Monocyte subsets and MPAs were detected by four-color platform (CDl4-APC, CDl6-PE-Cy7, CD86-PE and CD41-Alexa Fluor R 488). According to the expression of the platelet surface marker CD41, the number of monocyte subsets and MPAs was analyzed using the fluorescent microspheres of absolute counting tube. The Wilcoxon rank sum test and receiver operating characteristic (ROC) curve analysis were performed. Results CD14 + CD16 ++ monocytes in intraprocedural thrombotic events (IPTE) group were significantly fewer than those in non-IPTE group, and the percentage in total mononuclear cells decreased. Compared with non-IPTE group, MPA binding ratio and monocyte subset MPA binding ratio were significantly higher in IPTE group. ROC analysis showed that MPA binding ratio and subgroup MPA binding ratio had a better predictive value for IPTE in patients with AMI. Conclusion The CD14 + CD16 ++ monocytes in IPTE group were significantly fewer than those in the non-IPTE group. MPA binding ratio and MPA binding ratio of monocyte subsets were significantly higher in the IPTE group than in the non-IPTE group, so they have a good predictive value for IPTE in patients with AMI.

Improvement of selective removal of heavy metals in cyanobacteria by NaOH treatment.

PubMed

Nagase, Hiroyasu; Inthorn, Duangrat; Oda, Aiko; Nishimura, Jun; Kajiwara, Yumiko; Park, Myong-Oku; Hirata, Kazumasa; Miyamoto, Kazuhisa

2005-04-01

In the freshwater cyanobacterium, Tolypothrix tenuis, treatment with 0.1 M NaOH increased its Cd-selective adsorption ability in the presence of Ca(2+) or Mg(2+). The selective adsorption was also achieved by other alkaline treatments. Energy-distributed spectroscopy analysis revealed that Cd(2+) was found mainly on the surface of non-treated cells, whereas it was distributed throughout the cell after NaOH treatment. The alkaline treatment was effective in increasing the selective adsorption ability of the cyanobacterium for other bivalent heavy metals such as Cu(2+), Pb(2+) and Zn(2+). The treatment was also applicable to Anabaena variabilis and Microcystis aeruginosa, which are typical cyanobacteria causing algal blooms. The main binding site of Cd(2+) in NaOH-treated cells is assumed to be the carboxyl groups because the binding ability of the cells was diminished by the esterification of carboxyl groups. These results suggest that alkaline treatment of cyanobacteria is a useful technique for producing biosorbents having highly specific binding abilities for heavy metals.

Interleukin 4: signalling mechanisms and control of T cell differentiation.

PubMed

Paul, W E

1997-01-01

Interleukin 4 (IL-4) is a pleiotropic type I cytokine that controls both growth and differentiation among haemopoietic and non-haemopoietic cells. Its receptor is a heterodimer. One chain, the IL-4R alpha chain, binds IL-4 with high affinity and determines the nature of the biochemical signals that are induced. The second chain, gamma c, is required for the induction of such signals. IL-4-mediated growth depends upon activation events that involve phosphorylation of Y497 of IL-4R alpha, leading to the binding and phosphorylation of 4PS/IRS-2 in haemopoietic cells and of IRS-1 in non-haemopoietic cells. By contrast, IL-4-mediated differentiation events depend upon more distal regions of the IL-4R alpha chain that include a series of STAT-6 binding sites. The distinctive roles of these receptor domains was verified by receptor-reconstruction experiments. The 'growth' and 'differentiation' domains of the IL-4R alpha chain, independently expressed as chimeric structures with a truncated version of the IL-2R beta chain, were shown to convey their functions to the hybrid receptor. The critical role of STAT-6 in IL-4-mediated gene activation and differentiation was made clear by the finding that lymphocytes from STAT-6 knockout mice are strikingly deficient in these functions but have retained the capacity to grow, at least partially, in response to IL-4. IL-4 plays a central role in determining the phenotype of naive CD4+ T cells. In the presence of IL-4, newly primed naive T cells develop into IL-4 producers while in its absence they preferentially become gamma-interferon (IFN-gamma) producers. Recently, a specialized subpopulation of T cells, CD4+/NK1.1+ cells, has been shown to produce large amounts of IL-4 upon stimulation. Two examples of mice with deficiencies in these cells are described--beta 2-microglobulin knockout mice and SJL mice. Both show defects in the development of IL-4-producing cells and in the increase in serum IgE in response to stimulation with the polyclonal stimulant anti-IgD. Both sets of mice have major diminutions in the number of CD4+/ NK1.1+ T cells, strongly indicating an important role of these cells in some but not all IgE responses to physiologic stimuli.

Identification of the platelet-derived chemokine CXCL4/PF-4 as a broad-spectrum HIV-1 inhibitor

PubMed Central

Auerbach, David J.; Lin, Yin; Miao, Huiyi; Cimbro, Raffaello; DiFiore, Michelle J.; Gianolini, Monica E.; Furci, Lucinda; Biswas, Priscilla; Fauci, Anthony S.; Lusso, Paolo

2012-01-01

The natural history of HIV-1 infection is highly variable in different individuals, spanning from a rapidly progressive course to a long-term asymptomatic infection. A major determinant of the pace of disease progression is the in vivo level of HIV-1 replication, which is regulated by a complex network of cytokines and chemokines expressed by immune and inflammatory cells. The chemokine system is critically involved in the control of HIV-1 replication by virtue of the role played by specific chemokine receptors, most notably CCR5 and CXCR4, as cell-surface coreceptors for HIV-1 entry; hence, the chemokines that naturally bind such coreceptors act as endogenous inhibitors of HIV-1. Here, we show that the CXC chemokine CXCL4 (PF-4), the most abundant protein contained within the α-granules of platelets, is a broad-spectrum inhibitor of HIV-1 infection. Unlike other known HIV-suppressive chemokines, CXCL4 inhibits infection by the majority of primary HIV-1 isolates regardless of their coreceptor-usage phenotype or genetic subtype. Consistent with the lack of viral phenotype specificity, blockade of HIV-1 infection occurs at the level of virus attachment and entry via a unique mechanism that involves direct interaction of CXCL4 with the major viral envelope glycoprotein, gp120. The binding site for CXCL4 was mapped to a region of the gp120 outer domain proximal to the CD4-binding site. The identification of a platelet-derived chemokine as an endogenous antiviral factor may have relevance for the pathogenesis and treatment of HIV-1 infection. PMID:22645343

Identification of the platelet-derived chemokine CXCL4/PF-4 as a broad-spectrum HIV-1 inhibitor.

PubMed

Auerbach, David J; Lin, Yin; Miao, Huiyi; Cimbro, Raffaello; Difiore, Michelle J; Gianolini, Monica E; Furci, Lucinda; Biswas, Priscilla; Fauci, Anthony S; Lusso, Paolo

2012-06-12

The natural history of HIV-1 infection is highly variable in different individuals, spanning from a rapidly progressive course to a long-term asymptomatic infection. A major determinant of the pace of disease progression is the in vivo level of HIV-1 replication, which is regulated by a complex network of cytokines and chemokines expressed by immune and inflammatory cells. The chemokine system is critically involved in the control of HIV-1 replication by virtue of the role played by specific chemokine receptors, most notably CCR5 and CXCR4, as cell-surface coreceptors for HIV-1 entry; hence, the chemokines that naturally bind such coreceptors act as endogenous inhibitors of HIV-1. Here, we show that the CXC chemokine CXCL4 (PF-4), the most abundant protein contained within the α-granules of platelets, is a broad-spectrum inhibitor of HIV-1 infection. Unlike other known HIV-suppressive chemokines, CXCL4 inhibits infection by the majority of primary HIV-1 isolates regardless of their coreceptor-usage phenotype or genetic subtype. Consistent with the lack of viral phenotype specificity, blockade of HIV-1 infection occurs at the level of virus attachment and entry via a unique mechanism that involves direct interaction of CXCL4 with the major viral envelope glycoprotein, gp120. The binding site for CXCL4 was mapped to a region of the gp120 outer domain proximal to the CD4-binding site. The identification of a platelet-derived chemokine as an endogenous antiviral factor may have relevance for the pathogenesis and treatment of HIV-1 infection.

Synthesis of water-soluble polyamine derivatives effective as N-methyl-D-aspartate receptor antagonists.

PubMed

Masuko, Takashi; Yoshida, Shuhei; Metori, Koichi; Kizawa, Yasuo; Kusama, Tadashi; Miyake, Muneharu

2010-06-01

The novel water-soluble N-methyl-D-aspartate (NMDA) receptor antagonists, N-{4-[4-(4-Guanidinobutylamino)butylamino]butyl}-p-toluenesulfonamide trihydrochloride (1a, TsHSPMG), N-{4-[4-(4-Guanidinobutylamino)butylamino]butyl}butane-1-sulfonamide trihydrochloride (1b, BsHSPMG), N-{3-[4-(3-Guanidinopropylamino)butylamino]propyl}-p-toluenesulfonamide trihydrochroride (2a, TsSPMG) and N-{3-[4-(3-Guanidinopropylamino)butylamino]propyl}butane-1-sulfonamide trihydrochroride (2b, BsSPMG), were synthesized, and the effects of these polyamine derivatives on NMDA receptors were studied using voltage-clamp recordings of recombinant NMDA receptors expressed in Xenopus oocytes. Although spermine potentiates 153% and 310% of NMDA (NR1A/NR2B) receptors in the presence of saturated and unsaturated glycine, respectively, all the novel polyamine derivatives, TsHSPMG (1a), BsHSPMG (1b), TsSPMG (2a) and BsSPMG (2b), significantly inhibited NR1A/NR2B receptors in both conditions. The degree of NMDA receptor inhibition by TsHSPMG (1a) and BsHSPMG (1b) was stronger than that by TsSPMG (2a) and BsSPMG (2b).

Molecular Bases of cyclodextrin Adapter Interactions with Engineered Protein Nanopores

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

Banerjee, A.; Mikhailova, E; Cheley, S

2010-01-01

Engineered protein pores have several potential applications in biotechnology: as sensor elements in stochastic detection and ultrarapid DNA sequencing, as nanoreactors to observe single-molecule chemistry, and in the construction of nano- and micro-devices. One important class of pores contains molecular adapters, which provide internal binding sites for small molecules. Mutants of the {alpha}-hemolysin ({alpha}HL) pore that bind the adapter {beta}-cyclodextrin ({beta}CD) {approx}10{sup 4} times more tightly than the wild type have been obtained. We now use single-channel electrical recording, protein engineering including unnatural amino acid mutagenesis, and high-resolution x-ray crystallography to provide definitive structural information on these engineered protein nanoporesmore » in unparalleled detail.« less

Endothelins activate Ca(2+)-gated K(+) channels via endothelin B receptors in CD-1 mouse erythrocytes.

PubMed

Rivera, A; Rotter, M A; Brugnara, C

1999-10-01

Cell dehydration mediated by Ca(2+)-activated K(+) channels plays an important role in the pathogenesis of sickle cell disease. CD-1 mouse erythrocytes possess a Ca(2+)-activated K(+) channel (Gardos channel) with maximal velocity (V(max)) of 0.154 +/- 0.02 mmol. l cells(-1). min(-1) and an affinity constant (K(0.5)) for Ca(2+) of 286 +/- 83 nM in the presence of A-23187. Cells pretreated with 500 nM endothelin-1 (ET-1) increased their V(max) by 88 +/- 9% (n = 8) and decreased their K(0.5) for Ca(2+) to 139 +/- 63 nM (P < 0.05; n = 4). Activation of the Gardos channel resulted in an EC(50) of 75 +/- 20 nM for ET-1 and 374 +/- 97 nM for ET-3. Analysis of the affinity of unlabeled ET-1 for its receptor showed two classes of binding sites with apparent dissociation constants of 167 +/- 51 and 785 +/- 143 nM and with capacity of binding sites of 298 +/- 38 and 1,568 +/- 211 sites/cell, respectively. The Gardos channel was activated by the endothelin B (ET(B)) receptor agonist IRL 1620 and inhibited by BQ-788, demonstrating the involvement of ET(B) receptors. Calphostin C inhibited 73% of ET-1-induced Gardos activation and 84% of the ET-1-induced membrane protein kinase C activity. Thus endothelins regulate erythrocyte Gardos channels via ET(B) receptors and a calphostin-sensitive mechanism.

Structural analysis of a highly glycosylated and unliganded gp120-based antigen using mass spectrometry†

PubMed Central

Wang, Liwen; Qin, Yali; Ilchenko, Serguei; Bohon, Jen; Shi, Wuxian; Cho, Michael W.; Takamoto, Keiji; Chance, Mark R.

2010-01-01

Structural characterization of the HIV envelope protein gp120 is very important to provide an understanding of the protein's immunogenicity and it's binding to cell receptors. So far, crystallographic structure determination of gp120 with an intact V3 loop (in the absence of CD4 co-receptor or antibody) has not been achieved. The third variable region (V3) of the gp120 is immunodominant and contains glycosylation signatures that are essential for co-receptor binding and viral entry to T-cells. In this study, we characterized the structure of the outer domain of gp120 with an intact V3 loop (gp120-OD8) purified from Drosophila S2 cells utilizing mass spectrometry-based approaches. We mapped the glycosylation sites and calculated glycosylation occupancy of gp120-OD8; eleven sites from fifteen glycosylation motifs were determined as having high mannose or hybrid glycosylation structures. The specific glycan moieties of nine glycosylation sites from eight unique glycopeptides were determined by a combination of ECD and CID MS approaches. Hydroxyl radical-mediated protein footprinting coupled with mass spectrometry analysis was employed to provide detailed information on protein structure of gp120-OD8 by directly identifying accessible and hydroxyl radical-reactive side chain residues. Comparison of gp120-OD8 experimental footprinting data with a homology model derived from the ligated CD4/ gp120-OD8 crystal structure revealed a flexible V3 loop structure where the V3 tip may provide contacts with the rest of the protein while residues in the V3 base remain solvent accessible. In addition, the data illustrate interactions between specific sugar moieties and amino acid side chains potentially important to the gp120-OD8 structure. PMID:20825246

Preventing subclinical necrotic enteritis through Lactobacillus johnsonii BS15 by ameliorating lipid metabolism and intestinal microflora in broiler chickens.

PubMed

Qing, Xiaodan; Zeng, Dong; Wang, Hesong; Ni, Xueqin; Liu, Lei; Lai, Jing; Khalique, Abdul; Pan, Kangcheng; Jing, Bo

2017-12-01

Increasing studies have focused on the beneficial effects of Lactobacillus johnsonii in certain diseases. Here, we studied the prevention ability of a probiotic strain, L. johnsonii BS15 on subclinical necrotic enteritis (SNE), and its underlying mechanism. 180 male Cobb 500 chicks were randomly allotted into three groups and administrated with BS15 (1 × 10 6 cfu/g) or Man Rogosa Sharpe liquid medium throughout a 28-day experimental period. With the exception of the normal group, SNE infection was treated for the remaining experimental period after the chicks were fed with normal diet 14 days. Results showed that BS15 notably suppressed the SNE-induced loss of average daily gain and liver functional abnormality. Additionally, BS15 facilitated lipid metabolism of SNE boilers when the contents of peroxisome proliferator activated receptor γ and adipose triglyceride lipase in adipose tissue and serum high-density lipoprotein cholesterol decreased. BS15 also attenuated the hepatic lipid accumulation of stricken chicks by suppressing the genes expression of acetyl-CoA carboxylase, fatty acid synthase and sterol regulatory element binding protein-1c as well as stimulating the genes expression of peroxisome proliferator activated receptor α and carnitine palmitoyltransferase-1. Moreover, BS15 enhanced the development of SNE gut by improving the intestinal development and digestion as well as adjusting the gut microflora. Therefore, BS15 may provide a promising natural preventative strategy against SNE, which may be contributed to the amelioration of lipid metabolism and intestinal microflora.

Prime, Shock, and Kill: Priming CD4 T Cells from HIV Patients with a BCL-2 Antagonist before HIV Reactivation Reduces HIV Reservoir Size

PubMed Central

Cummins, Nathan W.; Sainski, Amy M.; Dai, Haiming; Natesampillai, Sekar; Pang, Yuan-Ping; Bren, Gary D.; de Araujo Correia, Maria Cristina Miranda; Sampath, Rahul; Rizza, Stacey A.; O'Brien, Daniel; Yao, Joseph D.

2016-01-01

ABSTRACT Understanding how some HIV-infected cells resist the cytotoxicity of HIV replication is crucial to enabling HIV cure efforts. HIV killing of CD4 T cells that replicate HIV can involve HIV protease-mediated cleavage of procaspase 8 to generate a fragment (Casp8p41) that directly binds and activates the mitochondrial proapoptotic protein BAK. Here, we demonstrate that Casp8p41 also binds with nanomolar affinity to the antiapoptotic protein Bcl-2, which sequesters Casp8p41 and prevents apoptosis. Further, we show that central memory CD4 T cells (TCM) from HIV-infected individuals have heightened expression of BCL-2 relative to procaspase 8, possibly explaining the persistence of HIV-infected TCM despite generation of Casp8p41. Consistent with this hypothesis, the selective BCL-2 antagonist venetoclax induced minimal killing of uninfected CD4 T cells but markedly increased the death of CD4 T cells and diminished cell-associated HIV DNA when CD4 T cells from antiretroviral therapy (ART)-suppressed HIV patients were induced with αCD3/αCD28 to reactivate HIV ex vivo. Thus, priming CD4 T cells from ART suppressed HIV patients with a BCL-2 antagonist, followed by HIV reactivation, achieves reductions in cell-associated HIV DNA, whereas HIV reactivation alone does not. IMPORTANCE HIV infection is incurable due to a long-lived reservoir of HIV+ memory CD4 T cells, and no clinically relevant interventions have been identified that reduce the number of these HIV DNA-containing cells. Since postintegration HIV replication can result in HIV protease generation of Casp8p41, which activates BAK, causing infected CD4 T cell death, we sought to determine whether this occurs in memory CD4 T cells. Here, we demonstrate that memory CD4 T cells can generate Casp8p41 and yet are intrinsically resistant to death induced by diverse stimuli, including Casp8p41. Furthermore, BCL-2 expression is relatively increased in these cells and directly binds and inhibits Casp8p41's proapoptotic effects. Antagonizing BCL-2 with venetoclax derepresses this antagonism, resulting in death, preferentially in HIV DNA containing cells, since only these cells generate Casp8p41. Thus, BCL-2 antagonism is a clinically relevant intervention with the potential to reduce HIV reservoir size in patients. PMID:26842479

The localisation of the heparin binding sites of human and murine interleukin-12 within the carboxyterminal domain of the P40 subunit.

PubMed

Garnier, Pascale; Mummery, Rosemary; Forster, Mark J; Mulloy, Barbara; Gibbs, Roslyn V; Rider, Christopher C

2018-05-09

We have previously shown that the heterodimeric cytokine interleukin-12, and the homodimer of its larger subunit p40, both bind to heparin and heparan sulfate with relatively high affinity. In the present study we characterised these interactions using a series of chemically modified heparins as competitive inhibitors. Human interleukin-12 and p40 homodimer show indistinguishable binding profiles with a panel of heparin derivatives, but that of murine interleukin-12 is distinct. Heparin markedly protects the human and murine p40 subunits, but not the p35 subunits, from cleavage by the bacterial endoprotease LysC, further implicating the larger subunit as the location of the heparin binding site. Moreover the essential role of the carboxyterminal D3 domain in heparin binding is established by the failure of a truncated construct of the p40 subunit lacking this domain to bind. Predictive docking calculations indicate that a cluster of basic residues at the tip of the exposed C'D' loop within D3 is important in heparin binding. However since the human and murine C'D' loops differ considerably in length, the mode and three dimensional orientation of heparin binding are likely to differ substantially between the human and murine p40s. Thus overall the binding of IL-12 via its p40 subunit to heparin-related polysaccharides of the extracellular matrix appears to be functionally important since it has been conserved across mammalian species despite this structural divergence. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

The novel benzopyran class of selective cyclooxygenase-2 inhibitors. Part 2: The second clinical candidate having a shorter and favorable human half-life

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

Wang, Jane L.; Limburg, David; Graneto, Matthew J.

2012-05-29

In this Letter, we provide the structure-activity relationships, optimization of design, testing criteria, and human half-life data for a series of selective COX-2 inhibitors. During the course of our structure-based drug design efforts, we discovered two distinct binding modes within the COX-2 active site for differently substituted members of this class. The challenge of a undesirably long human half-life for the first clinical candidate 1t{sub 1/2} = 360 h was addressed by multiple strategies, leading to the discovery of 29b-(S) (SC-75416) with t{sub 1/2} = 34 h.

Stable coordination of the inhibitory Ca2+ ion at MIDAS in integrin CD11b/CD18 by an antibody-derived ligand aspartate: Implications for integrin regulation and structure-based drug design

PubMed Central

Mahalingam, Bhuvaneshwari; Ajroud, Kaouther; Alonso, Jose Luis; Anand, Saurabh; Adair, Brian; Horenstein, Alberto L; Malavasi, Fabio; Xiong, Jian-Ping; Arnaout, M. Amin

2011-01-01

A central feature of integrin interaction with physiologic ligands is the monodentate binding of a ligand carboxylate to a Mg2+ ion hexacoordinated at the metal-ion-dependent-adhesion site (MIDAS) in the integrin A-domain. This interaction stabilizes the A-domain in the high-affinity state, which is distinguished from the default low-affinity state by tertiary changes in the domain that culminate in cell adhesion. Small molecule ligand-mimetic integrin antagonists act as partial agonists, eliciting similar activating conformational changes in the A-domain, which has contributed to paradoxical adhesion and increased patient mortality in large clinical trials. As with other ligand-mimetic integrin antagonists, the function-blocking monoclonal antibody (mAb) 107 binds MIDAS of integrin CD11b/CD18 A-domain (CD11bA), but in contrast, it favors the inhibitory Ca2+ ion over Mg2+ at MIDAS. We determined the crystal structures of the Fab fragment of mAb 107 complexed to the low- and high-affinity states of CD11bA. Favored binding of Ca2+ at MIDAS is caused by the unusual symmetric bidentate ligation of a Fab-derived ligand Asp to a heptacoordinated MIDAS Ca2+. Binding of Fab 107 to CD11bA did not trigger the activating tertiary changes in the domain or in the full-length integrin. These data show that denticity of the ligand Asp/Glu can modify divalent cation selectivity at MIDAS and hence integrin function. Stabilizing the Ca2+ ion at MIDAS by bidentate ligation to a ligand Asp/Glu may provide one approach for designing pure integrin antagonists. PMID:22095715

Interaction between Saikosaponin D, Paeoniflorin, and Human Serum Albumin.

PubMed

Liang, Guo-Wu; Chen, Yi-Cun; Wang, Yi; Wang, Hong-Mei; Pan, Xiang-Yu; Chen, Pei-Hong; Niu, Qing-Xia

2018-01-27

Saikosaponin D (SSD) and paeoniflorin (PF) are the major active constituents of Bupleuri Radix and Paeonia lactiflora Pall , respectively, and have been widely used in China to treat liver and other diseases for many centuries. We explored the binding of SSD/PF to human serum albumin (HSA) by using fluorospectrophotometry, circular dichroism (CD) and molecular docking. Both SSD and PF produced a conformational change in HSA. Fluorescence quenching was accompanied by a blue shift in the fluorescence spectra. Co-binding of PF and SSD also induced quenching and a conformational change in HSA. The Stern-Volmer equation showed that quenching was dominated by static quenching. The binding constant for ternary interaction was below that for binary interaction. Site-competitive experiments demonstrated that SSD/PF bound to site I (subdomain IIA) and site II (subdomain IIIA) in HSA. Analysis of thermodynamic parameters indicated that hydrogen bonding and van der Waals forces were mostly responsible for the binary association. Also, there was energy transfer upon binary interaction. Molecular docking supported the experimental findings in conformation, binding sites and binding forces.

Technetium-99m-labeled rituximab for use as a specific tracer of sentinel lymph node biopsy: a translational research study.

PubMed

Wang, Xuejuan; Yang, Zhi; Lin, Baohe; Zhang, Yan; Zhai, Shizhen; Zhao, Qichao; Xie, Qing; Liu, Fei; Han, Xuedi; Li, Jinfeng; Ouyang, Tao

2016-06-21

We aimed to develop and translate a CD20-antigen-targeted radiopharmaceutical, Technetium-99 m-labeled (99mTc) rituximab, for sentinel lymph node (SLN) detection. 99mTc-rituximab was synthesized and tested for stability in human serum. The binding affinity to CD20 was evaluated in Raji cells by flow cytometric analysis. Biodistribution and sentinel node mapping were carried out in bal b/c mice. Eighty-five patients with breast cancer participated in this study. Dynamic sentinel lymphoscintigraphy was first assessed in 12 patients before planar lymphoscintigraphy was assessed in a larger cohort. All patients underwent sentinel lymph node biopsy (SLNB), followed by axillary lymph node dissection. The cell-binding study showed that 99mTc-rituximab possessed compatible affinity to human CD20. In the mechanism study, 99mTc-labeled anti-mouse CD20 monoclonal antibodies could bind to mouse CD20 and accumulate in the SLN with 2.62±1.25 % of the percentage of injected activity, which could be blocked by excessive unlabeled antibody. Low uptake of non-sentinel nodes and fast clearance from the injection site were observed in the mice. Sentinel nodes were identified in 82 of 85 breast cancer patients (96.5%) by lymphoscintigraphy and SLNB. The sensitivity, specificity, and accuracy were 96.8% (30/31), 100% (51/51), and 98.8% (81/82), respectively. 99mTc-rituximab, specifically binding to CD20, met most of the requirements of an ideal sentinel mapping agent for use in clinical settings.

4Bs or Not 4Bs: Bricks, Bytes, Brains, and Bandwidth

ERIC Educational Resources Information Center

Treat, Tod

2011-01-01

The effective integration of planning to include bricks, bytes, brains, and bandwidth (the 4Bs) represents an opportunity for community colleges to extend their capacity as knowledge-intensive organizations, coupling knowledge, technology, and learning. Integration is important to ensure that the interplay among organizations, agents within them,…

Effect of Sulfometuron Methyl on Ground Water and Stream Quality in Coastal Plain Forest Watersheds

Treesearch

D.G. Neary; J.L. Michael

1989-01-01

Sulfometuron methyl [methyl 2-[[[[(4,6-dimethyl-2-pyrimidinyl)a-mino]carbonyl]amino]sulfonyl]benzoate] was applied by a ground sprayer at a maximum labeled rate of 0.42 kg ha-1 a.i. to a 4 ha Coastal Plain flatwoods watershed BS site preperation for tree planting. Herbicide residues were detected in streamflow for only seven days after...

A Particle Batch Smoother Approach to Snow Water Equivalent Estimation

NASA Technical Reports Server (NTRS)

Margulis, Steven A.; Girotto, Manuela; Cortes, Gonzalo; Durand, Michael

2015-01-01

This paper presents a newly proposed data assimilation method for historical snow water equivalent SWE estimation using remotely sensed fractional snow-covered area fSCA. The newly proposed approach consists of a particle batch smoother (PBS), which is compared to a previously applied Kalman-based ensemble batch smoother (EnBS) approach. The methods were applied over the 27-yr Landsat 5 record at snow pillow and snow course in situ verification sites in the American River basin in the Sierra Nevada (United States). This basin is more densely vegetated and thus more challenging for SWE estimation than the previous applications of the EnBS. Both data assimilation methods provided significant improvement over the prior (modeling only) estimates, with both able to significantly reduce prior SWE biases. The prior RMSE values at the snow pillow and snow course sites were reduced by 68%-82% and 60%-68%, respectively, when applying the data assimilation methods. This result is encouraging for a basin like the American where the moderate to high forest cover will necessarily obscure more of the snow-covered ground surface than in previously examined, less-vegetated basins. The PBS generally outperformed the EnBS: for snow pillows the PBSRMSE was approx.54%of that seen in the EnBS, while for snow courses the PBSRMSE was approx.79%of the EnBS. Sensitivity tests show relative insensitivity for both the PBS and EnBS results to ensemble size and fSCA measurement error, but a higher sensitivity for the EnBS to the mean prior precipitation input, especially in the case where significant prior biases exist.

A new type of natural bispecific antibody with potential protective effect in Hashimoto thyroiditis.

PubMed

Li, Wenli; Fan, Gaowei; Chen, Lida; Zhang, Rui; Zhang, Kuo; Sun, Yu; Lin, Guigao; Xie, Jiehong; Wang, Lunan; Li, Jinming

2014-09-01

As a new antibody concept, natural bispecific antibodies (nBsAbs) have been detected in long-term passive immunization and some diseases, but their potential immunomodulatory role remains unclear. Hashimoto thyroiditis (HT) appears to fulfill the condition for nBsAb production but has not yet been characterized. The objective of the study was to identify a new nBsAb against thyroid peroxidase (TPO) and thyroglobulin (Tg) in HT patients and to preliminarily explore its immunomodulatory role. Serum samples were obtained from 136 HT patients, 92 diseased controls, and 99 healthy controls for anti-TPO/Tg nBsAb detection. The relationship between anti-TPO/Tg nBsAb and other clinical parameters was also analyzed. The anti-TPO/Tg nBsAb was detected using a double-antigen sandwich ELISA. Higher nBsAb levels were found to be associated with decreased inflammation in HT patients. The prevalence of anti-TPO/Tg nBsAb in HT was 44.9% (61 of 136), significantly higher than that of diseased controls (2.2%, 2 of 92) (P < .0001) and healthy controls (0%, 0 of 99) (P < .0001). HT patients who were nBsAb positive were prone to have significantly lower levels of serum C-reactive protein and TNF-α compared with the nBsAb-negative individuals (P < .05). The serum amyloid A and interferon-γ levels also showed a similar trend in the two groups. The IgG subclass of anti-TPO/Tg nBsAb was IgG4. Further analysis showed a negative correlation between anti-TPO/Tg nBsAb and serum total IgG4 (r = -0.697, P = .025) in IgG4 thyroiditis patients. A new type of nBsAb against TPO and Tg in HT patients is identified. Our data also indicate a protective effect of anti-TPO/Tg nBsAb in the pathogenesis of HT and extend prior knowledge about nBsAb in diseases.

Steered Molecular Dynamics for Investigating the Interactions Between Insulin Receptor Tyrosine Kinase (IRK) and Variants of Protein Tyrosine Phosphatase 1B (PTP1B).

PubMed

Nguyen, Hung; Do, Nhat; Phan, Tuyn; Pham, Tri

2018-02-01

The aim of this study is to use steered molecular dynamics to investigate the dissociation process between IRK and PTP1Bs for wild type and five mutants (consisting of p.D181E, p.D181A, p.Q262A, p.D181A-Y46F, and p.D181A-Q262A). The gained results are observed not only the unbinding mechanism of IRK-PTP1B complexes came from pulling force profile, number of hydrogen bonds, and interaction energy between IRK and PTP1Bs but also described PTP1B's point mutations could variably change its binding affinity towards IRK. Additionally, the binding free energy calculated by Molecular Mechanics/Poisson-Boltzmann Surface Area (MM-PBSA) is also revealed that electrostatic energy and polar solvation energy mainly made up the binding free energy of PTP1B-IRK complexes.

First observation of the decay Bs0-->Ds-Ds+ and measurement of its branching ratio.

PubMed

Aaltonen, T; Adelman, J; Akimoto, T; Albrow, M G; Alvarez González, B; Amerio, S; Amidei, D; Anastassov, A; Annovi, A; Antos, J; Aoki, M; Apollinari, G; Apresyan, A; Arisawa, T; Artikov, A; Ashmanskas, W; Attal, A; Aurisano, A; Azfar, F; Azzi-Bacchetta, P; Azzurri, P; Bacchetta, N; Badgett, W; Barbaro-Galtieri, A; Barnes, V E; Barnett, B A; Baroiant, S; Bartsch, V; Bauer, G; Beauchemin, P-H; Bedeschi, F; Bednar, P; Behari, S; Bellettini, G; Bellinger, J; Belloni, A; Benjamin, D; Beretvas, A; Beringer, J; Berry, T; Bhatti, A; Binkley, M; Bisello, D; Bizjak, I; Blair, R E; Blocker, C; Blumenfeld, B; Bocci, A; Bodek, A; Boisvert, V; Bolla, G; Bolshov, A; Bortoletto, D; Boudreau, J; Boveia, A; Brau, B; Bridgeman, A; Brigliadori, L; Bromberg, C; Brubaker, E; Budagov, J; Budd, H S; Budd, S; Burkett, K; Busetto, G; Bussey, P; Buzatu, A; Byrum, K L; Cabrera, S; Campanelli, M; Campbell, M; Canelli, F; Canepa, A; Carlsmith, D; Carosi, R; Carrillo, S; Carron, S; Casal, B; Casarsa, M; Castro, A; Catastini, P; Cauz, D; Cavalli-Sforza, M; Cerri, A; Cerrito, L; Chang, S H; Chen, Y C; Chertok, M; Chiarelli, G; Chlachidze, G; Chlebana, F; Cho, K; Chokheli, D; Chou, J P; Choudalakis, G; Chuang, S H; Chung, K; Chung, W H; Chung, Y S; Ciobanu, C I; Ciocci, M A; Clark, A; Clark, D; Compostella, G; Convery, M E; Conway, J; Cooper, B; Copic, K; Cordelli, M; Cortiana, G; Crescioli, F; Cuenca Almenar, C; Cuevas, J; Culbertson, R; Cully, J C; Dagenhart, D; Datta, M; Davies, T; de Barbaro, P; De Cecco, S; Deisher, A; De Lentdecker, G; De Lorenzo, G; Dell'Orso, M; Demortier, L; Deng, J; Deninno, M; De Pedis, D; Derwent, P F; Di Giovanni, G P; Dionisi, C; Di Ruzza, B; Dittmann, J R; D'Onofrio, M; Donati, S; Dong, P; Donini, J; Dorigo, T; Dube, S; Efron, J; Erbacher, R; Errede, D; Errede, S; Eusebi, R; Fang, H C; Farrington, S; Fedorko, W T; Feild, R G; Feindt, M; Fernandez, J P; Ferrazza, C; Field, R; Flanagan, G; Forrest, R; Forrester, S; Franklin, M; Freeman, J C; Furic, I; Gallinaro, M; Galyardt, J; Garberson, F; Garcia, J E; Garfinkel, A F; Gerberich, H; Gerdes, D; Giagu, S; Giakoumopolou, V; Giannetti, P; Gibson, K; Gimmell, J L; Ginsburg, C M; Giokaris, N; Giordani, M; Giromini, P; Giunta, M; Glagolev, V; Glenzinski, D; Gold, M; Goldschmidt, N; Golossanov, A; Gomez, G; Gomez-Ceballos, G; Goncharov, M; González, O; Gorelov, I; Goshaw, A T; Goulianos, K; Gresele, A; Grinstein, S; Grosso-Pilcher, C; Grundler, U; Guimaraes da Costa, J; Gunay-Unalan, Z; Haber, C; Hahn, K; Hahn, S R; Halkiadakis, E; Hamilton, A; Han, B-Y; Han, J Y; Handler, R; Happacher, F; Hara, K; Hare, D; Hare, M; Harper, S; Harr, R F; Harris, R M; Hartz, M; Hatakeyama, K; Hauser, J; Hays, C; Heck, M; Heijboer, A; Heinemann, B; Heinrich, J; Henderson, C; Herndon, M; Heuser, J; Hewamanage, S; Hidas, D; Hill, C S; Hirschbuehl, D; Hocker, A; Hou, S; Houlden, M; Hsu, S-C; Huffman, B T; Hughes, R E; Husemann, U; Huston, J; Incandela, J; Introzzi, G; Iori, M; Ivanov, A; Iyutin, B; James, E; Jayatilaka, B; Jeans, D; Jeon, E J; Jindariani, S; Johnson, W; Jones, M; Joo, K K; Jun, S Y; Jung, J E; Junk, T R; Kamon, T; Kar, D; Karchin, P E; Kato, Y; Kephart, R; Kerzel, U; Khotilovich, V; Kilminster, B; Kim, D H; Kim, H S; Kim, J E; Kim, M J; Kim, S B; Kim, S H; Kim, Y K; Kimura, N; Kirsch, L; Klimenko, S; Klute, M; Knuteson, B; Ko, B R; Koay, S A; Kondo, K; Kong, D J; Konigsberg, J; Korytov, A; Kotwal, A V; Kraus, J; Kreps, M; Kroll, J; Krumnack, N; Kruse, M; Krutelyov, V; Kubo, T; Kuhlmann, S E; Kuhr, T; Kulkarni, N P; Kusakabe, Y; Kwang, S; Laasanen, A T; Lai, S; Lami, S; Lammel, S; Lancaster, M; Lander, R L; Lannon, K; Lath, A; Latino, G; Lazzizzera, I; Lecompte, T; Lee, J; Lee, J; Lee, Y J; Lee, S W; Lefèvre, R; Leonardo, N; Leone, S; Levy, S; Lewis, J D; Lin, C; Lin, C S; Linacre, J; Lindgren, M; Lipeles, E; Lister, A; Litvintsev, D O; Liu, T; Lockyer, N S; Loginov, A; Loreti, M; Lovas, L; Lu, R-S; Lucchesi, D; Lueck, J; Luci, C; Lujan, P; Lukens, P; Lungu, G; Lyons, L; Lys, J; Lysak, R; Lytken, E; Mack, P; Macqueen, D; Madrak, R; Maeshima, K; Makhoul, K; Maki, T; Maksimovic, P; Malde, S; Malik, S; Manca, G; Manousakis, A; Margaroli, F; Marino, C; Marino, C P; Martin, A; Martin, M; Martin, V; Martínez, M; Martínez-Ballarín, R; Maruyama, T; Mastrandrea, P; Masubuchi, T; Mattson, M E; Mazzanti, P; McFarland, K S; McIntyre, P; McNulty, R; Mehta, A; Mehtala, P; Menzemer, S; Menzione, A; Merkel, P; Mesropian, C; Messina, A; Miao, T; Miladinovic, N; Miles, J; Miller, R; Mills, C; Milnik, M; Mitra, A; Mitselmakher, G; Miyake, H; Moed, S; Moggi, N; Moon, C S; Moore, R; Morello, M; Movilla Fernandez, P; Mülmenstädt, J; Mukherjee, A; Muller, Th; Mumford, R; Murat, P; Mussini, M; Nachtman, J; Nagai, Y; Nagano, A; Naganoma, J; Nakamura, K; Nakano, I; Napier, A; Necula, V; Neu, C; Neubauer, M S; Nielsen, J; Nodulman, L; Norman, M; Norniella, O; Nurse, E; Oh, S H; Oh, Y D; Oksuzian, I; Okusawa, T; Oldeman, R; Orava, R; Osterberg, K; Pagan Griso, S; Pagliarone, C; Palencia, E; Papadimitriou, V; Papaikonomou, A; Paramonov, A A; Parks, B; Pashapour, S; Patrick, J; Pauletta, G; Paulini, M; Paus, C; Pellett, D E; Penzo, A; Phillips, T J; Piacentino, G; Piedra, J; Pinera, L; Pitts, K; Plager, C; Pondrom, L; Portell, X; Poukhov, O; Pounder, N; Prakoshyn, F; Pronko, A; Proudfoot, J; Ptohos, F; Punzi, G; Pursley, J; Rademacker, J; Rahaman, A; Ramakrishnan, V; Ranjan, N; Redondo, I; Reisert, B; Rekovic, V; Renton, P; Rescigno, M; Richter, S; Rimondi, F; Ristori, L; Robson, A; Rodrigo, T; Rogers, E; Rolli, S; Roser, R; Rossi, M; Rossin, R; Roy, P; Ruiz, A; Russ, J; Rusu, V; Saarikko, H; Safonov, A; Sakumoto, W K; Salamanna, G; Saltó, O; Santi, L; Sarkar, S; Sartori, L; Sato, K; Savoy-Navarro, A; Scheidle, T; Schlabach, P; Schmidt, E E; Schmidt, M A; Schmidt, M P; Schmitt, M; Schwarz, T; Scodellaro, L; Scott, A L; Scribano, A; Scuri, F; Sedov, A; Seidel, S; Seiya, Y; Semenov, A; Sexton-Kennedy, L; Sfyria, A; Shalhout, S Z; Shapiro, M D; Shears, T; Shepard, P F; Sherman, D; Shimojima, M; Shochet, M; Shon, Y; Shreyber, I; Sidoti, A; Sinervo, P; Sisakyan, A; Slaughter, A J; Slaunwhite, J; Sliwa, K; Smith, J R; Snider, F D; Snihur, R; Soderberg, M; Soha, A; Somalwar, S; Sorin, V; Spalding, J; Spinella, F; Spreitzer, T; Squillacioti, P; Stanitzki, M; St Denis, R; Stelzer, B; Stelzer-Chilton, O; Stentz, D; Strologas, J; Stuart, D; Suh, J S; Sukhanov, A; Sun, H; Suslov, I; Suzuki, T; Taffard, A; Takashima, R; Takeuchi, Y; Tanaka, R; Tecchio, M; Teng, P K; Terashi, K; Thom, J; Thompson, A S; Thompson, G A; Thomson, E; Tipton, P; Tiwari, V; Tkaczyk, S; Toback, D; Tokar, S; Tollefson, K; Tomura, T; Tonelli, D; Torre, S; Torretta, D; Tourneur, S; Trischuk, W; Tu, Y; Turini, N; Ukegawa, F; Uozumi, S; Vallecorsa, S; van Remortel, N; Varganov, A; Vataga, E; Vázquez, F; Velev, G; Vellidis, C; Veszpremi, V; Vidal, M; Vidal, R; Vila, I; Vilar, R; Vine, T; Vogel, M; Volobouev, I; Volpi, G; Würthwein, F; Wagner, P; Wagner, R G; Wagner, R L; Wagner-Kuhr, J; Wagner, W; Wakisaka, T; Wallny, R; Wang, S M; Warburton, A; Waters, D; Weinberger, M; Wester, W C; Whitehouse, B; Whiteson, D; Wicklund, A B; Wicklund, E; Williams, G; Williams, H H; Wilson, P; Winer, B L; Wittich, P; Wolbers, S; Wolfe, C; Wright, T; Wu, X; Wynne, S M; Yagil, A; Yamamoto, K; Yamaoka, J; Yamashita, T; Yang, C; Yang, U K; Yang, Y C; Yao, W M; Yeh, G P; Yoh, J; Yorita, K; Yoshida, T; Yu, G B; Yu, I; Yu, S S; Yun, J C; Zanello, L; Zanetti, A; Zaw, I; Zhang, X; Zheng, Y; Zucchelli, S

2008-01-18

We report the observation of the exclusive decay Bs0-->Ds-Ds+ at the 7.5 standard deviation level using 355 pb(-1) of data collected by the CDF II detector in pp collisions at sqrt[s]=1.96 TeV at the Fermilab Tevatron. We measure the relative branching ratio B(Bs0-->Ds-Ds+)/B(B0-->D-Ds+)=1.44(-0.44)(+0.48). Using the world average value for B(B0-->D-Ds+), we find B(Bs0-->Ds-Ds+)=(9.4(-4.2)(+4.4))x10(-3). This provides a lower bound DeltaGammasCP/Gammas>or=2B(Bs0-->Ds-Ds+)>1.2x10(-2) at 95% C.L.

Predicting the Binding Mode of 2-Hydroxypropyl-β-cyclodextrin to Cholesterol by Means of the MD Simulation and the 3D-RISM-KH Theory.

PubMed

Hayashino, Yuji; Sugita, Masatake; Arima, Hidetoshi; Irie, Tetsumi; Kikuchi, Takeshi; Hirata, Fumio

2018-03-19

It has been found that a cyclodextrin derivative, 2-hydroxypropyl-β-cyclodextrin (HPβCD), has reasonable therapeutic effect on Niemann-Pick disease type C, which is caused by abnormal accumulation of unesterified cholesterol and glycolipids in the lysosomes and shortage of esterified cholesterol in other cellular compartments. We study the binding affinity and mode of HPβCD with cholesterol to elucidate the possible mechanism of HPβCD for removing cholesterol from the lysosomes. The dominant binding mode of HPβCD with cholesterol is found based on the molecular dynamics simulation and a statistical mechanics theory of liquids, or the three-dimensional reference interaction site model theory with Kovalenko-Hirata closure relation. We examine the two types of complexes between HPβCD and cholesterol, namely, one-to-one (1:1) and two-to-one (2:1). It is predicted that the 1:1 complex makes two or three types of stable binding mode in solution, in which the βCD ring tends to be located at the edge of the steroid skeleton. For the 2:1 complex, there are four different types of the complex conceivable, depending on the orientation between the two HPβCDs: head-to-head (HH), head-to-tail (HT), tail-to-head (TH), and tail-to-tail (TT). The HT and HH cyclodextrin dimers show higher affinity to cholesterol compared to the other dimers and to all the binding modes of 1:1 complexes. The physical reason why the HT and HH dimers have higher affinity compared to the other complexes is discussed based on the consistency with the 1:1 complex. On the one hand, in case of the HT and HH dimers, the position of each CD in the dimer along the cholesterol chain comes right on or close to one of the positions where a single CD makes a stable complex. On the other hand, one of the CD molecules is located on unstable region along the cholesterol chain, for the case of TH and TT dimers.

Phosphatidylinositol 4,5-Bisphosphate Clusters the Cell Adhesion Molecule CD44 and Assembles a Specific CD44-Ezrin Heterocomplex, as Revealed by Small Angle Neutron Scattering

DOE PAGES

Khajeh, Jahan Ali; Ju, Jeong Ho; Gupta, Yogesh K.; ...

2015-01-08

The cell adhesion molecule CD44 regulates diverse cellular functions, including cell-cell and cell-matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co-localizes with the membrane-cytoskeleton adapter protein Ezrin, which links the CD44 assembled receptor signaling complexes to the cytoskeletal actin and organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered and adopts an autoinhibited conformation, which prevents CD44ct from binding directly to activated Ezrin in solution. Binding to the signaling lipid phosphatidylinositol 4,5-biphosphlate (PIP2) disrupts autoinhibition in CD44ct, and activates CD44ct to associate with Ezrin.more » Further, using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific hetero-tetramer complex of CD44ct with Ezrin. This study reveals a novel autoregulation mechanism in the cytoplasmic tail of CD44 and the role of PIP2 in mediating the assembly of multimeric CD44ct-Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of multimeric PIP2-CD44-Ezrin complexes.« less

Binding affinities of Schiff base Fe(II) complex with BSA and calf-thymus DNA: Spectroscopic investigations and molecular docking analysis

NASA Astrophysics Data System (ADS)

Rudra, Suparna; Dasmandal, Somnath; Patra, Chiranjit; Kundu, Arjama; Mahapatra, Ambikesh

2016-09-01

The binding interaction of a synthesized Schiff base Fe(II) complex with biological macromolecules viz., bovine serum albumin (BSA) and calf thymus(ct)-DNA have been investigated using different spectroscopic techniques coupled with viscosity measurements at physiological pH and 298 K. Regular amendments in emission intensities of BSA upon the action of the complex indicate significant interaction between them, and the binding interaction have been characterized by Stern Volmer plots and thermodynamic binding parameters. On the basis of this quenching technique one binding site with binding constant (Kb = (7.6 ± 0.21) × 105) between complex and protein have been obtained at 298 K. Time-resolved fluorescence studies have also been encountered to understand the mechanism of quenching induced by the complex. Binding affinities of the complex to the fluorophores of BSA namely tryptophan (Trp) and tyrosine (Tyr) have been judged by synchronous fluorescence studies. Secondary structural changes of BSA rooted by the complex has been revealed by CD spectra. On the other hand, hypochromicity of absorption spectra of the complex with the addition of ct-DNA and the gradual reduction in emission intensities of ethidium bromide bound ct-DNA in presence of the complex indicate noticeable interaction between ct-DNA and the complex with the binding constant (4.2 ± 0.11) × 106 M- 1. Life-time measurements have been studied to determine the relative amplitude of binding of the complex to ct-DNA base pairs. Mode of binding interaction of the complex with ct-DNA has been deciphered by viscosity measurements. CD spectra have also been used to understand the changes in ct-DNA structure upon binding with the metal complex. Density functional theory (DFT) and molecular docking analysis have been employed in highlighting the interactive phenomenon and binding location of the complex with the macromolecules.

Cell membrane-bound CD200 signals both via an extracellular domain and following nuclear translocation of a cytoplasmic fragment.

PubMed

Chen, Zhiqi; Kapus, Andras; Khatri, Ismat; Kos, Olha; Zhu, Fang; Gorczynski, Reginald M

2018-06-01

In previous studies we had reported that the immunosuppressive cell membrane bound molecule CD200 is released from the cell following cleavage by matrix metalloproteases, with the released soluble CD200 acting as an immunosuppressant following binding to, and signaling through, its cognate receptor CD200R expressed on target cells. We now show that although the intracellular cytoplasmic tail (CD200 C-tail ) of CD200 has no consensus sites for adapter molecules which might signal the CD200 + cell directly, cleavage of the CD200 C-tail from the membrane region of CD200 by a consensus γ-secretase, leads to nuclear translocation and DNA binding (identified by chromatin immunoprecipitation followed by sequencing, Chip-sequencing) of the CD200 C-tail . Subsequently there occurs an altered expression of a limited number of genes, many of which are transcription factors (TFs) known to be associated with regulation of cell proliferation. Altered expression of these TFs was also prominent following transfection of CD200 + B cell lines and fresh patient CLL cells with a vector construct containing the CD200 C-tail . Artificial transfection of non CD200 + Hek293 cells with this CD200 C-tail construct resulted in altered expression of most of these same genes. Introduction of a siRNA for one of these TFs, POTEA, reversed CD200 C-tail regulation of altered cell proliferation. Copyright © 2018 Elsevier Ltd. All rights reserved.