Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.

PubMed

Zielonka, Stefan; Weber, Niklas; Becker, Stefan; Doerner, Achim; Christmann, Andreas; Christmann, Christine; Uth, Christina; Fritz, Janine; Schäfer, Elena; Steinmann, Björn; Empting, Martin; Ockelmann, Pia; Lierz, Michael; Kolmar, Harald

2014-12-10

A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold. Copyright © 2014 Elsevier B.V. All rights reserved.

The influence of antibody fragment format on phage display based affinity maturation of IgG

PubMed Central

Steinwand, Miriam; Droste, Patrick; Frenzel, Andrè; Hust, Michael; Dübel, Stefan; Schirrmann, Thomas

2014-01-01

Today, most approved therapeutic antibodies are provided as immunoglobulin G (IgG), whereas small recombinant antibody formats are required for in vitro antibody generation and engineering during drug development. Particularly, single chain (sc) antibody fragments like scFv or scFab are well suited for phage display and bacterial expression, but some have been found to lose affinity during conversion into IgG.   In this study, we compared the influence of the antibody format on affinity maturation of the CD30-specific scFv antibody fragment SH313-F9, with the overall objective being improvement of the IgG. The variable genes of SH313-F9 were randomly mutated and then cloned into libraries encoding different recombinant antibody formats, including scFv, Fab, scFabΔC, and FabΔC. All tested antibody formats except Fab allowed functional phage display of the parental antibody SH313-F9, and the corresponding mutated antibody gene libraries allowed isolation of candidates with enhanced CD30 binding. Moreover, scFv and scFabΔC antibody variants retained improved antigen binding after subcloning into the single gene encoded IgG-like formats scFv-Fc or scIgG, but lost affinity after conversion into IgGs. Only affinity maturation using the Fab-like FabΔC format, which does not contain the carboxy terminal cysteines, allowed successful selection of molecules with improved binding that was retained after conversion to IgG. Thus, affinity maturation of IgGs is dependent on the antibody format employed for selection and screening. In this study, only FabΔC resulted in the efficient selection of IgG candidates with higher affinity by combination of Fab-like conformation and improved phage display compared with Fab. PMID:24262918

Development of an affinity-matured humanized anti-epidermal growth factor receptor antibody for cancer immunotherapy.

PubMed

Nakanishi, Takeshi; Maru, Takamitsu; Tahara, Kazuhiro; Sanada, Hideaki; Umetsu, Mitsuo; Asano, Ryutaro; Kumagai, Izumi

2013-02-01

We showed previously that humanization of 528, a murine anti-epidermal growth factor receptor (EGFR) antibody, causes reduced affinity for its target. Here, to improve the affinity of the humanized antibody for use in cancer immunotherapy, we constructed phage display libraries focused on the complementarity-determining regions (CDRs) of the antibody and carried out affinity selection. Two-step selections using libraries constructed in a stepwise manner enabled a 32-fold affinity enhancement of humanized 528 (h528). Thermodynamic analysis of the interactions between the variable domain fragment of h528 (h528Fv) mutants and the soluble extracellular domain of EGFR indicated that the h528Fv mutants obtained from the first selection showed a large increase in negative enthalpy change due to binding, resulting in affinity enhancement. Furthermore, mutants from the second selection showed a decrease in entropy loss, which led to further affinity maturation. These results suggest that a single mutation in the heavy chain variable domain (i.e. Tyr(52) to Trp) enthalpically contributed for overcoming the energetic barrier to the antigen-antibody interaction, which was a major hurdle for the in vitro affinity maturation of h528. We reported previously that the humanized bispecific diabody hEx3 Db, which targets EGFR and CD3, shows strong anti-tumor activity. hEx3 Db mutants, in which the variable domains of h528 were replaced with those of the affinity-enhanced mutants, were prepared and characterized. In a growth inhibition assay of tumor cells, the hEx3 Db mutants showed stronger anti-tumor activity than that of hEx3 Db, suggesting that affinity enhancement of h528Fv enhances the anti-tumor activity of the bispecific diabody.

Random mutagenesis of two complementarity determining region amino acids yields an unexpectedly high frequency of antibodies with increased affinity for both cognate antigen and autoantigen

PubMed Central

1995-01-01

To gain insight into the mechanism and limitations of antibody affinity maturation leading to memory B cell formation, we generated a phage display library of random mutants at heavy chain variable (V) complementarity determining region 2 positions 58 and 59 of an anti-p- azophenylarsonate (Ars) Fab. Single amino acid substitutions at these positions resulting from somatic hypermutation are recurrent products of affinity maturation in vivo. Most of the ex vivo mutants retained specificity for Ars. Among the many mutants displaying high Ars-binding activity, only one contained a position 58 and 59 amino acid combination that has been previously observed among the monoclonal antibodies (mAbs) derived from Ars-immunized mice. Affinity measurements on 14 of the ex vivo mutants with high Ars-binding activity showed that 11 had higher intrinsic affinities for Ars that the wild-type V region. However, nine of these Fabs also bound strongly to denatured DNA, a property neither displayed by the wild-type V region nor observed among the mutants characteristic of in vivo affinity maturation. These data suggest that ex vivo enhancement of mAb affinity via site-directed and random mutagenesis approaches may often lead to a reduction in antibody specificity that could complicate the use of the resulting mAbs for diagnostic and therapeutic applications. Moreover, the data are compatible with a hypothesis proposing that increased specificity for antigen, rather than affinity per se, is the driving force for formation of the memory B cell compartment. PMID:7650481

Enhanced ADCC Activity of Affinity Maturated and Fc-Engineered Mini-Antibodies Directed against the AML Stem Cell Antigen CD96

PubMed Central

Kellner, Christian; Bräutigam, Joachim; Staudinger, Matthias; Schub, Natalie; Peipp, Matthias; Gramatzki, Martin; Humpe, Andreas

2012-01-01

CD96, a cell surface antigen recently described to be preferentially expressed on acute myeloid leukemia (AML) leukemic stem cells (LSC) may represent an interesting target structure for the development of antibody-based therapeutic approaches. The v-regions from the CD96-specific hybridoma TH-111 were isolated and used to generate a CD96-specific single chain fragment of the variable regions (scFv). An affinity maturated variant resulting in 4-fold enhanced CD96-binding was generated by random mutagenesis and stringent selection using phage display. The affinity maturated scFv CD96-S32F was used to generate bivalent mini-antibodies by genetically fusing an IgG1 wild type Fc region or a variant with enhanced CD16a binding. Antibody dependent cell-mediated cytotoxicity (ADCC) experiments revealed that Fc engineering was essential to trigger significant effector cell-mediated lysis when the wild type scFv was used. The mini-antibody variant generated by fusing the affinity-maturated scFv with the optimized Fc variant demonstrated the highest ADCC activity (2.3-fold enhancement in efficacy). In conclusion, our data provide proof of concept that CD96 could serve as a target structure for effector cell-mediated lysis and demonstrate that both enhancing affinity for CD96 and for CD16a resulted in mini-antibodies with the highest cytolytic potential. PMID:22879978

Plasma cell output from germinal centers is regulated by signals from Tfh and stromal cells

PubMed Central

George, Laura A.; Acs, Andreas; Durrett, Russell E.

2018-01-01

Germinal centers (GCs) are the sites where B cells undergo affinity maturation. The regulation of cellular output from the GC is not well understood. Here, we show that from the earliest stages of the GC response, plasmablasts emerge at the GC–T zone interface (GTI). We define two main factors that regulate this process: Tfh-derived IL-21, which supports production of plasmablasts from the GC, and TNFSF13 (APRIL), which is produced by a population of podoplanin+ CD157high fibroblastic reticular cells located in the GTI that are also rich in message for IL-6 and chemokines CXCL12, CCL19, and CCL21. Plasmablasts in the GTI express the APRIL receptor TNFRSF13B (TACI), and blocking TACI interactions specifically reduces the numbers of plasmablasts appearing in the GTI. Plasma cells generated in the GTI may provide an early source of affinity-matured antibodies that may neutralize pathogens or provide feedback regulating GC B cell selection. PMID:29549115

Immunoglobulin D (IgD)-deficient mice reveal an auxiliary receptor function for IgD in antigen-mediated recruitment of B cells

PubMed Central

1993-01-01

To assess the role of immunoglobulin D (IgD) in vivo we generated IgD- deficient mice by gene targeting and studied B cell development and function in the absence of IgD expression. In the mutant animals, conventional and CD5-positive (B1) B cells are present in normal numbers, and the expression of the surface markers CD22 and CD23 in the compartment of conventional B cells indicates acquisition of a mature phenotype. As in wild-type animals, most of the peripheral B cells are resting cells. The IgD-deficient mice respond well to T cell- independent and -dependent antigens. However, in heterozygous mutant animals, B cells expressing the wild type IgH locus are overrepresented in the peripheral B cell pool, and T cell-dependent IgG1 responses are further dominated by B cells expressing the wild-type allele. Similarly, in homozygous mutant (IgD-deficient) animals, affinity maturation is delayed in the early primary response compared to control animals, although the mutants are capable of generating high affinity B cell memory. Thus, rather than being involved in major regulatory processes as had been suggested, IgD seems to function as an antigen receptor optimized for efficient recruitment of B cells into antigen- driven responses. The IgD-mediated acceleration of affinity maturation in the early phase of the T cell-dependent primary response may confer to the animal a critical advantage in the defense against pathogens. PMID:8418208

First molecular and biochemical analysis of in vivo affinity maturation in an ectothermic vertebrate.

PubMed

Dooley, Helen; Stanfield, Robyn L; Brady, Rebecca A; Flajnik, Martin F

2006-02-07

The cartilaginous fish are the oldest phylogenetic group in which Igs have been found. Sharks produce a unique Ig isotype, IgNAR, a heavy-chain homodimer that does not associate with light chains. Instead, the variable (V) regions of IgNAR bind antigen as soluble single domains. Our group has shown that IgNAR plays an integral part in the humoral response of nurse sharks (Ginglymostoma cirratum) upon antigen challenge. Here, we generated phage-displayed libraries of IgNAR V regions from an immunized animal and found a family of clones derived from the same rearrangement event but differentially mutated during expansion. Because of the cluster organization of shark Ig genes and the paucicopy nature of IgNAR, we were able to construct the putative ancestor of this family. By studying mutations in the context of clone affinities, we found evidence that affinity maturation occurs for this isotype. Subsequently, we were able to identify mutations important in the affinity improvement of this family. Because the family clones were all obtained after immunization, they provide insight into the in vivo maturation mechanisms, in general, and for single-domain antibody fragments.

pMHC affinity controls duration of CD8+ T cell–DC interactions and imprints timing of effector differentiation versus expansion

PubMed Central

Sharpe, James; Zehn, Dietmar; Kreutzfeldt, Mario

2016-01-01

During adaptive immune responses, CD8+ T cells with low TCR affinities are released early into the circulation before high-affinity clones become dominant at later time points. How functional avidity maturation is orchestrated in lymphoid tissue and how low-affinity cells contribute to host protection remains unclear. In this study, we used intravital imaging of reactive lymph nodes (LNs) to show that T cells rapidly attached to dendritic cells irrespective of TCR affinity, whereas one day later, the duration of these stable interactions ceased progressively with lowering peptide major histocompatibility complex (pMHC) affinity. This correlated inversely BATF (basic leucine zipper transcription factor, ATF-like) and IRF4 (interferon-regulated factor 4) induction and timing of effector differentiation, as low affinity–primed T cells acquired cytotoxic activity earlier than high affinity–primed ones. After activation, low-affinity effector CD8+ T cells accumulated at efferent lymphatic vessels for egress, whereas high affinity–stimulated CD8+ T cells moved to interfollicular regions in a CXCR3-dependent manner for sustained pMHC stimulation and prolonged expansion. The early release of low-affinity effector T cells led to rapid target cell elimination outside reactive LNs. Our data provide a model for affinity-dependent spatiotemporal orchestration of CD8+ T cell activation inside LNs leading to functional avidity maturation and uncover a role for low-affinity effector T cells during early microbial containment. PMID:27799622

Selection and maturation of antibodies by phage display through fusion to pIX.

PubMed

Tornetta, Mark; Reddy, Ramachandra; Wheeler, John C

2012-09-01

Antibody discovery and optimization by M13 phage display have evolved significantly over the past twenty years. Multiple methods of antibody display and selection have been developed - direct display on pIII or indirect display through a Cysteine disulfide linkage or a coiled-coil adapter protein. Here we describe display of Fab libraries on the smaller pIX protein at the opposite end of the virion and its application to discovery of novel antibodies from naive libraries. Antibody selection based on pIX-mediated display produces results comparable to other in vitro methods and uses an efficient direct infection of antigen-bound phages, eliminating any chemical dissociation step(s). Additionally, some evidence suggests that pIX-mediated display can be more efficient than pIII-mediated display in affinity selections. Functional assessment of phage-derived antibodies can be hindered by insufficient affinities or lack of epitopic diversity. Here we describe an approach to managing primary hits from our Fab phage libraries into epitope bins and subsequent high-throughput maturation of clones to isolate epitope- and sequence-diverse panels of high affinity binders. Use of the Octet biosensor was done to examine Fab binding in a facile label-free method and determine epitope competition groups. A receptor extracellular domain and chemokine were subjected to this method of binning and affinity maturation. Parental clones demonstrated improvement in affinity from 1-100nM to 10-500pM. Copyright © 2012 Elsevier Inc. All rights reserved.

The challenges of modelling antibody repertoire dynamics in HIV infection

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

Luo, Shishi; Perelson, Alan S.

Antibody affinity maturation by somatic hypermutation of B-cell immunoglobulin variable region genes has been studied for decades in various model systems using well-defined antigens. While much is known about the molecular details of the process, our understanding of the selective forces that generate affinity maturation are less well developed, particularly in the case of a co-evolving pathogen such as HIV. Despite this gap in understanding, high-throughput antibody sequence data are increasingly being collected to investigate the evolutionary trajectories of antibody lineages in HIV-infected individuals. Here, we review what is known in controlled experimental systems about the mechanisms underlying antibody selectionmore » and compare this to the observed temporal patterns of antibody evolution in HIV infection. In addition, we describe how our current understanding of antibody selection mechanisms leaves questions about antibody dynamics in HIV infection unanswered. Without a mechanistic understanding of antibody selection in the context of a co-evolving viral population, modelling and analysis of antibody sequences in HIV-infected individuals will be limited in their interpretation and predictive ability.« less

The challenges of modelling antibody repertoire dynamics in HIV infection

DOE PAGES

Luo, Shishi; Perelson, Alan S.

2015-07-20

Antibody affinity maturation by somatic hypermutation of B-cell immunoglobulin variable region genes has been studied for decades in various model systems using well-defined antigens. While much is known about the molecular details of the process, our understanding of the selective forces that generate affinity maturation are less well developed, particularly in the case of a co-evolving pathogen such as HIV. Despite this gap in understanding, high-throughput antibody sequence data are increasingly being collected to investigate the evolutionary trajectories of antibody lineages in HIV-infected individuals. Here, we review what is known in controlled experimental systems about the mechanisms underlying antibody selectionmore » and compare this to the observed temporal patterns of antibody evolution in HIV infection. In addition, we describe how our current understanding of antibody selection mechanisms leaves questions about antibody dynamics in HIV infection unanswered. Without a mechanistic understanding of antibody selection in the context of a co-evolving viral population, modelling and analysis of antibody sequences in HIV-infected individuals will be limited in their interpretation and predictive ability.« less

Inhibiting HER3-mediated tumor cell growth with affibody molecules engineered to low picomolar affinity by position-directed error-prone PCR-like diversification.

PubMed

Malm, Magdalena; Kronqvist, Nina; Lindberg, Hanna; Gudmundsdotter, Lindvi; Bass, Tarek; Frejd, Fredrik Y; Höidén-Guthenberg, Ingmarie; Varasteh, Zohreh; Orlova, Anna; Tolmachev, Vladimir; Ståhl, Stefan; Löfblom, John

2013-01-01

The HER3 receptor is implicated in the progression of various cancers as well as in resistance to several currently used drugs, and is hence a potential target for development of new therapies. We have previously generated Affibody molecules that inhibit heregulin-induced signaling of the HER3 pathways. The aim of this study was to improve the affinity of the binders to hopefully increase receptor inhibition efficacy and enable a high receptor-mediated uptake in tumors. We explored a novel strategy for affinity maturation of Affibody molecules that is based on alanine scanning followed by design of library diversification to mimic the result from an error-prone PCR reaction, but with full control over mutated positions and thus less biases. Using bacterial surface display and flow-cytometric sorting of the maturation library, the affinity for HER3 was improved more than 30-fold down to 21 pM. The affinity is among the higher that has been reported for Affibody molecules and we believe that the maturation strategy should be generally applicable for improvement of affinity proteins. The new binders also demonstrated an improved thermal stability as well as complete refolding after denaturation. Moreover, inhibition of ligand-induced proliferation of HER3-positive breast cancer cells was improved more than two orders of magnitude compared to the previously best-performing clone. Radiolabeled Affibody molecules showed specific targeting of a number of HER3-positive cell lines in vitro as well as targeting of HER3 in in vivo mouse models and represent promising candidates for future development of targeted therapies and diagnostics.

Natural and man-made V-gene repertoires for antibody discovery

PubMed Central

Finlay, William J. J.; Almagro, Juan C.

2012-01-01

Antibodies are the fastest-growing segment of the biologics market. The success of antibody-based drugs resides in their exquisite specificity, high potency, stability, solubility, safety, and relatively inexpensive manufacturing process in comparison with other biologics. We outline here the structural studies and fundamental principles that define how antibodies interact with diverse targets. We also describe the antibody repertoires and affinity maturation mechanisms of humans, mice, and chickens, plus the use of novel single-domain antibodies in camelids and sharks. These species all utilize diverse evolutionary solutions to generate specific and high affinity antibodies and illustrate the plasticity of natural antibody repertoires. In addition, we discuss the multiple variations of man-made antibody repertoires designed and validated in the last two decades, which have served as tools to explore how the size, diversity, and composition of a repertoire impact the antibody discovery process. PMID:23162556

Sequence intrinsic somatic mutation mechanisms contribute to affinity maturation of VRC01-class HIV-1 broadly neutralizing antibodies

PubMed Central

Hwang, Joyce K.; Wang, Chong; Du, Zhou; Meyers, Robin M.; Kepler, Thomas B.; Neuberg, Donna; Kwong, Peter D.; Mascola, John R.; Joyce, M. Gordon; Bonsignori, Mattia; Haynes, Barton F.; Yeap, Leng-Siew; Alt, Frederick W.

2017-01-01

Variable regions of Ig chains provide the antigen recognition portion of B-cell receptors and derivative antibodies. Ig heavy-chain variable region exons are assembled developmentally from V, D, J gene segments. Each variable region contains three antigen-contacting complementarity-determining regions (CDRs), with CDR1 and CDR2 encoded by the V segment and CDR3 encoded by the V(D)J junction region. Antigen-stimulated germinal center (GC) B cells undergo somatic hypermutation (SHM) of V(D)J exons followed by selection for SHMs that increase antigen-binding affinity. Some HIV-1–infected human subjects develop broadly neutralizing antibodies (bnAbs), such as the potent VRC01-class bnAbs, that neutralize diverse HIV-1 strains. Mature VRC01-class bnAbs, including VRC-PG04, accumulate very high SHM levels, a property that hinders development of vaccine strategies to elicit them. Because many VRC01-class bnAb SHMs are not required for broad neutralization, high overall SHM may be required to achieve certain functional SHMs. To elucidate such requirements, we used a V(D)J passenger allele system to assay, in mouse GC B cells, sequence-intrinsic SHM-targeting rates of nucleotides across substrates representing maturation stages of human VRC-PG04. We identify rate-limiting SHM positions for VRC-PG04 maturation, as well as SHM hotspots and intrinsically frequent deletions associated with SHM. We find that mature VRC-PG04 has low SHM capability due to hotspot saturation but also demonstrate that generation of new SHM hotspots and saturation of existing hotspot regions (e.g., CDR3) does not majorly influence intrinsic SHM in unmutated portions of VRC-PG04 progenitor sequences. We discuss implications of our findings for bnAb affinity maturation mechanisms. PMID:28747530

Maturation of Shark Single-Domain (IgNAR) Antibodies: Evidence for Induced-Fit Binding

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

Stanfield, R.L.; Dooley, H.; Verdino, P.

2007-07-13

Sharks express an unusual heavy-chain isotype called IgNAR, whose variable regions bind antigen as independent soluble domains. To further probe affinity maturation of the IgNAR response, we structurally characterized the germline and somatically matured versions of a type II variable (V) region, both in the presence and absence of its antigen, hen egg-white lysozyme. Despite a disulfide bond linking complementarity determining regions (CDRs) 1 and 3, both germline and somatically matured V regions displayed significant structural changes in these CDRs upon complex formation with antigen. Somatic mutations in the IgNAR V region serve to increase the number of contacts withmore » antigen, as reflected by a tenfold increase in affinity, and one of these mutations appears to stabilize the CDR3 region. In addition, a residue in the HV4 loop plays an important role in antibody-antigen interaction, consistent with the high rate of somatic mutations in this non-CDR loop.« less

Mutation in Fas Ligand Impairs Maturation of Thymocytes Bearing Moderate Affinity T Cell Receptors

PubMed Central

Boursalian, Tamar E.; Fink, Pamela J.

2003-01-01

Fas ligand, best known as a death-inducer, is also a costimulatory molecule required for maximal proliferation of mature antigen-specific CD4+ and CD8+ T cells. We now extend the role of Fas ligand by showing that it can also influence thymocyte development. T cell maturation in some, but not all, strains of TCR transgenic mice is severely impaired in thymocytes expressing mutant Fas ligand incapable of interacting with Fas. Mutant Fas ligand inhibits neither negative selection nor death by neglect. Instead, it appears to modulate positive selection of thymocytes expressing both class I– and class II–restricted T cell receptors of moderate affinity for their positively selecting ligands. Fas ligand is therefore an inducer of death, a costimulator of peripheral T cell activation, and an accessory molecule in positive selection. PMID:12860933

Highly sensitive and unbiased approach for elucidating antibody repertoires

PubMed Central

Lin, Sherry G.; Ba, Zhaoqing; Du, Zhou; Zhang, Yu; Hu, Jiazhi; Alt, Frederick W.

2016-01-01

Developing B lymphocytes undergo V(D)J recombination to assemble germ-line V, D, and J gene segments into exons that encode the antigen-binding variable region of Ig heavy (H) and light (L) chains. IgH and IgL chains associate to form the B-cell receptor (BCR), which, upon antigen binding, activates B cells to secrete BCR as an antibody. Each of the huge number of clonally independent B cells expresses a unique set of IgH and IgL variable regions. The ability of V(D)J recombination to generate vast primary B-cell repertoires results from a combinatorial assortment of large numbers of different V, D, and J segments, coupled with diversification of the junctions between them to generate the complementary determining region 3 (CDR3) for antigen contact. Approaches to evaluate in depth the content of primary antibody repertoires and, ultimately, to study how they are further molded by secondary mutation and affinity maturation processes are of great importance to the B-cell development, vaccine, and antibody fields. We now describe an unbiased, sensitive, and readily accessible assay, referred to as high-throughput genome-wide translocation sequencing-adapted repertoire sequencing (HTGTS-Rep-seq), to quantify antibody repertoires. HTGTS-Rep-seq quantitatively identifies the vast majority of IgH and IgL V(D)J exons, including their unique CDR3 sequences, from progenitor and mature mouse B lineage cells via the use of specific J primers. HTGTS-Rep-seq also accurately quantifies DJH intermediates and V(D)J exons in either productive or nonproductive configurations. HTGTS-Rep-seq should be useful for studies of human samples, including clonal B-cell expansions, and also for following antibody affinity maturation processes. PMID:27354528

Isolation of high-affinity, neutralizing anti-idiotype antibodies by phage and ribosome display for application in immunogenicity and pharmacokinetic analyses.

PubMed

Chin, Stacey E; Ferraro, Franco; Groves, Maria; Liang, Meina; Vaughan, Tristan J; Dobson, Claire L

2015-01-01

Anti-idiotype antibodies against a therapeutic antibody are key reagents for the development of immunogenicity and pharmacokinetic (PK) assays during pre-clinical and clinical development. Here we have used a combination of phage and ribosome display to isolate a panel of monoclonal anti-idiotype antibodies with sub-nanomolar affinity and high specificity to a human anti-IgE monoclonal antibody. Anti-idiotype antibodies were enriched from scFv libraries using phage display, and a biochemical epitope competition assay was used to identify anti-idiotypes which neutralized IgE binding, which was essential for the intended use of the anti-idiotypes as positive controls in neutralizing anti-drug antibody (Nab) assays. The phage display-derived anti-idiotype antibodies were rapidly affinity-matured using a random point mutagenesis approach in ribosome display. Ten anti-idiotype antibodies with improved neutralizing activity relative to the parent antibodies displayed sub-nanomolar affinity for the anti-IgE antibody, representing up to 20-fold improvements in affinity from just two rounds of affinity-based selection. The optimized anti-idiotype antibodies retained the specificity of the parent antibodies, and importantly, were fit for purpose for use in PK and anti-drug antibody (ADA) assays. The approach we describe here for generation of anti-idiotype antibodies to an anti-IgE antibody is generically applicable for the rapid isolation and affinity maturation of anti-idiotype antibodies to any antibody-based drug candidate. Copyright © 2014 Elsevier B.V. All rights reserved.

The WAVE2 complex regulates T cell receptor signaling to integrins via Abl- and CrkL-C3G-mediated activation of Rap1.

PubMed

Nolz, Jeffrey C; Nacusi, Lucas P; Segovis, Colin M; Medeiros, Ricardo B; Mitchell, Jason S; Shimizu, Yoji; Billadeau, Daniel D

2008-09-22

WAVE2 regulates T cell receptor (TCR)-stimulated actin cytoskeletal dynamics leading to both integrin clustering and affinity maturation. Although WAVE2 mediates integrin affinity maturation by recruiting vinculin and talin to the immunological synapse in an Arp2/3-dependent manner, the mechanism by which it regulates integrin clustering is unclear. We show that the Abl tyrosine kinase associates with the WAVE2 complex and TCR ligation induces WAVE2-dependent membrane recruitment of Abl. Furthermore, we show that WAVE2 regulates TCR-mediated activation of the integrin regulatory guanosine triphosphatase Rap1 via the recruitment and activation of the CrkL-C3G exchange complex. Moreover, we demonstrate that although Abl does not regulate the recruitment of CrkL-C3G into the membrane, it does affect the tyrosine phosphorylation of C3G, which is required for its guanine nucleotide exchange factor activity toward Rap1. This signaling node regulates not only TCR-stimulated integrin clustering but also affinity maturation. These findings identify a previously unknown mechanism by which the WAVE2 complex regulates TCR signaling to Rap1 and integrin activation.

The WAVE2 complex regulates T cell receptor signaling to integrins via Abl- and CrkL–C3G-mediated activation of Rap1

PubMed Central

Nolz, Jeffrey C.; Nacusi, Lucas P.; Segovis, Colin M.; Medeiros, Ricardo B.; Mitchell, Jason S.; Shimizu, Yoji; Billadeau, Daniel D.

2008-01-01

WAVE2 regulates T cell receptor (TCR)–stimulated actin cytoskeletal dynamics leading to both integrin clustering and affinity maturation. Although WAVE2 mediates integrin affinity maturation by recruiting vinculin and talin to the immunological synapse in an Arp2/3-dependent manner, the mechanism by which it regulates integrin clustering is unclear. We show that the Abl tyrosine kinase associates with the WAVE2 complex and TCR ligation induces WAVE2-dependent membrane recruitment of Abl. Furthermore, we show that WAVE2 regulates TCR-mediated activation of the integrin regulatory guanosine triphosphatase Rap1 via the recruitment and activation of the CrkL–C3G exchange complex. Moreover, we demonstrate that although Abl does not regulate the recruitment of CrkL–C3G into the membrane, it does affect the tyrosine phosphorylation of C3G, which is required for its guanine nucleotide exchange factor activity toward Rap1. This signaling node regulates not only TCR-stimulated integrin clustering but also affinity maturation. These findings identify a previously unknown mechanism by which the WAVE2 complex regulates TCR signaling to Rap1 and integrin activation. PMID:18809728

Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

PubMed Central

Nilvebrant, Johan; Åstrand, Mikael; Georgieva-Kotseva, Maria; Björnmalm, Mattias; Löfblom, John; Hober, Sophia

2014-01-01

The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein. PMID:25089830

Developing recombinant antibodies for biomarker detection

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

Baird, Cheryl L.; Fischer, Christopher J.; Pefaur, Noah B.

2010-10-01

Monoclonal antibodies (mAbs) have an essential role in biomarker validation and diagnostic assays. A barrier to pursuing these applications is the reliance on immunization and hybridomas to produce mAbs, which is time-consuming and may not yield the desired mAb. We recommend a process flow for affinity reagent production that utilizes combinatorial protein display systems (eg, yeast surface display or phage display) rather than hybridomas. These systems link a selectable phenotype-binding conferred by an antibody fragment-with a means for recovering the encoding gene. Recombinant libraries obtained from immunizations can produce high-affinity antibodies (<10 nM) more quickly than other methods. Non-immune librariesmore » provide an alternate route when immunizations are not possible, or when suitable mAbs are not recovered from an immune library. Directed molecular evolution (DME) is an integral part of optimizing mAbs obtained from combinatorial protein display, but can also be used on hybridoma-derived mAbs. Variants can easily be obtained and screened to increase the affinity of the parent mAb (affinity maturation). We discuss examples where DME has been used to tailor affinity reagents to specific applications. Combinatorial protein display also provides an accessible method for identifying antibody pairs, which are necessary for sandwich-type diagnostic assays.« less

Subcellular distribution of Lck during CD4 T-cell maturation in the thymic medulla regulates the T-cell activation threshold.

PubMed

Stephen, Tom Li; Wilson, Bridget S; Laufer, Terri M

2012-05-08

Mature peripheral T cells respond to foreign but not to self-antigens. During development in the thymus, deletion of high-affinity self-reactive immature thymocytes contributes to tolerance of mature T cells. However, double-positive thymocytes are positively selected to survive if they respond to self-peptide-MHC complexes; thus, there must be mechanisms to prevent overt reactivity to those same complexes in the periphery. "Developmental tuning" is the active process through which T-cell receptor (TCR)-associated signaling pathways of single-positive (SP) thymocytes are attenuated to respond appropriately to self-peptide-MHC complexes in the periphery. We previously showed that MHC class II expression in the thymic medulla was necessary to tune CD4(+) SP (CD4 SP) thymocytes. CD4 SP thymocytes from mice lacking medullary MHC class II expression had inappropriately enhanced proximal TCR signaling to low-affinity self-ligands that was associated with altered cellular distribution of the tyrosine kinase Lck. Now, we report that activation of both tuned and untuned CD4 SP thymocytes is Lck-dependent. Untuned CD4 SP cells contain a pool of Lck with increased basal phosphorylation that is not associated with the CD4 coreceptor. Phosphorylation of this pool of Lck decreases with tuning. Immunogold transmission electron microscopy of membrane sheets permitted direct visualization of Lck. In the absence of tuning, a significant proportion of Lck and the TCR subunit CD3ζ are expressed on the same protein island; this close association of Lck and the TCR probably explains the enhanced activation of untuned CD4 SP cells. Thus, changes in membrane topography during thymic maturation determine the set point for TCR responsiveness.

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

Daud, A.I.; Bumpus, F.M.; Husain, A.

Ovarian angiotensin I (Ang I)-converting enzyme (ACE), estimated by the specific binding of the ACE inhibitor (125I)iodo-MK-351A, is localized on multiple ovarian structures, including follicular granulosa cells, corpora lutea, terminal epithelium, and ovarian blood vessels, but total ovarian ACE does not display a cyclic pattern of variation during the rat estrous cycle. We have previously shown that ACE is localized on the granulosa cell layer of a subpopulation of rat ovarian follicles. Our present study shows that ovarian granulosa cells contain high affinity (binding site affinity (Kd), approximately 90 pM) and low capacity (binding site density (Bmax), approximately 12 fmol/2.5more » X 10(5) cells) (125I)iodo-MK-351A-binding sites and convert (125I)iodo-Ang I to (125I)iodo-Ang II (greater than 85% of this conversion was inhibited by the ACE inhibitor captopril). Throughout the rat estrous cycle, 94-100% of developing follicles and 89-96% of atretic follicles contained high levels of ACE; however, ACE was either not observed or its levels were very low in preovulatory follicles. These findings indicate the presence of high levels of biologically active ACE on the surface of granulosa cells and suggest a potential role for follicular ACE in early stages of follicular maturation and atresia. Although ACE is known to process a variety of peptides found within the ovary, and these peptides may have opposing effects on follicular maturation, we attempted to define the cumulative effect of ACE inhibition on follicular maturation.« less

Aberrant antibody affinity selection in SHIP-deficient B cells.

PubMed

Leung, Wai-Hang; Tarasenko, Tatiana; Biesova, Zuzana; Kole, Hemanta; Walsh, Elizabeth R; Bolland, Silvia

2013-02-01

The strength of the Ag receptor signal influences development and negative selection of B cells, and it might also affect B-cell survival and selection in the GC. Here, we have used mice with B-cell-specific deletion of the 5'-inositol phosphatase SHIP as a model to study affinity selection in cells that are hyperresponsive to Ag and cytokine receptor stimulation. In the absence of SHIP, B cells have lower thresholds for Ag- and interferon (IFN)-induced activation, resulting in augmented negative selection in the BM and enhanced B-cell maturation in the periphery. Despite a tendency to spontaneously downregulate surface IgM expression, SHIP deficiency does not alter anergy induction in response to soluble hen-egg lysozyme Ag in the MDA4 transgenic model. SHIP-deficient B cells spontaneously produce isotype-switched antibodies; however, they are poor responders in immunization and infection models. While SHIP-deficient B cells form GCs and undergo mutation, they are not properly selected for high-affinity antibodies. These results illustrate the importance of negative regulation of B-cell responses, as lower thresholds for B-cell activation promote survival of low affinity and deleterious receptors to the detriment of optimal Ab affinity maturation. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Using Genotype Abundance to Improve Phylogenetic Inference

PubMed Central

Mesin, Luka; Victora, Gabriel D; Minin, Vladimir N; Matsen, Frederick A

2018-01-01

Abstract Modern biological techniques enable very dense genetic sampling of unfolding evolutionary histories, and thus frequently sample some genotypes multiple times. This motivates strategies to incorporate genotype abundance information in phylogenetic inference. In this article, we synthesize a stochastic process model with standard sequence-based phylogenetic optimality, and show that tree estimation is substantially improved by doing so. Our method is validated with extensive simulations and an experimental single-cell lineage tracing study of germinal center B cell receptor affinity maturation. PMID:29474671

A fragment of alpha-actinin promotes monocyte/macrophage maturation in vitro.

PubMed

Luikart, S; Wahl, D; Hinkel, T; Masri, M; Oegema, T

1999-02-01

Conditioned media (CM) from cultures of HL-60 myeloid leukemia cells grown on extracellular bone marrow matrix contains a factor that induces macrophage-like maturation of HL-60 cells. This factor was purified from the CM of HL-60 cells grown on bone marrow stroma by ammonium sulfate precipitation, then sequential chromatography on DEAE, affi-gel blue affinity, gel exclusion, and wheat germ affinity columns, followed by C-4 reverse phase HPLC, and SDS-PAGE. The maturation promoting activity of the CM was identified in a single 31 kD protein. Amino acid sequence analysis of four internal tryptic peptides of this protein confirmed significant homology with amino acid residues 48-60, 138-147, 215-220, and 221-236 of human cytoskeletal alpha-actinin. An immunoaffinity purified rabbit polyclonal anti-chicken alpha-actinin inhibited the activity of HL-60 conditioned media. A 27 kD amino-terminal fragment of alpha-actinin produced by thermolysin digestion of chicken gizzard alpha-actinin, but not intact alpha-actinin, had maturation promoting activity on several cell types, including blood monocytes, as measured by lysozyme secretion and tartrate-resistant acid phosphatase staining. We conclude that an extracellular alpha-actinin fragment can promote monocyte/macrophage maturation. This represents the first example of a fragment of a cytoskeletal component, which may be released during tissue remodeling and repair, playing a role in phagocyte maturation.

Functional integrins from normal and glycosylation-deficient baby hamster kidney cells. Terminal processing of asparagine-linked oligosaccharides is not correlated with fibronectin-binding activity.

PubMed

Koyama, T; Hughes, R C

1992-12-25

We have examined the properties of the alpha 5 beta 1 integrin of baby hamster kidney (BHK) cells, a ricin-resistant variant Ric14 lacking N-acetylglucosaminyl transferase I, and hence unable to complete assembly of hybrid- or complex-type N-glycans, and BHK cells treated with 1-deoxymannojirimycin (dMM), an inhibitor of Golgi mannosidases involved in the initial processing of N-glycan precursors. Comparable amounts of alpha 5 beta 1 integrin were isolated from these cells by chromatography of detergent extracts on a fibronectin cell-binding fragment affinity column and elution with EDTA. The alpha 5 beta 1 integrin obtained from normal BHK cells by fibronectin affinity chromatography contained mainly endoglycosidase H-resistant oligosaccharides, whereas in RicR14 cells or dMM-treated BHK cells these were entirely endoglycosidase H-sensitive. Analysis of lactoperoxidase labeled or long term biosynthetically 35S-labeled proteins from cultures of normal or glycosylation deficient cells showed similar steady state levels of alpha 5 beta 1 integrin and expression at the cell surface. Pulse-chase experiments in normal BHK cells showed rapid conversion of the alpha 5 subunit into a mature form containing oligosaccharides resistant to endoglycosidase H and slower maturation of a precursor beta 1 subunit, as in other cell types. In Ric14 cells the precursor beta 1 subunit was found to carry glycans larger than the fully processed Man5GlcNAc2 glycan of the mature subunit, indicating that the bulk precursor pool had not been translocated into the cis-Golgi compartment containing mannosidase I. We conclude that in BHK cells terminal oligosaccharide processing of alpha 5 beta 1 integrin subunits is not required for dimer formation, surface expression, and fibronectin binding, and that expression of the glycosylation defect of Ric14 cells on the alpha 5 beta 1 integrin does not account for the reduced adhesiveness of these cells on fibronectin compared with normal and dMM-treated BHK cells.

Imprinting the Fate of Antigen-Reactive B Cells through the Affinity of the B Cell Receptor

PubMed Central

O'Connor, Brian P.; Vogel, Laura A.; Zhang, Weijun; Loo, William; Shnider, Danielle; Lind, Evan F.; Ratliff, Michelle; Noelle, Randolph J.; Erickson, Loren D.

2010-01-01

Long-lived plasma cells (PCs) and memory B cells (Bmem) constitute the cellular components of enduring humoral immunity, whereas short-lived PCs that rapidly produce Ig correspond to the host's need for immediate protection against pathogens. In this study we show that the innate affinity of the BCR for Ag imprints upon naive B cells their differentiation fate to become short-or long-lived PCs and Bmem. Using BCR transgenic mice with varying affinities for Ag, naive B cells with high affinity lose their capacity to form germinal centers (GCs), develop neither Bmem nor long-lived PCs, and are destined to a short-lived PC fate. Moderate affinity interactions result in hastened GC responses, and differentiation to long-lived PCs, but Bmem remain extinct. In contrast, lower affinity interactions show tempered GCs, producing Bmem and affinity-matured, long-lived PCs. Thus, a continuum of elementary to comprehensive humoral immune responses exists that is controlled by inherent BCR affinity. PMID:17114443

Transgenic mouse strains as platforms for the successful discovery and development of human therapeutic monoclonal antibodies.

PubMed

Green, Larry L

2014-03-01

Transgenic mice have yielded seven of the ten currently-approved human antibody drugs, making them the most successful platform for the discovery of fully human antibody therapeutics. The use of the in vivo immune system helps drive this success by taking advantage of the natural selection process that produces antibodies with desirable characteristics. Appropriately genetically-engineered mice act as robust engines for the generation of diverse repertoires of affinity- matured fully human variable regions with intrinsic properties necessary for successful antibody drug development including high potency, specificity, manufacturability, solubility and low risk of immunogenicity. A broad range of mAb drug targets are addressable in these mice, comprising both secreted and transmembrane targets, including membrane multi-spanning targets, as well as human target antigens that share high sequence identity with their mouse orthologue. Transgenic mice can routinely yield antibodies with sub-nanomolar binding affinity for their antigen, with lead candidate mAbs frequently possessing affinities for binding to their target of less than 100 picomolar, without requiring any ex vivo affinity optimization. While the originator transgenic mice platforms are no longer broadly available, a new generation of transgenic platforms is in development for discovery of the next wave of human therapeutic antibodies.

Thinking about Winnicott and the origins of the self.

PubMed

Gaddini, Renata

2004-01-01

Reading Rodman's "Winnicott: Life and Work" has induced some considerations in the author who for years has shared Winnicott's "research analysis" in the field of potential space. Among these considerations is the rarely remarked affinity between Erikson and Winnicott in their view of human nature as well as in their seeing basic trust as the essence of good growth. For Winnicott particularly maternal functions have great maturational value, facilitating the process which takes infants from their first sensory experiences to mentalization, where father will exist in reality. The significance of illusion at different maturational stages is briefly considered as opposed to reality. To the latter a distant origin (possibly in foetal movements, which in utero meet resistance) has been attributed by Winnicott as mentioned in one of his last letter. "Meeting resistance one feels real" was one of his sayings (Gaddini, 2003).

Optimized Expression and Purification for High-Activity Preparations of Algal [FeFe]-Hydrogenase

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

Yacoby, I.; Tegler, L. T.; Pochekailov, S.

2012-04-01

Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies. We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus,more » led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin. In combination, this process resulted in HydA1 purification yields of 5 mg L{sup -1} of culture from E. coli with specific activities of 1000 U (U = 1 {micro}mol hydrogen evolved mg{sup -1} min{sup -1}). The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required.« less

Variable domain glycosylation of ACPA-IgG: A missing link in the maturation of the ACPA response?

PubMed

Kempers, Ayla C; Hafkenscheid, Lise; Scherer, Hans Ulrich; Toes, René E M

2018-01-01

Anti-citrullinated Protein Antibodies (ACPA) are excellent markers for Rheumatoid arthritis (RA) and are postulated to have a pathogenic role in the disease process. A multistep model for the evolution of the ACPA response in RA was proposed in which an initial break of tolerance causes, as "first hit", "silent" production of ACPA without any clinical symptoms. The model further proposes that the ACPA immune response matures upon a certain (unknown) trigger, a "second hit", which leads to epitope spreading, an increase in ACPA titres and extended isotype usage before clinical RA manifestations. These occurrences are indicative of an expansion of the citrulline-specific B cell response, though ACPA remain of low avidity even in established disease. This persistence of low avidity is puzzling, as the typical signs of maturation of the immune response seem to be uncoupled from the classical process of affinity maturation. In fact, it suggests that B cells expressing ACPA could bypass selection mechanisms that otherwise control the expansion of auto-reactive B cells. In the established, chronic phase, we recently found that ACPA-IgG are extensively glycosylated in the variable (Fab) domain. More than 90% of ACPA-IgG molecules carry Fab glycans that are highly sialylated. This molecular feature is striking and may provide a missing link in our understanding of the maturation of the ACPA immune response. This review, therefore, describes the current knowledge about ACPA Fab glycosylation in the pathogenesis of RA. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

T follicular helper cell differentiation, function, and roles in disease

PubMed Central

Crotty, Shane

2014-01-01

Summary Follicular helper T (Tfh) cells are specialized providers of T cell help to B cells, and are essential for germinal center formation, affinity maturation, and the development of most high affinity antibodies and memory B cells. Tfh cell differentiation is a multi-stage, multi-factorial process involving B cell lymphoma 6 (Bcl6) and other transcription factors. This article reviews understanding of Tfh cell biology, including their differentiation, migration, transcriptional regulation, and B cell help functions. Tfh cells are critical components of many protective immune responses against pathogens. As such, there is strong interest in harnessing Tfh cells to improve vaccination strategies. Tfh cells also have roles in a range of other diseases, particularly autoimmune diseases. Overall, there have been dramatic advances in this young field, but there is much to be learned about Tfh cell biology in the interest of applying that knowledge to biomedical needs. PMID:25367570

Computational design of protein interactions: designing proteins that neutralize influenza by inhibiting its hemagglutinin surface protein

NASA Astrophysics Data System (ADS)

Fleishman, Sarel

2012-02-01

Molecular recognition underlies all life processes. Design of interactions not seen in nature is a test of our understanding of molecular recognition and could unlock the vast potential of subtle control over molecular interaction networks, allowing the design of novel diagnostics and therapeutics for basic and applied research. We developed the first general method for designing protein interactions. The method starts by computing a region of high affinity interactions between dismembered amino acid residues and the target surface and then identifying proteins that can harbor these residues. Designs are tested experimentally for binding the target surface and successful ones are affinity matured using yeast cell surface display. Applied to the conserved stem region of influenza hemagglutinin we designed two unrelated proteins that, following affinity maturation, bound hemagglutinin at subnanomolar dissociation constants. Co-crystal structures of hemagglutinin bound to the two designed binders were within 1Angstrom RMSd of their models, validating the accuracy of the design strategy. One of the designed proteins inhibits the conformational changes that underlie hemagglutinin's cell-invasion functions and blocks virus infectivity in cell culture, suggesting that such proteins may in future serve as diagnostics and antivirals against a wide range of pathogenic influenza strains. We have used this method to obtain experimentally validated binders of several other target proteins, demonstrating the generality of the approach. We discuss the combination of modeling and high-throughput characterization of design variants which has been key to the success of this approach, as well as how we have used the data obtained in this project to enhance our understanding of molecular recognition. References: Science 332:816 JMB, in press Protein Sci 20:753

Miniature protein ligands for EVH1 domains: Interplay between affinity, specificity, and cell motility⊥

PubMed Central

Holtzman, Jennifer H.; Woronowicz, Kamil; Golemi-Kotra, Dasantila; Schepartz, Alanna

2008-01-01

Dynamic rearrangements of the actin cytoskeleton power cell motility in contexts ranging from intracellular microbial pathogenesis to axon guidance. The Ena/VASP family proteins--Mena, VASP, and Evl--are believed to control cell motility by serving as a direct link between signaling events and the actin cytoskeleton. Our lab has previously reported a novel miniature protein, pGolemi, which binds with high affinity to the EVH1 domain of Mena (Mena1-112) but not to those of VASP (VASP1-115) or Evl (Evl1-115) and also causes an unusual defect in actin-driven L. monocytogenes motility. Here, we use scanning mutagenesis to examine the effects of single amino acid changes within pGolemi on EVH1 domain affinity and specificity, miniature protein secondary structure, and L. monocytogenes motility. The data suggest that pGolemi contains the expected aPP-like fold and binds Mena1-112 in a manner highly analogous to the proline-rich repeat region of L. monocytogenes ActA protein. Residues throughout pGolemi contribute to both EVH1 domain affinity and paralog specificity. Moreover, the affinities of pGolemi variants for Mena1-112 correlate with selectivity against the EVH1 domains of VASP and Evl. In L. monocytogenes motility assays, speed and speed variability correlate strongly with EVH1 paralog specificity, suggesting that the Ena/VASP paralogs do not play equivalent roles in the process of L. monocytogenes actin tail maturation. PMID:17973491

Structure-based affinity maturation of a chimeric anti-ricin antibody C4C13.

PubMed

Luo, Longlong; Luo, Qun; Guo, Leiming; Lv, Ming; Lin, Zhou; Geng, Jing; Li, Xinying; Li, Yan; Shen, Beifen; Qiao, Chunxia; Feng, Jiannan

2014-01-01

Ricin is a highly lethal toxin. Anti-ricin chimeric monoclonal antibody (mAb) C4C13 was prepared in our lab; however, its binding affinity was much weaker than that of the parent antibody 4C13. In this study, based on the computer-guided homology modeling and conformational optimization methods, the 3-D structure of C4C13 variable regions Fv was constructed and optimized. Using molecular docking and dynamics simulation methods, the 3-D complex structure of ricin and C4C13 Fv was obtained. Considering the orientation property, surface electrostatic distribution, residues chemical and physical character and intermolecular hydrogen bond, the binding mode and key residues were predicted. According to C4C13 Fv fragment and ricin complementary binding surface, electrostatic attraction periphery and van der Waals interaction interface, three mutants (i.e., M1 (N(H102)F, W(H103)Y); M2 (W(H103)Y) and M3 (R(L90)G)) were designed, in which M1 and M2 were predicted to possess higher antigen-binding activity than C4C13, while M3 was weaker. The relative affinity assays by ELISA showed that M1 and M2 mutations had higher affinity (9.6 and 18.3 nmol/L) than C4C13 (130 nmol/L) and M3 had weaker affinity (234.5 nmol/L) than C4C13. The results showed that the modeling complex structure of the antigen (ricin) and antibody (C4C13) is reasonable. Our work offered affinity maturated antibodies by site mutations, which were beneficial for valuable anti-ricin antibody design and preparation in future.

Somatic Hypermutation-Induced Changes in the Structure and Dynamics of HIV-1 Broadly Neutralizing Antibodies.

PubMed

Davenport, Thaddeus M; Gorman, Jason; Joyce, M Gordon; Zhou, Tongqing; Soto, Cinque; Guttman, Miklos; Moquin, Stephanie; Yang, Yongping; Zhang, Baoshan; Doria-Rose, Nicole A; Hu, Shiu-Lok; Mascola, John R; Kwong, Peter D; Lee, Kelly K

2016-08-02

Antibody somatic hypermutation (SHM) and affinity maturation enhance antigen recognition by modifying antibody paratope structure to improve its complementarity with the target epitope. SHM-induced changes in paratope dynamics may also contribute to antibody maturation, but direct evidence of this is limited. Here, we examine two classes of HIV-1 broadly neutralizing antibodies (bNAbs) for SHM-induced changes in structure and dynamics, and delineate the effects of these changes on interactions with the HIV-1 envelope glycoprotein (Env). In combination with new and existing structures of unmutated and affinity matured antibody Fab fragments, we used hydrogen/deuterium exchange with mass spectrometry to directly measure Fab structural dynamics. Changes in antibody structure and dynamics were positioned to improve complementarity with Env, with changes in dynamics primarily observed at the paratope peripheries. We conclude that SHM optimizes paratope complementarity to conserved HIV-1 epitopes and restricts the mobility of paratope-peripheral residues to minimize clashes with variable features on HIV-1 Env. Copyright © 2016 Elsevier Ltd. All rights reserved.

A Listeria monocytogenes RNA helicase essential for growth and ribosomal maturation at low temperatures uses its C terminus for appropriate interaction with the ribosome.

PubMed

Netterling, Sakura; Vaitkevicius, Karolis; Nord, Stefan; Johansson, Jörgen

2012-08-01

Listeria monocytogenes, a Gram-positive food-borne human pathogen, is able to grow at temperatures close to 0°C and is thus of great concern for the food industry. In this work, we investigated the physiological role of one DExD-box RNA helicase in Listeria monocytogenes. The RNA helicase Lmo1722 was required for optimal growth at low temperatures, whereas it was dispensable at 37°C. A Δlmo1722 strain was less motile due to downregulation of the major subunit of the flagellum, FlaA, caused by decreased flaA expression. By ribosomal fractionation experiments, it was observed that Lmo1722 was mainly associated with the 50S subunit of the ribosome. Absence of Lmo1722 decreased the fraction of 50S ribosomal subunits and mature 70S ribosomes and affected the processing of the 23S precursor rRNA. The ribosomal profile could be restored to wild-type levels in a Δlmo1722 strain expressing Lmo1722. Interestingly, the C-terminal part of Lmo1722 was redundant for low-temperature growth, motility, 23S rRNA processing, and appropriate ribosomal maturation. However, Lmo1722 lacking the C terminus showed a reduced affinity for the 50S and 70S fractions, suggesting that the C terminus is important for proper guidance of Lmo1722 to the 50S subunit. Taken together, our results show that the Listeria RNA helicase Lmo1722 is essential for growth at low temperatures, motility, and rRNA processing and is important for ribosomal maturation, being associated mainly with the 50S subunit of the ribosome.

Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding.

PubMed

Munck Petersen, C; Nielsen, M S; Jacobsen, C; Tauris, J; Jacobsen, L; Gliemann, J; Moestrup, S K; Madsen, P

1999-02-01

We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP). The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain. We now show that the truncation results from cellular processing. Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide. We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin. The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity. Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation.

Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding.

PubMed Central

Munck Petersen, C; Nielsen, M S; Jacobsen, C; Tauris, J; Jacobsen, L; Gliemann, J; Moestrup, S K; Madsen, P

1999-01-01

We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP). The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain. We now show that the truncation results from cellular processing. Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide. We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin. The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity. Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation. PMID:9927419

Precursor-product discrimination by La protein during tRNA metabolism

PubMed Central

Bayfield, Mark A.; Maraia, Richard J.

2009-01-01

SUMMARY La proteins bind pre-tRNAs at their UUU-3'OH ends, facilitating their maturation. While the mechanism by which La binds pre-tRNA 3' trailers is known, the function of the RNA-binding β-sheet surface of RRM1 is unknown. How La dissociates from UUU-3'OH-containing trailers after 3' processing is also unknown. La preferentially binds pre-tRNAs over processed tRNAs or 3' trailer products through coupled use of two sites: one on the La motif and another on the RRM1 β surface that binds elsewhere on tRNA. Two sites provide stable pre-tRNA binding while processed tRNA and 3' trailer are released from their single sites relatively fast. RRM1 loop-3 mutations decrease affinity for pre-tRNA and tRNA but not UUU-3'OH trailer, and impair tRNA maturation in vivo. We propose that RRM1 functions in activities that are more complex than UUU-3'OH binding. Accordingly, the RRM1 mutations also impair a RNA chaperone activity of La. The results suggest how La distinguishes precursor from product RNAs, allowing it to recycle onto a new pre-tRNA. PMID:19287396

Next-Generation Sequencing of Antibody Display Repertoires

PubMed Central

Rouet, Romain; Jackson, Katherine J. L.; Langley, David B.; Christ, Daniel

2018-01-01

In vitro selection technology has transformed the development of therapeutic monoclonal antibodies. Using methods such as phage, ribosome, and yeast display, high affinity binders can be selected from diverse repertoires. Here, we review strategies for the next-generation sequencing (NGS) of phage- and other antibody-display libraries, as well as NGS platforms and analysis tools. Moreover, we discuss recent examples relating to the use of NGS to assess library diversity, clonal enrichment, and affinity maturation. PMID:29472918

Affinity Maturation of a Cyclic Peptide Handle for Therapeutic Antibodies Using Deep Mutational Scanning*

PubMed Central

van Rosmalen, Martijn; Janssen, Brian M. G.; Hendrikse, Natalie M.; van der Linden, Ardjan J.; Pieters, Pascal A.; Wanders, Dave; de Greef, Tom F. A.; Merkx, Maarten

2017-01-01

Meditopes are cyclic peptides that bind in a specific pocket in the antigen-binding fragment of a therapeutic antibody such as cetuximab. Provided their moderate affinity can be enhanced, meditope peptides could be used as specific non-covalent and paratope-independent handles in targeted drug delivery, molecular imaging, and therapeutic drug monitoring. Here we show that the affinity of a recently reported meditope for cetuximab can be substantially enhanced using a combination of yeast display and deep mutational scanning. Deep sequencing was used to construct a fitness landscape of this protein-peptide interaction, and four mutations were identified that together improved the affinity for cetuximab 10-fold to 15 nm. Importantly, the increased affinity translated into enhanced cetuximab-mediated recruitment to EGF receptor-overexpressing cancer cells. Although in silico Rosetta simulations correctly identified positions that were tolerant to mutation, modeling did not accurately predict the affinity-enhancing mutations. The experimental approach reported here should be generally applicable and could be used to develop meditope peptides with low nanomolar affinity for other therapeutic antibodies. PMID:27974464

BRILIA: Integrated Tool for High-Throughput Annotation and Lineage Tree Assembly of B-Cell Repertoires.

PubMed

Lee, Donald W; Khavrutskii, Ilja V; Wallqvist, Anders; Bavari, Sina; Cooper, Christopher L; Chaudhury, Sidhartha

2016-01-01

The somatic diversity of antigen-recognizing B-cell receptors (BCRs) arises from Variable (V), Diversity (D), and Joining (J) (VDJ) recombination and somatic hypermutation (SHM) during B-cell development and affinity maturation. The VDJ junction of the BCR heavy chain forms the highly variable complementarity determining region 3 (CDR3), which plays a critical role in antigen specificity and binding affinity. Tracking the selection and mutation of the CDR3 can be useful in characterizing humoral responses to infection and vaccination. Although tens to hundreds of thousands of unique BCR genes within an expressed B-cell repertoire can now be resolved with high-throughput sequencing, tracking SHMs is still challenging because existing annotation methods are often limited by poor annotation coverage, inconsistent SHM identification across the VDJ junction, or lack of B-cell lineage data. Here, we present B-cell repertoire inductive lineage and immunosequence annotator (BRILIA), an algorithm that leverages repertoire-wide sequencing data to globally improve the VDJ annotation coverage, lineage tree assembly, and SHM identification. On benchmark tests against simulated human and mouse BCR repertoires, BRILIA correctly annotated germline and clonally expanded sequences with 94 and 70% accuracy, respectively, and it has a 90% SHM-positive prediction rate in the CDR3 of heavily mutated sequences; these are substantial improvements over existing methods. We used BRILIA to process BCR sequences obtained from splenic germinal center B cells extracted from C57BL/6 mice. BRILIA returned robust B-cell lineage trees and yielded SHM patterns that are consistent across the VDJ junction and agree with known biological mechanisms of SHM. By contrast, existing BCR annotation tools, which do not account for repertoire-wide clonal relationships, systematically underestimated both the size of clonally related B-cell clusters and yielded inconsistent SHM frequencies. We demonstrate BRILIA's utility in B-cell repertoire studies related to VDJ gene usage, mechanisms for adenosine mutations, and SHM hot spot motifs. Furthermore, we show that the complete gene usage annotation and SHM identification across the entire CDR3 are essential for studying the B-cell affinity maturation process through immunosequencing methods.

The Splicing ATPase Prp43p Is a Component of Multiple Preribosomal Particles

PubMed Central

Lebaron, Simon; Froment, Carine; Fromont-Racine, Micheline; Rain, Jean-Christophe; Monsarrat, Bernard; Caizergues-Ferrer, Michèle; Henry, Yves

2005-01-01

Prp43p is a putative helicase of the DEAH family which is required for the release of the lariat intron from the spliceosome. Prp43p could also play a role in ribosome synthesis, since it accumulates in the nucleolus. Consistent with this hypothesis, we find that depletion of Prp43p leads to accumulation of 35S pre-rRNA and strongly reduces levels of all downstream pre-rRNA processing intermediates. As a result, the steady-state levels of mature rRNAs are greatly diminished following Prp43p depletion. We present data arguing that such effects are unlikely to be solely due to splicing defects. Moreover, we demonstrate by a combination of a comprehensive two-hybrid screen, tandem-affinity purification followed by mass spectrometry, and Northern analyses that Prp43p is associated with 90S, pre-60S, and pre-40S ribosomal particles. Prp43p seems preferentially associated with Pfa1p, a novel specific component of pre-40S ribosomal particles. In addition, Prp43p interacts with components of the RNA polymerase I (Pol I) transcription machinery and with mature 18S and 25S rRNAs. Hence, Prp43p might be delivered to nascent 90S ribosomal particles during pre-rRNA transcription and remain associated with preribosomal particles until their final maturation steps in the cytoplasm. Our data also suggest that the ATPase activity of Prp43p is required for early steps of pre-rRNA processing and normal accumulation of mature rRNAs. PMID:16227579

Bioreactor Transient Exposure Activates Specific Neurotrophic Pathway in Cortical Neurons

NASA Astrophysics Data System (ADS)

Zimmitti, V.; Benedetti, E.; Caracciolo, V.; Sebastiani, P.; Di Loreto, S.

2010-02-01

Altered gravity forces might influence neuroplasticity and can provoke changes in biochemical mechanisms. In this contest, neurotrophins have a pivotal role, particularly nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF). A suspension of dissociated cortical cells from rat embryos was exposed to 24 h of microgravity before plating in normal adherent culture system. Expression and transductional signalling pathways of NGF and BDNF were assessed at the end of maturational process (8-10 days in vitro). Rotating wall vessel bioreactor (RWV) pre-exposition did not induce changes in NGF expression and its high affinity receptor TrkA. On the contrary both BDNF expression and its high affinity receptor TrkB were strongly up-regulated, inducing Erk-5, but not Erk-1/2 activation and, in turn, MEF2C over-expression and activation. According to our previous and present results, we postulate that relatively short microgravitational stimuli, applied to neural cells during the developmental stage, exert a long time activation of specific neurotrophic pathways.

Affinity maturation in an HIV broadly neutralizing B-cell lineage through reorientation of variable domains.

PubMed

Fera, Daniela; Schmidt, Aaron G; Haynes, Barton F; Gao, Feng; Liao, Hua-Xin; Kepler, Thomas B; Harrison, Stephen C

2014-07-15

Rapidly evolving pathogens, such as human immunodeficiency and influenza viruses, escape immune defenses provided by most vaccine-induced antibodies. Proposed strategies to elicit broadly neutralizing antibodies require a deeper understanding of antibody affinity maturation and evolution of the immune response to vaccination or infection. In HIV-infected individuals, viruses and B cells evolve together, creating a virus-antibody "arms race." Analysis of samples from an individual designated CH505 has illustrated the interplay between an antibody lineage, CH103, and autologous viruses at various time points. The CH103 antibodies, relatively broad in their neutralization spectrum, interact with the CD4 binding site of gp120, with a contact dominated by CDRH3. We show by analyzing structures of progenitor and intermediate antibodies and by correlating them with measurements of binding to various gp120s that there was a shift in the relative orientation of the light- and heavy-chain variable domains during evolution of the CH103 lineage. We further show that mutations leading to this conformational shift probably occurred in response to insertions in variable loop 5 (V5) of the HIV envelope. The shift displaced the tips of the light chain away from contact with V5, making room for the inserted residues, which had allowed escape from neutralization by the progenitor antibody. These results, which document the selective mechanism underlying this example of a virus-antibody arms race, illustrate the functional significance of affinity maturation by mutation outside the complementarity determining region surface of the antibody molecule.

Improved antibody-based ricin neutralization by affinity maturation is correlated with slower off-rate values.

PubMed

Rosenfeld, Ronit; Alcalay, Ron; Mechaly, Adva; Lapidoth, Gideon; Epstein, Eyal; Kronman, Chanoch; J Fleishman, Sarel; Mazor, Ohad

2017-09-01

While potent monoclonal antibodies against ricin were introduced over the years, the question whether increasing antibody affinity enables better toxin neutralization was not fully addressed yet. The aim of this study was to characterize the contribution of antibody affinity to the ricin neutralization potential of the antibody. cHD23 monoclonal antibody that targets the toxin B-subunit and interferes with its binding to membranal receptors, was isolated. In order to create antibody clones with improved affinity toward ricin, a scFv-phage display library containing mutated versions of the variable regions of cHD23 was constructed and clones with improved binding of ricin were isolated. Structural modeling of these mutants suggests that the inserted mutations may increase the antibody conformational flexibility thus improving its ability to bind ricin. While it was found that the selected clones exhibited improved neutralization of ricin, the correlation between the KD values and potency was only minor (r = 0.55). However, a positive correlation (r = 0.84) exist between the off-rate values (koff) of the affinity matured clones and their ability to neutralize ricin. As cell membranes display inordinately large amounts of potential surface binding sites for ricin, it is suggested that antibodies with improved off-rate values block the ability of the toxin to bind to target receptors, in a highly efficient manner. Currently, antibody-based therapy is the most effective treatment for ricin intoxication and it is anticipated that the findings of this study will provide useful information and a possible strategy to design an improved antibody-based therapy for the toxin. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

[B lymphocyte clonal evolution of human reactive lymph nodes revealed by lineage tree analysis].

PubMed

Tabibian-Keissar, Hilla; Schiby, Ginette; Azogui-Rosenthal, Noemie; Hazanov, Helena; Rakovsky, Aviya Shapira; Michaeli, Miri; Rosenblatt, Kinneret; Mehr, Ramit; Barshack, Iris

2013-06-01

Hypermutation and selection processes, characterizing T-dependent B cell responses taking place in germinal centers of lymph nodes, lead to B cell receptor affinity maturation. Those immune responses lead to the development of memory B cells and plasma cells that secrete high amounts of antibody molecules. The dynamics of B cell clonal evolution during affinity maturation has significant importance in infectious and autoimmune diseases, malignancies and aging. Immunoglobulin (Ig) gene mutational Lineage tree construction by comparing variable regions of Ig-gene sequences to the Ig germline gene is an interesting approach for studying B cell cLonal evolution. Lineage tree shapes and Ig gene mutations can be evaluated not only qualitatively and intuitively, but also quantitatively, and thus reveal important information related to hypermutation and selection. In this paper we describe the experimental protocols that we used for PCR amplification of Igvariable region genes from human formalin fixed paraffin embedded reactive lymph node tissues and the subsequent bioinformatical analyses of sequencing data using Ig mutational lineage trees. B cell populations of three out of four reactive Lymph node tissues were composed of several clones. Most of the Ig gene mutational lineage trees were small and narrow. Significant differences were not detected by quantification of Lineage trees. B lymphocyte clones that were detected in human reactive lymph node tissues represent major responding clones in normal polyclonal immune response. This result is in line with the polyclonal profile of B Lymphocyte populations that reside in reactive lymph node tissues.

Characterization of the molecular basis of group II intron RNA recognition by CRS1-CRM domains.

PubMed

Keren, Ido; Klipcan, Liron; Bezawork-Geleta, Ayenachew; Kolton, Max; Shaya, Felix; Ostersetzer-Biran, Oren

2008-08-22

CRM (chloroplast RNA splicing and ribosome maturation) is a recently recognized RNA-binding domain of ancient origin that has been retained in eukaryotic genomes only within the plant lineage. Whereas in bacteria CRM domains exist as single domain proteins involved in ribosome maturation, in plants they are found in a family of proteins that contain between one and four repeats. Several members of this family with multiple CRM domains have been shown to be required for the splicing of specific plastidic group II introns. Detailed biochemical analysis of one of these factors in maize, CRS1, demonstrated its high affinity and specific binding to the single group II intron whose splicing it facilitates, the plastid-encoded atpF intron RNA. Through its association with two intronic regions, CRS1 guides the folding of atpF intron RNA into its predicted "catalytically active" form. To understand how multiple CRM domains cooperate to achieve high affinity sequence-specific binding to RNA, we analyzed the RNA binding affinity and specificity associated with each individual CRM domain in CRS1; whereas CRM3 bound tightly to the RNA, CRM1 associated specifically with a unique region found within atpF intron domain I. CRM2, which demonstrated only low binding affinity, also seems to form specific interactions with regions localized to domains I, III, and IV. We further show that CRM domains share structural similarities and RNA binding characteristics with the well known RNA recognition motif domain.

Specific Fluorine Labeling of the HyHEL10 Antibody Affects Antigen Binding and Dynamics

PubMed Central

Acchione, Mauro; Lee, Yi-Chien; DeSantis, Morgan E.; Lipschultz, Claudia A.; Wlodawer, Alexander; Li, Mi; Shanmuganathan, Aranganathan; Walter, Richard L.; Smith-Gill, Sandra; Barchi, Joseph J.

2012-01-01

To more fully understand the molecular mechanisms responsible for variations in binding affinity with antibody maturation, we explored the use of site specific fluorine labeling and 19F nuclear magnetic resonance (NMR). Several single-chain (scFv) antibodies, derived from an affinity-matured series of anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either complete or individual replacement of tryptophan residues with 5-fluorotryptophan (5FW). An array of biophysical techniques was used to gain insight into the impact of fluorine substitution on the overall protein structure and antigen binding. SPR measurements indicated that 5FW incorporation lowered binding affinity for the HEL antigen. The degree of analogue impact was residue-dependent, and the greatest decrease in affinity was observed when 5FW was substituted for residues near the binding interface. In contrast, corresponding crystal structures in complex with HEL were essentially indistinguishable from the unsubstituted antibody. 19F NMR analysis showed severe overlap of signals in the free fluorinated protein that was resolved upon binding to antigen, suggesting very distinct chemical environments for each 5FW in the complex. Preliminary relaxation analysis suggested the presence of chemical exchange in the antibody–antigen complex that could not be observed by X-ray crystallography. These data demonstrate that fluorine NMR can be an extremely useful tool for discerning structural changes in scFv antibody–antigen complexes with altered function that may not be discernible by other biophysical techniques. PMID:22769726

Structural correlates of affinity in fetal versus adult endplate nicotinic receptors

NASA Astrophysics Data System (ADS)

Nayak, Tapan Kumar; Chakraborty, Srirupa; Zheng, Wenjun; Auerbach, Anthony

2016-04-01

Adult-type nicotinic acetylcholine receptors (AChRs) mediate signalling at mature neuromuscular junctions and fetal-type AChRs are necessary for proper synapse development. Each AChR has two neurotransmitter binding sites located at the interface of a principal and a complementary subunit. Although all agonist binding sites have the same core of five aromatic amino acids, the fetal site has ~30-fold higher affinity for the neurotransmitter ACh. Here we use molecular dynamics simulations of adult versus fetal homology models to identify complementary-subunit residues near the core that influence affinity, and use single-channel electrophysiology to corroborate the results. Four residues in combination determine adult versus fetal affinity. Simulations suggest that at lower-affinity sites, one of these unsettles the core directly and the others (in loop E) increase backbone flexibility to unlock a key, complementary tryptophan from the core. Swapping only four amino acids is necessary and sufficient to exchange function between adult and fetal AChRs.

Synthesis of Novel Caspase Inhibitors for Characterization of the Active Caspase Proteome in Vitro and in Vivo

PubMed Central

Henzing, Alexander J.; Dodson, Helen; Reid, Joel M.; Kaufmann, Scott H.; Baxter, Robert L.; Earnshaw, William C.

2008-01-01

Caspases are cysteine proteases that are essential for cytokine maturation and apoptosis. To facilitate the dissection of caspase function in vitro and in vivo, we have synthesized irreversible caspase inhibitors with biotin attached via linker arms of various lengths (12a–d) and a 2,4-dinitrophenyl labeled inhibitor (13). Affinity labeling of apoptotic extracts followed by blotting reveals that these affinity probes detect active caspases. Using the strong affinity of avidin for biotin, we have isolated affinity-labeled caspase-6 from apoptotic cytosolic extracts of cells overexpressing procaspase 6 by treatment with 12c, which contains biotin attached to the Nε-lysine of the inhibitor by a 22.5 Å linker arm, followed by affinity purification on monomeric avidin-Sepharose beads. 13 has proven sufficiently cell permeable to rescue cells from apoptotic execution. These novel caspase inhibitors should provide powerful probes for the study of the active caspase proteome during apoptosis both in vitro and in vivo. PMID:17181147

Defect in IgV gene somatic hypermutation in common variable immuno-deficiency syndrome.

PubMed

Levy, Y; Gupta, N; Le Deist, F; Garcia, C; Fischer, A; Weill, J C; Reynaud, C A

1998-10-27

Common Variable Immuno-Deficiency (CVID) is the most common symptomatic primary antibody-deficiency syndrome, but the basic immunologic defects underlying this syndrome are not well defined. We report here that among eight patients studied (six CVID and two hypogammaglobulinemic patients with recurrent infections), there is in two CVID patients a dramatic reduction in Ig V gene somatic hypermutation with 40-75% of IgG transcripts totally devoid of mutations in the circulating memory B cell compartment. Functional assays of the T cell compartment point to an intrinsic B cell defect in the process of antibody affinity maturation in these two cases.

The effects analysis of two neonicotinoid insecticides on in vitro maturation of porcine oocytes using hanging drop monoculture method.

PubMed

Ishikawa, Sadamasa; Hiraga, Kou; Hiradate, Yuuki; Tanemura, Kentaro

2015-06-01

Acetamiprid (ACE) and imidacroprid (IMI) are known neonicotinoid insecticides with strong affinities for the insect-selective nicotinic acetylcholine receptor. These provide insect control by hyperstimulating insect nerves and are used for agricultural pest management. However, it has also been reported that ACE and IMI affect mammalian reproductive function. We determined the effects of ACE and IMI on the in vitro maturation of porcine oocytes. Significant decreases in nuclear maturation rates were observed in the ACE or IMI-exposed groups. Also, in matured oocytes from the ACE or IMI-exposed groups, irregular chromosomes were observed. Our results suggest that ACE and IMI exposure was detrimental to porcine oocytes and the extent of the effects depends on the concentration of exposure.

Toxic metals (Ni2+, Pb2+, Hg2+) binding affinity of dissolved organic matter (DOM) derived from different ages municipal landfill leachate

NASA Astrophysics Data System (ADS)

Rikta, S. Y.; Tareq, Shafi M.; Uddin, M. Khabir

2018-03-01

Solid waste production is rapidly increasing in Bangladesh and landfill leachate is the consequence of the decomposition of this waste. These leachates contain heavy metals and significant amount of dissolved organic matter (DOM). DOM is known to have considerable role in heavy metals speciation. Hence, it is important to characterize DOM/leachate and evaluate toxic metals binding affinity of DOM. The objectives of this study were to characterize the DOM in landfill leachate through physico-chemical and optical analyses and to investigate the toxic metals (Ni2+, Pb2+ and Hg2+) binding affinity of three different ages (fresh sample L-1, young sample L-2 and mature sample L-3) DOM samples. Results suggested that leachate is a potential pollutant which contained very high organic pollutant load. Conditional stability constant (Log K) and percentages of fluorophores that correspond to metal binding (% f) values indicated that young DOM sample (L-2) had the highest binding affinity to all the three metals ions. In general, DOM samples showed the following order affinity to the metal ions; Ni2+ binding affinity: L-2 > L-3 > L-1, Pb2+ binding affinity: L-2 > L-3 > L-1 and Hg2+ binding affinity: L-2 > L-1 > L-3.

Targeted inhibition of oncogenic miR-21 maturation with designed RNA-binding proteins

PubMed Central

Chen, Yu; Yang, Fan; Zubovic, Lorena; Pavelitz, Tom; Yang, Wen; Godin, Katherine; Walker, Matthew; Zheng, Suxin; Macchi, Paolo; Varani, Gabriele

2016-01-01

The RNA Recognition Motif (RRM) is the largest family of eukaryotic RNA-binding proteins. Engineered RRMs with new specificity would provide valuable tools and an exacting test of our understanding of specificity. We have achieved the first successful re-design of the specificity of an RRM using rational methods and demonstrated re-targeting of activity in cells. We engineered the conserved RRM of human Rbfox proteins to specifically bind to the terminal loop of miR-21 precursor with high affinity and inhibit its processing by Drosha and Dicer. We further engineered Giardia Dicer by replacing its PAZ domain with the designed RRM. The reprogrammed enzyme degrades pre-miR-21 specifically in vitro and suppresses mature miR-21 levels in cells, which results in increased expression of PDCD4 and significantly decreased viability for cancer cells. The results demonstrate the feasibility of engineering the sequence-specificity of RRMs and of using this ubiquitous platform for diverse biological applications. PMID:27428511

Precursor-product discrimination by La protein during tRNA metabolism.

PubMed

Bayfield, Mark A; Maraia, Richard J

2009-04-01

La proteins bind pre-tRNAs at their UUU-3'OH ends, facilitating their maturation. Although the mechanism by which La binds pre-tRNA 3' trailers is known, the function of the RNA binding beta-sheet surface of the RNA-recognition motif (RRM1) is unknown. How La dissociates from UUU-3'OH-containing trailers after 3' processing is also unknown. Here we show that La preferentially binds pre-tRNAs over processed tRNAs or 3' trailer products through coupled use of two sites: one on the La motif and another on the RRM1 beta-surface that binds elsewhere on tRNA. Two sites provide stable pre-tRNA binding, whereas the processed tRNA and 3' trailer are released from their single sites relatively fast. RRM1 loop-3 mutations decrease affinity for pre-tRNA and tRNA, but not for the UUU-3'OH trailer, and impair tRNA maturation in vivo. We propose that RRM1 functions in activities that are more complex than UUU-3'OH binding. Accordingly, the RRM1 mutations also impair an RNA chaperone activity of La. The results suggest how La distinguishes precursor from product RNAs, allowing it to recycle onto a new pre-tRNA.

An affinity/avidity model of peripheral T cell regulation

PubMed Central

Jiang, Hong; Wu, Yilun; Liang, Bitao; Zheng, Zongyu; Tang, Guomei; Kanellopoulos, Jean; Soloski, Mark; Winchester, Robert; Goldstein, Itamar; Chess, Leonard

2005-01-01

We show in these studies that Qa-1–dependent CD8+ T cells are involved in the establishment and maintenance of peripheral self tolerance as well as facilitating affinity maturation of CD4+ T cells responding to foreign antigen. We provide experimental evidence that the strategy used by the Qa-1–dependent CD8+ T cells to accomplish both these tasks in vivo is to selectively downregulate T cell clones that respond to both self and foreign antigens with intermediate, not high or low, affinity/avidity. Thus, the immune system evolved to regulate peripheral immunity using a unified mechanism that efficiently and effectively permits the system to safeguard peripheral self tolerance yet promote the capacity to deal with foreign invaders. PMID:15668735

In Planta Processing and Glycosylation of a Nematode CLAVATA3/ENDOSPERM SURROUNDING REGION-Like Effector and Its Interaction with a Host CLAVATA2-Like Receptor to Promote Parasitism1[OPEN

PubMed Central

Chen, Shiyan; Lang, Ping; Chronis, Demosthenis; Zhang, Sheng; De Jong, Walter S.; Mitchum, Melissa G.

2015-01-01

Like other biotrophic plant pathogens, plant-parasitic nematodes secrete effector proteins into host cells to facilitate infection. Effector proteins that mimic plant CLAVATA3/ENDOSPERM SURROUNDING REGION-related (CLE) proteins have been identified in several cyst nematodes, including the potato cyst nematode (PCN); however, the mechanistic details of this cross-kingdom mimicry are poorly understood. Plant CLEs are posttranslationally modified and proteolytically processed to function as bioactive ligands critical to various aspects of plant development. Using ectopic expression coupled with nanoliquid chromatography-tandem mass spectrometry analysis, we show that the in planta mature form of proGrCLE1, a multidomain CLE effector secreted by PCN during infection, is a 12-amino acid arabinosylated glycopeptide (named GrCLE1-1Hyp4,7g) with striking structural similarity to mature plant CLE peptides. This glycopeptide is more resistant to hydrolytic degradation and binds with higher affinity to a CLAVATA2-like receptor (StCLV2) from potato (Solanum tuberosum) than its nonglycosylated forms. We further show that StCLV2 is highly up-regulated at nematode infection sites and that transgenic potatoes with reduced StCLV2 expression are less susceptible to PCN infection, indicating that interference of the CLV2-mediated signaling pathway confers nematode resistance in crop plants. These results strongly suggest that phytonematodes have evolved to utilize host cellular posttranslational modification and processing machinery for the activation of CLE effectors following secretion into plant cells and highlight the significance of arabinosylation in regulating nematode CLE effector activity. Our finding also provides evidence that multidomain CLEs are modified and processed similarly to single-domain CLEs, adding new insight into CLE maturation in plants. PMID:25416475

Insight into the growth dynamics and systematic affinities of the Late Cretaceous Gargantuavis from bone microstructure

NASA Astrophysics Data System (ADS)

Chinsamy, Anusuya; Buffetaut, Eric; Canoville, Aurore; Angst, Delphine

2014-05-01

Enigmatic avialan remains of Gargantuavis philoinos from the Ibero-Armorican island of the Late Cretaceous European archipelago (Southern France) led to a debate concerning its taxonomic affinities. Here, we show that the bone microstructure of Gargantuavis resembles that of Apteryx, the extinct emeids and Megalapteryx from New Zealand, and indicates that like these slow-growing terrestrial birds, it took several years to attain skeletal maturity. Our findings suggest that the protracted cyclical growth in these ornithurines may have been in response to insular evolution.

Dietary starch breakdown product sensing mobilizes and apically activates α-glucosidases in small intestinal enterocytes.

PubMed

Chegeni, Mohammad; Amiri, Mahdi; Nichols, Buford L; Naim, Hassan Y; Hamaker, Bruce R

2018-02-20

Dietary starch is finally converted to glucose for absorption by the small intestine mucosal α-glucosidases (sucrase-isomaltase [SI] and maltase-glucoamylase), and control of this process has health implications. Here, the molecular mechanisms were analyzed associated with starch-triggered maturation and transport of SI. Biosynthetic pulse-chase in Caco-2 cells revealed that the high MW SI species (265 kDa) induced by maltose (an α-amylase starch digestion product) had a higher rate of early trafficking and maturation compared with a glucose-induced SI (245 kDa). The maltose-induced SI was found to have higher affinity to lipid rafts, which are associated with enhanced targeting to the apical membrane and higher activity. Accordingly, in situ maltose-hydrolyzing action was enhanced in the maltose-treated cells. Thus, starch digestion products at the luminal surface of small intestinal enterocytes are sensed and accelerate the intracellular processing of SI to enhance starch digestion capacity in the intestinal lumen.-Chegeni, M., Amiri, M., Nichols, B. L., Naim, H. Y., Hamaker, B. R. Dietary starch breakdown product sensing mobilizes and apically activates α-glucosidases in small intestinal enterocytes.

Shark immunity bites back: affinity maturation and memory response in the nurse shark, Ginglymostoma cirratum.

PubMed

Dooley, Helen; Flajnik, Martin F

2005-03-01

The cartilaginous fish are the oldest phylogenetic group in which all of the molecular components of the adaptive immune system have been found. Although early studies clearly showed that sharks could produce an IgM-based response following immunization, evidence for memory, affinity maturation and roles for the other isotypes (notably IgNAR) in this group remained inconclusive. The data presented here illustrate that the nurse shark (Ginglymostoma cirratum) is able to produce not only an IgM response, but we also show for the first time a highly antigen-specific IgNAR response. Additionally, under appropriate conditions, a memory response for both isotypes can be elicited. Analysis of the response shows differential expression of pentameric and monomeric IgM. Pentameric IgM provides the 'first line of defense' through high-avidity, low-affinity interaction with antigen. In contrast, monomeric IgM and IgNAR seem responsible for the specific, antigen-driven response. We propose the presence of distinct lineages of B cells in sharks. As there is no conventional isotype switching, each lineage seems pre-determined to express a single isotype (IgM versus IgNAR). However, our data suggest that there may also be specific lineages for the different forms (pentameric versus monomeric) of the IgM isotype.

In silico design of fragment-based drug targeting host processing α-glucosidase i for dengue fever

NASA Astrophysics Data System (ADS)

Toepak, E. P.; Tambunan, U. S. F.

2017-02-01

Dengue is a major health problem in the tropical and sub-tropical regions. The development of antiviral that targeting dengue’s host enzyme can be more effective and efficient treatment than the viral enzyme. Host enzyme processing α-glucosidase I has an important role in the maturation process of dengue virus envelope glycoprotein. The inhibition of processing α-glucosidase I can become a promising target for dengue fever treatment. The antiviral approach using in silico fragment-based drug design can generate drug candidates with high binding affinity. In this research, 198.621 compounds were obtained from ZINC15 Biogenic Database. These compounds were screened to find the favorable fragments according to Rules of Three and pharmacological properties. The screening fragments were docked into the active site of processing α-glucosidase I. The potential fragment candidates from the molecular docking simulation were linked with castanospermine (CAST) to generate ligands with a better binding affinity. The Analysis of ligand - enzyme interaction showed ligands with code LRS 22, 28, and 47 have the better binding free energy than the standard ligand. Ligand LRS 28 (N-2-4-methyl-5-((1S,3S,6S,7R,8R,8aR)-1,6,7,8-tetrahydroxyoctahydroindolizin-3-yl) pentyl) indolin-1-yl) propionamide) itself among the other ligands has the lowest binding free energy. Pharmacological properties prediction also showed the ligands LRS 22, 28, and 47 can be promising as the dengue fever drug candidates.

Processing of N-linked oligosaccharides from precursor- to mature-form herpes simplex virus type 1 glycoprotein gC.

PubMed

Serafini-Cessi, F; Dall'Olio, F; Pereira, L; Campadelli-Fiume, G

1984-09-01

Immature and mature forms of glycoprotein gC were purified by immunoadsorbent from herpes simplex virus type 1-infected BHK cells labeled with [3H]mannose for a 20-min pulse or for 11 h followed by a 3-h chase. The nature of N-asparagine-linked oligosaccharides carried by the immature form, pgC (molecular weight = 92,000), and the mature gC (molecular weight = 120,000) has been investigated. All pronase-digested glycopeptides of pgC were susceptible to endo-beta-N-acetylglucosaminidase H treatment; thus they have a high-mannose structure. Using thin-layer chromatography to separate endo-beta-N-acetylglucosaminidase H-cleaved oligosaccharides, polymannosyl chains of different sizes, ranging from Man9GlcNAc to Man5GlcNAc, were separated. The major components were Man8GlcNAc and Man7GlcNAc, suggesting that pgC labeled in a 20-min pulse represents the form of glycoprotein already routed to the Golgi apparatus. Analysis of glycopeptides of mature gC showed that the majority (95%) of N-linked glycans were converted to complex-type glycans. Ion-exchange chromatography and affinity chromatography on concanavalin A-Sepharose and leucoagglutinin-agarose revealed that diantennary and triantennary glycans predominated, whereas tetrantennary chains were not present. Parts of the di- and triantennary chains were not fully sialylated. The high heterogeneity of complex-type chains found in mature gC may be related to the high number of N-glycosylation sites of the glycoprotein as predicted by DNA sequencing studies (Frink et al., J. Virol. 45:634-647, 1983).

Humanization of Antibodies Using Heavy Chain Complementarity-determining Region 3 Grafting Coupled with in Vitro Somatic Hypermutation*

PubMed Central

Bowers, Peter M.; Neben, Tamlyn Y.; Tomlinson, Geoffery L.; Dalton, Jennifer L.; Altobell, Larry; Zhang, Xue; Macomber, John L.; Wu, Betty F.; Toobian, Rachelle M.; McConnell, Audrey D.; Verdino, Petra; Chau, Betty; Horlick, Robert A.; King, David J.

2013-01-01

A method for simultaneous humanization and affinity maturation of monoclonal antibodies has been developed using heavy chain complementarity-determining region (CDR) 3 grafting combined with somatic hypermutation in vitro. To minimize the amount of murine antibody-derived antibody sequence used during humanization, only the CDR3 region from a murine antibody that recognizes the cytokine hβNGF was grafted into a nonhomologous human germ line V region. The resulting CDR3-grafted HC was paired with a CDR-grafted light chain, displayed on the surface of HEK293 cells, and matured using in vitro somatic hypermutation. A high affinity humanized antibody was derived that was considerably more potent than the parental antibody, possessed a low pm dissociation constant, and demonstrated potent inhibition of hβNGF activity in vitro. The resulting antibody contained half the heavy chain murine donor sequence compared with the same antibody humanized using traditional methods. PMID:23355464

Specific Fluorine Labeling of the HyHEL10 Antibody Affects Antigen Binding and Dynamics

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

Acchione, Mauro; Lee, Yi-Chien; DeSantis, Morgan E.

To more fully understand the molecular mechanisms responsible for variations in binding affinity with antibody maturation, we explored the use of site specific fluorine labeling and {sup 19}F nuclear magnetic resonance (NMR). Several single-chain (scFv) antibodies, derived from an affinity-matured series of anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either complete or individual replacement of tryptophan residues with 5-fluorotryptophan ({sup 5F}W). An array of biophysical techniques was used to gain insight into the impact of fluorine substitution on the overall protein structure and antigen binding. SPR measurements indicated that {sup 5F}W incorporation lowered binding affinity for themore » HEL antigen. The degree of analogue impact was residue-dependent, and the greatest decrease in affinity was observed when {sup 5F}W was substituted for residues near the binding interface. In contrast, corresponding crystal structures in complex with HEL were essentially indistinguishable from the unsubstituted antibody. {sup 19}F NMR analysis showed severe overlap of signals in the free fluorinated protein that was resolved upon binding to antigen, suggesting very distinct chemical environments for each {sup 5F}W in the complex. Preliminary relaxation analysis suggested the presence of chemical exchange in the antibody-antigen complex that could not be observed by X-ray crystallography. These data demonstrate that fluorine NMR can be an extremely useful tool for discerning structural changes in scFv antibody-antigen complexes with altered function that may not be discernible by other biophysical techniques.« less

IA-2 autoantibody affinity in children at risk for type 1 diabetes.

PubMed

Krause, Stephanie; Chmiel, Ruth; Bonifacio, Ezio; Scholz, Marlon; Powell, Michael; Furmaniak, Jadwiga; Rees Smith, Bernard; Ziegler, Anette-G; Achenbach, Peter

2012-12-01

Autoantibodies to insulinoma-associated protein 2 (IA-2A) are associated with increased risk for type 1 diabetes. Here we examined IA-2A affinity and epitope specificity to assess heterogeneity in response intensity in relation to pathogenesis and diabetes risk in 50 children who were prospectively followed from birth. At first IA-2A appearance, affinity ranged from 10(7) to 10(11)L/mol and was high (>1.0×10(9)L/mol) in 41 (82%) children. IA-2A affinity was not associated with epitope specificity or HLA class II haplotype. On follow-up, affinity increased or remained high, and IA-2A were commonly against epitopes within the protein tyrosine phosphatase-like IA-2 domain and the homologue protein IA-2β. IA-2A were preceded or accompanied by other islet autoantibodies in 49 (98%) children, of which 34 progressed to diabetes. IA-2A affinity did not stratify diabetes risk. In conclusion, the IA-2A response in children is intense with rapid maturation against immunogenic epitopes and a strong association with diabetes development. Copyright © 2012 Elsevier Inc. All rights reserved.

Optimization of affinity, specificity and function of designed influenza inhibitors using deep sequencing

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

Whitehead, Timothy A.; Chevalier, Aaron; Song, Yifan

2012-06-19

We show that comprehensive sequence-function maps obtained by deep sequencing can be used to reprogram interaction specificity and to leapfrog over bottlenecks in affinity maturation by combining many individually small contributions not detectable in conventional approaches. We use this approach to optimize two computationally designed inhibitors against H1N1 influenza hemagglutinin and, in both cases, obtain variants with subnanomolar binding affinity. The most potent of these, a 51-residue protein, is broadly cross-reactive against all influenza group 1 hemagglutinins, including human H2, and neutralizes H1N1 viruses with a potency that rivals that of several human monoclonal antibodies, demonstrating that computational design followedmore » by comprehensive energy landscape mapping can generate proteins with potential therapeutic utility.« less

The activation threshold of CD4+ T cells is defined by TCR/peptide-MHC class II interactions in the thymic medulla.

PubMed

Stephen, Tom Li; Tikhonova, Anastasia; Riberdy, Janice M; Laufer, Terri M

2009-11-01

Immature thymocytes that are positively selected based upon their response to self-peptide-MHC complexes develop into mature T cells that are not overtly reactive to those same complexes. Developmental tuning is the active process through which TCR-associated signaling pathways of single-positive thymocytes are attenuated to respond appropriately to the peptide-MHC molecules that will be encountered in the periphery. In this study, we explore the mechanisms that regulate the tuning of CD4(+) single-positive T cells to MHC class II encountered in the thymic medulla. Experiments with murine BM chimeras demonstrate that tuning can be mediated by MHC class II expressed by either thymic medullary epithelial cells or thymic dendritic cells. Tuning does not require the engagement of CD4 by MHC class II on stromal cells. Rather, it is mediated by interactions between MHC class II and the TCR. To understand the molecular changes that distinguish immature hyperactive T cells from tuned mature CD4(+) T cells, we compared their responses to TCR stimulation. The altered response of mature CD4 single-positive thymocytes is characterized by the inhibition of ERK activation by low-affinity self-ligands and increased expression of the inhibitory tyrosine phosphatase SHP-1. Thus, persistent TCR engagement by peptide-MHC class II on thymic medullary stroma inhibits reactivity to self-Ags and prevents autoreactivity in the mature repertoire.

Promotion of tyrosinase folding in COS 7 cells by calnexin.

PubMed

Toyofuku, K; Wada, I; Hirosaki, K; Park, J S; Hori, Y; Jimbow, K

1999-01-01

To understand the process of expression of tyrosinase, a key enzyme of melanogenesis, we examined its maturation in the endoplasmic reticulum (ER) by using a heterogeneous expression system. When human tyrosinase cDNA was introduced into COS 7 cells, tyrosinase activity was minimally detected. Immunofluorescence study revealed that tyrosinase was immunolocalized in the nuclear rim, the reticular network, and the punctuated structures. Because a cytoplasmic tail of tyrosinase-gene family protein functions as a lysosomal targeting signal in non-melanocytic cells, and immature and/or misfolded molecules are selectively retained in the ER, the observed localization suggested the inefficient maturation in the COS 7 cells. We thus examined if supplementation of calnexin, a membrane-bound chaperone with affinity for oligosaccharide-processing intermediates containing monoglucose, could improve the process. As expected, the activity was enhanced approximately 2-fold by co-transfection of cDNA encoding calnexin. In contrast, co-transfection of the cytosolic tail-free calnexin, which inhibits calnexin function by allowing premature egress of its ligands from the ER, suppressed expression of this enhanced tyrosinase activity. When alpha-glucosidase activity, which is required for calnexin function, was inhibited by castanospermine (CST) treatment, expression of tyrosinase activity was completely abolished. To confirm the direct involvement of calnexin in tyrosinase maturation, the interaction of calnexin with tyrosinase was examined. Immunoprecipitation of calnexin from extracts of [35S]methionine labeled cells with anti-calnexin antibody revealed that the association is highest immediately after the pulse and that nascent tyrosinase is gradually dissociated upon chase. The association was completely inhibited when CST was included in the medium. Hence, we suggest that the proper folding of tyrosinase is largely dependent on its direct interaction with calnexin for the determined duration in the ER.

Affinity Maturation of an Anti-V Antigen IgG Expressed In Situ Via Adenovirus Gene Delivery Confers Enhanced Protection Against Yersinia pestis Challenge

PubMed Central

Van Blarcom, Thomas J.; Sofer-Podesta, Carolina; Ang, John; Boyer, Julie L.; Crystal, Ronald G.; Georgiou, George

2013-01-01

Genetic transfer of neutralizing antibodies has been shown to confer strong and persistent protection against bacterial and viral infectious agents. While it is well established that for many exogenous neutralizing antibodies increased antigen affinity correlates with protection, the effect of antigen affinity on antibodies produced in situ following adenoviral gene transfer has not been examined. The mouse IgG2b monoclonal antibody 2C12.4 recognizes the Yersinia pestis Type III secretion apparatus protein LcrV (V antigen) and confers protection in mice when administered as an IgG intraperitoneally or, following genetic immunization with engineered, replication-defective serotype 5 human adenovirus (Ad) 1. 2C12.4 was expressed as a scFv fragment in E. coli and was shown to display a KD=3.5 nM by surface plasmon resonance (SPR) analysis. The 2C12.4 scFv was subjected to random mutagenesis and variants with increased affinity were isolated by flow cytometry using the Anchored Periplasmic Expression (APEx) bacterial display system. After a single round of mutagenesis, variants displaying up to 35-fold lower KD values (H8, KD=100 pM) were isolated. The variable domains of the H8 scFv were used to replace those of the parental 2C12.4 IgG encoded in the Ad vector, AdαV giving rise to AdαV.H8. The two adenoviral vectors resulted in similar titers of anti-V antigen antibodies 3 days post-immunization with 109, 1010 or 1011 particle units. Following intranasal challenge with 363 LD50Y. pestis CO92, 54% of the mice immunized with 1010 pu of AdαV.H8 survived at the 14 day end point compared to only 15% survivors for the group immunized with AdαV expressing the lower affinity 2C12.4 (P<0.04, AdαV versus AdαV.H8). These results indicate that affinity maturation of a neutralizing antibody delivered by genetic transfer may confer increased protection not only for Y. pestis challenge but possibly for other pathogens. PMID:20393511

von Willebrand factor (VWF) propeptide binding to VWF D'D3 domain attenuates platelet activation and adhesion.

PubMed

Madabhushi, Sri R; Shang, Chengwei; Dayananda, Kannayakanahalli M; Rittenhouse-Olson, Kate; Murphy, Mary; Ryan, Thomas E; Montgomery, Robert R; Neelamegham, Sriram

2012-05-17

Noncovalent association between the von Willebrand factor (VWF) propeptide (VWFpp) and mature VWF aids N-terminal multimerization and protein compartmentalization in storage granules. This association is currently thought to dissipate after secretion into blood. In the present study, we examined this proposition by quantifying the affinity and kinetics of VWFpp binding to mature VWF using surface plasmon resonance and by developing novel anti-VWF D'D3 mAbs. Our results show that the only binding site for VWFpp in mature VWF is in its D'D3 domain. At pH 6.2 and 10mM Ca(2+), conditions mimicking intracellular compartments, VWFpp-VWF binding occurs with high affinity (K(D) = 0.2nM, k(off) = 8 × 10(-5) s(-1)). Significant, albeit weaker, binding (K(D) = 25nM, k(off) = 4 × 10(-3) s(-1)) occurs under physiologic conditions of pH 7.4 and 2.5mM Ca(2+). This interaction was also observed in human plasma (K(D) = 50nM). The addition of recombinant VWFpp in both flow-chamber-based platelet adhesion assays and viscometer-based shear-induced platelet aggregation and activation studies reduced platelet adhesion and activation partially. Anti-D'D3 mAb DD3.1, which blocks VWFpp binding to VWF-D'D3, also abrogated platelet adhesion, as shown by shear-induced platelet aggregation and activation studies. Our data demonstrate that VWFpp binding to mature VWF occurs in the circulation, which can regulate the hemostatic potential of VWF by reducing VWF binding to platelet GpIbα.

Structure-guided engineering of Anticalins with improved binding behavior and biochemical characteristics for application in radio-immuno imaging and/or therapy.

PubMed

Eggenstein, E; Eichinger, A; Kim, H-J; Skerra, A

2014-02-01

Modern strategies in radio-immuno therapy and in vivo imaging require robust, small, and specific ligand-binding proteins. In this context we have previously developed artificial lipocalins, so-called Anticalins, with high binding activity toward rare-earth metal-chelate complexes using combinatorial protein design. Here we describe further improvement of the Anticalin C26 via in vitro affinity maturation to yield CL31, which has a fourfold slower dissociation half-life above 2h. Also, we present the crystallographic analyses of both the initial and the improved Anticalin, providing insight into the molecular mechanism of chelated metal binding and the role of amino acid substitutions during the step-wise affinity maturation. Notably, one of the four structurally variable loops that form the ligand pocket in the lipocalin scaffold undergoes a significant conformational change from C26 to CL31, acting as a lid that closes over the accommodated metal-chelate ligand. A systematic mutational study indicated that further improvement of ligand affinity is difficult to achieve while providing clues on the contribution of relevant side chains in the engineered binding pocket. Unexpectedly, some of the amino acid replacements led to strong increases - more then 10-fold - in the yield of soluble protein from periplasmic secretion in Escherichia coli. Copyright © 2013 Elsevier Inc. All rights reserved.

The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6

PubMed Central

Boschert, V.; Frisch, C.; Back, J. W.; van Pee, K.; Weidauer, S. E.; Muth, E.-M.; Schmieder, P.; Beerbaum, M.; Knappik, A.; Timmerman, P.

2016-01-01

The glycoprotein sclerostin has been identified as a negative regulator of bone growth. It exerts its function by interacting with the Wnt co-receptor LRP5/6, blocks the binding of Wnt factors and thereby inhibits Wnt signalling. Neutralizing anti-sclerostin antibodies are able to restore Wnt activity and enhance bone growth thereby presenting a new osteoanabolic therapy approach for diseases such as osteoporosis. We have generated various Fab antibodies against human and murine sclerostin using a phage display set-up. Biochemical analyses have identified one Fab developed against murine sclerostin, AbD09097 that efficiently neutralizes sclerostin's Wnt inhibitory activity. In vitro interaction analysis using sclerostin variants revealed that this neutralizing Fab binds to sclerostin's flexible second loop, which has been shown to harbour the LRP5/6 binding motif. Affinity maturation was then applied to AbD09097, providing a set of improved neutralizing Fab antibodies which particularly bind human sclerostin with enhanced affinity. Determining the crystal structure of AbD09097 provides first insights into how this antibody might recognize and neutralize sclerostin. Together with the structure–function relationship derived from affinity maturation these new data will foster the rational design of new and highly efficient anti-sclerostin antibodies for the therapy of bone loss diseases such as osteoporosis. PMID:27558933

Processing of N-linked oligosaccharides from precursor- to mature-form herpes simplex virus type 1 glycoprotein gC.

PubMed Central

Serafini-Cessi, F; Dall'Olio, F; Pereira, L; Campadelli-Fiume, G

1984-01-01

Immature and mature forms of glycoprotein gC were purified by immunoadsorbent from herpes simplex virus type 1-infected BHK cells labeled with [3H]mannose for a 20-min pulse or for 11 h followed by a 3-h chase. The nature of N-asparagine-linked oligosaccharides carried by the immature form, pgC (molecular weight = 92,000), and the mature gC (molecular weight = 120,000) has been investigated. All pronase-digested glycopeptides of pgC were susceptible to endo-beta-N-acetylglucosaminidase H treatment; thus they have a high-mannose structure. Using thin-layer chromatography to separate endo-beta-N-acetylglucosaminidase H-cleaved oligosaccharides, polymannosyl chains of different sizes, ranging from Man9GlcNAc to Man5GlcNAc, were separated. The major components were Man8GlcNAc and Man7GlcNAc, suggesting that pgC labeled in a 20-min pulse represents the form of glycoprotein already routed to the Golgi apparatus. Analysis of glycopeptides of mature gC showed that the majority (95%) of N-linked glycans were converted to complex-type glycans. Ion-exchange chromatography and affinity chromatography on concanavalin A-Sepharose and leucoagglutinin-agarose revealed that diantennary and triantennary glycans predominated, whereas tetrantennary chains were not present. Parts of the di- and triantennary chains were not fully sialylated. The high heterogeneity of complex-type chains found in mature gC may be related to the high number of N-glycosylation sites of the glycoprotein as predicted by DNA sequencing studies (Frink et al., J. Virol. 45:634-647, 1983). Images PMID:6088806

SRC-like adaptor protein regulates B cell development and function.

PubMed

Dragone, Leonard L; Myers, Margaret D; White, Carmen; Sosinowski, Tomasz; Weiss, Arthur

2006-01-01

The avidity of BCRs and TCRs influences signal strength during processes of lymphocyte development. Avidity is determined by both the intrinsic affinity for Ag and surface levels of the Ag receptor. The Src-like adaptor protein (SLAP) is a regulator of TCR levels on thymocytes, and its deficiency alters thymocyte development. We hypothesized that SLAP, which is expressed in B cells, also is important in regulating BCR levels, signal strength, and B cell development. To test this hypothesis, we analyzed the B cell compartment in SLAP-deficient mice. We found increased splenic B cell numbers and decreased surface IgM levels on mature, splenic B cells deficient in SLAP. Immature bone marrow and splenic B cells from BCR-transgenic, SLAP-deficient mice were found to express higher surface levels of IgM. In contrast, mature splenic B cells from BCR-transgenic mice expressed decreased levels of surface BCR associated with decreased calcium flux and activation-induced markers, compared with controls. These data suggest that SLAP regulates BCR levels and signal strength during lymphocyte development.

Structure-Based Rational Design of a Toll-like Receptor 4 (TLR4) Decoy Receptor with High Binding Affinity for a Target Protein

PubMed Central

Lee, Sang-Chul; Hong, Seungpyo; Park, Keunwan; Jeon, Young Ho; Kim, Dongsup; Cheong, Hae-Kap; Kim, Hak-Sung

2012-01-01

Repeat proteins are increasingly attracting much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural features. Nonetheless, engineering interaction interface and understanding molecular basis for affinity maturation of repeat proteins still remain a challenge. Here, we present a structure-based rational design of a repeat protein with high binding affinity for a target protein. As a model repeat protein, a Toll-like receptor4 (TLR4) decoy receptor composed of leucine-rich repeat (LRR) modules was used, and its interaction interface was rationally engineered to increase the binding affinity for myeloid differentiation protein 2 (MD2). Based on the complex crystal structure of the decoy receptor with MD2, we first designed single amino acid substitutions in the decoy receptor, and obtained three variants showing a binding affinity (KD) one-order of magnitude higher than the wild-type decoy receptor. The interacting modes and contributions of individual residues were elucidated by analyzing the crystal structures of the single variants. To further increase the binding affinity, single positive mutations were combined, and two double mutants were shown to have about 3000- and 565-fold higher binding affinities than the wild-type decoy receptor. Molecular dynamics simulations and energetic analysis indicate that an additive effect by two mutations occurring at nearby modules was the major contributor to the remarkable increase in the binding affinities. PMID:22363519

Affinity maturation of a portable Fab–RNA module for chaperone-assisted RNA crystallography

PubMed Central

Koirala, Deepak; Shelke, Sandip A; Dupont, Marcel; Ruiz, Stormy; DasGupta, Saurja; Bailey, Lucas J; Benner, Steven A; Piccirilli, Joseph A

2018-01-01

Abstract Antibody fragments such as Fabs possess properties that can enhance protein and RNA crystallization and therefore can facilitate macromolecular structure determination. In particular, Fab BL3–6 binds to an AAACA RNA pentaloop closed by a GC pair with ∼100 nM affinity. The Fab and hairpin have served as a portable module for RNA crystallization. The potential for general application make it desirable to adjust the properties of this crystallization module in a manner that facilitates its use for RNA structure determination, such as ease of purification, surface entropy or binding affinity. In this work, we used both in vitro RNA selection and phage display selection to alter the epitope and paratope sides of the binding interface, respectively, for improved binding affinity. We identified a 5′-GNGACCC-3′ consensus motif in the RNA and S97N mutation in complimentarity determining region L3 of the Fab that independently impart about an order of magnitude improvement in affinity, resulting from new hydrogen bonding interactions. Using a model RNA, these modifications facilitated crystallization under a wider range of conditions and improved diffraction. The improved features of the Fab–RNA module may facilitate its use as an affinity tag for RNA purification and imaging and as a chaperone for RNA crystallography. PMID:29309709

FigA, a putative homolog of low-affinity calcium system member Fig1 in Saccharomyces cerevisiae, is involved in growth and asexual and sexual development in Aspergillus nidulans.

PubMed

Zhang, Shizhu; Zheng, Hailin; Long, Nanbiao; Carbó, Natalia; Chen, Peiying; Aguilar, Pablo S; Lu, Ling

2014-02-01

Calcium-mediated signaling pathways are widely employed in eukaryotes and are implicated in the regulation of diverse biological processes. In Saccharomyces cerevisiae, at least two different calcium uptake systems have been identified: the high-affinity calcium influx system (HACS) and the low-affinity calcium influx system (LACS). Compared to the HACS, the LACS in fungi is not well known. In this study, FigA, a homolog of the LACS member Fig1 from S. cerevisiae, was functionally characterized in the filamentous fungus Aspergillus nidulans. Loss of figA resulted in retardant hyphal growth and a sharp reduction of conidial production. Most importantly, FigA is essential for the homothallic mating (self-fertilization) process; further, FigA is required for heterothallic mating (outcrossing) in the absence of HACS midA. Interestingly, in a figA deletion mutant, adding extracellular Ca(2+) rescued the hyphal growth defects but could not restore asexual and sexual reproduction. Furthermore, quantitative PCR results revealed that figA deletion sharply decreased the expression of brlA and nsdD, which are known as key regulators during asexual and sexual development, respectively. In addition, green fluorescent protein (GFP) tagging at the C terminus of FigA (FigA::GFP) showed that FigA localized to the center of the septum in mature hyphal cells, to the location between vesicles and metulae, and between the junctions of metulae and phialides in conidiophores. Thus, our findings suggest that FigA, apart from being a member of a calcium uptake system in A. nidulans, may play multiple unexplored roles during hyphal growth and asexual and sexual development.

A truncated and dimeric format of an Affibody library on bacteria enables FACS-mediated isolation of amyloid-beta aggregation inhibitors with subnanomolar affinity.

PubMed

Lindberg, Hanna; Härd, Torleif; Löfblom, John; Ståhl, Stefan

2015-09-01

The amyloid hypothesis suggests that accumulation of amyloid β (Aβ) peptides in the brain is involved in development of Alzheimer's disease. We previously generated a small dimeric affinity protein that inhibited Aβ aggregation by sequestering the aggregation prone parts of the peptide. The affinity protein is originally based on the Affibody scaffold, but is evolved to a distinct interaction mechanism involving complex structural rearrangement in both the Aβ peptide and the affinity proteins upon binding. The aim of this study was to decrease the size of the dimeric affinity protein and significantly improve its affinity for the Aβ peptide to increase its potential as a future therapeutic agent. We combined a rational design approach with combinatorial protein engineering to generate two different affinity maturation libraries. The libraries were displayed on staphylococcal cells and high-affinity Aβ-binding molecules were isolated using flow-cytometric sorting. The best performing candidate binds Aβ with a KD value of around 300 pM, corresponding to a 50-fold improvement in affinity relative to the first-generation binder. The new dimeric Affibody molecule was shown to capture Aβ1-42 peptides from spiked E. coli lysate. Altogether, our results demonstrate successful engineering of this complex binder for increased affinity to the Aβ peptide. © 2015 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution-Non-Commercial-NoDerivs Licence, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

The High-Affinity Phosphate Transporter GmPT5 Regulates Phosphate Transport to Nodules and Nodulation in Soybean1[W][OA

PubMed Central

Qin, Lu; Zhao, Jing; Tian, Jiang; Chen, Liyu; Sun, Zhaoan; Guo, Yongxiang; Lu, Xing; Gu, Mian; Xu, Guohua; Liao, Hong

2012-01-01

Legume biological nitrogen (N) fixation is the most important N source in agroecosystems, but it is also a process requiring a considerable amount of phosphorus (P). Therefore, developing legume varieties with effective N2 fixation under P-limited conditions could have profound significance for improving agricultural sustainability. We show here that inoculation with effective rhizobial strains enhanced soybean (Glycine max) N2 fixation and P nutrition in the field as well as in hydroponics. Furthermore, we identified and characterized a nodule high-affinity phosphate (Pi) transporter gene, GmPT5, whose expression was elevated in response to low P. Yeast heterologous expression verified that GmPT5 was indeed a high-affinity Pi transporter. Localization of GmPT5 expression based on β-glucuronidase staining in soybean composite plants with transgenic roots and nodules showed that GmPT5 expression occurred principally in the junction area between roots and young nodules and in the nodule vascular bundles for juvenile and mature nodules, implying that GmPT5 might function in transporting Pi from the root vascular system into nodules. Overexpression or knockdown of GmPT5 in transgenic composite soybean plants altered nodulation and plant growth performance, which was partially dependent on P supply. Through both in situ and in vitro 33P uptake assays using transgenic soybean roots and nodules, we demonstrated that GmPT5 mainly functions in transporting Pi from roots to nodules, especially under P-limited conditions. We conclude that the high-affinity Pi transporter, GmPT5, controls Pi entry from roots to nodules, is critical for maintaining Pi homeostasis in nodules, and subsequently regulates soybean nodulation and growth performance. PMID:22740613

Integrity of immunoglobulin variable regions is supported by GANP during AID-induced somatic hypermutation in germinal center B cells

PubMed Central

Eid, Mohammed Mansour Abbas; Shimoda, Mayuko; Singh, Shailendra Kumar; Almofty, Sarah Ameen; Pham, Phuong; Goodman, Myron F.; Maeda, Kazuhiko; Sakaguchi, Nobuo

2017-01-01

Abstract Immunoglobulin affinity maturation depends on somatic hypermutation (SHM) in immunoglobulin variable (IgV) regions initiated by activation-induced cytidine deaminase (AID). AID induces transition mutations by C→U deamination on both strands, causing C:G→T:A. Error-prone repairs of U by base excision and mismatch repairs (MMRs) create transversion mutations at C/G and mutations at A/T sites. In Neuberger’s model, it remained to be clarified how transition/transversion repair is regulated. We investigate the role of AID-interacting GANP (germinal center-associated nuclear protein) in the IgV SHM profile. GANP enhances transition mutation of the non-transcribed strand G and reduces mutation at A, restricted to GYW of the AID hotspot motif. It reduces DNA polymerase η hotspot mutations associated with MMRs followed by uracil-DNA glycosylase. Mutation comparison between IgV complementary and framework regions (FWRs) by Bayesian statistical estimation demonstrates that GANP supports the preservation of IgV FWR genomic sequences. GANP works to maintain antibody structure by reducing drastic changes in the IgV FWR in affinity maturation. PMID:28541550

Structure-function analyses unravel distinct effects of allosteric inhibitors of HIV-1 integrase on viral maturation and integration.

PubMed

Bonnard, Damien; Le Rouzic, Erwann; Eiler, Sylvia; Amadori, Céline; Orlov, Igor; Bruneau, Jean-Michel; Brias, Julie; Barbion, Julien; Chevreuil, Francis; Spehner, Danièle; Chasset, Sophie; Ledoussal, Benoit; Moreau, François; Saïb, Ali; Klaholz, Bruno P; Emiliani, Stéphane; Ruff, Marc; Zamborlini, Alessia; Benarous, Richard

2018-04-20

Recently, a new class of HIV-1 integrase (IN) inhibitors with a dual mode of action, called IN-LEDGF/p75 allosteric inhibitors (INLAIs), was described. Designed to interfere with the IN-LEDGF/p75 interaction during viral integration, unexpectedly, their major impact was on virus maturation. This activity has been linked to induction of aberrant IN multimerization, whereas inhibition of the IN-LEDGF/p75 interaction accounts for weaker antiretroviral effect at integration. Because these dual activities result from INLAI binding to IN at a single binding site, we expected that these activities co-evolved together, driven by the affinity for IN. Using an original INLAI, MUT-A, and its activity on an Ala-125 (A125) IN variant, we found that these two activities on A125-IN can be fully dissociated: MUT-A-induced IN multimerization and the formation of eccentric condensates in viral particles, which are responsible for inhibition of virus maturation, were lost, whereas inhibition of the IN-LEDGF/p75 interaction and consequently integration was fully retained. Hence, the mere binding of INLAI to A125 IN is insufficient to promote the conformational changes of IN required for aberrant multimerization. By analyzing the X-ray structures of MUT-A bound to the IN catalytic core domain (CCD) with or without the Ala-125 polymorphism, we discovered that the loss of IN multimerization is due to stabilization of the A125-IN variant CCD dimer, highlighting the importance of the CCD dimerization energy for IN multimerization. Our study reveals that affinity for the LEDGF/p75-binding pocket is not sufficient to induce INLAI-dependent IN multimerization and the associated inhibition of viral maturation. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

In planta processing and glycosylation of a nematode CLAVATA3/ENDOSPERM SURROUNDING REGION-like effector and its interaction with a host CLAVATA2-like receptor to promote parasitism.

PubMed

Chen, Shiyan; Lang, Ping; Chronis, Demosthenis; Zhang, Sheng; De Jong, Walter S; Mitchum, Melissa G; Wang, Xiaohong

2015-01-01

Like other biotrophic plant pathogens, plant-parasitic nematodes secrete effector proteins into host cells to facilitate infection. Effector proteins that mimic plant CLAVATA3/ENDOSPERM SURROUNDING REGION-related (CLE) proteins have been identified in several cyst nematodes, including the potato cyst nematode (PCN); however, the mechanistic details of this cross-kingdom mimicry are poorly understood. Plant CLEs are posttranslationally modified and proteolytically processed to function as bioactive ligands critical to various aspects of plant development. Using ectopic expression coupled with nanoliquid chromatography-tandem mass spectrometry analysis, we show that the in planta mature form of proGrCLE1, a multidomain CLE effector secreted by PCN during infection, is a 12-amino acid arabinosylated glycopeptide (named GrCLE1-1Hyp4,7g) with striking structural similarity to mature plant CLE peptides. This glycopeptide is more resistant to hydrolytic degradation and binds with higher affinity to a CLAVATA2-like receptor (StCLV2) from potato (Solanum tuberosum) than its nonglycosylated forms. We further show that StCLV2 is highly up-regulated at nematode infection sites and that transgenic potatoes with reduced StCLV2 expression are less susceptible to PCN infection, indicating that interference of the CLV2-mediated signaling pathway confers nematode resistance in crop plants. These results strongly suggest that phytonematodes have evolved to utilize host cellular posttranslational modification and processing machinery for the activation of CLE effectors following secretion into plant cells and highlight the significance of arabinosylation in regulating nematode CLE effector activity. Our finding also provides evidence that multidomain CLEs are modified and processed similarly to single-domain CLEs, adding new insight into CLE maturation in plants. © 2015 American Society of Plant Biologists. All Rights Reserved.

Identification of proteins associated with the yeast mitochondrial RNA polymerase by tandem affinity purification

PubMed Central

Markov, Dmitriy A; Savkina, Maria; Anikin, Michael; Del Campo, Mark; Ecker, Karen; Lambowitz, Alan M; De Gnore, Jon P; McAllister, William T

2009-01-01

The abundance of mitochondrial (mt) transcripts varies under different conditions, and is thought to depend upon rates of transcription initiation, transcription termination/attenuation and RNA processing/degradation. The requirement to maintain the balance between RNA synthesis and processing may involve coordination between these processes; however, little is known about factors that regulate the activity of mtRNA polymerase (mtRNAP). Recent attempts to identify mtRNAP–protein interactions in yeast by means of a generalized tandem affinity purification (TAP) protocol were not successful, most likely because they involved a C-terminal mtRNAP–TAP fusion (which is incompatible with mtRNAP function) and because of the use of whole-cell solubilization protocols that did not preserve the integrity of mt protein complexes. Based upon the structure of T7 RNAP (to which mtRNAPs show high sequence similarity), we identified positions in yeast mtRNAP that allow insertion of a small affinity tag, confirmed the mature N-terminus, constructed a functional N-terminal TAP–mtRNAP fusion, pulled down associated proteins, and identified them by LC–MS–MS. Among the proteins found in the pull-down were a DEAD-box protein (Mss116p) and an RNA-binding protein (Pet127p). Previous genetic experiments suggested a role for these proteins in linking transcription and RNA degradation, in that a defect in the mt degradadosome could be suppressed by overexpression of either of these proteins or, independently, by mutations in either mtRNAP or its initiation factor Mtf1p. Further, we found that Mss116p inhibits transcription by mtRNAP in vitro in a steady-state reaction. Our results support the hypothesis that Mss116p and Pet127p are involved in modulation of mtRNAP activity. Copyright © 2009 John Wiley & Sons, Ltd. PMID:19536766

Random mutagenesis of BoNT/E Hc nanobody to construct a secondary phage-display library.

PubMed

Shahi, B; Mousavi Gargari, S L; Rasooli, I; Rajabi Bazl, M; Hoseinpoor, R

2014-08-01

To construct secondary mutant phage-display library of recombinant single variable domain (VHH) against botulinum neurotoxin E by error-prone PCR. The gene coding for specific VHH derived from the camel immunized with binding domain of botulinum neurotoxin E (BoNT/E) was amplified by error-prone PCR. Several biopanning rounds were used to screen the phage-displaying BoNT/E Hc nanobodies. The final nanobody, SHMR4, with increased affinity recognized BoNT/E toxin with no cross-reactivity with other antigens especially with related BoNT toxins. The constructed nanobody could be a suitable candidate for VHH-based biosensor production to detect the Clostridium botulinum type E. Diagnosis and treatment of botulinum neurotoxins are important. Generation of high-affinity antibodies based on the construction of secondary libraries using affinity maturation step leads to the development of reagents for precise diagnosis and therapy. © 2014 The Society for Applied Microbiology.

ESCRT-III-Associated Protein ALIX Mediates High-Affinity Phosphate Transporter Trafficking to Maintain Phosphate Homeostasis in Arabidopsis

PubMed Central

Cardona-López, Ximena; Cuyas, Laura; Marín, Elena; Irigoyen, María Luisa; Gil, Erica; Puga, María Isabel; Bligny, Richard; Nussaume, Laurent; Geldner, Niko; Paz-Ares, Javier

2015-01-01

Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate-sufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life. PMID:26342016

The glycocalyx promotes cooperative binding and clustering of adhesion receptors.

PubMed

Xu, Guang-Kui; Qian, Jin; Hu, Jinglei

2016-05-18

Cell adhesion plays a pivotal role in various biological processes, e.g., immune responses, cancer metastasis, and stem cell differentiation. The adhesion behaviors depend subtly on the binding kinetics of receptors and ligands restricted at the cell-substrate interfaces. Although much effort has been directed toward investigating the kinetics of adhesion molecules, the role of the glycocalyx, anchored on cell surfaces as an exterior layer, is still unclear. In this paper, we propose a theoretical approach to study the collective binding kinetics of a few and a large number of binders in the presence of the glycocalyx, representing the cases of initial and mature adhesions of cells, respectively. The analytical results are validated by finding good agreement with our Monte Carlo simulations. In the force loading case, the on-rate and affinity increase as more bonds form, whereas this cooperative effect is not observed in the displacement loading case. The increased thickness and stiffness of the glycocalyx tend to decrease the affinity for a few bonds, while they have less influence on the affinity for a large number of bonds. Moreover, for a flexible membrane with thermally-excited shape fluctuations, the glycocalyx is exhibited to promote the formation of bond clusters, mainly due to the cooperative binding of binders. This study helps to understand the cooperative kinetics of adhesion receptors under physiologically relevant loading conditions and sheds light on the novel role of the glycocalyx in cell adhesion.

Optimization of the production and purification processes of carnobacteriocins Cbn BM1 and Cbn B2 from Carnobacterium maltaromaticum CP5 by heterologous expression in Escherichia coli.

PubMed

Jasniewski, Jordane; Cailliez-Grimal, Catherine; Gelhaye, Eric; Revol-Junelles, Anne-Marie

2008-04-01

An optimization of the production and purification processes of carnobacteriocins Cbn BM1 and Cbn B2 from Carnobacterium maltaromaticum CP5, by heterologous expression in Escherichia coli is described. The genes encoding mature bacteriocin were cloned into an E. coli expression system and expressed as a fusion protein with a thermostable thioredoxin. Recombinant E. coli were cultivated following a fed-batch fermentation process with pH, temperature and oxygenation regulation. The overexpression of the fusion proteins was improved by replacing IPTG by lactose. The fusion proteins were purified by thermal coagulation followed by affinity chromatography. The thioredoxin fusion protein was removed by using CNBr instead of enterokinase and the carnobacteriocins were recovered by reverse-phase chromatography. These optimizations led us to produce up to 320 mg of pure protein per liter of culture, which is four to ten fold higher than what is described for other heterologous expression systems.

A new species of Erythrostemon (Leguminosae, Caesalpinioideae) from the western Río Balsas Depression, Mexico.

PubMed

Sotuyo, Solange; Contreras-Jiménez, José Luis; Lewis, Gwilym P

2017-01-01

A new legume species from a seasonally dry forest of the Western Río Balsas Depression, in the states of Guerrero and Michoacán, Mexico, Erythrostemon guevarafeferii , is herein described and illustrated. The new species shows morphological affinities with Erythrostemon hintonii , from which it is distinguished in having fewer leaflets per pinna, mature leaflets disposed toward the upper half of the pinnae rachises, long inflorescences on curved slender peduncles, abundant red glands on its flowers and inflorescences, and its fruit glabrous with red stipitate glands at maturity. A taxonomic key to the Río Balsas Depression species of Erythrostemon is included.

Development of a 'mouse and human cross-reactive' affinity-matured exosite inhibitory human antibody specific to TACE (ADAM17) for cancer immunotherapy.

PubMed

Kwok, Hang Fai; Botkjaer, Kenneth A; Tape, Christopher J; Huang, Yanchao; McCafferty, John; Murphy, Gillian

2014-06-01

We previously showed that a human anti-TACE antibody, D1(A12), is a potent inhibitor of TNF-α converting enzyme (TACE) ectodomain proteolysis and has pharmacokinetic properties suitable for studies of the inhibition of TACE-dependent growth factor shedding in relation to possible therapeutic applications. However, the lack of murine TACE immunoreactivity limits pre-clinical in vivo studies to human xenograft models which are poor analogies to in situ pathology and are not considered clinically predictive. Here, to overcome these limitations, we set out to develop a 'mouse and human cross-reactive' specific anti-TACE antibody. We first re-investigated the originally selected anti-TACE ectodomain phage-display clones, and isolated a lead 'mouse-human cross-reactive' anti-TACE scFv, clone A9. We reformatted scFv-A9 into an IgG2 framework for comprehensive biochemical and cellular characterization and further demonstrated that A9 is an exosite TACE inhibitor. However, surface plasmon resonance analysis and quenched-fluorescent (QF) peptide assay indicated that IgG reformatting of A9 caused low binding affinity and an 80-fold reduction in TACE ectodomain inhibition, severely limiting its efficacy. To address this, we constructed second generation phage-display randomization libraries focused on the complementarity-determining region 3, and carried out affinity selections shuffling between human and mouse TACE ectodomain as antigen in addition to an off-rate selection to increase the chance of affinity improvement. The bespoke 'three-step' selections enabled a 100-fold affinity enhancement of A9 IgG, and also improved its IC50 in a QF peptide assay to 0.2 nM. In human and mouse cancer cell assays, matured A9 IgG showed significant cell-surface TACE inhibition as a monotherapy or combination therapy with chemotherapeutic agent. Collectively, these data suggest that we successfully developed an exosite inhibitor of TACE with sub-nanomolar affinity, which possesses both murine and human immunoreactive properties that can be used for in vivo application in murine pre-clinical cancer models. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Expression in Escherichia coli, purification, refolding and antifungal activity of an osmotin from Solanum nigrum

PubMed Central

Campos, Magnólia de A; Silva, Marilia S; Magalhães, Cláudio P; Ribeiro, Simone G; Sarto, Rafael PD; Vieira, Eduardo A; Grossi de Sá, Maria F

2008-01-01

Background Heterologous protein expression in microorganisms may contribute to identify and demonstrate antifungal activity of novel proteins. The Solanum nigrum osmotin-like protein (SnOLP) gene encodes a member of pathogenesis-related (PR) proteins, from the PR-5 sub-group, the last comprising several proteins with different functions, including antifungal activity. Based on deduced amino acid sequence of SnOLP, computer modeling produced a tertiary structure which is indicative of antifungal activity. Results To validate the potential antifungal activity of SnOLP, a hexahistidine-tagged mature SnOLP form was overexpressed in Escherichia coli M15 strain carried out by a pQE30 vector construction. The urea solubilized His6-tagged mature SnOLP protein was affinity-purified by immobilized-metal (Ni2+) affinity column chromatography. As SnOLP requires the correct formation of eight disulfide bonds, not correctly formed in bacterial cells, we adapted an in vitro method to refold the E. coli expressed SnOLP by using reduced:oxidized gluthatione redox buffer. This method generated biologically active conformations of the recombinant mature SnOLP, which exerted antifungal action towards plant pathogenic fungi (Fusarium solani f. sp.glycines, Colletotrichum spp., Macrophomina phaseolina) and oomycete (Phytophthora nicotiana var. parasitica) under in vitro conditions. Conclusion Since SnOLP displays activity against economically important plant pathogenic fungi and oomycete, it represents a novel PR-5 protein with promising utility for biotechnological applications. PMID:18334031

Generation of single domain antibody fragments derived from camelids and generation of manifold constructs.

PubMed

Vincke, Cécile; Gutiérrez, Carlos; Wernery, Ulrich; Devoogdt, Nick; Hassanzadeh-Ghassabeh, Gholamreza; Muyldermans, Serge

2012-01-01

Immunizing a camelid (camels and llamas) with soluble, properly folded proteins raises an affinity-matured immune response in the unique camelid heavy-chain only antibodies (HCAbs). The peripheral blood lymphocytes of the immunized animal are used to clone the antigen-binding antibody fragment from the HCAbs in a phage display vector. A representative aliquot of the library of these antigen-binding fragments is used to retrieve single domain antigen-specific binders by successive rounds of panning. These single domain antibody fragments are cloned in tandem to generate manifold constructs (bivalent, biparatopic or bispecific constructs) to increase their functional affinity, to increase specificity, or to connect two independent antigen molecules.

Eugenol prevents amyloid formation of proteins and inhibits amyloid-induced hemolysis

NASA Astrophysics Data System (ADS)

Dubey, Kriti; Anand, Bibin G.; Shekhawat, Dolat Singh; Kar, Karunakar

2017-02-01

Eugenol has attracted considerable attention because of its potential for many pharmaceutical applications including anti-inflammatory, anti-tumorigenic and anti-oxidant properties. Here, we have investigated the effect of eugenol on amyloid formation of selected globular proteins. We find that both spontaneous and seed-induced aggregation processes of insulin and serum albumin (BSA) are significantly suppressed in the presence of eugenol. Isothermal titration calorimetric data predict a single binding site for eugenol-insulin complex confirming the affinity of eugenol for native soluble insulin species. We also find that eugenol suppresses amyloid-induced hemolysis. Our findings reveal the inherent ability of eugenol to stabilize native proteins and to delay the conversion of protein species of native conformation into β-sheet assembled mature fibrils, which seems to be crucial for its inhibitory effect.

Analysis of Structural Features Contributing to Weak Affinities of Ubiquitin/Protein Interactions.

PubMed

Cohen, Ariel; Rosenthal, Eran; Shifman, Julia M

2017-11-10

Ubiquitin is a small protein that enables one of the most common post-translational modifications, where the whole ubiquitin molecule is attached to various target proteins, forming mono- or polyubiquitin conjugations. As a prototypical multispecific protein, ubiquitin interacts non-covalently with a variety of proteins in the cell, including ubiquitin-modifying enzymes and ubiquitin receptors that recognize signals from ubiquitin-conjugated substrates. To enable recognition of multiple targets and to support fast dissociation from the ubiquitin modifying enzymes, ubiquitin/protein interactions are characterized with low affinities, frequently in the higher μM and lower mM range. To determine how structure encodes low binding affinity of ubiquitin/protein complexes, we analyzed structures of more than a hundred such complexes compiled in the Ubiquitin Structural Relational Database. We calculated various structure-based features of ubiquitin/protein binding interfaces and compared them to the same features of general protein-protein interactions (PPIs) with various functions and generally higher affinities. Our analysis shows that ubiquitin/protein binding interfaces on average do not differ in size and shape complementarity from interfaces of higher-affinity PPIs. However, they contain fewer favorable hydrogen bonds and more unfavorable hydrophobic/charge interactions. We further analyzed how binding interfaces change upon affinity maturation of ubiquitin toward its target proteins. We demonstrate that while different features are improved in different experiments, the majority of the evolved complexes exhibit better shape complementarity and hydrogen bond pattern compared to wild-type complexes. Our analysis helps to understand how low-affinity PPIs have evolved and how they could be converted into high-affinity PPIs. Copyright © 2017 Elsevier Ltd. All rights reserved.

In silico maturation of binding-specificity of DNA aptamers against Proteus mirabilis.

PubMed

Savory, Nasa; Lednor, Danielle; Tsukakoshi, Kaori; Abe, Koichi; Yoshida, Wataru; Ferri, Stefano; Jones, Brian V; Ikebukuro, Kazunori

2013-10-01

Proteus mirabilis is a prominent cause of catheter-associated urinary tract infections (CAUTIs) among patients undergoing long-term bladder catheterization. There are currently no effective means of preventing P. mirabilis infections, and strategies for prophylaxis and rapid early diagnosis are urgently required. Aptamers offer significant potential for development of countermeasures against P. mirabilis CAUTI and are an ideal class of molecules for the development of diagnostics and therapeutics. Here we demonstrate the application of Cell-SELEX to identify DNA aptamers that show high affinity for P. mirabilis. While the aptamers identified displayed high affinity for P. mirabilis cells in dot blotting assays, they also bound to other uropathogenic bacteria. To improve aptamer specificity for P. mirabilis, an in silico maturation (ISM) approach was employed. Two cycles of ISM allowed the identification of an aptamer showing 36% higher specificity, evaluated as a ratio of binding signal for P. mirabilis to that for Escherichia coli (also a cause of CAUTI and the most common urinary tract pathogen). Aptamers that specifically recognize P. mirabilis would have diagnostic and therapeutic values and constitute useful tools for studying membrane-associated proteins in this organism. Copyright © 2013 Wiley Periodicals, Inc.

Integrity of immunoglobulin variable regions is supported by GANP during AID-induced somatic hypermutation in germinal center B cells.

PubMed

Eid, Mohammed Mansour Abbas; Shimoda, Mayuko; Singh, Shailendra Kumar; Almofty, Sarah Ameen; Pham, Phuong; Goodman, Myron F; Maeda, Kazuhiko; Sakaguchi, Nobuo

2017-05-01

Immunoglobulin affinity maturation depends on somatic hypermutation (SHM) in immunoglobulin variable (IgV) regions initiated by activation-induced cytidine deaminase (AID). AID induces transition mutations by C→U deamination on both strands, causing C:G→T:A. Error-prone repairs of U by base excision and mismatch repairs (MMRs) create transversion mutations at C/G and mutations at A/T sites. In Neuberger's model, it remained to be clarified how transition/transversion repair is regulated. We investigate the role of AID-interacting GANP (germinal center-associated nuclear protein) in the IgV SHM profile. GANP enhances transition mutation of the non-transcribed strand G and reduces mutation at A, restricted to GYW of the AID hotspot motif. It reduces DNA polymerase η hotspot mutations associated with MMRs followed by uracil-DNA glycosylase. Mutation comparison between IgV complementary and framework regions (FWRs) by Bayesian statistical estimation demonstrates that GANP supports the preservation of IgV FWR genomic sequences. GANP works to maintain antibody structure by reducing drastic changes in the IgV FWR in affinity maturation. © The Author 2017. Published by Oxford University Press on behalf of The Japanese Society for Immunology.

A new species of Erythrostemon (Leguminosae, Caesalpinioideae) from the western Río Balsas Depression, Mexico

PubMed Central

Sotuyo, Solange; Contreras-Jiménez, José Luis; Lewis, Gwilym P.

2017-01-01

Abstract A new legume species from a seasonally dry forest of the Western Río Balsas Depression, in the states of Guerrero and Michoacán, Mexico, Erythrostemon guevarafeferii, is herein described and illustrated. The new species shows morphological affinities with Erythrostemon hintonii, from which it is distinguished in having fewer leaflets per pinna, mature leaflets disposed toward the upper half of the pinnae rachises, long inflorescences on curved slender peduncles, abundant red glands on its flowers and inflorescences, and its fruit glabrous with red stipitate glands at maturity. A taxonomic key to the Río Balsas Depression species of Erythrostemon is included. PMID:28228685

Water channel in the binding site of a high affinity anti-methotrexate antibody.

PubMed

Gayda, Susan; Longenecker, Kenton L; Manoj, Sharmila; Judge, Russell A; Saldana, Sylvia C; Ruan, Qiaoqiao; Swift, Kerry M; Tetin, Sergey Y

2014-06-17

In the present study, we report the structure of the free and drug-bound Fab fragment of a high affinity anti-methotrexate antibody and perform a thermodynamic analysis of the binding process. The anti-methotrexate Fab fragment features a remarkably rigid tunnel-like binding site that extends into a water channel serving as a specialized route to move solvent out and into the site upon ligand binding and dissociation. This new finding in antibody structure-function relationships directly relates to the fast association (1 × 10⁷ M⁻¹ s⁻¹) and slow dissociation (4 × 10⁻⁵ s⁻¹) rates determined for mAb ADD056, resulting in a very strong binding with a K(D) ~ 3.6 pM at 20 °C. As follows from the X-ray data analysis, the methotrexate-antibody complex is stabilized by an extended network of hydrogen bonds and stacking interactions. The analysis also shows structural involvement of the CDR H3 in formation of the water channel revealing another important role of this hypervariable region. This suggests a new direction in natural affinity maturation and opens a new possibility in antibody engineering. Methotrexate is a widely used therapeutic agent for many malignant diseases and inflammatory disorders. Unfortunately, it may also interfere with central aspects of metabolism and thereby cause inevitable side effects. Therefore, methotrexate therapy requires careful monitoring of drug blood levels, which is traditionally done by immunoassays. An understanding of the structure-function properties of antibodies selected for drug monitoring substantiates the performance and robustness of such tests.

Perspectives on Intra- and Intercellular Trafficking of Hedgehog for Tissue Patterning

PubMed Central

Simon, Eléanor; Aguirre-Tamaral, Adrián; Aguilar, Gustavo; Guerrero, Isabel

2016-01-01

Intercellular communication is a fundamental process for correct tissue development. The mechanism of this process involves, among other things, the production and secretion of signaling molecules by specialized cell types and the capability of these signals to reach the target cells in order to trigger specific responses. Hedgehog (Hh) is one of the best-studied signaling pathways because of its importance during morphogenesis in many organisms. The Hh protein acts as a morphogen, activating its targets at a distance in a concentration-dependent manner. Post-translational modifications of Hh lead to a molecule covalently bond to two lipid moieties. These lipid modifications confer Hh high affinity to lipidic membranes, and intense studies have been carried out to explain its release into the extracellular matrix. This work reviews Hh molecule maturation, the intracellular recycling needed for its secretion and the proposed carriers to explain Hh transportation to the receiving cells. Special focus is placed on the role of specialized filopodia, also named cytonemes, in morphogen transport and gradient formation. PMID:29615597

Perspectives on Intra- and Intercellular Trafficking of Hedgehog for Tissue Patterning.

PubMed

Simon, Eléanor; Aguirre-Tamaral, Adrián; Aguilar, Gustavo; Guerrero, Isabel

2016-12-02

Intercellular communication is a fundamental process for correct tissue development. The mechanism of this process involves, among other things, the production and secretion of signaling molecules by specialized cell types and the capability of these signals to reach the target cells in order to trigger specific responses. Hedgehog (Hh) is one of the best-studied signaling pathways because of its importance during morphogenesis in many organisms. The Hh protein acts as a morphogen, activating its targets at a distance in a concentration-dependent manner. Post-translational modifications of Hh lead to a molecule covalently bond to two lipid moieties. These lipid modifications confer Hh high affinity to lipidic membranes, and intense studies have been carried out to explain its release into the extracellular matrix. This work reviews Hh molecule maturation, the intracellular recycling needed for its secretion and the proposed carriers to explain Hh transportation to the receiving cells. Special focus is placed on the role of specialized filopodia, also named cytonemes, in morphogen transport and gradient formation.

Altering the spectrum of immunoglobulin V gene somatic hypermutation by modifying the active site of AID.

PubMed

Wang, Meng; Rada, Cristina; Neuberger, Michael S

2010-01-18

High-affinity antibodies are generated by somatic hypermutation with nucleotide substitutions introduced into the IgV in a semirandom fashion, but with intrinsic mutational hotspots strategically located to optimize antibody affinity maturation. The process is dependent on activation-induced deaminase (AID), an enzyme that can deaminate deoxycytidine in DNA in vitro, where its activity is sensitive to the identity of the 5'-flanking nucleotide. As a critical test of whether such DNA deamination activity underpins antibody diversification and to gain insight into the extent to which the antibody mutation spectrum is dependent on the intrinsic substrate specificity of AID, we investigated whether it is possible to change the IgV mutation spectrum by altering AID's active site such that it prefers a pyrimidine (rather than a purine) flanking the targeted deoxycytidine. Consistent with the DNA deamination mechanism, B cells expressing the modified AID proteins yield altered IgV mutation spectra (exhibiting a purine-->pyrimidine shift in flanking nucleotide preference) and altered hotspots. However, AID-catalyzed deamination of IgV targets in vitro does not yield the same degree of hotspot dominance to that observed in vivo, indicating the importance of features beyond AID's active site and DNA local sequence environment in determining in vivo hotspot dominance.

Impact of Chronic Viral Infection on T-Cell Dependent Humoral Immune Response.

PubMed

Rodriguez, Stéphane; Roussel, Mikaël; Tarte, Karin; Amé-Thomas, Patricia

2017-01-01

During the last decades, considerable efforts have been done to decipher mechanisms supported by microorganisms or viruses involved in the development, differentiation, and function of immune cells. Pathogens and their associated secretome as well as the continuous inflammation observed in chronic infection are shaping both innate and adaptive immunity. Secondary lymphoid organs are functional structures ensuring the mounting of adaptive immune response against microorganisms and viruses. Inside these organs, germinal centers (GCs) are the specialized sites where mature B-cell differentiation occurs leading to the release of high-affinity immunoglobulin (Ig)-secreting cells. Different steps are critical to complete B-cell differentiation process, including proliferation, somatic hypermutations in Ig variable genes, affinity-based selection, and class switch recombination. All these steps require intense interactions with cognate CD4 + helper T cells belonging to follicular helper lineage. Interestingly, pathogens can disturb this subtle machinery affecting the classical adaptive immune response. In this review, we describe how viruses could act directly on GC B cells, either through B-cell infection or by their contribution to B-cell cancer development and maintenance. In addition, we depict the indirect impact of viruses on B-cell response through infection of GC T cells and stromal cells, leading to immune response modulation.

Nickel binding and [NiFe]-hydrogenase maturation by the metallochaperone SlyD with a single metal-binding site in Escherichia coli.

PubMed

Kaluarachchi, Harini; Altenstein, Matthias; Sugumar, Sonia R; Balbach, Jochen; Zamble, Deborah B; Haupt, Caroline

2012-03-16

SlyD (sensitive to lysis D) is a nickel metallochaperone involved in the maturation of [NiFe]-hydrogenases in Escherichia coli (E. coli) and specifically contributes to the nickel delivery step during enzyme biosynthesis. This protein contains a C-terminal metal-binding domain that is rich in potential metal-binding residues that enable SlyD to bind multiple nickel ions with high affinity. The SlyD homolog from Thermus thermophilus does not contain the extended cysteine- and histidine-rich C-terminal tail of the E. coli protein, yet it binds a single Ni(II) ion tightly. To investigate whether a single metal-binding motif can functionally replace the full-length domain, we generated a truncation of E. coli SlyD, SlyD155. Ni(II) binding to SlyD155 was investigated by using isothermal titration calorimetry, NMR and electrospray ionization mass spectrometry measurements. This in vitro characterization revealed that SlyD155 contains a single metal-binding motif with high affinity for nickel. Structural characterization by X-ray absorption spectroscopy and NMR indicated that nickel was coordinated in an octahedral geometry with at least two histidines as ligands. Heterodimerization between SlyD and another hydrogenase accessory protein, HypB, is essential for optimal hydrogenase maturation and was confirmed for SlyD155 via cross-linking experiments and NMR titrations, as were conserved chaperone and peptidyl-prolyl isomerase activities. Although these properties of SlyD are preserved in the truncated version, it does not modulate nickel binding to HypB in vitro or contribute to the maturation of [NiFe]-hydrogenases in vivo, unlike the full-length protein. This study highlights the importance of the unusual metal-binding domain of E. coli SlyD in hydrogenase biogenesis. Copyright © 2012 Elsevier Ltd. All rights reserved.

Development of an interleukin (IL)-33 sandwich ELISA kit specific for mature IL-33.

PubMed

Kim, Eunsom; Kwak, Areum; Jhun, Hyunjhung; Lee, Siyoung; Jo, Seunghyun; Lee, Jongho; Kang, Tae-Bong; Her, Erk; Bae, Suyoung; Lee, Youngmin; Kim, Soohyun

2016-01-01

Interleukin (IL)-33 is an inflammatory cytokine and belongs to the IL-1 family of cytokines. There are eleven members of the IL-1 family of cytokines and all have important roles in host defense against infections. Their levels are increased during infection and in various auto-inflammatory diseases. IL-33 is also associated with autoimmune diseases such as asthma, atopic dermatitis, rheumatoid arthritis, and atherosclerosis. IL-33 receptors consist of IL-1R4 and IL-1R3 to induce both Th1 and Th2 type immune response. Here we present the development of monoclonal antibodies (mAbs) against human mature IL-33. Recombinant human mature IL-33 protein was expressed in E. coli and purified by multi-step affinity chromatography. The human IL-33 activity was examined in HMC-1 and Raw 264.7 cells. Mice were immunized with the biologically active mature IL-33 to generate mAb against IL-33. The anti-IL-33 mAb (clone/4) was used as a capture antibody for a sandwich enzyme-linked immunosorbent assay (ELISA). This assay detects mature IL-33 with a high sensitivity (80 pg/mL) but does not recognize the biologically inactive precursor IL-33. This article describes the methods for a newly developed IL-33 ELISA kit that is specific for mature IL-33 and may be used to analyze bioactive mature IL-33 in various immunological diseases.

Identification of inhibitory scFv antibodies targeting fibroblast activation protein utilizing phage display functional screens

PubMed Central

Zhang, Jiping; Valianou, Matthildi; Simmons, Heidi; Robinson, Matthew K.; Lee, Hyung-Ok; Mullins, Stefanie R.; Marasco, Wayne A.; Adams, Gregory P.; Weiner, Louis M.; Cheng, Jonathan D.

2013-01-01

Fibroblast activation protein (FAP) is a serine protease selectively expressed on tumor stromal fibroblasts in epithelial carcinomas and is important in cancer growth, adhesion, and metastases. As FAP enzymatic activity is a potent therapeutic target, we aimed to identify inhibitory antibodies. Using a competitive inhibition strategy, we used phage display techniques to identify 53 single-chain variable fragments (scFvs) after three rounds of panning against FAP. These scFvs were expressed and characterized for binding to FAP by surface plasmon resonance and flow cytometry. Functional assessment of these antibodies yielded an inhibitory scFv antibody, named E3, which could attenuate 35% of FAP cleavage of the fluorescent substrate Ala-Pro-7-amido-4-trifluoromethylcoumarin compared with nonfunctional scFv control. Furthermore, a mutant E3 scFv was identified by yeast affinity maturation. It had higher affinity (4-fold) and enhanced inhibitory effect on FAP enzyme activity (3-fold) than E3. The application of both inhibitory anti-FAP scFvs significantly affected the formation of 3-dimensional FAP-positive cell matrix, as demonstrated by reducing the fibronectin fiber orientation from 41.18% (negative antibody control) to 34.06% (E3) and 36.15% (mutant E3), respectively. Thus, we have identified and affinity-maturated the first scFv antibody capable of inhibiting FAP function. This scFv antibody has the potential to disrupt the role of FAP in tumor invasion and metastasis.—Zhang, J., Valianou, M., Simmons, H., Robinson, M. K., Lee, H.-O., Mullins, S. R., Marasco, W. A., Adams, G. P., Weiner, L. M., Cheng, J. D. Identification of inhibitory ScFv antibodies targeting fibroblast activation protein utilizing phage display functional screens. PMID:23104982

Evaluation of affinity and pseudo-affinity adsorption processes for penicillin acylase purification.

PubMed

Fonseca, L P; Cabral, J M

1996-01-01

Affinity ligand (6-Aminopenicillanic acid, Amoxycillin, Ampicillin, Benzylpenicillin and 4-Phenylbutylanzine) of penicillin acylase (EC 3.5.1.11) were attached to hydrophilic gels like Sepharose 4B-CNBr and Minileak 'medium'. Ampicillin and 4-Phenylbutylamine were the affinity ligands that presented the higher concentrations attached to both gels. Penicillin acylase adsorption on these affinity gels was mainly dependent on the activated group of the gel, the affinity ligand attached and the experimental conditions of enzyme adsorption. Under affinity conditions only the ligands Amoxycillin, Ampicillin and 4-Phenylbutylamine, immobilized on Minileak, adsorbed the enzyme from osmotic shock extracts at different pH values. These affinity ligand systems were characterized by low adsorption capacities of penicillin acylase activity (1.2-2.1 IU mL-1 gel) and specific activity (1.5-2.9 IU mg-1 prot). Under pseudo-affinity conditions all the ligands attached both activated to gels (Sepharose 4B-CNBr and Minileak) adsorbed the enzyme. The affinity gels were characterized by higher values of adsorption capacity (3.7 and 55.6 IU mL-1 gel) and adsorbed specific activity (2.0 and 6.1 IU mg-1 prot) than those observed under affinity conditions. The space arm of Minileak gel, shown to be fundamental to enzyme adsorption under affinity conditions, preferentially adsorbed proteins in relation to the enzyme under pseudo-affinity conditions. However, this effect was partially minimized when the gel was derivatized by the affinity ligands at concentrations higher than 6 mumol mL-1 gel. Ampicillin was the affinity ligand that presented the best results for specific adsorption of penicillin acylase under affinity and pseudo-affinity adsorption processes. The Sepharose 4B-CNBr derivatized gel also presented a good adsorption capacity of enzyme activity (26.8 IU mL-1 gel) under pseudo-affinity adsorption processes.

Effects of metal on the biochemical properties of Helicobacter pylori HypB, a maturation factor of [NiFe]-hydrogenase and urease.

PubMed

Sydor, Andrew M; Liu, Jenny; Zamble, Deborah B

2011-03-01

The biosyntheses of the [NiFe]-hydrogenase and urease enzymes in Helicobacter pylori require several accessory proteins for proper construction of the nickel-containing metallocenters. The hydrogenase accessory proteins HypA and HypB, a GTPase, have been implicated in the nickel delivery steps of both enzymes. In this study, the metal-binding properties of H. pylori HypB were characterized, and the effects of metal binding on the biochemical behavior of the protein were examined. The protein can bind stoichiometric amounts of Zn(II) or Ni(II), each with nanomolar affinity. Mutation of Cys106 and His107, which are located between two major GTPase motifs, results in undetectable Ni(II) binding, and the Zn(II) affinity is weakened by 2 orders of magnitude. These two residues are also required for the metal-dependent dimerization observed in the presence of Ni(II) but not Zn(II). The addition of metals to the protein has distinct impacts on GTPase activity, with zinc significantly reducing GTP hydrolysis to below detectable levels and nickel only slightly altering the k(cat) and K(m) of the reaction. The regulation of HypB activities by metal binding may contribute to the maturation of the nickel-containing enzymes.

Scarcity of autoreactive human blood IgA+ memory B cells

PubMed Central

Prigent, Julie; Lorin, Valérie; Kök, Ayrin; Hieu, Thierry; Bourgeau, Salomé

2016-01-01

Class‐switched memory B cells are key components of the “reactive” humoral immunity, which ensures a fast and massive secretion of high‐affinity antigen‐specific antibodies upon antigenic challenge. In humans, IgA class‐switched (IgA+) memory B cells and IgA antibodies are abundant in the blood. Although circulating IgA+ memory B cells and their corresponding secreted immunoglobulins likely possess major protective and/or regulatory immune roles, little is known about their specificity and function. Here, we show that IgA+ and IgG+ memory B‐cell antibodies cloned from the same healthy humans share common immunoglobulin gene features. IgA and IgG memory antibodies have comparable lack of reactivity to vaccines, common mucosa‐tropic viruses and commensal bacteria. However, the IgA+ memory B‐cell compartment contains fewer polyreactive clones and importantly, only rare self‐reactive clones compared to IgG+ memory B cells. Self‐reactivity of IgAs is acquired following B‐cell affinity maturation but not antibody class switching. Together, our data suggest the existence of different regulatory mechanisms for removing autoreactive clones from the IgG+ and IgA+ memory B‐cell repertoires, and/or different maturation pathways potentially reflecting the distinct nature and localization of the cognate antigens recognized by individual B‐cell populations. PMID:27469325

In vitro maturation of Drosophila melanogaster Spätzle protein with refolded Easter reveals a novel cleavage site within the prodomain.

PubMed

Ursel, Christian; Fandrich, Uwe; Hoffmann, Anita; Sieg, Torsten; Ihling, Christian; Stubbs, Milton T

2013-08-01

Dorsoventral patterning during Drosophila melanogaster embryogenesis is mediated by a well-defined gradient of the mature NGF-like ligand Spätzle. Easter, the ultimate protease of a ventrally-restricted serine protease cascade, plays a key role in the regulation of the morphogenic gradient, catalyzing the activation cleavage of proSpätzle. As a result of alternative splicing, proSpätzle exists in multiple isoforms, almost all of which differ only in their prodomain. Although this domain is unstructured in isolation, it has a stabilizing influence on the mature cystine knot domain and is involved in the binding to the Toll receptor. Here, we report the expression and refolding of Easter, and show that the renatured enzyme performs the activation cleavage of two Spätzle isoforms. We determine the affinity of the prodomain for the cystine knot domain, and show that Easter performs a previously unknown secondary cleavage in each prodomain.

Small leucine-rich repeat proteoglycans associated with mature insoluble elastin serve as binding sites for galectins.

PubMed

Itoh, Aiko; Nonaka, Yasuhiro; Ogawa, Takashi; Nakamura, Takanori; Nishi, Nozomu

2017-11-01

We previously reported that galectin-9 (Gal-9), an immunomodulatory animal lectin, could bind to insoluble collagen preparations and exerted direct cytocidal effects on immune cells. In the present study, we found that mature insoluble elastin is capable of binding Gal-9 and other members of the human galectin family. Lectin blot analysis of a series of commercial water-soluble elastin preparations, PES-(A) ~ PES-(E), revealed that only PES-(E) contained substances recognized by Gal-9. Gal-9-interacting substances in PES-(E) were affinity-purified, digested with trypsin and then analyzed by reversed-phase HPLC. Peptide fragments derived from five members of the small leucine-rich repeat proteoglycan family, versican, lumican, osteoglycin/mimecan, prolargin, and fibromodulin, were identified by N-terminal amino acid sequence analysis. The results indicate that Gal-9 and possibly other galectins recognize glycans attached to small leucine-rich repeat proteoglycans associated with insoluble elastin and also indicate the possibility that mature insoluble elastin serves as an extracellular reservoir for galectins.

Comparative temporospatial expression profiling of murine amelotin protein during amelogenesis.

PubMed

Somogyi-Ganss, Eszter; Nakayama, Yohei; Iwasaki, Kengo; Nakano, Yukiko; Stolf, Daiana; McKee, Marc D; Ganss, Bernhard

2012-01-01

Tooth enamel is formed in a typical biomineralization process under the guidance of specific organic components. Amelotin (AMTN) is a recently identified, secreted protein that is transcribed predominantly during the maturation stage of enamel formation, but its protein expression profile throughout amelogenesis has not been described in detail. The main objective of this study was to define the spatiotemporal expression profile of AMTN during tooth development in comparison with other known enamel proteins. A peptide antibody against AMTN was raised in rabbits, affinity purified and used for immunohistochemical analyses on sagittal and transverse paraffin sections of decalcified mouse hemimandibles. The localization of AMTN was compared to that of known enamel proteins amelogenin, ameloblastin, enamelin, odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4. Three-dimensional images of AMTN localization in molars at selected ages were reconstructed from serial stained sections, and transmission electron microscopy was used for ultrastructural localization of AMTN. AMTN was detected in ameloblasts of molars in a transient fashion, declining at the time of tooth eruption. Prominent expression in maturation stage ameloblasts of the continuously erupting incisor persisted into adulthood. In contrast, amelogenin, ameloblastin and enamelin were predominantly found during the early secretory stage, while odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4 expression in maturation stage ameloblasts paralleled that of AMTN. Secreted AMTN was detected at the interface between ameloblasts and the mineralized enamel. Recombinant AMTN protein did not mediate cell attachment in vitro. These results suggest a primary role for AMTN in the late stages of enamel mineralization. Copyright © 2011 S. Karger AG, Basel.

Defective Guanine Nucleotide Exchange in the Elongation Factor-like 1 (EFL1) GTPase by Mutations in the Shwachman-Diamond Syndrome Protein*

PubMed Central

García-Márquez, Adrián; Gijsbers, Abril; de la Mora, Eugenio; Sánchez-Puig, Nuria

2015-01-01

Ribosome biogenesis is orchestrated by the action of several accessory factors that provide time and directionality to the process. One such accessory factor is the GTPase EFL1 involved in the cytoplasmic maturation of the ribosomal 60S subunit. EFL1 and SBDS, the protein mutated in the Shwachman-Diamond syndrome (SBDS), release the anti-association factor eIF6 from the surface of the ribosomal subunit 60S. Here we report a kinetic analysis of fluorescent guanine nucleotides binding to EFL1 alone and in the presence of SBDS using fluorescence stopped-flow spectroscopy. Binding kinetics of EFL1 to both GDP and GTP suggests a two-step mechanism with an initial binding event followed by a conformational change of the complex. Furthermore, the same behavior was observed in the presence of the SBDS protein irrespective of the guanine nucleotide evaluated. The affinity of EFL1 for GTP is 10-fold lower than that calculated for GDP. Association of EFL1 to SBDS did not modify the affinity for GTP but dramatically decreased that for GDP by increasing the dissociation rate of the nucleotide. Thus, SBDS acts as a guanine nucleotide exchange factor (GEF) for EFL1 promoting its activation by the release of GDP. Finally, fluorescence anisotropy measurements showed that the S143L mutation present in the Shwachman-Diamond syndrome altered a surface epitope for EFL1 and largely decreased the affinity for it. These results suggest that loss of interaction between these proteins due to mutations in the disease consequently prevents the nucleotide exchange regulation the SBDS exerts on EFL1. PMID:25991726

Kinetic analysis of pre-ribosome structure in vivo

PubMed Central

Swiatkowska, Agata; Wlotzka, Wiebke; Tuck, Alex; Barrass, J. David; Beggs, Jean D.; Tollervey, David

2012-01-01

Pre-ribosomal particles undergo numerous structural changes during maturation, but their high complexity and short lifetimes make these changes very difficult to follow in vivo. In consequence, pre-ribosome structure and composition have largely been inferred from purified particles and analyzed in vitro. Here we describe techniques for kinetic analyses of the changes in pre-ribosome structure in living cells of Saccharomyces cerevisiae. To allow this, in vivo structure probing by DMS modification was combined with affinity purification of newly synthesized 20S pre-rRNA over a time course of metabolic labeling with 4-thiouracil. To demonstrate that this approach is generally applicable, we initially analyzed the accessibility of the region surrounding cleavage site D site at the 3′ end of the mature 18S rRNA region of the pre-rRNA. This revealed a remarkably flexible structure throughout 40S subunit biogenesis, with little stable RNA–protein interaction apparent. Analysis of folding in the region of the 18S central pseudoknot was consistent with previous data showing U3 snoRNA–18S rRNA interactions. Dynamic changes in the structure of the hinge between helix 28 (H28) and H44 of pre-18S rRNA were consistent with recently reported interactions with the 3′ guide region of U3 snoRNA. Finally, analysis of the H18 region indicates that the RNA structure matures early, but additional protection appears subsequently, presumably reflecting protein binding. The structural analyses described here were performed on total, affinity-purified, newly synthesized RNA, so many classes of RNA and RNA–protein complex are potentially amenable to this approach. PMID:23093724

Functional identification of activity-regulated, high-affinity glutamine transport in hippocampal neurons inhibited by riluzole.

PubMed

Erickson, Jeffrey D

2017-07-01

Glutamine (Gln) is considered the preferred precursor for the neurotransmitter pool of glutamate (Glu), the major excitatory transmitter in the mammalian CNS. Here, an activity-regulated, high-affinity Gln transport system is described in developing and mature neuron-enriched hippocampal cultures that is potently inhibited by riluzole (IC 50 1.3 ± 0.5 μM), an anti-glutamatergic drug, and is blocked by low concentrations of 2-(methylamino)isobutyrate (MeAIB), a system A transport inhibitor. K + -stimulated MeAIB transport displays an affinity (K m ) for MeAIB of 37 ± 1.2 μM, saturates at ~ 200 μM, is dependent on extracellular Ca 2+ , and is blocked by inhibition of voltage-gated Ca 2+ channels. Spontaneous MeAIB transport is also dependent on extracellullar Ca 2+ and voltage-gated calcium channels, but is also blocked by the Na + channel blocker tetrodotoxin, by Glu receptor antagonists, and by GABA indicating its dependence on intact neural circuits driven by endogenous glutamatergic activity. The transport of MeAIB itself does not rely on Ca 2+ , but on Na + ions, and is pH sensitive. Activity-regulated, riluzole-sensitive spontaneous and K + -stimulated transport is minimal at 7-8 days in vitro, coordinately induced during the next 2 weeks and is maximally expressed by days in vitro > 20; the known period for maturation of the Glu/Gln cycle and regulated pre-synaptic Glu release. Competition analyses with various amino acids indicate that Gln is the most likely physiological substrate. Activity-regulated Gln/MeAIB transport is not observed in astrocytes. The functional identification of activity-regulated, high-affinity, riluzole-sensitive Gln/MeAIB transport in hippocampal neurons may have important ramifications in the neurobiology of activity-stimulated pre-synaptic Glu release, the Glu/Gln cycle between astrocytes and neurons, and neuronal Glu-induced excitotoxicity. Cover Image for this issue: doi: 10.1111/jnc.13805. © 2017 International Society for Neurochemistry.

High-throughput sequencing of natively paired antibody chains provides evidence for original antigenic sin shaping the antibody response to influenza vaccination.

PubMed

Tan, Yann-Chong; Blum, Lisa K; Kongpachith, Sarah; Ju, Chia-Hsin; Cai, Xiaoyong; Lindstrom, Tamsin M; Sokolove, Jeremy; Robinson, William H

2014-03-01

We developed a DNA barcoding method to enable high-throughput sequencing of the cognate heavy- and light-chain pairs of the antibodies expressed by individual B cells. We used this approach to elucidate the plasmablast antibody response to influenza vaccination. We show that >75% of the rationally selected plasmablast antibodies bind and neutralize influenza, and that antibodies from clonal families, defined by sharing both heavy-chain VJ and light-chain VJ sequence usage, do so most effectively. Vaccine-induced heavy-chain VJ regions contained on average >20 nucleotide mutations as compared to their predicted germline gene sequences, and some vaccine-induced antibodies exhibited higher binding affinities for hemagglutinins derived from prior years' seasonal influenza as compared to their affinities for the immunization strains. Our results show that influenza vaccination induces the recall of memory B cells that express antibodies that previously underwent affinity maturation against prior years' seasonal influenza, suggesting that 'original antigenic sin' shapes the antibody response to influenza vaccination. Published by Elsevier Inc.

TFH-IgA responses keep microbiota in check.

PubMed

Honda, Kenya

2015-02-11

In this issue of Cell Host & Microbe, Kubinak et al. (2015) demonstrate that gut microbiota-mediated signaling through MyD88 in CD4+ T cells induces their differentiation into T follicular helper (TFH) cells that promote affinity-matured microbiota antigen-specific immunoglobulin A (IgA) responses. These TFH-driven IgA responses are essential for maintaining gut bacterial diversity and a healthy microbiota. Copyright © 2015 Elsevier Inc. All rights reserved.

Affinity+: Semi-Structured Brainstorming on Large Displays

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

Burtner, Edwin R.; May, Richard A.; Scarberry, Randall E.

2013-04-27

Affinity diagraming is a powerful method for encouraging and capturing lateral thinking in a group environment. The Affinity+ Concept was designed to improve the collaborative brainstorm process through the use of large display surfaces in conjunction with mobile devices like smart phones and tablets. The system works by capturing the ideas digitally and allowing users to sort and group them on a large touch screen manually. Additionally, Affinity+ incorporates theme detection, topic clustering, and other processing algorithms that help bring structured analytic techniques to the process without requiring explicit leadership roles and other overhead typically involved in these activities.

Human Fanconi Anemia Complementation Group A Protein Stimulates the 5’ Flap Endonuclease Activity of FEN1

PubMed Central

Qian, Liangyue; Yuan, Fenghua; Rodriguez-Tello, Paola; Padgaonkar, Suyog; Zhang, Yanbin

2013-01-01

In eukaryotic cells, Flap endonuclease 1 (FEN1) is a major structure-specific endonuclease that processes 5’ flapped structures during maturation of lagging strand DNA synthesis, long patch base excision repair, and rescue of stalled replication forks. Here we report that fanconi anemia complementation group A protein (FANCA), a protein that recognizes 5’ flap structures and is involved in DNA repair and maintenance of replication forks, constantly stimulates FEN1-mediated incision of both DNA and RNA flaps. Kinetic analyses indicate that FANCA stimulates FEN1 by increasing the turnover rate of FEN1 and altering its substrate affinity. More importantly, six pathogenic FANCA mutants are significantly less efficient than the wild-type at stimulating FEN1 endonuclease activity, implicating that regulation of FEN1 by FANCA contributes to the maintenance of genomic stability. PMID:24349332

Removal of dissolved textile dyes from wastewater by a compost sorbent

USGS Publications Warehouse

Tsui, L.S.; Roy, W.R.; Cole, M.A.

2003-01-01

The objective of this study was to evaluate the potential for treating dye-contaminated waste streams by sorption using compost as a low-cost sorbent. A mature, thermophilic compost sample was used to sorb CI Acid Black 24, CI Acid Orange 74, CI Basic Blue 9, CI Basic Green 4, CI Direct Blue 71, CI Direct Orange 39, CI Reactive Orange 16 and CI Reactive Red 2 from solution using a batch-sorption method. With the exception of the two reactive dyes, the sorption kinetics were favourable for a continuous-flow treatment process with the compost-dye mixtures reaching a steady state within 3-5 h. Based on limited comparisons, the affinity of the compost for each dye appeared to be competitive with other non-activated carbon sorbents. The results suggest that additional research on using compost as a sorbent for dye-contaminated solutions is warranted.

[Influence of different processing methods and mature stages on 3,29-dibenzoyl rarounitriol of Trichosanthes kirilowii seeds].

PubMed

Liu, Jin-Na; Xie, Xiao-Liang; Yang, Tai-Xin; Zhang, Cun-Li; Jia, Dong-Sheng; Liu, Ming; Wen, Chun-Xiu

2014-04-01

To study the different mature stages and the best processing methods on the quality of Trichosanthes kirilowii seeds. The content of 3,29-dibenzoyl rarounitriol in Trichosanthes kirilowii seeds was determined by HPLC. The sample of different mature stages such as immature, near mature and fully mature and processed by different methods were studied. Fully mature Trichosanthes kirilowii seeds were better than the immatured, and the best processing method was dried under 60degrees C, the content of 3,29-dibenzoyl rarounitriol reached up to 131.63microlg/mL. Different processing methods and different mature stages had a significant influence on the quality of Trichosanthes kirilowii seeds.

Analysis of the HLA-DR peptidome from human dendritic cells reveals high affinity repertoires and nonconventional pathways of peptide generation.

PubMed

Ciudad, M Teresa; Sorvillo, Nicoletta; van Alphen, Floris P; Catalán, Diego; Meijer, Alexander B; Voorberg, Jan; Jaraquemada, Dolores

2017-01-01

Dendritic cells (DCs) are the major professional APCs of the immune system; however, their MHC-II-associated peptide repertoires have been hard to analyze, mostly because of their scarce presence in blood and tissues. In vitro matured human monocyte-derived DCs (MoDCs) are widely used as professional APCs in experimental systems. In this work, we have applied mass spectrometry to identify the HLA-DR-associated self-peptide repertoires from small numbers of mature MoDCs (∼5 × 10 6 cells), derived from 7 different donors. Repertoires of 9 different HLA-DR alleles were defined from analysis of 1319 peptides, showing the expected characteristics of MHC-II-associated peptides. Most peptides identified were predicted high binders for their respective allele, formed nested sets, and belonged to endo-lysosomal pathway-degraded proteins. Approximately 20% of the peptides were derived from cytosolic and nuclear proteins, a recurrent finding in HLA-DR peptide repertoires. Of interest, most of these peptides corresponded to single sequences, did not form nested sets, and were located at the C terminus of the parental protein, which suggested alternative processing. Analysis of cleavage patterns for terminal peptides predominantly showed aspartic acid before the cleavage site of both C- and N-terminal peptides and proline immediately after the cleavage site in C-terminal peptides. Proline was also frequent next to the cut sites of internal peptides. These data provide new insights into the Ag processing capabilities of DCs. The relevance of these processing pathways and their contribution to response to infection, tolerance induction, or autoimmunity deserve further analysis. © Society for Leukocyte Biology.

On the origin of C3 nephritic factor (antibody to the alternative pathway C3 convertase): evidence for the Adam and Eve concept of autoantibody production.

PubMed

Spitzer, R E; Stitzel, A E; Tsokos, G

1992-09-01

The antibody to the alternative pathway C3 convertase, designated C3 nephritic factor or C3NeF, is an autoantibody that is produced in everyone from the time of birth. The elaboration of C3NeF utilizes germline V-region genes which undergo antigen-driven affinity maturation, resulting in an autoantibody that is produced in large amounts with high affinity and narrow specificity. Our data also suggest that under normal conditions, the idiotypic network may play an important part in the control of this autoantibody. Further, a defect in the network with loss of control or inappropriate stimulation may be an underlying mechanism in the unrestricted production of C3NeF in patients with membranoproliferative glomerulonephritis.

Alteration of Electrostatic Surface Potential Enhances Affinity and Tumor Killing Properties of Anti-ganglioside GD2 Monoclonal Antibody hu3F8.

PubMed

Zhao, Qi; Ahmed, Mahiuddin; Guo, Hong-fen; Cheung, Irene Y; Cheung, Nai-Kong V

2015-05-22

Ganglioside GD2 is highly expressed on neuroectodermal tumors and an attractive therapeutic target for antibodies that have already shown some clinical efficacy. To further improve the current antibodies, which have modest affinity, we sought to improve affinity by using a combined method of random mutagenesis and in silico assisted design to affinity-mature the anti-GD2 monoclonal antibody hu3F8. Using yeast display, mutants in the Fv with enhanced binding over the parental clone were FACS-sorted and cloned. In silico modeling identified the minimal key interacting residues involved in the important charged interactions with the sialic acid groups of GD2. Two mutations, D32H (L-CDR1) and E1K (L-FR1) altered the electrostatic surface potential of the antigen binding site, allowing for an increase in positive charge to enhance the interaction with the negatively charged GD2-pentasaccharide headgroup. Purified scFv and IgG mutant forms were then tested for antigen specificity by ELISA, for tissue specificity by immunohistochemistry, for affinity by BIACORE, for antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity in vitro, and for anti-tumor efficacy in xenografted humanized mice. The nearly 7-fold improvement in affinity of hu3F8 with a single D32H (L-CDR1) mutation translated into a ∼12-fold improvement in NK92MI-transfected CD16-mediated ADCC, a 6-fold improvement in CD32-mediated ADCC, and a 2.5-fold improvement in complement-mediated cytotoxicity while maintaining restricted normal tissue cross-reactivity and achieving substantial improvement in tumor ablation in vivo. Despite increasing GD2 affinity, the double mutation D32H (L-CDR1) and E1K (L-FR1) did not further improve anti-tumor efficacy. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Somatic immunoglobulin hypermutation

PubMed Central

Diaz, Marilyn; Casali, Paolo

2015-01-01

Immunoglobulin hypermutation provides the structural correlate for the affinity maturation of the antibody response. Characteristic modalities of this mechanism include a preponderance of point-mutations with prevalence of transitions over transversions, and the mutational hotspot RGYW sequence. Recent evidence suggests a mechanism whereby DNA-breaks induce error-prone DNA synthesis in immunoglobulin V(D)J regions by error-prone DNA polymerases. The nature of the targeting mechanism and the trans-factors effecting such breaks and their repair remain to be determined. PMID:11869898

Somatic mutations and affinity maturation are impaired by excessive numbers of T follicular helper cells and restored by Treg cells or memory T cells.

PubMed

Preite, Silvia; Baumjohann, Dirk; Foglierini, Mathilde; Basso, Camilla; Ronchi, Francesca; Fernandez Rodriguez, Blanca M; Corti, Davide; Lanzavecchia, Antonio; Sallusto, Federica

2015-11-01

We previously reported that Cd3e-deficient mice adoptively transferred with CD4(+) T cells generate high numbers of T follicular helper (Tfh) cells, which go on to induce a strong B-cell and germinal center (GC) reaction. Here, we show that in this system, GC B cells display an altered distribution between the dark and light zones, and express low levels of activation-induced cytidine deaminase. Furthermore, GC B cells from Cd3e(-/-) mice accumulate fewer somatic mutations as compared with GC B cells from wild-type mice, and exhibit impaired affinity maturation and reduced differentiation into long-lived plasma cells. Reconstitution of Cd3e(-/-) mice with regulatory T (Treg) cells restored Tfh-cell numbers, GC B-cell numbers and B-cell distribution within dark and light zones, and the rate of antibody somatic mutations. Tfh-cell numbers and GC B-cell numbers and dynamics were also restored by pre-reconstitution of Cd3e(-/-) mice with Cxcr5(-/-) Treg cells or non-regulatory, memory CD4(+) T cells. Taken together, these findings underline the importance of a quantitatively regulated Tfh-cell response for an efficient and long-lasting serological response. © 2015 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Immunological Control of Viral Infections in Bats and the Emergence of Viruses Highly Pathogenic to Humans

PubMed Central

Schountz, Tony; Baker, Michelle L.; Butler, John; Munster, Vincent

2017-01-01

Bats are reservoir hosts of many important viruses that cause substantial disease in humans, including coronaviruses, filoviruses, lyssaviruses, and henipaviruses. Other than the lyssaviruses, they do not appear to cause disease in the reservoir bats, thus an explanation for the dichotomous outcomes of infections of humans and bat reservoirs remains to be determined. Bats appear to have a few unusual features that may account for these differences, including evidence of constitutive interferon (IFN) activation and greater combinatorial diversity in immunoglobulin genes that do not undergo substantial affinity maturation. We propose these features may, in part, account for why bats can host these viruses without disease and how they may contribute to the highly pathogenic nature of bat-borne viruses after spillover into humans. Because of the constitutive IFN activity, bat-borne viruses may be shed at low levels from bat cells. With large naive antibody repertoires, bats may control the limited virus replication without the need for rapid affinity maturation, and this may explain why bats typically have low antibody titers to viruses. However, because bat viruses have evolved in high IFN environments, they have enhanced countermeasures against the IFN response. Thus, upon infection of human cells, where the IFN response is not constitutive, the viruses overwhelm the IFN response, leading to abundant virus replication and pathology. PMID:28959255

Sexually dimorphic development and binding characteristics of NMDA receptors in the brain of the platyfish

NASA Technical Reports Server (NTRS)

Flynn, K. M.; Schreibman, M. P.; Yablonsky-Alter, E.; Banerjee, S. P.

1999-01-01

This study investigated age- and gender-specific variations in properties of the glutamate N-methyl-d-aspartate receptor (NMDAR) in a freshwater teleost, the platyfish (Xiphophorus maculatus). Prior localization of the immunoreactive (ir)-R1 subunit of the NMDAR protein (R1) in cells of the nucleus olfactoretinalis (NOR), a primary gonadotropin-releasing hormone (GnRH)-containing brain nucleus in the platyfish, suggests that NMDAR, as in mammals, is involved in modulation of the platyfish brain-pituitary-gonad (BPG) axis. The current study shows that the number of cells in the NOR displaying ir-R1 is significantly increased in pubescent and mature female platyfish when compared to immature and senescent animals. In males, there is no significant change in ir-R1 expression in the NOR at any time in their lifespan. The affinity of the noncompetitive antagonist ((3)H)MK-801 for the NMDAR is significantly increased in pubescent females while maximum binding of ((3)H)MK-801 to the receptor reaches a significant maximum in mature females. In males, both MK-801 affinity and maximum binding remain unchanged throughout development. This is the first report of gender differences in the association of NMDA receptors with neuroendocrine brain areas during development. It is also the first report to suggest NMDA receptor involvement in the development of the BPG axis in a nonmammalian vertebrate. Copyright 1999 Academic Press.

Immunocytochemical localization of actin in epithelial cells of rat small intestine by light and electron microscopy.

PubMed

Hagen, S J; Trier, J S

1988-07-01

We used post-embedding immunocytochemical techniques and affinity-purified anti-actin antibody to evaluate localization of actin in epithelial cells of small intestine by fluorescence and electron microscopy. Small intestine was fixed with 2% formaldehyde-0.1% glutaraldehyde and embedded in Lowicryl K4M. One-micron or thin sections were stained with antibody followed by rhodamine- or colloidal gold-labeled goat anti-rabbit IgG, respectively. Label was present overlying microvilli, the apical terminal web, and the cytoplasm directly adjacent to occluding and intermediate junctions. Label was associated with outer mitochondrial membranes of all cells and the supranuclear Golgi region of goblet cells. Lateral cytoplasmic interdigitations between mature cells and subplasmalemmal filaments next to intrusive cells were densely labeled. The cytoplasm adjacent to unplicated domains of lateral membrane was focally labeled. Label was prominent over organized filament bundles within the subplasmalemmal web at the base of mature cells, whereas there was focal labeling of the cytoplasm adjacent to the basal membrane of undifferentiated cells. Basolateral epithelial cell processes were labeled. Label was focally present overlying the cellular ground substance. Our results demonstrate that actin is distributed in a distinctive fashion within intestinal epithelial cells. This distribution suggests that in addition to its function as a structural protein, actin may participate in regulation of epithelial tight junction permeability, in motile processes including migration of cells from the crypt to the villus tip, in accommodation of intrusive intraepithelial cells and in adhesion of cells to one another and to their substratum.

The presence of high-affinity, low-capacity estradiol-17β binding in rainbow trout scale indicates a possible endocrine route for the regulation of scale resorption

USGS Publications Warehouse

Persson, Petra; Shrimpton, J.M.; McCormick, S.D.; Bjornsson, Bjorn Thrandur

2000-01-01

High-affinity, low-capacity estradiol-17β (E2) binding is present in rainbow trout scale. The Kd and Bmax of the scale E2 binding are similar to those of the liver E2 receptor (Kd is 1.6 ± 0.1 and 1.4 ± 0.1 nM, and Bmax is 9.1 ± 1.2 and 23.1 ± 2.2 fmol x mg protein-1, for scale and liver, respectively), but different from those of the high-affinity, low-capacity E2 binding in plasma (Kd is 4.0 ± 0.4 nM and Bmax is 625.4 ± 63.1 fmol x mg protein-1). The E2 binding in scale was displaced by testosterone, but not by diethylstilbestrol. Hence, the ligand binding specificity is different from that of the previously characterized liver E2 receptor, where E2 is displaced by diethylstilbestrol, but not by testosterone. The putative scale E2 receptor thus appears to bind both E2 and testosterone, and it is proposed that the increased scale resorption observed during sexual maturation in both sexes of several salmonid species may be mediated by this receptor. No high-affinity, low-capacity E2 binding could be detected in rainbow trout gill or skin.

Boca-dependent maturation of β-propeller/EGF modules in low-density lipoprotein receptor proteins

PubMed Central

Culi, Joaquim; Springer, Timothy A; Mann, Richard S

2004-01-01

The extracellular portions of cell surface receptor proteins are often comprised of independently folding protein domains. As they are translated into the endoplasmic reticulum (ER), some of these domains require protein chaperones to assist in their folding. Members of the low-density lipoprotein receptor (LDLR) family require the chaperone called Boca in Drosophila or its ortholog, Mesoderm development, in the mouse. All LDLRs have at least one six-bladed β-propeller domain, which is immediately followed by an epidermal growth factor (EGF) repeat. We show here that Boca is specifically required for the maturation of these β-propeller/EGF modules through the secretory pathway, but is not required for other LDLR domains. Protein interaction data suggest that as LDLRs are translated into the ER, Boca binds to the β-propeller. Subsequently, once the EGF repeat is translated, the β-propeller/EGF module achieves a more mature state that has lower affinity for Boca. We also show that Boca-dependent β-propeller/EGF modules are found not only throughout the LDLR family but also in the precursor to the mammalian EGF ligand. PMID:15014448

Involvement of estradiol-17beta and its membrane receptor, G protein coupled receptor 30 (GPR30) in regulation of oocyte maturation in zebrafish, Danio rario.

PubMed

Pang, Yefei; Thomas, Peter

2009-03-01

The orphan G protein coupled receptor, GPR30, has the characteristics of a high affinity, specific estrogen membrane receptor on Atlantic croaker oocytes and mediates estrogen inhibition of oocyte maturation in this perciform fish. In order to determine the broad applicability of these findings to other teleosts, similar experiments were conducted in a cyprinid fish, zebrafish, in the present study. GPR30 mRNA expression was detected in zebrafish oocytes but not in the ovarian follicular cells. Both spontaneous and 17, 20beta-dihyroxy-4-pregnen-3-one (DHP)-induced maturation of follicle-enclosed zebrafish oocytes was significantly decreased when they were incubated with either estradiol-17beta, or the GPR30 agonists, ICI 182 780 and tamoxifen, or with the GPR30 specific agonist G-1. On the other hand spontaneous oocyte maturation increased two-fold when zebrafish ovarian follicles were incubated with an aromatase inhibitor, ATD. Moreover, the stimulatory effects of ATD on germinal vesicle breakdown (GVBD) were partially reversed by co-treatment with 100 nM of E2 or G-1. These results suggest that endogenous estrogens acting through GPR30 are involved in maintaining meiotic arrest of zebrafish oocytes.

Erythroid pyrimidine 5'-nucleotidase: cloning, developmental expression, and regulation by cAMP and in vivo hypoxia.

PubMed

Mass, Markus; Simo, Erika; Dragon, Stefanie

2003-12-01

A characteristic process of terminal erythroid differentiation is the degradation of ribosomal RNA into mononucleotides. The pyrimidine mononucleotides can be dephosphorylated by pyrimidine 5'-nucleotidase (P5N-I). In humans, a lack of this enzyme causes hemolytic anemia with ribosomal structures and trinucleotides retained in the red blood cells (RBCs). Although the protein/nucleotide sequence of P5N-I is known in mammals, the onset and regulation of P5N-I during erythroid maturation is unknown. However, in circulating chicken embryonic RBCs, the enzyme is induced together with carbonic anhydrase (CAII) and 2,3-bisphosphoglycerate (2,3-BPG) by norepinephrine (NE) and adenosine, which are released by the embryo under hypoxic conditions. Here, we present the chicken P5N-I sequence and the gene expression of P5N-I during RBC maturation; the profile of gene expression follows the enzyme activity with a rise between days 13 and 16 of embryonic development. The p5n-I expression is induced (1) in definitive but not primitive RBCs by stimulation of beta-adrenergic/adenosine receptors, and (2) in definitive RBCs by hypoxic incubation of the chicken embryo. Since embryonic RBCs increase their hemoglobin-oxygen affinity by degradation of nucleotides such as uridine triphosphate (UTP) and cytidine triphosphate (CTP), the induction of p5n-I expression can be seen as an adaptive response to hypoxia.

Second Generation Grp94-Selective Inhibitors Provide Opportunities for the Inhibition of Metastatic Cancer

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

Crowley, Vincent M.; Huard, Dustin J. E.; Lieberman, Raquel L.

Glucose regulated protein 94 (Grp94) is the endoplasmic reticulum (ER) resident isoform of the 90 kDa heat shock protein (Hsp90) family and its inhibition represents a promising therapeutic target for the treatment of many diseases. Modification of the first generation cis-amide bioisostere imidazole to alter the angle between the resorcinol ring and the benzyl side chain via cis-amide replacements produced compounds with improved Grp94 affinity and selectivity. Structure–activity relationship studies led to the discovery of compound 30, which exhibits 540 nm affinity and 73-fold selectivity towards Grp94. Grp94 is responsible for the maturation and trafficking of proteins associated with cellmore » signaling and motility, including select integrins. The Grp94-selective inhibitor 30 was shown to exhibit potent anti-migratory effects against multiple aggressive and metastatic cancers.« less

Animal-Friendly Affinity Reagents: Replacing the Needless in the Haystack.

PubMed

Gray, A C; Sidhu, S S; Chandrasekera, P C; Hendriksen, C F M; Borrebaeck, C A K

2016-12-01

The multibillion-dollar global antibody industry produces an indispensable resource but that is generated using millions of animals. Despite the irrefutable maturation and availability of animal-friendly affinity reagents (AFAs) employing naïve B lymphocyte or synthetic recombinant technologies expressed by phage display, animal immunisation is still authorised for antibody production. Remarkably, replacement opportunities have been overlooked, despite the enormous potential reduction in animal use. Directive 2010/63/EU requires that animals are not used where alternatives exist. To ensure its implementation, we have engaged in discussions with the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) and the Directorate General for Environment to carve out an EU-led replacement strategy. Measures must be imposed to avoid outsourcing, regulate commercial production, and ensure that antibody producers are fully supported. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Knock-Down of a Tonoplast Localized Low-Affinity Nitrate Transporter OsNPF7.2 Affects Rice Growth under High Nitrate Supply

PubMed Central

Hu, Rui; Qiu, Diyang; Chen, Yi; Miller, Anthony J.; Fan, Xiaorong; Pan, Xiaoping; Zhang, Mingyong

2016-01-01

The large nitrate transporter 1/peptide transporter family (NPF) has been shown to transport diverse substrates, including nitrate, amino acids, peptides, phytohormones, and glucosinolates. However, the rice (Oryza sativa) root-specific family member OsNPF7.2 has not been functionally characterized. Here, our data show that OsNPF7.2 is a tonoplast localized low-affinity nitrate transporter, that affects rice growth under high nitrate supply. Expression analysis showed that OsNPF7.2 was mainly expressed in the elongation and maturation zones of roots, especially in the root sclerenchyma, cortex and stele. It was also induced by high concentrations of nitrate. Subcellular localization analysis showed that OsNPF7.2 was localized on the tonoplast of large and small vacuoles. Heterologous expression in Xenopus laevis oocytes suggested that OsNPF7.2 was a low-affinity nitrate transporter. Knock-down of OsNPF7.2 retarded rice growth under high concentrations of nitrate. Therefore, we deduce that OsNPF7.2 plays a role in intracellular allocation of nitrate in roots, and thus influences rice growth under high nitrate supply. PMID:27826301

Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site.

PubMed

Hatoum-Aslan, Asma; Maniv, Inbal; Marraffini, Luciano A

2011-12-27

Precise RNA processing is fundamental to all small RNA-mediated interference pathways. In prokaryotes, clustered, regularly interspaced, short palindromic repeats (CRISPR) loci encode small CRISPR RNAs (crRNAs) that protect against invasive genetic elements by antisense targeting. CRISPR loci are transcribed as a long precursor that is cleaved within repeat sequences by CRISPR-associated (Cas) proteins. In many organisms, this primary processing generates crRNA intermediates that are subject to additional nucleolytic trimming to render mature crRNAs of specific lengths. The molecular mechanisms underlying this maturation event remain poorly understood. Here, we defined the genetic requirements for crRNA primary processing and maturation in Staphylococcus epidermidis. We show that changes in the position of the primary processing site result in extended or diminished maturation to generate mature crRNAs of constant length. These results indicate that crRNA maturation occurs by a ruler mechanism anchored at the primary processing site. We also show that maturation is mediated by specific cas genes distinct from those genes involved in primary processing, showing that this event is directed by CRISPR/Cas loci.

Neuropeptides shaping the central nervous system development: Spatiotemporal actions of VIP and PACAP through complementary signaling pathways.

PubMed

Maduna, Tando; Lelievre, Vincent

2016-12-01

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are neuropeptides with wide, complementary, and overlapping distributions in the central and peripheral nervous systems, where they exert important regulatory roles in many physiological processes. VIP and PACAP display a large range of biological cellular targets and functions in the adult nervous system including regulation of neurotransmission and neuroendocrine secretion and neuroprotective and neuroimmune responses. As the main focus of the present review, VIP and PACAP also have been long implicated in nervous system development and maturation through their interaction with the seven transmembrane domain G protein-coupled receptors, PAC1, VPAC1, and VPAC2, initiating multiple signaling pathways. Compared with PAC1, which solely binds PACAP with very high affinity, VPACs exhibit high affinities for both VIP and PACAP but differ from each other because of their pharmacological profile for both natural accessory peptides and synthetic or chimeric molecules, with agonistic and antagonistic properties. Complementary to initial pharmacological studies, transgenic animals lacking these neuropeptides or their receptors have been used to further characterize the neuroanatomical, electrophysiological, and behavioral roles of PACAP and VIP in the developing central nervous system. In this review, we recapitulate the critical steps and processes guiding/driving neurodevelopment in vertebrates and superimposing the potential contribution of PACAP and VIP receptors on the given timeline. We also describe how alterations in VIP/PACAP signaling may contribute to both (neuro)developmental and adult pathologies and suggest that tuning of VIP/PACAP signaling in a spatiotemporal manner may represent a novel avenue for preventive therapies of neurological and psychiatric disorders. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Identity, Affinity, Reality: Making the Case for Affinity Groups in Elementary School

ERIC Educational Resources Information Center

Parsons, Julie; Ridley, Kimberly

2012-01-01

Affinity groups are places where students build connections and process "ouch" moments from their classes. Children talk about the isolation they sometimes feel. The relationships students gain through race-based affinity groups enable them to feel less alone with their emotions and help them build a stronger sense of self. At the same…

BDNF Binds Its Pro-Peptide with High Affinity and the Common Val66Met Polymorphism Attenuates the Interaction

PubMed Central

Uegaki, Koichi; Kumanogoh, Haruko; Mizui, Toshiyuki; Hirokawa, Takatsugu; Ishikawa, Yasuyuki; Kojima, Masami

2017-01-01

Most growth factors are initially synthesized as precursors then cleaved into bioactive mature domains and pro-domains, but the biological roles of pro-domains are poorly understood. In the present study, we investigated the pro-domain (or pro-peptide) of brain-derived neurotrophic factor (BDNF), which promotes neuronal survival, differentiation and synaptic plasticity. The BDNF pro-peptide is a post-processing product of the precursor BDNF. Using surface plasmon resonance and biochemical experiments, we first demonstrated that the BDNF pro-peptide binds to mature BDNF with high affinity, but not other neurotrophins. This interaction was more enhanced at acidic pH than at neutral pH, suggesting that the binding is significant in intracellular compartments such as trafficking vesicles rather than the extracellular space. The common Val66Met BDNF polymorphism results in a valine instead of a methionine in the pro-domain, which affects human brain functions and the activity-dependent secretion of BDNF. We investigated the influence of this variation on the interaction between BDNF and the pro-peptide. Interestingly, the Val66Met polymorphism stabilized the heterodimeric complex of BDNF and its pro-peptide. Furthermore, compared with the Val-containing pro-peptide, the complex with the Met-type pro-peptide was more stable at both acidic and neutral pH, suggesting that the Val66Met BDNF polymorphism forms a more stable complex. A computational modeling provided an interpretation to the role of the Val66Met mutation in the interaction of BDNF and its pro-peptide. Lastly, we performed electrophysiological experiments, which indicated that the BDNF pro-peptide, when pre-incubated with BDNF, attenuated the ability of BDNF to inhibit hippocampal long-term depression (LTD), suggesting a possibility that the BDNF pro-peptide may interact directly with BDNF and thereby inhibit its availability. It was previously reported that the BDNF pro-domain exerts a chaperone-like function and assists the folding of the BDNF protein. However, our results suggest a new role for the BDNF pro-domain (or pro-peptide) following proteolytic cleave of precursor BDNF, and provide insight into the Val66Met polymorphism. PMID:28498321

Bisphenol A and Related Alkylphenols Exert Nongenomic Estrogenic Actions Through a G Protein-Coupled Estrogen Receptor 1 (Gper)/Epidermal Growth Factor Receptor (Egfr) Pathway to Inhibit Meiotic Maturation of Zebrafish Oocytes1

PubMed Central

Fitzgerald, Amanda C.; Peyton, Candace; Dong, Jing; Thomas, Peter

2015-01-01

Xenobiotic estrogens, such as bisphenol A (BPA), disrupt a wide variety of genomic estrogen actions, but their nongenomic estrogen actions remain poorly understood. We investigated nongenomic estrogenic effects of low concentrations of BPA and three related alkylphenols on the inhibition of zebrafish oocye maturation (OM) mediated through a G protein-coupled estrogen receptor 1 (Gper)-dependent epidermal growth factor receptor (Egfr) pathway. BPA (10–100 nM) treatment for 3 h mimicked the effects of estradiol-17beta (E2) and EGF, decreasing spontaneous maturation of defolliculated zebrafish oocytes, an effect not blocked by coincubation with actinomycin D, but blocked by coincubation with a Gper antibody. BPA displayed relatively high binding affinity (15.8% that of E2) for recombinant zebrafish Gper. The inhibitory effects of BPA were attenuated by inhibition of upstream regulators of Egfr, intracellular tyrosine kinase (Src) with PP2, and matrix metalloproteinase with ilomastat. Treatment with an inhibitor of Egfr transactivation, AG1478, and an inhibitor of the mitogen-activated protein kinase (MAPK) 3/1 pathway, U0126, increased spontaneous OM and blocked the inhibitory effects of BPA, E2, and the selective GPER agonist, G-1. Western blot analysis showed that BPA (10–200 nM) mimicked the stimulatory effects of E2 and EGF on Mapk3/1 phosphorylation. Tetrabromobisphenol A, 4-nonylphenol, and tetrachlorobisphenol A (5–100 nM) also inhibited OM, an effect blocked by cotreatment with AG1478, as well as with the GPER antagonist, G-15, and displayed similar binding affinities as BPA to zebrafish Gper. The results suggest that BPA and related alkylphenols disrupt zebrafish OM by a novel nongenomic estrogenic mechanism involving activation of the Gper/Egfr/Mapk3/1 pathway. PMID:26490843

Bisphenol A and Related Alkylphenols Exert Nongenomic Estrogenic Actions Through a G Protein-Coupled Estrogen Receptor 1 (Gper)/Epidermal Growth Factor Receptor (Egfr) Pathway to Inhibit Meiotic Maturation of Zebrafish Oocytes.

PubMed

Fitzgerald, Amanda C; Peyton, Candace; Dong, Jing; Thomas, Peter

2015-12-01

Xenobiotic estrogens, such as bisphenol A (BPA), disrupt a wide variety of genomic estrogen actions, but their nongenomic estrogen actions remain poorly understood. We investigated nongenomic estrogenic effects of low concentrations of BPA and three related alkylphenols on the inhibition of zebrafish oocye maturation (OM) mediated through a G protein-coupled estrogen receptor 1 (Gper)-dependent epidermal growth factor receptor (Egfr) pathway. BPA (10-100 nM) treatment for 3 h mimicked the effects of estradiol-17beta (E2) and EGF, decreasing spontaneous maturation of defolliculated zebrafish oocytes, an effect not blocked by coincubation with actinomycin D, but blocked by coincubation with a Gper antibody. BPA displayed relatively high binding affinity (15.8% that of E2) for recombinant zebrafish Gper. The inhibitory effects of BPA were attenuated by inhibition of upstream regulators of Egfr, intracellular tyrosine kinase (Src) with PP2, and matrix metalloproteinase with ilomastat. Treatment with an inhibitor of Egfr transactivation, AG1478, and an inhibitor of the mitogen-activated protein kinase (MAPK) 3/1 pathway, U0126, increased spontaneous OM and blocked the inhibitory effects of BPA, E2, and the selective GPER agonist, G-1. Western blot analysis showed that BPA (10-200 nM) mimicked the stimulatory effects of E2 and EGF on Mapk3/1 phosphorylation. Tetrabromobisphenol A, 4-nonylphenol, and tetrachlorobisphenol A (5-100 nM) also inhibited OM, an effect blocked by cotreatment with AG1478, as well as with the GPER antagonist, G-15, and displayed similar binding affinities as BPA to zebrafish Gper. The results suggest that BPA and related alkylphenols disrupt zebrafish OM by a novel nongenomic estrogenic mechanism involving activation of the Gper/Egfr/Mapk3/1 pathway. © 2015 by the Society for the Study of Reproduction, Inc.

Enhanced stability of monomer fold correlates with extreme drug resistance of HIV-1 protease.

PubMed

Louis, John M; Tözsér, József; Roche, Julien; Matúz, Krisztina; Aniana, Annie; Sayer, Jane M

2013-10-29

During treatment, mutations in HIV-1 protease (PR) are selected rapidly that confer resistance by decreasing affinity to clinical protease inhibitors (PIs). As these unique drug resistance mutations can compromise the fitness of the virus to replicate, mutations that restore conformational stability and activity while retaining drug resistance are selected on further evolution. Here we identify several compensating mechanisms by which an extreme drug-resistant mutant bearing 20 mutations (PR20) with >5-fold increased Kd and >4000-fold decreased affinity to the PI darunavir functions. (1) PR20 cleaves, albeit poorly, Gag polyprotein substrates essential for viral maturation. (2) PR20 dimer, which exhibits distinctly enhanced thermal stability, has highly attenuated autoproteolysis, thus likely prolonging its lifetime in vivo. (3) The enhanced stability of PR20 results from stabilization of the monomer fold. Both monomeric PR20(T26A) and dimeric PR20 exhibit Tm values 6-7.5 °C higher than those for their PR counterparts. Two specific mutations in PR20, L33F and L63P at sites of autoproteolysis, increase the Tm of monomeric PR(T26A) by ~8 °C, similar to PR20(T26A). However, without other compensatory mutations as seen in PR20, L33F and L63P substitutions, together, neither restrict autoproteolysis nor significantly reduce binding affinity to darunavir. To determine whether dimer stability contributes to binding affinity for inhibitors, we examined single-chain dimers of PR and PR(D25N) in which the corresponding identical monomer units were covalently linked by GGSSG sequence. Linking of the subunits did not appreciably change the ΔTm on inhibitor binding; thus stabilization by tethering appears to have little direct effect on enhancing inhibitor affinity.

Enrichment of high affinity subclasses and glycoforms from serum-derived IgG using FcγRs as affinity ligands.

PubMed

Boesch, Austin W; Kappel, James H; Mahan, Alison E; Chu, Thach H; Crowley, Andrew R; Osei-Owusu, Nana Y; Alter, Galit; Ackerman, Margaret E

2018-05-01

As antibodies continue to gain predominance in drug discovery and development pipelines, efforts to control and optimize their activity in vivo have matured to incorporate sophisticated abilities to manipulate engagement of specific Fc binding partners. Such efforts to promote diverse functional outcomes include modulating IgG-Fc affinity for FcγRs to alternatively potentiate or reduce effector functions, such as antibody-dependent cellular cytotoxicity and phagocytosis. While a number of natural and engineered Fc features capable of eliciting variable effector functions have been demonstrated in vitro and in vivo, elucidation of these important functional relationships has taken significant effort through use of diverse genetic, cellular and enzymatic techniques. As an orthogonal approach, we demonstrate use of FcγR as chromatographic affinity ligands to enrich and therefore simultaneously identify favored binding species from a complex mixture of serum-derived pooled polycloncal human IgG, a load material that contains the natural repertoire of Fc variants and post-translational modifications. The FcγR-enriched IgG was characterized for subclass and glycoform composition and the impact of this bioseparation step on antibody activity was measured in cell-based effector function assays including Natural Killer cell activation and monocyte phagocytosis. This work demonstrates a tractable means to rapidly distinguish complex functional relationships between two or more interacting biological agents by leveraging affinity chromatography followed by secondary analysis with high-resolution biophysical and functional assays and emphasizes a platform capable of surveying diverse natural post-translational modifications that may not be easily produced with high purity or easily accessible with recombinant expression techniques. © 2018 Wiley Periodicals, Inc.

T Cell Receptor Engineering and Analysis Using the Yeast Display Platform

PubMed Central

Smith, Sheena N.; Harris, Daniel T.; Kranz, David M.

2017-01-01

The αβ heterodimeric T cell receptor (TCR) recognizes peptide antigens that are transported to the cell surface as a complex with a protein encoded by the major histocompatibility complex (MHC). T cells thus evolved a strategy to sense these intracellular antigens, and to respond either by eliminating the antigen-presenting cell (e.g. a virus-infected cell) or by secreting factors that recruit the immune system to the site of the antigen. The central role of the TCR in the binding of antigens as peptide-MHC (pepMHC) ligands has now been studied thoroughly. Interestingly, despite their exquisite sensitivity (e.g. T cell activation by as few as 1 to 3 pepMHC complexes on a single target cell), TCRs are known to have relatively low affinities for pepMHC, with KD values in the micromolar range. There has been interest in engineering the affinity of TCRs in order to use this class of molecules in ways similar to now done with antibodies. By doing so, it would be possible to harness the potential of TCRs as therapeutics against a much wider array of antigens that include essentially all intracellular targets. To engineer TCRs, and to analyze their binding features more rapidly, we have used a yeast display system as a platform. Expression and engineering of a single-chain form of the TCR, analogous to scFv fragments from antibodies, allow the TCR to be affinity matured with a variety of possible pepMHC ligands. In addition, the yeast display platform allows one to rapidly generate TCR variants with diverse binding affinities and to analyze specificity and affinity without the need for purification of soluble forms of the TCRs. The present chapter describes the methods for engineering and analyzing single-chain TCRs using yeast display. PMID:26060072

Isolation and characterization of an IgNAR variable domain specific for the human mitochondrial translocase receptor Tom70.

PubMed

Nuttall, Stewart D; Krishnan, Usha V; Doughty, Larissa; Pearson, Kylie; Ryan, Michael T; Hoogenraad, Nicholas J; Hattarki, Meghan; Carmichael, Jennifer A; Irving, Robert A; Hudson, Peter J

2003-09-01

The new antigen receptor (IgNAR) from sharks is a disulphide bonded dimer of two protein chains, each containing one variable and five constant domains, and functions as an antibody. In order to assess the antigen-binding capabilities of isolated IgNAR variable domains (VNAR), we have constructed an in vitro library incorporating synthetic CDR3 regions of 15-18 residues in length. Screening of this library against the 60 kDa cytosolic domain of the 70 kDa outer membrane translocase receptor from human mitochondria (Tom70) resulted in one dominant antigen-specific clone (VNAR 12F-11) after four rounds of in vitro selection. VNAR 12F-11 was expressed into the Escherichia coli periplasm and purified by anti-FLAG affinity chromatography at yields of 3 mg x L(-1). Purified protein eluted from gel filtration columns as a single monomeric protein and CD spectrum analysis indicated correct folding into the expected beta-sheet conformation. Specific binding to Tom70 was demonstrated by ELISA and BIAcore (Kd = 2.2 +/- 0.31 x 10(-9) m-1) indicating that these VNAR domains can be efficiently displayed as bacteriophage libraries, and selected against target antigens with an affinity and stability equivalent to that obtained for other single domain antibodies. As an initial step in producing 'intrabody' variants of 12F-11, the impact of modifying or removing the conserved immunoglobulin intradomain disulphide bond was assessed. High affinity binding was only retained in the wild-type protein, which combined with our inability to affinity mature 12F-11, suggests that this particular VNAR is critically dependent upon precise CDR loop conformations for its binding affinity.

Identification of Useful Nanobodies by Phage Display of Immune Single Domain Libraries Derived from Camelid Heavy Chain Antibodies.

PubMed

Romao, Ema; Morales-Yanez, Francisco; Hu, Yaozhong; Crauwels, Maxine; De Pauw, Pieter; Hassanzadeh, Gholamreza Ghassanzadeh; Devoogdt, Nick; Ackaert, Chloe; Vincke, Cecile; Muyldermans, Serge

2016-01-01

The discovery of functional heavy chain-only antibodies devoid of light chains in sera of camelids and sharks in the early nineties provided access to the generation of minimal-sized, single-domain, in vivo affinity-matured, recombinant antigenbinding fragments, also known as Nanobodies. Recombinant DNA technology and adaptation of phage display vectors form the basis to construct large naïve, synthetic or medium sized immune libraries from where multiple Nanobodies have been retrieved. Alternative selection methods (i.e. bacterial display, bacterial two-hybrid, Cis-display and ribosome display) have also been developed to identify Nanobodies. The antigen affinity, stability, expression yields and structural details of the Nanobodies have been determined by standard technology. Nanobodies were subsequently engineered for higher stability and affinity, to have a sequence closer to that of human immunoglobulin domains, or to add designed effector functions. Antigen specific Nanobodies recognizing with high affinity their cognate antigen were retrieved from various libraries. High expression yields are obtained from microorganisms, even when expressed in the cytoplasm. The purified Nanobodies are shown to possess beneficial biochemical and biophysical properties. The crystal structure of Nanobody::antigen complexes reveal the preference of Nanobodies for cavities on the antigen surface. Thanks to the properties described above, Nanobodies became a highly valued and versatile tool for biomolecular research. Moreover, numerous diagnostic and therapeutic Nanobody-based applications have been developed in the past decade. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

The presence of high-affinity, low-capacity estradiol-17β binding in rainbow trout scale indicates a possible endocrine route for the regulation of scale resorption

USGS Publications Warehouse

Persson, Petra; Shrimpton, J. Mark; McCormick, Stephen D.; Bjornsson, Bjorn Thrandur

2000-01-01

High-affinity, low-capacity estradiol-17β (E2) binding is present in rainbow trout scale. The Kd and Bmax of the scale E2 binding are similar to those of the liver E2 receptor (Kd is 1.6 ± 0.1 and 1.4 ± 0.1 nM, and Bmax is 9.1 ± 1.2 and 23.1 ± 2.2 fmol × mg protein-1, for scale and liver, respectively), but different from those of the high-affinity, low-capacity E2 binding in plasma (Kd is 4.0 ± 0.4 nM and Bmax is 625.4 ± 63.1 fmol × mg protein−1). The E2 binding in scale was displaced by testosterone, but not by diethylstilbestrol. Hence, the ligand binding specificity is different from that of the previously characterized liver E2 receptor, where E2 is displaced by diethylstilbestrol, but not by testosterone. The putative scale E2 receptor thus appears to bind both E2 and testosterone, and it is proposed that the increased scale resorption observed during sexual maturation in both sexes of several salmonid species may be mediated by this receptor. No high-affinity, low-capacity E2 binding could be detected in rainbow trout gill or skin.

Environmental sensing by mature B cells is controlled by the transcription factors PU.1 and SpiB.

PubMed

Willis, Simon N; Tellier, Julie; Liao, Yang; Trezise, Stephanie; Light, Amanda; O'Donnell, Kristy; Garrett-Sinha, Lee Ann; Shi, Wei; Tarlinton, David M; Nutt, Stephen L

2017-11-10

Humoral immunity requires B cells to respond to multiple stimuli, including antigen, membrane and soluble ligands, and microbial products. Ets family transcription factors regulate many aspects of haematopoiesis, although their functions in humoral immunity are difficult to decipher as a result of redundancy between the family members. Here we show that mice lacking both PU.1 and SpiB in mature B cells do not generate germinal centers and high-affinity antibody after protein immunization. PU.1 and SpiB double-deficient B cells have a survival defect after engagement of CD40 or Toll-like receptors (TLR), despite paradoxically enhanced plasma cell differentiation. PU.1 and SpiB regulate the expression of many components of the B cell receptor signaling pathway and the receptors for CD40L, BAFF and TLR ligands. Thus, PU.1 and SpiB enable B cells to appropriately respond to environmental cues.

Human Immune Disorder Arising from Mutation of the α Chain of the Interleukin-2 Receptor

NASA Astrophysics Data System (ADS)

Sharfe, Nigel; Dadi, Harjit K.; Shahar, Michal; Roifman, Chaim M.

1997-04-01

Profound cellular immunodeficiency occurs as the result of mutations in proteins involved in both the differentiation and function of mature lymphoid cells. We describe here a novel human immune aberration arising from a truncation mutation of the interleukin-2 receptor α chain (CD25), a subunit of the tripartite high-affinity receptor for interleukin 2. This immunodeficiency is characterized by decreased numbers of peripheral T cells displaying abnormal proliferation but normal B cell development. Extensive lymphocytic infiltration of tissues, including lung, liver, gut, and bone, is observed, accompanied by tissue atrophy and inflammation. Although mature T cells are present, the absence of CD25 does affect the differentiation of thymocytes. While displaying normal development of CD2, CD3, CD4, and CD8 expression, CD25-deficient cortical thymocytes do not express CD1, and furthermore they fail to normally down-regulate levels of the anti-apoptotic protein bcl-2.

Focused Evolution of HIV-1 Neutralizing Antibodies Revealed by Structures and Deep Sequencing

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

Wu, Xueling; Zhou, Tongqing; Zhu, Jiang

2013-03-04

Antibody VRC01 is a human immunoglobulin that neutralizes about 90% of HIV-1 isolates. To understand how such broadly neutralizing antibodies develop, we used x-ray crystallography and 454 pyrosequencing to characterize additional VRC01-like antibodies from HIV-1-infected individuals. Crystal structures revealed a convergent mode of binding for diverse antibodies to the same CD4-binding-site epitope. A functional genomics analysis of expressed heavy and light chains revealed common pathways of antibody-heavy chain maturation, confined to the IGHV1-2*02 lineage, involving dozens of somatic changes, and capable of pairing with different light chains. Broadly neutralizing HIV-1 immunity associated with VRC01-like antibodies thus involves the evolution ofmore » antibodies to a highly affinity-matured state required to recognize an invariant viral structure, with lineages defined from thousands of sequences providing a genetic roadmap of their development.« less

A baculovirus polyhedron envelope protein: immunogold localization in infected cells and mature polyhedra.

PubMed

Russell, R L; Rohrmann, G F

1990-01-01

A polyclonal antiserum against a trpE fusion protein containing the complete open reading frame of the polyhedron envelope (PE) protein from the nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV) was used for immunogold staining and electron microscopic examination of polyhedra, isolated polyhedron envelopes, and infected insect cells at selected times postinfection. The antiserum specifically stained the peripheral envelope of mature polyhedra and also stained the envelope structure which remained after polyhedra were dissolved in dilute alkaline solutions. In OpMNPV-infected Lymantria dispar cells, the PE protein was detected by 48 hr postinfection (hr p.i.) but specific localization and staining of developing polyhedra were not evident. However, by 72 hr p.i. substantial and preferential staining of the periphery of developing polyhedra was evident even though a distinct polyhedron envelope was not yet observed. In addition, the periphery of fibrillar structures was stained by the PE antiserum. By 96 hr p.i., mature envelopes surrounded polyhedra and these polyhedron envelopes were stained with the PE antibody. The progression of PE protein staining during polyhedron morphogenesis indicates that the PE protein accumulates and becomes associated with developing polyhedra in the nucleus between 48 and 72 hr p.i. Very late in infection the mature polyhedron envelope forms on the polyhedron surface. The apparent affinity of the PE protein for the surface of maturing polyhedra suggests that it may be a major component of the polyhedron envelope or may form the matrix for the deposition of other components which contribute to the mature envelope. Immunogold staining and protease digestion experiments indicate that protein is an essential component of the polyhedron envelope.

High Fidelity Processing and Activation of the Human α-Defensin HNP1 Precursor by Neutrophil Elastase and Proteinase 3

PubMed Central

Tongaonkar, Prasad; Golji, Amir E.; Tran, Patti; Ouellette, André J.; Selsted, Michael E.

2012-01-01

The azurophilic granules of human neutrophils contain four α-defensins called human neutrophil peptides (HNPs 1–4). HNPs are tridisulfide-linked antimicrobial peptides involved in the intracellular killing of organisms phagocytosed by neutrophils. The peptides are produced as inactive precursors (proHNPs) which are processed to active microbicides by as yet unidentified convertases. ProHNP1 was expressed in E. coli and the affinity-purified propeptide isolated as two species, one containing mature HNP1 sequence with native disulfide linkages (“folded proHNP1”) and the other containing non-native disulfide linked proHNP1 conformers (misfolded proHNP1). Native HNP1, liberated by CNBr treatment of folded proHNP1, was microbicidal against Staphylococcus aureus, but the peptide derived from misfolded proHNP1 was inactive. We hypothesized that neutrophil elastase (NE), proteinase 3 (PR3) or cathepsin G (CG), serine proteases that co-localize with HNPs in azurophil granules, are proHNP1 activating convertases. Folded proHNP1 was converted to mature HNP1 by both NE and PR3, but CG generated an HNP1 variant with an N-terminal dipeptide extension. NE and PR3 cleaved folded proHNP1 to produce a peptide indistinguishable from native HNP1 purified from neutrophils, and the microbicidal activities of in vitro derived and natural HNP1 peptides were equivalent. In contrast, misfolded proHNP1 conformers were degraded extensively under the same conditions. Thus, NE and PR3 possess proHNP1 convertase activity that requires the presence of the native HNP1 disulfide motif for high fidelity activation of the precursor in vitro. PMID:22448222

High fidelity processing and activation of the human α-defensin HNP1 precursor by neutrophil elastase and proteinase 3.

PubMed

Tongaonkar, Prasad; Golji, Amir E; Tran, Patti; Ouellette, André J; Selsted, Michael E

2012-01-01

The azurophilic granules of human neutrophils contain four α-defensins called human neutrophil peptides (HNPs 1-4). HNPs are tridisulfide-linked antimicrobial peptides involved in the intracellular killing of organisms phagocytosed by neutrophils. The peptides are produced as inactive precursors (proHNPs) which are processed to active microbicides by as yet unidentified convertases. ProHNP1 was expressed in E. coli and the affinity-purified propeptide isolated as two species, one containing mature HNP1 sequence with native disulfide linkages ("folded proHNP1") and the other containing non-native disulfide linked proHNP1 conformers (misfolded proHNP1). Native HNP1, liberated by CNBr treatment of folded proHNP1, was microbicidal against Staphylococcus aureus, but the peptide derived from misfolded proHNP1 was inactive. We hypothesized that neutrophil elastase (NE), proteinase 3 (PR3) or cathepsin G (CG), serine proteases that co-localize with HNPs in azurophil granules, are proHNP1 activating convertases. Folded proHNP1 was converted to mature HNP1 by both NE and PR3, but CG generated an HNP1 variant with an N-terminal dipeptide extension. NE and PR3 cleaved folded proHNP1 to produce a peptide indistinguishable from native HNP1 purified from neutrophils, and the microbicidal activities of in vitro derived and natural HNP1 peptides were equivalent. In contrast, misfolded proHNP1 conformers were degraded extensively under the same conditions. Thus, NE and PR3 possess proHNP1 convertase activity that requires the presence of the native HNP1 disulfide motif for high fidelity activation of the precursor in vitro.

Compression force sensing regulates integrin αIIbβ3 adhesive function on diabetic platelets.

PubMed

Ju, Lining; McFadyen, James D; Al-Daher, Saheb; Alwis, Imala; Chen, Yunfeng; Tønnesen, Lotte L; Maiocchi, Sophie; Coulter, Brianna; Calkin, Anna C; Felner, Eric I; Cohen, Neale; Yuan, Yuping; Schoenwaelder, Simone M; Cooper, Mark E; Zhu, Cheng; Jackson, Shaun P

2018-03-14

Diabetes is associated with an exaggerated platelet thrombotic response at sites of vascular injury. Biomechanical forces regulate platelet activation, although the impact of diabetes on this process remains ill-defined. Using a biomembrane force probe (BFP), we demonstrate that compressive force activates integrin α IIb β 3 on discoid diabetic platelets, increasing its association rate with immobilized fibrinogen. This compressive force-induced integrin activation is calcium and PI 3-kinase dependent, resulting in enhanced integrin affinity maturation and exaggerated shear-dependent platelet adhesion. Analysis of discoid platelet aggregation in the mesenteric circulation of mice confirmed that diabetes leads to a marked enhancement in the formation and stability of discoid platelet aggregates, via a mechanism that is not inhibited by therapeutic doses of aspirin and clopidogrel, but is eliminated by PI 3-kinase inhibition. These studies demonstrate the existence of a compression force sensing mechanism linked to α IIb β 3 adhesive function that leads to a distinct prothrombotic phenotype in diabetes.

Lunar sample studies. [breccias basalts, and anorthosites

NASA Technical Reports Server (NTRS)

1977-01-01

Lunar samples discussed and the nature of their analyses are: (1) an Apollo 15 breccia which is thoroughly analyzed as to the nature of the mature regolith from which it derived and the time and nature of the lithification process, (2) two Apollo 11 and one Apollo 12 basalts analyzed in terms of chemistry, Cross-Iddings-Pirsson-Washington norms, mineralogy, and petrography, (3) eight Apollo 17 mare basalts, also analyzed in terms of chemistry, Cross-Iddings-Pirsson-Washington norms, mineralogy, and petrography. The first seven are shown to be chemically similar although of two main textural groups; the eighth is seen to be distinct in both chemistry and mineralogy, (4) a troctolitic clast from a Fra Mauro breccia, analyzed and contrasted with other high-temperature lunar mineral assemblages. Two basaltic clasts from the same breccia are shown to have affinities with rock 14053, and (5) the uranium-thorium-lead systematics of three Apollo 16 samples are determined; serious terrestrial-lead contamination of the first two samples is attributed to bandsaw cutting in the lunar curatorial facility.

A systems analysis of the erythropoietic responses to weightlessness. Volume 2: Description of the model of erythropoiesis regulation. Part A: Model for regulation of erythropoiesis. Part B: Detailed description of the model for regulation of erythropoiesis

NASA Technical Reports Server (NTRS)

Leonard, J. I.

1985-01-01

A mathematical model of the erythropoiesis on total red blood cell mass is presented. The loss of red cell mass has been a consistent finding during space flight. Computer simulation of this phenomenon required a model that could account for oxygen transport, red cell production, and red cell destruction. The elements incorporated into the feedback regulation loop of the model are based on the accepted concept that erythrocyte production is governed by the balance between oxygen supply and demand in the body. The mechanisms and pathways of the control circuit include oxygenation of hemoglobin and oxygenation of tissues by blood transport and diffusional processes. Other features of the model include a variable oxygen-hemoglobin affinity, and time delays which represent time for erythropoietin (erythrocyte-stimulating hormone) distribution in plasma, and time for maturation of the erythrocytes in bone marrow.

Establishment of a simple and quantitative immunospot assay for detecting anti-type II collagen antibody using an infrared fluorescence imaging system (IFIS).

PubMed

Ota, Shusuke; Kanazawa, Satoshi; Kobayashi, Masaaki; Otsuka, Takanobu; Okamoto, Takashi

2005-04-01

Antibodies to type II collagen (col II) have been detected in patients with rheumatoid arthritis and in animal models of collagen induced arthritis. Here, we describe a novel method to detect anti-col II antibodies using an immunospot assay with an infrared fluorescence imaging system. This method showed very high sensitivity and specificity, and was simple, with low background levels. It also showed higher reproducibility and linearity, with a dynamic range of approximately 500-fold, than the conventional immunospot assay with enhanced chemiluminescence detection. Using this method we were able to demonstrate the antibody affinity maturation process in mice immunized with col II. In these immunized mice, although cross-reactive antibodies reacting with other collagen species were detected in earlier stages of immunization, the titers of cross-reactive antibodies rapidly diminished after the antigen boost, concomitantly with the elevation of the anti-col II antibody. The method and its possible applications are discussed.

Computing the binding affinity of a ligand buried deep inside a protein with the hybrid steered molecular dynamics

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

Villarreal, Oscar D.; Yu, Lili; Department of Laboratory Medicine, Yancheng Vocational Institute of Health Sciences, Yancheng, Jiangsu 224006

Computing the ligand-protein binding affinity (or the Gibbs free energy) with chemical accuracy has long been a challenge for which many methods/approaches have been developed and refined with various successful applications. False positives and, even more harmful, false negatives have been and still are a common occurrence in practical applications. Inevitable in all approaches are the errors in the force field parameters we obtain from quantum mechanical computation and/or empirical fittings for the intra- and inter-molecular interactions. These errors propagate to the final results of the computed binding affinities even if we were able to perfectly implement the statistical mechanicsmore » of all the processes relevant to a given problem. And they are actually amplified to various degrees even in the mature, sophisticated computational approaches. In particular, the free energy perturbation (alchemical) approaches amplify the errors in the force field parameters because they rely on extracting the small differences between similarly large numbers. In this paper, we develop a hybrid steered molecular dynamics (hSMD) approach to the difficult binding problems of a ligand buried deep inside a protein. Sampling the transition along a physical (not alchemical) dissociation path of opening up the binding cavity- -pulling out the ligand- -closing back the cavity, we can avoid the problem of error amplifications by not relying on small differences between similar numbers. We tested this new form of hSMD on retinol inside cellular retinol-binding protein 1 and three cases of a ligand (a benzylacetate, a 2-nitrothiophene, and a benzene) inside a T4 lysozyme L99A/M102Q(H) double mutant. In all cases, we obtained binding free energies in close agreement with the experimentally measured values. This indicates that the force field parameters we employed are accurate and that hSMD (a brute force, unsophisticated approach) is free from the problem of error amplification suffered by many sophisticated approaches in the literature.« less

Thermodynamic Bounds on the Ultra- and Infra-affinity of Hsp70 for Its Substrates

NASA Astrophysics Data System (ADS)

Nguyen, Basile; Hartich, David; Seifert, Udo; Rios, Paolo De Los

2017-07-01

The 70 kDa Heat Shock Proteins Hsp70 have several essential functions in living systems, such as protecting cells against protein aggregation, assisting protein folding, remodeling protein complexes and driving the translocation into organelles. These functions require high affinity for non-specific amino-acid sequences that are ubiquitous in proteins. It has been recently shown that this high affinity, called ultra-affinity, depends on a process driven out of equilibrium by ATP hydrolysis. Here we establish the thermodynamic bounds for ultra-affinity, and further show that the same reaction scheme can in principle be used both to strengthen and to weaken affinities (leading in this case to infra-affinity). We show that cofactors are essential to achieve affinity beyond the equilibrium range. Finally, biological implications are discussed.

Puberty and dispersal in a wild primate population

PubMed Central

Onyango, Patrick O.; Gesquiere, Laurence R.; Altmann, Jeanne; Alberts, Susan C.

2013-01-01

The onset of reproduction is preceded by a host of organismal adjustments and transformations, involving morphological, physiological, and behavioral changes. In highly social mammals, including humans and most nonhuman primates, the timing and nature of maturational processes is affected by the animal’s social milieu as well as its ecology. Here, we review a diverse set of findings on how maturation unfolds in wild baboons in the Amboseli basin of southern Kenya, and we place these findings in the context of other reports of maturational processes in primates and other mammals. First, we describe the series of events and processes that signal maturation in female and male baboons. Sex differences in age at both sexual maturity and first reproduction documented for this species are consistent with expectations of life history theory; males mature later than females and exhibit an adolescent growth spurt that is absent or minimal in females. Second, we summarize what we know about sources of variance in the timing of maturational processes including natal dispersal. In Amboseli, individuals in a food-enhanced group mature earlier than their wild-feeding counterparts, and offspring of high-ranking females mature earlier than offspring of low-ranking females. We also report on how genetic admixture, which occurs in Amboseli between two closely related baboon taxa, affects individual maturation schedules. PMID:23998668

Related Mechanisms of Antibody Somatic Hypermutation and Class Switch Recombination

PubMed Central

HWANG, JOYCE K.; ALT, FREDERICK W.; YEAP, LENG-SIEW

2015-01-01

The primary antibody repertoire is generated by mechanisms involving the assembly of the exons that encode the antigen-binding variable regions of immunoglobulin heavy (IgH) and light (IgL) chains during the early development of B lymphocytes. After antigen-dependent activation, mature B lymphocytes can further alter their IgH and IgL variable region exons by the process of somatic hypermutation (SHM), which allows the selection of B cells in which SHMs resulted in the production of antibodies with increased antigen affinity. In addition, during antigen-dependent activation, B cells can also change the constant region of their IgH chain through a DNA double-strand-break (DSB) dependent process referred to as IgH class switch recombination (CSR), which generates B cell progeny that produce antibodies with different IgH constant region effector functions that are best suited for a elimination of a particular pathogen or in a particular setting. Both the mutations that underlie SHM and the DSBs that underlie CSR are initiated in target genes by activation-induced cytidine deaminase (AID). This review describes in depth the processes of SHM and CSR with a focus on mechanisms that direct AID cytidine deamination in activated B cells and mechanisms that promote the differential outcomes of such cytidine deamination. PMID:26104555

Epigenetic and gene expression changes in the adolescent brain: What have we learned from animal models?

PubMed

Mychasiuk, Richelle; Metz, Gerlinde A S

2016-11-01

Adolescence is defined as the gradual period of transition between childhood and adulthood that is characterized by significant brain maturation, growth spurts, sexual maturation, and heightened social interaction. Although originally believed to be a uniquely human aspect of development, rodent and non-human primates demonstrate maturational patterns that distinctly support an adolescent stage. As epigenetic processes are essential for development and differentiation, but also transpire in mature cells in response to environmental influences, they are an important aspect of adolescent brain maturation. The purpose of this review article was to examine epigenetic programming in animal models of brain maturation during adolescence. The discussion focuses on animal models to examine three main concepts; epigenetic processes involved in normal adolescent brain maturation, the influence of fetal programming on adolescent brain development and the epigenome, and finally, postnatal experiences such as exercise and drugs that modify epigenetic processes important for adolescent brain maturation. This corollary emphasizes the utility of animal models to further our understanding of complex processes such as epigenetic regulation and brain development. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effect of hybrid, maturity, and mechanical processing of corn silage on intake and digestibility by beef cattle.

PubMed

Andrae, J G; Hunt, C W; Pritchard, G T; Kennington, L R; Harrison, J H; Kezar, W; Mahanna, W

2001-09-01

A study involving a 2 x 2 x 2 factorial arrangement of treatments was conducted to evaluate effects of hybrid (Pioneer 3335 and 3489), maturity (half milkline and blacklayer), and mechanical processing (field chopper with and without on-board rollers engaged) on intake and digestibility of corn silage. Forty Angus steers (322 +/- 5.2 kg BW) were assigned to the eight silage treatments (five steers per treatment) and individually fed using electronic gates. Diets consisted of 60% corn silage and 40% chopped alfalfa hay (DM basis). Following a 5-d adaptation period, intake was measured for 7 d and subsequently fecal samples were collected for 5 d. Chromic oxide (5 g/d) was fed beginning 7 d before fecal sample collection and digestibility was determined by the ratio of Cr in the feed and feces. Steers were reallocated to treatments and these procedures were repeated, providing 10 observations per treatment. In addition, all silages were ruminally incubated in six mature cows for 0, 8, 16, 24, 48, and 96 h to determine extent and rate of DM, starch, NDF, and ADF disappearance. Processing increased DMI of hybrid 3489 but did not affect DMI of hybrid 3335 (hybrid x processing; P < 0.06). Total tract digestibility of DM, starch, NDF, and ADF decreased (P < 0.01) as plant maturity increased. Maturity tended to decrease starch digestibility more for hybrid 3489 than for hybrid 3335 (hybrid x maturity; P < 0.10). Processing increased (P < 0.01) starch digestibility but decreased (P < 0.01) NDF and ADF digestibility, resulting in no processing effect on DM digestibility. There was a numerical trend for processing to increase starch digestibility more for latethan for early-maturity corn silage (maturity x processing; P = 0.11). Processing increased in situ rates of DM and starch disappearance and maturity decreased in situ disappearance rates of starch and fiber. These data indicate that hybrid, maturity, and processing all affect corn silage digestibility. Mechanical processing of corn silage increased starch digestibility, which may have been associated with the observed decreased fiber digestibility.

Reconfiguration of the proteasome during chaperone-mediated assembly

PubMed Central

Park, Soyeon; Li, Xueming; Kim, Ho Min; Singh, Chingakham Ranjit; Tian, Geng; Hoyt, Martin A.; Lovell, Scott; Battaile, Kevin P.; Zolkiewski, Michal; Coffino, Philip; Roelofs, Jeroen; Cheng, Yifan; Finley, Daniel

2013-01-01

The proteasomal ATPase ring, comprising Rpt1-Rpt6, associates with the heptameric α ring of the proteasome core particle (CP) in the mature proteasome, with the Rpt C-terminal tails inserting into pockets of the α ring1–4. Rpt ring assembly is mediated by four chaperones, each binding a distinct Rpt subunit5–10. We report that the base subassembly of the proteasome, which includes the Rpt ring, forms a high affinity complex with the CP. This complex is subject to active dissociation by the chaperones Hsm3, Nas6, and Rpn14. Chaperone-mediated dissociation was abrogated by a nonhydrolyzable ATP analog, indicating that chaperone action is coupled to nucleotide hydrolysis by the Rpt ring. Unexpectedly, synthetic Rpt tail peptides bound α pockets with poor specificity, except for Rpt6, which uniquely bound the α2/α3 pocket. Although the Rpt6 tail is not visualized within an α pocket in mature proteasomes2–4, it inserts into the α2/α3 pocket in the base-CP complex and is important for complex formation. Thus, the Rpt-CP interface is reconfigured when the lid complex joins the nascent proteasome to form the mature holoenzyme. PMID:23644457

G-protein coupled estrogen receptor (GPER) inhibits final oocyte maturation in common carp, Cyprinus carpio.

PubMed

Majumder, Suravi; Das, Sumana; Moulik, Sujata Roy; Mallick, Buddhadev; Pal, Puja; Mukherjee, Dilip

2015-01-15

GPR-30, now named as GPER (G protein-coupled estrogen receptor) was first identified as an orphan receptor and subsequently shown to be required for estrogen-mediated signaling in certain cancer cells. Later studies demonstrated that GPER has the characteristics of a high affinity estrogen membrane receptor on Atlantic croaker and zebra fish oocytes and mediates estrogen inhibition of oocyte maturation in these two distantly related teleost. To determine the broad application of these findings to other teleost, expression of GPER mRNA and its involvement in 17β-estradiol mediated inhibition of oocyte maturation in other cyprinid, Cyprinus carpio was investigated. Carp oocytes at pre-vitellogenic, late-vitellogenic and post-vitellogenic stages of development contained GPER mRNA and its transcribed protein with a maximum at late-vitellogenic oocytes. Ovarian follicular cells did not express GPER mRNA. Carp oocytes GPER mRNA was essentially identical to that found in other perciformes and cyprinid fish oocytes. Both spontaneous and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P)-induced oocyte maturation in carp was significantly decreased when they were incubated with either E2, or GPER agonist G-1. On the other hand spontaneous oocyte maturation was significantly increased when carp ovarian follicles were incubated with an aromatase inhibitor, fadrozole, GPER antagonist, G-15 and enzymatic removal of the ovarian follicle cell layers. This increase in oocyte maturation was partially reversed by co-treatment with E2. Consistent with previous findings with human and fish GPR30, E2 treatment in carp oocytes caused increase in cAMP production and simultaneously decrease in oocyte maturation, which was inhibited by the addition of 17,20β-P. The results suggest that E2 and GPER play a critical role in regulating re-entry in to meiotic cell cycle in carp oocytes. Copyright © 2014 Elsevier Inc. All rights reserved.

Sultr4;1 mutant seeds of Arabidopsis have an enhanced sulphate content and modified proteome suggesting metabolic adaptations to altered sulphate compartmentalization

PubMed Central

2010-01-01

Background Sulphur is an essential macronutrient needed for the synthesis of many cellular components. Sulphur containing amino acids and stress response-related compounds, such as glutathione, are derived from reduction of root-absorbed sulphate. Sulphate distribution in cell compartments necessitates specific transport systems. The low-affinity sulphate transporters SULTR4;1 and SULTR4;2 have been localized to the vacuolar membrane, where they may facilitate sulphate efflux from the vacuole. Results In the present study, we demonstrated that the Sultr4;1 gene is expressed in developing Arabidopsis seeds to a level over 10-fold higher than the Sultr4;2 gene. A characterization of dry mature seeds from a Sultr4;1 T-DNA mutant revealed a higher sulphate content, implying a function for this transporter in developing seeds. A fine dissection of the Sultr4;1 seed proteome identified 29 spots whose abundance varied compared to wild-type. Specific metabolic features characteristic of an adaptive response were revealed, such as an up-accumulation of various proteins involved in sugar metabolism and in detoxification processes. Conclusions This study revealed a role for SULTR4;1 in determining sulphate content of mature Arabidopsis seeds. Moreover, the adaptive response of sultr4;1 mutant seeds as revealed by proteomics suggests a function of SULTR4;1 in redox homeostasis, a mechanism that has to be tightly controlled during development of orthodox seeds. PMID:20426829

In vitro protease cleavage and computer simulations reveal the HIV-1 capsid maturation pathway

NASA Astrophysics Data System (ADS)

Ning, Jiying; Erdemci-Tandogan, Gonca; Yufenyuy, Ernest L.; Wagner, Jef; Himes, Benjamin A.; Zhao, Gongpu; Aiken, Christopher; Zandi, Roya; Zhang, Peijun

2016-12-01

HIV-1 virions assemble as immature particles containing Gag polyproteins that are processed by the viral protease into individual components, resulting in the formation of mature infectious particles. There are two competing models for the process of forming the mature HIV-1 core: the disassembly and de novo reassembly model and the non-diffusional displacive model. To study the maturation pathway, we simulate HIV-1 maturation in vitro by digesting immature particles and assembled virus-like particles with recombinant HIV-1 protease and monitor the process with biochemical assays and cryoEM structural analysis in parallel. Processing of Gag in vitro is accurate and efficient and results in both soluble capsid protein and conical or tubular capsid assemblies, seemingly converted from immature Gag particles. Computer simulations further reveal probable assembly pathways of HIV-1 capsid formation. Combining the experimental data and computer simulations, our results suggest a sequential combination of both displacive and disassembly/reassembly processes for HIV-1 maturation.

E-learning process maturity level: a conceptual framework

NASA Astrophysics Data System (ADS)

Rahmah, A.; Santoso, H. B.; Hasibuan, Z. A.

2018-03-01

ICT advancement is a sure thing with the impact influencing many domains, including learning in both formal and informal situations. It leads to a new mindset that we should not only utilize the given ICT to support the learning process, but also improve it gradually involving a lot of factors. These phenomenon is called e-learning process evolution. Accordingly, this study attempts to explore maturity level concept to provide the improvement direction gradually and progression monitoring for the individual e-learning process. Extensive literature review, observation, and forming constructs are conducted to develop a conceptual framework for e-learning process maturity level. The conceptual framework consists of learner, e-learning process, continuous improvement, evolution of e-learning process, technology, and learning objectives. Whilst, evolution of e-learning process depicted as current versus expected conditions of e-learning process maturity level. The study concludes that from the e-learning process maturity level conceptual framework, it may guide the evolution roadmap for e-learning process, accelerate the evolution, and decrease the negative impact of ICT. The conceptual framework will be verified and tested in the future study.

The capsid-spacer peptide 1 Gag processing intermediate is a dominant-negative inhibitor of HIV-1 maturation.

PubMed

Checkley, Mary Ann; Luttge, Benjamin G; Soheilian, Ferri; Nagashima, Kunio; Freed, Eric O

2010-04-25

The human immunodeficiency virus type 1 (HIV-1) maturation inhibitor bevirimat disrupts virus replication by inhibiting the cleavage of the capsid-spacer peptide 1 (CA-SP1) Gag processing intermediate to mature CA. The observation that bevirimat delays but does not completely block CA-SP1 processing suggests that the presence of uncleaved CA-SP1 may disrupt the maturation process in trans. In this study, we validate this hypothesis by using a genetic approach to demonstrate that a non-cleavable CA-SP1 mutant exerts a dominant-negative effect on maturation of wild-type HIV-1. In contrast, a mutant in which cleavage can occur internally within SP1 is significantly less potent as a dominant-negative inhibitor. We also show that bevirimat blocks processing at both the major CA-SP1 cleavage site and the internal site. These data underscore the importance of full CA-SP1 processing for HIV-1 maturation and highlight the therapeutic potential of inhibitors that target this Gag cleavage event. Published by Elsevier Inc.

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding

PubMed Central

Koenig, Patrick; Lee, Chingwei V.; Walters, Benjamin T.; Janakiraman, Vasantharajan; Stinson, Jeremy; Patapoff, Thomas W.; Fuh, Germaine

2017-01-01

Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function. The resulting mutational landscape demonstrates that large portions of antibody variable domain positions are open to mutation, and that beneficial mutations can be found throughout the variable domains. We determine the role of one antigen-distal light chain position 83, demonstrating that mutation at this site optimizes both antigen affinity and thermostability by modulating the interdomain conformational dynamics of the antigen-binding fragment. Furthermore, by analyzing a large number of human antibody sequences and structures, we demonstrate that somatic mutations occur frequently at position 83, with corresponding domain conformations observed for G6.31. Therefore, the modulation of interdomain dynamics represents an important mechanism during antibody maturation in vivo. PMID:28057863

CD8+ recent thymic emigrants exhibit increased responses to low affinity ligands and improved access to peripheral sites of inflammation

PubMed Central

Berkley, Amy M.; Fink, Pamela J.

2014-01-01

To explore the TCR sensitivity of recent thymic emigrants (RTEs), we triggered T cells with altered peptide ligands (APLs). Upon peptide stimulation in vitro, RTEs exhibited increased TCR signal transduction, and following infection in vivo with APL-expressing bacteria, CD8 RTEs expanded to a greater extent in response to low affinity antigens than their mature T cell counterparts. RTEs skewed to short-lived effector cells in response to all APLs but were also characterized by diminished cytokine production. RTEs responding to infection expressed increased levels of VLA-4, with consequent improved entry into inflamed tissue and pathogen clearance. These positive outcomes were offset by the capacity of RTEs to elicit autoimmunity. Overall, salient features of CD8 RTE biology should inform strategies to improve neonatal vaccination and therapies for cancer and HIV, as RTEs make up a large proportion of the T cells in lymphodepleted environments. PMID:25172492

Cutting edge: CD8+ recent thymic emigrants exhibit increased responses to low-affinity ligands and improved access to peripheral sites of inflammation.

PubMed

Berkley, Amy M; Fink, Pamela J

2014-10-01

To explore the TCR sensitivity of recent thymic emigrants (RTEs), we triggered T cells with altered peptide ligands (APLs). Upon peptide stimulation in vitro, RTEs exhibited increased TCR signal transduction, and following infection in vivo with APL-expressing bacteria, CD8 RTEs expanded to a greater extent in response to low-affinity Ags than did their mature T cell counterparts. RTEs skewed to short-lived effector cells in response to all APLs but also were characterized by diminished cytokine production. RTEs responding to infection expressed increased levels of VLA-4, with consequent improved entry into inflamed tissue and pathogen clearance. These positive outcomes were offset by the capacity of RTEs to elicit autoimmunity. Overall, salient features of CD8 RTE biology should inform strategies to improve neonatal vaccination and therapies for cancer and HIV, because RTEs make up a large proportion of the T cells in lymphodepleted environments. Copyright © 2014 by The American Association of Immunologists, Inc.

Autophagy proteins are not universally required for phagosome maturation.

PubMed

Cemma, Marija; Grinstein, Sergio; Brumell, John H

2016-09-01

Phagocytosis plays a central role in immunity and tissue homeostasis. After internalization of cargo into single-membrane phagosomes, these compartments undergo a maturation sequences that terminates in lysosome fusion and cargo degradation. Components of the autophagy pathway have recently been linked to phagosome maturation in a process called LC3-associated phagocytosis (LAP). In this process, autophagy machinery is thought to conjugate LC3 directly onto the phagosomal membrane to promote lysosome fusion. However, a recent study has suggested that ATG proteins may in fact impair phagosome maturation to promote antigen presentation. Here, we examined the impact of ATG proteins on phagosome maturation in murine cells using FCGR2A/FcγR-dependent phagocytosis as a model. We show that phagosome maturation is not affected in Atg5-deficient mouse embryonic fibroblasts, or in Atg5- or Atg7-deficient bone marrow-derived macrophages using standard assays of phagosome maturation. We propose that ATG proteins may be required for phagosome maturation under some conditions, but are not universally required for this process.

Seafood-Processing Sludge Composting: Changes to Microbial Communities and Physico-Chemical Parameters of Static Treatment versus for Turning during the Maturation Stage

PubMed Central

Alves, David; Mato, Salustiano

2016-01-01

In general, in composting facilities the active, or intensive, stage of the process is done separately from the maturation stage, using a specific technology and time. The pre-composted material to be matured can contain enough biodegradable substrates to cause microbial proliferation, which in turn can cause temperatures to increase. Therefore, not controlling the maturation period during waste management at an industrial level can result in undesired outcomes. The main hypothesis of this study is that controlling the maturation stage through turning provides one with an optimized process when compared to the static approach. The waste used was sludge from a seafood-processing plant, mixed with shredded wood (1:2, v/v). The composting system consists of an intensive stage in a 600L static reactor, followed by maturation in triplicate in 200L boxes for 112 days. Two tests were carried out with the same process in reactor and different treatments in boxes: static maturation and turning during maturation when the temperature went above 55°C. PLFAs, organic matter, pH, electrical conductivity, forms of nitrogen and carbon, hydrolytic enzymes and respiratory activity were periodically measured. Turning significantly increased the duration of the thermophilic phase and consequently increased the organic-matter degradation. PCA differentiated significantly the two treatments in function of tracking parameters, especially pH, total carbon, forms of nitrogen and C/N ratio. So, stability and maturity optimum values for compost were achieved in less time with turnings. Whereas turning resulted in microbial-group stabilization and a low mono/sat ratio, static treatment produced greater variability in microbial groups and a high mono/sat ratio, the presence of more degradable substrates causes changes in microbial communities and their study during maturation gives an approach of the state of organic-matter degradation. Obtaining quality compost and optimizing the composting process requires using turning as a control mechanism during maturation. PMID:28002444

A conceptual framework and classification of capability areas for business process maturity

NASA Astrophysics Data System (ADS)

Van Looy, Amy; De Backer, Manu; Poels, Geert

2014-03-01

The article elaborates on business process maturity, which indicates how well an organisation can perform based on its business processes, i.e. on its way of working. This topic is of paramount importance for managers who try to excel in today's competitive world. Hence, business process maturity is an emerging research field. However, no consensus exists on the capability areas (or skills) needed to excel. Moreover, their theoretical foundation and synergies with other fields are frequently neglected. To overcome this gap, our study presents a conceptual framework with six main capability areas and 17 sub areas. It draws on theories regarding the traditional business process lifecycle, which are supplemented by recognised organisation management theories. The comprehensiveness of this framework is validated by mapping 69 business process maturity models (BPMMs) to the identified capability areas, based on content analysis. Nonetheless, as a consensus neither exists among the collected BPMMs, a classification of different maturity types is proposed, based on cluster analysis and discriminant analysis. Consequently, the findings contribute to the grounding of business process literature. Possible future avenues are evaluating existing BPMMs, directing new BPMMs or investigating which combinations of capability areas (i.e. maturity types) contribute more to performance than others.

Does implied volatility of currency futures option imply volatility of exchange rates?

NASA Astrophysics Data System (ADS)

Wang, Alan T.

2007-02-01

By investigating currency futures options, this paper provides an alternative economic implication for the result reported by Stein [Overreactions in the options market, Journal of Finance 44 (1989) 1011-1023] that long-maturity options tend to overreact to changes in the implied volatility of short-maturity options. When a GARCH process is assumed for exchange rates, a continuous-time relationship is developed. We provide evidence that implied volatilities may not be the simple average of future expected volatilities. By comparing the term-structure relationship of implied volatilities with the process of the underlying exchange rates, we find that long-maturity options are more consistent with the exchange rates process. In sum, short-maturity options overreact to the dynamics of underlying assets rather than long-maturity options overreacting to short-maturity options.

Top 10 Research Questions Related to Growth and Maturation of Relevance to Physical Activity, Performance, and Fitness

ERIC Educational Resources Information Center

Malina, Robert M.

2014-01-01

Growth, maturation, and development dominate the daily lives of children and adolescents for approximately the first 2 decades of life. Growth and maturation are biological processes, while development is largely a behavioral process. The 3 processes occur simultaneously and interact. They can be influenced by physical activity and also can…

7 CFR 51.1313 - Mature.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 2 2010-01-01 2010-01-01 false Mature. 51.1313 Section 51.1313 Agriculture... Standards for Winter Pears 1 Definitions § 51.1313 Mature. (a) Mature means that the pear has reached the stage of maturity which will insure the proper completion of the ripening process. (b) Before a mature...

7 CFR 51.1313 - Mature.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 2 2012-01-01 2012-01-01 false Mature. 51.1313 Section 51.1313 Agriculture... Standards for Winter Pears 1 Definitions § 51.1313 Mature. (a) Mature means that the pear has reached the stage of maturity which will insure the proper completion of the ripening process. (b) Before a mature...

7 CFR 51.1313 - Mature.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 2 2011-01-01 2011-01-01 false Mature. 51.1313 Section 51.1313 Agriculture... Standards for Winter Pears 1 Definitions § 51.1313 Mature. (a) Mature means that the pear has reached the stage of maturity which will insure the proper completion of the ripening process. (b) Before a mature...

Dual Affinity Heparin-Based Hydrogels Achieve Pro-Regenerative Immunomodulation and Microvascular Remodeling.

PubMed

Ogle, Molly E; Krieger, Jack R; Tellier, Liane E; McFaline-Figueroa, Jennifer; Temenoff, Johnna S; Botchwey, Edward A

2018-04-09

The immune response to biomaterial implants critically regulates functional outcomes such as vascularization, transplant integration/survival, and fibrosis. To create "immunologically smart" materials, the host-material response may be engineered to optimize the recruitment of pro-regenerative leukocyte subsets which mature into corresponding wound-healing macrophages. We have recently identified a unique feature of pro-regenerative Ly6C low monocytes that is a higher expression of both the bioactive lipid receptor sphingosine-1-phosphate receptor 3 (S1PR3) and the stromal derived factor-1α (SDF-1α) receptor CXCR4. Therefore, we designed a bifunctional hydrogel to harnesses a mechanistic synergy between these signaling axes to enhance the recruitment of endogenous pro-regenerative monocytes. To overcome the challenge of codelivering two physiochemically distinct molecules-a large hydrophilic protein and hydrophobic small molecule-we engineered a dual affinity hydrogel that exploits the growth factor affinity of a heparin derivative (Hep -N ) and lipid chaperone activity of albumin. The sphingosine analog FTY720 and SDF-1α are successfully loaded and coreleased from the Hep -N -functionalized PEG-DA hydrogels while maintaining bioactivity. Placement of these hydrogels into a murine partial thickness skin wound demonstrates that corelease of FTY720 and SDF-1α yields superior recruitment of myeloid cells to the implant interface compared to either factor alone. Although in vivo delivery of FTY720 or SDF-1α individually promotes the enhanced recruitment of Ly-6C low anti-inflammatory monocytes, codelivery enhances the early accumulation and persistence of the differentiated wound healing CD206 + macrophages in the tissue surrounding the gel. Co-delivery similarly promoted the synergistic expansion of vasculature adjacent to the implant, a key step in tissue healing. Taken together, these findings suggest that the combination of chemotactic molecules may provide additional maturation signals to the infiltrating leukocytes to facilitate macrophage transition and vascular network expansion, thus, ultimately, potentiating tissue repair. The coupling of multiple pro-regenerative biological cues provides a foundation for more fine-tuned immunoregenerative modulation to facilitate tissue repair.

Nucleic acid chaperone activity of retroviral Gag proteins.

PubMed

Rein, Alan

2010-01-01

Retrovirus particles in which the Gag protein has not yet been cleaved by the viral protease are termed immature particles. The viral RNA within these particles shows clear evidence of the action of a nucleic acid chaperone (NAC): the genomic RNA is dimeric, and a cellular tRNA molecule is annealed, by its 3' 18 nucleotides, to a complementary stretch in the viral RNA, in preparation for priming reverse transcription in the next round of infection. It seems very likely that the NAC that has catalyzed dimerization and tRNA annealing is the NC domain of the Gag protein itself. However, neither the dimeric linkage nor the tRNA:viral RNA complex has the same structure as those in mature virus particles: thus the conformational effects of Gag within the particles are not equivalent to those of the free NC protein present in mature particles. It is not known whether these dissimilarities reflect intrinsic differences in the NAC activities of Gag and NC, or limitations on Gag imposed by the structure of the immature particle. Analysis of the interactions of recombinant Gag proteins with nucleic acids is complicated by the fact that they result in assembly of virus-like particles. Nevertheless, the available data indicates that the affinity of Gag for nucleic acids can be considerably higher than that of free NC. This enhanced affinity may be due to contributions of the matrix domain, a positively charged region at the N-terminus of Gag; interactions of neighboring Gag molecules with each other may also increase the affinity due to cooperativity of the binding. Recombinant HIV-1 Gag protein clearly exhibits NAC activity. In two well-studied experimental systems, Gag was more efficient than NC, as its NAC effects could be detected at a significantly lower molar ratio of protein to nucleotide than with NC. In one system, binding of nucleic acid by the matrix domain of Gag retarded the Gag-induced annealing of two RNAs; this effect could be ameliorated by the competitive binding of inositol hexakisphosphate to the matrix domain.

Gene duplication and neo-functionalization in the evolutionary and functional divergence of the metazoan copper transporters Ctr1 and Ctr2.

PubMed

Logeman, Brandon L; Wood, L Kent; Lee, Jaekwon; Thiele, Dennis J

2017-07-07

Copper is an essential element for proper organismal development and is involved in a range of processes, including oxidative phosphorylation, neuropeptide biogenesis, and connective tissue maturation. The copper transporter (Ctr) family of integral membrane proteins is ubiquitously found in eukaryotes and mediates the high-affinity transport of Cu + across both the plasma membrane and endomembranes. Although mammalian Ctr1 functions as a Cu + transporter for Cu acquisition and is essential for embryonic development, a homologous protein, Ctr2, has been proposed to function as a low-affinity Cu transporter, a lysosomal Cu exporter, or a regulator of Ctr1 activity, but its functional and evolutionary relationship to Ctr1 is unclear. Here we report a biochemical, genetic, and phylogenetic comparison of metazoan Ctr1 and Ctr2, suggesting that Ctr2 arose over 550 million years ago as a result of a gene duplication event followed by loss of Cu + transport activity. Using a random mutagenesis and growth selection approach, we identified amino acid substitutions in human and mouse Ctr2 proteins that support copper-dependent growth in yeast and enhance copper accumulation in Ctr1 -/- mouse embryonic fibroblasts. These mutations revert Ctr2 to a more ancestral Ctr1-like state while maintaining endogenous functions, such as stimulating Ctr1 cleavage. We suggest key structural aspects of metazoan Ctr1 and Ctr2 that discriminate between their biological roles, providing mechanistic insights into the evolutionary, biochemical, and functional relationships between these two related proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Smooth affine shear tight frames: digitization and applications

NASA Astrophysics Data System (ADS)

Zhuang, Xiaosheng

2015-08-01

In this paper, we mainly discuss one of the recent developed directional multiscale representation systems: smooth affine shear tight frames. A directional wavelet tight frame is generated by isotropic dilations and translations of directional wavelet generators, while an affine shear tight frame is generated by anisotropic dilations, shears, and translations of shearlet generators. These two tight frames are actually connected in the sense that the affine shear tight frame can be obtained from a directional wavelet tight frame through subsampling. Consequently, an affine shear tight frame indeed has an underlying filter bank from the MRA structure of its associated directional wavelet tight frame. We call such filter banks affine shear filter banks, which can be designed completely in the frequency domain. We discuss the digitization of affine shear filter banks and their implementations: the forward and backward digital affine shear transforms. Redundancy rate and computational complexity of digital affine shear transforms are also investigated in this paper. Numerical experiments and comparisons in image/video processing show the advantages of digital affine shear transforms over many other state-of-art directional multiscale representation systems.

Autocatalytic maturation, physical/chemical properties, and crystal structure of group N HIV-1 protease: relevance to drug resistance.

PubMed

Sayer, Jane M; Agniswamy, Johnson; Weber, Irene T; Louis, John M

2010-11-01

The mature protease from Group N human immunodeficiency virus Type 1 (HIV-1) (PR1(N)) differs in 20 amino acids from the extensively studied Group M protease (PR1(M)) at positions corresponding to minor drug-resistance mutations (DRMs). The first crystal structure (1.09 Å resolution) of PR1(N) with the clinical inhibitor darunavir (DRV) reveals the same overall structure as PR1(M), but with a slightly larger inhibitor-binding cavity. Changes in the 10s loop and the flap hinge propagate to shift one flap away from the inhibitor, whereas L89F and substitutions in the 60s loop perturb inhibitor-binding residues 29-32. However, kinetic parameters of PR1(N) closely resemble those of PR1(M), and calorimetric results are consistent with similar binding affinities for DRV and two other clinical PIs, suggesting that minor DRMs coevolve to compensate for the detrimental effects of drug-specific major DRMs. A miniprecursor (TFR 1-61-PR1(N)) comprising the transframe region (TFR) fused to the N-terminus of PR1(N) undergoes autocatalytic cleavage at the TFR/PR1(N) site concomitant with the appearance of catalytic activity characteristic of the dimeric, mature enzyme. This cleavage is inhibited at an equimolar ratio of precursor to DRV (∼6 μM), which partially stabilizes the precursor dimer from a monomer. However, cleavage at L34/W35 within the TFR, which precedes the TFR 1-61/PR1(N) cleavage at pH ≤ 5, is only partially inhibited. Favorable properties of PR1(N) relative to PR1(M) include its suitability for column fractionation by size under native conditions and >10-fold higher dimer dissociation constant (150 nM). Exploiting these properties may facilitate testing of potential dimerization inhibitors that perturb early precursor processing steps.

Leveraging People-Related Maturity Issues for Achieving Higher Maturity and Capability Levels

NASA Astrophysics Data System (ADS)

Buglione, Luigi

During the past 20 years Maturity Models (MM) become a buzzword in the ICT world. Since the initial Crosby's idea in 1979, plenty of models have been created in the Software & Systems Engineering domains, addressing various perspectives. By analyzing the content of the Process Reference Models (PRM) in many of them, it can be noticed that people-related issues have little weight in the appraisals of the capabilities of organizations while in practice they are considered as significant contributors in traditional process and organizational performance appraisals, as stressed instead in well-known Performance Management models such as MBQA, EFQM and BSC. This paper proposes some ways for leveraging people-related maturity issues merging HR practices from several types of maturity models into the organizational Business Process Model (BPM) in order to achieve higher organizational maturity and capability levels.

Bistability in cell signaling: How to make continuous processes discontinuous, and reversible processes irreversible

NASA Astrophysics Data System (ADS)

Ferrell, James E.; Xiong, Wen

2001-03-01

Xenopus oocyte maturation is an example of an all-or-none, irreversible cell fate induction process. In response to a submaximal concentration of the steroid hormone progesterone, a given oocyte may either mature or not mature, but it can exist in intermediate states only transiently. Moreover, once an oocyte has matured, it will remain arrested in the mature state even after the progesterone is removed. It has been hypothesized that the all-or-none character of oocyte maturation, and some aspects of the irreversibility of maturation, arise out of the bistability of the signal transduction system that triggers maturation. The bistability, in turn, is hypothesized to arise from the way the signal transducers are organized into a signaling circuit that includes positive feedback (which makes it so that the system cannot rest in intermediate states) and ultrasensitivity (which filters small stimuli out of the feedback loop, allowing the system to have a stable off-state). Here we review two simple graphical methods that are commonly used to analyze bistable systems, discuss the experimental evidence for bistability in oocyte maturation, and suggest that bistability may be a common means of producing all-or-none responses and a type of biochemical memory.

Characterization of a novel Y2K-type dehydrin VrDhn1 from Vigna radiata.

PubMed

Lin, Chia-Hui; Peng, Po-Hsin; Ko, Chia-Yun; Markhart, Albert H; Lin, Tsai-Yun

2012-05-01

A novel dehydrin gene (VrDhn1) was isolated from an embryo cDNA library of Vigna radiata (L.) Wilczek (mungbean) variety VC1973A. The intronless VrDhn1 gene encodes a protein belonging to the Y(2)K-type dehydrin family. VrDhn1 protein accumulated in embryos and cotyledons during seed maturation and disappeared 2 days after seed imbibition (DAI). The expression of VrDhn1 mRNA and accumulation of VrDhn1 protein were at high levels in mature seeds, but neither mRNA nor protein was detected in mungbean vegetative tissues under normal growth conditions. The VrDhn1 mRNA level was extremely high in mature seeds and decreased to ∼30% at 1 DAI, and was not detectable at ~7 DAI. Tissue dehydration, salinity and exogenous ABA markedly induced VrDhn1 transcripts in plants as measured by quantitative real-time reverse transcription-PCR (qRT-PCR). VrDhn1 protein was not detected using immunoblots in seedlings under stress treatments. In mature seeds or 1 DAI seedlings, VrDhn1 proteins were immunolocalized in the nucleus and cytoplasm. VrDhn1 exhibited low affinity for non-specific interaction with DNA using electrophoretic mobility shift assays (EMSAs), and the exogenous addition of Zn(2+) or Ni(2+) stimulated interaction. The His-tagged VrDhn1 (30.17 kDa) protein showed a molecular mass of 63.1 kDa on gel filtration, suggesting a dimer form. This is the first report showing that a Y(2)K-type VrDhn1 enters the nucleus and interacts with DNA during seed maturation.

Initial differences in lipid processing leading to pig-and beef-derived mature adipocyte differentiation

USDA-ARS?s Scientific Manuscript database

Clonal cultures of pig-derived mature adipocytes are capable of dedifferentiating and forming proliferative-competent progeny cells in vitro. Initial lipid processing, is different to that observed in cultures of beef-derived adipocytes. Mature pig adipocytes extrude lipid before proliferation, wher...

“Velcro” Engineering of High Affinity CD47 Ectodomain as Signal Regulatory Protein α (SIRPα) Antagonists That Enhance Antibody-dependent Cellular Phagocytosis*

PubMed Central

Ho, Chia Chi M.; Guo, Nan; Sockolosky, Jonathan T.; Ring, Aaron M.; Weiskopf, Kipp; Özkan, Engin; Mori, Yasuo; Weissman, Irving L.; Garcia, K. Christopher

2015-01-01

CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the “don't-eat-me” signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined “Velcro” engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that “Velcro” engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy. PMID:25837251

Isolation, nucleotide sequence and expression of a cDNA encoding feline granulocyte colony-stimulating factor.

PubMed

Dunham, S P; Onions, D E

2001-06-21

A cDNA encoding feline granulocyte colony stimulating factor (fG-CSF) was cloned from alveolar macrophages using the reverse transcriptase-polymerase chain reaction. The cDNA is 949 bp in length and encodes a predicted mature protein of 174 amino acids. Recombinant fG-CSF was expressed as a glutathione S-transferase fusion and purified by affinity chromatography. Biological activity of the recombinant protein was demonstrated using the murine myeloblastic cell line GNFS-60, which showed an ED50 for fG-CSF of approximately 2 ng/ml. Copyright 2001 Academic Press.

Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms

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

Ford, Nicole R.; Hecht, Karen A.; Hu, Dehong

2016-01-08

The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins incorporating a tetracysteine tag for site-directed labeling with biarsenical affinity probes and either EGFP or single chain antibody to test colocalization of probes with the EGFP-tagged recombinant protein or binding of biosilica-immobilized antibodies to large and small molecule antigens, respectively. Site-directed labeling with the biarsenical probes demonstrated colocalization with EGFP-encoded proteins in nascent and mature biosilica, supporting their use in studying biosilica maturation. Isolated biosilica transformed with a single chain antibody against either the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT) effectively boundmore » the respective antigens. A marked increase in fluorescence lifetime of the TNT surrogate Alexa Fluor 555-trinitrobenzene reflected the high binding specificity of the transformed isolated biosilica. These results demonstrated the potential use of biosilica-immobilized single chain antibodies as binders for large and small molecule antigens in sensing and therapeutics.« less

Portal protein functions akin to a DNA-sensor that couples genome-packaging to icosahedral capsid maturation

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

Lokareddy, Ravi K.; Sankhala, Rajeshwer S.; Roy, Ankoor

Tailed bacteriophages and herpesviruses assemble infectious particles via an empty precursor capsid (or ‘procapsid’) built by multiple copies of coat and scaffolding protein and by one dodecameric portal protein. Genome packaging triggers rearrangement of the coat protein and release of scaffolding protein, resulting in dramatic procapsid lattice expansion. Here, we provide structural evidence that the portal protein of the bacteriophage P22 exists in two distinct dodecameric conformations: an asymmetric assembly in the procapsid (PC-portal) that is competent for high affinity binding to the large terminase packaging protein, and a symmetric ring in the mature virion (MV-portal) that has negligible affinitymore » for the packaging motor. Modelling studies indicate the structure of PC-portal is incompatible with DNA coaxially spooled around the portal vertex, suggesting that newly packaged DNA triggers the switch from PC- to MV-conformation. Thus, we propose the signal for termination of ‘Headful Packaging’ is a DNA-dependent symmetrization of portal protein.« less

Viral receptor-binding site antibodies with diverse germline origins.

PubMed

Schmidt, Aaron G; Therkelsen, Matthew D; Stewart, Shaun; Kepler, Thomas B; Liao, Hua-Xin; Moody, M Anthony; Haynes, Barton F; Harrison, Stephen C

2015-05-21

Vaccines for rapidly evolving pathogens will confer lasting immunity if they elicit antibodies recognizing conserved epitopes, such as a receptor-binding site (RBS). From characteristics of an influenza-virus RBS-directed antibody, we devised a signature motif to search for similar antibodies. We identified, from three vaccinees, over 100 candidates encoded by 11 different VH genes. Crystal structures show that antibodies in this class engage the hemagglutinin RBS and mimic binding of the receptor, sialic acid, by supplying a critical dipeptide on their projecting, heavy-chain third complementarity determining region. They share contacts with conserved, receptor-binding residues but contact different residues on the RBS periphery, limiting the likelihood of viral escape when several such antibodies are present. These data show that related modes of RBS recognition can arise from different germline origins and mature through diverse affinity maturation pathways. Immunogens focused on an RBS-directed response will thus have a broad range of B cell targets. Copyright © 2015 Elsevier Inc. All rights reserved.

Activity Enhancement Based on the Chemical Equilibrium of Multiple-Subunit Nitrile Hydratase from Bordetella petrii.

PubMed

Liu, Yi; Liu, Ping; Lin, Lu; Zhao, Yueqin; Zhong, Wenjuan; Wu, Lunjie; Zhou, Zhemin; Sun, Weifeng

2016-09-01

The maturation mechanism of nitrile hydratase (NHase) of Pseudomonas putida NRRL-18668 was discovered and named as "self-subunit swapping." Since the NHase of Bordetella petrii DSM 12804 is similar to that of P. putida, the NHase maturation of B. petrii is proposed to be the same as that of P. putida. However, there is no further information on the application of NHase according to these findings. We successfully rapidly purified NHase and its activator through affinity his tag, and found that the cell extracts of NHase possessed multiple types of protein ingredients including α, β, α2β2, and α(P14K)2 who were in a state of chemical equilibrium. Furthermore, the activity was significantly enhanced through adding extra α(P14K)2 to the cell extracts of NHase according to the chemical equilibrium. Our findings are useful for the activity enhancement of multiple-subunit enzyme and for the first time significantly increased the NHase activity according to the chemical equilibrium.

Pulsed Dilution Method for the Recovery of Aggregated Mouse TNF-α.

PubMed

Mahmoodi, Merat; Ghodsi, Maryam; Moghadam, Malihe; Sankian, Mojtaba

2017-04-01

The expression of mouse tumor necrosis factor alpha (TNF-α) in Escherichia coli is a favorable way to get high yield of protein; however, the formation of cytoplasmic inclusion bodies, which is the consequence of insoluble accumulated proteins, is a major obstacle in this system. To overcome this obstacle, we used a pulsed dilution method to convert the product to its native conformation. Reducing agent and guanidine hydrochloride were used to solubilize inclusion bodies formed after TNF-(α) expression. Then, the refolding procedure was performed by pulsed dilution of the denatured protein into a refolding buffer. The properly-folded protein was purified by metal affinity chromatography. SDS-PAGE showed a 19.9 kDa band related to the mature TNF-(α) protein. The protein was recognized by anti-mouse TNF-(α) on western blots. The final concentration of the purified recombinant TNF-(α) was 62.5 µg/mL. Our study demonstrates the efficiency of this method to produce a high yield of folded mature TNF- (α).

Color back projection for fruit maturity evaluation

NASA Astrophysics Data System (ADS)

Zhang, Dong; Lee, Dah-Jye; Desai, Alok

2013-12-01

In general, fruits and vegetables such as tomatoes and dates are harvested before they fully ripen. After harvesting, they continue to ripen and their color changes. Color is a good indicator of fruit maturity. For example, tomatoes change color from dark green to light green and then pink, light red, and dark red. Assessing tomato maturity helps maximize its shelf life. Color is used to determine the length of time the tomatoes can be transported. Medjool dates change color from green to yellow, and the orange, light red and dark red. Assessing date maturity helps determine the length of drying process to help ripen the dates. Color evaluation is an important step in the processing and inventory control of fruits and vegetables that directly affects profitability. This paper presents an efficient color back projection and image processing technique that is designed specifically for real-time maturity evaluation of fruits. This color processing method requires very simple training procedure to obtain the frequencies of colors that appear in each maturity stage. This color statistics is used to back project colors to predefined color indexes. Fruit maturity is then evaluated by analyzing the reprojected color indexes. This method has been implemented and used for commercial production.

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

Liu, Ren; Trindade, Alexandre; Instituto Gulbenkian de Ciencia, Oeiras

Highlights: Black-Right-Pointing-Pointer Low dose Dll4-Fc increases vascular proliferation and overall perfusion. Black-Right-Pointing-Pointer Low dose Dll4-Fc helps vascular injury recovery in hindlimb ischemia model. Black-Right-Pointing-Pointer Low dose Dll4-Fc helps vascular injury recovery in skin flap model. Black-Right-Pointing-Pointer Dll4 heterozygous deletion promotes vascular injury recovery. Black-Right-Pointing-Pointer Dll4 overexpression delays vascular injury recovery. -- Abstract: Notch pathway regulates vessel development and maturation. Dll4, a high-affinity ligand for Notch, is expressed predominantly in the arterial endothelium and is induced by hypoxia among other factors. Inhibition of Dll4 has paradoxical effects of reducing the maturation and perfusion in newly forming vessels while increasing the densitymore » of vessels. We hypothesized that partial and/or intermittent inhibition of Dll4 may lead to increased vascular response and still allow vascular maturation to occur. Thus tissue perfusion can be restored rapidly, allowing quicker recovery from ischemia or tissue injury. Our studies in two different models (hindlimb ischemia and skin flap) show that inhibition of Dll4 at low dose allows faster recovery from vascular and tissue injury. This opens a new possibility for Dll4 blockade's therapeutic application in promoting recovery from vascular injury and restoring blood supply to ischemic tissues.« less

An inorganic boronate affinity in-needle monolithic device for specific capture of cis-diol containing compounds.

PubMed

Jin, Shanxia; Zhang, Wei; Yang, Qin; Dai, Lili; Zhou, Ping

2018-02-01

In this work, inorganic boronate affinity monolith was prepared by in situ synthesis in 0.33mm i.d. stainless steel needle through sol-gel process using tetraethoxysilane and tetrabutyl orthotitanate as the co-precursors. The morphology, structure and composition of the monolith were characterized. In contrast to conventional boronate affinity materials, inorganic boric acid was used as affinity ligand. Different compounds were used for the evaluation of the boronate affinity of this inorganic monolithic material. The monolith exhibited good selectivity towards cis-diol containing compounds. Recovery of greater than 90% was achieved for in-needle extraction of catechol under neutral conditions. Owing to the hydrophilic property of the monolith, the procedure of affinity chromatography could be performed in aqueous solution. This monolithic in-needle device will be useful for boronate affinity extraction of small-volume samples. Copyright © 2017 Elsevier B.V. All rights reserved.

p75 neurotrophin receptor and pro-BDNF promote cell survival and migration in clear cell renal cell carcinoma

PubMed Central

Sánchez-Prieto, Ricardo; Saada, Sofiane; Naves, Thomas; Guillaudeau, Angélique; Perraud, Aurélie; Sindou, Philippe; Lacroix, Aurélie; Descazeaud, Aurélien; Lalloué, Fabrice; Jauberteau, Marie-Odile

2016-01-01

p75NTR, a member of TNF receptor family, is the low affinity receptor common to several mature neurotrophins and the high affinity receptor for pro-neurotrophins. Brain-Derived Neurotrophic Factor (BDNF), a member of neurotrophin family has been described to play an important role in development and progression of several cancers, through its binding to a high affinity tyrosine kinase receptor B (TrkB) and/or p75NTR. However, the functions of these two receptors in renal cell carcinoma (RCC) have never been investigated. An overexpression of p75NTR, pro-BDNF, and to a lesser extent for TrkB and sortilin, was detected by immunohistochemistry in a cohort of 83 clear cell RCC tumors. p75NTR, mainly expressed in tumor tissues, was significantly associated with higher Fuhrman grade in multivariate analysis. In two derived-RCC lines, 786-O and ACHN cells, we demonstrated that pro-BDNF induced cell survival and migration, through p75NTR as provided by p75NTR RNA silencing or blocking anti-p75NTR antibody. This mechanism is independent of TrkB activation as demonstrated by k252a, a tyrosine kinase inhibitor for Trk neurotrophin receptors. Taken together, these data highlight for the first time an important role for p75NTR in renal cancer and indicate a putative novel target therapy in RCC. PMID:27120782

Evaluation of Phage Display Discovered Peptides as Ligands for Prostate-Specific Membrane Antigen (PSMA)

PubMed Central

Edwards, W. Barry

2013-01-01

The aim of this study was to identify potential ligands of PSMA suitable for further development as novel PSMA-targeted peptides using phage display technology. The human PSMA protein was immobilized as a target followed by incubation with a 15-mer phage display random peptide library. After one round of prescreening and two rounds of screening, high-stringency screening at the third round of panning was performed to identify the highest affinity binders. Phages which had a specific binding activity to PSMA in human prostate cancer cells were isolated and the DNA corresponding to the 15-mers were sequenced to provide three consensus sequences: GDHSPFT, SHFSVGS and EVPRLSLLAVFL as well as other sequences that did not display consensus. Two of the peptide sequences deduced from DNA sequencing of binding phages, SHSFSVGSGDHSPFT and GRFLTGGTGRLLRIS were labeled with 5-carboxyfluorescein and shown to bind and co-internalize with PSMA on human prostate cancer cells by fluorescence microscopy. The high stringency requirements yielded peptides with affinities KD∼1 µM or greater which are suitable starting points for affinity maturation. While these values were less than anticipated, the high stringency did yield peptide sequences that apparently bound to different surfaces on PSMA. These peptide sequences could be the basis for further development of peptides for prostate cancer tumor imaging and therapy. PMID:23935860

Reversion of autocrine transformation by a dominant negative platelet-derived growth factor mutant.

PubMed Central

Vassbotn, F S; Andersson, M; Westermark, B; Heldin, C H; Ostman, A

1993-01-01

A non-receptor-binding mutant of the platelet-derived growth factor (PDGF) A chain, PDGF-0, was generated by exchanging 7 amino acids in the sequence. The mutant chains formed dimers that were similar to wild-type PDGF-AA with regard to stability and rate of processing to the mature 30-kDa secreted forms. Moreover, the mutant chains formed disulfide-bonded heterodimers with the PDGF B chain in NIH 3T3 cells heterodimer underwent the same processing and secretion as PDGF-AB. Transfection of c-sis-expressing 3T3 cells with PDGF-0 significantly inhibited the transformed phenotype of these cells, as determined by the following criteria. (i) Compared with PDGF-0-negative clones, PDGF-0-producing clones showed a reverted morphology. (ii) Clones producing PDGF-0 grew more slowly than PDGF-0-negative clones, with a fivefold difference in cell number after 14 days in culture. (iii) The expression of PDGF-0 completely inhibited the ability of the c-sis-expressing 3T3 cells to form colonies in soft agar; this inhibition was overcome by the addition of recombinant PDGF-BB to the culture medium, showing that the lack of colony formation of these cells was not due to a general unresponsiveness to PDGF. The specific expression of a PDGF-0/PDGF wild-type heterodimer in COS cells revealed that the affinity of the mutant heterodimer for the PDGF alpha receptor was decreased by approximately 50-fold compared with that of PDGF-AA. Thus, we show that a non-receptor-binding PDGF A-chain mutant neutralizes in a trans-dominant manner the autocrine transforming potential of the c-sis/PDGF B chain by forming low-affinity heterodimers with wild-type PDGF chains. This method of specifically antagonizing the effect of PDGF may be useful in investigations of the role of PDGF in normal and pathological conditions. Images PMID:8321214

Reversion of autocrine transformation by a dominant negative platelet-derived growth factor mutant.

PubMed

Vassbotn, F S; Andersson, M; Westermark, B; Heldin, C H; Ostman, A

1993-07-01

A non-receptor-binding mutant of the platelet-derived growth factor (PDGF) A chain, PDGF-0, was generated by exchanging 7 amino acids in the sequence. The mutant chains formed dimers that were similar to wild-type PDGF-AA with regard to stability and rate of processing to the mature 30-kDa secreted forms. Moreover, the mutant chains formed disulfide-bonded heterodimers with the PDGF B chain in NIH 3T3 cells heterodimer underwent the same processing and secretion as PDGF-AB. Transfection of c-sis-expressing 3T3 cells with PDGF-0 significantly inhibited the transformed phenotype of these cells, as determined by the following criteria. (i) Compared with PDGF-0-negative clones, PDGF-0-producing clones showed a reverted morphology. (ii) Clones producing PDGF-0 grew more slowly than PDGF-0-negative clones, with a fivefold difference in cell number after 14 days in culture. (iii) The expression of PDGF-0 completely inhibited the ability of the c-sis-expressing 3T3 cells to form colonies in soft agar; this inhibition was overcome by the addition of recombinant PDGF-BB to the culture medium, showing that the lack of colony formation of these cells was not due to a general unresponsiveness to PDGF. The specific expression of a PDGF-0/PDGF wild-type heterodimer in COS cells revealed that the affinity of the mutant heterodimer for the PDGF alpha receptor was decreased by approximately 50-fold compared with that of PDGF-AA. Thus, we show that a non-receptor-binding PDGF A-chain mutant neutralizes in a trans-dominant manner the autocrine transforming potential of the c-sis/PDGF B chain by forming low-affinity heterodimers with wild-type PDGF chains. This method of specifically antagonizing the effect of PDGF may be useful in investigations of the role of PDGF in normal and pathological conditions.

Post-thymic maturation: young T cells assert their individuality.

PubMed

Fink, Pamela J; Hendricks, Deborah W

2011-07-22

T cell maturation was once thought to occur entirely within the thymus. Now, evidence is mounting that the youngest peripheral T cells in both mice and humans comprise a distinct population from their more mature, yet still naive, counterparts. These cells, termed recent thymic emigrants (RTEs), undergo a process of post-thymic maturation that can be monitored at the levels of cell phenotype and immune function. Understanding this final maturation step in the process of generating useful and safe T cells is of clinical relevance, given that RTEs are over-represented in neonates and in adults recovering from lymphopenia. Post-thymic maturation may function to ensure T cell fitness and self tolerance.

Interaction between phloretin and the red blood cell membrane

PubMed Central

1976-01-01

Phloretin binding to red blood cell components has been characterized at pH6, where binding and inhibitory potency are maximal. Binding to intact red cells and to purified hemoglobin are nonsaturated processes approximately equal in magnitude, which strongly suggests that most of the red cell binding may be ascribed to hemoglobin. This conclusion is supported by the fact that homoglobin-free red cell ghosts can bind only 10% as much phloretin as an equivalent number of red cells. The permeability of the red cell membrane to phloretin has been determined by a direct measurement at the time-course of the phloretin uptake. At a 2% hematocrit, the half time for phloretin uptake is 8.7s, corresponding to a permeability coefficient of 2 x 10(-4) cm/s. The concentration dependence of the binding to ghosts reveals two saturable components. Phloretin binds with high affinity (K diss = 1.5 muM) to about 2.5 x 10(6) sites per cell; it also binds with lower affinity (Kdiss = 54 muM) to a second (5.5 x 10(7) per cell) set of sites. In sonicated total lipid extracts of red cell ghosts, phloretin binding consists of a single, saturable component. Its affinity and total number of sites are not significantly different from those of the low affinity binding process in ghosts. No high affinity binding of phloretin is exhibited by the red cell lipid extracts. Therefore, the high affinity phloretin binding sites are related to membrane proteins, and the low affinity sites result from phloretin binding to lipid. The identification of these two types of binding sites allows phloretin effects on protein-mediated transport processes to be distinguished from effects on the lipid region of the membrane. PMID:5575

Microscopic dynamics research on the "mature" process of dye-sensitized solar cells after injection of highly concentrated electrolyte.

PubMed

Liang, Zhongguan; Liu, Weiqing; Chen, Jun; Hu, Linhua; Dai, Songyuan

2015-01-21

After injection of electrolyte, the internal three-dimensional solid-liquid penetration system of dye-sensitized solar cells (DSCs) can take a period of time to reach "mature" state. This paper studies the changes of microscopic processes of DSCs including TiO2 energy-level movement, localized state distribution, charge accumulation, electron transport, and recombination dynamics, from the beginning of electrolyte injection to the time of reached mature state. The results show that the microscopic dynamics process of DSCs exhibited a time-dependent behavior and achieved maturity ∼12 h after injecting the electrolyte into DSCs. Within 0-12 h, several results were observed: (1) the conduction band edge of TiO2 moved slightly toward negative potential direction; (2) the localized states in the band gap of TiO2 was reduced according to the same distribution law; (3) the transport resistance in TiO2 film increased, and electron transport time was prolonged as the time of maturity went on, which indicated that the electron transport process is impeded gradually; (4) the recombination resistance at the TiO2/electrolyte (EL) interface increases, and electron lifetime gradually extends, therefore, the recombination process is continuously suppressed. Furthermore, results suggest that the parameters of EL/Pt-transparent conductive oxide (TCO) interface including the interfacial capacitance, electron-transfer resistance, and transfer time constant would change with time of maturity, indicating that the EL/Pt-TCO interface is a potential factor affecting the mature process of DSCs.

A Community Standard Format for the Representation of Protein Affinity Reagents*

PubMed Central

Gloriam, David E.; Orchard, Sandra; Bertinetti, Daniela; Björling, Erik; Bongcam-Rudloff, Erik; Borrebaeck, Carl A. K.; Bourbeillon, Julie; Bradbury, Andrew R. M.; de Daruvar, Antoine; Dübel, Stefan; Frank, Ronald; Gibson, Toby J.; Gold, Larry; Haslam, Niall; Herberg, Friedrich W.; Hiltke, Tara; Hoheisel, Jörg D.; Kerrien, Samuel; Koegl, Manfred; Konthur, Zoltán; Korn, Bernhard; Landegren, Ulf; Montecchi-Palazzi, Luisa; Palcy, Sandrine; Rodriguez, Henry; Schweinsberg, Sonja; Sievert, Volker; Stoevesandt, Oda; Taussig, Michael J.; Ueffing, Marius; Uhlén, Mathias; van der Maarel, Silvère; Wingren, Christer; Woollard, Peter; Sherman, David J.; Hermjakob, Henning

2010-01-01

Protein affinity reagents (PARs), most commonly antibodies, are essential reagents for protein characterization in basic research, biotechnology, and diagnostics as well as the fastest growing class of therapeutics. Large numbers of PARs are available commercially; however, their quality is often uncertain. In addition, currently available PARs cover only a fraction of the human proteome, and their cost is prohibitive for proteome scale applications. This situation has triggered several initiatives involving large scale generation and validation of antibodies, for example the Swedish Human Protein Atlas and the German Antibody Factory. Antibodies targeting specific subproteomes are being pursued by members of Human Proteome Organisation (plasma and liver proteome projects) and the United States National Cancer Institute (cancer-associated antigens). ProteomeBinders, a European consortium, aims to set up a resource of consistently quality-controlled protein-binding reagents for the whole human proteome. An ultimate PAR database resource would allow consumers to visit one on-line warehouse and find all available affinity reagents from different providers together with documentation that facilitates easy comparison of their cost and quality. However, in contrast to, for example, nucleotide databases among which data are synchronized between the major data providers, current PAR producers, quality control centers, and commercial companies all use incompatible formats, hindering data exchange. Here we propose Proteomics Standards Initiative (PSI)-PAR as a global community standard format for the representation and exchange of protein affinity reagent data. The PSI-PAR format is maintained by the Human Proteome Organisation PSI and was developed within the context of ProteomeBinders by building on a mature proteomics standard format, PSI-molecular interaction, which is a widely accepted and established community standard for molecular interaction data. Further information and documentation are available on the PSI-PAR web site. PMID:19674966

Impact of M36I polymorphism on the interaction of HIV-1 protease with its substrates: insights from molecular dynamics

PubMed Central

2014-01-01

Background Over the last decades, a vast structural knowledge has been gathered on the HIV-1 protease (PR). Noticeably, most of the studies focused the B-subtype, which has the highest prevalence in developed countries. Accordingly, currently available anti-HIV drugs target this subtype, with considerable benefits for the corresponding patients. However, in developing countries, there is a wide variety of HIV-1 subtypes carrying PR polymorphisms related to reduced drug susceptibility. The non-active site mutation, M36I, is the most frequent polymorphism, and is considered as a non-B subtype marker. Yet, the structural impact of this substitution on the PR structure and on the interaction with natural substrates remains poorly documented. Results Herein, we used molecular dynamics simulations to investigate the role of this polymorphism on the interaction of PR with six of its natural cleavage-sites substrates. Free energy analyses by MMPB/SA calculations showed an affinity decrease of M36I-PR for the majority of its substrates. The only exceptions were the RT-RH, with equivalent affinity, and the RH-IN, for which an increased affinity was found. Furthermore, molecular simulations suggest that, unlike other peptides, RH-IN induced larger structural fluctuations in the wild-type enzyme than in the M36I variant. Conclusions With multiple approaches and analyses we identified structural and dynamical determinants associated with the changes found in the binding affinity of the M36I variant. This mutation influences the flexibility of both PR and its complexed substrate. The observed impact of M36I, suggest that combination with other non-B subtype polymorphisms, could lead to major effects on the interaction with the 12 known cleavage sites, which should impact the virion maturation. PMID:25573486

Optimal immunization cocktails can promote induction of broadly neutralizing Abs against highly mutable pathogens.

PubMed

Shaffer, J Scott; Moore, Penny L; Kardar, Mehran; Chakraborty, Arup K

2016-10-24

Strategies to elicit Abs that can neutralize diverse strains of a highly mutable pathogen are likely to result in a potent vaccine. Broadly neutralizing Abs (bnAbs) against HIV have been isolated from patients, proving that the human immune system can evolve them. Using computer simulations and theory, we study immunization with diverse mixtures of variant antigens (Ags). Our results show that particular choices for the number of variant Ags and the mutational distances separating them maximize the probability of inducing bnAbs. The variant Ags represent potentially conflicting selection forces that can frustrate the Darwinian evolutionary process of affinity maturation. An intermediate level of frustration maximizes the chance of evolving bnAbs. A simple model makes vivid the origin of this principle of optimal frustration. Our results, combined with past studies, suggest that an appropriately chosen permutation of immunization with an optimally designed mixture (using the principles that we describe) and sequential immunization with variant Ags that are separated by relatively large mutational distances may best promote the evolution of bnAbs.

AID Biology: A pathological and clinical perspective.

PubMed

Choudhary, Meenal; Tamrakar, Anubhav; Singh, Amit Kumar; Jain, Monika; Jaiswal, Ankit; Kodgire, Prashant

2018-01-02

Activation-induced cytidine deaminase (AID), primarily expressed in activated mature B lymphocytes in germinal centers, is the key factor in adaptive immune response against foreign antigens. AID is responsible for producing high-affinity and high-specificity antibodies against an infectious agent, through the physiological DNA alteration processes of antibody genes by somatic hypermutation (SHM) and class-switch recombination (CSR) and functions by deaminating deoxycytidines (dC) to deoxyuridines (dU), thereby introducing point mutations and double-stranded chromosomal breaks (DSBs). The beneficial physiological role of AID in antibody diversification is outweighed by its detrimental role in the genesis of several chronic immune diseases, under non-physiological conditions. This review offers a comprehensive and better understanding of AID biology and its pathological aspects, as well as addresses the challenges involved in AID-related cancer therapeutics, based on various recent advances and evidence available in the literature till date. In this article, we discuss ways through which our interpretation of AID biology may reflect upon novel clinical insights, which could be successfully translated into designing clinical trials and improving patient prognosis and disease management.

Optimal immunization cocktails can promote induction of broadly neutralizing Abs against highly mutable pathogens

PubMed Central

Shaffer, J. Scott; Moore, Penny L.; Kardar, Mehran; Chakraborty, Arup K.

2016-01-01

Strategies to elicit Abs that can neutralize diverse strains of a highly mutable pathogen are likely to result in a potent vaccine. Broadly neutralizing Abs (bnAbs) against HIV have been isolated from patients, proving that the human immune system can evolve them. Using computer simulations and theory, we study immunization with diverse mixtures of variant antigens (Ags). Our results show that particular choices for the number of variant Ags and the mutational distances separating them maximize the probability of inducing bnAbs. The variant Ags represent potentially conflicting selection forces that can frustrate the Darwinian evolutionary process of affinity maturation. An intermediate level of frustration maximizes the chance of evolving bnAbs. A simple model makes vivid the origin of this principle of optimal frustration. Our results, combined with past studies, suggest that an appropriately chosen permutation of immunization with an optimally designed mixture (using the principles that we describe) and sequential immunization with variant Ags that are separated by relatively large mutational distances may best promote the evolution of bnAbs. PMID:27791170

Purification and characterization of a milk-clotting aspartic proteinase from globe artichoke (Cynara scolymus L.).

PubMed

Llorente, Berta E; Brutti, Cristina B; Caffini, Néstor O

2004-12-29

The study of proteinase expression in crude extracts from different organs of the globe artichoke (Cynara scolymus L.) disclosed that enzymes with proteolytic and milk-clotting activity are mainly located in mature flowers. Maximum proteolytic activity was recorded at pH 5.0, and inhibition studies showed that only pepstatin, specific for aspartic proteinases, presented a significant inhibitory effect. Such properties, in addition to easy enzyme inactivation by moderate heating, make this crude protease extract potentially useful for cheese production. Adsorption with activated carbon, together with anion exchange and affinity chromatography, led to the isolation of a heterodimeric milk-clotting proteinase consisting of 30- and 15-kDa subunits. MALDI-TOF MS of the 15-kDa chain determined a 15.358-Da mass, and the terminal amino sequence presented 96% homology with the smaller cardosin A subunit. The amino terminal sequence of the 30-kDa chain proved to be identical to the larger cardosin A subunit. Electrophoresis evidenced proteinase self-processing that was confirmed by immunoblots presenting 62-, 30-, and 15-kDa bands.

Development and validation of an affinity chromatography step using a peptide ligand for cGMP production of factor VIII.

PubMed

Kelley, Brian D; Tannatt, Molly; Magnusson, Robert; Hagelberg, Sigrid; Booth, James

2004-08-05

An affinity chromatography step was developed for purification of recombinant B-Domain Deleted Factor VIII (BDDrFVIII) using a peptide ligand selected from a phage display library. The peptide library had variegated residues, contained both within a disulfide bond-constrained ring and flanking the ring. The peptide ligand binds to BDDrFVIII with a dissociation constant of approximately 1 microM both in free solution and when immobilized on a chromatographic resin. The peptide is chemically synthesized and the affinity resin is produced by coupling the peptide to an agarose matrix preactivated with N-hydroxysuccinimide. Coupling conditions were optimized to give consistent and complete ligand incorporation and validated with a robustness study that tested various combinations of processing limits. The peptide affinity chromatographic operation employs conditions very similar to an immunoaffinity chromatography step currently in use for BDDrFVIII manufacture. The process step provides excellent recovery of BDDrFVIII from a complex feed stream and reduces host cell protein and DNA by 3-4 logs. Process validation studies established resin reuse over 26 cycles without changes in product recovery or purity. A robustness study using a factorial design was performed and showed that the step was insensitive to small changes in process conditions that represent normal variation in commercial manufacturing. A scaled-down model of the process step was qualified and used for virus removal studies. A validation package addressing the safety of the leached peptide included leaching rate measurements under process conditions, testing of peptide levels in product pools, demonstration of robust removal downstream by spiking studies, end product testing, and toxicological profiling of the ligand. The peptide ligand affinity step was scaled up for cGMP production of BDDrFVIII for clinical trials.

miRNA Enriched in Human Neuroblast Nuclei Bind the MAZ Transcription Factor and Their Precursors Contain the MAZ Consensus Motif.

PubMed

Goldie, Belinda J; Fitzsimmons, Chantel; Weidenhofer, Judith; Atkins, Joshua R; Wang, Dan O; Cairns, Murray J

2017-01-01

While the cytoplasmic function of microRNA (miRNA) as post-transcriptional regulators of mRNA has been the subject of significant research effort, their activity in the nucleus is less well characterized. Here we use a human neuronal cell model to show that some mature miRNA are preferentially enriched in the nucleus. These molecules were predominantly primate-specific and contained a sequence motif with homology to the consensus MAZ transcription factor binding element. Precursor miRNA containing this motif were shown to have affinity for MAZ protein in nuclear extract. We then used Ago1/2 RIP-Seq to explore nuclear miRNA-associated mRNA targets. Interestingly, the genes for Ago2-associated transcripts were also significantly enriched with MAZ binding sites and neural function, whereas Ago1-transcripts were associated with general metabolic processes and localized with SC35 spliceosomes. These findings suggest the MAZ transcription factor is associated with miRNA in the nucleus and may influence the regulation of neuronal development through Ago2-associated miRNA induced silencing complexes. The MAZ transcription factor may therefore be important for organizing higher order integration of transcriptional and post-transcriptional processes in primate neurons.

The chemical basis for the origin of the genetic code and the process of protein synthesis

NASA Technical Reports Server (NTRS)

1982-01-01

The major thrust is to understand just how the process of protein synthesis, including that very important aspect, genetic coding, came to be. Two aspects of the problem: the chemistry of active aminoacyl species; and affinities between amino acids and nucleotides, and specifically, how these affinities might affect the chemistry between the two are stressed.

Evolution of microbial dynamics during the maturation phase of the composting of different types of waste.

PubMed

Villar, Iria; Alves, David; Garrido, Josefina; Mato, Salustiano

2016-08-01

During composting, facilities usually exert greater control over the bio-oxidative phase of the process, which uses a specific technology and generally has a fixed duration. After this phase, the material is deposited to mature, with less monitoring during the maturation phase. While there has been considerable study of biological parameters during the thermophilic phase, there is less research on the stabilization and maturation phase. This study evaluates the effects of the type of starting material on the evolution of microbial dynamics during the maturation phase of composting. Three waste types were used: sludge from the fish processing industry, municipal sewage sludge and pig manure, each independently mixed with shredded pine wood as bulking agent. The composting system for each waste type comprised a static reactor with capacity of 600L for the bio-oxidative phase followed by stabilization and maturation phase in triplicate 200L boxes for 112days. Phospholipid fatty acids, enzyme activities and physico-chemical parameters were measured throughout the maturation phase. The evolution of the total microbial biomass, Gram + bacteria, Gram - bacteria, fungi and enzymatic activities (β-glucosidase, cellulase, protease, acid and alkaline phosphatase) depended significantly on the waste type (p<0.001). The predominant microbial community for each waste type remained present throughout the maturation process, indicating that the waste type determines the microorganisms that are able to develop at this stage. While fungi predominated during fish sludge maturation, manure and municipal sludge were characterized by a greater proportion of bacteria. Both the structure of the microbial community and enzymatic activities provided important information for monitoring the composting process. More attention should be paid to the maturation phase in order to optimize composting. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

S-Glutathionylation of estrogen receptor α affects dendritic cell function.

PubMed

Zhang, Jie; Ye, Zhi-Wei; Chen, Wei; Manevich, Yefim; Mehrotra, Shikhar; Ball, Lauren; Janssen-Heininger, Yvonne; Tew, Kenneth D; Townsend, Danyelle M

2018-03-23

Glutathione S -transferase Pi (GSTP) is a thiolase that catalyzes the addition of glutathione (GSH) to receptive cysteines in target proteins, producing an S -glutathionylated residue. Accordingly, previous studies have reported that S -glutathionylation is constitutively decreased in cells from mice lacking GSTP ( Gstp1 / p2 -/- ). Here, we found that bone marrow-derived dendritic cells (BMDDCs) from Gstp1 / p2 -/- mice have proliferation rates that are greater than those in their WT counterparts ( Gstp1 / p2 +/+ ). Moreover, Gstp1 / p2 -/- BMDDCs had increased reactive oxygen species (ROS) levels and decreased GSH:glutathione disulfide (GSSG) ratios. Estrogen receptor α (ERα) is linked to myeloproliferation and differentiation, and we observed that its steady-state levels are elevated in Gstp1 / p2 -/- BMDDCs, indicating a link between GSTP and ERα activities. BMDDCs differentiated by granulocyte-macrophage colony-stimulating factor had elevated ERα levels, which were more pronounced in Gstp1 / p2 -/- than WT mice. When stimulated with lipopolysaccharide for maturation, Gstp1 / p2 -/- BMDDCs exhibited augmented endocytosis, maturation rate, cytokine secretion, and T-cell activation; heightened glucose uptake and glycolysis; increased Akt signaling (in the mTOR pathway); and decreased AMPK-mediated phosphorylation of proteins. Of note, GSTP formed a complex with ERα, stimulating ERα S -glutathionylation at cysteines 221, 245, 417, and 447; altering ERα's binding affinity for estradiol; and reducing overall binding potential (receptor density and affinity) 3-fold. Moreover, in Gstp1 / p2 -/- BMDDCs, ERα S -glutathionylation was constitutively decreased. Taken together, these findings suggest that GSTP-mediated S -glutathionylation of ERα controls BMDDC differentiation and affects metabolic function in dendritic cells. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Dengue Virus NS2B/NS3 Protease Inhibitors Exploiting the Prime Side.

PubMed

Lin, Kuan-Hung; Ali, Akbar; Rusere, Linah; Soumana, Djade I; Kurt Yilmaz, Nese; Schiffer, Celia A

2017-05-15

The mosquito-transmitted dengue virus (DENV) infects millions of people in tropical and subtropical regions. Maturation of DENV particles requires proper cleavage of the viral polyprotein, including processing of 8 of the 13 substrate cleavage sites by dengue virus NS2B/NS3 protease. With no available direct-acting antiviral targeting DENV, NS2/NS3 protease is a promising target for inhibitor design. Current design efforts focus on the nonprime side of the DENV protease active site, resulting in highly hydrophilic and nonspecific scaffolds. However, the prime side also significantly modulates DENV protease binding affinity, as revealed by engineering the binding loop of aprotinin, a small protein with high affinity for DENV protease. In this study, we designed a series of cyclic peptides interacting with both sides of the active site as inhibitors of dengue virus protease. The design was based on two aprotinin loops and aimed to leverage both key specific interactions of substrate sequences and the entropic advantage driving aprotinin's high affinity. By optimizing the cyclization linker, length, and amino acid sequence, the tightest cyclic peptide achieved a K i value of 2.9 μM against DENV3 wild-type (WT) protease. These inhibitors provide proof of concept that both sides of DENV protease active site can be exploited to potentially achieve specificity and lower hydrophilicity in the design of inhibitors targeting DENV. IMPORTANCE Viruses of the flaviviral family, including DENV and Zika virus transmitted by Aedes aegypti , continue to be a threat to global health by causing major outbreaks in tropical and subtropical regions, with no available direct-acting antivirals for treatment. A better understanding of the molecular requirements for the design of potent and specific inhibitors against flaviviral proteins will contribute to the development of targeted therapies for infections by these viruses. The cyclic peptides reported here as DENV protease inhibitors provide novel scaffolds that enable exploiting the prime side of the protease active site, with the aim of achieving better specificity and lower hydrophilicity than those of current scaffolds in the design of antiflaviviral inhibitors. Copyright © 2017 American Society for Microbiology.

Using the Assessment Process to Overcome Imposter Syndrome in Mature Students

ERIC Educational Resources Information Center

Chapman, Amanda

2017-01-01

This research draws on the experience of a group of mature students' studies during their first year at university. All experienced varying degrees of Imposter Syndrome, feelings of fraudulence and a lack of confidence in their ability. The process of "becoming" a mature student is one of identity change and risk. Gaining a sense of…

Capability Maturity Model (CMM) for Software Process Improvements

NASA Technical Reports Server (NTRS)

Ling, Robert Y.

2000-01-01

This slide presentation reviews the Avionic Systems Division's implementation of the Capability Maturity Model (CMM) for improvements in the software development process. The presentation reviews the process involved in implementing the model and the benefits of using CMM to improve the software development process.

Antibodies elicited by the first non-viral prophylactic cancer vaccine show tumor-specificity and immunotherapeutic potential

PubMed Central

Lohmueller, Jason J.; Sato, Shuji; Popova, Lana; Chu, Isabel M.; Tucker, Meghan A.; Barberena, Roberto; Innocenti, Gregory M.; Cudic, Mare; Ham, James D.; Cheung, Wan Cheung; Polakiewicz, Roberto D.; Finn, Olivera J.

2016-01-01

MUC1 is a shared tumor antigen expressed on >80% of human cancers. We completed the first prophylactic cancer vaccine clinical trial based on a non-viral antigen, MUC1, in healthy individuals at-risk for colon cancer. This trial provided a unique source of potentially effective and safe immunotherapeutic drugs, fully-human antibodies affinity-matured in a healthy host to a tumor antigen. We purified, cloned, and characterized 13 IgGs specific for several tumor-associated MUC1 epitopes with a wide range of binding affinities. These antibodies bind hypoglycosylated MUC1 on human cancer cell lines and tumor tissues but show no reactivity against fully-glycosylated MUC1 on normal cells and tissues. We found that several antibodies activate complement-mediated cytotoxicity and that T cells carrying chimeric antigen receptors with the antibody variable regions kill MUC1+ target cells, express activation markers, and produce interferon gamma. Fully-human and tumor-specific, these antibodies are candidates for further testing and development as immunotherapeutic drugs. PMID:27545199

Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses.

PubMed

Pardi, Norbert; Hogan, Michael J; Naradikian, Martin S; Parkhouse, Kaela; Cain, Derek W; Jones, Letitia; Moody, M Anthony; Verkerke, Hans P; Myles, Arpita; Willis, Elinor; LaBranche, Celia C; Montefiori, David C; Lobby, Jenna L; Saunders, Kevin O; Liao, Hua-Xin; Korber, Bette T; Sutherland, Laura L; Scearce, Richard M; Hraber, Peter T; Tombácz, István; Muramatsu, Hiromi; Ni, Houping; Balikov, Daniel A; Li, Charles; Mui, Barbara L; Tam, Ying K; Krammer, Florian; Karikó, Katalin; Polacino, Patricia; Eisenlohr, Laurence C; Madden, Thomas D; Hope, Michael J; Lewis, Mark G; Lee, Kelly K; Hu, Shiu-Lok; Hensley, Scott E; Cancro, Michael P; Haynes, Barton F; Weissman, Drew

2018-06-04

T follicular helper (Tfh) cells are required to develop germinal center (GC) responses and drive immunoglobulin class switch, affinity maturation, and long-term B cell memory. In this study, we characterize a recently developed vaccine platform, nucleoside-modified, purified mRNA encapsulated in lipid nanoparticles (mRNA-LNPs), that induces high levels of Tfh and GC B cells. Intradermal vaccination with nucleoside-modified mRNA-LNPs encoding various viral surface antigens elicited polyfunctional, antigen-specific, CD4 + T cell responses and potent neutralizing antibody responses in mice and nonhuman primates. Importantly, the strong antigen-specific Tfh cell response and high numbers of GC B cells and plasma cells were associated with long-lived and high-affinity neutralizing antibodies and durable protection. Comparative studies demonstrated that nucleoside-modified mRNA-LNP vaccines outperformed adjuvanted protein and inactivated virus vaccines and pathogen infection. The incorporation of noninflammatory, modified nucleosides in the mRNA is required for the production of large amounts of antigen and for robust immune responses. © 2018 Pardi et al.

The Impact of Maturity Stage on Cell Membrane Integrity and Enzymatic Browning Reactions in High Pressure Processed Peaches (Prunus persica).

PubMed

Techakanon, Chukwan; Gradziel, Thomas M; Zhang, Lu; Barrett, Diane M

2016-09-28

Fruit maturity is an important factor associated with final product quality, and it may have an effect on the level of browning in peaches that are high pressure processed (HPP). Peaches from three different maturities, as determined by firmness (M1 = 50-55 N, M2 = 35-40 N, and M3 = 15-20 N), were subjected to pressure levels at 0.1, 200, and 400 MPa for 10 min. The damage from HPP treatment results in loss of fruit integrity and the development of browning during storage. Increasing pressure levels of HPP treatment resulted in greater damage, particularly in the more mature peaches, as determined by shifts in transverse relaxation time (T2) of the vacuolar component and by light microscopy. The discoloration of peach slices of different maturities processed at the same pressure was comparable, indicating that the effect of pressure level is greater than that of maturity in the development of browning.

Latent myostatin has significant activity and this activity is controlled more efficiently by WFIKKN1 than by WFIKKN2

PubMed Central

Szláma, György; Trexler, Mária; Patthy, László

2013-01-01

Myostatin, a negative regulator of skeletal muscle growth, is produced from myostatin precursor by multiple steps of proteolytic processing. After cleavage by a furin-type protease, the propeptide and growth factor domains remain associated, forming a noncovalent complex, the latent myostatin complex. Mature myostatin is liberated from latent myostatin by bone morphogenetic protein 1/tolloid proteases. Here, we show that, in reporter assays, latent myostatin preparations have significant myostatin activity, as the noncovalent complex dissociates at an appreciable rate, and both mature and semilatent myostatin (a complex in which the dimeric growth factor domain interacts with only one molecule of myostatin propeptide) bind to myostatin receptor. The interaction of myostatin receptor with semilatent myostatin is efficiently blocked by WAP, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 1 or growth and differentiation factor-associated serum protein 2 (WFIKKN1), a large extracellular multidomain protein that binds both mature myostatin and myostatin propeptide [Kondás et al. (2008) J Biol Chem 283, 23677–23684]. Interestingly, the paralogous protein WAP, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 2 or growth and differentiation factor-associated serum protein 1 (WFIKKN2) was less efficient than WFIKKN1 as an antagonist of the interactions of myostatin receptor with semilatent myostatin. Our studies have shown that this difference is attributable to the fact that only WFIKKN1 has affinity for the propeptide domain, and this interaction increases its potency in suppressing the receptor-binding activity of semilatent myostatin. As the interaction of WFIKKN1 with various forms of myostatin permits tighter control of myostatin activity until myostatin is liberated from latent myostatin by bone morphogenetic protein 1/tolloid proteases, WFIKKN1 may have greater potential as an antimyostatic agent than WFIKKN2. Structured digital abstract Furin cleaves Promyostatin by protease assay (View interaction) myostatin binds to PRO by surface plasmon resonance (View interaction) BMP-1 cleaves Promyostatin by protease assay (View interaction) ACR IIB physically interacts with Latent Myostatin by surface plasmon resonance (View interaction) Promyostatin and Promyostatin bind by comigration in gel electrophoresis (View interaction) WFIKKN1 binds to Latent Myostatin by pull down (View interaction) ACR IIB binds to Mature Myostatin by surface plasmon resonance (View Interaction: 1, 2, 3) WFIKKN1 binds to Myostatin Prodomain by surface plasmon resonance (View Interaction: 1, 2, 3) PMID:23829672

Neural classifier in the estimation process of maturity of selected varieties of apples

NASA Astrophysics Data System (ADS)

Boniecki, P.; Piekarska-Boniecka, H.; Koszela, K.; Zaborowicz, M.; Przybył, K.; Wojcieszak, D.; Zbytek, Z.; Ludwiczak, A.; Przybylak, A.; Lewicki, A.

2015-07-01

This paper seeks to present methods of neural image analysis aimed at estimating the maturity state of selected varieties of apples which are popular in Poland. An identification of the degree of maturity of selected varieties of apples has been conducted on the basis of information encoded in graphical form, presented in the digital photos. The above process involves the application of the BBCH scale, used to determine the maturity of apples. The aforementioned scale is widely used in the EU and has been developed for many species of monocotyledonous plants and dicotyledonous plants. It is also worth noticing that the given scale enables detailed determinations of development stage of a given plant. The purpose of this work is to identify maturity level of selected varieties of apples, which is supported by the use of image analysis methods and classification techniques represented by artificial neural networks. The analysis of graphical representative features based on image analysis method enabled the assessment of the maturity of apples. For the utilitarian purpose the "JabVis 1.1" neural IT system was created, in accordance with requirements of the software engineering dedicated to support the decision-making processes occurring in broadly understood production process and processing of apples.

Insertion and assembly of the precursor of subunit II into the photosystem I complex may precede its processing.

PubMed Central

Cohen, Y; Steppuhn, J; Herrmann, R G; Yalovsky, S; Nechushtai, R

1992-01-01

The biogenesis and assembly of subunit II of photosystem I (PSI) (psaD gene product) were studied and characterized. The precursor and the mature form were produced in vitro and incubated with intact plastids or isolated thylakoids. Following import of the precursor into isolated plastids, mostly the mature form of subunit II was found in the thylakoids. However, when the processing activity was inhibited only the precursor form was present in the membranes. The precursor was processed by a stromal peptidase and processing could occur before or after insertion of the precursor into the thylakoids. Following insertion into isolated thylakoids, both the precursor and the mature form of subunit II were confined to the PSI complex. Insertion of the mature form of subunit II was much less efficient than that of the precursor. Kinetic studies showed that the precursor was inserted into the membrane. Only at a later stage, the mature form began to accumulate. These results suggest that in vivo the precursor of subunit II is inserted and embedded in the thylakoids, as part of the PSI complex. Only later, it is processed to the mature form through the action of a stromal peptidase. Images PMID:1740118

Optimizing IV and V for Mature Organizations

NASA Technical Reports Server (NTRS)

Fuhman, Christopher

2003-01-01

NASA is intending for its future software development agencies to have at least a Level 3 rating in the Carnegie Mellon University Capability Maturity Model (CMM). The CMM has built-in Verification and Validation (V&V) processes that support higher software quality. Independent Verification and Validation (IV&V) of software developed by mature agencies can be therefore more effective than for software developed by less mature organizations. How is Independent V&V different with respect to the maturity of an organization? Knowing a priori the maturity of an organization's processes, how can IV&V planners better identify areas of need choose IV&V activities, etc? The objective of this research is to provide a complementary set of guidelines and criteria to assist the planning of IV&V activities on a project using a priori knowledge of the measurable levels of maturity of the organization developing the software.

Retrovirus maturation-an extraordinary structural transformation.

PubMed

Mattei, Simone; Schur, Florian Km; Briggs, John Ag

2016-06-01

Retroviruses such as HIV-1 assemble and bud from infected cells in an immature, non-infectious form. Subsequently, a series of proteolytic cleavages catalysed by the viral protease leads to a spectacular structural rearrangement of the viral particle into a mature form that is competent to fuse with and infect a new cell. Maturation involves changes in the structures of protein domains, in the interactions between protein domains, and in the architecture of the viral components that are assembled by the proteins. Tight control of proteolytic cleavages at different sites is required for successful maturation, and the process is a major target of antiretroviral drugs. Here we will describe what is known about the structures of immature and mature retrovirus particles, and about the maturation process by which one transitions into the other. Despite a wealth of available data, fundamental questions about retroviral maturation remain unanswered. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Ca2+/Calmodulin-dependent kinase II signaling causes skeletal overgrowth and premature chondrocyte maturation.

PubMed

Taschner, Michael J; Rafigh, Mehran; Lampert, Fabienne; Schnaiter, Simon; Hartmann, Christine

2008-05-01

The long bones of vertebrate limbs originate from cartilage templates and are formed by the process of endochondral ossification. This process requires that chondrocytes undergo a progressive maturation from proliferating to postmitotic prehypertrophic to mature, hypertrophic chondrocytes. Coordinated control of proliferation and maturation regulates growth of the skeletal elements. Various signals and pathways have been implicated in orchestrating these processes, but the underlying intracellular molecular mechanisms are often not entirely known. Here we demonstrated in the chick using replication-competent retroviruses that constitutive activation of Calcium/Calmodulin-dependent kinase II (CaMKII) in the developing wing resulted in elongation of skeletal elements associated with premature differentiation of chondrocytes. The premature maturation of chondrocytes was a cell-autonomous effect of constitutive CaMKII signaling associated with down-regulation of cell-cycle regulators and up-regulation of chondrocyte maturation markers. In contrast, the elongation of the skeletal elements resulted from a non-cell autonomous up-regulation of the Indian hedgehog responsive gene encoding Parathyroid-hormone-related peptide. Reduction of endogenous CaMKII activity by overexpressing an inhibitory peptide resulted in shortening of the skeletal elements associated with a delay in chondrocyte maturation. Thus, CaMKII is an essential component of intracellular signaling pathways regulating chondrocyte maturation.

Camelid VHH affinity ligands enable separation of closely related biopharmaceuticals

PubMed Central

Pabst, Timothy M.; Wendeler, Michaela; Wang, Xiangyang; Bezemer, Sandra; Hermans, Pim

2016-01-01

Abstract Interest in new and diverse classes of molecules such as recombinant toxins, enzymes, and blood factors continues to grow for use a biotherapeutics. Compared to monoclonal antibodies, these novel drugs typically lack a commercially available affinity chromatography option, which leads to greater process complexity, longer development timelines, and poor platformability. To date, for both monoclonal antibodies and novel molecules, affinity chromatography has been mostly reserved for separation of process‐related impurities such as host cell proteins and DNA. Reports of affinity purification of closely related product variants and modified forms are much rarer. In this work we describe custom affinity chromatography development using camelid VHH antibody fragments as "tunable" immunoaffinity ligands for separation of product‐related impurities. One example demonstrates high selectivity for a recombinant immunotoxin where no binding was observed for an undesired deamidated species. Also discussed is affinity purification of a coagulation factor through specific recognition of the gamma‐carboxylglutamic acid domain. PMID:27677057

Native Mass Spectrometry in Fragment-Based Drug Discovery.

PubMed

Pedro, Liliana; Quinn, Ronald J

2016-07-28

The advent of native mass spectrometry (MS) in 1990 led to the development of new mass spectrometry instrumentation and methodologies for the analysis of noncovalent protein-ligand complexes. Native MS has matured to become a fast, simple, highly sensitive and automatable technique with well-established utility for fragment-based drug discovery (FBDD). Native MS has the capability to directly detect weak ligand binding to proteins, to determine stoichiometry, relative or absolute binding affinities and specificities. Native MS can be used to delineate ligand-binding sites, to elucidate mechanisms of cooperativity and to study the thermodynamics of binding. This review highlights key attributes of native MS for FBDD campaigns.

Electroculture for crop enhancement by air anions

NASA Astrophysics Data System (ADS)

Pohl, H. A.; Todd, G. W.

1981-12-01

Electroculture, the practice of applying strong electric fields or other sources of small air ions to growing plants, has potential to markedly increase crop production and to speed crop growth. The considerable evidence for its effectiveness, and the studies of the mechanisms for its actions are discussed. A mild current of air anions (4 pA/cm2) stimulates bean crop growth and also earlier blossoming and increased growth in the annual, Exacum affine (Persian violet), as well as in seedling geraniums. The present results would indicate that the growing period required until the plants reach a saleable stage of maturity can be shortened by about two weeks under greenhouse conditions.

High Affinity Antibodies against Influenza Characterize the Plasmablast Response in SLE Patients After Vaccination

PubMed Central

Kaur, Kaval; Zheng, Nai-Ying; Smith, Kenneth; Huang, Min; Li, Lie; Pauli, Noel T.; Henry Dunand, Carole J.; Lee, Jane-Hwei; Morrissey, Michael; Wu, Yixuan; Joachims, Michelle L.; Munroe, Melissa E.; Lau, Denise; Qu, Xinyan; Krammer, Florian; Wrammert, Jens; Palese, Peter; Ahmed, Rafi; James, Judith A.; Wilson, Patrick C.

2015-01-01

Breakdown of B cell tolerance is a cardinal feature of systemic lupus erythematosus (SLE). Increased numbers of autoreactive mature naïve B cells have been described in SLE patients and autoantibodies have been shown to arise from autoreactive and non-autoreactive precursors. How these defects, in the regulation of B cell tolerance and selection, influence germinal center (GC) reactions that are directed towards foreign antigens has yet to be investigated. Here, we examined the characteristics of post-GC foreign antigen-specific B cells from SLE patients and healthy controls by analyzing monoclonal antibodies generated from plasmablasts induced specifically by influenza vaccination. We report that many of the SLE patients had anti-influenza antibodies with higher binding affinity and neutralization capacity than those from controls. Although overall frequencies of autoreactivity in the influenza-specific plasmablasts were similar for SLE patients and controls, the variable gene repertoire of influenza-specific plasmablasts from SLE patients was altered, with increased usage of JH6 and long heavy chain CDR3 segments. We found that high affinity anti-influenza antibodies generally characterize the plasmablast responses of SLE patients with low levels of autoreactivity; however, certain exceptions were noted. The high-avidity antibody responses in SLE patients may also be correlated with cytokines that are abnormally expressed in lupus. These findings provide insights into the effects of dysregulated immunity on the quality of antibody responses following influenza vaccination and further our understanding of the underlying abnormalities of lupus. PMID:25951191

Synthetic Human Monoclonal Antibodies toward Staphylococcal Enterotoxin B (SEB) Protective against Toxic Shock Syndrome*

PubMed Central

Karauzum, Hatice; Chen, Gang; Abaandou, Laura; Mahmoudieh, Mahta; Boroun, Atefeh R.; Shulenin, Sergey; Devi, V. Sathya; Stavale, Eric; Warfield, Kelly L.; Zeitlin, Larry; Roy, Chad J.; Sidhu, Sachdev S.; Aman, M. Javad

2012-01-01

Staphylococcal enterotoxin B (SEB) is a potent toxin that can cause toxic shock syndrome and act as a lethal and incapacitating agent when used as a bioweapon. There are currently no vaccines or immunotherapeutics available against this toxin. Using phage display technology, human antigen-binding fragments (Fabs) were selected against SEB, and proteins were produced in Escherichia coli cells and characterized for their binding affinity and their toxin neutralizing activity in vitro and in vivo. Highly protective Fabs were converted into full-length IgGs and produced in mammalian cells. Additionally, the production of anti-SEB antibodies was explored in the Nicotiana benthamiana plant expression system. Affinity maturation was performed to produce optimized lead anti-SEB antibody candidates with subnanomolar affinities. IgGs produced in N. benthamiana showed characteristics comparable with those of counterparts produced in mammalian cells. IgGs were tested for their therapeutic efficacy in the mouse toxic shock model using different challenge doses of SEB and a treatment with 200 μg of IgGs 1 h after SEB challenge. The lead candidates displayed full protection from lethal challenge over a wide range of SEB challenge doses. Furthermore, mice that were treated with anti-SEB IgG had significantly lower IFNγ and IL-2 levels in serum compared with mock-treated mice. In summary, these anti-SEB monoclonal antibodies represent excellent therapeutic candidates for further preclinical and clinical development. PMID:22645125

Characterization of 16S rRNA Processing with Pre-30S Subunit Assembly Intermediates from E. coli.

PubMed

Smith, Brian A; Gupta, Neha; Denny, Kevin; Culver, Gloria M

2018-06-08

Ribosomal RNA (rRNA) is a major component of ribosomes and is fundamental to the process of translation. In bacteria, 16S rRNA is a component of the small ribosomal subunit and plays a critical role in mRNA decoding. rRNA maturation entails the removal of intervening spacer sequences contained within the pre-rRNA transcript by nucleolytic enzymes. Enzymatic activities involved in maturation of the 5'-end of 16S rRNA have been identified, but those involved in 3'-end maturation of 16S rRNA are more enigmatic. Here, we investigate molecular details of 16S rRNA maturation using purified in vivo-formed small subunit (SSU) assembly intermediates (pre-SSUs) from wild-type Escherichia coli that contain precursor 16S rRNA (17S rRNA). Upon incubation of pre-SSUs with E. coli S100 cell extracts or purified enzymes implicated in 16S rRNA processing, the 17S rRNA is processed into additional intermediates and mature 16S rRNA. These results illustrate that exonucleases RNase R, RNase II, PNPase, and RNase PH can process the 3'-end of pre-SSUs in vitro. However, the endonuclease YbeY did not exhibit nucleolytic activity with pre-SSUs under these conditions. Furthermore, these data demonstrate that multiple pathways facilitate 16S rRNA maturation with pre-SSUs in vitro, with the dominant pathways entailing complete processing of the 5'-end of 17S rRNA prior to 3'-end maturation or partial processing of the 5'-end with concomitant processing of the 3'-end. These results reveal the multifaceted nature of SSU biogenesis and suggest that E. coli may be able to escape inactivation of any one enzyme by using an existing complementary pathway. Copyright © 2018 Elsevier Ltd. All rights reserved.

cld and lec23 are disparate mutations that affect maturation of lipoprotein lipase in the endoplasmic reticulum.

PubMed

Briquet-Laugier, V; Ben-Zeev, O; White, A; Doolittle, M H

1999-11-01

The mutations cld (combined lipase deficiency) and lec23 disrupt in a similar manner the expression of lipoprotein lipase (LPL). Whereas cld affects an unknown gene, lec23 abolishes the activity of alpha-glucosidase I, an enzyme essential for proper folding and assembly of nascent glycoproteins. The hypothesis that cld, like lec23, affects the folding/assembly of nascent LPL was confirmed by showing that in cell lines homozygous for these mutations (Cld and Lec23, respectively), the majority of LPL was inactive, displayed heterogeneous aggregation, and had a decreased affinity for heparin. While inactive LPL was retained in the ER, a small amount of LPL that had attained a native conformation was transported through the Golgi and secreted. Thus, Cld and Lec23 cells recognized and retained the majority of LPL as misfolded, maintaining the standard of quality control. Examination of candidate factors affecting protein maturation, such as glucose addition and trimming, proteins involved in lectin chaperone cycling, and other abundant ER chaperones, revealed that calnexin levels were dramatically reduced in livers from cld/cld mice; this finding was also confirmed in Cld cells. We conclude that cld may affect components in the ER, such as calnexin, that play a role in protein maturation. Whether the reduced calnexin levels per se contribute to the LPL deficiency awaits confirmation.

Sedimentation properties in density gradients correspond with levels of sperm DNA fragmentation, chromatin compaction and binding affinity to hyaluronic acid.

PubMed

Torabi, Forough; Binduraihem, Adel; Miller, David

2017-03-01

Mature spermatozoa bind hyaluronic acid in the extracellular matrix via hyaladherins. Immature spermatozoa may be unable to interact because they do not express the appropriate hyaladherins on their surface. Fresh human semen samples were fractionated using differential density gradient centrifugation (DDGC) and the ability of these fractions to bind hyaluronic acid was evaluated. The presence of sperm hyaladherins was also assessed. CD44 was located mainly on the acrosome and equatorial segment and became more restricted to the equatorial segment in capacitated spermatozoa. Hyaluronic acid-TRITC (hyaluronic acid conjugated with tetramethylrhodamine isothiocyanante), a generic hyaluronic-acid-binding reagent, labelled the membrane and the neck region, particularly after capacitation. Sperm populations obtained after DDGC or after interaction with hyaluronic acid were assessed for DNA fragmentation and chromatin maturity. Strong relationships between both measures and sperm sedimentation and hyaluronic-acid-binding profiles were revealed. Capacitation enhanced hyaluronic acid binding of both DDGC-pelleted sperm and sperm washed free of seminal fluid. In conclusion, hyaladherins were detected on human sperm and a higher capacity for sperm hyaluronic-acid-binding was shown to correspond with their DDGC sedimentation profiles and with lower levels of DNA fragmentation and better chromatin maturity. Capacitation induced changes in the distribution and presence of hyaladherins may enhance hyaluronic-acid-binding. Copyright © 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Summary of evidence for an anticodonic basis for the origin of the genetic code

NASA Technical Reports Server (NTRS)

Lacey, J. C., Jr.; Mullins, D. W., Jr.

1981-01-01

This article summarizes data supporting the hypothesis that the genetic code origin was based on relationships (probably affinities) between amino acids and their anticodon nucleotides. Selective activation seems to follow from selective affinity and consequently, incorporation of amino acids into peptides can also be selective. It is suggested that these selectivities in affinity and activation, coupled with the base pairing specificities, allowed the origin of the code and the process of translation.

Analysis of Biological Interactions by Affinity Chromatography: Clinical and Pharmaceutical Applications

PubMed Central

Hage, David S.

2017-01-01

BACKGROUND The interactions between biochemical and chemical agents in the body are important in many clinical processes. Affinity chromatography and high-performance affinity chromatography (HPAC), in which a column contains an immobilized biologically-related binding agent, are two methods that can be used to study these interactions. CONTENT This review looks at various approaches that can be used in affinity chromatography and HPAC to characterize the strength or rate of a biological interaction, the number and types of sites that are involved in this process, and the interactions between multiple solutes for the same binding agent. A number of applications for these methods are examined, with an emphasis on recent developments and high-performance affinity methods. These applications include the use of these techniques for fundamental studies of biological interactions, high-throughput screening of drugs, work with modified proteins, tools for personalized medicine, and studies of drug-drug competition for a common binding agent. SUMMARY The wide range of formats and detection methods that can be used with affinity chromatography and HPAC for examining biological interactions makes these tools attractive for various clinical and pharmaceutical applications. Future directions in the development of small-scale columns and the coupling of these methods with other techniques, such as mass spectrometry or other separation methods, should continue to increase the flexibility and ease with which these approaches can be used in work involving clinical or pharmaceutical samples. PMID:28396561

Compound immobilization and drug-affinity chromatography.

PubMed

Rix, Uwe; Gridling, Manuela; Superti-Furga, Giulio

2012-01-01

Bioactive small molecules act through modulating a yet unpredictable number of targets. It is therefore of critical importance to define the cellular target proteins of a compound as an entry point to understanding its mechanism of action. Often, this can be achieved in a direct fashion by chemical proteomics. As with any affinity chromatography, immobilization of the bait to a solid support is one of the earliest and most crucial steps in the process. Interfering with structural features that are important for identification of a target protein will be detrimental to binding affinity. Also, many molecules are sensitive to heat or to certain chemicals, such as acid or base, and might be destroyed during the process of immobilization, which therefore needs to be not only efficient, but also mild. The subsequent affinity chromatography step needs to preserve molecular and conformational integrity of both bait compound and proteins in order to result in the desired specific enrichment while ensuring a high level of compatibility with downstream analysis by mass spectrometry. Thus, the right choice of detergent, buffer, and protease inhibitors is also essential. This chapter describes a widely applicable procedure for the immobilization of small molecule drugs and for drug-affinity chromatography with subsequent protein identification by mass spectrometry.

The Cutting Edge of Affinity Electrophoresis Technology

PubMed Central

Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

2015-01-01

Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years. PMID:28248262

Molecular Hybridization of Potent and Selective γ-Hydroxybutyric Acid (GHB) Ligands: Design, Synthesis, Binding Studies, and Molecular Modeling of Novel 3-Hydroxycyclopent-1-enecarboxylic Acid (HOCPCA) and trans-γ-Hydroxycrotonic Acid (T-HCA) Analogs.

PubMed

Krall, Jacob; Jensen, Claus Hatt; Bavo, Francesco; Falk-Petersen, Christina Birkedahl; Haugaard, Anne Stæhr; Vogensen, Stine Byskov; Tian, Yongsong; Nittegaard-Nielsen, Mia; Sigurdardóttir, Sara Björk; Kehler, Jan; Kongstad, Kenneth Thermann; Gloriam, David E; Clausen, Rasmus Prætorius; Harpsøe, Kasper; Wellendorph, Petrine; Frølund, Bente

2017-11-09

γ-Hydroxybutyric acid (GHB) is a neuroactive substance with specific high-affinity binding sites. To facilitate target identification and ligand optimization, we herein report a comprehensive structure-affinity relationship study for novel ligands targeting these binding sites. A molecular hybridization strategy was used based on the conformationally restricted 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) and the linear GHB analog trans-4-hydroxycrotonic acid (T-HCA). In general, all structural modifications performed on HOCPCA led to reduced affinity. In contrast, introduction of diaromatic substituents into the 4-position of T-HCA led to high-affinity analogs (medium nanomolar K i ) for the GHB high-affinity binding sites as the most high-affinity analogs reported to date. The SAR data formed the basis for a three-dimensional pharmacophore model for GHB ligands, which identified molecular features important for high-affinity binding, with high predictive validity. These findings will be valuable in the further processes of both target characterization and ligand identification for the high-affinity GHB binding sites.

Affinity precipitation of human serum albumin using a thermo-response polymer with an L-thyroxin ligand.

PubMed

Ding, Zhaoyang; Cao, Xuejun

2013-12-17

Affinity precipitation has been reported as a potential technology for the purification of proteins at the early stage of downstream processing. The technology could be achieved using reversible soluble-insoluble polymers coupled with an affinity ligand to purify proteins from large volumes of dilute solution material such as fermentation broths or plasma. In this study, a thermo-response polymer was synthesized using N-methylol acrylamide, N-isopropyl acrylamide and butyl acrylate as monomers. The molecular weight of the polymer measured by the viscosity method was 3.06 × 104 Da and the lower critical solution temperature (LCST) was 28.0°C.The recovery of the polymer above the LCST was over 95.0%. Human serum albumin (HSA) is the most abundant protein in the human serum system, and it has important functions in the human body. High purity HSA is required in pharmaceuticals. Safe and efficient purification is a crucial process during HSA production. A thermo-response polymer was synthesized and L-thyroxin immobilized on the polymer as an affinity ligand to enable affinity precipitation of HSA. The LCST of the affinity polymer was 31.0°C and the recovery was 99.6% of its original amount after recycling three times. The optimal adsorption condition was 0.02 M Tris-HCl buffer (pH 7.0) and the HSA adsorption capacity was 14.9 mg/g polymer during affinity precipitation. Circular dichroism spectra and a ForteBio Octet system were used to analyze the interactions between the affinity polymer and HSA during adsorption and desorption. The recovery of total HSA by elution with 1.0 mol/L NaSCN was 93.6%. When the affinity polymer was applied to purification of HSA from human serum, HSA could be purified to single-band purity according to SDS-PAGE. A thermo-response polymer was synthesized and L-thyroxin was attached to the polymer. Affinity precipitation was used to purify HSA from human serum.

Affinity precipitation of human serum albumin using a thermo-response polymer with an L-thyroxin ligand

PubMed Central

2013-01-01

Background Affinity precipitation has been reported as a potential technology for the purification of proteins at the early stage of downstream processing. The technology could be achieved using reversible soluble-insoluble polymers coupled with an affinity ligand to purify proteins from large volumes of dilute solution material such as fermentation broths or plasma. In this study, a thermo-response polymer was synthesized using N-methylol acrylamide, N-isopropyl acrylamide and butyl acrylate as monomers. The molecular weight of the polymer measured by the viscosity method was 3.06 × 104 Da and the lower critical solution temperature (LCST) was 28.0°C.The recovery of the polymer above the LCST was over 95.0%. Human serum albumin (HSA) is the most abundant protein in the human serum system, and it has important functions in the human body. High purity HSA is required in pharmaceuticals. Safe and efficient purification is a crucial process during HSA production. Results A thermo-response polymer was synthesized and L-thyroxin immobilized on the polymer as an affinity ligand to enable affinity precipitation of HSA. The LCST of the affinity polymer was 31.0°C and the recovery was 99.6% of its original amount after recycling three times. The optimal adsorption condition was 0.02 M Tris–HCl buffer (pH 7.0) and the HSA adsorption capacity was 14.9 mg/g polymer during affinity precipitation. Circular dichroism spectra and a ForteBio Octet system were used to analyze the interactions between the affinity polymer and HSA during adsorption and desorption. The recovery of total HSA by elution with 1.0 mol/L NaSCN was 93.6%. When the affinity polymer was applied to purification of HSA from human serum, HSA could be purified to single-band purity according to SDS-PAGE. Conclusion A thermo-response polymer was synthesized and L-thyroxin was attached to the polymer. Affinity precipitation was used to purify HSA from human serum. PMID:24341315

Proteins feel more than they see: fine-tuning of binding affinity by properties of the non-interacting surface.

PubMed

Kastritis, Panagiotis L; Rodrigues, João P G L M; Folkers, Gert E; Boelens, Rolf; Bonvin, Alexandre M J J

2014-07-15

Protein-protein complexes orchestrate most cellular processes such as transcription, signal transduction and apoptosis. The factors governing their affinity remain elusive however, especially when it comes to describing dissociation rates (koff). Here we demonstrate that, next to direct contributions from the interface, the non-interacting surface (NIS) also plays an important role in binding affinity, especially polar and charged residues. Their percentage on the NIS is conserved over orthologous complexes indicating an evolutionary selection pressure. Their effect on binding affinity can be explained by long-range electrostatic contributions and surface-solvent interactions that are known to determine the local frustration of the protein complex surface. Including these in a simple model significantly improves the affinity prediction of protein complexes from structural models. The impact of mutations outside the interacting surface on binding affinity is supported by experimental alanine scanning mutagenesis data. These results enable the development of more sophisticated and integrated biophysical models of binding affinity and open new directions in experimental control and modulation of biomolecular interactions. Copyright © 2014. Published by Elsevier Ltd.

Purification and Properties of Cytidine Deaminase from Normal and Leukemic Granulocytes

PubMed Central

Chabner, Bruce A.; Johns, David G.; Coleman, C. Norman; Drake, James C.; Evans, Warren H.

1974-01-01

Cytidine deaminase, an enzyme that catalyses the deamination of both cytidine and its nucleoside analogues including the antineoplastic agents cytosine arabinoside (ara-C) and 5-azacytidine (5-azaC), has been partially purified from normal and leukemic human granulocytes. The purification procedure included heat precipitation at 70°C, ammonium sulfate precipitation, calcium phosphate gel ion exchange, and Sephadex G-150 gel filtration. The enzyme has mol wt 51,000, isoelectric pH of 4.8, and maximum activity over a broad pH range of 5-9.5. The enzyme is stabilized by the presence of the sulfhydryl reagent, dithiothreitol. Cytidine deaminase from normal human granulocytes has a greater affinity for its physiologic substrate cytidine (Km = 1.1 × 10−5 M) than for ara-C (8.8 × 10−5 M) or 5-azaC (4.3 × 10−4 M). Halogenated analogues such as 5-fluorocytidine and 5-bromo-2′-deoxycytidine also exhibited substrate activity, with maximum velocities greater than that of the physiologic substrates cytidine and deoxycytidine. No activity was observed with nucleotides or deoxynucleotides. The relative maximum velocity of the enzyme for cytidine and its nucleoside analogues remained constant during purification, indicating that a single enzyme was responsible for deamination of these substrates. Tetrahydrouridine (THU) was found to be a strong competitive inhibitor of partially purified deaminase with a Ki of 5.4 × 10−8 M. The biochemical properties of partially purified preparations of cytidine deaminase from normal and leukemic cells were compared with respect to isoelectric pH, molecular weight, and substrate and inhibitor kinetic parameters, and no differences were observed. However, normal circulating granulocytes contained a significantly greater concentration of cytidine deaminase (3.52±1.86 × 103/mg protein) than chronic myelocytic leukemia (CML) cells (1.40±0.70 × 103 U/mg protein) or acute myelocytic leukemia (AML) cells (0.19±0.17 × 103 U/mg protein). To explain these differences in enzyme levels in leukemic versus normal cells, the changes in cytidine deaminase levels associated with maturation of normal granulocytes were studied in normal human bone marrow. Myeloid precursors obtained from bone marrow aspirates were separated into mature and immature fractions by Ficoll density centrifugation. Deaminase activity in lysates of mature granulocytes was 3.55-14.2 times greater than the activity found in the lysates of immature cells. Decreased enzyme activity was also found in immature myeloid cells from a patient with CML as compared to mature granulocytes from the same patient. These observations support the conclusion that the greater specific activity of cytidine deaminase in normal mature granulocytes as compared to leukemic cells is related to the process of granulocyte maturation rather than a specific enzymatic defect in leukemic cells. PMID:4521417

In vitro suppression of dendritic cells by Helicobacter pylori OipA.

PubMed

Teymournejad, Omid; Mobarez, Ashraf M; Hassan, Zuhair M; Moazzeni, Seyed M; Ahmadabad, Hassan N

2014-04-01

Outer inflammatory protein A (OipA) has an important role in Helicobacter pylori pathogenesis. In this study, we purified the outer membrane protein and evaluated the effects of this protein on maturation and cytokine production by dendritic cells (DCs). The oipA gene was inserted into pET28a, and this construct was transformed into Escherichia coli BL21 (DE3). Purification of the recombinant protein was performed by Ni-NTA affinity chromatography. Immature DCs were purified from spleen of C57BL/6 mice with more than 90% purity and were treated with several concentrations of OipA (1-20 μg/mL) overnight. Expression of maturation markers (CD86, CD40, and MHC-II) on the surface of DCs and production of IL-10 and IL-12 were assessed by flow cytometry and ELISA, respectively. The expression of DC maturation markers CD40, CD86, and MHC-II was downregulated on the surface of OipA-treated DCs at concentrations of 10 and 20 μg/mL compared with negative control. Production of IL-10 decreases with increasing OipA concentration at a concentration of 5 μg/mL, but we detected no change in IL-12 production. Inability to eliminate H. pylori from stomach is partly due to the evasion of the bacteria from the immune response. DCs are central mediators between innate and adaptive immunity, and DC cytokines direct the types of adaptive immune response. This study indicated that OipA of H. pylori is a DC maturation suppression factor. Previous studies have shown that H. pylori manage tolerogenic programming in DCs leading to long-time gastric colonization. In conclusion, H. pylori OipA helps the establishment of chronic infection with reduction in IL-10 and suppression of DC maturation. © 2014 John Wiley & Sons Ltd.

TBC-8, a putative RAB-2 GAP, regulates dense core vesicle maturation in Caenorhabditis elegans.

PubMed

Hannemann, Mandy; Sasidharan, Nikhil; Hegermann, Jan; Kutscher, Lena M; Koenig, Sabine; Eimer, Stefan

2012-01-01

Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2-specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation.

Moral Judgment Maturity of Process and Reactive Schizophrenics.

ERIC Educational Resources Information Center

Herron, William G.; And Others

1983-01-01

Premorbid adjustment, paranoid symptomatology, and orientation were examined as major predictors of moral judgment maturity in 40 schizophrenics. Results suggest the importance of cognitive and social skills in the development of schizophrenics' moral judgment maturity. (Author/RH)

The pivotal role of abscisic acid signaling during transition from seed maturation to germination.

PubMed

Yan, An; Chen, Zhong

2017-05-01

Seed maturation and germination are two continuous developmental processes that link two distinct generations in spermatophytes; the precise genetic control of these two processes is, therefore, crucially important for the survival of the next generation. Pieces of experimental evidence accumulated so far indicate that a concerted action of endogenous signals and environmental cues is required to govern these processes. Plant hormone abscisic acid (ABA) has been suggested to play a predominant role in directing seed maturation and maintaining seed dormancy under unfavorable environmental conditions until antagonized by gibberellins (GA) and certain environmental cues to allow the commencement of seed germination when environmental conditions are favorable; therefore, the balance of ABA and GA is a major determinant of the timing of seed germination. Due to the advent of new technologies and system biology approaches, molecular studies are beginning to draw a picture of the sophisticated genetic network that drives seed maturation during the past decade, though the picture is still incomplete and many details are missing. In this review, we summarize recent advances in ABA signaling pathway in the regulation of seed maturation as well as the transition from seed maturation to germination, and highlight the importance of system biology approaches in the study of seed maturation.

An Innovative Cloning Platform Enables Large-Scale Production and Maturation of an Oxygen-Tolerant [NiFe]-Hydrogenase from Cupriavidus necator in Escherichia coli

PubMed Central

Schiffels, Johannes; Pinkenburg, Olaf; Schelden, Maximilian; Aboulnaga, El-Hussiny A. A.; Baumann, Marcus E. M.; Selmer, Thorsten

2013-01-01

Expression of multiple heterologous genes in a dedicated host is a prerequisite for approaches in synthetic biology, spanning from the production of recombinant multiprotein complexes to the transfer of tailor-made metabolic pathways. Such attempts are often exacerbated, due in most cases to a lack of proper directional, robust and readily accessible genetic tools. Here, we introduce an innovative system for cloning and expression of multiple genes in Escherichia coli BL21 (DE3). Using the novel methodology, genes are equipped with individual promoters and terminators and subsequently assembled. The resulting multiple gene cassettes may either be placed in one vector or alternatively distributed among a set of compatible plasmids. We demonstrate the effectiveness of the developed tool by production and maturation of the NAD+reducing soluble [NiFe]-hydrogenase (SH) from Cupriavidus necator H16 (formerly Ralstonia eutropha H16) in E. coli BL21Star™ (DE3). The SH (encoded in hoxFUYHI) was successfully matured by co-expression of a dedicated set of auxiliary genes, comprising seven hyp genes (hypC1D1E1A2B2F2X) along with hoxW, which encodes a specific endopeptidase. Deletion of genes involved in SH maturation reduced maturation efficiency substantially. Further addition of hoxN1, encoding a high-affinity nickel permease from C. necator, considerably increased maturation efficiency in E. coli. Carefully balanced growth conditions enabled hydrogenase production at high cell-densities, scoring mg·(Liter culture)−1 yields of purified functional SH. Specific activities of up to 7.2±1.15 U·mg−1 were obtained in cell-free extracts, which is in the range of the highest activities ever determined in C. necator extracts. The recombinant enzyme was isolated in equal purity and stability as previously achieved with the native form, yielding ultrapure preparations with anaerobic specific activities of up to 230 U·mg−1. Owing to the combinatorial power exhibited by the presented cloning platform, the system might represent an important step towards new routes in synthetic biology. PMID:23861944

Functional maturation of motor nerve terminals in the avian iris: ultrastructure, transmitter metabolism and synaptic reliability

PubMed Central

Pilar, Guillermo; Tuttle, Jeremy; Vaca, Ken

1981-01-01

1. The transformation of easily fatigued embryonic neuromuscular junctions into highly reliable mature terminals was examined by studying functional and morphological changes during development of the avian iris. The mature ability to follow repetitive electrical nerve stimulation was correlated with the rate of acetylcholine (ACh) synthesis and choline uptake, and with the fine structure of the nerve terminals and the post-synaptic elements. 2. The terminals of the ciliary nerve of the chick initially form functional synaptic contacts with the iris muscle at embryonic St. 34-40. At the onset of this period, no Na+-dependent high affinity choline uptake can be demonstrated, and the low level of ACh synthesis present is sensitive to Na+ removal. At St. 36 [3H]ACh synthesis begins to increase, the increment being Na+-dependent. 3. ACh synthesis in the embryonic iris was insensitive to a conditioning [K+]o depolarization even as late as St. 43. Just before hatching, depolarization elicits some augmentation in synthesis, but by 2 days ex ovo this release-induced response has increased by an order of magnitude. 4. Concurrently with the acquisition of the ability to respond to depolarization with accelerated synthesis, neuromuscular transmission in the iris becomes reliable and secure during stimulation at 20 Hz. Embryonic junctions rapidly block during such stimulation, and the failure is shown to be presynaptic in origin, resulting most probably from failure to sustain adequate levels of transmitter release. 5. Ultrastructural examination of the developing ciliary terminals revealed few synaptic vesicles at early stages, and a dearth of other specializations. The sequence of development from these small structurally undistinguished endings to large en plaque junctions completely filled with vesicles was reconstructed and compared to other neuromuscular junctions. Morphological maturation appears progressive with little evidence of discontinuity signalling functional status, but it is only after the terminals enlarge and become closely packed with vesicles that mature synaptic reliability is found. 6. The temporal correlation between responsiveness of transmitter synthesis to depolarization and reliable neuromuscular transmission suggests that modulation of neurotransmitter metabolism in response to demand signals the achievement of junctional maturity. ImagesABPlate 2Plate 3Plate 4 PMID:6279822

Mapping protein-protein interactions with phage-displayed combinatorial peptide libraries.

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

Kay, B. K.; Castagnoli, L.; Biosciences Division

This unit describes the process and analysis of affinity selecting bacteriophage M13 from libraries displaying combinatorial peptides fused to either a minor or major capsid protein. Direct affinity selection uses target protein bound to a microtiter plate followed by purification of selected phage by ELISA. Alternatively, there is a bead-based affinity selection method. These methods allow one to readily isolate peptide ligands that bind to a protein target of interest and use the consensus sequence to search proteomic databases for putative interacting proteins.

Induced maturation of human immunodeficiency virus.

PubMed

Mattei, Simone; Anders, Maria; Konvalinka, Jan; Kräusslich, Hans-Georg; Briggs, John A G; Müller, Barbara

2014-12-01

HIV-1 assembles at the plasma membrane of virus-producing cells as an immature, noninfectious particle. Processing of the Gag and Gag-Pol polyproteins by the viral protease (PR) activates the viral enzymes and results in dramatic structural rearrangements within the virion--termed maturation--that are a prerequisite for infectivity. Despite its fundamental importance for viral replication, little is currently known about the regulation of proteolysis and about the dynamics and structural intermediates of maturation. This is due mainly to the fact that HIV-1 release and maturation occur asynchronously both at the level of individual cells and at the level of particle release from a single cell. Here, we report a method to synchronize HIV-1 proteolysis in vitro based on protease inhibitor (PI) washout from purified immature virions, thereby temporally uncoupling virus assembly and maturation. Drug washout resulted in the induction of proteolysis with cleavage efficiencies correlating with the off-rate of the respective PR-PI complex. Proteolysis of Gag was nearly complete and yielded the correct products with an optimal half-life (t(1/2)) of ~5 h, but viral infectivity was not recovered. Failure to gain infectivity following PI washout may be explained by the observed formation of aberrant viral capsids and/or by pronounced defects in processing of the reverse transcriptase (RT) heterodimer associated with a lack of RT activity. Based on our results, we hypothesize that both the polyprotein processing dynamics and the tight temporal coupling of immature particle assembly and PR activation are essential for correct polyprotein processing and morphological maturation and thus for HIV-1 infectivity. Cleavage of the Gag and Gag-Pol HIV-1 polyproteins into their functional subunits by the viral protease activates the viral enzymes and causes major structural rearrangements essential for HIV-1 infectivity. This proteolytic maturation occurs concomitant with virus release, and investigation of its dynamics is hampered by the fact that virus populations in tissue culture contain particles at all stages of assembly and maturation. Here, we developed an inhibitor washout strategy to synchronize activation of protease in wild-type virus. We demonstrated that nearly complete Gag processing and resolution of the immature virus architecture are accomplished under optimized conditions. Nevertheless, most of the resulting particles displayed irregular morphologies, Gag-Pol processing was not faithfully reconstituted, and infectivity was not recovered. These data show that HIV-1 maturation is sensitive to the dynamics of processing and also that a tight temporal link between virus assembly and PR activation is required for correct polyprotein processing. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Persistent Low-Level Replication of SIVΔnef Drives Maturation of Antibody and CD8 T Cell Responses to Induce Protective Immunity against Vaginal SIV Infection.

PubMed

Adnan, Sama; Reeves, R Keith; Gillis, Jacqueline; Wong, Fay E; Yu, Yi; Camp, Jeremy V; Li, Qingsheng; Connole, Michelle; Li, Yuan; Piatak, Michael; Lifson, Jeffrey D; Li, Wenjun; Keele, Brandon F; Kozlowski, Pamela A; Desrosiers, Ronald C; Haase, Ashley T; Johnson, R Paul

2016-12-01

Defining the correlates of immune protection conferred by SIVΔnef, the most effective vaccine against SIV challenge, could enable the design of a protective vaccine against HIV infection. Here we provide a comprehensive assessment of immune responses that protect against SIV infection through detailed analyses of cellular and humoral immune responses in the blood and tissues of rhesus macaques vaccinated with SIVΔnef and then vaginally challenged with wild-type SIV. Despite the presence of robust cellular immune responses, animals at 5 weeks after vaccination displayed only transient viral suppression of challenge virus, whereas all macaques challenged at weeks 20 and 40 post-SIVΔnef vaccination were protected, as defined by either apparent sterile protection or significant suppression of viremia in infected animals. Multiple parameters of CD8 T cell function temporally correlated with maturation of protection, including polyfunctionality, phenotypic differentiation, and redistribution to gut and lymphoid tissues. Importantly, we also demonstrate the induction of a tissue-resident memory population of SIV-specific CD8 T cells in the vaginal mucosa, which was dependent on ongoing low-level antigenic stimulation. Moreover, we show that vaginal and serum antibody titers inversely correlated with post-challenge peak viral load, and we correlate the accumulation and affinity maturation of the antibody response to the duration of the vaccination period as well as to the SIVΔnef antigenic load. In conclusion, maturation of SIVΔnef-induced CD8 T cell and antibody responses, both propelled by viral persistence in the gut mucosa and secondary lymphoid tissues, results in protective immune responses that are able to interrupt viral transmission at mucosal portals of entry as well as potential sites of viral dissemination.

Mammalian enamel maturation: Crystallographic changes prior to tooth eruption

PubMed Central

Kallistová, Anna; Horáček, Ivan; Šlouf, Miroslav; Skála, Roman; Fridrichová, Michaela

2017-01-01

Using the distal molar of a minipig as a model, we studied changes in the microstructural characteristics of apatite crystallites during enamel maturation (16-23 months of postnatal age), and their effects upon the mechanical properties of the enamel coat. The slow rate of tooth development in a pig model enabled us to reveal essential heterochronies in particular components of the maturation process. The maturation changes began along the enamel-dentine junction (EDJ) of the trigonid, spreading subsequently to the outer layers of the enamel coat to appear at the surface zone with a 2-month delay. Correspondingly, at the distal part of the tooth the timing of maturation processes is delayed by 3-5 month compared to the mesial part of the tooth. The early stage of enamel maturation (16-20 months), when the enamel coat is composed almost exclusively of radial prismatic enamel, is characterized by a gradual increase in crystallite thickness (by a mean monthly increment of 3.8 nm); and an increase in the prism width and thickness of crystals composed of elementary crystallites. The late stage of maturation (the last two months prior to tooth eruption), marked with the rapid appearance of the interprismatic matrix (IPM) during which the crystals densely infill spaces between prisms, is characterized by an abrupt decrease in microstrain and abrupt changes in the micromechanical properties of the enamel: a rapid increase in its ability to resist long-term load and its considerable hardening. The results suggest that in terms of crystallization dynamics the processes characterizing the early and late stage of mammalian enamel maturation represent distinct entities. In regards to common features with enamel formation in the tribosphenic molar we argue that the separation of these processes could be a common apomorphy of mammalian amelogenetic dynamics in general. PMID:28196135

Mammalian enamel maturation: Crystallographic changes prior to tooth eruption.

PubMed

Kallistová, Anna; Horáček, Ivan; Šlouf, Miroslav; Skála, Roman; Fridrichová, Michaela

2017-01-01

Using the distal molar of a minipig as a model, we studied changes in the microstructural characteristics of apatite crystallites during enamel maturation (16-23 months of postnatal age), and their effects upon the mechanical properties of the enamel coat. The slow rate of tooth development in a pig model enabled us to reveal essential heterochronies in particular components of the maturation process. The maturation changes began along the enamel-dentine junction (EDJ) of the trigonid, spreading subsequently to the outer layers of the enamel coat to appear at the surface zone with a 2-month delay. Correspondingly, at the distal part of the tooth the timing of maturation processes is delayed by 3-5 month compared to the mesial part of the tooth. The early stage of enamel maturation (16-20 months), when the enamel coat is composed almost exclusively of radial prismatic enamel, is characterized by a gradual increase in crystallite thickness (by a mean monthly increment of 3.8 nm); and an increase in the prism width and thickness of crystals composed of elementary crystallites. The late stage of maturation (the last two months prior to tooth eruption), marked with the rapid appearance of the interprismatic matrix (IPM) during which the crystals densely infill spaces between prisms, is characterized by an abrupt decrease in microstrain and abrupt changes in the micromechanical properties of the enamel: a rapid increase in its ability to resist long-term load and its considerable hardening. The results suggest that in terms of crystallization dynamics the processes characterizing the early and late stage of mammalian enamel maturation represent distinct entities. In regards to common features with enamel formation in the tribosphenic molar we argue that the separation of these processes could be a common apomorphy of mammalian amelogenetic dynamics in general.

Kaposi's Sarcoma-Associated Herpesvirus MicroRNA Single-Nucleotide Polymorphisms Identified in Clinical Samples Can Affect MicroRNA Processing, Level of Expression, and Silencing Activity

PubMed Central

Han, Soo-Jin; Marshall, Vickie; Barsov, Eugene; Quiñones, Octavio; Ray, Alex; Labo, Nazzarena; Trivett, Matthew; Ott, David; Renne, Rolf

2013-01-01

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 12 pre-microRNAs that can produce 25 KSHV mature microRNAs. We previously reported single-nucleotide polymorphisms (SNPs) in KSHV-encoded pre-microRNA and mature microRNA sequences from clinical samples (V. Marshall et al., J. Infect. Dis., 195:645–659, 2007). To determine whether microRNA SNPs affect pre-microRNA processing and, ultimately, mature microRNA expression levels, we performed a detailed comparative analysis of (i) mature microRNA expression levels, (ii) in vitro Drosha/Dicer processing, and (iii) RNA-induced silencing complex-dependent targeting of wild-type (wt) and variant microRNA genes. Expression of pairs of wt and variant pre-microRNAs from retroviral vectors and measurement of KSHV mature microRNA expression by real-time reverse transcription-PCR (RT-PCR) revealed differential expression levels that correlated with the presence of specific sequence polymorphisms. Measurement of KSHV mature microRNA expression in a panel of primary effusion lymphoma cell lines by real-time RT-PCR recapitulated some observed expression differences but suggested a more complex relationship between sequence differences and expression of mature microRNA. Furthermore, in vitro maturation assays demonstrated significant SNP-associated changes in Drosha/DGCR8 and/or Dicer processing. These data demonstrate that SNPs within KSHV-encoded pre-microRNAs are associated with differential microRNA expression levels. Given the multiple reports on the involvement of microRNAs in cancer, the biological significance of these phenotypic and genotypic variants merits further studies in patients with KSHV-associated malignancies. PMID:24006441

Searching for DNA Lesions: Structural Evidence for Lower- and Higher-Affinity DNA Binding Conformations of Human Alkyladenine DNA Glycosylase

PubMed Central

2011-01-01

To efficiently repair DNA, human alkyladenine DNA glycosylase (AAG) must search the million-fold excess of unmodified DNA bases to find a handful of DNA lesions. Such a search can be facilitated by the ability of glycosylases, like AAG, to interact with DNA using two affinities: a lower-affinity interaction in a searching process and a higher-affinity interaction for catalytic repair. Here, we present crystal structures of AAG trapped in two DNA-bound states. The lower-affinity depiction allows us to investigate, for the first time, the conformation of this protein in the absence of a tightly bound DNA adduct. We find that active site residues of AAG involved in binding lesion bases are in a disordered state. Furthermore, two loops that contribute significantly to the positive electrostatic surface of AAG are disordered. Additionally, a higher-affinity state of AAG captured here provides a fortuitous snapshot of how this enzyme interacts with a DNA adduct that resembles a one-base loop. PMID:22148158

Top 10 research questions related to growth and maturation of relevance to physical activity, performance, and fitness.

PubMed

Malina, Robert M

2014-06-01

Growth, maturation, and development dominate the daily lives of children and adolescents for approximately the first 2 decades of life. Growth and maturation are biological processes, while development is largely a behavioral process. The 3 processes occur simultaneously and interact. They can be influenced by physical activity and also can influence activity, performance, and fitness. Allowing for these potential interactions, 10 questions on growth and maturation that have relevance to physical activity, performance, and fitness are presented. The questions are not mutually exclusive and address several broadly defined topical areas: exercise and growth, body weight status (body mass index, adiposity rebound, "unhealthy weight gain"), movement proficiency (hypothesized barrier, role in obesity), individual differences, tracking, maturity-associated variation in performance, and corresponding variation in physical activity. Central to the discussion of each is the need for a biocultural approach recognizing the interactions of biology and behavior as potential influences on the variables of interest.

Effect of organic matter properties, clay mineral type and thermal maturity on gas adsorption in organic-rich shale systems

USGS Publications Warehouse

Zhang, Tongwei; Ellis, Geoffrey S.; Ruppel, Stephen C.; Milliken, Kitty; Lewan, Mike; Sun, Xun; Baez, Luis; Beeney, Ken; Sonnenberg, Steve

2013-01-01

A series of CH4 adsorption experiments on natural organic-rich shales, isolated kerogen, clay-rich rocks, and artificially matured Woodford Shale samples were conducted under dry conditions. Our results indicate that physisorption is a dominant process for CH4 sorption, both on organic-rich shales and clay minerals. The Brunauer–Emmett–Teller (BET) surface area of the investigated samples is linearly correlated with the CH4 sorption capacity in both organic-rich shales and clay-rich rocks. The presence of organic matter is a primary control on gas adsorption in shale-gas systems, and the gas-sorption capacity is determined by total organic carbon (TOC) content, organic-matter type, and thermal maturity. A large number of nanopores, in the 2–50 nm size range, were created during organic-matter thermal decomposition, and they significantly contributed to the surface area. Consequently, methane-sorption capacity increases with increasing thermal maturity due to the presence of nanopores produced during organic-matter decomposition. Furthermore, CH4 sorption on clay minerals is mainly controlled by the type of clay mineral present. In terms of relative CH4 sorption capacity: montmorillonite ≫ illite – smectite mixed layer > kaolinite > chlorite > illite. The effect of rock properties (organic matter content, type, maturity, and clay minerals) on CH4 adsorption can be quantified with the heat of adsorption and the standard entropy, which are determined from adsorption isotherms at different temperatures. For clay-mineral rich rocks, the heat of adsorption (q) ranges from 9.4 to 16.6 kJ/mol. These values are considerably smaller than those for CH4 adsorption on kerogen (21.9–28 kJ/mol) and organic-rich shales (15.1–18.4 kJ/mol). The standard entropy (Δs°) ranges from -64.8 to -79.5 J/mol/K for clay minerals, -68.1 to -111.3 J/mol/K for kerogen, and -76.0 to -84.6 J/mol/K for organic-rich shales. The affinity of CH4 molecules for sorption on organic matter is stronger than for most common clay minerals. Thus, it is expected that CH4 molecules may preferentially occupy surface sites on organic matter. However, active sites on clay mineral surfaces are easily blocked by water. As a consequence, organic-rich shales possess a larger CH4-sorption capacity than clay-rich rocks lacking organic matter. The thermodynamic parameters obtained in this study can be incorporated into model predictions of the maximum Langmuir pressure and CH4- sorption capacity of shales under reservoir temperature and pressure conditions.

Mathematical analysis of frontal affinity chromatography in particle and membrane configurations.

PubMed

Tejeda-Mansir, A; Montesinos, R M; Guzmán, R

2001-10-30

The scaleup and optimization of large-scale affinity-chromatographic operations in the recovery, separation and purification of biochemical components is of major industrial importance. The development of mathematical models to describe affinity-chromatographic processes, and the use of these models in computer programs to predict column performance is an engineering approach that can help to attain these bioprocess engineering tasks successfully. Most affinity-chromatographic separations are operated in the frontal mode, using fixed-bed columns. Purely diffusive and perfusion particles and membrane-based affinity chromatography are among the main commercially available technologies for these separations. For a particular application, a basic understanding of the main similarities and differences between particle and membrane frontal affinity chromatography and how these characteristics are reflected in the transport models is of fundamental relevance. This review presents the basic theoretical considerations used in the development of particle and membrane affinity chromatography models that can be applied in the design and operation of large-scale affinity separations in fixed-bed columns. A transport model for column affinity chromatography that considers column dispersion, particle internal convection, external film resistance, finite kinetic rate, plus macropore and micropore resistances is analyzed as a framework for exploring further the mathematical analysis. Such models provide a general realistic description of almost all practical systems. Specific mathematical models that take into account geometric considerations and transport effects have been developed for both particle and membrane affinity chromatography systems. Some of the most common simplified models, based on linear driving-force (LDF) and equilibrium assumptions, are emphasized. Analytical solutions of the corresponding simplified dimensionless affinity models are presented. Particular methods for estimating the parameters that characterize the mass-transfer and adsorption mechanisms in affinity systems are described.

Distilling the Verification Process for Prognostics Algorithms

NASA Technical Reports Server (NTRS)

Roychoudhury, Indranil; Saxena, Abhinav; Celaya, Jose R.; Goebel, Kai

2013-01-01

The goal of prognostics and health management (PHM) systems is to ensure system safety, and reduce downtime and maintenance costs. It is important that a PHM system is verified and validated before it can be successfully deployed. Prognostics algorithms are integral parts of PHM systems. This paper investigates a systematic process of verification of such prognostics algorithms. To this end, first, this paper distinguishes between technology maturation and product development. Then, the paper describes the verification process for a prognostics algorithm as it moves up to higher maturity levels. This process is shown to be an iterative process where verification activities are interleaved with validation activities at each maturation level. In this work, we adopt the concept of technology readiness levels (TRLs) to represent the different maturity levels of a prognostics algorithm. It is shown that at each TRL, the verification of a prognostics algorithm depends on verifying the different components of the algorithm according to the requirements laid out by the PHM system that adopts this prognostics algorithm. Finally, using simplified examples, the systematic process for verifying a prognostics algorithm is demonstrated as the prognostics algorithm moves up TRLs.

System Maturity and Architecture Assessment Methods, Processes, and Tools

DTIC Science & Technology

2012-03-02

Deshmukh , and M. Sarfaraz. Development of Systems Engineering Maturity Models and Management Tools. Systems Engineering Research Center Final Technical...Ramirez- Marquez, D. Nowicki, A. Deshmukh , and M. Sarfaraz. Development of Systems Engineering Maturity Models and Management Tools. Systems Engineering

New definitions for cotton fiber maturity ratio

USDA-ARS?s Scientific Manuscript database

Cotton fiber maturity affects fiber physical, mechanical, and chemical properties, as well as the processability and qualities of yarn and fabrics. New definitions of cotton fiber maturity ratio are introduced. The influences of sampling, sample preparation, measurement method, and correlations am...

The Roles of Family B and D DNA Polymerases in Thermococcus Species 9°N Okazaki Fragment Maturation*

PubMed Central

Greenough, Lucia; Kelman, Zvi; Gardner, Andrew F.

2015-01-01

During replication, Okazaki fragment maturation is a fundamental process that joins discontinuously synthesized DNA fragments into a contiguous lagging strand. Efficient maturation prevents repeat sequence expansions, small duplications, and generation of double-stranded DNA breaks. To address the components required for the process in Thermococcus, Okazaki fragment maturation was reconstituted in vitro using purified proteins from Thermococcus species 9°N or cell extracts. A dual color fluorescence assay was developed to monitor reaction substrates, intermediates, and products. DNA polymerase D (polD) was proposed to function as the replicative polymerase in Thermococcus replicating both the leading and the lagging strands. It is shown here, however, that it stops before the previous Okazaki fragments, failing to rapidly process them. Instead, Family B DNA polymerase (polB) was observed to rapidly fill the gaps left by polD and displaces the downstream Okazaki fragment to create a flap structure. This flap structure was cleaved by flap endonuclease 1 (Fen1) and the resultant nick was ligated by DNA ligase to form a mature lagging strand. The similarities to both bacterial and eukaryotic systems and evolutionary implications of archaeal Okazaki fragment maturation are discussed. PMID:25814667

Development of model for analysing respective collections of intended hematopoietic stem cells and harvests of unintended mature cells in apheresis for autologous hematopoietic stem cell collection.

PubMed

Hequet, O; Le, Q H; Rodriguez, J; Dubost, P; Revesz, D; Clerc, A; Rigal, D; Salles, G; Coiffier, B

2014-04-01

Hematopoietic stem cells (HSCs) required to perform peripheral hematopoietic autologous stem cell transplantation (APBSCT) can be collected by processing several blood volumes (BVs) in leukapheresis sessions. However, this may cause granulocyte harvest in graft and decrease in patient's platelet blood level. Both consequences may induce disturbances in patient. One apheresis team's current purpose is to improve HSC collection by increasing HSC collection and prevent increase in granulocyte and platelet harvests. Before improving HSC collection it seemed important to know more about the way to harvest these types of cells. The purpose of our study was to develop a simple model for analysing respective collections of intended CD34+ cells among HSC (designated here as HSC) and harvests of unintended platelets or granulocytes among mature cells (designated here as mature cells) considering the number of BVs processed and factors likely to influence cell collection or harvest. For this, we processed 1, 2 and 3 BVs in 59 leukapheresis sessions and analysed corresponding collections and harvests with a referent device (COBE Spectra). First we analysed the amounts of HSC collected and mature cells harvested and second the evolution of the respective shares of HSC and mature cells collected or harvested throughout the BV processes. HSC collections and mature cell harvests increased globally (p<0.0001) and their respective shares remained stable throughout the BV processes (p non-significant). We analysed the role of intrinsic (patient's features) and extrinsic (features before starting leukapheresis sessions) factors in collections and harvests, which showed that only pre-leukapheresis blood levels (CD34+cells and platelets) influenced both cell collections and harvests (CD34+cells and platelets) (p<0.001) and shares of HSC collections and mature unintended cells harvests (p<0.001) throughout the BV processes. Altogether, our results suggested that the main factors likely to influence intended HSC collections or unintended mature cell harvests were pre-leukapheresis blood cell levels. Our model was meant to assist apheresis teams in analysing shares of HSC collected and mature cells harvested with new devices or with new types of HSC mobilization. Copyright © 2014 Elsevier Ltd. All rights reserved.

Proteomic analysis of 'Zaosu' pear (Pyrus bretschneideri Rehd.) and its early-maturing bud sport.

PubMed

Liu, Xueting; Zhai, Rui; Feng, Wenting; Zhang, Shiwei; Wang, Zhigang; Qiu, Zonghao; Zhang, Junke; Ma, Fengwang; Xu, Lingfei

2014-07-01

Maturation of fruits involves a series of physiological, biochemical, and organoleptic changes that eventually make fleshy fruits attractive, palatable, and nutritional. In order to understand the mature mechanism of the early-maturing bud sport of 'Zaosu' pear, we analyzed the differences of proteome expression between the both pears in different mature stages by the methods of a combination of two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. Seventy-five differential expressed protein spots (p<0.05) were obtained between 'Zaosu' pear and its early-maturing bud sport, but only sixty-eight were demonstratively identified in the database of NCBI and uniprot. The majority of proteins were linked to metabolism, energy, stress response/defense and cell structure. Additionally, our data confirmed an increase of proteins related to cell-wall modification, oxidative stress and pentose phosphate metabolism and a decrease of proteins related to photosynthesis and glycolysis during the development process of both pears, but all these proteins increased or decreased faster in the early-maturing bud sport. This comparative analysis between both pears showed that these proteins were closely associated with maturation and could provide more detailed characteristics of the maturation process of both pears. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Antigenic Structure of the Human Muscle Nicotinic Acetylcholine Receptor Main Immunogenic Region

PubMed Central

Luo, Jie; Lindstrom, Jon

2009-01-01

The main immunogenic region on the α1 subunits of muscle nicotinic acetylcholine receptors provokes half or more of the autoantibodies in myasthenia gravis and its animal model. Many of these autoantibodies depend on the native conformation of the receptor for their ability to bind with high affinity. We mapped this region and explained the conformation-dependence of its epitopes by making chimeras in which sequences of human muscle α1 subunits were replaced in human neuronal α7 subunits or Aplysia acetylcholine binding protein. These chimeras also revealed that the main immunogenic region can play a major role in promoting conformational maturation, and, consequently, assembly of receptor subunits. PMID:19705087

Oxidation in the complementarity-determining regions differentially influences the properties of therapeutic antibodies

PubMed Central

Dashivets, Tetyana; Stracke, Jan; Dengl, Stefan; Knaupp, Alexander; Pollmann, Jan; Buchner, Johannes; Schlothauer, Tilman

2016-01-01

ABSTRACT Therapeutic antibodies can undergo a variety of chemical modification reactions in vitro. Depending on the site of modification, either antigen binding or Fc-mediated functions can be affected. Oxidation of tryptophan residues is one of the post-translational modifications leading to altered antibody functionality. In this study, we examined the structural and functional properties of a therapeutic antibody construct and 2 affinity matured variants thereof. Two of the 3 antibodies carry an oxidation-prone tryptophan residue in the complementarity-determining region of the VL domain. We demonstrate the differences in the stability and bioactivity of the 3 antibodies, and reveal differential degradation pathways for the antibodies susceptible to oxidation. PMID:27612038

Shared antigenicity between the polar filaments of myxosporeans and other Cnidaria.

PubMed

Ringuette, Maurice J; Koehler, Anne; Desser, Sherwin S

2011-02-01

Nematocysts containing coiled polar filaments are a distinguishing feature of members of the phylum Cnidaria. As a first step to characterizing the molecular structure of polar filaments, a polyclonal antiserum was raised in rabbits against a cyanogen bromide-resistant protein extract of mature cysts containing spores of Myxobolus pendula. The antiserum reacted only with proteins associated with extruded polar filaments. Western blot and whole-mount immunohistochemical analyses indicated a conservation of polar filament epitopes between M. pendula and 2 related cnidarians, i.e., the anthozoan, Nematostella vectensis, and the hydrozoan, Hydra vulgaris. This conservation of polar filament epitopes lends further support to a shared affinity between Myxozoa and cnidarians.

People Capability Maturity Model. SM.

DTIC Science & Technology

1995-09-01

People Capability Maturity Model SM .^^^^_ -——’ Bill Curtis William E. ] Sally Mille] Hefley r Accesion For t NTIS DTIC...People CMM The P-CMM adapts the architecture and the maturity framework underlying the CMM for use with people-related improvement issues. The CMM...focuses on helping organizations improve their software development processes. By adapting the maturity framework and the CMM architecture

Structural Maturation of HIV-1 Reverse Transcriptase—A Metamorphic Solution to Genomic Instability

PubMed Central

London, Robert E.

2016-01-01

Human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT)—a critical enzyme of the viral life cycle—undergoes a complex maturation process, required so that a pair of p66 precursor proteins can develop conformationally along different pathways, one evolving to form active polymerase and ribonuclease H (RH) domains, while the second forms a non-functional polymerase and a proteolyzed RH domain. These parallel maturation pathways rely on the structural ambiguity of a metamorphic polymerase domain, for which the sequence–structure relationship is not unique. Recent nuclear magnetic resonance (NMR) studies utilizing selective labeling techniques, and structural characterization of the p66 monomer precursor have provided important insights into the details of this maturation pathway, revealing many aspects of the three major steps involved: (1) domain rearrangement; (2) dimerization; and (3) subunit-selective RH domain proteolysis. This review summarizes the major structural changes that occur during the maturation process. We also highlight how mutations, often viewed within the context of the mature RT heterodimer, can exert a major influence on maturation and dimerization. It is further suggested that several steps in the RT maturation pathway may provide attractive targets for drug development. PMID:27690082

Occupational health and safety of workers in agriculture and horticulture.

PubMed

Lundqvist, P

2000-01-01

Working in agriculture and horticulture gives considerable job satisfaction. The tasks are often interesting; you can see the result of your own work, watch your crop grow and mature; you have an affinity with nature and can follow the changes in the seasons. However, today it is a dangerous work environment fraught with occupational injuries and diseases due to hazardous situations and to physiological, physical, biological, chemical, psychological, and sociological factors. The ongoing rapid development may, on the other hand, bring about many changes during the next decades with more farmers and growers switching to organic production. Moreover, increased awareness of animal welfare also may lead to improved working conditions. Large-scale operations with fewer family-operated agricultural businesses might mean fewer injuries among children and older farmers. A consequence of large-scale operations may also be better regulation of working conditions. The greater use of automation technology eliminates many harmful working postures and movements when milking cows and carrying out other tasks. Information technology offers people the opportunity to gain more knowledge about their work. Labeling food produced in a worker-friendly work environment may give the consumers a chance to be involved in the process.

Germinal-center development of memory B cells driven by IL-9 from follicular helper T cells.

PubMed

Wang, Yifeng; Shi, Jingwen; Yan, Jiacong; Xiao, Zhengtao; Hou, Xiaoxiao; Lu, Peiwen; Hou, Shiyue; Mao, Tianyang; Liu, Wanli; Ma, Yuanwu; Zhang, Lianfeng; Yang, Xuerui; Qi, Hai

2017-08-01

Germinal centers (GCs) support high-affinity, long-lived humoral immunity. How memory B cells develop in GCs is not clear. Through the use of a cell-cycle-reporting system, we identified GC-derived memory precursor cells (GC-MP cells) that had quit cycling and reached G0 phase while in the GC, exhibited memory-associated phenotypes with signs of affinity maturation and localized toward the GC border. After being transferred into adoptive hosts, GC-MP cells reconstituted a secondary response like genuine memory B cells. GC-MP cells expressed the interleukin 9 (IL-9) receptor and responded to IL-9. Acute treatment with IL-9 or antibody to IL-9 accelerated or retarded the positioning of GC-MP cells toward the GC edge and exit from the GC, and enhanced or inhibited the development of memory B cells, which required B cell-intrinsic responsiveness to IL-9. Follicular helper T cells (T FH cells) produced IL-9, and deletion of IL-9 from T cells or, more specifically, from GC T FH cells led to impaired memory formation of B cells. Therefore, the GC development of memory B cells is promoted by T FH cell-derived IL-9.

Drop-out phagemid vector for switching from phage displayed affinity reagents to expression formats.

PubMed

Pershad, Kritika; Sullivan, Mark A; Kay, Brian K

2011-05-15

Affinity reagents that are generated by phage display are typically subcloned into an expression vector for further biochemical characterization. This insert transfer process is time consuming and laborious especially if many inserts are to be subcloned. To simplify the transfer process, we have constructed a "drop-out" phagemid vector that can be rapidly converted to an expression vector by a simple restriction enzyme digestion with MfeI (to "drop-out" the gene III coding sequence), which generates alkaline phosphatase (AP) fusions of the affinity reagents on religation. Subsequently, restriction digestion with AscI drops out the AP coding region and religation generates affinity reagents with a C-terminal six-histidine tag. To validate the usefulness of this vector, four different human single chain Fragments of variable regions (scFv) were tested, three of which show specific binding to three zebrafish (Danio rerio) proteins, namely suppression of tumorigenicity 13, recoverin, and Ppib and the fourth binds to human Lactoferrin protein. For each of the constructs tested, the gene III and AP drop-out efficiency was between 90% and 100%. This vector is especially useful in speeding up the downstream screening of affinity reagents and bypassing the time-consuming subcloning experiments. Copyright © 2011 Elsevier Inc. All rights reserved.

[Comparison of acetonitrile, ethanol and chromatographic column to eliminate high-abundance proteins in human serum].

PubMed

Li, Yin; Liao, Ming; He, Xiao; Zhou, Yi; Luo, Rong; Li, Hongtao; Wang, Yun; He, Min

2012-11-01

To compare the effects of acetonitrile precipitation, ethanol precipitation and multiple affinity chromatography column Human 14 removal to eliminate high-abundance proteins in human serum. Elimination of serum high-abundance proteins performed with acetonitrile precipitation, ethanol precipitation and multiple affinity chromatography column Human 14 removal. Bis-Tris Mini Gels electrophoresis and two-dimensional gel electrophoresis to detect the effect. Grey value analysis from 1-DE figure showed that after serum processed by acetonitrile method, multiple affinity chromatography column Human 14 removal method and ethanol method, the grey value of albumin changed into 157.2, 40.8 and 8.2 respectively from the original value of 19. 2-DE analysis results indicated that using multiple affinity chromatography column Human 14 method, the protein points noticeable increased by 137 compared to the original serum. After processed by acetonitrile method and ethanol method, the protein point reduced, but the low abundance protein point emerged. The acetonitrile precipitation could eliminate the vast majority of high abundance proteins in serum and gain more proteins of low molecular weight, ethanol precipitation could eliminate part of high abundance proteins in serum, but low abundance proteins less harvested, and multiple affinity chromatography column Human 14 method could effectively removed the high abundance proteins, and keep a large number of low abundance proteins.

Structure-kinetic relationships--an overlooked parameter in hit-to-lead optimization: a case of cyclopentylamines as chemokine receptor 2 antagonists.

PubMed

Vilums, Maris; Zweemer, Annelien J M; Yu, Zhiyi; de Vries, Henk; Hillger, Julia M; Wapenaar, Hannah; Bollen, Ilse A E; Barmare, Farhana; Gross, Raymond; Clemens, Jeremy; Krenitsky, Paul; Brussee, Johannes; Stamos, Dean; Saunders, John; Heitman, Laura H; Ijzerman, Adriaan P

2013-10-10

Preclinical models of inflammatory diseases (e.g., neuropathic pain, rheumatoid arthritis, and multiple sclerosis) have pointed to a critical role of the chemokine receptor 2 (CCR2) and chemokine ligand 2 (CCL2). However, one of the biggest problems of high-affinity inhibitors of CCR2 is their lack of efficacy in clinical trials. We report a new approach for the design of high-affinity and long-residence-time CCR2 antagonists. We developed a new competition association assay for CCR2, which allows us to investigate the relation of the structure of the ligand and its receptor residence time [i.e., structure-kinetic relationship (SKR)] next to a traditional structure-affinity relationship (SAR). By applying combined knowledge of SAR and SKR, we were able to re-evaluate the hit-to-lead process of cyclopentylamines as CCR2 antagonists. Affinity-based optimization yielded compound 1 with good binding (Ki = 6.8 nM) but very short residence time (2.4 min). However, when the optimization was also based on residence time, the hit-to-lead process yielded compound 22a, a new high-affinity CCR2 antagonist (3.6 nM), with a residence time of 135 min.

Carrier-mediated γ-aminobutyric acid transport across the basolateral membrane of human intestinal Caco-2 cell monolayers.

PubMed

Nielsen, Carsten Uhd; Carstensen, Mette; Brodin, Birger

2012-06-01

The aim of the present study was to investigate the transport of γ-aminobutyric acid (GABA) across the basolateral membrane of intestinal cells. The proton-coupled amino acid transporter, hPAT1, mediates the influx of GABA and GABA mimetic drug substances such as vigabatrin and gaboxadol and the anticancer prodrug δ-aminolevulinic acid across the apical membrane of small intestinal enterocytes. Little is however known about the basolateral transport of these substances. We investigated basolateral transport of GABA in mature Caco-2 cell monolayers using isotope studies. Here we report that, at least two transporters seem to be involved in the basolateral transport of GABA. The basolateral uptake consisted of a high-affinity system with a K(m) of 290 μM and V(max) of 75 pmol cm(-2) min(-1) and a low affinity system with a K(m) of approximately 64 mM and V(max) of 1.6 nmol cm(-2) min(-1). The high-affinity transporter is Na(+) and Cl(-) dependent. The substrate specificity of the high-affinity transporter was further studied and Gly-Sar, Leucine, gaboxadol, sarcosine, lysine, betaine, 5-hydroxythryptophan, proline and glycine reduced the GABA uptake to approximately 44-70% of the GABA uptake in the absence of inhibitor. Other substances such as β-alanine, GABA, 5-aminovaleric acid, taurine and δ-aminolevulinic acid reduced the basolateral GABA uptake to 6-25% of the uptake in the absence of inhibitor. Our results indicate that the distance between the charged amino- and acid-groups is particular important for inhibition of basolateral GABA uptake. Thus, there seems to be a partial substrate overlap between the basolateral GABA transporter and hPAT1, which may prove important for understanding drug interactions at the level of intestinal transport. Copyright © 2012 Elsevier B.V. All rights reserved.

TBC-8, a Putative RAB-2 GAP, Regulates Dense Core Vesicle Maturation in Caenorhabditis elegans

PubMed Central

Hannemann, Mandy; Sasidharan, Nikhil; Hegermann, Jan; Kutscher, Lena M.; Koenig, Sabine; Eimer, Stefan

2012-01-01

Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2–specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation. PMID:22654674

Lipid raft dynamics linked to sperm competency for fertilization in mice.

PubMed

Watanabe, Hitomi; Takeda, Rie; Hirota, Keiji; Kondoh, Gen

2017-05-01

It is well known that mammalian sperm acquires fertilization ability after several maturation processes, particularly within the female reproductive tract. In a previous study, we found that both glycosylphosphatidylinositol (GPI)-anchored protein (GPI-AP) release and lipid raft movement occur during the sperm maturation process. In several genetic studies, release of GPI-AP is a crucial step for sperm fertilization ability in the mouse. Here, we show that lipid raft movement is also fundamental for sperm to be competent for fertilization by comparing the sperm maturation process of two mouse inbred strains, C57BL/6 and BALB/c. We found that ganglioside GM1 movement was exclusively reduced in BALB/c compared with C57BL/6 among other examined sperm maturation parameters, such as GPI-AP release, sperm migration to the oviduct, cholesterol efflux, protein tyrosine phosphorylation and acrosome reaction, and was strongly linked to sperm fertility phenotype. The relationship between GM1 movement and in vitro fertilization ability was confirmed in other mouse strains, suggesting that lipid raft movement is one of the important steps for completing the sperm maturation process. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Molecular changes and signaling events occurring in sperm during epididymal maturation

PubMed Central

Gervasi, Maria Gracia; Visconti, Pablo E.

2017-01-01

After leaving the testis, sperm have not yet acquired the ability to move progressively and are unable to fertilize oocytes. To become fertilization-competent they must go through an epididymal maturation process in the male, and capacitation in the female tract. Epididymal maturation can be defined as those changes occurring to sperm in the epididymis that render the sperm the ability to capacitate in the female tract. As part of this process, sperm cells undergo a series of biochemical and physiological changes that require incorporation of new molecules derived from the epididymal epithelium, as well as post-translational modifications of endogenous proteins synthesized during spermiogenesis in the testis. This review will focus on epididymal maturation events, with emphasis in recent advances in the understanding of the molecular basis of this process. PMID:28297559

Toward the Decision Tree for Inferring Requirements Maturation Types

NASA Astrophysics Data System (ADS)

Nakatani, Takako; Kondo, Narihito; Shirogane, Junko; Kaiya, Haruhiko; Hori, Shozo; Katamine, Keiichi

Requirements are elicited step by step during the requirements engineering (RE) process. However, some types of requirements are elicited completely after the scheduled requirements elicitation process is finished. Such a situation is regarded as problematic situation. In our study, the difficulties of eliciting various kinds of requirements is observed by components. We refer to the components as observation targets (OTs) and introduce the word “Requirements maturation.” It means when and how requirements are elicited completely in the project. The requirements maturation is discussed on physical and logical OTs. OTs Viewed from a logical viewpoint are called logical OTs, e.g. quality requirements. The requirements of physical OTs, e.g., modules, components, subsystems, etc., includes functional and non-functional requirements. They are influenced by their requesters' environmental changes, as well as developers' technical changes. In order to infer the requirements maturation period of each OT, we need to know how much these factors influence the OTs' requirements maturation. According to the observation of actual past projects, we defined the PRINCE (Pre Requirements Intelligence Net Consideration and Evaluation) model. It aims to guide developers in their observation of the requirements maturation of OTs. We quantitatively analyzed the actual cases with their requirements elicitation process and extracted essential factors that influence the requirements maturation. The results of interviews of project managers are analyzed by WEKA, a data mining system, from which the decision tree was derived. This paper introduces the PRINCE model and the category of logical OTs to be observed. The decision tree that helps developers infer the maturation type of an OT is also described. We evaluate the tree through real projects and discuss its ability to infer the requirements maturation types.

Application of Mean of Absolute Deviation Method for the Selection of Best Nonlinear Component Based on Video Encryption

NASA Astrophysics Data System (ADS)

Anees, Amir; Khan, Waqar Ahmad; Gondal, Muhammad Asif; Hussain, Iqtadar

2013-07-01

The aim of this work is to make use of the mean of absolute deviation (MAD) method for the evaluation process of substitution boxes used in the advanced encryption standard. In this paper, we use the MAD technique to analyze some popular and prevailing substitution boxes used in encryption processes. In particular, MAD is applied to advanced encryption standard (AES), affine power affine (APA), Gray, Lui J., Residue Prime, S8 AES, SKIPJACK, and Xyi substitution boxes.

Deciphering the Binding between Nupr1 and MSL1 and Their DNA-Repairing Activity

PubMed Central

Doménech, Rosa; Pantoja-Uceda, David; Gironella, Meritxell; Santoro, Jorge; Velázquez-Campoy, Adrián; Neira, José L.; Iovanna, Juan L.

2013-01-01

The stress protein Nupr1 is a highly basic, multifunctional, intrinsically disordered protein (IDP). MSL1 is a histone acetyl transferase-associated protein, known to intervene in the dosage compensation complex (DCC). In this work, we show that both Nupr1 and MSL1 proteins were recruited and formed a complex into the nucleus in response to DNA-damage, which was essential for cell survival in reply to cisplatin damage. We studied the interaction of Nupr1 and MSL1, and their binding affinities to DNA by spectroscopic and biophysical methods. The MSL1 bound to Nupr1, with a moderate affinity (2.8 µM) in an entropically-driven process. MSL1 did not bind to non-damaged DNA, but it bound to chemically-damaged-DNA with a moderate affinity (1.2 µM) also in an entropically-driven process. The Nupr1 protein bound to chemically-damaged-DNA with a slightly larger affinity (0.4 µM), but in an enthalpically-driven process. Nupr1 showed different interacting regions in the formed complexes with Nupr1 or DNA; however, they were always disordered (“fuzzy”), as shown by NMR. These results underline a stochastic description of the functionality of the Nupr1 and its other interacting partners. PMID:24205110

78 FR 5516 - Self-Regulatory Organizations; The Depository Trust Company; Notice of Filing Advance Notice To...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-01-25

... modify its Rules as they relate to the Issuing/Paying Agent's (``IPA's'') refusal to pay process. DTC is proposing not to process a reversal of a transaction initiated by an IPA when issuances of Money Market... morning for MMIs maturing that day. The automatic process electronically sweeps all maturing positions of...

Constrained Combinatorial Libraries of Gp2 Proteins Enhance Discovery of PD-L1 Binders.

PubMed

Kruziki, Max A; Sarma, Vidur; Hackel, Benjamin J

2018-06-05

Engineered protein ligands are used for molecular therapy, diagnostics, and industrial biotechnology. The Gp2 domain is a 45-amino acid scaffold that has been evolved for specific, high-affinity binding to multiple targets by diversification of two solvent-exposed loops. Inspired by sitewise enrichment of select amino acids, including cysteine pairs, in earlier Gp2 discovery campaigns, we hypothesized that the breadth and efficiency of de novo Gp2 discovery will be aided by sitewise amino acid constraint within combinatorial library design. We systematically constructed eight libraries and comparatively evaluated their efficacy for binder discovery via yeast display against a panel of targets. Conservation of a cysteine pair at the termini of the first diversified paratope loop increased binder discovery 16-fold ( p < 0.001). Yet two other libraries with conserved cysteine pairs, within the second loop or an interloop pair, did not aid discovery thereby indicating site-specific impact. Via a yeast display protease resistance assay, Gp2 variants from the loop one cysteine pair library were 3.3 ± 2.1-fold ( p = 0.005) more stable than nonconstrained variants. Sitewise constraint of noncysteine residues-guided by previously evolved binders, natural Gp2 homology, computed stability, and structural analysis-did not aid discovery. A panel of binders to programmed death ligand 1 (PD-L1), a key target in cancer immunotherapy, were discovered from the loop 1 cysteine constraint library. Affinity maturation via loop walking resulted in strong, specific cellular PD-L1 affinity ( K d = 6-9 nM).

Multiple Length Peptide-Pheromone Variants Produced by Streptococcus pyogenes Directly Bind Rgg Proteins to Confer Transcriptional Regulation*

PubMed Central

Aggarwal, Chaitanya; Jimenez, Juan Cristobal; Nanavati, Dhaval; Federle, Michael J.

2014-01-01

Streptococcus pyogenes, a human-restricted pathogen, accounts for substantial mortality related to infections worldwide. Recent studies indicate that streptococci produce and respond to several secreted peptide signaling molecules (pheromones), including those known as short hydrophobic peptides (SHPs), to regulate gene expression by a quorum-sensing mechanism. Upon transport into the bacterial cell, pheromones bind to and modulate activity of receptor proteins belonging to the Rgg family of transcription factors. Previously, we reported biofilm regulation by the Rgg2/3 quorum-sensing circuit in S. pyogenes. The aim of this study was to identify the composition of mature pheromones from cell-free culture supernatants that facilitate biofilm formation. Bioluminescent reporters were employed to detect active pheromones in culture supernatants fractionated by reverse-phase chromatography, and mass spectrometry was used to characterize their properties. Surprisingly, multiple SHPs that varied by length were detected. Synthetic peptides of each variant were tested individually using bioluminescence reporters and biofilm growth assays, and although activities differed widely among the group, peptides comprising the C-terminal eight amino acids of the full-length native peptide were most active. Direct Rgg/SHP interactions were determined using a fluorescence polarization assay that utilized FITC-labeled peptide ligands. Peptide receptor affinities were seen to be as low as 500 nm and their binding affinities directly correlated with observed bioactivity. Revelation of naturally produced pheromones along with determination of their affinity for cognate receptors are important steps forward in designing compounds whose purpose is positioned for future therapeutics aimed at treating infections through the interference of bacterial communication. PMID:24958729

[Maturity Levels of Quality and Risk Management at the University Hospital Schleswig-Holstein].

PubMed

Jussli-Melchers, Jill; Hilbert, Carsten; Jahnke, Iris; Wehkamp, Kai; Rogge, Annette; Freitag-Wolf, Sandra; Kahla-Witzsch, Heike A; Scholz, Jens; Petzina, Rainer

2018-05-16

Quality and risk management in hospitals are not only required by law but also for an optimal patient-centered and process-optimized patient care. To evaluate the maturity levels of quality and risk management at the University Hospital Schleswig-Holstein (UKSH), a structured analytical tool was developed for easy and efficient application. Four criteria concerning quality management - quality assurance (QS), critical incident reporting system (CIRS), complaint management (BM) and process management (PM) - were evaluated with a structured questionnaire. Self-assessment and external assessment were performed to classify the maturity levels at the UKSH (location Kiel and Lübeck). Every quality item was graded into four categories from "A" (fully implemented) to "D" (not implemented at all). First of all, an external assessment was initiated by the head of the department of quality and risk management. Thereafter, a self-assessment was performed by 46 clinical units of the UKSH. Discrepancies were resolved in a collegial dialogue. Based on these data, overall maturity levels were obtained for every clinical unit. The overall maturity level "A" was reached by three out of 46 (6.5%) clinical units. No unit was graded with maturity level "D". 50% out of all units reached level "B" and 43.5% level "C". The distribution of the four different quality criteria revealed a good implementation of complaint management (maturity levels "A" and "B" in 78.3%), whereas the levels for CIRS were "C" and "D" in 73.9%. Quality assurance and process management showed quite similar distributions for the levels of maturity "B" and "C" (87% QS; 91% PM). The structured analytical tool revealed maturity levels of 46 clinical units of the UKSH and defined the maturity levels of four relevant quality criteria (QS, CIRS, BM, PM). As a consequence, extensive procedures were implemented to raise the standard of quality and risk management. In future, maturity levels will be reevaluated every two years. This qualitative maturity level model enables in a simple and efficient way precise statements concerning presence, manifestation and development of quality and risk management. © Georg Thieme Verlag KG Stuttgart · New York.

Viewing pre-60S maturation at a minute’s timescale

PubMed Central

Zisser, Gertrude; Ohmayer, Uli; Mauerhofer, Christina; Mitterer, Valentin; Klein, Isabella; Rechberger, Gerald N; Wolinski, Heimo; Prattes, Michael; Pertschy, Brigitte; Milkereit, Philipp

2018-01-01

Abstract The formation of ribosomal subunits is a highly dynamic process that is initiated in the nucleus and involves more than 200 trans-acting factors, some of which accompany the pre-ribosomes into the cytoplasm and have to be recycled into the nucleus. The inhibitor diazaborine prevents cytoplasmic release and recycling of shuttling pre-60S maturation factors by inhibiting the AAA-ATPase Drg1. The failure to recycle these proteins results in their depletion in the nucleolus and halts the pathway at an early maturation step. Here, we made use of the fast onset of inhibition by diazaborine to chase the maturation path in real-time from 27SA2 pre-rRNA containing pre-ribosomes localized in the nucleolus up to nearly mature 60S subunits shortly after their export into the cytoplasm. This allows for the first time to put protein assembly and disassembly reactions as well as pre-rRNA processing into a chronological context unraveling temporal and functional linkages during ribosome maturation. PMID:29294095

Individual heterogeneity in growth and age at sexual maturity: A gamma process analysis of capture–mark–recapture data

USGS Publications Warehouse

Link, William; Hesed, Kyle Miller

2015-01-01

Knowledge of organisms’ growth rates and ages at sexual maturity is important for conservation efforts and a wide variety of studies in ecology and evolutionary biology. However, these life history parameters may be difficult to obtain from natural populations: individuals encountered may be of unknown age, information on age at sexual maturity may be uncertain and interval-censored, and growth data may include both individual heterogeneity and measurement errors. We analyzed mark–recapture data for Red-backed Salamanders (Plethodon cinereus) to compare sex-specific growth rates and ages at sexual maturity. Aging of individuals was made possible by the use of a von Bertalanffy model of growth, complemented with models for interval-censored and imperfect observations at sexual maturation. Individual heterogeneity in growth was modeled through the use of Gamma processes. Our analysis indicates that female P. cinereus mature earlier and grow more quickly than males, growing to nearly identical asymptotic size distributions as males.

High affinity ligands from in vitro selection: Complex targets

PubMed Central

Morris, Kevin N.; Jensen, Kirk B.; Julin, Carol M.; Weil, Michael; Gold, Larry

1998-01-01

Human red blood cell membranes were used as a model system to determine if the systematic evolution of ligands by exponential enrichment (SELEX) methodology, an in vitro protocol for isolating high-affinity oligonucleotides that bind specifically to virtually any single protein, could be used with a complex mixture of potential targets. Ligands to multiple targets were generated simultaneously during the selection process, and the binding affinities of these ligands for their targets are comparable to those found in similar experiments against pure targets. A secondary selection scheme, deconvolution-SELEX, facilitates rapid isolation of the ligands to targets of special interest within the mixture. SELEX provides high-affinity compounds for multiple targets in a mixture and might allow a means for dissecting complex biological systems. PMID:9501188

Three cell recognition changes accompany the ingression of sea urchin primary mesenchyme cells.

PubMed

Fink, R D; McClay, D R

1985-01-01

At gastrulation the primary mesenchyme cells of sea urchin embryos lose contact with the extracellular hyaline layer and with neighboring blastomeres as they pass through the basal lamina and enter the blastocoel. This delamination process was examined using a cell-binding assay to follow changes in affinities between mesenchyme cells and their three substrates: hyalin, early gastrula cells, and basal lamina. Sixteen-cell-stage micromeres (the precursors of primary mesenchyme cells), and mesenchyme cells obtained from mesenchyme-blastula-stage embryos were used in conjunction with micromeres raised in culture to intermediate ages. The micromeres exhibited an affinity for hyalin, but the affinity was lost at the time of mesenchyme ingression in vivo. Similarly, micromeres had an affinity for monolayers of gastrula cells but the older mesenchyme cells lost much of their cell-to-cell affinity. Presumptive ectoderm and endoderm cells tested against the gastrula monolayers showed no decrease in binding over the same time interval. When micromeres and primary mesenchyme cells were tested against basal lamina preparations, there was an increase in affinity that was associated with developmental time. Presumptive ectoderm and endoderm cells showed no change in affinity over the same interval. Binding measurements using isolated basal laminar components identified fibronectin as one molecule for which the wandering primary mesenchyme cells acquired a specific affinity. The data indicate that as the presumptive mesenchyme cells leave the vegetal plate of the embryo they lose affinities for hyalin and for neighboring cells, and gain an affinity for fibronectin associated with the basal lamina and extracellular matrix that lines the blastocoel.

Genomics of Mature and Immature Olfactory Sensory Neurons

PubMed Central

Nickell, Melissa D.; Breheny, Patrick; Stromberg, Arnold J.; McClintock, Timothy S.

2014-01-01

The continuous replacement of neurons in the olfactory epithelium provides an advantageous model for investigating neuronal differentiation and maturation. By calculating the relative enrichment of every mRNA detected in samples of mature mouse olfactory sensory neurons (OSNs), immature OSNs, and the residual population of neighboring cell types, and then comparing these ratios against the known expression patterns of >300 genes, enrichment criteria that accurately predicted the OSN expression patterns of nearly all genes were determined. We identified 847 immature OSN-specific and 691 mature OSN-specific genes. The control of gene expression by chromatin modification and transcription factors, and neurite growth, protein transport, RNA processing, cholesterol biosynthesis, and apoptosis via death domain receptors, were overrepresented biological processes in immature OSNs. Ion transport (ion channels), presynaptic functions, and cilia-specific processes were overrepresented in mature OSNs. Processes overrepresented among the genes expressed by all OSNs were protein and ion transport, ER overload response, protein catabolism, and the electron transport chain. To more accurately represent gradations in mRNA abundance and identify all genes expressed in each cell type, classification methods were used to produce probabilities of expression in each cell type for every gene. These probabilities, which identified 9,300 genes expressed in OSNs, were 96% accurate at identifying genes expressed in OSNs and 86% accurate at discriminating genes specific to mature and immature OSNs. This OSN gene database not only predicts the genes responsible for the major biological processes active in OSNs, but also identifies thousands of never before studied genes that support OSN phenotypes. PMID:22252456

Determining Roles of Accessory Genes in Denitrification by Mutant Fitness Analyses

PubMed Central

Vaccaro, Brian J.; Thorgersen, Michael P.; Lancaster, W. Andrew; Price, Morgan N.; Wetmore, Kelly M.; Poole, Farris L.; Deutschbauer, Adam; Arkin, Adam P.

2015-01-01

Enzymes of the denitrification pathway play an important role in the global nitrogen cycle, including release of nitrous oxide, an ozone-depleting greenhouse gas. In addition, nitric oxide reductase, maturation factors, and proteins associated with nitric oxide detoxification are used by pathogens to combat nitric oxide release by host immune systems. While the core reductases that catalyze the conversion of nitrate to dinitrogen are well understood at a mechanistic level, there are many peripheral proteins required for denitrification whose basic function is unclear. A bar-coded transposon DNA library from Pseudomonas stutzeri strain RCH2 was grown under denitrifying conditions, using nitrate or nitrite as an electron acceptor, and also under molybdenum limitation conditions, with nitrate as the electron acceptor. Analysis of sequencing results from these growths yielded gene fitness data for 3,307 of the 4,265 protein-encoding genes present in strain RCH2. The insights presented here contribute to our understanding of how peripheral proteins contribute to a fully functioning denitrification pathway. We propose a new low-affinity molybdate transporter, OatABC, and show that differential regulation is observed for two MoaA homologs involved in molybdenum cofactor biosynthesis. We also propose that NnrS may function as a membrane-bound NO sensor. The dominant HemN paralog involved in heme biosynthesis is identified, and a CheR homolog is proposed to function in nitrate chemotaxis. In addition, new insights are provided into nitrite reductase redundancy, nitric oxide reductase maturation, nitrous oxide reductase maturation, and regulation. PMID:26452555

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

Vaccaro, Brian J.; Thorgersen, Michael P.; Lancaster, W. Andrew

Enzymes of the denitrification pathway play an important role in the global nitrogen cycle, including release of nitrous oxide, an ozone-depleting greenhouse gas. In addition, nitric oxide reductase, maturation factors, and proteins associated with nitric oxide detoxification are used by pathogens to combat nitric oxide release by host immune systems. While the core reductases that catalyze the conversion of nitrate to dinitrogen are well understood at a mechanistic level, there are many peripheral proteins required for denitrification whose basic function is unclear. A bar-coded transposon DNA library fromPseudomonas stutzeristrain RCH2 was grown under denitrifying conditions, using nitrate or nitrite asmore » an electron acceptor, and also under molybdenum limitation conditions, with nitrate as the electron acceptor. Analysis of sequencing results from these growths yielded gene fitness data for 3,307 of the 4,265 protein-encoding genes present in strain RCH2. The insights presented here contribute to our understanding of how peripheral proteins contribute to a fully functioning denitrification pathway. We propose a new low-affinity molybdate transporter, OatABC, and show that differential regulation is observed for two MoaA homologs involved in molybdenum cofactor biosynthesis. We also propose that NnrS may function as a membrane-bound NO sensor. Finally, the dominant HemN paralog involved in heme biosynthesis is identified, and a CheR homolog is proposed to function in nitrate chemotaxis. In addition, new insights are provided into nitrite reductase redundancy, nitric oxide reductase maturation, nitrous oxide reductase maturation, and regulation.« less

Bone morphogenetic protein 9 (BMP9) controls lymphatic vessel maturation and valve formation

PubMed Central

Levet, Sandrine; Ciais, Delphine; Merdzhanova, Galina; Mallet, Christine; Zimmers, Teresa A.; Lee, Se-Jin; Navarro, Fabrice P.; Texier, Isabelle; Feige, Jean-Jacques; Bailly, Sabine

2013-01-01

Lymphatic vessels are critical for the maintenance of tissue fluid homeostasis and their dysfunction contributes to several human diseases. The activin receptor-like kinase 1 (ALK1) is a transforming growth factor-β family type 1 receptor that is expressed on both blood and lymphatic endothelial cells (LECs). Its high-affinity ligand, bone morphogenetic protein 9 (BMP9), has been shown to be critical for retinal angiogenesis. The aim of this work was to investigate whether BMP9 could play a role in lymphatic development. We found that Bmp9 deficiency in mice causes abnormal lymphatic development. Bmp9-knockout (KO) pups presented hyperplastic mesenteric collecting vessels that maintained LYVE-1 expression. In accordance with this result, we found that BMP9 inhibited LYVE-1 expression in LECs in an ALK1-dependent manner. Bmp9-KO pups also presented a significant reduction in the number and in the maturation of mesenteric lymphatic valves at embryonic day 18.5 and at postnatal days 0 and 4. Interestingly, the expression of several genes known to be involved in valve formation (Foxc2, Connexin37, EphrinB2, and Neuropilin1) was upregulated by BMP9 in LECS. Finally, we demonstrated that Bmp9-KO neonates and adult mice had decreased lymphatic draining efficiency. These data identify BMP9 as an important extracellular regulator in the maturation of the lymphatic vascular network affecting valve development and lymphatic vessel function. PMID:23741013

Over-Expression, Purification and Crystallization of Human Dihydrolipoamide Dehydrogenase

NASA Technical Reports Server (NTRS)

Hong, Y. S.; Ciszak, Ewa; Patel, Mulchand

2000-01-01

Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 1.8.1.4) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For crystallization, E3 samples were prepared with and without His-tag. To minimize the aggregation of E3, apo- and holo- forms of E3s were tested, as well as a mutated E3. Dynamic light scattering measurements revealed that the E3 preparations without His-tag and substrate are highly monodispersive with regard to homodimers. Consequent crystallization trials of this E3 preparation led to single crystals of E3 grown by the vapor diffusion method. Crystals were obtained within a few days from solution containing poly (ethylene glycol) monomethyl ether 5000 as a precipitant. Autoindexing and integration of the X-ray diffraction data showed that E3 crystals belong to an orthorhombic system with unit cell parameters a-- 123. 1, b= 165.3 and c=214.3A. Further optimization of protein preparation and crystallization experiments for the structural determination will be discussed.

SELMAP - SELEX affinity landscape MAPping of transcription factor binding sites using integrated microfluidics

PubMed Central

Chen, Dana; Orenstein, Yaron; Golodnitsky, Rada; Pellach, Michal; Avrahami, Dorit; Wachtel, Chaim; Ovadia-Shochat, Avital; Shir-Shapira, Hila; Kedmi, Adi; Juven-Gershon, Tamar; Shamir, Ron; Gerber, Doron

2016-01-01

Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities. Current methods to study sequence-specific binding preferences suffer from limited dynamic range, sequence bias, lack of specificity and limited throughput. We have developed a microfluidic-based device for SELEX Affinity Landscape MAPping (SELMAP) of TF binding, which allows high-throughput measurement of 16 proteins in parallel. We used it to measure the relative affinities of Pho4, AtERF2 and Btd full-length proteins to millions of different DNA binding sites, and detected both high and low-affinity interactions in equilibrium conditions, generating a comprehensive landscape of the relative TF affinities to all possible DNA 6-mers, and even DNA10-mers with increased sequencing depth. Low quantities of both the TFs and DNA oligomers were sufficient for obtaining high-quality results, significantly reducing experimental costs. SELMAP allows in-depth screening of hundreds of TFs, and provides a means for better understanding of the regulatory processes that govern gene expression. PMID:27628341

7 CFR 51.312 - Mature.

Code of Federal Regulations, 2013 CFR

2013-01-01

..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Apples Definitions § 51.312 Mature. “Mature” means that the apples have reached the stage of development which will insure the proper completion of the ripening process. Before a mature apple becomes overripe it will show varying degrees of firmness...

7 CFR 51.312 - Mature.

Code of Federal Regulations, 2014 CFR

2014-01-01

..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Apples Definitions § 51.312 Mature. “Mature” means that the apples have reached the stage of development which will insure the proper completion of the ripening process. Before a mature apple becomes overripe it will show varying degrees of firmness...

Properties of the [NiFe]-hydrogenase maturation protein HypD.

PubMed

Blokesch, Melanie; Böck, August

2006-07-24

A mutational screen of amino acid residues of hydrogenase maturation protein HypD from Escherichia coli disclosed that seven conserved cysteine residues located in three different motifs in HypD are essential. Evidence is presented for potential functions of these motifs in the maturation process.

Simultaneous measurements of Cotton fiber maturity, fineness, ribbon width, and micronaire

USDA-ARS?s Scientific Manuscript database

Maturity (degree of secondary wall development) and fineness (linear density) are important cotton quality and processing properties, but their direct measurement is often difficult and/or expensive to perform. An indirect but critical measurement of maturity and fineness is micronaire, which is on...

Functional Interactions between Newborn and Mature Neurons Leading to Integration into Established Neuronal Circuits.

PubMed

Boulanger-Weill, Jonathan; Candat, Virginie; Jouary, Adrien; Romano, Sebastián A; Pérez-Schuster, Verónica; Sumbre, Germán

2017-06-19

From development up to adulthood, the vertebrate brain is continuously supplied with newborn neurons that integrate into established mature circuits. However, how this process is coordinated during development remains unclear. Using two-photon imaging, GCaMP5 transgenic zebrafish larvae, and sparse electroporation in the larva's optic tectum, we monitored spontaneous and induced activity of large neuronal populations containing newborn and functionally mature neurons. We observed that the maturation of newborn neurons is a 4-day process. Initially, newborn neurons showed undeveloped dendritic arbors, no neurotransmitter identity, and were unresponsive to visual stimulation, although they displayed spontaneous calcium transients. Later on, newborn-labeled neurons began to respond to visual stimuli but in a very variable manner. At the end of the maturation period, newborn-labeled neurons exhibited visual tuning curves (spatial receptive fields and direction selectivity) and spontaneous correlated activity with neighboring functionally mature neurons. At this developmental stage, newborn-labeled neurons presented complex dendritic arbors and neurotransmitter identity (excitatory or inhibitory). Removal of retinal inputs significantly perturbed the integration of newborn neurons into the functionally mature tectal network. Our results provide a comprehensive description of the maturation of newborn neurons during development and shed light on potential mechanisms underlying their integration into a functionally mature neuronal circuit. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Assessing spectral and temporal processing in children and adults using temporal modulation transfer function (TMTF), Iterated Ripple Noise (IRN) perception, and spectral ripple discrimination (SRD).

PubMed

Peter, Varghese; Wong, Kogo; Narne, Vijaya Kumar; Sharma, Mridula; Purdy, Suzanne C; McMahon, Catherine

2014-02-01

There are many clinically available tests for the assessment of auditory processing skills in children and adults. However, there is limited data available on the maturational effects on the performance on these tests. The current study investigated maturational effects on auditory processing abilities using three psychophysical measures: temporal modulation transfer function (TMTF), iterated ripple noise (IRN) perception, and spectral ripple discrimination (SRD). A cross-sectional study. Three groups of subjects were tested: 10 adults (18-30 yr), 10 older children (12-18 yr), and 10 young children (8-11 yr) Temporal envelope processing was measured by obtaining thresholds for amplitude modulation detection as a function of modulation frequency (TMTF; 4, 8, 16, 32, 64, and 128 Hz). Temporal fine structure processing was measured using IRN, and spectral processing was measured using SRD. The results showed that young children had significantly higher modulation thresholds at 4 Hz (TMTF) compared to the other two groups and poorer SRD scores compared to adults. The results on IRN did not differ across groups. The results suggest that different aspects of auditory processing mature at different age periods and these maturational effects need to be considered while assessing auditory processing in children. American Academy of Audiology.

The roles of family B and D DNA polymerases in Thermococcus species 9°N Okazaki fragment maturation.

PubMed

Greenough, Lucia; Kelman, Zvi; Gardner, Andrew F

2015-05-15

During replication, Okazaki fragment maturation is a fundamental process that joins discontinuously synthesized DNA fragments into a contiguous lagging strand. Efficient maturation prevents repeat sequence expansions, small duplications, and generation of double-stranded DNA breaks. To address the components required for the process in Thermococcus, Okazaki fragment maturation was reconstituted in vitro using purified proteins from Thermococcus species 9°N or cell extracts. A dual color fluorescence assay was developed to monitor reaction substrates, intermediates, and products. DNA polymerase D (polD) was proposed to function as the replicative polymerase in Thermococcus replicating both the leading and the lagging strands. It is shown here, however, that it stops before the previous Okazaki fragments, failing to rapidly process them. Instead, Family B DNA polymerase (polB) was observed to rapidly fill the gaps left by polD and displaces the downstream Okazaki fragment to create a flap structure. This flap structure was cleaved by flap endonuclease 1 (Fen1) and the resultant nick was ligated by DNA ligase to form a mature lagging strand. The similarities to both bacterial and eukaryotic systems and evolutionary implications of archaeal Okazaki fragment maturation are discussed. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Biosynthesis and intracellular movement of the melanosomal membrane glycoprotein gp75, the human b (brown) locus product

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

Vijayasaradhi, S.; Doskoch, P.M.; Houghton, A.N.

1991-10-01

A 75-kDa melanosomal glycoprotein (gp75) is the product of a gene that maps to the b (brown) locus, a genetic locus that determines coat color in the mouse. The b locus is conserved (88% identity) between mouse and human. The mouse monoclonal antibody TA99 was used to study the biosynthesis and processing of gp75. gp75 was synthesized as a 55-kDa polypeptide, glycosylated by addition and processing of five or more Asn-linked carbohydrate chains through the cis and trans Golgi, and transported to melanosomes as a mature 75-kDa form. Synthesis and processing of gp75 was rapid (T{sub 1/2} < 30 min),more » and early steps in processing were required for efficient export of gp75 was quite stable in the melanosome. Studies with inhibitors of steps in oligosaccharide processing showed that alternative forms of gp75 were generated during trimming reactions by mannosidase IA/IB and that further maturation resulted in the two mature forms of gp75. The authors purpose that the kinetics of biosynthesis and processing reflect events in the biogenesis and maturation of melanosomes.« less

An Environmental Management Maturity Model of Construction Programs Using the AHP-Entropy Approach.

PubMed

Bai, Libiao; Wang, Hailing; Huang, Ning; Du, Qiang; Huang, Youdan

2018-06-23

The accelerating process of urbanization in China has led to considerable opportunities for the development of construction projects, however, environmental issues have become an important constraint on the implementation of these projects. To quantitatively describe the environmental management capabilities of such projects, this paper proposes a 2-dimensional Environmental Management Maturity Model of Construction Program (EMMMCP) based on an analysis of existing projects, group management theory and a management maturity model. In this model, a synergetic process was included to compensate for the lack of consideration of synergies in previous studies, and it was involved in the construction of the first dimension, i.e., the environmental management index system. The second dimension, i.e., the maturity level of environment management, was then constructed by redefining the hierarchical characteristics of construction program (CP) environmental management maturity. Additionally, a mathematical solution to this proposed model was derived via the Analytic Hierarchy Process (AHP)-entropy approach. To verify the effectiveness and feasibility of this proposed model, a computational experiment was conducted, and the results show that this approach could not only measure the individual levels of different processes, but also achieve the most important objective of providing a reference for stakeholders when making decisions on the environmental management of construction program, which reflects this model is reasonable for evaluating the level of environmental management maturity in CP. To our knowledge, this paper is the first study to evaluate the environmental management maturity levels of CP, which would fill the gap between project program management and environmental management and provide a reference for relevant management personnel to enhance their environmental management capabilities.

Genomic profiling of bovine corpus luteum maturation

PubMed Central

Wigoda, Noa; Ben-Dor, Shifra; Orr, Irit; Meidan, Rina

2018-01-01

To unveil novel global changes associated with corpus luteum (CL) maturation, we analyzed transcriptome data for the bovine CL on days 4 and 11, representing the developing vs. mature gland. Our analyses revealed 681 differentially expressed genes (363 and 318 on day 4 and 11, respectively), with ≥2 fold change and FDR of <5%. Different gene ontology (GO) categories were represented prominently in transcriptome data at these stages (e.g. days 4: cell cycle, chromosome, DNA metabolic process and replication and on day 11: immune response; lipid metabolic process and complement activation). Based on bioinformatic analyses, select genes expression in day 4 and 11 CL was validated with quantitative real-time PCR. Cell specific expression was also determined in enriched luteal endothelial and steroidogenic cells. Genes related to the angiogenic process such as NOS3, which maintains dilated vessels and MMP9, matrix degrading enzyme, were higher on day 4. Importantly, our data suggests day 11 CL acquire mechanisms to prevent blood vessel sprouting and promote their maturation by expressing NOTCH4 and JAG1, greatly enriched in luteal endothelial cells. Another endothelial specific gene, CD300LG, was identified here in the CL for the first time. CD300LG is an adhesion molecule enabling lymphocyte migration, its higher levels at mid cycle are expected to support the transmigration of immune cells into the CL at this stage. Together with steroidogenic genes, most of the genes regulating de-novo cholesterol biosynthetic pathway (e.g HMGCS, HMGCR) and cholesterol uptake from plasma (LDLR, APOD and APOE) were upregulated in the mature CL. These findings provide new insight of the processes involved in CL maturation including blood vessel growth and stabilization, leucocyte transmigration as well as progesterone synthesis as the CL matures. PMID:29590145

Organo luminescent semiconductor nanocrystal probes for biological applications and process for making and using such probes

DOEpatents

Weiss, Shimon [Pinole, CA; Bruchez, Jr., Marcel; Alivisatos, Paul [Oakland, CA

2008-01-01

A semiconductor nanocrystal compound is described capable of linking to an affinity molecule. The compound comprises (1) a semiconductor nanocrystal capable of emitting electromagnetic radiation and/or absorbing energy, and/or scattering or diffracting electromagnetic radiation--when excited by an electromagnetic radiation source or a particle beam; and (2) an affinity molecule linked to the semiconductor nanocrystal. The semiconductor nanocrystal is linked to an affinity molecule to form a semiconductor nanocrystal probe capable of bonding with a detectable substance. Exposure of the semiconductor nanocrystal to excitation energy will excite the semiconductor nanocrystal causing the emission of electromagnetic radiation. Further described are processes for respectively: making the luminescent semiconductor nanocrystal compound; making the semiconductor nanocrystal probe; and using the probe to determine the presence of a detectable substance in a material.

Mature Students Speak Up: Career Exploration and the Working Alliance

ERIC Educational Resources Information Center

Pott, Terilyn

2015-01-01

This exploratory study was undertaken to learn more about how mature students perceive the career counselling process in a post-secondary institution. Through the use of critical incident technique this study examined how three mature students interpret their relationship between themselves and their counsellors. Significant factors identified as…

Physical abrasion of mafic minerals and basalt grains: application to Martian aeolian deposits

USGS Publications Warehouse

Cornwall, Carin; Bandfield, Joshua L.; Titus, Timothy N.; Schreiber, B. C.; Montgomery, D.R.

2015-01-01

Sediment maturity, or the mineralogical and physical characterization of sediment deposits, has been used to locate sediment source, transport medium and distance, weathering processes, and paleoenvironments on Earth. Mature terrestrial sands are dominated by quartz, which is abundant in source lithologies on Earth and is physically and chemically stable under a wide range of conditions. Immature sands, such as those rich in feldspars or mafic minerals, are composed of grains that are easily physically weathered and highly susceptible to chemical weathering. On Mars, which is predominately mafic in composition, terrestrial standards of sediment maturity are not applicable. In addition, the martian climate today is cold, dry and sediments are likely to be heavily influenced by physical weathering rather than chemical weathering. Due to these large differences in weathering processes and composition, martian sediments require an alternate maturity index. Abrason tests have been conducted on a variety of mafic materials and results suggest that mature martian sediments may be composed of well sorted, well rounded, spherical basalt grains. In addition, any volcanic glass present is likely to persist in a mechanical weathering environment while chemically altered products are likely to be winnowed away. A modified sediment maturity index is proposed that can be used in future studies to constrain sediment source, paleoclimate, mechanisms for sediment production, and surface evolution. This maturity index may also provide details about erosional and sediment transport systems and preservation processes of layered deposits.

Effects of inhibitors on 1-methyladenine induced maturation of starfish oocytes

NASA Astrophysics Data System (ADS)

Lee, Harold H.; Xu, Quanhan

1986-12-01

1-methladenine (1-MA) induces starfish oocytes maturation via surface reaction followed by the appearance of a cytoplasmic maturation factor which in turn induces germinal vesicle breakdown (GVBD) to resume meiosis. Cellular mechanisms involved in GVBD were investigated by microinjection of metabolic inhibitors. Colchicine (Co) inhibited maturation, cytochalasin-B (CB) delayed GVBD and actinomycin-D-(Act-D) and puromycin (Pu) had no effect. It appears that the microtubule and the microfilament systems are associated with the nuclear membrane dissolution during the process of oocyte maturation of starfish.

IFN-γ regulates CD8+ memory T cell differentiation and survival in response to weak, but not strong, TCR signals.

PubMed

Stoycheva, Diana; Deiser, Katrin; Stärck, Lilian; Nishanth, Gopala; Schlüter, Dirk; Uckert, Wolfgang; Schüler, Thomas

2015-01-15

In response to primary Ag contact, naive mouse CD8(+) T cells undergo clonal expansion and differentiate into effector T cells. After pathogen clearance, most effector T cells die, and only a small number of memory T cell precursors (TMPs) survive to form a pool of long-lived memory T cells (TMs). Although high- and low-affinity CD8(+) T cell clones are recruited into the primary response, the TM pool consists mainly of high-affinity clones. It remains unclear whether the more efficient expansion of high-affinity clones and/or cell-intrinsic processes exclude low-affinity T cells from the TM pool. In this article, we show that the lack of IFN-γR signaling in CD8(+) T cells promotes TM formation in response to weak, but not strong, TCR agonists. The IFN-γ-sensitive accumulation of TMs correlates with reduced mammalian target of rapamycin activation and the accumulation of long-lived CD62L(hi)Bcl-2(hi)Eomes(hi) TMPs. Reconstitution of mammalian target of rapamycin or IFN-γR signaling is sufficient to block this process. Hence, our data suggest that IFN-γR signaling actively blocks the formation of TMPs responding to weak TCR agonists, thereby promoting the accumulation of high-affinity T cells finally dominating the TM pool. Copyright © 2015 by The American Association of Immunologists, Inc.

Localization of trefoil factor family peptide 3 (TFF3) in epithelial tissues originating from the three germ layers of developing mouse embryo.

PubMed

Bijelić, Nikola; Belovari, Tatjana; Tolušić Levak, Maja; Baus Lončar, Mirela

2017-08-20

Trefoil factor family (TFF) peptides are involved in the maintenance of epithelial integrity and epithelial restitution. Mature epithelial tissues originate from different embryonic germ layers. The objective of this research was to explore the presence and localization of TFF3 peptide in mouse embryonic epithelia and to examine if the occurrence of TFF3 peptide is germ layer-dependent. Mouse embryos (14-18 days old) were fixed in 4% paraformaldehyde and embedded in paraffin. Immunohistochemistry was performed with affinity purified rabbit anti-TFF3 antibody, goat anti-rabbit biotinylated secondary antibody and streptavidin-horseradish peroxidase, followed by 3,3'-diaminobenzidine. TFF3 peptide was present in the gastric and intestinal mucosa, respiratory mucosa in the upper and lower airways, pancreas, kidney tubules, epidermis, and oral cavity. The presence and localization of TFF3 peptide was associated with the embryonic stage and tissue differentiation. TFF3 peptide distribution specific to the germ layers was not observed. The role of TFF3 peptide in cell migration and differentiation, immune response, and apoptosis might be associated with specific embryonic epithelial cells. TFF3 peptide may also be considered as a marker for mucosal maturation.

AcsF Catalyzes the ATP-dependent Insertion of Nickel into the Ni,Ni-[4Fe4S] Cluster of Acetyl-CoA Synthase*

PubMed Central

Gregg, Christina M.; Goetzl, Sebastian; Jeoung, Jae-Hun

2016-01-01

Acetyl-CoA synthase (ACS) catalyzes the reversible condensation of CO, CoA, and a methyl-cation to form acetyl-CoA at a unique Ni,Ni-[4Fe4S] cluster (the A-cluster). However, it was unknown which proteins support the assembly of the A-cluster. We analyzed the product of a gene from the cluster containing the ACS gene, cooC2 from Carboxydothermus hydrogenoformans, named AcsFCh, and showed that it acts as a maturation factor of ACS. AcsFCh and inactive ACS form a stable 2:1 complex that binds two nickel ions with higher affinity than the individual components. The nickel-bound ACS-AcsFCh complex remains inactive until MgATP is added, thereby converting inactive to active ACS. AcsFCh is a MinD-type ATPase and belongs to the CooC protein family, which can be divided into homologous subgroups. We propose that proteins of one subgroup are responsible for assembling the Ni,Ni-[4Fe4S] cluster of ACS, whereas proteins of a second subgroup mature the [Ni4Fe4S] cluster of carbon monoxide dehydrogenases. PMID:27382049

Alternative Affinity Ligands for Immunoglobulins.

PubMed

Kruljec, Nika; Bratkovič, Tomaž

2017-08-16

The demand for recombinant therapeutic antibodies and Fc-fusion proteins is expected to increase in the years to come. Hence, extensive efforts are concentrated on improving the downstream processing. In particular, the development of better-affinity chromatography matrices, supporting robust time- and cost-effective antibody purification, is warranted. With the advances in molecular design and high-throughput screening approaches from chemical and biological combinatorial libraries, novel affinity ligands representing alternatives to bacterial immunoglobulin (Ig)-binding proteins have entered the scene. Here, we review the design, development, and properties of diverse classes of alternative antibody-binding ligands, ranging from engineered versions of Ig-binding proteins, to artificial binding proteins, peptides, aptamers, and synthetic small-molecular-weight compounds. We also provide examples of applications for the novel affinity matrices in chromatography and beyond.

Defective postsecretory maturation of MUC5B mucin in cystic fibrosis airways

PubMed Central

Abdullah, Lubna H.; Evans, Jessica R.; Wang, T. Tiffany; Ford, Amina A.; Makhov, Alexander M.; Nguyen, Kristine; Coakley, Raymond D.; Griffith, Jack D.; Davis, C. William; Ballard, Stephen T.

2017-01-01

In cystic fibrosis (CF), airway mucus becomes thick and viscous, and its clearance from the airways is impaired. The gel-forming mucins undergo an ordered “unpacking/maturation” process after granular release that requires an optimum postsecretory environment, including hydration and pH. We hypothesized that this unpacking process is compromised in the CF lung due to abnormal transepithelial fluid transport that reduces airway surface hydration and alters ionic composition. Using human tracheobronchial epithelial cells derived from non-CF and CF donors and mucus samples from human subjects and domestic pigs, we investigated the process of postsecretory mucin unfolding/maturation, how these processes are defective in CF airways, and the probable mechanism underlying defective unfolding. First, we found that mucins released into a normal lung environment transform from a compact granular form to a linear form. Second, we demonstrated that this maturation process is defective in the CF airway environment. Finally, we demonstrated that independent of HCO3− and pH levels, airway surface dehydration was the major determinant of this abnormal unfolding process. This defective unfolding/maturation process after granular release suggests that the CF extracellular environment is ion/water depleted and likely contributes to abnormal mucus properties in CF airways prior to infection and inflammation. PMID:28352653

Precursor processing for plant peptide hormone maturation by subtilisin-like serine proteinases.

PubMed

Schardon, Katharina; Hohl, Mathias; Graff, Lucile; Pfannstiel, Jens; Schulze, Waltraud; Stintzi, Annick; Schaller, Andreas

2016-12-23

Peptide hormones that regulate plant growth and development are derived from larger precursor proteins by proteolytic processing. Our study addressed the role of subtilisin-like proteinases (SBTs) in this process. Using tissue-specific expression of proteinase inhibitors as a tool to overcome functional redundancy, we found that SBT activity was required for the maturation of IDA (INFLORESCENCE DEFICIENT IN ABSCISSION), a peptide signal for the abscission of floral organs in Arabidopsis We identified three SBTs that process the IDA precursor in vitro, and this processing was shown to be required for the formation of mIDA (the mature and bioactive form of IDA) as the endogenous signaling peptide in vivo. Hence, SBTs act as prohormone convertases in plants, and several functionally redundant SBTs contribute to signal biogenesis. Copyright © 2016, American Association for the Advancement of Science.

Fault detection for piecewise affine systems with application to ship propulsion systems.

PubMed

Yang, Ying; Linlin, Li; Ding, Steven X; Qiu, Jianbin; Peng, Kaixiang

2017-09-09

In this paper, the design approach of non-synchronized diagnostic observer-based fault detection (FD) systems is investigated for piecewise affine processes via continuous piecewise Lyapunov functions. Considering that the dynamics of piecewise affine systems in different regions can be considerably different, the weighting matrices are used to weight the residual of each region, so as to optimize the fault detectability. A numerical example and a case study on a ship propulsion system are presented in the end to demonstrate the effectiveness of the proposed results. Copyright © 2017 ISA. Published by Elsevier Ltd. All rights reserved.

Recognition of genetically modified product based on affinity propagation clustering and terahertz spectroscopy

NASA Astrophysics Data System (ADS)

Liu, Jianjun; Kan, Jianquan

2018-04-01

In this paper, based on the terahertz spectrum, a new identification method of genetically modified material by support vector machine (SVM) based on affinity propagation clustering is proposed. This algorithm mainly uses affinity propagation clustering algorithm to make cluster analysis and labeling on unlabeled training samples, and in the iterative process, the existing SVM training data are continuously updated, when establishing the identification model, it does not need to manually label the training samples, thus, the error caused by the human labeled samples is reduced, and the identification accuracy of the model is greatly improved.

Analysis of epididymal sperm maturation by MALDI profiling and top-down mass spectrometry.

PubMed

Labas, Valérie; Spina, Lucie; Belleannee, Clémence; Teixeira-Gomes, Ana-Paula; Gargaros, Audrey; Dacheux, Françoise; Dacheux, Jean-Louis

2015-01-15

The fertilization ability of male gametes is achieved after their transit through the epididymis where important post-gonadal differentiation occurs in different cellular compartments. Most of these maturational modifications occur at the protein level. The epididymal sperm maturation process was investigated using the ICM-MS (Intact Cell MALDI-TOF MS) approach on boar spermatozoa isolated from four different epididymal regions (immature to mature stage). Differential and quantitative MALDI-TOF profiling for whole cells or sub-cellular fractions was combined with targeted top-down MS in order to identify endogenous biomolecules. Using this approach, 172m/z peaks ranging between 2 and 20kDa were found to be modified during maturation of sperm. Using top-down MS, 62m/z were identified corresponding to peptidoforms/proteoforms with post-translational modifications (MS data are available via ProteomeXchange with identifier PXD001303). Many of the endogenous peptides were characterized as N-, C-terminal sequences or internal fragments of proteins presenting specific cleavages, suggesting the presence of sequential protease activities in the spermatozoa. This is the first time that such proteolytic activities could be evidenced for various sperm proteins through quantification of their proteolytic products. ICM-MS/top-down MS thus proved to be a valid approach for peptidome/degradome studies and provided new contributions to understanding of the maturation process of the male gamete involved in the development of male fertility. This peptidomic study (i) characterized the peptidome of epididymal spermatozoa from boar (Sus scrofa); (ii) established characteristic molecular phenotypes distinguishing degrees of maturation of spermatozoa during epididymal transit, and (iii) revealed that protease activities were at the origin of numerous peptides from known and unknown proteins involved in sperm maturation and/or fertility processes. Copyright © 2014 Elsevier B.V. All rights reserved.

Risk Quantification of Systems Engineering Documents Improves Probability of DOD Project Success

DTIC Science & Technology

2009-09-01

comprehensive risk model for DoD milestone review documentation as well as recommended changes to the Capability Maturity Model Integration ( CMMI ) Project...Milestone Documentation, Project Planning, Rational Frame, Political Frame, CMMI Project Planning Process Area, CMMI Risk Management Process Area...well as recommended changes to the Capability Maturity Model Integration ( CMMI ) Project Planning and Risk Management process areas. The intent is to

Additive effect of multiple pharmacological chaperones on maturation of CFTR processing mutants

PubMed Central

Wang, Ying; Loo, Tip W.; Bartlett, M. Claire; Clarke, David M.

2007-01-01

The most common cause of CF (cystic fibrosis) is the deletion of Phe508 (ΔF508) in the CFTR [CF TM (transmembrane) conductance regulator] chloride channel. One major problem with ΔF508 CFTR is that the protein is defective in folding so that little mature protein is delivered to the cell surface. Expression of ΔF508 CFTR in the presence of small molecules known as correctors or pharmacological chaperones can increase the level of mature protein. Unfortunately, the efficiency of corrector-induced maturation of ΔF508 CFTR is probably too low to have therapeutic value and approaches are needed to increase maturation efficiency. We postulated that expression of ΔF508 CFTR in the presence of multiple correctors that bound to different sites may have an additive effect on maturation. In support of this mechanism, we found that expression of P-glycoprotein (CFTR's sister protein) processing mutants in the presence of two compounds that bind to different sites (rhodamine B and Hoechst 33342) had an additive effect on maturation. Therefore we tested whether expression of ΔF508 CFTR in the presence of combinations of three different classes of corrector molecules would increase its maturation efficiency. It was found that the combination of the quinazoline VRT-325 together with the thiazole corr-2b or bisaminomethylbithiazole corr-4a doubled the steady-state maturation efficiency of ΔF508 CFTR (approx. 40% of total CFTR was mature protein) compared with expression in the presence of a single compound. The additive effect of the correctors on ΔF508 CFTR maturation suggests that they directly interact at different sites of the protein. PMID:17535157

Measuring the electron affinity of organic solids: an indispensable new tool for organic electronics.

PubMed

Yoshida, Hiroyuki

2014-04-01

Electron affinity is a fundamental energy parameter of materials. In organic semiconductors, the electron affinity is closely related to electron conduction. It is not only important to understand fundamental electronic processes in organic solids, but it is also indispensable for research and development of organic semiconductor devices such as organic light-emitting diodes and organic photovoltaic cells. However, there has been no experimental technique for examining the electron affinity of organic materials that meets the requirements of such research. Recently, a new method, called low-energy inverse-photoemission spectroscopy, has been developed. A beam of low-energy electrons is focused onto the sample surface, and photons emitted owing to the radiative transition to unoccupied states are then detected. From the onset of the spectral intensity, the electron affinity is determined within an uncertainty of 0.1 eV. Unlike in conventional inverse-photoemission spectroscopy, sample damage is negligible and the resolution is improved by a factor of 2. The principle of the method and several applications are reported.

A dynamic T cell–limited checkpoint regulates affinity-dependent B cell entry into the germinal center

PubMed Central

Schwickert, Tanja A.; Victora, Gabriel D.; Fooksman, David R.; Kamphorst, Alice O.; Mugnier, Monica R.; Gitlin, Alexander D.; Dustin, Michael L.

2011-01-01

The germinal center (GC) reaction is essential for the generation of the somatically hypermutated, high-affinity antibodies that mediate adaptive immunity. Entry into the GC is limited to a small number of B cell clones; however, the process by which this limited number of clones is selected is unclear. In this study, we demonstrate that low-affinity B cells intrinsically capable of seeding a GC reaction fail to expand and become activated in the presence of higher-affinity B cells even before GC coalescence. Live multiphoton imaging shows that selection is based on the amount of peptide–major histocompatibility complex (pMHC) presented to cognate T cells within clusters of responding B and T cells at the T–B border. We propose a model in which T cell help is restricted to the B cells with the highest amounts of pMHC, thus allowing for a dynamic affinity threshold to be imposed on antigen-binding B cells. PMID:21576382

Nanoparticle Addition to Enhance the Mechanical Response of Magnesium Alloys Including Nanoscale Deformation Mechanisms

NASA Astrophysics Data System (ADS)

Paramsothy, Muralidharan; Gupta, Manoj

In this study, various magnesium alloy nanocomposites derived from AZ (Aluminium-Zinc) or ZK (Zinc-Zirconium) series matrices and containing Al2O3, Si3N4, TiC or carbon nanotube (CNT) nanoparticle reinforcement (representative oxide, nitride, carbide or carbon nanoparticle reinforcement, respectively) were fabricated using solidification processing followed by hot extrusion. The main aim here was to simultaneously enhance tensile strength and ductility of each alloy using nanoparticles. The magnesium-oxygen strong affinity and magnesium-carbon weak affinity (comparison of extremes in affinity) are both well known in the context of magnesium composite processing. However, an approach to possibly quantify this affinity in magnesium nanocomposite processing is not clear. In this study accordingly, Nanoscale Electro Negative Interface Density or NENID quantifies the nanoparticle-alloy matrix interfacial area per unit volume in the magnesium alloy nanocomposite taking into consideration the electronegativity of the nanoparticle reinforcement. The beneficial (as well as comparative) effect of the nanoparticles on each alloy is discussed in this article. Regarding the mechanical performance of the nanocomposites, it is important to understand the experimentally observed nanoparticle-matrix interactions during plastic deformation (nanoscale deformation mechanisms). Little is known in this area based on direct observations for metal matrix nanocomposites. Here, relevant multiple nanoscale phenomena includes the emanation of high strain zones (HSZs) from nanoparticle surfaces.

Organo Luminescent semiconductor nanocrystal probes for biological applications and process for making and using such probes

DOEpatents

Weiss, Shimon; Bruchez, Jr., Marcel; Alivisatos, Paul

1999-01-01

A luminescent semiconductor nanocrystal compound is described which is capable of linking to an affinity molecule. The compound comprises (1) a semiconductor nanocrystal capable of emitting electromagnetic radiation (luminescing) in a narrow wavelength band and/or absorbing energy, and/or scattering or diffracting electromagnetic radiation--when excited by an electromagnetic radiation source (of narrow or broad bandwidth) or a particle beam; and (2) at least one linking agent, having a first portion linked to the semiconductor nanocrystal and a second portion capable of linking to an affinity molecule. The luminescent semiconductor nanocrystal compound is linked to an affinity molecule to form an organo luminescent semiconductor nanocrystal probe capable of bonding with a detectable substance in a material being analyzed, and capable of emitting electromagnetic radiation in a narrow wavelength band and/or absorbing, scattering, or diffracting energy when excited by an electromagnetic radiation source (of narrow or broad bandwidth) or a particle beam. The probe is stable to repeated exposure to light in the presence of oxygen and/or other radicals. Further described is a process for making the luminescent semiconductor nanocrystal compound and for making the organo luminescent semiconductor nanocrystal probe comprising the luminescent semiconductor nanocrystal compound linked to an affinity molecule capable of bonding to a detectable substance. A process is also described for using the probe to determine the presence of a detectable substance in a material.

Spiritual Maturation and Religious Behaviors in Christian University Students

ERIC Educational Resources Information Center

Welch, Ronald D.; Mellberg, Kimberlee

2008-01-01

Spiritual maturation processes of internalization and questing were assessed at a Christian university to determine their relationship to year in school and certain religious behaviors. This was a first step toward the development of a new model of Christian higher education that will intentionally facilitate spiritual maturation. A group of 179…

Cone and Seed Maturation of Southern Pines

Treesearch

James P. Barnett

1976-01-01

If slightly reduced yields and viability are acceptable, loblolly and slash cone collections can begin 2 to 3 weeks before maturity if the cones are stored before processing. Longleaf(P. palestris Mill.) pine cones should be collected only when mature, as storage decreased germination of seeds from immature cones. Biochemical analyses to determine reducing sugar...

Pedagogical Formation Students' Resistance Behaviors towards Teaching-Learning Processes and Their Moral Maturity

ERIC Educational Resources Information Center

Argon, Türkan; Sezen-Gültekin, Gözde

2016-01-01

Moral maturity, defined as the competence in moral emotions, thoughts, judgments, attitudes and behaviors, is one of the most important qualities that the would-be teachers at Faculties of Education must possess. Teachers with moral maturity will train students with the qualities of reliability, responsibility, fairness, objectivity, consistency…

A review of factors influencing maturation of atlantic salmon salmo salar with focus on water recirculation aquaculture system environments

USDA-ARS?s Scientific Manuscript database

Maturation of Atlantic salmon Salmo salar is an extremely complex process, particularly in aquaculture systems, with many variables (known or otherwise) having the capacity to influence the timing and prevalence of maturation, and acting as promoters and/or inhibitors of sexual development. The vast...

Laboratory and outside the laboratory measurements of ginned and ot ginned cotton for fiber micronaire and maturity by portable Near Infrared (NIR) Instruments

USDA-ARS?s Scientific Manuscript database

Micronaire is a key cotton quality assessment property, impacting downstream fiber processing and dye consistency. A component of micronaire is fiber maturity (degree of secondary wall development). Historically, micronaire and maturity are measured in a laboratory under tight environmental condit...

A Maturity Model for Assessing the Use of ICT in School Education

ERIC Educational Resources Information Center

Solar, Mauricio; Sabattin, Jorge; Parada, Victor

2013-01-01

This article describes an ICT-based and capability-driven model for assessing ICT in education capabilities and maturity of schools. The proposed model, called ICTE-MM (ICT in School Education Maturity Model), has three elements supporting educational processes: information criteria, ICT resources, and leverage domains. Changing the traditional…

U.s. Geological survey core drilling on the atlantic shelf.

PubMed

Hathaway, J C; Poag, C W; Valentine, P C; Manheim, F T; Kohout, F A; Bothner, M H; Miller, R E; Schultz, D M; Sangrey, D A

1979-11-02

The first broad program of scientific shallow drilling on the U.S. Atlantic continental shelf has delineated rocks of Pleistocene to Late Cretaceous age, including phosphoritic Miocene strata, widespread Eocene carbonate deposits that serve as reflective seismic markers, and several regional unconformities. Two sites, off Maryland and New Jersey, showed light hydrocarbon gases having affinity to mature petroleum. Pore fluid studies showed that relatively fresh to brackish water occurs beneath much of the Atlantic continental shelf, whereas increases in salinity off Georgla and beneath the Florida-Hatteras slope suggest buried evaporitic strata. The sediment cores showed engineering properties that range from good foundation strength to a potential for severe loss of strength through interaction between sediments and man-made structures.

How Follicular Dendritic Cells Shape the B-Cell Antigenome

PubMed Central

Kranich, Jan; Krautler, Nike Julia

2016-01-01

Follicular dendritic cells (FDCs) are stromal cells residing in primary follicles and in germinal centers of secondary and tertiary lymphoid organs (SLOs and TLOs). There, they play a crucial role in B-cell activation and affinity maturation of antibodies. FDCs have the unique capacity to bind and retain native antigen in B-cell follicles for long periods of time. Therefore, FDCs shape the B-cell antigenome (the sum of all B-cell antigens) in SLOs and TLOs. In this review, we discuss recent findings that explain how this stromal cell type can arise in almost any tissue during TLO formation and, furthermore, focus on the mechanisms of antigen capture and retention involved in the generation of long-lasting antigen depots displayed on FDCs. PMID:27446069

Cytoskeletal changes in oocytes and early embryos during in vitro fertilization process in mice.

PubMed

Gumus, E; Bulut, H E; Kaloglu, C

2010-02-01

The cytoskeleton plays crucial roles in the development and fertilization of germ cells and in the early embryo development. The growth, maturation and fertilization of oocytes require an active movement and a correct localization of cellular organelles. This is performed by the re-organization of microtubules and actin filaments. Therefore, the aim of the present study was to determine the changes in cytoskeleton during in vitro fertilization process using appropriate immunofluorescence techniques. While the chromatin content was found to be scattered throughout the nucleus during the oocyte maturation period, it was seen only around nucleolus following the completion of the maturation. Microtubules, during oocyte maturation, were regularly distributed throughout the ooplasm which was then localized in the subcortical region of oocytes. Similarly microfilaments were scattered throughout the ooplasm during the oocyte maturation period whereas they were seen in the subcortical region around the polar body and above the meiotic spindle throughout the late developmental stages. In conclusion, those changes occurred in microtubules and microfilaments might be closely related to the re-organization of the genetic material during the oocyte maturation and early embryo development.

MHC drives TCR repertoire shaping, but not maturation, in recent thymic emigrants.

PubMed

Houston, Evan G; Fink, Pamela J

2009-12-01

After developing in the thymus, recent thymic emigrants (RTEs) enter the lymphoid periphery and undergo a maturation process as they transition into the mature naive (MN) T cell compartment. This maturation presumably shapes RTEs into a pool of T cells best fit to function robustly in the periphery without causing autoimmunity; however, the mechanism and consequences of this maturation process remain unknown. Using a transgenic mouse system that specifically labels RTEs, we tested the influence of MHC molecules, key drivers of intrathymic T cell selection and naive peripheral T cell homeostasis, in shaping the RTE pool in the lymphoid periphery. We found that the TCRs expressed by RTEs are skewed to longer CDR3 regions compared with those of MN T cells, suggesting that MHC does streamline the TCR repertoire of T cells as they transition from the RTE to the MN T cell stage. This conclusion is borne out in studies in which the representation of individual TCRs was followed as a function of time since thymic egress. Surprisingly, we found that MHC is dispensable for the phenotypic and functional maturation of RTEs.

Tomato seeds maturity detection system based on chlorophyll fluorescence

NASA Astrophysics Data System (ADS)

Li, Cuiling; Wang, Xiu; Meng, Zhijun

2016-10-01

Chlorophyll fluorescence intensity can be used as seed maturity and quality evaluation indicator. Chlorophyll fluorescence intensity of seed coats is tested to judge the level of chlorophyll content in seeds, and further to judge the maturity and quality of seeds. This research developed a detection system of tomato seeds maturity based on chlorophyll fluorescence spectrum technology, the system included an excitation light source unit, a fluorescent signal acquisition unit and a data processing unit. The excitation light source unit consisted of two high power LEDs, two radiators and two constant current power supplies, and it was designed to excite chlorophyll fluorescence of tomato seeds. The fluorescent signal acquisition unit was made up of a fluorescence spectrometer, an optical fiber, an optical fiber scaffolds and a narrowband filter. The data processing unit mainly included a computer. Tomato fruits of green ripe stage, discoloration stage, firm ripe stage and full ripe stage were harvested, and their seeds were collected directly. In this research, the developed tomato seeds maturity testing system was used to collect fluorescence spectrums of tomato seeds of different maturities. Principal component analysis (PCA) method was utilized to reduce the dimension of spectral data and extract principal components, and PCA was combined with linear discriminant analysis (LDA) to establish discriminant model of tomato seeds maturity, the discriminant accuracy was greater than 90%. Research results show that using chlorophyll fluorescence spectrum technology is feasible for seeds maturity detection, and the developed tomato seeds maturity testing system has high detection accuracy.

Global Sensitivity Analysis for the determination of parameter importance in bio-manufacturing processes.

PubMed

Chhatre, Sunil; Francis, Richard; Newcombe, Anthony R; Zhou, Yuhong; Titchener-Hooker, Nigel; King, Josh; Keshavarz-Moore, Eli

2008-10-01

The present paper describes the application of GSA (Global Sensitivity Analysis) techniques to mathematical models of bioprocesses in order to rank inputs such as feed titres, flow rates and matrix capacities for the relative influence that each exerts upon outputs such as yield or throughput. GSA enables quantification of both the impact of individual variables on process outputs, as well as their interactions. These data highlight those attributes of a bioprocess which offer the greatest potential for achieving manufacturing improvements. Whereas previous GSA studies have been limited to individual unit operations, this paper extends the treatment to an entire downstream process and illustrates its utility by application to the production of a Fab-based rattlesnake antivenom called CroFab [(Crotalidae Polyvalent Immune Fab (Ovine); Protherics U.K. Limited]. Initially, hyperimmunized ovine serum containing rattlesnake antivenom IgG (product), other antibodies and albumin is applied to a synthetic affinity ligand adsorbent column to separate the antibodies from the albumin. The antibodies are papain-digested into Fab and Fc fragments, before concentration by ultrafiltration. Fc, residual IgG and albumin are eliminated by an ion-exchanger and then CroFab-specific affinity chromatography is used to produce purified antivenom. Application of GSA to the model of this process showed that product yield was controlled by IgG feed concentration and the synthetic-material affinity column's capacity and flow rate, whereas product throughput was predominantly influenced by the synthetic material's capacity, the ultrafiltration concentration factor and the CroFab affinity flow rate. Such information provides a rational basis for identifying the most promising strategies for delivering improvements to commercial-scale biomanufacturing processes.

Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

PubMed

Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

2016-10-01

Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a cost-effective, rapid, and reliable avenue for the purification of recombinant proteins in heterologous hosts. Copyright © 2016 Elsevier Inc. All rights reserved.

Organo luminescent semiconductor nanocrystal probes for biological applications and process for making and using such probes

DOEpatents

Weiss, Shimon; Bruchez, Jr., Marcel; Alivisatos, Paul

2006-09-05

A semiconductor nanocrystal compound is described capable of linking to an affinity molecule. The compound comprises (1) a semiconductor nanocrystal capable of emitting electromagnetic radiation and/or absorbing energy, and/or scattering or diffracting electromagnetic radiation--when excited by an electromagnetic radiation source or a particle beam; and (2) at least one linking agent, having a first portion linked to the semiconductor nanocrystal and a second portion capable of linking to an affinity molecule. The compound is linked to an affinity molecule to form a semiconductor nanocrystal probe capable of bonding with a detectable substance. subsequent exposure to excitation energy will excite the semiconductor nanocrystal in the probe causing the emission of electromagnetic radiation. Further described are processes for respectively: making the luminescent semiconductor nanocrystal compound; making the semiconductor nanocrystal probe; and using the probe to determine the presence of a detectable substance in a material.

Organo luminescent semiconductor nanocrystal probes for biological applications and process for making and using such probes

DOEpatents

Weiss, Shimon [Pinole, CA; Bruchez, Jr., Marcel; Alivisatos, Paul [Oakland, CA

2004-03-02

A semiconductor nanocrystal compound is described capable of linking to an affinity molecule. The compound comprises (1) a semiconductor nanocrystal capable of emitting electromagnetic radiation and/or absorbing energy, and/or scattering or diffracting electromagnetic radiation--when excited by an electromagnetic radiation source or a particle beam; and (2) at least one linking agent, having a first portion linked to the semiconductor nanocrystal and a second portion capable of linking to an affinity molecule. The compound is linked to an affinity molecule to form a semiconductor nanocrystal probe capable of bonding with a detectable substance. Subsequent exposure to excitation energy will excite the semiconductor nanocrystal in the probe, causing the emission of electromagnetic radiation. Further described are processes for respectively: making the semiconductor nanocrystal compound; making the semiconductor nanocrystal probe; and using the probe to determine the presence of a detectable substance in a material.

Organo luminescent semiconductor nanocrystal probes for biological applications and process for making and using such probes

DOEpatents

Weiss, Shimon; Bruchez, Jr., Marcel; Alivisatos, Paul

2005-08-09

A semiconductor nanocrystal compound is described capable of linking to an affinity molecule. The compound comprises (1) a semiconductor nanocrystal capable of emitting electromagnetic radiation and/or absorbing energy, and/or scattering or diffracting electromagnetic radiation--when excited by an electromagnetic radiation source or a particle beam; and (2) at least one linking agent, having a first portion linked to the semiconductor nanocrystal and a second portion capable of linking to an affinity molecule. The compound is linked to an affinity molecule to form a semiconductor nanocrystal probe capable of bonding with a detectable substance. Subsequent exposure to excitation energy will excite the semiconductor nanocrystal in the probe causing the emission of electromagnetic radiation. Further described are processes for respectively: making the luminescent semiconductor nanocrystal compound; making the semiconductor nanocrystal probe; and using the probe to determine the presence of a detectable substance in a material.

Semiconductor nanocrystal probes for biological applications and process for making and using such probes

DOEpatents

Weiss, Shimon; Bruchez, Marcel; Alivisatos, Paul

2014-01-28

A semiconductor nanocrystal compound and probe are described. The compound is capable of linking to one or more affinity molecules. The compound comprises (1) one or more semiconductor nanocrystals capable of, in response to exposure to a first energy, providing a second energy, and (2) one or more linking agents, having a first portion linked to the one or more semiconductor nanocrystals and a second portion capable of linking to one or more affinity molecules. One or more semiconductor nanocrystal compounds are linked to one or more affinity molecules to form a semiconductor nanocrystal probe capable of bonding with one or more detectable substances in a material being analyzed, and capable of, in response to exposure to a first energy, providing a second energy. Also described are processes for respectively: making the semiconductor nanocrystal compound; making the semiconductor nanocrystal probe; and treating materials with the probe.

Semiconductor nanocrystal probes for biological applications and process for making and using such probes

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

Weiss, Shimon; Bruchez, Marcel; Alivisatos, Paul A.

2016-12-27

A semiconductor nanocrystal compound and probe are described. The compound is capable of linking to one or more affinity molecules. The compound comprises (1) one or more semiconductor nanocrystals capable of, in response to exposure to a first energy, providing a second energy, and (2) one or more linking agents, having a first portion linked to the one or more semiconductor nanocrystals and a second portion capable of linking to one or more affinity molecules. One or more semiconductor nanocrystal compounds are linked to one or more affinity molecules to form a semiconductor nanocrystal probe capable of bonding with onemore » or more detectable substances in a material being analyzed, and capable of, in response to exposure to a first energy, providing a second energy. Also described are processes for respectively: making the semiconductor nanocrystal compound; making the semiconductor nanocrystal probe; and treating materials with the probe.« less

Dual role for argonautes in microRNA processing and posttranscriptional regulation of microRNA expression.

PubMed

Diederichs, Sven; Haber, Daniel A

2007-12-14

MicroRNAs are small endogenous noncoding RNAs involved in posttranscriptional gene regulation. During microRNA biogenesis, Drosha and Dicer process the primary transcript (pri-miRNA) through a precursor hairpin (pre-miRNA) to the mature miRNA. The miRNA is incorporated into the RNA-Induced Silencing Complex (RISC) with Argonaute proteins, the effector molecules in RNA interference (RNAi). Here, we show that all Argonautes elevate mature miRNA expression posttranscriptionally, independent of RNase activity. Also, we identify a role for the RISC slicer Argonaute2 (Ago2) in cleaving the pre-miRNA to an additional processing intermediate, termed Ago2-cleaved precursor miRNA or ac-pre-miRNA. This endogenous, on-pathway intermediate results from cleavage of the pre-miRNA hairpin 12 nucleotides from its 3'-end. By analogy to siRNA processing, Ago2 cleavage may facilitate removal of the nicked passenger strand from RISC after maturation. The multiple roles of Argonautes in the RNAi effector phase and miRNA biogenesis and maturation suggest coordinate regulation of microRNA expression and function.

Structure and Uncoating of Immature Adenovirus

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

Perez-Berna, A.J.; Mangel, W.; Marabini, R.

2009-09-18

Maturation via proteolytic processing is a common trait in the viral world and is often accompanied by large conformational changes and rearrangements in the capsid. The adenovirus protease has been shown to play a dual role in the viral infectious cycle: (a) in maturation, as viral assembly starts with precursors to several of the structural proteins but ends with proteolytically processed versions in the mature virion, and (b) in entry, because protease-impaired viruses have difficulties in endosome escape and uncoating. Indeed, viruses that have not undergone proteolytic processing are not infectious. We studied the three-dimensional structure of immature adenovirus particlesmore » as represented by the adenovirus type 2 thermosensitive mutant ts1 grown under non-permissive conditions and compared it with the mature capsid. Our three-dimensional electron microscopy maps at subnanometer resolution indicate that adenovirus maturation does not involve large-scale conformational changes in the capsid. Difference maps reveal the locations of unprocessed peptides pIIIa and pVI and help define their role in capsid assembly and maturation. An intriguing difference appears in the core, indicating a more compact organization and increased stability of the immature cores. We have further investigated these properties by in vitro disassembly assays. Fluorescence and electron microscopy experiments reveal differences in the stability and uncoating of immature viruses, both at the capsid and core levels, as well as disassembly intermediates not previously imaged.« less

Characterizing informative sequence descriptors and predicting binding affinities of heterodimeric protein complexes.

PubMed

Srinivasulu, Yerukala Sathipati; Wang, Jyun-Rong; Hsu, Kai-Ti; Tsai, Ming-Ju; Charoenkwan, Phasit; Huang, Wen-Lin; Huang, Hui-Ling; Ho, Shinn-Ying

2015-01-01

Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein complexes. The characterization analysis revealed that the average numbers of beta turns and hydrogen bonds at protein-protein interfaces in high binding affinity complexes are more than those in low binding affinity complexes.

Characterizing informative sequence descriptors and predicting binding affinities of heterodimeric protein complexes

PubMed Central

2015-01-01

Background Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. Results This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. Conclusions The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein complexes. The characterization analysis revealed that the average numbers of beta turns and hydrogen bonds at protein-protein interfaces in high binding affinity complexes are more than those in low binding affinity complexes. PMID:26681483

Electrochemical Affinity Biosensors Based on Disposable Screen-Printed Electrodes for Detection of Food Allergens

PubMed Central

Vasilescu, Alina; Nunes, Gilvanda; Hayat, Akhtar; Latif, Usman; Marty, Jean-Louis

2016-01-01

Food allergens are proteins from nuts and tree nuts, fish, shellfish, wheat, soy, eggs or milk which trigger severe adverse reactions in the human body, involving IgE-type antibodies. Sensitive detection of allergens in a large variety of food matrices has become increasingly important considering the emergence of functional foods and new food manufacturing technologies. For example, proteins such as casein from milk or lysozyme and ovalbumin from eggs are sometimes used as fining agents in the wine industry. Nonetheless, allergen detection in processed foods is a challenging endeavor, as allergen proteins are degraded during food processing steps involving heating or fermentation. Detection of food allergens was primarily achieved via Enzyme-Linked Immuno Assay (ELISA) or by chromatographic methods. With the advent of biosensors, electrochemical affinity-based biosensors such as those incorporating antibodies and aptamers as biorecognition elements were also reported in the literature. In this review paper, we highlight the success achieved in the design of electrochemical affinity biosensors based on disposable screen-printed electrodes towards detection of protein allergens. We will discuss the analytical figures of merit for various disposable screen-printed affinity sensors in relation to methodologies employed for immobilization of bioreceptors on transducer surface. PMID:27827963

Electrochemical Affinity Biosensors Based on Disposable Screen-Printed Electrodes for Detection of Food Allergens.

PubMed

Vasilescu, Alina; Nunes, Gilvanda; Hayat, Akhtar; Latif, Usman; Marty, Jean-Louis

2016-11-05

Food allergens are proteins from nuts and tree nuts, fish, shellfish, wheat, soy, eggs or milk which trigger severe adverse reactions in the human body, involving IgE-type antibodies. Sensitive detection of allergens in a large variety of food matrices has become increasingly important considering the emergence of functional foods and new food manufacturing technologies. For example, proteins such as casein from milk or lysozyme and ovalbumin from eggs are sometimes used as fining agents in the wine industry. Nonetheless, allergen detection in processed foods is a challenging endeavor, as allergen proteins are degraded during food processing steps involving heating or fermentation. Detection of food allergens was primarily achieved via Enzyme-Linked Immuno Assay (ELISA) or by chromatographic methods. With the advent of biosensors, electrochemical affinity-based biosensors such as those incorporating antibodies and aptamers as biorecognition elements were also reported in the literature. In this review paper, we highlight the success achieved in the design of electrochemical affinity biosensors based on disposable screen-printed electrodes towards detection of protein allergens. We will discuss the analytical figures of merit for various disposable screen-printed affinity sensors in relation to methodologies employed for immobilization of bioreceptors on transducer surface.

Treatment of mature landfill leachate by internal micro-electrolysis integrated with coagulation: a comparative study on a novel sequencing batch reactor based on zero valent iron.

PubMed

Ying, Diwen; Peng, Juan; Xu, Xinyan; Li, Kan; Wang, Yalin; Jia, Jinping

2012-08-30

A comparative study of treating mature landfill leachate with various treatment processes was conducted to investigate whether the method of combined processes of internal micro-electrolysis (IME) without aeration and IME with full aeration in one reactor was an efficient treatment for mature landfill leachate. A specifically designed novel sequencing batch internal micro-electrolysis reactor (SIME) with the latest automation technology was employed in the experiment. Experimental data showed that combined processes obtained a high COD removal efficiency of 73.7 ± 1.3%, which was 15.2% and 24.8% higher than that of the IME with and without aeration, respectively. The SIME reactor also exhibited a COD removal efficiency of 86.1 ± 3.8% to mature landfill leachate in the continuous operation, which is much higher (p<0.05) than that of conventional treatments of electrolysis (22.8-47.0%), coagulation-sedimentation (18.5-22.2%), and the Fenton process (19.9-40.2%), respectively. The innovative concept behind this excellent performance is a combination effect of reductive and oxidative processes of the IME, and the integration electro-coagulation. Optimal operating parameters, including the initial pH, Fe/C mass ratio, air flow rate, and addition of H(2)O(2), were optimized. All results show that the SIME reactor is a promising and efficient technology in treating mature landfill leachate. Copyright © 2012 Elsevier B.V. All rights reserved.

The translocator protein gene is associated with symptom severity and cerebral pain processing in fibromyalgia.

PubMed

Kosek, Eva; Martinsen, Sofia; Gerdle, Björn; Mannerkorpi, Kaisa; Löfgren, Monika; Bileviciute-Ljungar, Indre; Fransson, Peter; Schalling, Martin; Ingvar, Martin; Ernberg, Malin; Jensen, Karin B

2016-11-01

The translocator protein (TSPO) is upregulated during glia activation in chronic pain patients. TSPO constitutes the rate-limiting step in neurosteroid synthesis, thus modulating synaptic transmission. Related serotonergic mechanisms influence if pro- or anti-nociceptive neurosteroids are produced. This study investigated the effects of a functional genetic polymorphism regulating the binding affinity to the TSPO, thus affecting symptom severity and cerebral pain processing in fibromyalgia patients. Gene-to-gene interactions with a functional polymorphism of the serotonin transporter gene were assessed. Fibromyalgia patients (n=126) were genotyped regarding the polymorphisms of the TSPO (rs6971) and the serotonin transporter (5-HTTLPR/rs25531). Functional magnetic resonance imaging (n=24) was used to study brain activation during individually calibrated pressure pain. Compared to mixed/low TSPO affinity binders, the high TSPO affinity binders rated more severe pain (p=0.016) and fibromyalgia symptoms (p=0.02). A significant interaction was found between the TSPO and the serotonin transporter polymorphisms regarding pain severity (p<0.0001). Functional connectivity analyses revealed that the TSPO high affinity binding group had more pronounced pain-evoked functional connectivity in the right frontoparietal network, between the dorsolateral prefrontal area and the parietal cortex. In conclusion, fibromyalgia patients with the TSPO high affinity binding genotype reported a higher pain intensity and more severe fibromyalgia symptoms compared to mixed/low affinity binders, and this was modulated by interaction with the serotonin transporter gene. To our knowledge this is the first evidence of functional genetic polymorphisms affecting pain severity in FM and our findings are in line with proposed glia-related mechanisms. Furthermore, the functional magnetic resonance findings indicated an effect of translocator protein on the affective-motivational components of pain perception. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Evaluation of protein-ligand affinity prediction using steered molecular dynamics simulations.

PubMed

Okimoto, Noriaki; Suenaga, Atsushi; Taiji, Makoto

2017-11-01

In computational drug design, ranking a series of compound analogs in a manner that is consistent with experimental affinities remains a challenge. In this study, we evaluated the prediction of protein-ligand binding affinities using steered molecular dynamics simulations. First, we investigated the appropriate conditions for accurate predictions in these simulations. A conic harmonic restraint was applied to the system for efficient sampling of work values on the ligand unbinding pathway. We found that pulling velocity significantly influenced affinity predictions, but that the number of collectable trajectories was less influential. We identified the appropriate pulling velocity and collectable trajectories for binding affinity predictions as 1.25 Å/ns and 100, respectively, and these parameters were used to evaluate three target proteins (FK506 binding protein, trypsin, and cyclin-dependent kinase 2). For these proteins using our parameters, the accuracy of affinity prediction was higher and more stable when Jarzynski's equality was employed compared with the second-order cumulant expansion equation of Jarzynski's equality. Our results showed that steered molecular dynamics simulations are effective for predicting the rank order of ligands; thus, they are a potential tool for compound selection in hit-to-lead and lead optimization processes.

High-affinity K+ uptake in pepper plants.

PubMed

Martínez-Cordero, M Angeles; Martínez, Vicente; Rubio, Francisco

2005-06-01

High-affinity K+ uptake is an essential process for plant nutrition under K+-limiting conditions. The results presented here demonstrate that pepper (Capsicum annuum) plants grown in the absence of NH4+ and starved of K+ show an NH4+-sensitive high-affinity K+ uptake that allows plant roots to deplete external K+ to values below 1 microM. When plants are grown in the presence of NH4+, high-affinity K+ uptake is not inhibited by NH4+. Although NH4+-grown plants deplete external K+ below 1 microM in the absence of NH4+, when 1 mM NH4+ is present they do not deplete external K+ below 10 microM. A K+ transporter of the HAK family, CaHAK1, is very likely mediating the NH4+-sensitive component of the high-affinity K+ uptake in pepper roots. CaHAK1 is strongly induced in the roots that show the NH4+-sensitive high-affinity K+ uptake and its induction is reduced in K+-starved plants grown in the presence of NH4+. The NH4+-insensitive K+ uptake may be mediated by an AKT1-like K+ channel.

Changes in canopy processes following whole-forest canopy nitrogen fertilization of a mature spruce-hemlock forest

Treesearch

E. Gaige; D.B. Dail; D.Y. Hollinger; E.A. Davidson; I.J. Fernandez; H. Sievering; A. White; W. Halteman

2007-01-01

Most experimental additions of nitrogen to forest ecosystems apply the N to the forest floor, bypassing important processes taking place in the canopy, including canopy retention of N and/or conversion of N from one form to another. To quantify these processes, we carried out a large-scale experiment and determined the fate of nitrogen applied directly to a mature...

New Developments in the Technology Readiness Assessment Process in US DOE-EM - 13247

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

Krahn, Steven; Sutter, Herbert; Johnson, Hoyt

2013-07-01

A Technology Readiness Assessment (TRA) is a systematic, metric-based process and accompanying report that evaluates the maturity of the technologies used in systems; it is designed to measure technology maturity using the Technology Readiness Level (TRL) scale pioneered by the National Aeronautics and Space Administration (NASA) in the 1980's. More recently, DoD has adopted and provided systematic guidance for performing TRAs and determining TRLs. In 2007 the GAO recommended that the DOE adopt the NASA/DoD methodology for evaluating technology maturity. Earlier, in 2006-2007, DOE-EM had conducted pilot TRAs on a number of projects at Hanford and Savannah River. In Marchmore » 2008, DOE-EM issued a process guide, which established TRAs as an integral part of DOE-EM's Project Management Critical Decision Process. Since the development of its detailed TRA guidance in 2008, DOE-EM has continued to accumulate experience in the conduct of TRAs and the process for evaluating technology maturity. DOE has developed guidance on TRAs applicable department-wide. DOE-EM's experience with the TRA process, the evaluations that led to recently developed proposed revisions to the DOE-EM TRA/TMP Guide; the content of the proposed changes that incorporate the above lessons learned and insights are described. (authors)« less

Identification of cellular compartments involved in processing of cathepsin E in primary cultures of rat microglia.

PubMed

Sastradipura, D F; Nakanishi, H; Tsukuba, T; Nishishita, K; Sakai, H; Kato, Y; Gotow, T; Uchiyama, Y; Yamamoto, K

1998-05-01

Cathepsin E is a major nonlysosomal, intracellular aspartic proteinase that localizes in various cellular compartments such as the plasma membrane, endosome-like organelles, and the endoplasmic reticulum (ER). To learn the segregation mechanisms of cathepsin E into its appropriate cellular destinations, the present studies were initiated to define the biosynthesis, processing, and intracellular localization as well as the site of proteolytic maturation of the enzyme in primary cultures of rat brain microglia. Immunohistochemical and immunoblot analyses revealed that cathepsin E was the most abundant in microglia among various brain cell types, where the enzyme existed predominantly as the mature enzyme. Immunoelectron microscopy studies showed the presence of the enzyme predominantly in the endosome-like vacuoles and partly in the vesicles located in the trans-Golgi area and the lumen of ER. In the primary cultured microglial cells labeled with [35S]methionine, >95% of labeled cathepsin E were represented by a 46-kDa polypeptide (reduced form) after a 30-min pulse. Most of it was proteolytically processed via a 44-kDa intermediate to a 42-kDa mature form within 4 h of chase. This processing was completely inhibited by bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase. Brefeldin A, a blocker for the traffic of secretory proteins from the ER to the Golgi complex, also inhibited the processing of procathepsin E and enhanced its degradation. Procathepsin E, after pulse-labeling, showed complete susceptibility to endoglycosidase H, whereas the mature enzyme almost acquired resistance to endoglycosidases H as well as F. The present studies provide the first evidence that cathepsin E in microglia is first synthesized as the inactive precursor bearing high-mannose oligosaccharides and processed to the active mature enzyme with complex-type oligosaccharides via the intermediate form and that the final proteolytic maturation step occurs in endosome-like acidic compartments.

A viral, transporter associated with antigen processing (TAP)-independent, high affinity ligand with alternative interactions endogenously presented by the nonclassical human leukocyte antigen E class I molecule.

PubMed

Lorente, Elena; Infantes, Susana; Abia, David; Barnea, Eilon; Beer, Ilan; García, Ruth; Lasala, Fátima; Jiménez, Mercedes; Mir, Carmen; Morreale, Antonio; Admon, Arie; López, Daniel

2012-10-12

The transporter associated with antigen processing (TAP) enables the flow of viral peptides generated in the cytosol by the proteasome and other proteases to the endoplasmic reticulum, where they complex with nascent human leukocyte antigen (HLA) class I. Later, these peptide-HLA class I complexes can be recognized by CD8(+) lymphocytes. Cancerous cells and infected cells in which TAP is blocked, as well as individuals with unusable TAP complexes, are able to present peptides on HLA class I by generating them through TAP-independent processing pathways. Here, we identify a physiologically processed HLA-E ligand derived from the D8L protein in TAP-deficient vaccinia virus-infected cells. This natural high affinity HLA-E class I ligand uses alternative interactions to the anchor motifs previously described to be presented on nonclassical HLA class I molecules. This octameric peptide was also presented on HLA-Cw1 with similar binding affinity on both classical and nonclassical class I molecules. In addition, this viral peptide inhibits HLA-E-mediated cytolysis by natural killer cells. Comparison between the amino acid sequences of the presenting HLA-E and HLA-Cw1 alleles revealed a shared structural motif in both HLA class molecules, which could be related to their observed similar cross-reactivity affinities. This motif consists of several residues located on the floor of the peptide-binding site. These data expand the role of HLA-E as an antigen-presenting molecule.

Climbing the ladder: capability maturity model integration level 3

NASA Astrophysics Data System (ADS)

Day, Bryce; Lutteroth, Christof

2011-02-01

This article details the attempt to form a complete workflow model for an information and communication technologies (ICT) company in order to achieve a capability maturity model integration (CMMI) maturity rating of 3. During this project, business processes across the company's core and auxiliary sectors were documented and extended using modern enterprise modelling tools and a The Open Group Architectural Framework (TOGAF) methodology. Different challenges were encountered with regard to process customisation and tool support for enterprise modelling. In particular, there were problems with the reuse of process models, the integration of different project management methodologies and the integration of the Rational Unified Process development process framework that had to be solved. We report on these challenges and the perceived effects of the project on the company. Finally, we point out research directions that could help to improve the situation in the future.

Localization and characterization of an alpha-thrombin-binding site on platelet glycoprotein Ib alpha.

PubMed

De Marco, L; Mazzucato, M; Masotti, A; Ruggeri, Z M

1994-03-04

Glycoprotein (GP) Ib alpha is required for expression of the highest affinity alpha-thrombin-binding site on platelets, possibly contributing to platelet activation through a pathway involving cleavage of a specific receptor. This function may be important for the initiation of hemostasis and may also play a role in the development of pathological vascular occlusion. We have now identified a discrete sequence in the extracytoplasmic domain of GP Ib alpha, including residues 271-284 of the mature protein, which appears to be part of the high affinity alpha-thrombin-binding site. Synthetic peptidyl mimetics of this sequence inhibit alpha-thrombin binding to GP Ib as well as platelet activation and aggregation induced by subnanomolar concentrations of the agonist; they also inhibit alpha-thrombin binding to purified glycocalicin, the isolated extracytoplasmic portion of GP Ib alpha. The inhibitory peptides interfere with the clotting of fibrinogen by alpha-thrombin but not with the amidolytic activity of the enzyme on a small synthetic substrate, a finding compatible with the concept that the identified GP Ib alpha sequence interacts with the anion-binding exosite of alpha-thrombin but not with its active proteolytic site. The crucial structural elements of this sequence necessary for thrombin binding appear to be a cluster of negatively charged residues as well as three tyrosine residues that, in the native protein, may be sulfated. GP Ib alpha has no significant overall sequence homology with the thrombin inhibitor, hirudin, nor with the specific thrombin receptor on platelets; all three molecules, however, possess a distinct region rich in negatively charged residues that appear to be involved in thrombin binding. This may represent a case of convergent evolution of unrelated proteins for high affinity interaction with the same ligand.

Multiple length peptide-pheromone variants produced by Streptococcus pyogenes directly bind Rgg proteins to confer transcriptional regulation.

PubMed

Aggarwal, Chaitanya; Jimenez, Juan Cristobal; Nanavati, Dhaval; Federle, Michael J

2014-08-08

Streptococcus pyogenes, a human-restricted pathogen, accounts for substantial mortality related to infections worldwide. Recent studies indicate that streptococci produce and respond to several secreted peptide signaling molecules (pheromones), including those known as short hydrophobic peptides (SHPs), to regulate gene expression by a quorum-sensing mechanism. Upon transport into the bacterial cell, pheromones bind to and modulate activity of receptor proteins belonging to the Rgg family of transcription factors. Previously, we reported biofilm regulation by the Rgg2/3 quorum-sensing circuit in S. pyogenes. The aim of this study was to identify the composition of mature pheromones from cell-free culture supernatants that facilitate biofilm formation. Bioluminescent reporters were employed to detect active pheromones in culture supernatants fractionated by reverse-phase chromatography, and mass spectrometry was used to characterize their properties. Surprisingly, multiple SHPs that varied by length were detected. Synthetic peptides of each variant were tested individually using bioluminescence reporters and biofilm growth assays, and although activities differed widely among the group, peptides comprising the C-terminal eight amino acids of the full-length native peptide were most active. Direct Rgg/SHP interactions were determined using a fluorescence polarization assay that utilized FITC-labeled peptide ligands. Peptide receptor affinities were seen to be as low as 500 nm and their binding affinities directly correlated with observed bioactivity. Revelation of naturally produced pheromones along with determination of their affinity for cognate receptors are important steps forward in designing compounds whose purpose is positioned for future therapeutics aimed at treating infections through the interference of bacterial communication. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in guarana fruit.

PubMed

Schimpl, Flávia Camila; Kiyota, Eduardo; Mayer, Juliana Lischka Sampaio; Gonçalves, José Francisco de Carvalho; da Silva, José Ferreira; Mazzafera, Paulo

2014-09-01

Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein. Copyright © 2014 Elsevier Ltd. All rights reserved.

A Maturity Model: Does It Provide a Path for Online Course Design?

ERIC Educational Resources Information Center

Neuhauser, Charlotte

2004-01-01

Maturity models are successfully used by organizations attempting to improve their processes, products, and delivery. As more faculty include online course design and teaching, a maturity model of online course design may serve as a tool in planning and assessing their courses for improvement based on best practices. This article presents such a…

Morphometry and Connectivity of the Fronto-Parietal Verbal Working Memory Network in Development

ERIC Educational Resources Information Center

Ostby, Ylva; Tamnes, Christian K.; Fjell, Anders M.; Walhovd, Kristine B.

2011-01-01

Two distinctly different maturational processes--cortical thinning and white matter maturation--take place in the brain as we mature from late childhood to adulthood. To what extent does each contribute to the development of complex cognitive functions like working memory? The independent and joint contributions of cortical thickness of regions of…

[Scars, physiology, classification and assessment].

PubMed

Roques, Claude

2013-01-01

A skin scar is the sign of tissue repair following damage to the skin. Once formed, it follows a process of maturation which, after several months, results in a mature scar. This can be pathological with functional and/or aesthetic consequences. It is important to assess the scar as it matures in order to adapt the treatment to its evolution.

Biological maturation of youth athletes: assessment and implications.

PubMed

Malina, Robert M; Rogol, Alan D; Cumming, Sean P; Coelho e Silva, Manuel J; Figueiredo, Antonio J

2015-07-01

The search for talent is pervasive in youth sports. Selection/exclusion in many sports follows a maturity-related gradient largely during the interval of puberty and growth spurt. As such, there is emphasis on methods for assessing maturation. Commonly used methods for assessing status (skeletal age, secondary sex characteristics) and estimating timing (ages at peak height velocity (PHV) and menarche) in youth athletes and two relatively recent anthropometric (non-invasive) methods (status-percentage of predicted near adult height attained at observation, timing-predicted maturity offset/age at PHV) are described and evaluated. The latter methods need further validation with athletes. Currently available data on the maturity status and timing of youth athletes are subsequently summarised. Selection for sport and potential maturity-related correlates are then discussed in the context of talent development and associated models. Talent development from novice to elite is superimposed on a constantly changing base-the processes of physical growth, biological maturation and behavioural development, which occur simultaneously and interact with each other. The processes which are highly individualised also interact with the demands of a sport per se and with involved adults (coaches, trainers, administrators, parents/guardians). Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Investigation of the Mechanism of Electron Capture and Electron Transfer Dissociation of Peptides with a Covalently Attached Free Radical Hydrogen Atom Scavenger.

PubMed

Sohn, Chang Ho; Yin, Sheng; Peng, Ivory; Loo, Joseph A; Beauchamp, J L

2015-11-15

The mechanisms of electron capture and electron transfer dissociation (ECD and ETD) are investigated by covalently attaching a free-radical hydrogen atom scavenger to a peptide. The 2,2,6,6-tetramethylpiperidin-l-oxyl (TEMPO) radical was chosen as the scavenger due to its high hydrogen atom affinity (ca. 280 kJ/mol) and low electron affinity (ca. 0.45 ev), and was derivatized to the model peptide, FQX TEMPO EEQQQTEDELQDK. The X TEMPO residue represents a cysteinyl residue derivatized with an acetamido-TEMPO group. The acetamide group without TEMPO was also examined as a control. The gas phase proton affinity (882 kJ/mol) of TEMPO is similar to backbone amide carbonyls (889 kJ/mol), minimizing perturbation to internal solvation and sites of protonation of the derivatized peptides. Collision induced dissociation (CID) of the TEMPO tagged peptide dication generated stable odd-electron b and y type ions without indication of any TEMPO radical induced fragmentation initiated by hydrogen abstraction. The type and abundance of fragment ions observed in the CID spectra of the TEMPO and acetamide tagged peptides are very similar. However, ECD of the TEMPO labeled peptide dication yielded no backbone cleavage. We propose that a labile hydrogen atom in the charge reduced radical ions is scavenged by the TEMPO radical moiety, resulting in inhibition of N-C α backbone cleavage processes. Supplemental activation after electron attachment (ETcaD) and CID of the charge-reduced precursor ion generated by electron transfer of the TEMPO tagged peptide dication produced a series of b + H (b H ) and y + H (y H ) ions along with some c ions having suppressed intensities, consistent with stable O-H bond formation at the TEMPO group. In summary, the results indicate that ECD and ETD backbone cleavage processes are inhibited by scavenging of a labile hydrogen atom by the localized TEMPO radical moiety. This observation supports the conjecture that ECD and ETD processes involve long-lived intermediates formed by electron capture/transfer in which a labile hydrogen atom is present and plays a key role with low energy processes leading to c and z ion formation. Ab initio and density functional calculations are performed to support our conclusion, which depends most importantly on the proton affinity, electron affinity and hydrogen atom affinity of the TEMPO moiety.

Incorporation of feedback during beat synchronization is an index of neural maturation and reading skills.

PubMed

Woodruff Carr, Kali; Fitzroy, Ahren B; Tierney, Adam; White-Schwoch, Travis; Kraus, Nina

2017-01-01

Speech communication involves integration and coordination of sensory perception and motor production, requiring precise temporal coupling. Beat synchronization, the coordination of movement with a pacing sound, can be used as an index of this sensorimotor timing. We assessed adolescents' synchronization and capacity to correct asynchronies when given online visual feedback. Variability of synchronization while receiving feedback predicted phonological memory and reading sub-skills, as well as maturation of cortical auditory processing; less variable synchronization during the presence of feedback tracked with maturation of cortical processing of sound onsets and resting gamma activity. We suggest the ability to incorporate feedback during synchronization is an index of intentional, multimodal timing-based integration in the maturing adolescent brain. Precision of temporal coding across modalities is important for speech processing and literacy skills that rely on dynamic interactions with sound. Synchronization employing feedback may prove useful as a remedial strategy for individuals who struggle with timing-based language learning impairments. Copyright © 2016 Elsevier Inc. All rights reserved.

Impact of socio-emotional context, brain development, and pubertal maturation on adolescent risk-taking.

PubMed

Smith, Ashley R; Chein, Jason; Steinberg, Laurence

2013-07-01

While there is little doubt that risk-taking is generally more prevalent during adolescence than before or after, the underlying causes of this pattern of age differences have long been investigated and debated. One longstanding popular notion is the belief that risky and reckless behavior in adolescence is tied to the hormonal changes of puberty. However, the interactions between pubertal maturation and adolescent decision making remain largely understudied. In the current review, we discuss changes in decision making during adolescence, focusing on the asynchronous development of the affective, reward-focused processing system and the deliberative, reasoned processing system. As discussed, differential maturation in the structure and function of brain systems associated with these systems leaves adolescents particularly vulnerable to socio-emotional influences and risk-taking behaviors. We argue that this asynchrony may be partially linked to pubertal influences on development and specifically on the maturation of the affective, reward-focused processing system. Copyright © 2013 Elsevier Inc. All rights reserved.

Elucidating the 16S rRNA 3' boundaries and defining optimal SD/aSD pairing in Escherichia coli and Bacillus subtilis using RNA-Seq data.

PubMed

Wei, Yulong; Silke, Jordan R; Xia, Xuhua

2017-12-15

Bacterial translation initiation is influenced by base pairing between the Shine-Dalgarno (SD) sequence in the 5' UTR of mRNA and the anti-SD (aSD) sequence at the free 3' end of the 16S rRNA (3' TAIL) due to: 1) the SD/aSD sequence binding location and 2) SD/aSD binding affinity. In order to understand what makes an SD/aSD interaction optimal, we must define: 1) terminus of the 3' TAIL and 2) extent of the core aSD sequence within the 3' TAIL. Our approach to characterize these components in Escherichia coli and Bacillus subtilis involves 1) mapping the 3' boundary of the mature 16S rRNA using high-throughput RNA sequencing (RNA-Seq), and 2) identifying the segment within the 3' TAIL that is strongly preferred in SD/aSD pairing. Using RNA-Seq data, we resolve previous discrepancies in the reported 3' TAIL in B. subtilis and recovered the established 3' TAIL in E. coli. Furthermore, we extend previous studies to suggest that both highly and lowly expressed genes favor SD sequences with intermediate binding affinity, but this trend is exclusive to SD sequences that complement the core aSD sequences defined herein.

Functional display of platelet-binding VWF fragments on filamentous bacteriophage.

PubMed

Yee, Andrew; Tan, Fen-Lai; Ginsburg, David

2013-01-01

von Willebrand factor (VWF) tethers platelets to sites of vascular injury via interaction with the platelet surface receptor, GPIb. To further define the VWF sequences required for VWF-platelet interaction, a phage library displaying random VWF protein fragments was screened against formalin-fixed platelets. After 3 rounds of affinity selection, DNA sequencing of platelet-bound clones identified VWF peptides mapping exclusively to the A1 domain. Aligning these sequences defined a minimal, overlapping segment spanning P1254-A1461, which encompasses the C1272-C1458 cystine loop. Analysis of phage carrying a mutated A1 segment (C1272/1458A) confirmed the requirement of the cystine loop for optimal binding. Four rounds of affinity maturation of a randomly mutagenized A1 phage library identified 10 and 14 unique mutants associated with enhanced platelet binding in the presence and absence of botrocetin, respectively, with 2 mutants (S1370G and I1372V) common to both conditions. These results demonstrate the utility of filamentous phage for studying VWF protein structure-function and identify a minimal, contiguous peptide that bind to formalin-fixed platelets, confirming the importance of the VWF A1 domain with no evidence for another independently platelet-binding segment within VWF. These findings also point to key structural elements within the A1 domain that regulate VWF-platelet adhesion.

Cation binding to a Bacillus (1,3-1,4)-beta-glucanase. Geometry, affinity and effect on protein stability.

PubMed

Keitel, T; Meldgaard, M; Heinemann, U

1994-05-15

The hybrid Bacillus (1,3-1,4)-beta-glucanase H(A16-M), consisting of 16 N-terminal amino acids derived from the mature form of the B. amyloliquefaciens enzyme and of 198 C-proximal amino acids from the B. macerans enzyme, binds a calcium ion at a site at its molecular surface remote from the active center [T. Keitel, O. Simon, R. Borriss & U. Heinemann (1993) Proc. Natl Acad. Sci. USA 90, 5287-5291]. X-ray diffraction analysis at 0.22-nm resolution of crystals grown in the absence of calcium and in the presence of EDTA shows this site to be occupied by a sodium ion. Whereas the calcium ion has six oxygen atoms in its coordination sphere, two of which are from water molecules, sodium is fivefold coordinated with a fifth ligand belonging to a symmetry-related protein molecule in the crystal lattice. The affinity of H(A16-M) for calcium over sodium has been determined calorimetrically. Calcium binding stabilizes the native three-dimensional structure of the protein as shown by guanidinium chloride unfolding and thermal inactivation experiments. The enhanced enzymic activity of Bacillus beta-glucanases at elevated temperatures in the presence of calcium ions is attributed to a general stabilizing effect by the cation.

Surface plasmon resonance measurements of plasma antibody avidity during primary and secondary responses to anthrax protective antigen

PubMed Central

Lynch, Heather E.; Stewart, Shelley M.; Kepler, Thomas B.; Sempowski, Gregory D.; Alam, S. Munir

2014-01-01

Establishment of humoral immunity against pathogens is dependent on events that occur in the germinal center and the subsequent induction of high-affinity neutralizing antibodies. Quantitative assays that allow monitoring of affinity maturation and duration of antibody responses can provide useful information regarding the efficacy of vaccines and adjuvants. Using an anthrax protective antigen (rPA) and alum model antigen/adjuvant system, we describe a methodology for monitoring antigen-specific serum antibody concentration and avidity by surface plasmon resonance during primary and secondary immune responses. Our analyses showed that following a priming dose in mice, rPA-specific antibody concentration and avidity increases over time and reaches a maximal response in about six weeks, but gradually declines in the absence of antigenic boost. Germinal center reactions were observed early with maximal development achieved during the primary response, which coincided with peak antibody avidity responses to primary immunization. Boosting with antigen resulted in a rapid increase in rPA-specific antibody concentration and five-fold increase in avidity, which was not dependent on sustained GC development. The described methodology couples surface plasmon resonance-based plasma avidity measurements with germinal center analysis and provides a novel way to monitor humoral responses that can play a role in facilitating vaccine and adjuvant development. PMID:24316020

Inhibition of initial adhesion of oral bacteria through a lectin from Bauhinia variegata L. var. variegata expressed in Escherichia coli.

PubMed

Klafke, G B; Borsuk, S; Gonçales, R A; Arruda, F V S; Carneiro, V A; Teixeira, E H; Coelho da Silva, A L; Cavada, B S; Dellagostin, O A; Pinto, L S

2013-11-01

The aim of the present work was to study the in vitro effect of native and recombinant Bauhinia variegata var. variegata lectins in inhibiting early adhesion of Streptococcus mutans, Streptococcus sanguis and Streptococcus sobrinus to experimentally acquired pellicle. Native lectin from B. variegata (BVL) was purified by affinity chromatography of extract of seeds. The recombinant lectin (rBVL-I) was expressed in E. coli strain BL21 (DE3) from a genomic clone encoding the mature B. variegata lectin gene using the vector pAE-bvlI. Recombinant protein deposited in inclusion bodies was solubilized and subsequently purified by affinity chromatography. The rBVL-I was compared to BVL for agglutination of erythrocytes and initial adherence of oral bacteria on a saliva-coated surface. The results revealed that rBVL-I acts similarly to BVL for agglutination of erythrocytes. Both lectins showed adhesion inhibition effect on Step. sanguis, Step. mutans and Step. sobrinus. We report, for the first time, the inhibition of early adhesion of oral bacteria by a recombinant lectin. Our results support the proposed biotechnological application of lectins in a strategy to reduce development of dental caries by inhibiting the initial adhesion and biofilm formation. © 2013 The Society for Applied Microbiology.

Identification and characterization of a gene encoding for a nucleotidase from Phaseolus vulgaris.

PubMed

Cabello-Díaz, Juan Miguel; Gálvez-Valdivieso, Gregorio; Caballo, Cristina; Lambert, Rocío; Quiles, Francisco Antonio; Pineda, Manuel; Piedras, Pedro

2015-08-01

Nucleotidases are phosphatases that catalyze the removal of phosphate from nucleotides, compounds with an important role in plant metabolism. A phosphatase enzyme, with high affinity for nucleotides monophosphate previously identified and purified in embryonic axes from French bean, has been analyzed by MALDI TOF/TOF and two internal peptides have been obtained. The information of these peptide sequences has been used to search in the genome database and only a candidate gene that encodes for the phosphatase was identified (PvNTD1). The putative protein contains the conserved domains (motif I-IV) for haloacid dehalogenase-like hydrolases superfamily. The residues involved in the catalytic activity are also conserved. A recombinant protein overexpressed in Escherichia coli has shown molybdate resistant phosphatase activity with nucleosides monophosphate as substrate, confirming that the identified gene encodes for the phosphatase with high affinity for nucleotides purified in French bean embryonic axes. The activity of the purified protein was inhibited by adenosine. The expression of PvNTD1 gene was induced at the specific moment of radicle protrusion in embryonic axes. The gene was also highly expressed in young leaves whereas the level of expression in mature tissues was minimal. Copyright © 2015 The Authors. Published by Elsevier GmbH.. All rights reserved.

[The importance of putrescine in the human body].

PubMed

Zdrojewicz, Zygmunt; Lachowski, Michał

2014-04-10

Putrescine plays a very important role in the regulation of division, differentiation and maturation of cells as well as apoptosis. As the polycationic molecule it stabilizes the structure of DNA and participates in the functioning of cell membranes. It is able to interact with series of ion channels and has affinity for many receptors. The article presents the participation of putrescine in the metabolism of iron and mechanism of its transport across biological membranes. Especially important for the homeostasis of putrescine has ornithine decarboxylase and availability of its substrate--ornithine. Affecting to this enzyme is the simplest and widely used method of controlling the concentration of putrescine. For this purpose its inhibitor-eflornithine is applied. There was also a number of other enzymes involved in the metabolism of putrescine that was presented. Current information about the clinical relevance of putrescine in infertility, embryonic development, hirsutism, epilepsy, Alzheimer's disease, Parkinson's disease, prevention of metastases and hemostasis was also described. These processes were presented, in which putrescine plays a major role and focused on the latest reports. Attention was drawn to the situations where it has beneficial effects and those in which it is the cause of the pathology. Some of the cited reports are in phase of speculation on the possible use of it, but a significant part is already confirmed and used in clinical practice. The facts presented in this article show how great is the meaning of putrescine and how important role this simple specimen plays in the metabolic processes of living organisms.

From the epididymis to the egg: participation of CRISP proteins in mammalian fertilization.

PubMed

Da Ros, Vanina G; Muñoz, Mariana Weigel; Battistone, Maria A; Brukman, Nicolás G; Carvajal, Guillermo; Curci, Ludmila; Gómez-ElIas, MatIas D; Cohen, D Bora J; Cuasnicu, Patricia S

2015-01-01

Mammalian fertilization is a complex process that involves different steps of interaction between the male and female gametes. In spite of its relevance, the molecular mechanisms underlying this process still remain to be elucidated. The present review describes the contribution of our laboratory to the understanding of mammalian fertilization using Cysteine-RIch Secretory Proteins (CRISP) as model molecules. Substantial evidence obtained from in vitro assays and knockout models shows that epididymal CRISP1 associates with the sperm surface with two different affinities during maturation, and participates in the regulation of signaling pathways during capacitation as well as in both sperm-zona pellucida interaction and gamete fusion. These observations can be extended to humans as judged by our findings showing that the human homolog of the rodent protein (hCRISP1) is also involved in both stages of fertilization. Evidence supports that other members of the CRISP family secreted in the testis (CRISP2), epididymis (CRISP3-4) or during ejaculation (CRISP3) are also involved in sperm-egg interaction, supporting the existence of a functional redundancy and cooperation between homolog proteins ensuring the success of fertilization. Together, our observations indicate that CRISP proteins accompany spermatozoa along their transit through both the male and female reproductive tracts. We believe these results not only contribute to a better mechanistic understanding of fertilization but also support CRISP proteins as excellent candidates for future research on infertility and contraception.

Structural Basis of Neutralization of the Major Toxic Component from the Scorpion Centruroides noxius Hoffmann by a Human-derived Single-chain Antibody Fragment

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

Canul-Tec, Juan Carlos; Riaño-Umbarila, Lidia; Rudiño-Piñera, Enrique

2011-08-09

It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 {angstrom} resolution. A 15-residue span of the toxin is recognized by the antibody through a cleft formed by residues from five of the complementarity-determining regions of the scFv. Analysis of the interface of the complexmore » reveals three features. First, the epitope of toxin Cn2 overlaps with essential residues for the binding of {beta}-toxins to its Na+ channel receptor site. Second, the putative recognition of Css2 involves mainly residues that are present in both Cn2 and Css2 toxins. Finally, the effect on the increase of affinity of previously reported key residues during the maturation process of different scFvs can be inferred from the structure. Taken together, these results provide the structural basis that explain the mechanism of the 9004G neutralizing activity and give insight into the process of directed evolution that gave rise to this family of neutralizing scFvs.« less

Stimulated Emission Depletion Nanoscopy Reveals Time-Course of Human Immunodeficiency Virus Proteolytic Maturation.

PubMed

Hanne, Janina; Göttfert, Fabian; Schimer, Jiří; Anders-Össwein, Maria; Konvalinka, Jan; Engelhardt, Johann; Müller, Barbara; Hell, Stefan W; Kräusslich, Hans-Georg

2016-09-27

Concomitant with human immunodeficiency virus type 1 (HIV-1) budding from a host cell, cleavage of the structural Gag polyproteins by the viral protease (PR) triggers complete remodeling of virion architecture. This maturation process is essential for virus infectivity. Electron tomography provided structures of immature and mature HIV-1 with a diameter of 120-140 nm, but information about the sequence and dynamics of structural rearrangements is lacking. Here, we employed super-resolution STED (stimulated emission depletion) fluorescence nanoscopy of HIV-1 carrying labeled Gag to visualize the virion architecture. The incomplete Gag lattice of immature virions was clearly distinguishable from the condensed distribution of mature protein subunits. Synchronized activation of PR within purified particles by photocleavage of a caged PR inhibitor enabled time-resolved in situ observation of the induction of proteolysis and maturation by super-resolution microscopy. This study shows the rearrangement of subviral structures in a super-resolution light microscope over time, outwitting phototoxicity and fluorophore bleaching through synchronization of a biological process by an optical switch.

Effect of covering composting piles with mature compost on ammonia emission and microbial community structure of composting process.

PubMed

Maeda, Koki; Morioka, Riki; Osada, Takashi

2009-01-01

To control ammonia (NH(3)) volatilization from the dairy cattle (Bos taurus) manure composting process, a compost pile was covered with mature compost and the gas emissions evaluated using the dynamic chamber system. The peak of NH(3) volatilization observed immediately after piling up of the compost was reduced from 196 to 62 mg/m(3) by covering the compost pile with mature compost. The accumulation of NH(4)-N to the covered mature compost was also observed. Covering and mixing the compost with mature compost had no effect on the microbial community structure. However, over time the microbial community structure changed because of a decrease in easily degradable organic compounds in the compost piles. The availability of volatile fatty acids (VFA) was considered to be important for microbial community structure in the compost. After the VFA had disappeared, the NO(3)-N concentration increased and the cellulose degrading bacteria such as Cytophaga increased in number.

ATP Depletion Blocks Herpes Simplex Virus DNA Packaging and Capsid Maturation

PubMed Central

Dasgupta, Anindya; Wilson, Duncan W.

1999-01-01

During herpes simplex virus (HSV) assembly, immature procapsids must expel their internal scaffold proteins, transform their outer shell to form mature polyhedrons, and become packaged with the viral double-stranded (ds) DNA genome. A large number of virally encoded proteins are required for successful completion of these events, but their molecular roles are poorly understood. By analogy with the dsDNA bacteriophage we reasoned that HSV DNA packaging might be an ATP-requiring process and tested this hypothesis by adding an ATP depletion cocktail to cells accumulating unpackaged procapsids due to the presence of a temperature-sensitive lesion in the HSV maturational protease UL26. Following return to permissive temperature, HSV capsids were found to be unable to package DNA, suggesting that this process is indeed ATP dependent. Surprisingly, however, the display of epitopes indicative of capsid maturation was also inhibited. We conclude that either formation of these epitopes directly requires ATP or capsid maturation is normally arrested by a proofreading mechanism until DNA packaging has been successfully completed. PMID:9971781

Cell type-specific glycoconjugates of collecting duct cells during maturation of the rat kidney.

PubMed

Holthöfer, H

1988-08-01

The ontogeny of lectin-positive epithelial cell types and the maturation of polarized expression of the glycocalyx of the collecting ducts (CD) of the rat kidney were studied from samples of 18th-day fetal and neonatal kidneys of various ages. Lectins from Dolichos biflorus (DBA) and Vicia villosa (VVA), with preferential affinity to principal cells, stained virtually all CD cells of the fetal kidneys. However, within two days postnatally, the number of cells positive for DBA and VVA decreased to amounts found in the adult kidneys. Moreover, a characteristic change occurred rapidly after birth in the intracellular polarization of the reactive glycoconjugates, from a uniform plasmalemmal to a preferentially apical staining. In contrast, lectins from Arachis hypogaea (PNA), Maclura pomifera (MPA) and Lotus tetragonolobus (LTA), reacting indiscriminatively with principal and intercalated cells of adult kidneys, stained most CD cells in the fetal kidneys, and failed to show any postnatal change in the amount of positive cells or in the intracellular polarization. The immunocytochemical tests for (Na + K)-ATPase and carbonic anhydrase (CA II) revealed the characteristic postnatal decrease in the amount of principal cells and simultaneous increase in the amount of CA II rich intercalated cells. DBA and VVA reactive cells also decreased postnatally, paralleling the changes observed in the (Na + K)-ATPase positive principal cells. The present results suggest that the expression of the cell type-specific glycocalyx of principal and intercalated cells is developmentally regulated, undergoes profound changes during maturation, and is most likely associated with electrolyte transport phenomena.

Determining Roles of Accessory Genes in Denitrification by Mutant Fitness Analyses

DOE PAGES

Vaccaro, Brian J.; Thorgersen, Michael P.; Lancaster, W. Andrew; ...

2015-10-09

Enzymes of the denitrification pathway play an important role in the global nitrogen cycle, including release of nitrous oxide, an ozone-depleting greenhouse gas. In addition, nitric oxide reductase, maturation factors, and proteins associated with nitric oxide detoxification are used by pathogens to combat nitric oxide release by host immune systems. While the core reductases that catalyze the conversion of nitrate to dinitrogen are well understood at a mechanistic level, there are many peripheral proteins required for denitrification whose basic function is unclear. A bar-coded transposon DNA library fromPseudomonas stutzeristrain RCH2 was grown under denitrifying conditions, using nitrate or nitrite asmore » an electron acceptor, and also under molybdenum limitation conditions, with nitrate as the electron acceptor. Analysis of sequencing results from these growths yielded gene fitness data for 3,307 of the 4,265 protein-encoding genes present in strain RCH2. The insights presented here contribute to our understanding of how peripheral proteins contribute to a fully functioning denitrification pathway. We propose a new low-affinity molybdate transporter, OatABC, and show that differential regulation is observed for two MoaA homologs involved in molybdenum cofactor biosynthesis. We also propose that NnrS may function as a membrane-bound NO sensor. Finally, the dominant HemN paralog involved in heme biosynthesis is identified, and a CheR homolog is proposed to function in nitrate chemotaxis. In addition, new insights are provided into nitrite reductase redundancy, nitric oxide reductase maturation, nitrous oxide reductase maturation, and regulation.« less

Maturation of the cytochrome cd1 nitrite reductase NirS from Pseudomonas aeruginosa requires transient interactions between the three proteins NirS, NirN and NirF

PubMed Central

Nicke, Tristan; Schnitzer, Tobias; Münch, Karin; Adamczack, Julia; Haufschildt, Kristin; Buchmeier, Sabine; Kucklick, Martin; Felgenträger, Undine; Jänsch, Lothar; Riedel, Katharina; Layer, Gunhild

2013-01-01

The periplasmic cytochrome cd1 nitrite reductase NirS occurring in denitrifying bacteria such as the human pathogen Pseudomonas aeruginosa contains the essential tetrapyrrole cofactors haem c and haem d1. Whereas the haem c is incorporated into NirS by the cytochrome c maturation system I, nothing is known about the insertion of the haem d1 into NirS. Here, we show by co-immunoprecipitation that NirS interacts with the potential haem d1 insertion protein NirN in vivo. This NirS–NirN interaction is dependent on the presence of the putative haem d1 biosynthesis enzyme NirF. Further, we show by affinity co-purification that NirS also directly interacts with NirF. Additionally, NirF is shown to be a membrane anchored lipoprotein in P. aeruginosa. Finally, the analysis by UV–visible absorption spectroscopy of the periplasmic protein fractions prepared from the P. aeruginosa WT (wild-type) and a P. aeruginosa ΔnirN mutant shows that the cofactor content of NirS is altered in the absence of NirN. Based on our results, we propose a potential model for the maturation of NirS in which the three proteins NirS, NirN and NirF form a transient, membrane-associated complex in order to achieve the last step of haem d1 biosynthesis and insertion of the cofactor into NirS. PMID:23683062

The Fc Region of an Antibody Impacts the Neutralization of West Nile Viruses in Different Maturation States

PubMed Central

Lee, Phong D.; Mukherjee, Swati; Edeling, Melissa A.; Dowd, Kimberly A.; Austin, S. Kyle; Manhart, Carolyn J.; Diamond, Michael S.; Fremont, Daved H.

2013-01-01

Flavivirus-infected cells secrete a structurally heterogeneous population of viruses because of an inefficient virion maturation process. Flaviviruses assemble as noninfectious, immature virions composed of trimers of envelope (E) and precursor membrane (prM) protein heterodimers. Cleavage of prM is a required process during virion maturation, although this often remains incomplete for infectious virus particles. Previous work demonstrated that the efficiency of virion maturation could impact antibody neutralization through changes in the accessibility of otherwise cryptic epitopes on the virion. In this study, we show that the neutralization potency of monoclonal antibody (MAb) E33 is sensitive to the maturation state of West Nile virus (WNV), despite its recognition of an accessible epitope, the domain III lateral ridge (DIII-LR). Comprehensive epitope mapping studies with 166 E protein DIII-LR variants revealed that the functional footprint of MAb E33 on the E protein differs subtly from that of the well-characterized DIII-LR MAb E16. Remarkably, aromatic substitutions at E protein residue 306 ablated the maturation state sensitivity of E33 IgG, and the neutralization efficacy of E33 Fab fragments was not affected by changes in the virion maturation state. We propose that E33 IgG binding on mature virions orients the Fc region in a manner that impacts subsequent antibody binding to nearby sites. This Fc-mediated steric constraint is a novel mechanism by which the maturation state of a virion modulates the efficacy of the humoral immune response to flavivirus infection. PMID:24109224

Effects of drying process on the physicochemical properties of nopal cladodes at different maturity stages.

PubMed

Contreras-Padilla, Margarita; Gutiérrez-Cortez, Elsa; Valderrama-Bravo, María Del Carmen; Rojas-Molina, Isela; Espinosa-Arbeláez, Diego Germán; Suárez-Vargas, Raúl; Rodríguez-García, Mario Enrique

2012-03-01

Chemical proximate analysis was done in order to determine the changes of nutritional characteristics of nopal powders from three different maturity stages 50, 100, and 150 days and obtained by three different drying processes: freeze dried, forced air oven, and tunnel. Results indicate that nopal powder obtained by the process of freeze dried retains higher contents of protein, soluble fiber, and fat than the other two processes. Also, freeze dried process had less effect on color hue variable. No changes were observed in insoluble fiber content, chroma and lightness with the three different drying processes. Furthermore, the soluble fibers decreased with the age of nopal while insoluble fibers and ash content shows an opposite trend. In addition, the luminosity and hue values did not show differences among the maturity stages studied. The high content of dietary fibers of nopal pad powder could to be an interesting source of these important components for human diets and also could be used in food, cosmetics and pharmaceutical industry.

Adolescent Judgement As Evidenced In Response To Poetry

ERIC Educational Resources Information Center

Mason, J. S.

1974-01-01

In this article, the nature of adolescent judgement was investigated by means of two components: the maturation of adolescent mental processes and the interaction of poetry in that maturation. (Author/RK)

Rapid measurement of cotton fiber maturity and fineness by image analysis microscopy using the Cottonscope®

USDA-ARS?s Scientific Manuscript database

Two of the important cotton fiber quality and processing parameters are fiber maturity and fineness. Fiber maturity is the degree of development of the fiber’s secondary wall, and fiber fineness is a measure of the fiber’s linear density and can be expressed as mass per unit length. A well-known m...

A Quantitative Assessment of the Structure and Functions of a Mature Bottomland Hardwood Community: The Iatt Creek Ecosystem Site

Treesearch

Calvin E. Meier; John A. Stanturf; Emile S. Gardiner; Paul B. Hamel; Melvin L. Warren

1999-01-01

We report our efforts, initiated in 1995, to quantify ecological processes and functions in a relatively undisturbed, mature hardwood forest. The 320-ha site is located in central Louisiana on the upper reaches of Iatt Creek, an anastomosing minor stream bottom. The forest is a mature sweetgum (Liquidambar styraciflua L.)-cherrybark oak (

Generating and repairing genetically programmed DNA breaks during immunoglobulin class switch recombination

PubMed Central

Nicolas, Laura; Cols, Montserrat; Choi, Jee Eun; Chaudhuri, Jayanta; Vuong, Bao

2018-01-01

Adaptive immune responses require the generation of a diverse repertoire of immunoglobulins (Igs) that can recognize and neutralize a seemingly infinite number of antigens. V(D)J recombination creates the primary Ig repertoire, which subsequently is modified by somatic hypermutation (SHM) and class switch recombination (CSR). SHM promotes Ig affinity maturation whereas CSR alters the effector function of the Ig. Both SHM and CSR require activation-induced cytidine deaminase (AID) to produce dU:dG mismatches in the Ig locus that are transformed into untemplated mutations in variable coding segments during SHM or DNA double-strand breaks (DSBs) in switch regions during CSR. Within the Ig locus, DNA repair pathways are diverted from their canonical role in maintaining genomic integrity to permit AID-directed mutation and deletion of gene coding segments. Recently identified proteins, genes, and regulatory networks have provided new insights into the temporally and spatially coordinated molecular interactions that control the formation and repair of DSBs within the Ig locus. Unravelling the genetic program that allows B cells to selectively alter the Ig coding regions while protecting non-Ig genes from DNA damage advances our understanding of the molecular processes that maintain genomic integrity as well as humoral immunity. PMID:29744038

Metal–organic framework with optimally selective xenon adsorption and separation

DOE PAGES

Banerjee, Debasis; Simon, Cory M.; Plonka, Anna M.; ...

2016-06-13

Nuclear energy is considered among the most viable alternatives to our current fossil fuel based energy economy.1 The mass-deployment of nuclear energy as an emissions-free source requires the reprocessing of used nuclear fuel to mitigate the waste.2 One of the major concerns with reprocessing used nuclear fuel is the release of volatile radionuclides such as Xe and Kr. The most mature process for removing these radionuclides is energy- and capital-intensive cryogenic distillation. Alternatively, porous materials such as metal-organic frameworks (MOFs) have demonstrated the ability to selectively adsorb Xe and Kr at ambient conditions.3-8 High-throughput computational screening of large databases ofmore » porous materials has identified a calcium-based nanoporous MOF, SBMOF-1, as the most selective for Xe over Kr.9,10 Here, we affirm this prediction and report that SBMOF-1 exhibits by far the highest Xe adsorption capacity and a remarkable Xe/Kr selectivity under relevant nuclear reprocessing conditions. The exceptional selectivity of SBMOF-1 is attributed to its pore size tailored to Xe and its dense wall of atoms that constructs a binding site with a high affinity for Xe, as evident by single crystal X-ray diffraction and molecular simulation.« less

The molecular composition of ambers

USGS Publications Warehouse

Grimalt, J.O.; Simoneit, B.R.T.; Hatcher, P.G.; Nissenbaum, A.

1988-01-01

Bulk (elemental composition, IR, CP/MAS 13C NMR) and molecular (GC-MS) analyses have been performed on a series of ambers and resins derived from different locations (Dominican Republic, Philippines, Canada, Israel, New Zealand, Chile) having diverse botanical affinities (Araucariaceae, Hymenaea) and variable age (from Holocene to Early Cretaceous). No major differences have been observed from the elemental composition and the spectroscopic data; however, the molecular analyses of the solvent extractable fraction show that a specific mixture of components is present in each sample. These are mainly diterpenoid products that in general are also found abundantly in the higher plants from which the ambers and resins originate. Nevertheless, a direct relationship between major terpenoid constituents in fossil resins and precursor plant materials can only be established for the younger samples. Irrespective of the geographical or botanical origin of the ambers and resins, several common age-dependent molecular transformation trends can be recognized: (1) progressive loss of olefinic bonds (especially those located in exocyclic positions), (2) decrease of functionalized products, and (3) increasing proportion of aromatized components. However, even in the samples of older age (Cretaceous) the degree of aromatization is very low when compared with that of other higher-plant related materials such as fossilized woods or low rank coals. This indicates that maturation must involve essentially olefin polymerization processes instead of extensive aromatization. ?? 1988.

Unexpected trend in the compositional maturity of second-cycle sand

USGS Publications Warehouse

Solano-Acosta, W.; Dutta, P.K.

2005-01-01

It is generally accepted that recycling of sandstone generates relatively more mature sand than its parent sandstone. Such maturity is accomplished mainly through chemical weathering as the chemically unstable minerals are eliminated. Because chemical weathering is ubiquitous on the Earth's surface, maturity due to recycling is expected in most geological settings. However, contrary to one's expectation, second-cycle Holocene sand, exclusively derived from sandy facies of the first-cycle Pennsylvanian-Permian Cutler Formation, is actually less mature than its first-cycle parent near Gateway, Colorado. Both the Cutler sandstone and Holocene sand were the products of similar geological processes that controlled their respective composition. In spite of such similarities, a significant difference in composition is observed. We propose that the unexpected immaturity in second-cycle Holocene sand may be due to mechanical disintegration of coarse-grained feldspar and feldspar-rich rock fragments into relatively smaller fractions. Results presented in this paper are the first quantitative estimation of recycling of parent sandstone into daughter sand, and the first observed reverse maturity trend in second-cycle sand. These unexpected results suggest the need for further research to quantitatively understand the recycling process. ?? 2005 Elsevier B.V. All rights reserved.

Explanation of the Reaction of Monoclonal Antibodies with Candida Albicans Cell Surface in Terms of Compound Poisson Process

NASA Astrophysics Data System (ADS)

Dudek, Mirosław R.; Mleczko, Józef

Surprisingly, still very little is known about the mathematical modeling of peaks in the binding affinities distribution function. In general, it is believed that the peaks represent antibodies directed towards single epitopes. In this paper, we refer to fluorescence flow cytometry experiments and show that even monoclonal antibodies can display multi-modal histograms of affinity distribution. This result take place when some obstacles appear in the paratope-epitope reaction such that the process of reaching the specific epitope ceases to be a point Poisson process. A typical example is the large area of cell surface, which could be unreachable by antibodies leading to the heterogeneity of the cell surface repletion. In this case the affinity of cells to bind the antibodies should be described by a more complex process than the pure-Poisson point process. We suggested to use a doubly stochastic Poisson process, where the points are replaced by a binomial point process resulting in the Neyman distribution. The distribution can have a strongly multinomial character, and with the number of modes depending on the concentration of antibodies and epitopes. All this means that there is a possibility to go beyond the simplified theory, one response towards one epitope. As a consequence, our description provides perspectives for describing antigen-antibody reactions, both qualitatively and quantitavely, even in the case when some peaks result from more than one binding mechanism.

Caring for a Seriously Ill Child

MedlinePlus

... medical situation, but also your child's age and maturity level. It's important to know, if possible, what ... process when possible. Depending on their ages and maturity level, visiting the hospital, meeting the nursing and ...

Olive cultivar and maturation process on the oviposition preference of Bactrocera oleae (Rossi) (Diptera: Tephritidae).

PubMed

Malheiro, R; Casal, S; Pinheiro, L; Baptista, P; Pereira, J A

2018-02-21

The olive fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), is a key-pest in the main olives producing areas worldwide, and displays distinct preference to different olive cultivars. The present work intended to study oviposition preference towards three Portuguese cultivars (Cobrançosa, Madural, and Verdeal Transmontana) at different maturation indexes. Multiple oviposition bioassays (multiple-choice and no-choice) were conducted to assess cultivar preference. No-choice bioassays were conducted to assess the influence of different maturation indexes (MI 2; MI 3, and MI 4) in single cultivars. The longevity of olive fly adults according to the cultivar in which its larvae developed was also evaluated through survival assays. Cultivar and maturation are crucial aspects in olive fly preference. Field and laboratory assays revealed a preference towards cv. Verdeal Transmontana olives and a lower susceptibility to cv. Cobrançosa olives. A higher preference was observed for olives at MI 2 and MI 3. The slower maturation process in cv. Verdeal Transmontana (still green while the other cultivars are reddish or at black stage) seems to have an attractive effect on olive fly females, thus increasing its infestation levels. Olive fly adults from both sexes live longer if emerged from pupae developed from cv. Verdeal Transmontana fruits and live less if emerged from cv. Cobrançosa. Therefore, olive cultivar and maturation process are crucial aspects in olive fly preference, also influencing the longevity of adults.

Longitudinal Growth Curves of Brain Function Underlying Inhibitory Control through Adolescence

PubMed Central

Foran, William; Velanova, Katerina; Luna, Beatriz

2013-01-01

Neuroimaging studies suggest that developmental improvements in inhibitory control are primarily supported by changes in prefrontal executive function. However, studies are contradictory with respect to how activation in prefrontal regions changes with age, and they have yet to analyze longitudinal data using growth curve modeling, which allows characterization of dynamic processes of developmental change, individual differences in growth trajectories, and variables that predict any interindividual variability in trajectories. In this study, we present growth curves modeled from longitudinal fMRI data collected over 302 visits (across ages 9 to 26 years) from 123 human participants. Brain regions within circuits known to support motor response control, executive control, and error processing (i.e., aspects of inhibitory control) were investigated. Findings revealed distinct developmental trajectories for regions within each circuit and indicated that a hierarchical pattern of maturation of brain activation supports the gradual emergence of adult-like inhibitory control. Mean growth curves of activation in motor response control regions revealed no changes with age, although interindividual variability decreased with development, indicating equifinality with maturity. Activation in certain executive control regions decreased with age until adolescence, and variability was stable across development. Error-processing activation in the dorsal anterior cingulate cortex showed continued increases into adulthood and no significant interindividual variability across development, and was uniquely associated with task performance. These findings provide evidence that continued maturation of error-processing abilities supports the protracted development of inhibitory control over adolescence, while motor response control regions provide early-maturing foundational capacities and suggest that some executive control regions may buttress immature networks as error processing continues to mature. PMID:24227721

The integrated simulation and assessment of the impacts of process change in biotherapeutic antibody production.

PubMed

Chhatre, Sunil; Jones, Carl; Francis, Richard; O'Donovan, Kieran; Titchener-Hooker, Nigel; Newcombe, Anthony; Keshavarz-Moore, Eli

2006-01-01

Growing commercial pressures in the pharmaceutical industry are establishing a need for robust computer simulations of whole bioprocesses to allow rapid prediction of the effects of changes made to manufacturing operations. This paper presents an integrated process simulation that models the cGMP manufacture of the FDA-approved biotherapeutic CroFab, an IgG fragment used to treat rattlesnake envenomation (Protherics U.K. Limited, Blaenwaun, Ffostrasol, Llandysul, Wales, U.K.). Initially, the product is isolated from ovine serum by precipitation and centrifugation, before enzymatic digestion of the IgG to produce FAB and FC fragments. These are purified by ion exchange and affinity chromatography to remove the FC and non-specific FAB fragments from the final venom-specific FAB product. The model was constructed in a discrete event simulation environment and used to determine the potential impact of a series of changes to the process, such as increasing the step efficiencies or volumes of chromatographic matrices, upon product yields and process times. The study indicated that the overall FAB yield was particularly sensitive to changes in the digestive and affinity chromatographic step efficiencies, which have a predicted 30% greater impact on process FAB yield than do the precipitation or centrifugation stages. The study showed that increasing the volume of affinity matrix has a negligible impact upon total process time. Although results such as these would require experimental verification within the physical constraints of the process and the facility, the model predictions are still useful in allowing rapid "what-if" scenario analysis of the likely impacts of process changes within such an integrated production process.

Analysis of solute-protein interactions and solute-solute competition by zonal elution affinity chromatography.

PubMed

Tao, Pingyang; Poddar, Saumen; Sun, Zuchen; Hage, David S; Chen, Jianzhong

2018-02-02

Many biological processes involve solute-protein interactions and solute-solute competition for protein binding. One method that has been developed to examine these interactions is zonal elution affinity chromatography. This review discusses the theory and principles of zonal elution affinity chromatography, along with its general applications. Examples of applications that are examined include the use of this method to estimate the relative extent of solute-protein binding, to examine solute-solute competition and displacement from proteins, and to measure the strength of these interactions. It is also shown how zonal elution affinity chromatography can be used in solvent and temperature studies and to characterize the binding sites for solutes on proteins. In addition, several alternative applications of zonal elution affinity chromatography are discussed, which include the analysis of binding by a solute with a soluble binding agent and studies of allosteric effects. Other recent applications that are considered are the combined use of immunoextraction and zonal elution for drug-protein binding studies, and binding studies that are based on immobilized receptors or small targets. Copyright © 2018 Elsevier Inc. All rights reserved.

High affinity IgM(+) memory B cells are generated through a germinal center-dependent pathway.

PubMed

Hara, Yasushi; Tashiro, Yasuyuki; Murakami, Akikazu; Nishimura, Miyuki; Shimizu, Takeyuki; Kubo, Masato; Burrows, Peter D; Azuma, Takachika

2015-12-01

During a T cell-dependent immune response, B cells undergo clonal expansion and selection and the induction of isotype switching and somatic hypermutation (SHM). Although somatically mutated IgM(+) memory B cells have been reported, it has not been established whether they are really high affinity B cells. We tracked (4-hydroxy-3-nitrophenyl) acetyl hapten-specific GC B cells from normal immunized mice based on affinity of their B cell receptor (BCR) and performed BCR sequence analysis. SHM was evident by day 7 postimmunization and increased with time, such that high affinity IgM(+) as well as IgG(+) memory B cells continued to be generated up to day 42. In contrast, class-switch recombination (CSR) was almost completed by day 7 and then the ratio of IgG1(+)/IgM(+) GC B cells remained unchanged. Together these findings suggest that IgM(+) B cells undergo SHM in the GC to generate high affinity IgM(+) memory cells and that this process continues even after CSR is accomplished. Copyright © 2015 Elsevier Ltd. All rights reserved.

[Affine transformation-based automatic registration for peripheral digital subtraction angiography (DSA)].

PubMed

Kong, Gang; Dai, Dao-Qing; Zou, Lu-Min

2008-07-01

In order to remove the artifacts of peripheral digital subtraction angiography (DSA), an affine transformation-based automatic image registration algorithm is introduced here. The whole process is described as follows: First, rectangle feature templates are constructed with their centers of the extracted Harris corners in the mask, and motion vectors of the central feature points are estimated using template matching technology with the similarity measure of maximum histogram energy. And then the optimal parameters of the affine transformation are calculated with the matrix singular value decomposition (SVD) method. Finally, bilinear intensity interpolation is taken to the mask according to the specific affine transformation. More than 30 peripheral DSA registrations are performed with the presented algorithm, and as the result, moving artifacts of the images are removed with sub-pixel precision, and the time consumption is less enough to satisfy the clinical requirements. Experimental results show the efficiency and robustness of the algorithm.

Determining the role of missense mutations in the POU domain of HNF1A that reduce the DNA-binding affinity: A computational approach

PubMed Central

P., Sneha; D., Thirumal Kumar; C., George Priya Doss; R., Siva; Zayed, Hatem

2017-01-01

Maturity-onset diabetes of the young type 3 (MODY3) is a non-ketotic form of diabetes associated with poor insulin secretion. Over the past years, several studies have reported the association of missense mutations in the Hepatocyte Nuclear Factor 1 Alpha (HNF1A) with MODY3. Missense mutations in the POU homeodomain (POUH) of HNF1A hinder binding to the DNA, thereby leading to a dysfunctional protein. Missense mutations of the HNF1A were retrieved from public databases and subjected to a three-step computational mutational analysis to identify the underlying mechanism. First, the pathogenicity and stability of the mutations were analyzed to determine whether they alter protein structure and function. Second, the sequence conservation and DNA-binding sites of the mutant positions were assessed; as HNF1A protein is a transcription factor. Finally, the biochemical properties of the biological system were validated using molecular dynamic simulations in Gromacs 4.6.3 package. Two arginine residues (131 and 203) in the HNF1A protein are highly conserved residues and contribute to the function of the protein. Furthermore, the R131W, R131Q, and R203C mutations were predicted to be highly deleterious by in silico tools and showed lower binding affinity with DNA when compared to the native protein using the molecular docking analysis. Triplicate runs of molecular dynamic (MD) simulations (50ns) revealed smaller changes in patterns of deviation, fluctuation, and compactness, in complexes containing the R131Q and R131W mutations, compared to complexes containing the R203C mutant complex. We observed reduction in the number of intermolecular hydrogen bonds, compactness, and electrostatic potential, as well as the loss of salt bridges, in the R203C mutant complex. Substitution of arginine with cysteine at position 203 decreases the affinity of the protein for DNA, thereby destabilizing the protein. Based on our current findings, the MD approach is an important tool for elucidating the impact and affinity of mutations in DNA-protein interactions and understanding their function. PMID:28410371

Quantitative assessment of cervical vertebral maturation using cone beam computed tomography in Korean girls.

PubMed

Byun, Bo-Ram; Kim, Yong-Il; Yamaguchi, Tetsutaro; Maki, Koutaro; Son, Woo-Sung

2015-01-01

This study was aimed to examine the correlation between skeletal maturation status and parameters from the odontoid process/body of the second vertebra and the bodies of third and fourth cervical vertebrae and simultaneously build multiple regression models to be able to estimate skeletal maturation status in Korean girls. Hand-wrist radiographs and cone beam computed tomography (CBCT) images were obtained from 74 Korean girls (6-18 years of age). CBCT-generated cervical vertebral maturation (CVM) was used to demarcate the odontoid process and the body of the second cervical vertebra, based on the dentocentral synchondrosis. Correlation coefficient analysis and multiple linear regression analysis were used for each parameter of the cervical vertebrae (P < 0.05). Forty-seven of 64 parameters from CBCT-generated CVM (independent variables) exhibited statistically significant correlations (P < 0.05). The multiple regression model with the greatest R (2) had six parameters (PH2/W2, UW2/W2, (OH+AH2)/LW2, UW3/LW3, D3, and H4/W4) as independent variables with a variance inflation factor (VIF) of <2. CBCT-generated CVM was able to include parameters from the second cervical vertebral body and odontoid process, respectively, for the multiple regression models. This suggests that quantitative analysis might be used to estimate skeletal maturation status.

Proteomic changes during intestinal cell maturation in vivo

PubMed Central

Chang, Jinsook; Chance, Mark R.; Nicholas, Courtney; Ahmed, Naseem; Guilmeau, Sandra; Flandez, Marta; Wang, Donghai; Byun, Do-Sun; Nasser, Shannon; Albanese, Joseph M.; Corner, Georgia A.; Heerdt, Barbara G.; Wilson, Andrew J.; Augenlicht, Leonard H.; Mariadason, John M.

2008-01-01

Intestinal epithelial cells undergo progressive cell maturation as they migrate along the crypt-villus axis. To determine molecular signatures that define this process, proteins differentially expressed between the crypt and villus were identified by 2D-DIGE and MALDI-MS. Forty-six differentially expressed proteins were identified, several of which were validated by immunohistochemistry. Proteins upregulated in the villus were enriched for those involved in brush border assembly and lipid uptake, established features of differentiated intestinal epithelial cells. Multiple proteins involved in glycolysis were also upregulated in the villus, suggesting increased glycolysis is a feature of intestinal cell differentiation. Conversely, proteins involved in nucleotide metabolism, and protein processing and folding were increased in the crypt, consistent with functions associated with cell proliferation. Three novel paneth cell markers, AGR2, HSPA5 and RRBP1 were also identified. Notably, significant correlation was observed between overall proteomic changes and corresponding gene expression changes along the crypt-villus axis, indicating intestinal cell maturation is primarily regulated at the transcriptional level. This proteomic profiling analysis identified several novel proteins and functional processes differentially induced during intestinal cell maturation in vivo. Integration of proteomic, immunohistochemical, and parallel gene expression datasets demonstrate the coordinated manner in which intestinal cell maturation is regulated. PMID:18824147

Fidelity of metal insertion into hydrogenases.

PubMed

Magalon, A; Blokesch, M; Zehelein, E; Böck, A

2001-06-15

The fidelity of metal incorporation into the active center of hydrogenase 3 from Escherichia coli was studied by analyzing the inhibition of the maturation pathway by zinc and other transition metals. Hydrogenase maturation of wild-type cells was significantly affected only by concentrations of zinc or cadmium higher than 200 microM, whereas a mutant with a lesion in the nickel uptake system displayed a total blockade of the proteolytic processing of the precursor form into the mature form of the large subunit after growth in the presence of 10 microM Zn(2+). The precursor could not be processed in vitro by the maturation endopeptidase even in the presence of an excess of nickel ions. Evidence is presented that zinc does not interfere with the incorporation of iron into the metal center. Precursor of the large subunit accumulated in nickel proficient cells formed a transient substrate complex with the cognate endoprotease HycI whereas that of zinc-supplemented cells did not. The results show that zinc can intrude the nickel-dependent maturation pathway only when nickel uptake is blocked. Under this condition zinc appears to be incorporated at the nickel site of the large subunit and delivers a precursor not amenable to proteolytic processing since the interaction with the endoprotease is blocked.

Transcriptome and Small RNA Deep Sequencing Reveals Deregulation of miRNA Biogenesis in Human Glioma

PubMed Central

Moore, Lynette M.; Kivinen, Virpi; Liu, Yuexin; Annala, Matti; Cogdell, David; Liu, Xiuping; Liu, Chang-Gong; Sawaya, Raymond; Yli-Harja, Olli; Shmulevich, Ilya; Fuller, Gregory N.; Zhang, Wei; Nykter, Matti

2013-01-01

Altered expression of oncogenic and tumor-suppressing microRNAs (miRNAs) is widely associated with tumorigenesis. However, the regulatory mechanisms underlying these alterations are poorly understood. We sought to shed light on the deregulation of miRNA biogenesis promoting the aberrant miRNA expression profiles identified in these tumors. Using sequencing technology to perform both whole-transcriptome and small RNA sequencing of glioma patient samples, we examined precursor and mature miRNAs to directly evaluate the miRNA maturation process, and interrogated expression profiles for genes involved in the major steps of miRNA biogenesis. We found that ratios of mature to precursor forms of a large number of miRNAs increased with the progression from normal brain to low-grade and then to high-grade gliomas. The expression levels of genes involved in each of the three major steps of miRNA biogenesis (nuclear processing, nucleo-cytoplasmic transport, and cytoplasmic processing) were systematically altered in glioma tissues. Survival analysis of an independent data set demonstrated that the alteration of genes involved in miRNA maturation correlates with survival in glioma patients. Direct quantification of miRNA maturation with deep sequencing demonstrated that deregulation of the miRNA biogenesis pathway is a hallmark for glioma genesis and progression. PMID:23007860

Stability and maturity of biowaste composts derived by small municipalities: Correlation among physical, chemical and biological indices.

PubMed

Oviedo-Ocaña, E R; Torres-Lozada, P; Marmolejo-Rebellon, L F; Hoyos, L V; Gonzales, S; Barrena, R; Komilis, D; Sanchez, A

2015-10-01

Stability and maturity are important criteria to guarantee the quality of a compost that is applied to agriculture or used as amendment in degraded soils. Although different techniques exist to evaluate stability and maturity, the application of laboratory tests in municipalities in developing countries can be limited due to cost and application complexities. In the composting facilities of such places, some classical low cost on-site tests to monitor the composting process are usually implemented; however, such tests do not necessarily clearly identify conditions of stability and maturity. In this article, we have applied and compared results of stability and maturity tests that can be easily employed on site (i.e. temperature, pH, moisture, electrical conductivity [EC], odor and color), and of tests that require more complex laboratory techniques (volatile solids, C/N ratio, self-heating, respirometric index, germination index [GI]). The evaluation of the above was performed in the field scale using 2 piles of biowaste applied compost. The monitoring period was from day 70 to day 190 of the process. Results showed that the low-cost tests traditionally employed to monitor the composting process on-site, such as temperature, color and moisture, do not provide consistent determinations with the more complex laboratory tests used to assess stability (e.g. respiration index, self-heating, volatile solids). In the case of maturity tests (GI, pH, EC), both the on-site tests (pH, EC) and the laboratory test (GI) provided consistent results. Although, stability was indicated for most of the samples, the maturity tests indicated that products were consistently immature. Thus, a stable product is not necessarily mature. Conclusively, the decision on the quality of the compost in the installations located in developing countries requires the simultaneous use of a combination of tests that are performed both in the laboratory and on-site. Copyright © 2015 Elsevier Ltd. All rights reserved.

Proteomics: A valuable approach to elucidate spermatozoa post -testicular maturation in the endangered Acipenseridae family.

PubMed

Boccaletto, Pietro; Siddique, Mohammad Abdul Momin; Cosson, Jacky

2018-05-01

Proteomics techniques, such as two-dimensional polyacrylamide gel electrophoresis, mass spectrometry, and differential gel electrophoresis, have been extensively used to describe the protein composition of male gametes in different animals, mainly mammals. They have also provided a deeper understanding of protein functions involved in sperm processes, as in processes that in humans lead to male infertility. However, few studies focus on fish sperm proteomics and even fewer have tried to explore the proteomic profile of Sturgeon spermatozoa. Sturgeon is an endangered, ancient group of fish species exploited mostly for caviar. In this fish group, a part of the process that leads to final functional maturation of spermatozoa so as to have the capability to activate eggs during the fertilization process. This process has a broad similarity to post-testicular maturation in mammals; where spermatozoa leaving the testes must be mixed with seminal fluid along the transit through the Wolffian ducts to modify its surface membrane protein composition, leading to axonemal and acrosomal competence. The aim of this study was to review the current literature on various proteomic techniques, their usefulness in separating, identifying and studying the proteome composition of the fish spermatozoon, as well as their potential applications in studying the post-testicular maturation process in Sturgeon. Such understanding could lead to development of more sophisticated aquaculture techniques, favorable for sturgeon reproduction. Copyright © 2018 Elsevier B.V. All rights reserved.

How Much Binding Affinity Can be Gained by Filling a Cavity?

PubMed Central

Kawasaki, Yuko; Chufan, Eduardo E.; Lafont, Virginie; Hidaka, Koushi; Kiso, Yoshiaki; Amzel, L. Mario; Freire, Ernesto

2011-01-01

Binding affinity optimization is critical during drug development. Here we evaluate the thermodynamic consequences of filling a binding cavity with functionalities of increasing van der Waals radii (-H, -F, -Cl and CH3) that improve the geometric fit without participating in hydrogen bonding or other specific interactions. We observe a binding affinity increase of two orders of magnitude. There appears to be three phases in the process. The first phase is associated with the formation of stable van der Waals interactions. This phase is characterized by a gain in binding enthalpy and a loss in binding entropy, attributed to a loss of conformational degrees of freedom. For the specific case presented in this paper, the enthalpy gain amounts to −1.5 kcal/mol while the entropic losses amount to +0.9 kcal/mol resulting in a net 3.5-fold affinity gain. The second phase is characterized by simultaneous enthalpic and entropic gains. This phase improves the binding affinity 25-fold. The third phase represents the collapse of the trend and is triggered by the introduction of chemical functionalities larger than the binding cavity itself (CH(CH3)2). It is characterized by large enthalpy and affinity losses. The thermodynamic signatures associated with each phase provide guidelines for lead optimization. PMID:20028396

Selection is more intelligent than design: improving the affinity of a bivalent ligand through directed evolution.

PubMed

Ahmad, Kareem M; Xiao, Yi; Soh, H Tom

2012-12-01

Multivalent molecular interactions can be exploited to dramatically enhance the performance of an affinity reagent. The enhancement in affinity and specificity achieved with a multivalent construct depends critically on the effectiveness of the scaffold that joins the ligands, as this determines their positions and orientations with respect to the target molecule. Currently, no generalizable design rules exist for construction of an optimal multivalent ligand for targets with known structures, and the design challenge remains an insurmountable obstacle for the large number of proteins whose structures are not known. As an alternative to such design-based strategies, we report here a directed evolution-based method for generating optimal bivalent aptamers. To demonstrate this approach, we fused two thrombin aptamers with a randomized DNA sequence and used a microfluidic in vitro selection strategy to isolate scaffolds with exceptionally high affinities. Within five rounds of selection, we generated a bivalent aptamer that binds thrombin with an apparent dissociation constant (K(d)) <10 pM, representing a ∼200-fold improvement in binding affinity over the monomeric aptamers and a ∼15-fold improvement over the best designed bivalent construct. The process described here can be used to produce high-affinity multivalent aptamers and could potentially be adapted to other classes of biomolecules.

New Model of Information Technology Governance in the Government of Gorontalo City using Framework COBIT 4.1

NASA Astrophysics Data System (ADS)

Bouty, A. A.; Koniyo, M. H.; Novian, D.

2018-02-01

This study aims to determine the level of maturity of information technology governance in Gorontalo city government by applying the COBIT framework 4.1. The research method is the case study method, by conducting surveys and data collection at 25 institution in Gorontalo City. The results of this study is the analysis of information technology needs based on the measurement of maturity level. The results of the measurement of the maturity level of information technology governance shows that there are still many business processes running at lower level, from 9 existing business processes there are 4 processes at level 2 (repetitive but intuitive) and 3 processes at level 1 (Initial/Ad hoc). With these results, is expected that the government of Gorontalo city immediately make improvements to the governance of information technology so that it can run more effectively and efficiently.

MicroRNA-Mediated Down-Regulation of M-CSF Receptor Contributes to Maturation of Mouse Monocyte-Derived Dendritic Cells

PubMed Central

Riepsaame, Joey; van Oudenaren, Adri; den Broeder, Berlinda J. H.; van IJcken, Wilfred F. J.; Pothof, Joris; Leenen, Pieter J. M.

2013-01-01

Dendritic cell (DC) maturation is a tightly regulated process that requires coordinated and timed developmental cues. Here we investigate whether microRNAs are involved in this process. We identify microRNAs in mouse GM-CSF-generated, monocyte-related DC (GM-DC) that are differentially expressed during both spontaneous and LPS-induced maturation and characterize M-CSF receptor (M-CSFR), encoded by the Csf1r gene, as a key target for microRNA-mediated regulation in the final step toward mature DC. MicroRNA-22, -34a, and -155 are up-regulated in mature MHCIIhi CD86hi DC and mediate Csf1r mRNA and protein down-regulation. Experimental inhibition of Csf1r-targeting microRNAs in vitro results not only in sustained high level M-CSFR protein expression but also in impaired DC maturation upon stimulation by LPS. Accordingly, over-expression of Csf1r in GM-DC inhibits terminal differentiation. Taken together, these results show that developmentally regulated microRNAs control Csf1r expression, supplementing previously identified mechanisms that regulate its transcription and protein surface expression. Furthermore, our data indicate a novel function for Csf1r in mouse monocyte-derived DC, showing that down-regulation of M-CSFR expression is essential for final DC maturation. PMID:24198819

The Microanatomic Segregation of Selection by Apoptosis in the Germinal Center

PubMed Central

Mayer, Christian T.; Gazumyan, Anna; Kara, Ervin E.; Gitlin, Alexander D.; Golijanin, Jovana; Viant, Charlotte; Pai, Joy; Oliveira, Thiago Y.; Wang, Qiao; Escolano, Amelia; Medina-Ramirez, Max; Sanders, Rogier W.; Nussenzweig, Michel C.

2018-01-01

B cells undergo rapid cell division and affinity maturation in anatomically distinct sites in lymphoid organs called germinal centers (GCs). Homeostasis is maintained in part by B-cell apoptosis. However, the precise contribution of apoptosis to GC biology and selection is not well defined. We developed apoptosis-indicator mice and used them to visualize, purify, and characterize dying GC B cells. Apoptosis is prevalent in the GC with up to half of all GC B cells dying every 6h. Moreover, programmed cell death is differentially regulated in the light zone (LZ) and the dark zone (DZ): LZ B cells die by default if they are not positively selected, whereas DZ cells die when their antigen receptors are damaged by activation-induced cytidine deaminase (AID). PMID:28935768

Parallel selection of antibody libraries on phage and yeast surfaces via a cross-species display.

PubMed

Patel, Chirag A; Wang, Jinqing; Wang, Xinwei; Dong, Feng; Zhong, Pingyu; Luo, Peter P; Wang, Kevin C

2011-09-01

We created a cross-species display system that allows the display of the same antibody libraries on both prokaryotic phage and eukaryotic yeast without the need for molecular cloning. Using this cross-display system, a large, diverse library can be constructed once and subsequently used for display and selection in both phage and yeast systems. In this article, we performed the parallel phage and yeast selection of an antibody maturation library using this cross-display platform. This parallel selection allowed us to isolate more unique hits than single-species selection, with 162 unique clones from phage and 107 unique clones from yeast. In addition, we were able to shuttle yeast hits back to Escherichia coli cells for affinity characterization at a higher throughput.

Intracellular processing, glycosylation, and cell surface expression of human metapneumovirus attachment glycoprotein.

PubMed

Liu, Li; Bastien, Nathalie; Li, Yan

2007-12-01

The biosynthesis and posttranslational processing of human metapneumovirus attachment G glycoprotein were investigated. After pulse-labeling, the G protein accumulated as three species with molecular weights of 45,000, 50,000, and 53,000 (45K, 50K, and 53K, respectively). N-Glycosidase digestion indicated that these forms represent the unglycosylated precursor and N-glycosylated intermediate products, respectively. After an appropriate chase, these three naive forms were further processed to a mature 97K form. The presence of O-linked sugars in mature G protein was confirmed by O-glycanase digestion and lectin-binding assay using Arachis hypogaea (peanut agglutinin), an O-glycan-specific lectin. In addition, in the O-glycosylation-deficient cell line (CHO ldlD cell), the G protein could not be processed to the mature form unless the exogenous Gal and GalNAc were supplemented, which provided added evidence supporting the O-linked glycosylation of G protein. The maturation of G was completely blocked by monensin but was partially sensitive to brefeldin A (BFA), suggesting the O-linked glycosylation of G initiated in the trans-Golgi compartment and terminated in the trans-Golgi network. Enzymatic deglycosylation analysis confirmed that the BFA-G was a partial mature form containing N-linked oligosaccharides and various amounts of O-linked carbohydrate side chains. The expression of G protein at the cell surface could be detected by indirect immunofluorescence staining assay. Furthermore, cell surface immunoprecipitation displayed an efficient intracellular transport of G protein.

Characterization of Dedifferentiating Human Mature Adipocytes from the Visceral and Subcutaneous Fat Compartments: Fibroblast-Activation Protein Alpha and Dipeptidyl Peptidase 4 as Major Components of Matrix Remodeling

PubMed Central

Lessard, Julie; Pelletier, Mélissa; Biertho, Laurent; Biron, Simon; Marceau, Simon; Hould, Frédéric-Simon; Lebel, Stéfane; Moustarah, Fady; Lescelleur, Odette; Marceau, Picard; Tchernof, André

2015-01-01

Mature adipocytes can reverse their phenotype to become fibroblast-like cells. This is achieved by ceiling culture and the resulting cells, called dedifferentiated fat (DFAT) cells, are multipotent. Beyond the potential value of these cells for regenerative medicine, the dedifferentiation process itself raises many questions about cellular plasticity and the pathways implicated in cell behavior. This work has been performed with the objective of obtaining new information on adipocyte dedifferentiation, especially pertaining to new targets that may be involved in cellular fate changes. To do so, omental and subcutaneous mature adipocytes sampled from severely obese subjects have been dedifferentiated by ceiling culture. An experimental design with various time points along the dedifferentiation process has been utilized to better understand this process. Cell size, gene and protein expression as well as cytokine secretion were investigated. Il-6, IL-8, SerpinE1 and VEGF secretion were increased during dedifferentiation, whereas MIF-1 secretion was transiently increased. A marked decrease in expression of mature adipocyte transcripts (PPARγ2, C/EBPα, LPL and Adiponectin) was detected early in the process. In addition, some matrix remodeling transcripts (FAP, DPP4, MMP1 and TGFβ1) were rapidly and strongly up-regulated. FAP and DPP4 proteins were simultaneously induced in dedifferentiating mature adipocytes supporting a potential role for these enzymes in adipose tissue remodeling and cell plasticity. PMID:25816202

Affinity-based precipitation via a bivalent peptidic hapten for the purification of monoclonal antibodies.

PubMed

Handlogten, Michael W; Stefanick, Jared F; Deak, Peter E; Bilgicer, Basar

2014-09-07

In a previous study, we demonstrated a non-chromatographic affinity-based precipitation method, using trivalent haptens, for the purification of mAbs. In this study, we significantly improved this process by using a simplified bivalent peptidic hapten (BPH) design, which enables facile and rapid purification of mAbs while overcoming the limitations of the previous trivalent design. The improved affinity-based precipitation method (ABP(BPH)) combines the simplicity of salt-induced precipitation with the selectivity of affinity chromatography for the purification of mAbs. The ABP(BPH) method involves 3 steps: (i) precipitation and separation of protein contaminants larger than immunoglobulins with ammonium sulfate; (ii) selective precipitation of the target-antibody via BPH by inducing antibody-complex formation; (iii) solubilization of the antibody pellet and removal of BPH with membrane filtration resulting in the pure antibody. The ABP(BPH) method was evaluated by purifying the pharmaceutical antibody trastuzumab from common contaminants including CHO cell conditioned media, DNA, ascites fluid, other antibodies, and denatured antibody with >85% yield and >97% purity. Importantly, the purified antibody demonstrated native binding activity to cell lines expressing the target protein, HER2. Combined, the ABP(BPH) method is a rapid and scalable process for the purification of antibodies with the potential to improve product quality while decreasing purification costs.

Residual effects of low oxygen storage of mature green fruit on ripening processes and ester biosynthesis during ripening in bananas

USDA-ARS?s Scientific Manuscript database

Mature green banana (Musa sapientum L. cv. Cavendish) fruit were stored in 0.5%, 2 %, or 21% O2 for 7 days at 20 °C before ripening was initiated by ethylene. Residual effects of low O2 storage in mature green fruit on ripening and ester biosynthesis in fruit were investigated during ripening period...

Apical closure of mature molar roots with the use of calcium hydroxide.

PubMed

Rotstein, I; Friedman, S; Katz, J

1990-11-01

Calcium hydroxide may induce apical root closure in affected mature teeth as well as in immature teeth. Once an apical hard tissue barrier is formed, a permanent root canal filling can be safely condensed. Two cases are described in which calcium hydroxide induced apical root closure in mature molar teeth where the apical constriction was lost because of chronic inflammatory process.

A Developmental Model of Cross-Cultural Competence at the Tactical Level

DTIC Science & Technology

2010-11-01

components of 3C and describe how 3C develops in Soldiers. Five components of 3C were identified: Cultural Maturity , Cognitive Flexibility, Cultural...a result of the data analysis: Cultural Maturity , Cognitive Flexibility, Cultural Knowledge, Cultural Acuity, and Interpersonal Skills. These five...create regressions in the 3C development process. In short, KSAAs mature interdependently and simultaneously. Thus, development and transitions across

Role of the C-terminal and chitin insertion domains on enzymatic activity of endochitinase ChiA74 of Bacillus thuringiensis.

PubMed

Juárez-Hernández, Estefania O; Casados-Vázquez, Luz E; Bideshi, Dennis K; Salcedo-Hernández, R; Barboza-Corona, José E

2017-09-01

ChiA74 has modular structure that includes a secretion signal peptide (sp) sequence, and catalytic (CD), chitin insertion (CID), fibronectin type-III (FnIII) and chitin binding (CBD) domains. We described for the first time the existence of a putative CID in ChiA74. Mature ChiA74 lacking its sp sequence (rChiA74Δsp, ∼70kDa) and two truncated versions, rChiA74Δsp-60, rChiA74Δsp-50 lacking, respectively, CBD and CDB-FnIII were produced. rChiA74Δsp and rChiA74Δsp-60 are unstable and were processed to generate stable proteins of ∼50kDa. With colloidal chitin, rChiA74Δsp and rChiA74Δsp-50 had higher activity than rChiA74Δsp-60. rChiA74Δsp showed similar ability to bind chitin than rChiA74Δsp-50. The catalytic efficiencies (kcat/Km) of rChiA74Δsp and rChiA74Δsp-50 were higher, ∼ 21-fold than rChiA74Δsp-60, using chitin as the substrate. Optimal activity was detected at pH 7 and 40°C. Data suggest that the CBD in ChiA74 is important for binding to chitin, but not necessary as the presence of a CID together with the CD in a stable truncated version (i.e. ChiA74Δsp-50) has similar affinity and hydrolytic activity as the mature enzyme. The CID of ChiA74 showed identities of ∼ 55% with CIDs of other chitinases such as those from B. circulans and B. licheniformis, respectively, and conserved residues important for interacting with chitin. Copyright © 2017 Elsevier B.V. All rights reserved.

Sedimentology of Hirnantian glaciomarine deposits in the Balkan Terrane, western Bulgaria: Fixing a piece of the north peri-Gondwana jigsaw puzzle

NASA Astrophysics Data System (ADS)

Chatalov, Athanas

2017-04-01

Glaciomarine deposits of late Hirnantian age in the western part of the Palaeozoic Balkan Terrane have persistent thickness ( 7 m) and lateral uniformity in rock colour, bedding pattern, lithology, and sedimentary structures. Four lithofacies are distinguished from base to top: lonestone-bearing diamictites, interbedded structureless mudstones, crudely laminated diamictites, and finely laminated mudstones. The diamictites are clast-poor to clast-rich comprising muddy to sandy varieties. Their compositional maturity is evidenced by the very high amount of detrital quartz compared to the paucity of feldspar and unstable lithic grains. Other textural components include extraclasts derived from the local Ordovician basement, mudstone intraclasts, and sediment aggregates. Turbate structures, grain lineations, and soft sediment deformation of the matrix below larger grains are locally observed. Sedimentological analysis reveals that deposition occurred in an ice-intermediate to ice-distal, poorly agitated shelf environment by material supplied from meltwater buoyant plumes and rain-out from ice-rafted debris. Remobilization by mass-flow processes (cohesive debris flows and slumps) was an important mechanism particularly for the formation of massive diamictites. The glaciomarine deposits represent a typical deglaciation sequence reflecting retreat of the ice front (grounded or floating ice sheet), relative sea-level rise and gradually reduced sedimentation rate with increasing contribution from suspension fallout. This sequence was deposited on the non-glaciated shelf of the intracratonic North Gondwana platform along the southern margin of the Rheic Ocean. The Hirnantian strata of the Balkan Terrane can be correlated with similar glaciomarine deposits known from peri-Gondwana terranes elsewhere in Europe showing clear 'Armorican affinity'. Several lines of evidence suggest that the provenance of siliciclastic material was associated mainly with sedimentary recycling of mature sands which had been deposited across North Gondwana in Cambrian and pre-glacial Ordovician times.

Effects of the anti-androgen cyproterone acetate (CPA) on oocyte meiotic maturation in rainbow trout (Oncorhynchus mykiss).

PubMed

Rime, Hélène; Nguyen, Thaovi; Ombredane, Kevin; Fostier, Alexis; Bobe, Julien

2015-07-01

In the present study, we aimed at characterizing the effect of cyproterone acetate (CPA), an anti-androgenic compound, on oocyte meiotic maturation in a freshwater teleost fish species, the rainbow trout (Oncorhynchus mykiss). Fully-grown post-vitellogenic ovarian follicles were incubated in vitro with CPA, luteinizing hormone (Lh) or a combination of CPA and Lh. Incubations were also performed using a combination of Lh and testosterone (T). The occurrence of oocyte maturation (i.e., resumption of the meiotic process) was assessed by monitoring germinal vesicle breakdown (GVBD) after a 72h in vitro incubation. The effect of CPA on the production of 17,20β-dihydroxy-4-pregnen-3-one (17,20βP), the natural maturation-inducing steroid (MIS), was quantified by radioimmunoassay. Our results show that CPA dramatically inhibits Lh-induced oocyte maturation and MIS synthesis. We also observed a synergistic effect of Lh and T on oocyte maturation in highly competent oocytes (i.e., able to resume meiosis after stimulation by low doses of Lh). Our results also show that a combination of CPA and Lh inhibits phosphorylation of extracellular signal-regulated kinase (Erk), kinases that are associated with oocyte maturation in many species. As a whole, our results indicate that CPA has a potential to alter meiotic maturation in rainbow trout. Further analyses are, however, needed to determine the mechanisms by which this anti-androgen interferes with the meiotic process. Furthermore, the present study provides a framework for better understanding of the ecological consequences of exposure to anti-androgens and resulting meiotic maturation abnormalities observed in trout. Copyright © 2015 Elsevier B.V. All rights reserved.

Reverse engineering of an affinity-switchable molecular interaction characterized by atomic force microscopy single-molecule force spectroscopy.

PubMed

Anselmetti, Dario; Bartels, Frank Wilco; Becker, Anke; Decker, Björn; Eckel, Rainer; McIntosh, Matthew; Mattay, Jochen; Plattner, Patrik; Ros, Robert; Schäfer, Christian; Sewald, Norbert

2008-02-19

Tunable and switchable interaction between molecules is a key for regulation and control of cellular processes. The translation of the underlying physicochemical principles to synthetic and switchable functional entities and molecules that can mimic the corresponding molecular functions is called reverse molecular engineering. We quantitatively investigated autoinducer-regulated DNA-protein interaction in bacterial gene regulation processes with single atomic force microscopy (AFM) molecule force spectroscopy in vitro, and developed an artificial bistable molecular host-guest system that can be controlled and regulated by external signals (UV light exposure and thermal energy). The intermolecular binding functionality (affinity) and its reproducible and reversible switching has been proven by AFM force spectroscopy at the single-molecule level. This affinity-tunable optomechanical switch will allow novel applications with respect to molecular manipulation, nanoscale rewritable molecular memories, and/or artificial ion channels, which will serve for the controlled transport and release of ions and neutral compounds in the future.

Evaluation of IDA-PEVA hollow fiber membrane metal ion affinity chromatography for purification of a histidine-tagged human proinsulin.

PubMed

de Aquino, Luciana Cristina Lins; de Sousa, Heloisa Ribeiro Tunes; Miranda, Everson Alves; Vilela, Luciano; Bueno, Sônia Maria Alves

2006-04-13

Inabilities to process particulate material and to allow the use of high flow rates are limitations of conventional chromatography. Membranes have been suggested as matrix for affinity separation due to advantages such as allowing high flow rates and low-pressure drops. This work evaluated the feasibility of using an iminodiacetic acid linked poly(ethylenevinyl alcohol) membrane in the immobilized metal ion affinity chromatography (IMAC) purification of a human proinsulin(His)(6) of an industrial insulin production process. The screening of metal ions showed Ni(2+) as metal with higher selectivity and capacity among the Cu(2+), Ni(2+), Zn(2+) and Co(2+). The membrane showed to be equivalent to conventional chelating beads in terms of selectivity and had a lower capacity (3.68 mg/g versus 12.26 mg/g). The dynamic adsorption capacity for human proinsulin(His)(6) was unaffected by the mode of operation (dead-end and cross-flow filtration).

Aptamer-based polyhedral oligomeric silsesquioxane (POSS)-containing hybrid affinity monolith prepared via a "one-pot" process for selective extraction of ochratoxin A.

PubMed

Chen, Yiqiong; Chen, Maolin; Chi, Jinxin; Yu, Xia; Chen, Yongxuan; Lin, Xucong; Xie, Zenghong

2018-08-17

A novel aptamer-based polyhedral oligomeric silsesquioxane (POSS)-containing hybrid affinity monolith has been prepared with a facile "one-pot" process simultaneously via "free radical polymerization" and "thiol-ene" click reaction, and used for on-line selective extraction and practical analysis to trace ochratoxin A (OTA). By using the ternary porogenic mixture composed of water/DMF/PEG, a homogeneous polymerization mixture with POSS chemicals, acrylate-based monomers and aptamer aqueous solution was obtained, and the copolymerization of POSS chemicals, polymer monomers and aptamer aqueous solution was systematically studied. Characterizations such as the morphology, FT-IR and fluorescence spectra, mechanical stability, dynamic binding capacity, cross-reactivity and selectivity of the resultant affinity monolith were also evaluated. Attributed to the porous monolithic structure and aptamer-based affinity interaction, acceptable selective recognition and recovery yields towards trace OTA were obtained. With a 5-fold volume enrichment, the limit of detection (LOD) and limit of quantitation (LOQ) of OTA in fortified beer samples were gained at 0.025 ng/mL (S/N = 3) and 0.045 ng/mL (S/N = 10), respectively. It could be competent for the sensitive measure of actual OTA residues in real beer samples. In comparison with the previously reported strategies containing common "sol-gel" chemistry, the proposed protocol to fabricating aptamer-modified POSS-containing hybrid affinity monolith showed a simpler preparation with acceptable selectivity and higher recovery to trace OTA. Copyright © 2018 Elsevier B.V. All rights reserved.

A comparative study of family-specific protein-ligand complex affinity prediction based on random forest approach

NASA Astrophysics Data System (ADS)

Wang, Yu; Guo, Yanzhi; Kuang, Qifan; Pu, Xuemei; Ji, Yue; Zhang, Zhihang; Li, Menglong

2015-04-01

The assessment of binding affinity between ligands and the target proteins plays an essential role in drug discovery and design process. As an alternative to widely used scoring approaches, machine learning methods have also been proposed for fast prediction of the binding affinity with promising results, but most of them were developed as all-purpose models despite of the specific functions of different protein families, since proteins from different function families always have different structures and physicochemical features. In this study, we proposed a random forest method to predict the protein-ligand binding affinity based on a comprehensive feature set covering protein sequence, binding pocket, ligand structure and intermolecular interaction. Feature processing and compression was respectively implemented for different protein family datasets, which indicates that different features contribute to different models, so individual representation for each protein family is necessary. Three family-specific models were constructed for three important protein target families of HIV-1 protease, trypsin and carbonic anhydrase respectively. As a comparison, two generic models including diverse protein families were also built. The evaluation results show that models on family-specific datasets have the superior performance to those on the generic datasets and the Pearson and Spearman correlation coefficients ( R p and Rs) on the test sets are 0.740, 0.874, 0.735 and 0.697, 0.853, 0.723 for HIV-1 protease, trypsin and carbonic anhydrase respectively. Comparisons with the other methods further demonstrate that individual representation and model construction for each protein family is a more reasonable way in predicting the affinity of one particular protein family.

Tectonic affinities of the accreted basalts in southern Taiwan

NASA Astrophysics Data System (ADS)

Chen, Hsin-Yu; Yang, Huai-Jen; Liu, Yung-Hsin; Huang, Kuo-Fang; Takazawa, Eiichi

2018-06-01

Tectonic affinities of accreted basalts provide constraints on mass transport in convergent boundaries, improving our understandings on the evolution of regional geology. In this study, nineteen accreted basalts from the southernmost tip of Taiwan Island, which is on the convergent boundary between the Eurasian and Philippine Sea Plates, were analyzed for element concentrations as well as Sr, Nd, Hf, and Pb isotope ratios to investigate their tectonic affinities. All the samples contain > 3% LOI, reflecting post-magmatic alteration. LOI and Nb variation diagrams together with comparisons to oceanic basalt compositions indicated that the concentrations of most major elements and Rb, Sr, and Ba were modified by post-magmatic processes to varying extents, while P2O5, REE and HFSE remained immobile. Although some samples show Pb loss, most samples have Pb concentrations not affected by post-magmatic processes. Isotope ratios of Pb, Nd and Hf, generally reflect the mantle source characteristics. The εNd-εHf relationship and trace element abundance ratios indicated that the LREE-depleted samples were mostly scraped off the subducting South China Sea floor, reflecting the volumetric dominance of N-MORB on ocean floors. The overriding Philippine Sea Plate contributed both N-MORB and E-MORB to the accretionary prism. The tectonic affinities of the LREE-enriched samples, however, could not be unambiguously determined for the large geochemical variability of OIB from both subducting and overlying slabs. Based on our results, it is proposed that the tectonic affinity of the basalts in an accretionary prism can indicate the subduction polarity of the associated convergent boundary, providing a constraint for regional geology evolution.

The AGU Data Management Maturity Model Initiative

NASA Astrophysics Data System (ADS)

Bates, J. J.

2015-12-01

In September 2014, the AGU Board of Directors approved two initiatives to help the Earth and space sciences community address the growing challenges accompanying the increasing size and complexity of data. These initiatives are: 1) Data Science Credentialing: development of a continuing education and professional certification program to help scientists in their careers and to meet growing responsibilities and requirements around data science; and 2) Data Management Maturity (DMM) Model: development and implementation of a data management maturity model to assess process maturity against best practices, and to identify opportunities in organizational data management processes. Each of these has been organized within AGU as an Editorial Board and both Boards have held kick off meetings. The DMM model Editorial Board will recommend strategies for adapting and deploying a DMM model to the Earth and space sciences create guidance documents to assist in its implementation, and provide input on a pilot appraisal process. This presentation will provide an overview of progress to date in the DMM model Editorial Board and plans for work to be done over the upcoming year.

A novel function of the cell polarity-regulating kinase PAR-1/MARK in dendritic spines

PubMed Central

Hayashi, Kenji; Suzuki, Atsushi; Ohno, Shigeo

2011-01-01

Dendritic spines are postsynaptic structures that receive excitatory synaptic signals from presynaptic terminals in neurons. Because the morphology of spines has been considered to be a crucial factor for the efficiency of synaptic transmission, understanding the mechanisms regulating their morphology is important for neuroscience. Actin filaments and their regulatory proteins are known to actively maintain spine morphology; recent studies have also shown an essential role of microtubules (MTs). Live imaging of the plus-ends of MTs in mature neurons revealed that MTs stochastically enter spines and mediate accumulation of p140Cap, which regulates reorganization of actin filaments. However, the molecular mechanism by which MT dynamics is controlled has remained largely unknown. A cell polarity-regulating serine/threonine kinase, partitioning-defective 1 (PAR-1), phosphorylates classical MAPs and inhibits their binding to MTs. Because the interaction of MAPs with MTs can decrease MT dynamic instability, PAR-1 is supposed to activate MT dynamics through its MAP/MT affinity-regulating kinase (MARK) activity, although there is not yet any direct evidence for this. Here, we review recent findings on the localization of PAR-1b in the dendrites of mouse hippocampal neurons, and its novel function in the maintenance of mature spine morphology by regulating MT dynamics. PMID:22545177

A novel function of the cell polarity-regulating kinase PAR-1/MARK in dendritic spines.

PubMed

Hayashi, Kenji; Suzuki, Atsushi; Ohno, Shigeo

2011-11-01

Dendritic spines are postsynaptic structures that receive excitatory synaptic signals from presynaptic terminals in neurons. Because the morphology of spines has been considered to be a crucial factor for the efficiency of synaptic transmission, understanding the mechanisms regulating their morphology is important for neuroscience. Actin filaments and their regulatory proteins are known to actively maintain spine morphology; recent studies have also shown an essential role of microtubules (MTs). Live imaging of the plus-ends of MTs in mature neurons revealed that MTs stochastically enter spines and mediate accumulation of p140Cap, which regulates reorganization of actin filaments. However, the molecular mechanism by which MT dynamics is controlled has remained largely unknown. A cell polarity-regulating serine/threonine kinase, partitioning-defective 1 (PAR-1), phosphorylates classical MAPs and inhibits their binding to MTs. Because the interaction of MAPs with MTs can decrease MT dynamic instability, PAR-1 is supposed to activate MT dynamics through its MAP/MT affinity-regulating kinase (MARK) activity, although there is not yet any direct evidence for this. Here, we review recent findings on the localization of PAR-1b in the dendrites of mouse hippocampal neurons, and its novel function in the maintenance of mature spine morphology by regulating MT dynamics.

Immune and endocrine responses of adult spring Chinook salmon during freshwater migration and sexual maturation

USGS Publications Warehouse

Maule, A.G.; Schrock, R.M.; Slater, C.; Fitzpatrick, M.S.; Schreck, C. B.

1996-01-01

The immune –endocrine responses in spring chinook salmon (Oncorhynchus tshawytscha) were examined during their freshwater migration and final maturation. In 1990, migrating fish had high plasma cortisol titres (means 200 ng ml−1) and generated relatively few antibody-producing cells (APC) from peripheral blood leukocytes (PBL) (100 –200 per culture). After three weeks acclimation in constant environmental conditions, plasma cortisol was reduced and APC increased. There were no changes in number or affinity of glucocorticoid receptors. Concentrations of several sex steroids correlated with APC in females, but there were no such correlations in males. In 1993, fish in a hatchery had significantly greater cortisol concentrations in primary circulation than in secondary circulation, but sex steroid concentrations did not differ between circulations. Mean lysozyme activity in the primary and secondary circulation did not differ in June. In August, activity in the primary circulation was significantly less than that of the secondary, perhaps the result of acute stress associated with sampling. While some sex steroids correlated with lysozyme activity, the fact that in both years all endocrine and immune variables that correlated with each other also correlated with the date of sample, raises the question as to whether or not these are cause-and-effect relations.

Interactome Analyses of Mature γ-Secretase Complexes Reveal Distinct Molecular Environments of Presenilin (PS) Paralogs and Preferential Binding of Signal Peptide Peptidase to PS2*

PubMed Central

Jeon, Amy Hye Won; Böhm, Christopher; Chen, Fusheng; Huo, Hairu; Ruan, Xueying; Ren, Carl He; Ho, Keith; Qamar, Seema; Mathews, Paul M.; Fraser, Paul E.; Mount, Howard T. J.; St George-Hyslop, Peter; Schmitt-Ulms, Gerold

2013-01-01

γ-Secretase plays a pivotal role in the production of neurotoxic amyloid β-peptides (Aβ) in Alzheimer disease (AD) and consists of a heterotetrameric core complex that includes the aspartyl intramembrane protease presenilin (PS). The human genome codes for two presenilin paralogs. To understand the causes for distinct phenotypes of PS paralog-deficient mice and elucidate whether PS mutations associated with early-onset AD affect the molecular environment of mature γ-secretase complexes, quantitative interactome comparisons were undertaken. Brains of mice engineered to express wild-type or mutant PS1, or HEK293 cells stably expressing PS paralogs with N-terminal tandem-affinity purification tags served as biological source materials. The analyses revealed novel interactions of the γ-secretase core complex with a molecular machinery that targets and fuses synaptic vesicles to cellular membranes and with the H+-transporting lysosomal ATPase macrocomplex but uncovered no differences in the interactomes of wild-type and mutant PS1. The catenin/cadherin network was almost exclusively found associated with PS1. Another intramembrane protease, signal peptide peptidase, predominantly co-purified with PS2-containing γ-secretase complexes and was observed to influence Aβ production. PMID:23589300

RNA Mimicry by the Fap7 Adenylate Kinase in Ribosome Biogenesis

PubMed Central

Réty, Stéphane; Lebaron, Simon; Deschamps, Patrick; Bareille, Joseph; Jombart, Julie; Robert-Paganin, Julien; Delbos, Lila; Chardon, Florian; Zhang, Elodie; Charenton, Clément; Tollervey, David; Leulliot, Nicolas

2014-01-01

During biogenesis of the 40S and 60S ribosomal subunits, the pre-40S particles are exported to the cytoplasm prior to final cleavage of the 20S pre-rRNA to mature 18S rRNA. Amongst the factors involved in this maturation step, Fap7 is unusual, as it both interacts with ribosomal protein Rps14 and harbors adenylate kinase activity, a function not usually associated with ribonucleoprotein assembly. Human hFap7 also regulates Cajal body assembly and cell cycle progression via the p53–MDM2 pathway. This work presents the functional and structural characterization of the Fap7–Rps14 complex. We report that Fap7 association blocks the RNA binding surface of Rps14 and, conversely, Rps14 binding inhibits adenylate kinase activity of Fap7. In addition, the affinity of Fap7 for Rps14 is higher with bound ADP, whereas ATP hydrolysis dissociates the complex. These results suggest that Fap7 chaperones Rps14 assembly into pre-40S particles via RNA mimicry in an ATP-dependent manner. Incorporation of Rps14 by Fap7 leads to a structural rearrangement of the platform domain necessary for the pre-rRNA to acquire a cleavage competent conformation. PMID:24823650

Identification of CD22 Ligands on Bone Marrow Sinusoidal Endothelium Implicated in CD22-dependent Homing of Recirculating B Cells

PubMed Central

Nitschke, Lars; Floyd, Helen; Ferguson, David J.P.; Crocker, Paul R.

1999-01-01

CD22 is a B cell–specific transmembrane protein known to function as a negative regulator of B cell signaling. It has also been implicated in cell adhesion through recognition of α2,6-linked sialic acids on glycans of target cells. Previous studies showed that CD22-deficient mice had a strongly reduced population of mature recirculating B cells in the bone marrow despite normal B cell development. Using a soluble recombinant form of the receptor (CD22-Fc), we demonstrate here that sialylated ligands for CD22 are expressed on sinusoidal endothelial cells of murine bone marrow but not on endothelial cells in other tissues examined. Injection of CD22-Fc revealed that the CD22 ligands in the bone marrow were accessible to the circulation. Treatment of mice with either CD22-Fc or affinity-purified anti-CD22 antibody led to an ∼50% reduction in mature recirculating B cells in the bone marrow without affecting numbers in the spleen. Finally, consistent with the notion that CD22 is a homing receptor, we show that compared with wild-type mice, CD22-deficient animals have a lower number of immunoglobulin M–secreting plasma cells in the bone marrow. PMID:10224292

Protease-Mediated Maturation of HIV: Inhibitors of Protease and the Maturation Process.

PubMed

Adamson, Catherine S

2012-01-01

Protease-mediated maturation of HIV-1 virus particles is essential for virus infectivity. Maturation occurs concomitant with immature virus particle release and is mediated by the viral protease (PR), which sequentially cleaves the Gag and Gag-Pol polyproteins into mature protein domains. Maturation triggers a second assembly event that generates a condensed conical capsid core. The capsid core organizes the viral RNA genome and viral proteins to facilitate viral replication in the next round of infection. The fundamental role of proteolytic maturation in the generation of mature infectious particles has made it an attractive target for therapeutic intervention. Development of small molecules that target the PR active site has been highly successful and nine protease inhibitors (PIs) have been approved for clinical use. This paper provides an overview of their development and clinical use together with a discussion of problems associated with drug resistance. The second-half of the paper discusses a novel class of antiretroviral drug termed maturation inhibitors, which target cleavage sites in Gag not PR itself. The paper focuses on bevirimat (BVM) the first-in-class maturation inhibitor: its mechanism of action and the implications of naturally occurring polymorphisms that confer reduced susceptibility to BVM in phase II clinical trials.

Process Improvement Should Link to Security: SEPG 2007 Security Track Recap

DTIC Science & Technology

2007-09-01

the Systems Security Engineering Capability Maturity Model (SSE- CMM / ISO 21827) and its use in system software developments ...software development life cycle ( SDLC )? 6. In what ways should process improvement support security in the SDLC ? 1.2 10BPANEL RESOURCES For each... project management, and support practices through the use of the capability maturity models including the CMMI and the Systems Security

Maturity Assessment of Space Plug-and-Play Architecture

DTIC Science & Technology

2013-03-01

SSM SPA Service Module SRL System Readiness Level TAT Time-at-Tone TRA Technology Readiness Assessment TRL Technology Readiness Level USB Universal...maturity assessment—the Technology Readiness Level (TRL) process, the Integration Readiness Level (IRL) process, and the System Readiness Level ( SRL ...is an important hallmark of the SPA concept, and makes possible the composability and scalability of system designs that employ it. 14 4. SPA

Profiling Pre-MicroRNA and Mature MicroRNA Expressions Using a Single Microarray and Avoiding Separate Sample Preparation

PubMed Central

Gan, Lin; Denecke, Bernd

2013-01-01

Mature microRNA is a crucial component in the gene expression regulation network. At the same time, microRNA gene expression and procession is regulated in a precise and collaborated way. Pre-microRNAs mediate products during the microRNA transcription process, they can provide hints of microRNA gene expression regulation or can serve as alternative biomarkers. To date, little effort has been devoted to pre-microRNA expression profiling. In this study, three human and three mouse microRNA profile data sets, based on the Affymetrix miRNA 2.0 array, have been re-analyzed for both mature and pre-microRNA signals as a primary test of parallel mature/pre-microRNA expression profiling on a single platform. The results not only demonstrated a glimpse of pre-microRNA expression in human and mouse, but also the relationship of microRNA expressions between pre- and mature forms. The study also showed a possible application of currently available microRNA microarrays in profiling pre-microRNA expression in a time and cost effective manner. PMID:27605179

Proteomic analysis of mature and immature ejaculated spermatozoa from fertile men

PubMed Central

Cui, Zhihong; Sharma, Rakesh; Agarwal, Ashok

2016-01-01

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

Centriole maturation requires regulated Plk1 activity during two consecutive cell cycles.

PubMed

Kong, Dong; Farmer, Veronica; Shukla, Anil; James, Jana; Gruskin, Richard; Kiriyama, Shigeo; Loncarek, Jadranka

2014-09-29

Newly formed centrioles in cycling cells undergo a maturation process that is almost two cell cycles long before they become competent to function as microtubule-organizing centers and basal bodies. As a result, each cell contains three generations of centrioles, only one of which is able to form cilia. It is not known how this long and complex process is regulated. We show that controlled Plk1 activity is required for gradual biochemical and structural maturation of the centrioles and timely appendage assembly. Inhibition of Plk1 impeded accumulation of appendage proteins and appendage formation. Unscheduled Plk1 activity, either in cycling or interphase-arrested cells, accelerated centriole maturation and appendage and cilia formation on the nascent centrioles, erasing the age difference between centrioles in one cell. These findings provide a new understanding of how the centriole cycle is regulated and how proper cilia and centrosome numbers are maintained in the cells.

Centriole maturation requires regulated Plk1 activity during two consecutive cell cycles

PubMed Central

Kong, Dong; Farmer, Veronica; Shukla, Anil; James, Jana; Gruskin, Richard; Kiriyama, Shigeo

2014-01-01

Newly formed centrioles in cycling cells undergo a maturation process that is almost two cell cycles long before they become competent to function as microtubule-organizing centers and basal bodies. As a result, each cell contains three generations of centrioles, only one of which is able to form cilia. It is not known how this long and complex process is regulated. We show that controlled Plk1 activity is required for gradual biochemical and structural maturation of the centrioles and timely appendage assembly. Inhibition of Plk1 impeded accumulation of appendage proteins and appendage formation. Unscheduled Plk1 activity, either in cycling or interphase-arrested cells, accelerated centriole maturation and appendage and cilia formation on the nascent centrioles, erasing the age difference between centrioles in one cell. These findings provide a new understanding of how the centriole cycle is regulated and how proper cilia and centrosome numbers are maintained in the cells. PMID:25246616

R-Ras2 is required for germinal center formation to aid B cells during energetically demanding processes.

PubMed

Mendoza, Pilar; Martínez-Martín, Nuria; Bovolenta, Elena R; Reyes-Garau, Diana; Hernansanz-Agustín, Pablo; Delgado, Pilar; Diaz-Muñoz, Manuel D; Oeste, Clara L; Fernández-Pisonero, Isabel; Castellano, Ester; Martínez-Ruiz, Antonio; Alonso-Lopez, Diego; Santos, Eugenio; Bustelo, Xosé R; Kurosaki, Tomohiro; Alarcón, Balbino

2018-05-29

Upon antigen recognition within peripheral lymphoid organs, B cells interact with T cells and other immune cells to transiently form morphological structures called germinal centers (GCs), which are required for B cell clonal expansion, immunoglobulin class switching, and affinity maturation. This process, known as the GC response, is an energetically demanding process that requires the metabolic reprogramming of B cells. We showed that the Ras-related guanosine triphosphate hydrolase (GTPase) R-Ras2 (also known as TC21) plays an essential, nonredundant, and B cell-intrinsic role in the GC response. Both the conversion of B cells into GC B cells and their expansion were impaired in mice lacking R-Ras2, but not in those lacking a highly related R-Ras subfamily member or both the classic H-Ras and N-Ras GTPases. In the absence of R-Ras2, activated B cells did not exhibit increased oxidative phosphorylation or aerobic glycolysis. We showed that R-Ras2 was an effector of both the B cell receptor (BCR) and CD40 and that, in its absence, B cells exhibited impaired activation of the PI3K-Akt-mTORC1 pathway, reduced mitochondrial DNA replication, and decreased expression of genes involved in glucose metabolism. Because most human B cell lymphomas originate from GC B cells or B cells that have undergone the GC response, our data suggest that R-Ras2 may also regulate metabolism in B cell malignancies. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Genome-wide association study (GWAS) for growth rate and age at sexual maturation in Atlantic salmon (Salmo salar).

PubMed

Gutierrez, Alejandro P; Yáñez, José M; Fukui, Steve; Swift, Bruce; Davidson, William S

2015-01-01

Early sexual maturation is considered a serious drawback for Atlantic salmon aquaculture as it retards growth, increases production times and affects flesh quality. Although both growth and sexual maturation are thought to be complex processes controlled by several genetic and environmental factors, selection for these traits has been continuously accomplished since the beginning of Atlantic salmon selective breeding programs. In this genome-wide association study (GWAS) we used a 6.5K single-nucleotide polymorphism (SNP) array to genotype ∼ 480 individuals from the Cermaq Canada broodstock program and search for SNPs associated with growth and age at sexual maturation. Using a mixed model approach we identified markers showing a significant association with growth, grilsing (early sexual maturation) and late sexual maturation. The most significant associations were found for grilsing, with markers located in Ssa10, Ssa02, Ssa13, Ssa25 and Ssa12, and for late maturation with markers located in Ssa28, Ssa01 and Ssa21. A lower level of association was detected with growth on Ssa13. Candidate genes, which were linked to these genetic markers, were identified and some of them show a direct relationship with developmental processes, especially for those in association with sexual maturation. However, the relatively low power to detect genetic markers associated with growth (days to 5 kg) in this GWAS indicates the need to use a higher density SNP array in order to overcome the low levels of linkage disequilibrium observed in Atlantic salmon before the information can be incorporated into a selective breeding program.

[Role of hemoglobin affinity to oxygen in adaptation to hypoxemia].

PubMed

Kwasiborski, Przemysław Jerzy; Kowalczyk, Paweł; Zieliński, Jakub; Przybylski, Jacek; Cwetsch, Andrzej

2010-04-01

One of the basic mechanisms of adapting to hypoxemia is a decrease in the affinity of hemoglobin for oxygen. This process occurs mainly due to the increased synthesis of 2,3-diphosphoglycerate (2,3-DPG) in the erythrocytes, as well as through the Bohr effect. Hemoglobin with decreased affinity for oxygen increases the oxygenation of tissues, because it gives up oxygen more easily during microcirculation. In foetal circulation, however, at a partial oxygen pressure (pO2) of 25 mmHg in the umbilical vein, the oxygen carrier is type F hemoglobin which has a high oxygen affinity. The commonly accepted role for hemoglobin F is limited to facilitating diffusion through the placenta. Is fetal life the only moment when haemoglobin F is useful? THE AIM OF STUDY was to create a mathematical model, which would answer the question at what conditions an increase, rather than a decrease, in haemoglobin oxygen affinity is of benefit to the body. Using the kinetics of dissociation of oxygen from hemoglobin described by the Hill equation as the basis for further discussion, we created a mathematical model describing the pO2 value in the microcirculatory system and its dependence on arterial blood pO2. The calculations were performed for hemoglobin with low oxygen affinity (adult type) and high-affinity hemoglobin (fetal type). The modelling took into account both physiological and pathological ranges of acid-base equilibrium and tissue oxygen extraction parameters. It was shown that for the physiological range of acid-base equilibrium and the resting level of tissue oxygen extraction parameters, with an arterial blood pO2 of 26.8 mmHg, the higher-affinity hemoglobin becomes the more effective oxygen carrier. It was also demonstrated that the arterial blood pO2, below which the high-affinity hemoglobin becomes the more effective carrier, is dependent on blood pH and the difference between the arterial and venous oxygen saturation levels. Simulations performed for the pathological states showed that acidosis and increased tissue oxygen demand lead to a broadened arterial blood pO2 range, in which the high-affinity hemoglobin is more efficient. Contrary to the widely held view that the only response to hypoxemia is a decrease in haemoglobin oxygen affinity, it was shown that under extreme hypoxemic conditions, an increased haemoglobin oxygen affinity improves the oxygenation of tissues. It was also shown that the dominance of hemoglobin with a high oxygen affinity rapidly exceeds hemoglobin with low oxygen affinity in the case of acidosis with its accompanying high tissue oxygen extraction. In cases of extreme disruptions of the acid-base equilibrium, the dominance of high-oxygen-affinity hemoglobin spans over the entire possible range of pO2 in arterial blood.

Subcellular localization of calcium and Ca-ATPase activity during nuclear maturation in Bufo arenarum oocytes.

PubMed

Ramos, Inés; Cisint, Susana B; Crespo, Claudia A; Medina, Marcela F; Fernández, Silvia N

2009-08-01

The localization of calcium and Ca-ATPase activity in Bufo arenarum oocytes was investigated by ultracytochemical techniques during progesterone-induced nuclear maturation, under in vitro conditions. No Ca2+ deposits were detected in either control oocytes or progesterone-treated ones for 1-2 h. At the time when nuclear migration started, electron dense deposits of Ca2+ were visible in vesicles, endoplasmic reticulum cisternae and in the space between the annulate lamellae membranes. Furthermore, Ca-ATPase activity was also detected in these membrane structures. As maturation progressed, the cation deposits were observed in the cytomembrane structures, which underwent an important reorganization and redistribution. Thus, they moved from the subcortex and became located predominantly in the oocyte cortex area when nuclear maturation ended. Ca2+ stores were observed in vesicles surrounding or between the cortical granules, which are aligned close to the plasma membrane. The positive Ca-ATPase reaction in these membrane structures could indicate that the calcium deposit is an ATP-dependent process. Our results suggest that during oocyte maturation calcium would be stored in membrane structures where it remains available for release at the time of fertilization. Data obtained under our experimental conditions indicate that calcium from the extracellular medium would be important for the oocyte maturation process.

Vinyl sulfone silica: application of an open preactivated support to the study of transnitrosylation of plant proteins by S-nitrosoglutathione

PubMed Central

2013-01-01

Background S-nitrosylaton is implicated in the regulation of numerous signaling pathways with a diversity of regulatory roles. The high lability of the S-NO bond makes the study of proteins regulated by S-nitrosylation/denitrosylation a challenging task and most studies have focused on already S-nitrosylated proteins. We hypothesize that: i) S-nitrosoglutathione (GSNO) transnitrosylation is a feasible mechanism to account for the physiological S-nitrosylation of rather electropositive sulfur atoms from proteins, ii) affinity chromatography is a suitable approach to isolate proteins that are prone to undergo S-transnitrosylation and iii) vinyl sulfone silica is a suitable chromatographic bead. Results The combination of vinyl sulfone silica with GSNO yielded an affinity resin that withstood high ionic strength without shrinking or deforming and that it was suitable to isolate potential GSNO transnitrosylation target candidates. Fractions eluted at 1500 mM NaCl resulted in a symmetrical peak for both, protein and S-nitrosothiols, supporting the idea of transnitrosylation by GSNO as a selective process that involves strong and specific interactions with the target protein. Proteomic analysis led to the identification of 22 physiological significant enzymes that differ with the tissue analyzed, being regulatory proteins the most abundant group in hypocotyls. The identification of chloroplastidic FBPase, proteasome, GTP-binding protein, heat shock Hsp70, syntaxin, catalase I, thioredoxin peroxidase and cytochrome P450 that have already been reported as S-nitrosylated by other techniques can be considered as internal positive controls that validate our experimental approach. An additional validation was provided by the prediction of the S-nitrosylation sites in 19 of the GSNO transnitrosylation target candidates. Conclusions Vinyl sulfone silica is an open immobilization support that can be turned ad hoc and in a straightforward manner into an affinity resin. Its potential in omic sciences was successfully put to test in the context of the analysis of post-translational modification by S-nitrosylation with two different tissues: mature pea leaves and embryogenic sunflower hypocotyls. The identified proteins reveal an intriguing overlap among S-nitrosylation and both tyrosine nitration and thioredoxin regulation. Chloroplastidic FBPase is a paradigm of such overlap of post-translational modifications since it is reversible modified by thioredoxin and S-nitrosylation and irreversibly by tyrosine nitration. Our results suggest a complex interrelation among different modulation mechanisms mediated by NO-derived molecules. PMID:23586608

A mature and fusogenic form of the Nipah virus fusion protein requires proteolytic processing by cathepsin L

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

Pager, Cara Theresia; Craft, Willie Warren; Patch, Jared

2006-03-15

The Nipah virus fusion (F) protein is proteolytically processed to F{sub 1} + F{sub 2} subunits. We demonstrate here that cathepsin L is involved in this important maturation event. Cathepsin inhibitors ablated cleavage of Nipah F. Proteolytic processing of Nipah F and fusion activity was dramatically reduced in cathepsin L shRNA-expressing Vero cells. Additionally, Nipah virus F-mediated fusion was inhibited in cathepsin L-deficient cells, but coexpression of cathepsin L restored fusion activity. Both purified cathepsin L and B could cleave immunopurified Nipah F protein, but only cathepsin L produced products of the correct size. Our results suggest that endosomal cathepsinsmore » can cleave Nipah F, but that cathepsin L specifically converts Nipah F to a mature and fusogenic form.« less

Methods for Purifying Enzymes for Mycoremediation

NASA Technical Reports Server (NTRS)

Cullings, Kenneth W. (Inventor); DeSimone, Julia C. (Inventor); Paavola, Chad D. (Inventor)

2014-01-01

A process for purifying laccase from an ectomycorrhizal fruiting body is disclosed. The process includes steps of homogenization, sonication, centrifugation, filtration, affinity chromatography, ion exchange chromatography, and gel filtration. Purified laccase can also be separated into isomers.

Exploring the energy landscape of antibody-antigen complexes: protein dynamics, flexibility, and molecular recognition.

PubMed

Thielges, Megan C; Zimmermann, Jörg; Yu, Wayne; Oda, Masayuki; Romesberg, Floyd E

2008-07-08

The production of antibodies that selectively bind virtually any foreign compound is the hallmark of the immune system. While much is understood about how sequence diversity contributes to this remarkable feat of molecular recognition, little is known about how sequence diversity impacts antibody dynamics, which is also expected to contribute to molecular recognition. Toward this goal, we examined a panel of antibodies elicited to the chromophoric antigen fluorescein. On the basis of isothermal titration calorimetry, we selected six antibodies that bind fluorescein with diverse binding entropies, suggestive of varying contributions of dynamics to molecular recognition. Sequencing revealed that two pairs of antibodies employ homologous heavy chains that were derived from common germline genes, while the other two heavy chains and all six of the light chains were derived from different germline genes and are not homologous. Interestingly, more than half of all the somatic mutations acquired during affinity maturation among the six antibodies are located in positions unlikely to contact fluorescein directly. To quantify and compare the dynamics of the antibody-fluorescein complexes, three-pulse photon echo peak shift and transient grating spectroscopy were employed. All of the antibodies exhibited motions on three distinct time scales, ultrafast motions on the <100 fs time scale, diffusive motions on the picosecond time scale, and motions that occur on time scales longer than nanoseconds and thus appear static. However, the exact frequency of the picosecond time scale motion and the relative contribution of the different motions vary significantly among the antibody-chromophore complexes, revealing a high level of dynamic diversity. Using a hierarchical model, we relate the data to features of the antibodies' energy landscapes as well as their flexibility in terms of elasticity and plasticity. In all, the data provide a consistent picture of antibody flexibility, which interestingly appears to be correlated with binding entropy as well as with germline gene use and the mutations introduced during affinity maturation. The data also provide a gauge of the dynamic diversity of the antibody repertoire and suggest that this diversity might contribute to molecular recognition by facilitating the recognition of the broadest range of foreign molecules.

Generation and evaluation of antibody agents for molecular imaging of CD44v6-expressing cancers

PubMed Central

Haylock, Anna-Karin; Nilvebrant, Johan; Mortensen, Anja; Velikyan, Irina; Nestor, Marika; Falk, Ronny

2017-01-01

Aim The aim of this study was to generate and characterize scFv antibodies directed to human CD44v6, as well as to radiolabel and evaluate top candidates in vitro and in vivo for their potential use in CD44v6-targeted molecular imaging in cancer patients. Materials and methods Phage display selections were used to isolate CD44v6-specific scFvs. A chain shuffling strategy was employed for affinity maturation based on a set of CD44v6-specific first-generation clones. Two second-generation scFv clones were then chosen for labeling with 111In or 125I and assessed for CD44v6-specific binding on cultured tumor cells. In vivo uptake and distribution was evaluated in tumor-bearing mice using a dual tumor model. Finally, a proof-of-concept small animal PET-CT study was performed on one of the candidates labeled with 124I. Results Two affinity-matured clones, CD44v6-scFv-A11 and CD44v6-scFv-H12, displayed promising binding kinetics. Seven out of eight radiolabeled conjugates demonstrated CD44v6-specific binding. In vivo studies on selected candidates demonstrated very advantageous tumor-to-organ ratios, in particular for iodinated conjugates, where 125I-labeled scFvs exhibited favorable kinetics and tumor-to-blood ratios above five already at 24 hours p.i.. The small animal PET-CT study using 124I-labeled CD44v6-scFv-H12 was in line with the biodistribution data, clearly visualizing the high CD44v6-expressing tumor. Conclusion The single chain fragments, CD44v6-scFv-A11 and CD44v6-scFv-H12 specifically bind to CD44v6, and the radiolabeled counterparts provide high tumor-to-blood ratios and fast clearance from organs and blood. We conclude that radioiodinated CD44v6-scFv-A11 and CD44v6-scFv-H12 possess features highly suitable for stringent molecular imaging. PMID:29029420

Cutting edge: Contact with secondary lymphoid organs drives postthymic T cell maturation.

PubMed

Houston, Evan G; Nechanitzky, Robert; Fink, Pamela J

2008-10-15

T cell development, originally thought to be completed in the thymus, has recently been shown to continue for several weeks in the lymphoid periphery. The forces that drive this peripheral maturation are unclear. The use of mice transgenic for GFP driven by the RAG2 promoter has enabled the ready identification and analysis of recent thymic emigrants. Here, we show that recent thymic emigrant maturation is a progressive process and is promoted by T cell exit from the thymus. Further, we show that this maturation occurs within secondary lymphoid organs and does not require extensive lymphocyte recirculation.

Quantitative Assessment of Cervical Vertebral Maturation Using Cone Beam Computed Tomography in Korean Girls

PubMed Central

Byun, Bo-Ram; Kim, Yong-Il; Maki, Koutaro; Son, Woo-Sung

2015-01-01

This study was aimed to examine the correlation between skeletal maturation status and parameters from the odontoid process/body of the second vertebra and the bodies of third and fourth cervical vertebrae and simultaneously build multiple regression models to be able to estimate skeletal maturation status in Korean girls. Hand-wrist radiographs and cone beam computed tomography (CBCT) images were obtained from 74 Korean girls (6–18 years of age). CBCT-generated cervical vertebral maturation (CVM) was used to demarcate the odontoid process and the body of the second cervical vertebra, based on the dentocentral synchondrosis. Correlation coefficient analysis and multiple linear regression analysis were used for each parameter of the cervical vertebrae (P < 0.05). Forty-seven of 64 parameters from CBCT-generated CVM (independent variables) exhibited statistically significant correlations (P < 0.05). The multiple regression model with the greatest R 2 had six parameters (PH2/W2, UW2/W2, (OH+AH2)/LW2, UW3/LW3, D3, and H4/W4) as independent variables with a variance inflation factor (VIF) of <2. CBCT-generated CVM was able to include parameters from the second cervical vertebral body and odontoid process, respectively, for the multiple regression models. This suggests that quantitative analysis might be used to estimate skeletal maturation status. PMID:25878721

A Stiffness Switch in Human Immunodeficiency Virus

PubMed Central

Kol, Nitzan; Shi, Yu; Tsvitov, Marianna; Barlam, David; Shneck, Roni Z.; Kay, Michael S.; Rousso, Itay

2007-01-01

After budding from the cell, human immunodeficiency virus (HIV) and other retrovirus particles undergo a maturation process that is required for their infectivity. During maturation, HIV particles undergo a significant internal morphological reorganization, changing from a roughly spherically symmetric immature particle with a thick protein shell to a mature particle with a thin protein shell and conical core. However, the physical principles underlying viral particle production, maturation, and entry into cells remain poorly understood. Here, using nanoindentation experiments conducted by an atomic force microscope (AFM), we report the mechanical measurements of HIV particles. We find that immature particles are more than 14-fold stiffer than mature particles and that this large difference is primarily mediated by the HIV envelope cytoplasmic tail domain. Finite element simulation shows that for immature virions the average Young's modulus drops more than eightfold when the cytoplasmic tail domain is deleted (930 vs. 115 MPa). We also find a striking correlation between the softening of viruses during maturation and their ability to enter cells, providing the first evidence, to our knowledge, for a prominent role for virus mechanical properties in the infection process. These results show that HIV regulates its mechanical properties at different stages of its life cycle (i.e., stiff during viral budding versus soft during entry) and that this regulation may be important for efficient infectivity. Our report of this maturation-induced “stiffness switch” in HIV establishes the groundwork for mechanistic studies of how retroviral particles can regulate their mechanical properties to affect biological function. PMID:17158573

Bicarbonate Transport During Enamel Maturation.

PubMed

Yin, Kaifeng; Paine, Michael L

2017-11-01

Amelogenesis (tooth enamel formation) is a biomineralization process consisting primarily of two stages (secretory stage and maturation stage) with unique features. During the secretory stage, the inner epithelium of the enamel organ (i.e., the ameloblast cells) synthesizes and secretes enamel matrix proteins (EMPs) into the enamel space. The protein-rich enamel matrix forms a highly organized architecture in a pH-neutral microenvironment. As amelogenesis transitions to maturation stage, EMPs are degraded and internalized by ameloblasts through endosomal-lysosomal pathways. Enamel crystallite formation is initiated early in the secretory stage, however, during maturation stage the more rapid deposition of calcium and phosphate into the enamel space results in a rapid expansion of crystallite length and mineral volume. During maturation-stage amelogenesis, the pH value of enamel varies considerably from slightly above neutral to acidic. Extracellular acid-base balance during enamel maturation is tightly controlled by ameloblast-mediated regulatory networks, which include significant synthesis and movement of bicarbonate ions from both the enamel papillary layer cells and ameloblasts. In this review we summarize the carbonic anhydrases and the carbonate transporters/exchangers involved in pH regulation in maturation-stage amelogenesis. Proteins that have been shown to be instrumental in this process include CA2, CA6, CFTR, AE2, NBCe1, SLC26A1/SAT1, SLC26A3/DRA, SLC26A4/PDS, SLC26A6/PAT1, and SLC26A7/SUT2. In addition, we discuss the association of miRNA regulation with bicarbonate transport in tooth enamel formation.

Reconstructing life history of hominids and humans.

PubMed

Crews, Douglas E; Gerber, Linda M

2003-06-01

Aspects of life history, such as processes and timing of development, age at maturation, and life span are consistently associated with one another across the animal kingdom. Species that develop rapidly tend to mature and reproduce early, have many offspring, and exhibit shorter life spans (r-selection) than those that develop slowly, have extended periods of premature growth, mature later in life, reproduce later and less frequently, have few offspring and/or single births, and exhibit extended life spans (K-selection). In general, primates are among the most K-selected of species. A suite of highly derived life history traits characterizes humans. Among these are physically immature neonates, slowed somatic development both in utero and post-natally, late attainment of reproductive maturity and first birth, and extended post-mature survival. Exactly when, why, and through what types of evolutionary interactions this suite arose is currently the subject of much conjecture and debate. Humankind's biocultural adaptations have helped to structure human life history evolution in unique ways not seen in other animal species. Among all species, life history traits may respond rapidly to alterations in selective pressures through hormonal processes. Selective pressures on life history likely varied widely among hominids and humans over their evolutionary history. This suggests that current patterns of human growth, development, maturation, reproduction, and post-mature survival may be of recent genesis, rather then long-standing adaptations. Thus, life history patterns observed among contemporary human and chimpanzee populations may provide little insight to those that existed earlier in hominid/human evolution.

Successful stock production for forest regeneration: What foresters should ask nursery managers about their crops (and vice versa)

Treesearch

R.K. Dumroese; D.F. Jacobs; T.D. Landis

2005-01-01

Forest regeneration is a cyclic operation. Seeds are collected from mature trees and planted in nurseries so that the resulting seedlings can be outplanted to the forest after the mature trees are harvested. Similarly, the process of deciding upon, and growing, the best seedlings for that site should be a cyclic process between foresters and nursery managers. The ideal...

Supporting the Use of CERT (registered trademark) Secure Coding Standards in DoD Acquisitions

DTIC Science & Technology

2012-07-01

Capability Maturity Model IntegrationSM (CMMI®) [Davis 2009]. SM Team Software Process, TSP, and Capability Maturity Model Integration are service...STP Software Test Plan TEP Test and Evaluation Plan TSP Team Software Process V & V verification and validation CMU/SEI-2012-TN-016 | 47...Supporting the Use of CERT® Secure Coding Standards in DoD Acquisitions Tim Morrow ( Software Engineering Institute) Robert Seacord ( Software

Competitive Inhibition of the Endoplasmic Reticulum Signal Peptidase by Non-cleavable Mutant Preprotein Cargos*

PubMed Central

Cui, Jingqiu; Chen, Wei; Sun, Jinhong; Guo, Huan; Madley, Rachel; Xiong, Yi; Pan, Xingyi; Wang, Hongliang; Tai, Andrew W.; Weiss, Michael A.; Arvan, Peter; Liu, Ming

2015-01-01

Upon translocation across the endoplasmic reticulum (ER) membrane, secretory proteins are proteolytically processed to remove their signal peptide by signal peptidase (SPase). This process is critical for subsequent folding, intracellular trafficking, and maturation of secretory proteins. Prokaryotic SPase has been shown to be a promising antibiotic target. In contrast, to date, no eukaryotic SPase inhibitors have been reported. Here we report that introducing a proline immediately following the natural signal peptide cleavage site not only blocks preprotein cleavage but also, in trans, impairs the processing and maturation of co-expressed preproteins in the ER. Specifically, we find that a variant preproinsulin, pPI-F25P, is translocated across the ER membrane, where it binds to the catalytic SPase subunit SEC11A, inhibiting SPase activity in a dose-dependent manner. Similar findings were obtained with an analogous variant of preproparathyroid hormone, demonstrating that inhibition of the SPase does not depend strictly on the sequence or structure of the downstream mature protein. We further show that inhibiting SPase in the ER impairs intracellular processing of viral polypeptides and their subsequent maturation. These observations suggest that eukaryotic SPases (including the human ortholog) are, in principle, suitable therapeutic targets for antiviral drug design. PMID:26446786

Progressive and regressive developmental changes in neural substrates for face processing: Testing specific predictions of the Interactive Specialization account

PubMed Central

Joseph, Jane E.; Gathers, Ann D.; Bhatt, Ramesh S.

2010-01-01

Face processing undergoes a fairly protracted developmental time course but the neural underpinnings are not well understood. Prior fMRI studies have only examined progressive changes (i.e., increases in specialization in certain regions with age), which would be predicted by both the Interactive Specialization (IS) and maturational theories of neural development. To differentiate between these accounts, the present study also examined regressive changes (i.e., decreases in specialization in certain regions with age), which is predicted by the IS but not maturational account. The fMRI results show that both progressive and regressive changes occur, consistent with IS. Progressive changes mostly occurred in occipital-fusiform and inferior frontal cortex whereas regressive changes largely emerged in parietal and lateral temporal cortices. Moreover, inconsistent with the maturational account, all of the regions involved in face viewing in adults were active in children, with some regions already specialized for face processing by 5 years of age and other regions activated in children but not specifically for faces. Thus, neurodevelopment of face processing involves dynamic interactions among brain regions including age-related increases and decreases in specialization and the involvement of different regions at different ages. These results are more consistent with IS than maturational models of neural development. PMID:21399706

Loading direction regulates the affinity of ADP for kinesin.

PubMed

Uemura, Sotaro; Ishiwata, Shin'ichi

2003-04-01

Kinesin is an ATP-driven molecular motor that moves processively along a microtubule. Processivity has been explained as a mechanism that involves alternating single- and double-headed binding of kinesin to microtubules coupled to the ATPase cycle of the motor. The internal load imposed between the two bound heads has been proposed to be a key factor regulating the ATPase cycle in each head. Here we show that external load imposed along the direction of motility on a single kinesin molecule enhances the binding affinity of ADP for kinesin, whereas an external load imposed against the direction of motility decreases it. This coupling between loading direction and enzymatic activity is in accord with the idea that the internal load plays a key role in the unidirectional and cooperative movement of processive motors.

Affinity interactions between natural pigments and human whole saliva.

PubMed

Yao, Jiang-Wu; Lin, Feng; Tao, Tao; Lin, Chang-Jian

2011-03-01

The aim of the present study was to assess the null hypothesis that there are no differences of affinity between pigments and human whole saliva (WS), and the affinity is not influenced by the functional groups of pigments, temperatures, pH values, and salt concentrations. The affinity constants of interactions between WS and theaflavin (TF)/curcumin (Cur)/cyanidin (Cy) were determined by surface plasmon resonance (SPR) and fluorescence quenching. Mass-uptake at various temperatures, pH values, and salt concentrations was also carried out. The order of affinity of the pigments binding to WS is TF>Cur>Cy. A large number of complexes and precipitations of pigments/proteins were formed through a quick, strong, and almost irreversible binding process. The mass-uptake of pigments was affected not only by the functional groups, but also by molecular weight of pigments, temperatures, pH values, and salt concentrations. The complex of pigments may easily and rapidly deposit onto the WS film, and are difficult to remove from the WS surface. However, the complex of pigments can be reduced by properly regulating the physicochemical conditions, such as temperatures, pH values, and salt concentrations. Copyright © 2010 Elsevier Ltd. All rights reserved.

Metal-Chelate Affinity Precipitation with Thermo-Responsive Polymer for Purification of ε-Poly-L-Lysine.

PubMed

Li, Sipeng; Ding, Zhaoyang; Liu, Jifu; Cao, Xuejun

2017-12-01

ε-Poly-L-lysine (ε-PL) is a natural preservative for food processing industry. A thermo-responsive polymer, attached with Cu 2+ or Ni 2+ , was prepared for metal-chelate affinity precipitation for purification of ε-PL. The low critical solution temperatures (LCSTs) of these polymers were close to the room temperature (31.0-35.0 °C). The optimal adsorption conditions were as follows: pH 4.0, 0 mol/L NaCl, ligand density 75.00 μmol/g, and 120 min. The ligand Cu 2+ showed a stronger affinity interaction with ε-PL and the highest adsorption amount reached 251.93 mg/g polymer. The elution recovery of ε-PL could be 98.42% with 0.50 mol/L imidazole (pH = 8.0) as the eluent. The method could purify ε-PL from fermentation broth and the final product was proved as electrophoretic pure by SDS-PAGE. Moreover, these affinity polymers could be recycled after the purification of ε-PL and the recoveries were above 95.00%. Graphical Abstract Scheme for affinity precipitation of ε-PL.

A molecular determinant of phosphoinositide affinity in mammalian TRPV channels

NASA Astrophysics Data System (ADS)

Velisetty, Phanindra; Borbiro, Istvan; Kasimova, Marina A.; Liu, Luyu; Badheka, Doreen; Carnevale, Vincenzo; Rohacs, Tibor

2016-06-01

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is an important cofactor for ion channels. Affinity for this lipid is a major determinant of channel inhibition by depletion of PI(4,5)P2 upon phospholipase C (PLC) activation. Little is known about what determines PI(4,5)P2 affinity in mammalian ion channels. Here we report that two members of the Transient Receptor Potential Vanilloid (TRPV) ion channel family, TRPV5 and TRPV6 lack a positively charged residue in the TM4-TM5 loop that was shown to interact with PI(4,5)P2 in TRPV1, which shows high affinity for this lipid. When this positively charged residue was introduced to either TRPV6 or TRPV5, they displayed markedly higher affinities for PI(4,5)P2, and were largely resistant to inhibition by PI(4,5)P2 depletion. Furthermore, Ca2+-induced inactivation of TRPV6 was essentially eliminated in the G488R mutant, showing the importance of PLC-mediated PI(4,5)P2 depletion in this process. Computational modeling shows that the introduced positive charge interacts with PI(4,5)P2 in TRPV6.

Directed evolution of human T cell receptor CDR2 residues by phage display dramatically enhances affinity for cognate peptide-MHC without increasing apparent cross-reactivity

PubMed Central

Dunn, Steven M.; Rizkallah, Pierre J.; Baston, Emma; Mahon, Tara; Cameron, Brian; Moysey, Ruth; Gao, Feng; Sami, Malkit; Boulter, Jonathan; Li, Yi; Jakobsen, Bent K.

2006-01-01

The mammalian α/β T cell receptor (TCR) repertoire plays a pivotal role in adaptive immunity by recognizing short, processed, peptide antigens bound in the context of a highly diverse family of cell-surface major histocompatibility complexes (pMHCs). Despite the extensive TCR–MHC interaction surface, peptide-independent cross-reactivity of native TCRs is generally avoided through cell-mediated selection of molecules with low inherent affinity for MHC. Here we show that, contrary to expectations, the germ line-encoded complementarity determining regions (CDRs) of human TCRs, namely the CDR2s, which appear to contact only the MHC surface and not the bound peptide, can be engineered to yield soluble low nanomolar affinity ligands that retain a surprisingly high degree of specificity for the cognate pMHC target. Structural investigation of one such CDR2 mutant implicates shape complementarity of the mutant CDR2 contact interfaces as being a key determinant of the increased affinity. Our results suggest that manipulation of germ line CDR2 loops may provide a useful route to the production of high-affinity TCRs with therapeutic and diagnostic potential. PMID:16600963

Structural Basis of the High Affinity Interaction between the Alphavirus Nonstructural Protein-3 (nsP3) and the SH3 Domain of Amphiphysin-2.

PubMed

Tossavainen, Helena; Aitio, Olli; Hellman, Maarit; Saksela, Kalle; Permi, Perttu

2016-07-29

We show that a peptide from Chikungunya virus nsP3 protein spanning residues 1728-1744 binds the amphiphysin-2 (BIN1) Src homology-3 (SH3) domain with an unusually high affinity (Kd 24 nm). Our NMR solution complex structure together with isothermal titration calorimetry data on several related viral and cellular peptide ligands reveal that this exceptional affinity originates from interactions between multiple basic residues in the target peptide and the extensive negatively charged binding surface of amphiphysin-2 SH3. Remarkably, these arginines show no fixed conformation in the complex structure, indicating that a transient or fluctuating polyelectrostatic interaction accounts for this affinity. Thus, via optimization of such dynamic electrostatic forces, viral peptides have evolved a superior binding affinity for amphiphysin-2 SH3 compared with typical cellular ligands, such as dynamin, thereby enabling hijacking of amphiphysin-2 SH3-regulated host cell processes by these viruses. Moreover, our data show that the previously described consensus sequence PXRPXR for amphiphysin SH3 ligands is inaccurate and instead define it as an extended Class II binding motif PXXPXRpXR, where additional positive charges between the two constant arginine residues can give rise to extraordinary high SH3 binding affinity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Rational design of peptide affinity ligands for the purification of therapeutic enzymes.

PubMed

Trasatti, John P; Woo, James; Ladiwala, Asif; Cramer, Steven; Karande, Pankaj

2018-04-25

Non-mAb biologics represent a growing class of therapeutics under clinical development. Although affinity chromatography is a potentially attractive approach for purification, the development of platform technologies, such as Protein A for mAbs, has been challenging due to the inherent chemical and structural diversity of these molecules. Here, we present our studies on the rapid development of peptide affinity ligands for the purification of biologics using a prototypical enzyme therapeutic in clinical use. Employing a suite of de novo rational and combinatorial design strategies we designed and screened a library of peptides on microarray platforms for their ability to bind to the target with high affinity and selectivity in cell culture fluid. Lead peptides were evaluated on resin in batch conditions and compared with a commercially available resin to evaluate their efficacy. Two lead candidates identified from microarray studies provided high binding capacity to the target while demonstrating high selectivity against culture contaminants and product variants compared to a commercial resin system. These findings provide a proof-of-concept for developing affinity peptide-based bioseparations processes for a target biologic. Peptide affinity ligand design and screening approaches presented in this work can also be easily translated to other biologics of interest. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018. © 2018 American Institute of Chemical Engineers.

Binocular pattern deprivation interferes with the expression of proteins involved in primary visual cortex maturation in the cat.

PubMed

Laskowska-Macios, Karolina; Nys, Julie; Hu, Tjing-Tjing; Zapasnik, Monika; Van der Perren, Anke; Kossut, Malgorzata; Burnat, Kalina; Arckens, Lutgarde

2015-08-14

Binocular pattern deprivation from eye opening (early BD) delays the maturation of the primary visual cortex. This delay is more pronounced for the peripheral than the central visual field representation within area 17, particularly between the age of 2 and 4 months [Laskowska-Macios, Cereb Cortex, 2014]. In this study, we probed for related dynamic changes in the cortical proteome. We introduced age, cortical region and BD as principal variables in a 2-D DIGE screen of area 17. In this way we explored the potential of BD-related protein expression changes between central and peripheral area 17 of 2- and 4-month-old BD (2BD, 4BD) kittens as a valid parameter towards the identification of brain maturation-related molecular processes. Consistent with the maturation delay, distinct developmental protein expression changes observed for normal kittens were postponed by BD, especially in the peripheral region. These BD-induced proteomic changes suggest a negative regulation of neurite outgrowth, synaptic transmission and clathrin-mediated endocytosis, thereby implicating these processes in normal experience-induced visual cortex maturation. Verification of the expression of proteins from each of the biological processes via Western analysis disclosed that some of the transient proteomic changes correlate to the distinct behavioral outcome in adult life, depending on timing and duration of the BD period [Neuroscience 2013;255:99-109]. Taken together, the plasticity potential to recover from BD, in relation to ensuing restoration of normal visual input, appears to rely on specific protein expression changes and cellular processes induced by the loss of pattern vision in early life.

7 CFR 51.1158 - Mature.

Code of Federal Regulations, 2010 CFR

2010-01-01

... Citrus Industry, Part 1, Chapter 20-13 Market Classification, Maturity Standards and Processing or... 2065-S, 14th and Independence Ave., Washington, DC 20250 or at the National Archives and Records...: http://www.archives.gov/federal_register/code_of_federal_regulations/ibr_locations.html. ...

Aptamer-based downstream processing of his-tagged proteins utilizing magnetic beads.

PubMed

Kökpinar, Öznur; Walter, Johanna-Gabriela; Shoham, Yuval; Stahl, Frank; Scheper, Thomas

2011-10-01

Aptamers are synthetic nucleic acid-based high affinity ligands that are able to capture their corresponding target via molecular recognition. Here, aptamer-based affinity purification for His-tagged proteins was developed. Two different aptamers directed against the His-tag were immobilized on magnetic beads covalently. The resulting aptamer-modified magnetic beads were characterized and successfully applied for purification of different His-tagged proteins from complex E. coli cell lysates. Purification effects comparable to conventional immobilized metal affinity chromatography were achieved in one single purification step. Moreover, we have investigated the possibility to regenerate and reuse the aptamer-modified magnetic beads and have shown their long-term stability over a period of 6 months. Copyright © 2011 Wiley Periodicals, Inc.

Increasing the affinity of selective bZIP-binding peptides through surface residue redesign.

PubMed

Kaplan, Jenifer B; Reinke, Aaron W; Keating, Amy E

2014-07-01

The coiled-coil dimer is a prevalent protein interaction motif that is important for many cellular processes. The basic leucine-zipper (bZIP) transcription factors are one family of proteins for which coiled-coil mediated dimerization is essential for function, and misregulation of bZIPs can lead to disease states including cancer. This makes coiled coils attractive protein-protein interaction targets to disrupt using engineered molecules. Previous work designing peptides to compete with native coiled-coil interactions focused primarily on designing the core residues of the interface to achieve affinity and specificity. However, folding studies on the model bZIP GCN4 show that coiled-coil surface residues also contribute to binding affinity. Here we extend a prior study in which peptides were designed to bind tightly and specifically to representative members of each of 20 human bZIP families. These "anti-bZIP" peptides were designed with an emphasis on target-binding specificity, with contributions to design-target specificity and affinity engineered considering only the coiled-coil core residues. High-throughput testing using peptide arrays indicated many successes. We have now measured the binding affinities and specificities of anti-bZIPs that bind to FOS, XBP1, ATF6, and CREBZF in solution and tested whether redesigning the surface residues can increase design-target affinity. Incorporating residues that favor helix formation into the designs increased binding affinities in all cases, providing low-nanomolar binders of each target. However, changes in surface electrostatic interactions sometimes changed the binding specificity of the designed peptides. © 2014 The Protein Society.

Homeostatic signals do not drive post-thymic T cell maturation.

PubMed

Houston, Evan G; Boursalian, Tamar E; Fink, Pamela J

2012-01-01

Recent thymic emigrants, the youngest T cells in the lymphoid periphery, undergo a 3 week-long period of functional and phenotypic maturation before being incorporated into the pool of mature, naïve T cells. Previous studies indicate that this maturation requires T cell exit from the thymus and access to secondary lymphoid organs, but is MHC-independent. We now show that post-thymic T cell maturation is independent of homeostatic and costimulatory pathways, requiring neither signals delivered by IL-7 nor CD80/86. Furthermore, while CCR7/CCL19,21-regulated homing of recent thymic emigrants to the T cell zones within the secondary lymphoid organs is not required for post-thymic T cell maturation, an intact dendritic cell compartment modulates this process. It is thus clear that, unlike T cell development and homeostasis, post-thymic maturation is focused not on interrogating the T cell receptor or the cell's responsiveness to homeostatic or costimulatory signals, but on some as yet unrecognized property. Copyright © 2012 Elsevier Inc. All rights reserved.

Homeostatic Signals do not Drive Post-thymic T cell Maturation

PubMed Central

Houston, Evan G.; Boursalian, Tamar E.; Fink, Pamela J.

2012-01-01

Recent thymic emigrants, the youngest T cells in the lymphoid periphery, undergo a 3-week-long period of functional and phenotypic maturation before being incorporated into the pool of mature, naïve T cells. Previous studies indicate that this maturation requires T cell exit from the thymus and access to secondary lymphoid organs, but is MHC-independent. We now show that post-thymic T cell maturation is independent of homeostatic and costimulatory pathways, requiring neither signals delivered by IL-7 nor CD80/86. Furthermore, while CCR7/CCL19,21-regulated homing of recent thymic emigrants to the T cell zones within the secondary lymphoid organs is not required for post-thymic T cell maturation, an intact dendritic cell compartment modulates this process. It is thus clear that, unlike T cell development and homeostasis, post-thymic maturation is focused not on interrogating the T cell receptor or the cell’s responsiveness to homeostatic or costimulatory signals, but on some as yet unrecognized property. PMID:22398309

Knock-Down of CsNRT2.1, a Cucumber Nitrate Transporter, Reduces Nitrate Uptake, Root length, and Lateral Root Number at Low External Nitrate Concentration

PubMed Central

Li, Yang; Li, Juanqi; Yan, Yan; Liu, Wenqian; Zhang, Wenna; Gao, Lihong; Tian, Yongqiang

2018-01-01

Nitrogen (N) is a macronutrient that plays a crucial role in plant growth and development. Nitrate (NO3-) is the most abundant N source in aerobic soils. Plants have evolved two adaptive mechanisms such as up-regulation of the high-affinity transport system (HATS) and alteration of the root system architecture (RSA), allowing them to cope with the temporal and spatial variation of NO3-. However, little information is available regarding the nitrate transporter in cucumber, one of the most important fruit vegetables in the world. In this study we isolated a nitrate transporter named CsNRT2.1 from cucumber. Analysis of the expression profile of the CsNRT2.1 showed that CsNRT2.1 is a high affinity nitrate transporter which mainly located in mature roots. Subcellular localization analysis revealed that CsNRT2.1 is a plasma membrane transporter. In N-starved CsNRT2.1 knock-down plants, both of the constitutive HATS (cHATS) and inducible HATS (iHATS) were impaired under low external NO3- concentration. Furthermore, the CsNRT2.1 knock-down plants showed reduced root length and lateral root numbers. Together, our results demonstrated that CsNRT2.1 played a dual role in regulating the HATS and RSA to acquire NO3- effectively under N limitation. PMID:29911677