Simulation of B Cell Affinity Maturation Explains Enhanced Antibody Cross-Reactivity Induced by the Polyvalent Malaria Vaccine AMA1

DTIC Science & Technology

2014-07-01

cell decisions in lymphoid tissue. Mol. Cell . Biol. 28: 4040–4051. 22. Kosmrlj, A., A. K. Jha, E. S. Huseby, M. Kardar, and A. K. Chakraborty. 2008. How...JUL 2014 2. REPORT TYPE 3. DATES COVERED 00-00-2014 to 00-00-2014 4. TITLE AND SUBTITLE Simulation of B Cell Affinity Maturation Explains...8-98) Prescribed by ANSI Std Z39-18 The Journal of Immunology Simulation of B Cell Affinity Maturation Explains Enhanced Antibody Cross-Reactivity

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.

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.

Vaccine Efficacy and Affinity Maturation

NASA Astrophysics Data System (ADS)

Lee, Hayoun; Deem, Michael W.

2002-03-01

We propose macroscopic equations to describe variable vaccine efficacy between repeated vaccinee and first time vaccinee. The main ingredients are antigenic distance between epidemic strain and vaccne strain, and affinity maturation dynamics which differs in primary and second response. Increase of affinity by repeated vaccine leads to localization in immune space. This localization decreases the ability of the immune system to response to distant, but related epidemic strains.

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.

Cutting edge: double-stranded DNA breaks in the IgV region gene were detected at lower frequency in affinity-maturation impeded GANP-/- mice.

PubMed

Kawatani, Yousuke; Igarashi, Hideya; Matsui, Takeshi; Kuwahara, Kazuhiko; Fujimura, Satoru; Okamoto, Nobukazu; Takagi, Katsumasa; Sakaguchi, Nobuo

2005-11-01

Double-stranded DNA breaks (DSBs) at the IgV region (IgV) genes might be involved in somatic hypermutation and affinity-maturation of the B cell receptor in response to T cell-dependent Ag. By ligation-mediated PCR, we studied IgV DSBs that occurred in mature germinal center B cells in response to nitrophenyl-chicken gamma-globulin in a RAG1-independent, Ag-dependent, and IgV-selective manner. We quantified their levels in GANP-deficient B cells that have impaired generation of high-affinity Ab. GANP-/- B cells showed a decreased level of DSBs with blunt ends than control B cells and, on the contrary, the ganp gene transgenic (GANPTg) B cells showed an increased level. These results suggested that the level of IgV DSBs in germinal center B cells is associated with GANP expression, which is presumably required for B cell receptor affinity maturation.

Repertoire Analysis of Antibody CDR-H3 Loops Suggests Affinity Maturation Does Not Typically Result in Rigidification

PubMed Central

Jeliazkov, Jeliazko R.; Sljoka, Adnan; Kuroda, Daisuke; Tsuchimura, Nobuyuki; Katoh, Naoki; Tsumoto, Kouhei; Gray, Jeffrey J.

2018-01-01

Antibodies can rapidly evolve in specific response to antigens. Affinity maturation drives this evolution through cycles of mutation and selection leading to enhanced antibody specificity and affinity. Elucidating the biophysical mechanisms that underlie affinity maturation is fundamental to understanding B-cell immunity. An emergent hypothesis is that affinity maturation reduces the conformational flexibility of the antibody’s antigen-binding paratope to minimize entropic losses incurred upon binding. In recent years, computational and experimental approaches have tested this hypothesis on a small number of antibodies, often observing a decrease in the flexibility of the complementarity determining region (CDR) loops that typically comprise the paratope and in particular the CDR-H3 loop, which contributes a plurality of antigen contacts. However, there were a few exceptions and previous studies were limited to a small handful of cases. Here, we determined the structural flexibility of the CDR-H3 loop for thousands of recent homology models of the human peripheral blood cell antibody repertoire using rigidity theory. We found no clear delineation in the flexibility of naïve and antigen-experienced antibodies. To account for possible sources of error, we additionally analyzed hundreds of human and mouse antibodies in the Protein Data Bank through both rigidity theory and B-factor analysis. By both metrics, we observed only a slight decrease in the CDR-H3 loop flexibility when comparing affinity matured antibodies to naïve antibodies, and the decrease was not as drastic as previously reported. Further analysis, incorporating molecular dynamics simulations, revealed a spectrum of changes in flexibility. Our results suggest that rigidification may be just one of many biophysical mechanisms for increasing affinity. PMID:29545810

The protein-protein interface evolution acts in a similar way to antibody affinity maturation.

PubMed

Li, Bohua; Zhao, Lei; Wang, Chong; Guo, Huaizu; Wu, Lan; Zhang, Xunming; Qian, Weizhu; Wang, Hao; Guo, Yajun

2010-02-05

Understanding the evolutionary mechanism that acts at the interfaces of protein-protein complexes is a fundamental issue with high interest for delineating the macromolecular complexes and networks responsible for regulation and complexity in biological systems. To investigate whether the evolution of protein-protein interface acts in a similar way as antibody affinity maturation, we incorporated evolutionary information derived from antibody affinity maturation with common simulation techniques to evaluate prediction success rates of the computational method in affinity improvement in four different systems: antibody-receptor, antibody-peptide, receptor-membrane ligand, and receptor-soluble ligand. It was interesting to find that the same evolutionary information could improve the prediction success rates in all the four protein-protein complexes with an exceptional high accuracy (>57%). One of the most striking findings in our present study is that not only in the antibody-combining site but in other protein-protein interfaces almost all of the affinity-enhancing mutations are located at the germline hotspot sequences (RGYW or WA), indicating that DNA hot spot mechanisms may be widely used in the evolution of protein-protein interfaces. Our data suggest that the evolution of distinct protein-protein interfaces may use the same basic strategy under selection pressure to maintain interactions. Additionally, our data indicate that classical simulation techniques incorporating the evolutionary information derived from in vivo antibody affinity maturation can be utilized as a powerful tool to improve the binding affinity of protein-protein complex with a high accuracy.

Role of framework mutations and antibody flexibility in the evolution of broadly neutralizing antibodies

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

Ovchinnikov, Victor; Louveau, Joy E.; Barton, John P.

Eliciting antibodies that are cross reactive with surface proteins of diverse strains of highly mutable pathogens (e.g., HIV, influenza) could be key for developing effective universal vaccines. Mutations in the framework regions of such broadly neutralizing antibodies (bnAbs) have been reported to play a role in determining their properties. We used molecular dynamics simulations and models of affinity maturation to study specific bnAbs against HIV. Our results suggest that there are different classes of evolutionary lineages for the bnAbs. If germline B cells that initiate affinity maturation have high affinity for the conserved residues of the targeted epitope, framework mutationsmore » increase antibody rigidity as affinity maturation progresses to evolve bnAbs. If the germline B cells exhibit weak/moderate affinity for conserved residues, an initial increase in flexibility via framework mutations may be required for the evolution of bnAbs. Subsequent mutations that increase rigidity result in highly potent bnAbs. Implications of our results for immunogen design are discussed.« less

Role of framework mutations and antibody flexibility in the evolution of broadly neutralizing antibodies

DOE PAGES

Ovchinnikov, Victor; Louveau, Joy E.; Barton, John P.; ...

2018-02-14

Eliciting antibodies that are cross reactive with surface proteins of diverse strains of highly mutable pathogens (e.g., HIV, influenza) could be key for developing effective universal vaccines. Mutations in the framework regions of such broadly neutralizing antibodies (bnAbs) have been reported to play a role in determining their properties. We used molecular dynamics simulations and models of affinity maturation to study specific bnAbs against HIV. Our results suggest that there are different classes of evolutionary lineages for the bnAbs. If germline B cells that initiate affinity maturation have high affinity for the conserved residues of the targeted epitope, framework mutationsmore » increase antibody rigidity as affinity maturation progresses to evolve bnAbs. If the germline B cells exhibit weak/moderate affinity for conserved residues, an initial increase in flexibility via framework mutations may be required for the evolution of bnAbs. Subsequent mutations that increase rigidity result in highly potent bnAbs. Implications of our results for immunogen design are discussed.« less

Role of framework mutations and antibody flexibility in the evolution of broadly neutralizing antibodies

PubMed Central

2018-01-01

Eliciting antibodies that are cross reactive with surface proteins of diverse strains of highly mutable pathogens (e.g., HIV, influenza) could be key for developing effective universal vaccines. Mutations in the framework regions of such broadly neutralizing antibodies (bnAbs) have been reported to play a role in determining their properties. We used molecular dynamics simulations and models of affinity maturation to study specific bnAbs against HIV. Our results suggest that there are different classes of evolutionary lineages for the bnAbs. If germline B cells that initiate affinity maturation have high affinity for the conserved residues of the targeted epitope, framework mutations increase antibody rigidity as affinity maturation progresses to evolve bnAbs. If the germline B cells exhibit weak/moderate affinity for conserved residues, an initial increase in flexibility via framework mutations may be required for the evolution of bnAbs. Subsequent mutations that increase rigidity result in highly potent bnAbs. Implications of our results for immunogen design are discussed. PMID:29442996

Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging

PubMed Central

Orcutt, Kelly Davis; Slusarczyk, Adrian L; Cieslewicz, Maryelise; Ruiz-Yi, Benjamin; Bhushan, Kumar R; Frangioni, John V; Wittrup, K Dane

2014-01-01

Introduction In pretargeted radioimmunotherapy (PRIT), a bifunctional antibody is administered and allowed to pre-localize to tumor cells. Subsequently, a chelated radionuclide is administered and captured by cell-bound antibody while unbound hapten clears rapidly from the body. We aim to engineer high-affinity binders to DOTA chelates for use in PRIT applications. Methods We mathematically modeled antibody and hapten pharmacokinetics to analyze hapten tumor retention as a function of hapten binding affinity. Motivated by model predictions, we used directed evolution and yeast surface display to affinity mature the 2D12.5 antibody to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), reformatted as a single chain variable fragment (scFv). Results Modeling predicts that for high antigen density and saturating bsAb dose, a hapten binding affinity of 100 picomolar (pM) is needed for near-maximal hapten retention. We affinity matured 2D12.5 with an initial binding constant of about 10 nanomolar (nM) to DOTA-yttrium chelates. Affinity maturation resulted in a 1000-fold affinity improvement to biotinylated DOTA-yttrium, yielding an 8.2 ± 1.9 picomolar binder. The high-affinity scFv binds DOTA complexes of lutetium and gadolinium with similar picomolar affinity and indium chelates with low nanomolar affinity. When engineered into a bispecific antibody construct targeting carcinoembryonic antigen (CEA), pretargeted high-affinity scFv results in significantly higher tumor retention of a 111In-DOTA hapten compared to pretargeted wild-type scFv in a xenograft mouse model. Conclusions We have engineered a versatile, high-affinity DOTA-chelate-binding scFv. We anticipate it will prove useful in developing pretargeted imaging and therapy protocols to exploit the potential of a variety of radiometals. PMID:21315278

Structure-Guided Combinatorial Engineering Facilitates Affinity and Specificity Optimization of Anti-CD81 Antibodies.

PubMed

Nelson, Bryce; Adams, Jarrett; Kuglstatter, Andreas; Li, Zhijian; Harris, Seth F; Liu, Yang; Bohini, Sandya; Ma, Han; Klumpp, Klaus; Gao, Junjun; Sidhu, Sachdev S

2018-07-06

Hepatitis C viral infection is the major cause of chronic hepatitis that affects as many as 71 million people worldwide. Rather than target the rapidly shifting viruses and their numerous serotypes, four independent antibodies were made to target the host antigen CD81 and were shown to block hepatitis C viral entry. The single-chain variable fragment of each antibody was crystallized in complex with the CD81 large extracellular loop in order to guide affinity maturation of two distinct antibodies by phage display. Affinity maturation of antibodies using phage display has proven to be critical to therapeutic antibody development and typically involves modification of the paratope for increased affinity, improved specificity, enhanced stability or a combination of these traits. One antibody was engineered for increased affinity for human CD81 large extracellular loop that equated to increased efficacy, while the second antibody was engineered for cross-reactivity with cynomolgus CD81 to facilitate animal model testing. The use of structures to guide affinity maturation library design demonstrates the utility of combining structural analysis with phage display technologies. Copyright © 2018 Elsevier Ltd. All rights reserved.

In vitro fertilization of in vitro-matured equine oocytes: effect of maturation medium, duration of maturation, and sperm calcium ionophore treatment, and comparison with rates of fertilization in vivo after oviductal transfer.

PubMed

Hinrichs, K; Love, C C; Brinsko, S P; Choi, Y H; Varner, D D

2002-07-01

Three experiments were conducted to evaluate the effect of oocyte and sperm treatments on rates of in vitro fertilization (IVF) in the horse and to determine the capacity of in vitro-matured horse oocytes to be fertilized in vivo. There was no effect of duration of oocyte maturation (24 vs. 42 h) or calcium ionophore concentration during sperm capacitation (3 microM vs. 7.14 microM) on in vitro fertilization rates. Oocytes matured in 100% follicular fluid had significantly higher fertilization (13% to 24%) than did oocytes matured in maturation medium or in 20% follicular fluid (0% to 12%; P < 0.05). There was no significant difference in fertilization rate among 3 sperm treatments utilizing 7.14 microM calcium ionophore (12% to 21%). Of in vitro-matured oocytes recovered 40-44 h after transfer to the oviducts of inseminated mares, 77% showed normal fertilization (2 pronuclei to normal cleavage). Cleavage to 2 or more cells was seen in 22% of oocytes matured in follicular fluid and 63% of oocytes matured in maturation medium; this difference was significant (P < 0.05). We conclude that in vitro-matured horse oocytes are capable of being fertilized at high rates in the appropriate environment and that in vitro maturation of oocytes in follicular fluid increases fertilization rate in vitro but reduces embryo development after fertilization in vivo. Further work is needed to determine the optimum environment for sperm capacitation and IVF in the horse.

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.

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.

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.

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

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

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

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

Regulation of Fatty Acid Oxidation in Mouse Cumulus-Oocyte Complexes during Maturation and Modulation by PPAR Agonists

PubMed Central

Dunning, Kylie R.; Anastasi, Marie R.; Zhang, Voueleng J.; Russell, Darryl L.; Robker, Rebecca L.

2014-01-01

Fatty acid oxidation is an important energy source for the oocyte; however, little is known about how this metabolic pathway is regulated in cumulus-oocyte complexes. Analysis of genes involved in fatty acid oxidation showed that many are regulated by the luteinizing hormone surge during in vivo maturation, including acyl-CoA synthetases, carnitine transporters, acyl-CoA dehydrogenases and acetyl-CoA transferase, but that many are dysregulated when cumulus-oocyte complexes are matured under in vitro maturation conditions using follicle stimulating hormone and epidermal growth factor. Fatty acid oxidation, measured as production of 3H2O from [3H]palmitic acid, occurs in mouse cumulus-oocyte complexes in response to the luteinizing hormone surge but is significantly reduced in cumulus-oocyte complexes matured in vitro. Thus we sought to determine whether fatty acid oxidation in cumulus-oocyte complexes could be modulated during in vitro maturation by lipid metabolism regulators, namely peroxisome proliferator activated receptor (PPAR) agonists bezafibrate and rosiglitazone. Bezafibrate showed no effect with increasing dose, while rosiglitazone dose dependently inhibited fatty acid oxidation in cumulus-oocyte complexes during in vitro maturation. To determine the impact of rosiglitazone on oocyte developmental competence, cumulus-oocyte complexes were treated with rosiglitazone during in vitro maturation and gene expression, oocyte mitochondrial activity and embryo development following in vitro fertilization were assessed. Rosiglitazone restored Acsl1, Cpt1b and Acaa2 levels in cumulus-oocyte complexes and increased oocyte mitochondrial membrane potential yet resulted in significantly fewer embryos reaching the morula and hatching blastocyst stages. Thus fatty acid oxidation is increased in cumulus-oocyte complexes matured in vivo and deficient during in vitro maturation, a known model of poor oocyte quality. That rosiglitazone further decreased fatty acid oxidation during in vitro maturation and resulted in poor embryo development points to the developmental importance of fatty acid oxidation and the need for it to be optimized during in vitro maturation to improve this reproductive technology. PMID:24505284

The beneficial effects of cumulus cells and oocyte-cumulus cell gap junctions depends on oocyte maturation and fertilization methods in mice.

PubMed

Zhou, Cheng-Jie; Wu, Sha-Na; Shen, Jiang-Peng; Wang, Dong-Hui; Kong, Xiang-Wei; Lu, Angeleem; Li, Yan-Jiao; Zhou, Hong-Xia; Zhao, Yue-Fang; Liang, Cheng-Guang

2016-01-01

Cumulus cells are a group of closely associated granulosa cells that surround and nourish oocytes. Previous studies have shown that cumulus cells contribute to oocyte maturation and fertilization through gap junction communication. However, it is not known how this gap junction signaling affects in vivo versus in vitro maturation of oocytes, and their subsequent fertilization and embryonic development following insemination. Therefore, in our study, we performed mouse oocyte maturation and insemination using in vivo- or in vitro-matured oocyte-cumulus complexes (OCCs, which retain gap junctions between the cumulus cells and the oocytes), in vitro-matured, denuded oocytes co-cultured with cumulus cells (DCs, which lack gap junctions between the cumulus cells and the oocytes), and in vitro-matured, denuded oocytes without cumulus cells (DOs). Using these models, we were able to analyze the effects of gap junction signaling on oocyte maturation, fertilization, and early embryo development. We found that gap junctions were necessary for both in vivo and in vitro oocyte maturation. In addition, for oocytes matured in vivo, the presence of cumulus cells during insemination improved fertilization and blastocyst formation, and this improvement was strengthened by gap junctions. Moreover, for oocytes matured in vitro, the presence of cumulus cells during insemination improved fertilization, but not blastocyst formation, and this improvement was independent of gap junctions. Our results demonstrate, for the first time, that the beneficial effect of gap junction signaling from cumulus cells depends on oocyte maturation and fertilization methods.

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.

In vitro maturation of human oocytes for assisted reproduction.

PubMed

Jurema, Marcus W; Nogueira, Daniela

2006-11-01

To describe and evaluate the current practice of in vitro maturation of oocytes for assisted reproduction. Review of the available and relevant literature regarding in vitro maturation of oocytes. In vitro maturation of human oocytes retrieved from antral ovarian follicles is an emerging procedure quickly being incorporated into the realm of assisted reproductive technologies. This new technology has several potential advantages over traditional controlled ovarian hyperstimulation for IVF, such as reduction of costs by minimizing gonadotropin and GnRH analogue use, elimination of ovarian hyperstimulation syndrome, and simplicity of protocol. In vitro maturation of oocytes for assisted reproduction in human beings still is undergoing refinement but currently is providing efficacy and safety outcome comparable to that of traditional IVF in recent selected studies. Implementing in vitro maturation into an established IVF practice is feasible and requires only a few simple adjustments. Crucial to the advancement and optimization of the technology is a better understanding of how to maximize immature oocyte developmental competence and endometrial receptivity.

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.

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

A Compilation of In Vitro Rate and Affinity Values for Xenobiotic Biotransformation in Fish, Measured Under Physiological Conditions

EPA Science Inventory

This manuscript presents a summary of in vitro rate and affinity data for xenobiotic biotransformation enzymes in fish...One potential use of this data summary is to support in vitro to in vivo metabolism extrapolations which can be used as inputs to chemical kinetic models for f...

In vitro Maturation of Oocytes in High Altitude Women with Polycystic Ovaries.

PubMed

Tominaga, Luis A Vargas; Cáceres, Ricardo E Pella; Lechuga, Jose A Vargas; Durán, Livia S Bartolo; Vargas, Mariela Serrano

2015-05-01

Determine the effectiveness of in vitro maturation of oocytes in the infertility treatment in high altitude women with polycystic ovaries. descriptive and retrospective study. Women with polycystic ovaries and infertility. there were 11 women from locations above 7,546 feet above sea level with polycystic ovaries and infertility in which were performed in vitro maturation of oocytes, followed by intracytoplasmic sperm injection, culture and embryo vitrification. After that, the endometrium was prepared and the embryos were thawed and transferred. Main results mesurements: oocytes maturation, fertilization, clinical pregnancy and implantation rates. Oocytes maturation rate was 86.1%; fertilization rate 90.3%; clinical pregnancy rate 36.4% and implantation rate 17.4%. In vitro maturation of oocytes is an effective technique in the infertility treatment of high altitude women with polycystic ovaries.

Facile Affinity Maturation of Antibody Variable Domains Using Natural Diversity Mutagenesis

PubMed Central

Tiller, Kathryn E.; Chowdhury, Ratul; Li, Tong; Ludwig, Seth D.; Sen, Sabyasachi; Maranas, Costas D.; Tessier, Peter M.

2017-01-01

The identification of mutations that enhance antibody affinity while maintaining high antibody specificity and stability is a time-consuming and laborious process. Here, we report an efficient methodology for systematically and rapidly enhancing the affinity of antibody variable domains while maximizing specificity and stability using novel synthetic antibody libraries. Our approach first uses computational and experimental alanine scanning mutagenesis to identify sites in the complementarity-determining regions (CDRs) that are permissive to mutagenesis while maintaining antigen binding. Next, we mutagenize the most permissive CDR positions using degenerate codons to encode wild-type residues and a small number of the most frequently occurring residues at each CDR position based on natural antibody diversity. This mutagenesis approach results in antibody libraries with variants that have a wide range of numbers of CDR mutations, including antibody domains with single mutations and others with tens of mutations. Finally, we sort the modest size libraries (~10 million variants) displayed on the surface of yeast to identify CDR mutations with the greatest increases in affinity. Importantly, we find that single-domain (VHH) antibodies specific for the α-synuclein protein (whose aggregation is associated with Parkinson’s disease) with the greatest gains in affinity (>5-fold) have several (four to six) CDR mutations. This finding highlights the importance of sampling combinations of CDR mutations during the first step of affinity maturation to maximize the efficiency of the process. Interestingly, we find that some natural diversity mutations simultaneously enhance all three key antibody properties (affinity, specificity, and stability) while other mutations enhance some of these properties (e.g., increased specificity) and display trade-offs in others (e.g., reduced affinity and/or stability). Computational modeling reveals that improvements in affinity are generally not due to direct interactions involving CDR mutations but rather due to indirect effects that enhance existing interactions and/or promote new interactions between the antigen and wild-type CDR residues. We expect that natural diversity mutagenesis will be useful for efficient affinity maturation of a wide range of antibody fragments and full-length antibodies. PMID:28928732

Comparison of glucose metabolism in in vivo- and in vitro-matured tammar wallaby oocytes and its relationship to developmental potential following intracytoplasmic sperm injection.

PubMed

Magarey, Genevieve M; Mate, Karen E

2004-01-01

Although marsupial oocytes undergo nuclear maturation in vitro, there is, at present, no indication of their developmental potential, largely owing to the lack of in vitro fertilisation and related technologies for marsupials. Glucose metabolism has proven a useful indicator of oocyte cytoplasmic maturation and developmental potential in several eutherian species. Therefore, the aims of the present study were to compare: (1) the rates of glycolysis and glucose oxidation in immature, in vitro-matured and in vivo-matured tammar wallaby oocytes; and (2) the metabolic rate of individual oocytes with their ability to form pronuclei after intracytoplasmic sperm injection. The rates of glycolysis measured in immature (2.18 pmol oocyte(-1) h(-1)), in vitro- matured (0.93 pmol oocyte(-1) h(-1)) and in vivo-matured tammar wallaby oocytes (0.54 pmol oocyte(-1) h(-1)) were within a similar range to values obtained in eutherian species. However, unlike the trend observed in eutherian oocytes, the glycolytic rate was significantly higher in immature oocytes compared with either in vivo- or in vitro-matured oocytes (P < 0.001) and significantly higher in in vitro-matured oocytes compared with in vivo-matured oocytes (P < 0.001). No relationship was identified between glucose metabolism and the developmental capacity of oocytes after intracytoplasmic sperm injection when assessed after 17-19 h. Oocytes that became fertilised (two pronuclei) or activated (one or more pronucleus) were not distinguished from others by their metabolic rates. Longer culture after intracytoplasmic sperm injection (e.g. blastocyst stage) may show oocyte glucose metabolism to be predictive of developmental potential; however, culture to the single-cell stage did not reveal any significant differences in normally developing embryos.

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.

Progesterone improves porcine in vitro fertilisation system.

PubMed

Malo, Clara; Gil, Lydia; Cano, Rafael; Martinez, Felisa; Gonzalez, Noelia

2014-03-01

In an effort to improve the quality of in vitro produced porcine embryos, the effect of progestagens - progesterone analogues - on the in vitro developmental competence of porcine oocytes was studied. A total of 1421 in vitro matured oocytes, from 4 replicates, were inseminated with frozen-thawed spermatozoa. Progestagens were added to late maturation and embryo cultures (10 IU/ml). Fertilisation success (pre-maturation, penetration, monospermy and efficiency) and nuclear maturation were evaluated. There were no differences among prematuration rates between groups (P = 0.221). Penetration rates were higher (P < 0.001) in the presence of progestagens (75.0%) as compared to the control (51.7%). However, no differences were observed in monospermy percentages (P = 0.246). The results indicated that supplementation with progestagens increased the efficiency of the in vitro fertilisation system (P < 0.001). An additional beneficial effect was observed in nuclear maturation with progestagens (P = 0.035). In summary, progestagen supplementation is an important factor to improve the in vitro fertilisation procedure.

A feasibility study of prepubertal and over mature aged local goat in relation to results of In Vitro growth culture to obtain additional M-II oocyte resources

NASA Astrophysics Data System (ADS)

Ciptadi, Gatot; Ihsan, M. Nur; Rahayu, Sri; Widjaja, D. H. K.; Mudawamah, Mudawamah

2017-11-01

The aims of this research are to study the potential source of mature (M-II) oocytes of domestic animals using follicles isolated from prepubertal and over mature aged Indonesian local goats, resulting from an in vitro growth (IVG) method. This method of IVG could provide a new source of M-II oocytes for embryo production. In Indonesia, a very limited number of a good quality oocytes are available for research purposes, as there is a limited number of reproductive females slaughtered, which is dominated by prepubertal and old mature aged animals. IVG culture systems could be improved as an alternative method to provide a new source of a good quality oocytes for in vitro maturation of M-II oocytes. From a number of prepubertal and mature aged goats slaughtered in a local abattoir, the small oocytes in the preantral follicles were cultured in vitro to normal oocyte growth. The methods used in this research are experimental. Follicles were isolated, cultured in vitro for 14 days individually using a sticky medium containing 4% (w/v) polyvinylpyrrolidone in TCM 199 10% Fetal Bovine Serum supplemented with Follicle Stimulating Hormone, which was then evaluated for their follicle development and oocyte quality. The research results showed that a minimum follicle size and oocyte diameter is needed (>100 um) for early evaluation of maturation to be achieved, meanwhile oocytes recovered from IVG after being cultured in vitro for maturation resulted in a very low rate of maturation. However, in the future, IVG of the preantral follicles of Indonesian local goat could be considered as an alternative source of oocytes for both research purposes and embryo production in vitro.

Effect of ferulic acid supplementation on the developmental competence of porcine embryos during in vitro maturation.

PubMed

Tanihara, Fuminori; Hirata, Maki; Nhien, Nguyen Thi; Hirano, Takayuki; Kunihara, Toshiki; Otoi, Takeshige

2018-05-16

The value of laboratory and genetically-modified pigs is becoming increasingly clear; however, their in vitro development remains inefficient. Trans-ferulic acid (trans-FA) is an aromatic compound that is abundant in plant cell walls, and which exhibits antioxidant effects in vitro. Trans-FA is known to improve sperm viability and motility; however, its effects on porcine oocytes are unknown. Our aim was to investigate the effects of trans-FA supplementation during in vitro maturation on the meiotic and developmental competence of porcine oocytes. Oocytes were matured either without (control) or with trans-FA (10, 100, and 1,000 µM), fertilized, and cultured in vitro for 7 days. The maturation rate of oocytes cultured with 10 µM trans-FA (81.6%) was significantly higher than that of controls (65.0%; P<0.05). The fertilization rate of oocytes matured with 10 µM trans-FA (57.4%) was also significantly higher than that of controls (32.7%) and oocytes cultured with other concentrations (33.1% and 22.7% for 100 and 1,000 µM, respectively; P<0.05). Moreover, the blastocyst formation rate of oocytes matured with 10 µM trans-FA (6.9%) was significantly higher than that of controls (2.3%; P<0.05). Our results suggest that in vitro maturation with 10 µM trans-FA is beneficial for the in vitro production of porcine embryos and has the potential to improve production system.

Chlorogenic acid supplementation during in vitro maturation improves maturation, fertilization and developmental competence of porcine oocytes.

PubMed

Nguyen, T-V; Tanihara, F; Do, Ltk; Sato, Y; Taniguchi, M; Takagi, M; Van Nguyen, T; Otoi, T

2017-12-01

Chlorogenic acid (CGA) is a quinic acid conjugate of caffeic acid, and a phytochemical found in many fruits and beverages that acts as an antioxidant. The present study investigated the effects of CGA supplementation during in vitro maturation (IVM), on in vitro development of porcine oocytes, to improve the porcine in vitro production (IVP) system. Oocytes were matured either without (control) or with CGA (10, 50, 100 and 200 μM). Subsequently, the matured oocytes were fertilized and cultured in vitro for 7 day. The rates of maturation, fertilization and blastocyst formation of oocytes matured with 50 μM CGA were significantly (p < .05) higher than those of the control oocytes. Hydrogen peroxide (H 2 O 2 ) is one of the reactive oxygen species and induces DNA damage in porcine oocytes. When oocytes were matured with 1 mM H 2 O 2 to assess the protective effect of CGA, 50 μM CGA supplementation improved the maturation rate and the proportion of DNA-fragmented nuclei in oocytes compared with control oocytes matured without CGA. Moreover, when oocytes were matured with either 50 μM CGA (control) or caffeic acid (10, 50 and 100 μM), the rates of maturation, fertilization and the blastocyst formation of oocytes matured with 50 μM CGA were similar to those of oocytes matured with 10 and 50 μM caffeic acid. Our results suggest that CGA has comparable effects to caffeic acid, and IVM with 50 μM CGA is particularly beneficial to IVP of porcine embryos and protects oocytes from DNA damage induced by oxidative stress. Supplementation of CGA to the maturation medium has a potential to improve porcine IVP system. © 2017 Blackwell Verlag GmbH.

RAPID COMMUNICATION: Nerve growth factor influences cleavage rate and embryo development in sheep.

PubMed

Crispo, M; Dos Santos-Neto, P C; Vilariño, M; Mulet, A P; de León, A; Barbeito, L; Menchaca, A

2016-10-01

Recent information about Nerve growth factor (NGF), a protein traditionally associated to the nervous system that regulates survival and maturation of developing neurons, suggests that it may exert action also on different levels in the reproductive system. The aim of this study was to evaluate the effect of NGF added during in vitro oocyte maturation, fertilization or in vitro embryo development in sheep. Nerve growth factor was supplemented to the culture medium at 0, 100, or 1,000 ng/mL, during either in vitro maturation (Exp. 1), in vitro fertilization (Exp. 2), or in vitro culture (Exp. 3). In addition, NGF mRNA expression was determined in cumulus cells and oocytes. Nerve growth factor induced early cleavage when added during oocyte maturation or fertilization, improved embryo development when added during fertilization, and had no significant effect when added during embryo culture. In general, the effect was more evident with 100 rather than 1,000 ng/mL (P < 0.05). Expression of endogenous NGF was not detected in oocytes, and increased in cumulus cells when 1,000 ng/mL of NGF was added during fertilization, but not during maturation and embryo culture. In conclusion, the addition of NGF during oocyte maturation and fertilization affects in vitro cleavage and embryo development in sheep. We suggest a possible effect of this growth factor on oocyte maturation and mainly on the fertilization process.

Superovulation and in vitro oocyte maturation in three species of mice (Mus musculus, Mus spretus and Mus spicilegus).

PubMed

Martín-Coello, J; González, R; Crespo, C; Gomendio, M; Roldan, E R S

2008-10-01

Mouse oocytes can be obtained via superovulation or using in vitro maturation although several factors, including genetic background, may affect response. Our previous studies have identified various mouse species as models to understand the role of sexual selection on the evolution of sperm traits and function. In order to do comparative studies of sperm-oocyte interaction, we sought reliable methods for oocyte superovulation and in vitro maturation in mature females of three mouse species (genus Mus). When 5 IU pregnant mare's serum gonadotrophin (PMSG) and 5 IU human chorionic gonadotrophin (hCG) were injected 48 h apart, and oocytes collected 14 h post-hCG, good responses were obtained in Mus musculus (18+/-1.3 oocytes/female; mean+/-S.E.M.) and Mus spretus (12+/-0.8), but no ovulation was seen in Mus spicilegus. Changes in PMSG or hCG doses, or longer post-hCG intervals, did not improve results. Use of PMSG/luteinizing hormone (LH) resulted in good responses in M. musculus (19+/-1.2) and M. spretus (12+/-1.1) but not in M. spicilegus (5+/-0.9) with ovulation not increasing with higher LH doses. Follicular puncture 48 h after PMSG followed by in vitro maturation led to a high oocyte yield in the three species (M. musculus, 23+/-0.9; M. spretus, 17+/-1.1; M. spicilegus, 10+/-0.9) with a consistently high maturation rates. In vitro fertilization of both superovulated and in vitro matured oocytes resulted in a high proportion of fertilization (range: 83-87%) in the three species. Thus, in vitro maturation led to high yields in all three species. These results will allow future studies on gamete interaction in these closely related species and the role of sexual selection in gamete compatibility.

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

Increased Antibody Affinity Confers Broad In Vitro Protection against Escape Mutants of Severe Acute Respiratory Syndrome Coronavirus

PubMed Central

Rani, Mridula; Bolles, Meagan; Donaldson, Eric F.; Van Blarcom, Thomas; Baric, Ralph; Iverson, Brent

2012-01-01

Even though the effect of antibody affinity on neutralization potency is well documented, surprisingly, its impact on neutralization breadth and escape has not been systematically determined. Here, random mutagenesis and DNA shuffling of the single-chain variable fragment of the neutralizing antibody 80R followed by bacterial display screening using anchored periplasmic expression (APEx) were used to generate a number of higher-affinity variants of the severe acute respiratory syndrome coronavirus (SARS-CoV)-neutralizing antibody 80R with equilibrium dissociation constants (KD) as low as 37 pM, a >270-fold improvement relative to that of the parental 80R single-chain variable fragment (scFv). As expected, antigen affinity was shown to correlate directly with neutralization potency toward the icUrbani strain of SARS-CoV. Additionally, the highest-affinity antibody fragment displayed 10-fold-increased broad neutralization in vitro and completely protected against several SARS-CoV strains containing substitutions associated with antibody escape. Importantly, higher affinity also led to the suppression of viral escape mutants in vitro. Escape from the highest-affinity variant required reduced selective pressure and multiple substitutions in the binding epitope. Collectively, these results support the hypothesis that engineered antibodies with picomolar dissociation constants for a neutralizing epitope can confer escape-resistant protection. PMID:22696652

In vitro maturation and fertilization of oocytes from unstimulated ovaries in infertile women with polycystic ovary syndrome.

PubMed

Zhao, Jun-Zhao; Zhou, Wei; Zhang, Wei; Ge, Hong-Shan; Huang, Xue-Feng; Lin, Jin-Ju

2009-06-01

To evaluate the effects of in vitro maturation and fertilization of oocytes from unstimulated ovaries in infertile women with polycystic ovary syndrome (PCOS). Retrospective study. Reproductive Medicine Center, First Affiliated Hospital of Wenzhou Medical College, People's Republic of China. One hundred eighteen women with PCOS undergoing 152 cycles of in vitro maturation treatment. Oocyte retrieval was carried out by ultrasound-guided puncture on days 9-14 of the cycle. The oocytes were cultured in vitro using maturation culture medium, which consisted of M-199 + 20% fetal bovine serum (FBS) + 75 mIU/mL recombinant FSH +/- 0.5 IU/mL hCG. After the oocytes had matured in vitro, fertilization and embryo transfer were performed. Rates of clinical pregnancy, multiple pregnancies, and live birth. Relatively optimal laboratory results were obtained in this study. Embryo transfer was performed in 140 cycles, with a clinical pregnancy rate (PR) of 40.0% per transfer. Fifty-six babies have been born and there are 10 ongoing pregnancies. The overall multiple PR was 33.93%. Our results show that using in vitro matured oocytes from unstimulated ovaries could be offered as an alternative to conventional IVF in women with PCOS, and future work should address ways to decrease the incidence of multiple pregnancies.

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

Developmental competence of equine oocytes and embryos obtained by in vitro procedures ranging from in vitro maturation and ICSI to embryo culture, cryopreservation and somatic cell nuclear transfer.

PubMed

Galli, C; Colleoni, S; Duchi, R; Lagutina, I; Lazzari, G

2007-03-01

Development of assisted reproductive technologies in horses has been relatively slow compared to other domestic species, namely ruminants and pigs. The scarce availability of abattoir ovaries and the lack of interest from horse breeders and breed associations have been the main reasons for this delay. Progressively though, the technology of oocyte maturation in vitro has been established followed by the application of ICSI to achieve fertilization in vitro. Embryo culture was initially performed in vivo, in the mare oviduct or in the surrogate sheep oviduct, to achieve the highest embryo development, in the range of 18-36% of the fertilised oocytes. Subsequently, the parallel improvement of in vitro oocyte maturation conditions and embryo culture media has permitted high rates of embryo development from in vitro matured and in vitro cultured ICSI embryos, ranging from 5 to 10% in the early studies to up to 38% in the latest ones. From 2003, with the birth of the first cloned equids, the technology of somatic cell nuclear transfer has also become established due to improvement of the basic steps of embryo production in vitro, including cryopreservation. Pregnancy and foaling rates are still estimated based on a small number of in vitro produced equine embryos transferred to recipients. The largest set of data on non-surgical embryo transfer of in vitro produced embryos, from ICSI of both abattoir and in vitro-matured Ovum Pick Up (OPU) oocytes, and from somatic cell nuclear transfer, has been obtained in our laboratory. The data demonstrate that equine embryos produced by OPU and then cryopreserved can achieve up to 69% pregnancy rate with a foaling rate of 83%. These percentages are reduced to 11 and 23%, respectively, for cloned embryos. In conclusion, extensive evidence exists that in vitro matured equine oocytes can efficiently develop into viable embryos and offspring.

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

Structure-activity studies of dicationically substituted bis-benzimidazoles against Giardia lamblia: correlation of antigiardial activity with DNA binding affinity and giardial topoisomerase II inhibition.

PubMed Central

Bell, C A; Dykstra, C C; Naiman, N A; Cory, M; Fairley, T A; Tidwell, R R

1993-01-01

Nine dicationically substituted bis-benzimidazoles were examined for their in vitro activities against Giardia lamblia WB (ATCC 30957). The potential mechanisms of action of these compounds were evaluated by investigating the relationship among in vitro antigiardial activity and the affinity of the molecules for DNA and their ability to inhibit the activity of giardial topoisomerase II. Each compound demonstrated antigiardial activity, as measured by assessing the incorporation of [methyl-3H]thymidine by giardial trophozoites exposed to the test agents. Three compounds exhibited excellent in vitro antigiardial activities, with 50% inhibitory concentrations which compared very favorably with those of two currently used drugs, quinacrine HCl and metronidazole. Putative mechanisms of action for these compounds were suggested by the strong correlation observed among in vitro antigiardial activity and the affinity of the molecules for natural and synthetic DNA and their ability to inhibit the relaxation activity of giardial topoisomerase II. A strong correlation between the DNA binding affinity of these compounds and their inhibition of giardial topoisomerase II activity was also observed. Images PMID:8109934

The current challenges to efficient immature oocyte cryopreservation.

PubMed

Brambillasca, Fausta; Guglielmo, Maria Cristina; Coticchio, Giovanni; Mignini Renzini, Mario; Dal Canto, Mariabeatrice; Fadini, Rubens

2013-12-01

Oocyte cryopreservation represents an important tool for assisted reproductive technology. It offers the opportunity to preserve fertility in women at risk of loss of the ovarian function for various pathologies. It also represents a treatment alternative for couples that cannot benefit from embryo cryopreservation because of moral, religious, or legal constrains. On the other hand, in vitro oocyte maturation has a range of applications. It can be applied in patients with a contraindication to ovarian stimulation to prevent ovarian hyperstimulation syndrome or to eliminate the risk of stimulation of hormone-sensitive tumours in cancer patients. However, while mature oocyte cryopreservation has found wide-spread application and oocyte in vitro maturation has a place for the treatment of specific clinical conditions, data on the efficiency of freezing of immature or in vitro matured oocytes are poorer. In this review we will focus on the combination of oocyte in vitro maturation with oocyte cryopreservation with particular emphasis on the biological implications of the cryopreservation of immature or in vitro matured oocytes. The two cryopreservation approaches, slow freezing and vitrification, will be discussed in relation to possible cryodamage occurring to subcellular structures of the oocyte and the functional interaction between oocyte and cumulus cells.

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.

Effects of ghrelin on activation of Akt1 and ERK1/2 pathways during in vitro maturation of bovine oocytes.

PubMed

Chouzouris, Thomas-Markos; Dovolou, Eleni; Krania, Fotini; Pappas, Ioannis S; Dafopoulos, Konstantinos; Messinis, Ioannis E; Anifandis, George; Amiridis, Georgios S

2017-04-01

The purpose of this study was to investigate the possible molecular pathways through which ghrelin accelerates in vitro oocyte maturation. Bovine cumulus-oocyte complexes (COCs), after 18 or 24 h maturation in the absence or the presence of 800 pg ml-1 of acylated ghrelin were either assessed for nuclear maturation or underwent in vitro fertilization in standard media and putative zygotes were cultured in vitro for 8 days. In a subset of COCs the levels of phosphorylated Akt1 and ERK1/2 (MAPK1/3) were assessed at the 0th, 6th, 10th, 18th and 24th hours of in vitro maturation (IVM). At 18 and 24 h no difference existed in the proportion of matured oocytes in the ghrelin-treated group, while in the control group more (P < 0.05) matured oocyte were found at 24 h. Oocyte maturation for 24 h in the presence of ghrelin resulted in substantially reduced (P < 0.05) blastocyst yield(16.3%) in comparison with that obtained after 18 h (30.0%) or to both control groups (29.3% and 26.9%, for 18 and 24 h in maturation, respectively). Ghrelin-treated oocytes expressed lower Akt1 phosphorylation rate at the 10th hour of IVM, and higher ERK1/2 at the 6th and 10th hours of IVM compared with controls. In cumulus cells, at the 18th and 24th hours of IVM Akt1 phosphorylation rate was higher in ghrelin-treated oocytes. Our results imply that ghrelin acts in a different time-dependent manner on bovine oocytes and cumulus cells modulating Akt1 and ERK1/2 phosphorylation, which brings about acceleration of the oocyte maturation process.

Behaviour of the vitelline envelope in Bufo arenarum oocytes matured in vitro in blockade to polyspermy.

PubMed

Oterino, J; Sánchez Toranzo, G; Zelarayán, L; Ajmat, M T; Bonilla, F; Bühler, M I

2006-05-01

During activation of amphibian eggs, cortical granule exocytosis causes elaborate ultrastructural changes in the vitelline envelope. These changes involve modifications in the structure of the vitelline envelope and formation of a fertilization envelope (FE) that can no longer be penetrated by sperm. In Bufo arenarum, as the egg traverses the oviduct, the vitelline envelope is altered by a trypsin-like protease secreted by the oviduct, which induces an increased susceptibility of the vitelline envelope to sperm lysins. Full-grown oocytes of B. arenarum, matured in vitro by progesterone, are polyspermic, although cortical granule exocytosis seems to occur within a normal chronological sequence. These oocytes can be fertilized with or without trypsin treatment, suggesting that the vitelline envelope is totally sperm-permeable. Vitelline envelopes without trypsin treatment cannot retain either gp90 or gp96. This suggests that these glycoproteins are involved in the block to polyspermy and that trypsin treatment of matured in vitro oocytes before insemination is necessary to enable vitelline envelopes to block polyspermy. The loss of the binding capacity in vitelline envelopes isolated from B. arenarum oocytes matured in vitro with trypsin treatment and activated by electric shock suggests that previous trypsin treatment is a necessary step for sperm block to occur. When in vitro matured oocytes were incubated with the product of cortical granules obtained from in vitro matured oocytes (vCGP), vitelline envelopes with trypsin treatment were able to block sperm entry. These oocytes exhibited the characteristic signs of activation. These results support the idea that B. arenarum oocytes can be activated by external stimuli and suggest the presence of unknown oocyte surface receptors linked to the activation machinery in response to fertilization. Electrophoretic profiles obtained by SDS-PAGE of solubilized vitelline envelopes from oocytes matured in vitro revealed the conversion of gp40 (in vitro matured oocytes, without trypsin treatment) to gp38 (ascribable to trypsin activity or cortical granule product activity, CGP) and the conversion of gp70 to gp68 (ascribable to trypsin activity plus CGP activity). Taking into account that only the vitelline envelopes of in vitro matured oocytes with trypsin treatment and activated can block sperm entry, we may suggest that the conversion of gp70 to gp68 is related to the changes associated with sperm binding.

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

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

In vitro removal of human IgG autoantibodies by affinity filtration using immobilized L-histidine onto PEVA hollow fiber membranes.

PubMed

Ventura, R C; Zollner, R L; Legallais, C; Vijayalakshmi, M; Bueno, S M

2001-01-01

Histidine was immobilized onto PEVA membrane to obtain an affinity support for human IgG removal from serum with a view to clinical apheresis for the treatment of autoimmune diseases. These membranes were able to remove in vitro several autoantibodies from the serum of SLE patients.

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

Nanotechnology-Based Surface Plasmon Resonance Affinity Biosensors for In Vitro Diagnostics

PubMed Central

Antiochia, Riccarda; Bollella, Paolo; Favero, Gabriele

2016-01-01

In the last decades, in vitro diagnostic devices (IVDDs) became a very important tool in medicine for an early and correct diagnosis, a proper screening of targeted population, and also assessing the efficiency of a specific therapy. In this review, the most recent developments regarding different configurations of surface plasmon resonance affinity biosensors modified by using several nanostructured materials for in vitro diagnostics are critically discussed. Both assembly and performances of the IVDDs tested in biological samples are reported and compared. PMID:27594884

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.

“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

Sequence-specific DNA binding by MYC/MAX to low-affinity non-E-box motifs.

PubMed

Allevato, Michael; Bolotin, Eugene; Grossman, Mark; Mane-Padros, Daniel; Sladek, Frances M; Martinez, Ernest

2017-01-01

The MYC oncoprotein regulates transcription of a large fraction of the genome as an obligatory heterodimer with the transcription factor MAX. The MYC:MAX heterodimer and MAX:MAX homodimer (hereafter MYC/MAX) bind Enhancer box (E-box) DNA elements (CANNTG) and have the greatest affinity for the canonical MYC E-box (CME) CACGTG. However, MYC:MAX also recognizes E-box variants and was reported to bind DNA in a "non-specific" fashion in vitro and in vivo. Here, in order to identify potential additional non-canonical binding sites for MYC/MAX, we employed high throughput in vitro protein-binding microarrays, along with electrophoretic mobility-shift assays and bioinformatic analyses of MYC-bound genomic loci in vivo. We identified all hexameric motifs preferentially bound by MYC/MAX in vitro, which include the low-affinity non-E-box sequence AACGTT, and found that the vast majority (87%) of MYC-bound genomic sites in a human B cell line contain at least one of the top 21 motifs bound by MYC:MAX in vitro. We further show that high MYC/MAX concentrations are needed for specific binding to the low-affinity sequence AACGTT in vitro and that elevated MYC levels in vivo more markedly increase the occupancy of AACGTT sites relative to CME sites, especially at distal intergenic and intragenic loci. Hence, MYC binds diverse DNA motifs with a broad range of affinities in a sequence-specific and dose-dependent manner, suggesting that MYC overexpression has more selective effects on the tumor transcriptome than previously thought.

In vitro production of Sudanese camel (Camelus dromedarius) embryos from epididymal spermatozoa and follicular oocytes of slaughtered animals.

PubMed

Abdelkhalek, A E; Gabr, Sh A; Khalil, W A; Shamiah, Sh M; Pan, L; Qin, G; Farouk, M H

2017-03-28

Application of assisted reproductive technology in camelidea, such as artificial insemination (AI) and embryo transfer, has been slow in comparison to that for other livestock species. In Egypt, there are few attempts to establish in vitro maturation (IVM) and fertilization (IVF) techniques in dromedary camel. The present study was carried out to produce Sudanese camel embryos using in vitro matured oocytes and epididymal spermatozoa. Dromedary camel ovaries were collected from abattoirs and then, the oocytes were aspirated from all the visible follicles on the ovarian surface (~2-8 mm in a diameter). Meanwhile, Fetal Dromedary Camel Serum (FDCS) was obtained from camel fetuses after slaughtering. Thereafter, only Cumulus Oocyte Complexes (COCs) were matured in vitro in the Tissue Culture Medium (TCM-199) complemented with 10% FDCS. Spermatozoa required for in vitro fertilization were collected from testes (epididymal cauda) of the slaughtered camel bulls. The results clearly showed that the maturation rate of oocytes at metaphase II was about 59.5% while the fertilization rate was around 70.4%. Intriguingly, the embryo rates determined were 13.1%, in 2-cell; 0.0%, in 4-cell; 34.7%, in 8-16% cell; 39.1%, in morula and 13.1% in a blastocyst stage. This study represented a successful in vitro production of Sudanese dromedary camel embryos from epididymal sperm cells and in vitro matured oocytes recovered from slaughtered camels.

Oocyte cryopreservation and in vitro culture affect calcium signalling during human fertilization.

PubMed

Nikiforaki, D; Vanden Meerschaut, F; Qian, C; De Croo, I; Lu, Y; Deroo, T; Van den Abbeel, E; Heindryckx, B; De Sutter, P

2014-01-01

What are the precise patterns of calcium oscillations during the fertilization of human oocytes matured either in vivo or in vitro or aged in vitro and what is the effect of cryopreservation? Human oocytes matured in vivo exhibit a specific pattern of calcium oscillations, which is affected by in vitro maturation, in vitro ageing and cryopreservation. Oscillations in cytoplasmic calcium concentration are crucial for oocyte activation and further embryonic development. While several studies have described in detail the calcium oscillation pattern during fertilization in animal models, studies with human oocytes are scarce. This was a laboratory-based study using human MII oocytes matured in vivo or in vitro either fresh or after cryopreservation with slow freezing or vitrification. Altogether, 205 human oocytes were included in the analysis. In vivo and in vitro matured human oocytes were used for this research either fresh or following vitrification/warming (V/W) and slow freezing/thawing (F/T). Human oocytes were obtained following written informed consent from patients undergoing ovarian hyperstimulation. For the calcium pattern analysis, oocytes were loaded with the ratiometric calcium indicator fluorescent dye Fura-2. Following ICSI using sperm from a single donor, intracellular calcium was measured for 16 h at 37°C under 6% CO(2). The calcium oscillation parameters were calculated for all intact oocytes that showed calcium oscillations and were analyzed using the Mann-Whitney U-test. Human in vivo MII oocytes display a specific pattern of calcium oscillations following ICSI. This pattern is significantly affected by in vitro ageing, with the calcium oscillations occurring over a longer period of time and with a lower frequency, shorter duration and higher amplitude (P < 0.05). In vitro matured oocytes from the GV and MI stage exhibit a different pattern of calcium oscillations with calcium transients being of lower frequency and shorter duration compared with in vivo matured MII. In MI oocytes that reached the MII stage within 3 h the calcium oscillations additionally appear over a longer period of time (P < 0.05). In vivo MII oocytes show a different calcium oscillation pattern following V/W with calcium oscillations occurring over a longer period of time, with a higher amplitude and a lower frequency (P < 0.05). In vitro matured oocytes, either from the GV or the MI stage, also display an altered pattern of calcium oscillations after V/W and the parameters that were similarly affected in all these oocyte groups are the frequency and the amplitude of the calcium transients. Slow freezing/thawing differentially affects the calcium oscillation pattern of in vitro matured and in vitro aged oocytes. The relationship between a specific pattern of calcium oscillations and subsequent human embryonic development could not be evaluated since the calcium indicator used and the high-intensity excitation light impair development. Furthermore, all oocytes were derived from stimulated cycles and immature oocytes were denuded prior to in vitro maturation. Our data show for the first time how calcium signalling during human fertilization is affected by oocyte in vitro maturation, in vitro ageing as well as V/W and slow freezing/thawing. The analysis of calcium oscillations could be used as an oocyte quality indicator to evaluate in vitro culture and cryopreservation techniques of human oocytes. This work was supported by a clinical research mandate from the Flemish Foundation of Scientific Research (FWO-Vlaanderen, FWO09/ASP/063) to F.V.M, a fundamental clinical research mandate from the FWO-Vlaanderen (FWO05/FKM/001) to P.D.S and a Ghent University grant (KAN-BOF E/01321/01) to B.H. The authors have no conflict of interest to declare.

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

RNA-Seq transcriptome profiling of mouse oocytes after in vitro maturation and/or vitrification.

PubMed

Gao, Lei; Jia, Gongxue; Li, Ai; Ma, Haojia; Huang, Zhengyuan; Zhu, Shien; Hou, Yunpeng; Fu, Xiangwei

2017-10-16

In vitro maturation (IVM) and vitrification have been widely used to prepare oocytes before fertilization; however, potential effects of these procedures, such as expression profile changes, are poorly understood. In this study, mouse oocytes were divided into four groups and subjected to combinations of in vitro maturation and/or vitrification treatments. RNA-seq and in silico pathway analysis were used to identify differentially expressed genes (DEGs) that may be involved in oocyte viability after in vitro maturation and/or vitrification. Our results showed that 1) 69 genes were differentially expressed after IVM, 66 of which were up-regulated. Atp5e and Atp5o were enriched in the most significant gene ontology term "mitochondrial membrane part"; thus, these genes may be promising candidate biomarkers for oocyte viability after IVM. 2) The influence of vitrification on the transcriptome of oocytes was negligible, as no DEGs were found between vitrified and fresh oocytes. 3) The MII stage is more suitable for oocyte vitrification with respect to the transcriptome. This study provides a valuable new theoretical basis to further improve the efficiency of in vitro maturation and/or oocyte vitrification.

Effect of triiodothyronine on developmental competence of bovine oocytes.

PubMed

Costa, N N; Cordeiro, M S; Silva, T V G; Sastre, D; Santana, P P B; Sá, A L A; Sampaio, R V; Santos, S S D; Adona, P R; Miranda, M S; Ohashi, O M

2013-09-01

Developmental competence of in vitro-matured bovine oocytes is a limiting factor in production of embryos in vitro. Several studies have suggested a potential positive effect of thyroid hormones on cultured oocytes and/or their supporting cells. Therefore, the aim of the present study was to ascertain whether medium supplementation with triiodothyronine (T3) improved subsequent developmental competence of in vitro-matured bovine oocytes. For this purpose, we first documented (using reverse transcription PCR) that whereas bovine cumulus cells expressed both thyroid hormone receptor (TR)-α and TRβ, immature bovine oocytes expressed TRα only. Thereafter, to test the effects of TH on developmental competence, abattoir-derived oocytes were matured in vitro in a medium containing 0, 25, 50, or 100 nM T3 and subjected to in vitro fertilization. Embryo quality was evaluated by assessing cleavage and blastocyst rates, morphological quality, development kinetics, and total cell number on Day 8 of culture. Notably, addition of 50 or 100 nM T3 to the in vitro maturation medium increased (P < 0.05) the rate of hatched blastocysts on the eighth day of culture, as compared with other groups (62.4 ± 11.7, 53.1 ± 16.3, and 32.4 ± 5.3, respectively). Next, the relative expression levels of genes related to embryo quality POU-domain transcription factor (POU5F1) and glucose transporter-1 (GLUT 1) were compared between in vivo- and in vitro-produced blastocysts. On the basis of the previous experiments, IVP embryos originating from oocytes that were matured in vitro in the presence or absence of 50 nM T3 were evaluated. The treatment had no effect (P > 0.05) on gene expression. We concluded that supplementation of bovine oocyte in vitro maturation medium with T3 may have a beneficial effect on the kinetics of embryo development. Copyright © 2013 Elsevier Inc. All rights reserved.

Effect of sericin supplementation during in vitro maturation on the maturation, fertilization and development of porcine oocytes.

PubMed

Do, L T K; Namula, Z; Luu, V V; Sato, Y; Taniguchi, M; Isobe, T; Kikuchi, K; Otoi, T

2014-04-01

This study aimed to examine the effects of sericin supplementation during in vitro oocyte maturation on the nuclear maturation, fertilization and development of porcine oocytes. Cumulus-oocyte complexes (COCs) were cultured in maturation medium supplemented with 0 (control), 0.1, 0.5, 1.0, 2.5 or 5.0% sericin and were then subjected to in vitro fertilization and embryo culture. More COCs matured with 1.0% sericin underwent germinal vesicle breakdown and reached metaphase II compared with the control COCs matured without sericin (p < 0.01). The proportions of oocytes with DNA-fragmented nuclei did not differ between the groups, regardless of the sericin level. The total fertilization rate of oocytes matured with 1.0% sericin was higher (p < 0.05) than that of oocytes matured with 0.1%, 2.5% and 5.0% sericin. Supplementation with more than 1.0% sericin decreased the DNA fragmentation index of the blastocysts compared with the control group (p < 0.05). However, the supplementation of the maturation medium with sericin had no beneficial effects on the cleavage, development to the blastocyst stage and the total cell number of the embryos. Our findings indicate that supplementation with 1.0% sericin during maturation culture may improve the nuclear maturation and the quality of the embryos but does not affect blastocyst formation. © 2014 Blackwell Verlag GmbH.

Higher Nucleoporin-Importinβ Affinity at the Nuclear Basket Increases Nucleocytoplasmic Import

PubMed Central

Azimi, Mohammad; Mofrad, Mohammad R. K.

2013-01-01

Several in vitro studies have shown the presence of an affinity gradient in nuclear pore complex proteins for the import receptor Importinβ, at least partially contributing to nucleocytoplasmic transport, while others have historically argued against the presence of such a gradient. Nonetheless, the existence of an affinity gradient has remained an uncharacterized contributing factor. To shed light on the affinity gradient theory and better characterize how the existence of such an affinity gradient between the nuclear pore and the import receptor may influence the nucleocytoplasmic traffic, we have developed a general-purpose agent based modeling (ABM) framework that features a new method for relating rate constants to molecular binding and unbinding probabilities, and used our ABM approach to quantify the effects of a wide range of forward and reverse nucleoporin-Importinβ affinity gradients. Our results indicate that transport through the nuclear pore complex is maximized with an effective macroscopic affinity gradient of 2000 µM, 200 µM and 10 µM in the cytoplasmic, central channel and nuclear basket respectively. The transport rate at this gradient is approximately 10% higher than the transport rate for a comparable pore lacking any affinity gradient, which has a peak transport rate when all nucleoporins have an affinity of 200 µM for Importinβ. Furthermore, this optimal ratio of affinity gradients is representative of the ratio of affinities reported for the yeast nuclear pore complex – suggesting that the affinity gradient seen in vitro is highly optimized. PMID:24282617

Asymmetric ring structure of Vps4 required for ESCRT-III disassembly

NASA Astrophysics Data System (ADS)

Caillat, Christophe; Macheboeuf, Pauline; Wu, Yuanfei; McCarthy, Andrew A.; Boeri-Erba, Elisabetta; Effantin, Gregory; Göttlinger, Heinrich G.; Weissenhorn, Winfried; Renesto, Patricia

2015-12-01

The vacuolar protein sorting 4 AAA-ATPase (Vps4) recycles endosomal sorting complexes required for transport (ESCRT-III) polymers from cellular membranes. Here we present a 3.6-Å X-ray structure of ring-shaped Vps4 from Metallosphera sedula (MsVps4), seen as an asymmetric pseudohexamer. Conserved key interface residues are shown to be important for MsVps4 assembly, ATPase activity in vitro, ESCRT-III disassembly in vitro and HIV-1 budding. ADP binding leads to conformational changes within the protomer, which might propagate within the ring structure. All ATP-binding sites are accessible and the pseudohexamer binds six ATP with micromolar affinity in vitro. In contrast, ADP occupies one high-affinity and five low-affinity binding sites in vitro, consistent with conformational asymmetry induced on ATP hydrolysis. The structure represents a snapshot of an assembled Vps4 conformation and provides insight into the molecular motions the ring structure undergoes in a concerted action to couple ATP hydrolysis to ESCRT-III substrate disassembly.

What Are the Primary Limitations in B-Cell Affinity Maturation, and How Much Affinity Maturation Can We Drive with Vaccination?

PubMed Central

2017-01-01

A key goal of HIV-1 vaccine development is the induction of broadly neutralizing antibodies (bnAbs) targeted to the vulnerable regions of the HIV envelope. BnAbs develop overtime in ∼50%of HIV-1-infected individuals. However, to date, no vaccines have induced bnAbs and few or none of these vaccine-elicited HIV-1 antibodies carry the high frequencies of V(D)J mutations characteristic of bnAbs. Do the high frequencies of mutations characteristic of naturally induced bnAbs represent a fundamental barrier to the induction of bnAbs by vaccines? Recent studies suggest that high frequencies of V(D)J mutations can be achieved by serial vaccination strategies. Rather, it appears that, in the absence of HIV-1 infection, physiologic immune tolerance controls, including a germinal center process termed affinity reversion, may limit vaccine-driven bnAb development by clonal elimination or selecting for mutations incompatible with bnAb activity. PMID:28630077

Binding of /sup 125/I-hCG to rainbow trout (Salmo gairdneri) testis in vitro. [Human Chorionic Gonadotropin

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

Schlaghecke, R.

1983-02-01

Homogenates of maturing rainbow trout testes show specific binding sites for /sup 125/I-labeled hCG (. /sup 125/I-labeled hCG). The binding is competitively inhibited by unlabeled hCG and by a hypophyseal extract of rainbow trout. It could be demonstrated that the tissue /sup 125/I-hCG binding specificity is restricted to the gonadal preparation. The trout testis was characterized by determining affinity and capacity from Scatchard plot analysis giving a high constant of dissociation Kd 3.65 x 10(-10)/M and a low binding capacity of 0.88 x 10(-15) M/mg tissue. The test system is markedly dependent on temperature, incubation-time, and pH. The maximum bindingmore » was found at 37 degrees during 2 hr of incubation in a buffer of pH 7.5.« less

In Silico Identification of Novel APRIL Peptide Antagonists and Binding Insights by Molecular Modeling and Immunosorbent Assays.

PubMed

Silva, Joao H M da; Calmon-Hamaty, Flavia; Savino, Wilson; Hahne, Michael; Caffarena, Ernesto R

2015-01-01

The "A proliferation inducing ligand" protein (APRIL) is a cytokine over-expressed in many transformed and tumoral cells acting onto two distinct receptors of the Tumoral Necrosis Factor B cell maturation antigen (BCMA) and the transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI). We herein describe, through a detailed computational approach, the molecular interactions between TACI and its ligands APRIL and another structurally similar protein called B-cell activating factor (BAFF) by means of molecular dynamics. Dynamical analysis suggests R84 and D85 residues from TACI as possible mutation candidates, yielding increased affinity between TACI and APRIL. The association of computational simulations, site directed mutagenesis and peptide design could be a powerful tool, driving to better in vitro experiments. Our results contribute to the elucidation of APRIL signaling and help clarify the effects of blocking interaction between APRIL and its receptors through the use of particular peptides.

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.

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.

Changes in complementarity-determining regions significantly alter IgG binding to the neonatal Fc receptor (FcRn) and pharmacokinetics

PubMed Central

King, Amy C.; Kavosi, Mania; Wang, Mengmeng; O'Hara, Denise M.; Tchistiakova, Lioudmila; Katragadda, Madan

2018-01-01

ABSTRACT A large body of data exists demonstrating that neonatal Fc receptor (FcRn) binding of an IgG via its Fc CH2-CH3 interface trends with the pharmacokinetics (PK) of IgG. We have observed that PK of IgG molecules vary widely, even when they share identical Fc domains. This led us to hypothesize that domains distal from the Fc could contribute to FcRn binding and affect PK. In this study, we explored the role of these IgG domains in altering the affinity between IgG and FcRn. Using a surface plasmon resonance-based assay developed to examine the steady-state binding affinity (KD) of IgG molecules to FcRn, we dissected the contributions of IgG domains in modulating the affinity between FcRn and IgG. Through analysis of a broad collection of therapeutic antibodies containing more than 50 unique IgG molecules, we demonstrated that variable domains, and in particular complementarity-determining regions (CDRs), significantly alter binding affinity to FcRn in vitro. Furthermore, a panel of IgG molecules differing only by 1–5 mutations in CDRs altered binding affinity to FcRn in vitro, by up to 79-fold, and the affinity values correlated with calculated isoelectric point values of both variable domains and CDR-L3. In addition, tighter affinity values trend with faster in vivo clearance of a set of IgG molecules differing only by 1–3 mutations in human FcRn transgenic mice. Understanding the role of CDRs in modulation of IgG affinity to FcRn in vitro and their effect on PK of IgG may have far-reaching implications in the optimization of IgG therapeutics. PMID:28991504

Pregnancy and delivery after in vitro maturation of naked ICSI-GV oocytes with GH and transfer of a frozen thawed blastocyst: case report.

PubMed

Menezo, Yves J R; Nicollet, Bernard; Rollet, Jacques; Hazout, André

2006-01-01

To determine if GV oocytes, collected at the time of ICSI, can be matured in vitro and rescued for therapeutic treatment. A patient for whom all the collected oocytes at the GV stage after a classical COH protocol were matured in vitro with GH. All the naked oocytes were matured in a culture medium (ISM2) containing 15% patient serum +1.6 units of GH (Saizen) per millilitre. Oocytes were incubated overnight at 37 degrees C. The MII oocytes obtained were micro-injected. A fresh transfer was performed and a supernumerary blastocyst was frozen. The patient was pregnant and delivered a healthy girl after transfer of the frozen/thawed blastocyst. The baby girl is now 2 years old. In vitro maturation with GH allows rescuing naked GV oocytes collected at the time of ICSI. GH action does not pass through the cumulus cells. According to the possible lack of synchrony between the embryo and the uterus, we recommend to freeze the embryos obtained and to replace them in a controlled cycle.

Injurious effects of curcumin on maturation of mouse oocytes, fertilization and fetal development via apoptosis.

PubMed

Chen, Chia-Chi; Chan, Wen-Hsiung

2012-01-01

Curcumin, a common dietary pigment and spice, is a hydrophobic polyphenol derived from the rhizome of the herb Curcuma longa. Previously, we reported a cytotoxic effect of curcumin on mouse embryonic stem cells and blastocysts and its association with defects in subsequent development. In the present study, we further investigated the effects of curcumin on oocyte maturation and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, curcumin induced a significant reduction in the rate of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with curcumin during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased fetal weight. Experiments with an in vivo mouse model disclosed that consumption of drinking water containing 40 μM curcumin led to decreased oocyte maturation and in vitro fertilization as well as early embryonic developmental injury. Finally, pretreatment with a caspase-3-specific inhibitor effectively prevented curcumin-triggered injury effects, suggesting that embryo impairment by curcumin occurs mainly via a caspase-dependent apoptotic process.

Metal selectivity of the E. coli nickel metallochaperone, SlyD

PubMed Central

Kaluarachchi, Harini; Siebel, Judith F.; Kaluarachchi-Duffy, Supipi; Krecisz, Sandra; Sutherland, Duncan E. K.; Stillman, Martin J.; Zamble, Deborah B.

2012-01-01

SlyD is a Ni(II)-binding protein that contributes to nickel homeostasis in Escherichia coli. The C-terminal domain of SlyD contains a rich variety of metal-binding amino acids, suggesting broader metal-binding capabilities, and previous work demonstrated that the protein can coordinate several types of first row transition metals. However, the binding of SlyD to metals other than Ni(II) has not been previously characterized. To further our understanding of the in vitro metal-binding activity of SlyD and how it correlates with the in vivo function of this protein, the interactions between SlyD and the series of biologically relevant transition metals Mn(II), Fe(II), Co(II), Cu(I) and Zn(II) were examined by using a combination of optical spectroscopy and mass spectrometry. SlyD binding to Mn(II) or to Fe(II) ions was not detected but the protein coordinates multiple ions of Co(II), Zn(II) and Cu(I) with appreciable affinities (KD ≤ nM), highlighting the promiscuous nature of this protein. The order of affinities of SlyD for the metals examined is Mn(II), Fe(II) < Co(II) < Ni(II) ~ Zn(II) ≪ Cu(I). Although the purified protein is unable to overcome the large thermodynamic preference for Cu(I) and exclude Zn(II) chelation in the presence of Ni(II), in vivo studies reveal a Ni(II)-specific function for the protein. Furthermore, these latter experiments support a specific role for SlyD as a [NiFe]-hydrogenase enzyme maturation factor. The implications of the divergence between the metal selectivity of SlyD in vitro and the specific activity in vivo are discussed. PMID:22047179

Expression of G protein estrogen receptor (GPER) on membrane of mouse oocytes during maturation.

PubMed

Li, Yi-Ran; Ren, Chun-E; Zhang, Quan; Li, Ji-Chun; Chian, Ri-Cheng

2013-02-01

To determine expression of G-protein estrogen receptor (GPER) in mouse oocyte membrane during maturation. The expression of GPER from different maturation stages of oocytes, in vivo and in vitro matured oocytes as well as aging oocytes was examined by immune-fluorescence GPR30 antibody and the images were analyzed by laser scanning confocal microscope. Further confirmation was performed by Western blots for cell fractionation. Significant fluorescent signal was observed on the surface of mouse oocytes. The image expression was lower in germinal vesicle (GV) stage than mature metaphase-II (M-II) stage oocytes. There was high expression in in-vivo matured oocytes compared to in vitro matured oocytes. The highest expression was observed in aging oocytes compared with other oocytes. The changes of expression of GPER on mouse oocytes plasma membrane confirm oocyte membrane maturation, suggesting that those changes of GPER may be related to the functional role of oocyte maturation.

Scaffold design of trivalent chelator heads dictates high-affinity and stable His-tagged protein labeling in vitro and in cellulo.

PubMed

Gatterdam, Karl; Joest, Eike F; Gatterdam, Volker; Tampé, Robert

2018-05-29

Small chemical/biological interaction pairs are at the forefront in tracing proteins' function and interaction at high signal-to-background ratio in cellular pathways. Pharma ventures have eager plans to develop trisNTA probes for in vitro and in vivo screening of His-tagged protein targets. However, the optimal design of scaffold, linker, and chelator head yet deserves systematic investigations to achieve highest affinity and kinetic stability for in vitro and especially cell applications. In this study, we report on a library of N-nitrilotriacetic acid (NTA) based multivalent chelator heads (MCHs) built up on linear, cyclic, and dendritic scaffolds and contrast these with regard to their binding affinity and stability for labeling of cellular His-tagged proteins. Furthermore, we assign a new approach for tracing cellular target proteins at picomolar probe concentrations in cells. Finally, we describe fundamental differences between the MCH scaffold and define a cyclic trisNTA chelator, which displays the highest affinity and kinetic stability of all reversible, low-molecular weight interaction pairs. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

In vitro acute exposure to DEHP affects oocyte meiotic maturation, energy and oxidative stress parameters in a large animal model.

PubMed

Ambruosi, Barbara; Uranio, Manuel Filioli; Sardanelli, Anna Maria; Pocar, Paola; Martino, Nicola Antonio; Paternoster, Maria Stefania; Amati, Francesca; Dell'Aquila, Maria Elena

2011-01-01

Phthalates are ubiquitous environmental contaminants because of their use in plastics and other common consumer products. Di-(2-ethylhexyl) phthalate (DEHP) is the most abundant phthalate and it impairs fertility by acting as an endocrine disruptor. The aim of the present study was to analyze the effects of in vitro acute exposure to DEHP on oocyte maturation, energy and oxidative status in the horse, a large animal model. Cumulus cell (CC) apoptosis and oxidative status were also investigated. Cumulus-oocyte complexes from the ovaries of slaughtered mares were cultured in vitro in presence of 0.12, 12 and 1200 µM DEHP. After in vitro maturation (IVM), CCs were removed and evaluated for apoptosis (cytological assessment and TUNEL) and intracellular reactive oxygen species (ROS) levels. Oocytes were evaluated for nuclear chromatin configuration. Matured (Metaphase II stage; MII) oocytes were further evaluated for cytoplasmic energy and oxidative parameters. DEHP significantly inhibited oocyte maturation when added at low doses (0.12 µM; P<0.05). This effect was related to increased CC apoptosis (P<0.001) and reduced ROS levels (P<0.0001). At higher doses (12 and 1200 µM), DEHP induced apoptosis (P<0.0001) and ROS increase (P<0.0001) in CCs without affecting oocyte maturation. In DEHP-exposed MII oocytes, mitochondrial distribution patterns, apparent energy status (MitoTracker fluorescence intensity), intracellular ROS localization and levels, mt/ROS colocalization and total SOD activity did not vary, whereas increased ATP content (P<0.05), possibly of glycolytic origin, was found. Co-treatment with N-Acetyl-Cysteine reversed apoptosis and efficiently scavenged excessive ROS in DEHP-treated CCs without enhancing oocyte maturation. In conclusion, acute in vitro exposure to DEHP inhibits equine oocyte maturation without altering ooplasmic energy and oxidative stress parameters in matured oocytes which retain the potential to be fertilized and develop into embryos even though further studies are necessary to confirm this possibility.

In Vitro Acute Exposure to DEHP Affects Oocyte Meiotic Maturation, Energy and Oxidative Stress Parameters in a Large Animal Model

PubMed Central

Sardanelli, Anna Maria; Pocar, Paola; Martino, Nicola Antonio; Paternoster, Maria Stefania; Amati, Francesca; Dell'Aquila, Maria Elena

2011-01-01

Phthalates are ubiquitous environmental contaminants because of their use in plastics and other common consumer products. Di-(2-ethylhexyl) phthalate (DEHP) is the most abundant phthalate and it impairs fertility by acting as an endocrine disruptor. The aim of the present study was to analyze the effects of in vitro acute exposure to DEHP on oocyte maturation, energy and oxidative status in the horse, a large animal model. Cumulus cell (CC) apoptosis and oxidative status were also investigated. Cumulus-oocyte complexes from the ovaries of slaughtered mares were cultured in vitro in presence of 0.12, 12 and 1200 µM DEHP. After in vitro maturation (IVM), CCs were removed and evaluated for apoptosis (cytological assessment and TUNEL) and intracellular reactive oxygen species (ROS) levels. Oocytes were evaluated for nuclear chromatin configuration. Matured (Metaphase II stage; MII) oocytes were further evaluated for cytoplasmic energy and oxidative parameters. DEHP significantly inhibited oocyte maturation when added at low doses (0.12 µM; P<0.05). This effect was related to increased CC apoptosis (P<0.001) and reduced ROS levels (P<0.0001). At higher doses (12 and 1200 µM), DEHP induced apoptosis (P<0.0001) and ROS increase (P<0.0001) in CCs without affecting oocyte maturation. In DEHP-exposed MII oocytes, mitochondrial distribution patterns, apparent energy status (MitoTracker fluorescence intensity), intracellular ROS localization and levels, mt/ROS colocalization and total SOD activity did not vary, whereas increased ATP content (P<0.05), possibly of glycolytic origin, was found. Co-treatment with N-Acetyl-Cysteine reversed apoptosis and efficiently scavenged excessive ROS in DEHP-treated CCs without enhancing oocyte maturation. In conclusion, acute in vitro exposure to DEHP inhibits equine oocyte maturation without altering ooplasmic energy and oxidative stress parameters in matured oocytes which retain the potential to be fertilized and develop into embryos even though further studies are necessary to confirm this possibility. PMID:22076161

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

Bone Morphogenetic Protein 15 in the Pro-Mature Complex Form Enhances Bovine Oocyte Developmental Competence

PubMed Central

Sudiman, Jaqueline; Sutton-McDowall, Melanie L.; Ritter, Lesley J.; White, Melissa A.; Mottershead, David G.; Thompson, Jeremy G.; Gilchrist, Robert B.

2014-01-01

Developmental competence of in vitro matured (IVM) oocytes needs to be improved and this can potentially be achieved by adding recombinant bone morphogenetic protein 15 (BMP15) or growth differentiation factor (GDF9) to IVM. The aim of this study was to determine the effect of a purified pro-mature complex form of recombinant human BMP15 versus the commercially available bioactive forms of BMP15 and GDF9 (both isolated mature regions) during IVM on bovine embryo development and metabolic activity. Bovine cumulus oocyte complexes (COCs) were matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15, mature BMP15 or mature GDF9 +/− FSH. Metabolic measures of glucose uptake and lactate production from COCs and autofluorescence of NAD(P)H, FAD and GSH were measured in oocytes after IVM. Following in vitro fertilisation and embryo culture, day 8 blastocysts were stained for cell numbers. COCs matured in medium +/− FSH containing pro-mature BMP15 displayed significantly improved blastocyst development (57.7±3.9%, 43.5±4.2%) compared to controls (43.3±2.4%, 28.9±3.7%) and to mature GDF9+FSH (36.1±3.0%). The mature form of BMP15 produced intermediate levels of blastocyst development; not significantly different to control or pro-mature BMP15 levels. Pro-mature BMP15 increased intra-oocyte NAD(P)H, and reduced glutathione (GSH) levels were increased by both forms of BMP15 in the absence of FSH. Exogenous BMP15 in its pro-mature form during IVM provides a functional source of oocyte-secreted factors to improve bovine blastocyst development. This form of BMP15 may prove useful for improving cattle and human artificial reproductive technologies. PMID:25058588

Bone morphogenetic protein 15 in the pro-mature complex form enhances bovine oocyte developmental competence.

PubMed

Sudiman, Jaqueline; Sutton-McDowall, Melanie L; Ritter, Lesley J; White, Melissa A; Mottershead, David G; Thompson, Jeremy G; Gilchrist, Robert B

2014-01-01

Developmental competence of in vitro matured (IVM) oocytes needs to be improved and this can potentially be achieved by adding recombinant bone morphogenetic protein 15 (BMP15) or growth differentiation factor (GDF9) to IVM. The aim of this study was to determine the effect of a purified pro-mature complex form of recombinant human BMP15 versus the commercially available bioactive forms of BMP15 and GDF9 (both isolated mature regions) during IVM on bovine embryo development and metabolic activity. Bovine cumulus oocyte complexes (COCs) were matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15, mature BMP15 or mature GDF9 +/- FSH. Metabolic measures of glucose uptake and lactate production from COCs and autofluorescence of NAD(P)H, FAD and GSH were measured in oocytes after IVM. Following in vitro fertilisation and embryo culture, day 8 blastocysts were stained for cell numbers. COCs matured in medium +/- FSH containing pro-mature BMP15 displayed significantly improved blastocyst development (57.7±3.9%, 43.5±4.2%) compared to controls (43.3±2.4%, 28.9±3.7%) and to mature GDF9+FSH (36.1±3.0%). The mature form of BMP15 produced intermediate levels of blastocyst development; not significantly different to control or pro-mature BMP15 levels. Pro-mature BMP15 increased intra-oocyte NAD(P)H, and reduced glutathione (GSH) levels were increased by both forms of BMP15 in the absence of FSH. Exogenous BMP15 in its pro-mature form during IVM provides a functional source of oocyte-secreted factors to improve bovine blastocyst development. This form of BMP15 may prove useful for improving cattle and human artificial reproductive technologies.

A set of ligation-independent in vitro translation vectors for eukaryotic protein production.

PubMed

Bardóczy, Viola; Géczi, Viktória; Sawasaki, Tatsuya; Endo, Yaeta; Mészáros, Tamás

2008-03-27

The last decade has brought the renaissance of protein studies and accelerated the development of high-throughput methods in all aspects of proteomics. Presently, most protein synthesis systems exploit the capacity of living cells to translate proteins, but their application is limited by several factors. A more flexible alternative protein production method is the cell-free in vitro protein translation. Currently available in vitro translation systems are suitable for high-throughput robotic protein production, fulfilling the requirements of proteomics studies. Wheat germ extract based in vitro translation system is likely the most promising method, since numerous eukaryotic proteins can be cost-efficiently synthesized in their native folded form. Although currently available vectors for wheat embryo in vitro translation systems ensure high productivity, they do not meet the requirements of state-of-the-art proteomics. Target genes have to be inserted using restriction endonucleases and the plasmids do not encode cleavable affinity purification tags. We designed four ligation independent cloning (LIC) vectors for wheat germ extract based in vitro protein translation. In these constructs, the RNA transcription is driven by T7 or SP6 phage polymerase and two TEV protease cleavable affinity tags can be added to aid protein purification. To evaluate our improved vectors, a plant mitogen activated protein kinase was cloned in all four constructs. Purification of this eukaryotic protein kinase demonstrated that all constructs functioned as intended: insertion of PCR fragment by LIC worked efficiently, affinity purification of translated proteins by GST-Sepharose or MagneHis particles resulted in high purity kinase, and the affinity tags could efficiently be removed under different reaction conditions. Furthermore, high in vitro kinase activity testified of proper folding of the purified protein. Four newly designed in vitro translation vectors have been constructed which allow fast and parallel cloning and protein purification, thus representing useful molecular tools for high-throughput production of eukaryotic proteins.

The beneficial effect of repaglinide on in vitro maturation and development ability of immature mouse oocytes.

PubMed

Kalehoei, Eshrat; Azadbakht, Mehri

2017-08-01

Repaglinide is a hypoglycemic drug, causing depolarization of the cell membrane, opening the voltage-gated calcium channels, and then increasing intracellular calcium in the pancreatic B cells by inhibition of the K-ATP-sensitive channels. Oocyte in vitro maturation (IVM) is influenced by different factors such as calcium signaling. In this study, we examined the effects of repaglinide on in vitro maturation and fertilization ability of mouse oocyte. Immature oocytes were isolated from female Naval Medical Research Institute mice which are 6-8 wk old mechanically and then cultured in 30 μl droplets of T6 medium with different concentrations of repaglinide. The control group did not receive repaglinide (R 0 ). Treatment groups received different concentrations (5, 10, and 100 nM and 1 and 10 μM) of repaglinide (R 1 , R 2 , R 3 , R 4 , and R 5 , respectively). Oocyte in vitro maturation rate was assessed after 24 h. In vitro fertilization was performed using metaphase II oocytes obtained from R 0 and R 4 treatments. Embryo cleavage rate was calculated at 48 h post-IVF. Chi-square test was used for evaluating difference between control and treatment groups (p < 0.05). Oocyte maturation rate after 24 h in treatment groups R 2 , R 3 , R 4 , and R 5 was significantly higher than that in the control (p < 0.05). Supplementation of medium with 1 μM of repaglinide (R 4 ) during IVM significantly improved outcome of embryo cleavage rate than control at 48 h post-IVF (p < 0.05). In conclusion, repaglinide can be considered as an effective agent for in vitro oocyte maturation and embryo cleavage.

Oocyte quality and estradiol supplementation affect in vitro maturation success in the white-tailed deer (Odocoileus virginianus).

PubMed

Siriaroonrat, B; Comizzoli, P; Songsasen, N; Monfort, S L; Wildt, D E; Pukazhenthi, B S

2010-01-01

White-tailed deer oocyte biology is not well documented. The objective of this study was to determine (1) the influence of estradiol (E(2)) supplementation on meiotic resumption and the ability to "rescue" poorer quality (lower grade) oocytes and (2) the kinetics of oocyte nuclear maturation in vitro in the white-tailed deer. In Experiment 1, immature oocytes harvested during hunting-culling operations were cultured for 24h in the presence or absence of E(2). Incubation in 1mug/mL E(2) promoted nuclear maturation (to telophase I, TI; or to metaphase II, MII) in a higher proportion of Grade 1 oocytes ( approximately 77%; P<0.05) compared with that in Grade 2 or Grade 3 counterparts ( approximately 51%). For Grades 2 and 3 oocytes, there was no advantage (P>0.05) for E(2) supplementation in reaching TI/MII. In Experiment 2, Grade 1 oocytes were cultured in the presence of E(2) and nuclear status evaluated at 0, 3, 6, 12, and 24h of in vitro incubation. At 0h,>70% of oocytes already had undergone germinal vesicle breakdown. After 12h, approximately 70% of oocytes had reached metaphase I of nuclear maturation, with approximately 75% achieving TI/MII by 24h in vitro. In summary, adding E(2) to an in vitro maturation (IVM) culture system for white-tailed deer was advantageous, but only for the highest quality oocytes, with approximately 75% achieving nuclear maturation. In contrast, E(2) supplement did not benefit lower-grade oocytes, half of which will reach MII, with the other half failing. Under the described culture conditions, good-quality white-tailed deer oocytes achieve nuclear maturation over a time duration comparable with that reported in other ungulates.

Effects of Crocin Supplementation during In Vitro Maturation of Mouse Oocytes on Glutathione Synthesis and Cytoplasmic Maturation

PubMed Central

Mokhber Maleki, Elham; Eimani, Hussein; Bigdeli, Mohammad Reza; Golkar Narenji, Afsane; Abedi, Reyhane

2016-01-01

Background Crocin is an active ingredient of saffron (Crocus sativus L.) and its antioxidant properties have been previously investigated. This carotenoid scavenges free radicals and stimulates glutathione (GSH) synthesis; consequently, it may protect cells against oxidative stress. The aim of this research is to protect oocytes from oxidative stress by the addition of a natural source antioxidant. Materials and Methods In the present in vitro experimental study, we collected cumulus oocyte complexes (COCs) from mouse ovaries of euthanized, 6-8 week-old female Naval Medical Research Institute (NMRI) mice. Oocytes were subjected to in vitro maturation (IVM) in the presence of either crocin (5 or 10 μg/ml), 5 mM buthionine-[S-R]- sulfoximine (BSO), or the combination of crocin plus BSO. Oocytes that matured in vitro in a medium without crocin or BSO supplements were considered as controls. Following 16-18 hours of IVM, matured oocytes (n=631) were fertilized by capacitated sperm from NMRI male mice, and cultured in vitro for up to 96 hours to assess preimplantation embryonic development. The levels of GSH in metaphase II (MII) oocytes after IVM (n=240) were also assessed by the 5, 5-dithio-bis (2-nitrobenzoic acid) (DTNB)-GSH reductase recycling assay. Results Supplementation of IVM media with 10 µg/ml crocin significantly (P<0.05) increased nuclear maturation, preimplantation development and GSH concentrations compared with the control group. Maturation of oocytes in IVM medium supplemented with BSO alone or the combination of 5 µg/ml crocin and BSO drastically decreased GSH concentrations and subsequently resulted in low rates of maturation, fertilization and blastocyst development. However, the combination of 10 µg/ml crocin with 5 mM BSO increased the level of nuclear maturation which was comparable to the control group. Conclusion Supplementation of IVM media with crocin can improve nuclear maturation rates and subsequent developmental potential of mouse oocytes. This may occur by its beneficial effect in increasing GSH concentrations in MII oocytes. PMID:27123201

Mouse oocytes fertilised by ICSI during in vitro maturation retain the ability to be activated after refertilisation in metaphase II and can generate Ca2+ oscillations

PubMed Central

Jędrusik, Agnieszka; Ajduk, Anna; Pomorski, Paweł; Maleszewski, Marek

2007-01-01

Background At fertilisation, mammalian oocytes are activated by oscillations of intracellular Ca2+ ([Ca2+]i). Phospholipase Cζ, which is introduced by fertilising spermatozoon, triggers [Ca2+]i oscillations through the generation of inositol 1,4,5-triphosphate (IP3), which causes Ca2+ release by binding to IP3 receptors located on the endoplasmic reticulum (ER) of the oocyte. Ability to respond to this activating stimulus develops during meiotic maturation of the oocyte. Here we examine how the development of this ability is perturbed when a single spermatozoon is introduced into the oocyte prematurely, i.e. during oocyte maturation. Results Mouse oocytes during maturation in vitro were fertilised by ICSI (intracytoplasmic sperm injection) 1 – 4 h after germinal vesicle break-down (GVBD) and were subsequently cultured until they reached metaphase II (MII) stage. At MII stage they were fertilised in vitro for the second time (refertilisation). We observed that refertilised oocytes underwent activation with similar frequency as control oocytes, which also went through maturation in vitro, but were fertilised only once at MII stage (87% and 93%, respectively). Refertilised MII oocytes were able to develop [Ca2+]i oscillations in response to penetration by spermatozoa. We found however, that they generated a lower number of transients than control oocytes. We also showed that the oocytes, which were fertilised during maturation had a similar level of MPF activity as control oocytes, which were not subjected to ICSI during maturation, but had reduced level of IP3 receptors. Conclusion Mouse oocytes, which were experimentally fertilised during maturation retain the ability to generate repetitive [Ca2+]i transients, and to be activated after completion of maturation. PMID:17584490

Maturation capacity, morphology and morphometric assessment of human immature oocytes after vitrification and in-vitro maturation

PubMed Central

Nazari, Saeedeh; Khalili, Mohammad Ali; Esmaielzadeh, Forouzan; Mohsenzadeh, Mehdi

2011-01-01

Background: In general, 15% of oocytes collected in ART cycles are immature. These oocytes may be cryopreserved further for use in in-vitro maturation (IVM) program. Objective: The aim of this study was to determine maturation capacity, morphometric parameters and morphology of human immature oocytes in both fresh IVM (fIVM) and vitrified-IVM (vIVM) oocytes. Materials and Methods: 93 women who underwent controlled ovarian stimulation for ART were included. The immature oocytes (n=203) were divided into two groups: the first group (n=101) directly matured in vitro; and the second group (n=102) first vitrified, then matured in vitro. All oocytes underwent IVM in Ham’s F10 supplemented with LH+FSH and human follicular fluid. After 48h of incubation, the oocyte maturation rates, as well as morphometric and morphologic characteristics were assessed using cornus imaging and were compared. Results: Oocyte maturation rates were reduced in vIVM, (40.4%), in comparison with fIVM (59.4%, p<0.001). Following morphometric assessment, there was no difference in the mean oocyte diameters (µm) between fIVM and vIVM, 156.3±6.8 and 154.07±9.9, respectively. Other parameters of perimeters, egg areas, as well as oocyte and ooplasm volumes were similar in two groups. In addition, more morphologic abnormalities, such as, vacuole, and dark oocyte were observed in vIVM oocytes. Conclusion: fIVM was more successful than vIVM groups. No statistical differences were noticed in morphometry assessment in two groups. This suggests that morphometric parameters can not be applied as prognosis factor in oocyte maturation outcome in IVM program. PMID:26396566

Expression of apoptotic genes in immature and in vitro matured equine oocytes and cumulus cells.

PubMed

Leon, P M M; Campos, V F; Kaefer, C; Begnini, K R; McBride, A J A; Dellagostin, O A; Seixas, F K; Deschamps, J C; Collares, T

2013-08-01

The gene expression of Bax, Bcl-2, survivin and p53, following in vitro maturation of equine oocytes, was compared in morphologically distinct oocytes and cumulus cells. Cumulus-oocyte complexes (COC) were harvested and divided into two groups: G1 - morphologically healthy cells; and G2 - less viable cells or cells with some degree of atresia. Total RNA was isolated from both immature and in vitro matured COC and real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify gene expression. Our results showed there was significantly higher expression of survivin (P < 0.05) and lower expression of p53 (P < 0.01) in oocytes compared with cumulus cells in G1. No significant difference in gene expression was observed following in vitro maturation or in COC derived from G1 and G2. However, expression of the Bax gene was significantly higher in cumulus cells from G1 (P < 0.02).

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.

Injurious Effects of Emodin on Maturation of Mouse Oocytes, Fertilization and Fetal Development via Apoptosis

PubMed Central

Chang, Mei-Hui; Chang, Shao-Chung; Chan, Wen-Hsiung

2012-01-01

Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a major constituent of rhubarb, has a wide range of therapeutic applications. Previous studies have established that emodin induces apoptosis in the inner cell mass and trophectoderm of mouse blastocysts and leads to decreased embryonic development and viability, indicating a role as an injury risk factor for normal embryonic development. However, the mechanisms underlying its hazardous effects have yet to be characterized. In the current study, we further investigated the effects of emodin on oocyte maturation and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, emodin induced a significant reduction in the rates of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with emodin during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased fetal weight. Experiments using an in vivo mouse model disclosed that consumption of drinking water containing 20–40 μM emodin led to decreased oocyte maturation and in vitro fertilization, as well as early embryonic developmental injury. Notably, pretreatment with a caspase-3-specific inhibitor effectively prevented emodin-triggered injury effects, suggesting that impairment of embryo development occurs via a caspase-dependent apoptotic process. PMID:23203041

Injurious effects of emodin on maturation of mouse oocytes, fertilization and fetal development via apoptosis.

PubMed

Chang, Mei-Hui; Chang, Shao-Chung; Chan, Wen-Hsiung

2012-10-29

Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a major constituent of rhubarb, has a wide range of therapeutic applications. Previous studies have established that emodin induces apoptosis in the inner cell mass and trophectoderm of mouse blastocysts and leads to decreased embryonic development and viability, indicating a role as an injury risk factor for normal embryonic development. However, the mechanisms underlying its hazardous effects have yet to be characterized. In the current study, we further investigated the effects of emodin on oocyte maturation and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, emodin induced a significant reduction in the rates of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with emodin during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased fetal weight. Experiments using an in vivo mouse model disclosed that consumption of drinking water containing 20-40 μM emodin led to decreased oocyte maturation and in vitro fertilization, as well as early embryonic developmental injury. Notably, pretreatment with a caspase-3-specific inhibitor effectively prevented emodin-triggered injury effects, suggesting that impairment of embryo development occurs via a caspase-dependent apoptotic process.

Binding of ACE-inhibitors to in vitro and patient-derived amyloid-β fibril models.

PubMed

Bhavaraju, Manikanthan; Phillips, Malachi; Bowman, Deborah; Aceves-Hernandez, Juan M; Hansmann, Ulrich H E

2016-01-07

Currently, no drugs exist that can prevent or reverse Alzheimer's disease, a neurodegenerative disease associated with the presence, in the brain, of plaques that are composed of β-amyloid (Aβ) peptides. Recent studies suggest that angiotensin-converting enzyme (ACE) inhibitors, a set of drugs used to treat hypertension, may inhibit amyloid formation in vitro. In the present study, we investigate through computer simulations the binding of ACE inhibitors to patient-derived Aβ fibrils and contrast it with that of ACE inhibitors binding to in vitro generated fibrils. The binding affinities of the ACE inhibitors are compared with that of Congo red, a dye that is used to identify amyloid structures and that is known to be a weak inhibitor of Aβ aggregation. We find that ACE inhibitors have a lower binding affinity to the patient-derived fibrils than to in vitro generated ones. For patient-derived fibrils, their binding affinities are even lower than that of Congo red. Our observations raise doubts on the hypothesis that these drugs inhibit fibril formation in Alzheimer patients by interacting directly with the amyloids.

Ultrastructure changes in buffalo (Bubalus bubalis) oocytes before and after maturation in vitro with sericin.

PubMed

Gustina, Sri; Hasbi, Hasbi; Karja, Ni Wayan Kurniani; Setiadi, Mohamad Agus; Supriatna, Iman

2017-12-01

The aim of this research was to identify the changes in the cytoplasmic ultrastructure of immature and matured oocytes in buffalo (Bubalus bubalis). Oocytes were matured in vitro in tissue culture medium-199 with and without sericin, and then analyzed by light and transmission electron microscopy. The experiment result showed that the nuclear maturation rate of buffalo oocytes was significantly higher in the presence of sericin (80.6%) than without sericin (68.1%) (P < 0.05). The immature oocytes were characterized by cortical granule clusters in the ooplasm and the absence of perivitelline space (PVS). In contrast, the oocytes matured either with or without sericin showed the formation of PVS, erected microvilli, the migration of cortical granules to the cytoplasmic periphery, and the clear appearance of the mitochondria and vesicle in the oolemma. Interestingly, matured oocytes with sericin have smaller cortical granules than do immature oocytes (P < 0.05). In conclusion, supplementation of 0.05% sericin in the maturation medium can enhance the maturation rate of buffalo oocytes. Several cytoplasmic ultrastructures were relocated and modulated during the in vitro maturation process of buffalo oocytes: PVS development, cortical granules migration to periphery, and mitochondria and vesicles in the cortical region. The ultrastructure was similar between the groups with and without sericin. © 2017 Japanese Society of Animal Science.

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.

Evidence for a specific uptake and retention mechanism for 25-hydroxyvitamin D (25OHD) in skeletal muscle cells.

PubMed

Abboud, M; Puglisi, D A; Davies, B N; Rybchyn, M; Whitehead, N P; Brock, K E; Cole, L; Gordon-Thomson, C; Fraser, D R; Mason, R S

2013-09-01

Little is known about the mechanism for the prolonged residence time of 25-hydroxyvitamin D (25OHD) in blood. Several lines of evidence led us to propose that skeletal muscle could function as the site of an extravascular pool of 25OHD. In vitro studies investigated the capacity of differentiated C2 murine muscle cells to take up and release 25OHD, in comparison with other cell types and the involvement of the membrane protein megalin in these mechanisms. When C2 cells are differentiated into myotubes, the time-dependent uptake of labeled 25OHD is 2-3 times higher than in undifferentiated myoblasts or nonmuscle osteoblastic MG63 cells (P < .001). During in vitro release experiments (after 25OHD uptake), myotubes released only 32% ± 6% stored 25OHD after 4 hours, whereas this figure was 60% ± 2% for osteoblasts (P < .01). Using immunofluorescence, C2 myotubes and primary rat muscle fibers were, for the first time, shown to express megalin and cubilin, endocytotic receptors for the vitamin D binding protein (DBP), which binds nearly all 25OHD in the blood. DBP has a high affinity for actin in skeletal muscle. A time-dependent uptake of Alexafluor-488-labeled DBP into mature muscle cells was observed by confocal microscopy. Incubation of C2 myotubes (for 24 hours) with receptor-associated protein, a megalin inhibitor, led to a 40% decrease in 25OHD uptake (P < .01). These data support the proposal that 25OHD, after uptake into mature muscle cells, is held there by DBP, which has been internalized via membrane megalin and is retained by binding to actin.

Lipid characterization of individual porcine oocytes by dual mode DESI-MS and data fusion.

PubMed

Pirro, Valentina; Oliveri, Paolo; Ferreira, Christina Ramires; González-Serrano, Andrés Felipe; Machaty, Zoltan; Cooks, Robert Graham

2014-10-27

The development of sensitive measurements to analyze individual cells is of relevance to elucidate specialized roles or metabolic functions of each cell under physiological and pathological conditions. Lipids play multiple and critical roles in cellular functions and the application of analytical methods in the lipidomics area is of increasing interest. In this work, in vitro maturation of porcine oocytes was studied. Two independent sources of chemical information (represented by mass spectra in the positive and negative ion modes) from single oocytes (immature oocytes, 24-h and 44-h in vitro matured oocytes) were acquired by using desorption electrospray ionization-mass spectrometry (DESI-MS). Low and mid-level data fusion strategies are presented with the aim of better exploring the large amount of chemical information contained in the two mass spectrometric lipid profiles. Data were explored by principal component analysis (PCA) within the two multi-block approaches to include information on free fatty acids, phospholipids, cholesterol-related molecules, di- and triacylglycerols. After data fusion, clearer differences among immature and in vitro matured porcine oocytes were observed, which provide novel information regarding lipid metabolism throughout oocyte maturation. In particular, changes in TAG composition, as well as increase in fatty acid metabolism and membrane complexity were evidenced during the in vitro maturation process. This information can assist the improvement of in vitro embryo production for porcine species. Copyright © 2014 Elsevier B.V. All rights reserved.

iNKT cell cytotoxic responses control T-lymphoma growth in vitro and in vivo

PubMed Central

Bassiri, Hamid; Das, Rupali; Guan, Peng; Barrett, David M.; Brennan, Patrick J.; Banerjee, Pinaki P.; Wiener, Susan J.; Orange, Jordan S.; Brenner, Michael B.; Grupp, Stephan A.; Nichols, Kim E.

2013-01-01

Invariant natural killer T (iNKT) cells comprise a lineage of CD1d-restricted glycolipid-reactive T lymphocytes with important roles in host immunity to cancer. iNKT cells indirectly participate in antitumor responses by inducing dendritic cell maturation and producing cytokines that promote tumor clearance by CD8+ T and NK cells. Although iNKT cells thereby act as potent cellular adjuvants, it is less clear whether they directly control the growth of tumors. To gain insights into the direct contribution of iNKT cells to tumor immune surveillance, we developed in vitro and in vivo systems to selectively examine the antitumor activity of iNKT cells in the absence of other cytolytic effectors. Using the EL4 T-lymphoma cell line as a model, we find that iNKT cells exert robust and specific lysis of tumor cells in vitro in a manner that is differentially-induced by iNKT cell agonists of varying TCR affinities, such as OCH, α-galactosyl ceramide and PBS44. In vitro blockade of CD1d-mediated lipid antigen presentation, disruption of T cell receptor (TCR) signaling, or loss of perforin expression significantly reduce iNKT cell killing. Consistent with these findings, iNKT cell reconstitution of T, B, and NK cell-deficient mice slows EL4 growth in vivo via TCR-CD1d and perforin-dependent mechanisms. Together, these observations establish that iNKT cells are sufficient to control the growth of T-lymphoma in vitro and in vivo. They also suggest that the induction of iNKT cell cytotoxic responses in situ might serve as a more effective strategy to prevent and/or treat CD1d+ cancers, such as T-lymphoma. PMID:24563871

iNKT cell cytotoxic responses control T-lymphoma growth in vitro and in vivo .

PubMed

Bassiri, Hamid; Das, Rupali; Guan, Peng; Barrett, David M; Brennan, Patrick J; Banerjee, Pinaki P; Wiener, Susan J; Orange, Jordan S; Brenner, Michael B; Grupp, Stephan A; Nichols, Kim E

2014-01-01

Invariant natural killer T (iNKT) cells comprise a lineage of CD1d-restricted glycolipid-reactive T lymphocytes with important roles in host immunity to cancer. iNKT cells indirectly participate in antitumor responses by inducing dendritic cell maturation and producing cytokines that promote tumor clearance by CD8+ T and NK cells. Although iNKT cells thereby act as potent cellular adjuvants, it is less clear whether they directly control the growth of tumors. To gain insights into the direct contribution of iNKT cells to tumor immune surveillance, we developed in vitro and in vivo systems to selectively examine the antitumor activity of iNKT cells in the absence of other cytolytic effectors. Using the EL4 T-lymphoma cell line as a model, we found that iNKT cells exert robust and specific lysis of tumor cells in vitro in a manner that is differentially induced by iNKT cell agonists of varying T-cell receptor (TCR) affinities, such as OCH, α-galactosyl ceramide, and PBS44. In vitro blockade of CD1d-mediated lipid antigen presentation, disruption of TCR signaling, or loss of perforin expression significantly reduce iNKT cell killing. Consistent with these findings, iNKT cell reconstitution of T, B, and NK cell–deficient mice slows EL4 growth in vivo via TCR-CD1d and perforin-dependent mechanisms. Together, these observations establish that iNKT cells are sufficient to control the growth of T lymphoma in vitro and in vivo. They also suggest that the induction of iNKT cell cytotoxic responses in situ might serve as a more effective strategy to prevent and/or treat CD1d+ cancers, such as T lymphoma. ©2013 AACR.

The presence of acylated ghrelin during in vitro maturation of bovine oocytes induces cumulus cell DNA damage and apoptosis, and impairs early embryo development.

PubMed

Sirini, Matias A; Anchordoquy, Juan Mateo; Anchordoquy, Juan Patricio; Pascua, Ana M; Nikoloff, Noelia; Carranza, Ana; Relling, Alejandro E; Furnus, Cecilia C

2017-10-01

The aim of this study was to investigate the effects of acylated ghrelin supplementation during in vitro maturation (IVM) of bovine oocytes. IVM medium was supplemented with 20, 40 or 60 pM acylated ghrelin concentrations. Cumulus expansion area and oocyte nuclear maturation were studied as maturation parameters. Cumulus-oocyte complexes (COC) were assessed with the comet, apoptosis and viability assays. The in vitro effects of acylated ghrelin on embryo developmental capacity and embryo quality were also evaluated. Results demonstrated that acylated ghrelin did not affect oocyte nuclear maturation and cumulus expansion area. However, it induced cumulus cell (CC) death, apoptosis and DNA damage. The damage increased as a function of the concentration employed. Additionally, the percentages of blastocyst yield, hatching and embryo quality decreased with all acylated ghrelin concentrations tested. Our study highlights the importance of acylated ghrelin in bovine reproduction, suggesting that this metabolic hormone could function as a signal that prevents the progress to reproductive processes.

Supplementation with CTGF, SDF1, NGF, and HGF promotes ovine in vitro oocyte maturation and early embryo development.

PubMed

Wang, D H; Ren, J; Zhou, C J; Han, Z; Wang, L; Liang, C G

2018-05-17

The strategies for improving the in vitro maturation (IVM) of domestic animal oocytes focus on promoting nuclear and cytoplasmic maturation. The identification of paracrine factors and their supplementation in the culture medium represent effective approaches for oocyte maturation and embryo development. This study investigated the effects of paracrine factor supplementation including connective tissue growth factor (CTGF), nerve growth factor (NGF), hepatocyte growth factor (HGF), and stromal derived factor 1 (SDF1) on ovine oocytes and early parthenogenetic embryos using an in vitro culture system. First, we identified the optimal concentrations of CTGF (30 ng/mL), SDF1 (10 ng/mL), NGF (3 ng/mL), and HGF (100 ng/mL) for promoting oocyte maturation, which combined, induced nuclear maturation in 94.19% of oocytes. This combination also promoted cumulus cell expansion and inhibited oocyte/cumulus apoptosis, while enabling a larger proportion (33.04%) of embryos to develop into blastocysts than in the controls and prevented embryo apoptosis. These novel findings demonstrate that the paracrine factors CTGF, SDF1, NGF, and HGF facilitate ovine oocyte and early parthenogenetic embryo development in vitro. Thus, supplementation with these factors may help optimize the IVM of ovine oocytes and early parthenogenetic embryo development strategies. Copyright © 2018 Elsevier Inc. All rights reserved.

Comparison of survival rate of cleavage stage embryos produced from in vitro maturation cycles after slow freezing and after vitrification.

PubMed

Son, Weon-Young; Chung, Jin-Tae; Gidoni, Yariv; Holzer, Hananel; Levin, Dan; Chian, Ri-Cheng; Tan, Seang Lin

2009-09-01

Significantly more embryos survived the vitrification procedure compared to slow freezing (85.5% vs. 61.8%) in cleavage-stage human embryos produced from in vitro maturation cycles, suggesting that vitrification is more efficient than slow freezing for cryopreservation.

What Are the Primary Limitations in B-Cell Affinity Maturation, and How Much Affinity Maturation Can We Drive with Vaccination? Breaking through Immunity's Glass Ceiling.

PubMed

Kelsoe, Garnett; Haynes, Barton F

2018-05-01

A key goal of HIV-1 vaccine development is the induction of broadly neutralizing antibodies (bnAbs) targeted to the vulnerable regions of the HIV envelope. BnAbs develop over time in ∼50% of HIV-1-infected individuals. However, to date, no vaccines have induced bnAbs and few or none of these vaccine-elicited HIV-1 antibodies carry the high frequencies of V(D)J mutations characteristic of bnAbs. Do the high frequencies of mutations characteristic of naturally induced bnAbs represent a fundamental barrier to the induction of bnAbs by vaccines? Recent studies suggest that high frequencies of V(D)J mutations can be achieved by serial vaccination strategies. Rather, it appears that, in the absence of HIV-1 infection, physiologic immune tolerance controls, including a germinal center process termed affinity reversion, may limit vaccine-driven bnAb development by clonal elimination or selecting for mutations incompatible with bnAb activity. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Repair of osteochondral defects with in vitro engineered cartilage based on autologous bone marrow stromal cells in a swine model.

PubMed

He, Aijuan; Liu, Lina; Luo, Xusong; Liu, Yu; Liu, Yi; Liu, Fangjun; Wang, Xiaoyun; Zhang, Zhiyong; Zhang, Wenjie; Liu, Wei; Cao, Yilin; Zhou, Guangdong

2017-01-13

Functional reconstruction of large osteochondral defects is always a major challenge in articular surgery. Some studies have reported the feasibility of repairing articular osteochondral defects using bone marrow stromal cells (BMSCs) and biodegradable scaffolds. However, no significant breakthroughs have been achieved in clinical translation due to the instability of in vivo cartilage regeneration based on direct cell-scaffold construct implantation. To overcome the disadvantages of direct cell-scaffold construct implantation, the current study proposed an in vitro cartilage regeneration strategy, providing relatively mature cartilage-like tissue with superior mechanical properties. Our strategy involved in vitro cartilage engineering, repair of osteochondral defects, and evaluation of in vivo repair efficacy. The results demonstrated that BMSC engineered cartilage in vitro (BEC-vitro) presented a time-depended maturation process. The implantation of BEC-vitro alone could successfully realize tissue-specific repair of osteochondral defects with both cartilage and subchondral bone. Furthermore, the maturity level of BEC-vitro had significant influence on the repaired results. These results indicated that in vitro cartilage regeneration using BMSCs is a promising strategy for functional reconstruction of osteochondral defect, thus promoting the clinical translation of cartilage regeneration techniques incorporating BMSCs.

In vitro oocyte maturation, fertilization and culture after ovum pick-up in an endangered gazelle (Gazella dama mhorr).

PubMed

Berlinguer, F; González, R; Succu, S; del Olmo, A; Garde, J J; Espeso, G; Gomendio, M; Ledda, S; Roldan, E R S

2008-02-01

The recovery of immature oocytes followed by in vitro maturation, fertilization and culture (IVMFC) allows the rescue of biological material of great genetic value for the establishment of genetic resource banks of endangered species. Studies exist on sperm cryopreservation of endangered Mohor gazelle (Gazella dama mhorr), but no work has been carried out yet on oocyte collection, fertilization and culture in this or related species. The purpose of this study was to develop a protocol for ovarian stimulation for the recovery of oocytes and subsequent IVMFC in the Mohor gazelle using frozen-thawed spermatozoa. Ovum pick-up was performed after ovarian stimulation with a total dose of 5.28 mg of ovine FSH. A total of 35 oocytes were recovered from 56 punctured follicles (62%) (N=6 females). Out of 29 cumulus-oocyte complexes matured in vitro, 3% were found at germinal vesicle stage, 7% at metaphase I, 21% were degenerated, and 69% advanced to metaphase II. Fertilization and cleavage rates of matured oocytes were 40 and 30%, respectively. Embryos cleaved in vitro up to the 6-8 cell stage but none progressed to the blastocyst stage, suggesting the existence of a developmental block and the need to improve culture conditions. Although more studies are needed to improve hormonal stimulation and oocyte harvesting, as well as IVMFC conditions, this study demonstrates for the first time the feasibility of in vitro fertilization with frozen-thawed semen of in vitro matured oocytes collected by ovum pick-up from FSH-stimulated endangered gazelles.

Differential effects of herbicides atrazine and fenoxaprop-ethyl, and insecticides diazinon and malathion, on viability and maturation of porcine oocytes in vitro.

PubMed

Casas, Eduardo; Bonilla, Edmundo; Ducolomb, Yvonne; Betancourt, Miguel

2010-02-01

Exposure to pesticides may be a major cause of reproductive dysfunction in humans and animals. Atrazine and fenoxaprop-ethyl, widely used herbicides, and malathion and diazinon, organophosphate insecticides, are considered only slightly toxic to vertebrates; however, there is evidence of greater effects on reproductive function. The aim of this study was to evaluate the effect of these pesticides on oocyte viability and in vitro maturation. Gametes were matured in increasing concentrations of the pesticides and then stained with MTT to evaluate viability and bisbenzimide to assess the maturation stage, in the same oocyte. Atrazine had no effect on viability but maturation was significantly reduced, while fenoxaprop-ethyl affected both parameters. The insecticides affected viability and maturation but to a different degree. The four pesticides showed a more pronounced effect on maturation than on viability, due to a blockage at germinal vesicle stage.

Ultrasonographic-guided retrieval of cumulus oocyte complexes after super-stimulation in dromedary camel (Camelus dromedarius).

PubMed

Wani, N A; Skidmore, J A

2010-08-01

In Experiment 1, studies were conducted to apply the transvaginal ultrasound guided ovum pick-up (OPU) technique in dromedary camels after their ovarian super-stimulation and in vivo oocyte maturation. In Experiment 2, the developmental potential of two commonly used oocyte types, i.e., in vivo matured oocytes collected by OPU and abattoir derived in vitro-matured oocytes was compared after their chemical activation. In Experiment 3, developmental competence of oocytes collected from super-stimulated camels by OPU, matured either in vivo or in vitro, was compared after their chemical activation. Mature female dromedary camels super-stimulated with a combination of eCG and pFSH were given an injection of 20 microg of the GnRH analogue, buserelin 24, 26, or 28 h before the scheduled OPU. For collection of cumulus oocyte complexes (COCs) the transducer was guided through the vulva into the cranial most portion of the vagina and 17-gauge, 55 cm single-lumen needle was placed in the needle guide of the ultrasound probe and advanced through the vaginal fornix and into the follicle. Follicular fluid was aspirated using a regulated vacuum pump into tubes containing embryo-flushing media. Aspirates were searched for COCs using a stereomicroscope, and they were then denuded of cumulus cells by hyaluronidase and repeated pipetting. The oocytes were classified as mature (with a visible polar body), immature (with no visible polar body), activated (with divided or fragmented ooplasm) and others (degenerated and abnormal). Overall an average of 12.12 +/- 7.9 COCs were aspirated per animal with an oocyte recovery rate from the aspirated follicles of about 77%. The majority (> 90%) of the collected COCs by OPU were with loose and expanded cumulus cells. The proportion of matured oocytes obtained at 28-29 h (91.2 +/- 4.1) and 26-27 h (82.1 +/- 3.4) were higher (P < 0.005) when compared with those obtained at 24-25 h (40.4 +/- 16.3) after GnRH administration. In Experiment 2, a higher proportion (P < 0.05) of in vivo matured oocytes cleaved (84.6 +/- 2.1 vs. 60.9 +/- 6.6) and developed to blastocyst stages (52.4 +/- 4.1 vs. 30.5 +/- 3.3) when compared with in vitro matured oocytes collected from slaughterhouse ovaries. In Experiment 3, no difference was observed between the developmental competences of oocytes, collected from super stimulated camels, matured in vitro with those collected after their in vivo maturation. In conclusion, about 80-90% mature oocytes can be collected by ultrasound guided transvaginal ovum pick-up from super-stimulated dromedary camels 26-28 h after GnRH administration. The developmental response, to chemical activation, of in vivo matured oocytes collected by ultrasound guided transvaginal OPU is better than in vitro matured oocytes obtained from slaughterhouse ovaries. However, no difference was observed in the developmental competence of oocytes collected by OPU whether they were matured in vivo or in vitro. Copyright 2010 Elsevier Inc. All rights reserved.

Tumor-Targeting Anti-CD20 Antibodies Mediate In Vitro Expansion of Memory Natural Killer Cells: Impact of CD16 Affinity Ligation Conditions and In Vivo Priming.

PubMed

Capuano, Cristina; Battella, Simone; Pighi, Chiara; Franchitti, Lavinia; Turriziani, Ombretta; Morrone, Stefania; Santoni, Angela; Galandrini, Ricciarda; Palmieri, Gabriella

2018-01-01

Natural killer (NK) cells represent a pivotal player of innate anti-tumor immune responses. The impact of environmental factors in shaping the representativity of different NK cell subsets is increasingly appreciated. Human cytomegalovirus (HCMV) infection profoundly affects NK cell compartment, as documented by the presence of a CD94/NKG2C + FcεRIγ - long-lived "memory" NK cell subset, endowed with enhanced CD16-dependent functional capabilities, in a fraction of HCMV-seropositive subjects. However, the requirements for memory NK cell pool establishment/maintenance and activation have not been fully characterized yet. Here, we describe the capability of anti-CD20 tumor-targeting therapeutic monoclonal antibodies (mAbs) to drive the selective in vitro expansion of memory NK cells and we show the impact of donor' HCMV serostatus and CD16 affinity ligation conditions on this event. In vitro expanded memory NK cells maintain the phenotypic and functional signature of their freshly isolated counterpart; furthermore, our data demonstrate that CD16 affinity ligation conditions differently affect memory NK cell proliferation and functional activation, as rituximab-mediated low-affinity ligation represents a superior proliferative stimulus, while high-affinity aggregation mediated by glycoengineered obinutuzumab results in improved multifunctional responses. Our work also expands the molecular and functional characterization of memory NK cells, and investigates the possible impact of CD16 functional allelic variants on their in vivo and in vitro expansions. These results reveal new insights in Ab-driven memory NK cell responses in a therapeutic setting and may ultimately inspire new NK cell-based intervention strategies against cancer, in which the enhanced responsiveness to mAb-bound target could significantly impact therapeutic efficacy.

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.

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.

Treatment with acetyl-l-carnitine during in vitro maturation of buffalo oocytes improves oocyte quality and subsequent embryonic development.

PubMed

Xu, Hui-Yan; Yang, Xiao-Gan; Lu, Sheng-Sheng; Liang, Xing-Wei; Lu, Yang-Qing; Zhang, Ming; Lu, Ke-Huan

2018-06-01

Oocyte quality is one of the important factors in female fertility, in vitro maturation (IVM), and subsequent embryonic development. In the present study, we assessed whether acetyl-l-carnitine (ALC) supplementation during in vitro maturation of buffalo oocytes could improve oocyte quality and subsequent embryonic development. To determine the optimal level of ALC supplementation, we matured cumulus-oocyte complexes in maturation medium supplemented with 0, 2.5, and 5 mM ALC. The oocytes with a polar body were selected for parthenogenetic activation (PA) and in vitro fertilization (IVF). We found that oocytes matured in 2.5 mM ALC had significantly higher PA blastocyst rate (P < 0.05) and blastocyst cell number than those of unsupplemented oocytes (P < 0.05) and a significantly higher IVF blastocyst rate than that of oocytes matured in 5 mM ALC (P < 0.05). In all further experiments, we supplemented the maturation medium with 2.5 mM ALC. We then tested whether ALC supplementation could improve various markers of oocytes and cumulus cells. We compared cell proliferation; concentrations of reactive oxygen species (ROS), intracellular ATP, estradiol, and progesterone; mitochondrial distribution; mitochondrial DNA copy number (mtDNA); and expression levels of four genes encoding oocyte-derived factors (GDF9, BMP15) and steroid hormones (StAR, P450scc) between the supplemented and unsupplemented oocytes and cumulus cells. Cumulus cells matured with ALC supplementation were more prolific than those matured without ALC supplementation (P < 0.05). Oocytes treated with ALC had lower concentrations of intracellular ROS (P < 0.05) and a higher rate of diffuse mitochondrial distributions (P < 0.05) than those of untreated oocytes. Additionally, the mtDNA was higher in the ALC-treated oocytes (P < 0.05) and cumulus cells (P < 0.05) than that in the untreated cells. The ALC-treated maturation medium had a higher postmaturation concentration of estradiol than that of the untreated medium (P < 0.05). Finally, the gene expression levels of P450scc and GDF9 were greater in ALC-treated oocytes and cumulus cells than those in untreated cells (P < 0.05). Therefore, in buffalo, our results suggest that ALC affects mitochondrial function, regulates oocyte-derived paracrine factors, and increases the production of steroid hormones, leading to increased quality of matured oocytes and improved embryonic development in vitro. Copyright © 2018 Elsevier Inc. All rights reserved.

Abnormal chromosome behavior in human oocytes which remained unfertilized during human in vitro fertilization.

PubMed

Spielmann, H; Krüger, C; Stauber, M; Vogel, R

1985-09-01

Chromosomal abnormalities and abnormal embryonic development have previously been observed after human in vitro fertilization (IVF). Chromosomal abnormalities may arise not only after fertilization but even earlier during meiotic maturation of human oocytes in culture. Since chromosomal analysis is simple in oocytes during meiotic maturation, the chromosomal status was analyzed in oocytes which remained unfertilized in a human in vitro fertilization program. In 50 fertilization attempts the chromosomes of 62 unfertilized oocytes could be analyzed; 45 of them were in the process of meiotic maturation. In three oocytes two small polar bodies were observed 16-18 hr after insemination in the absence of fertilization. In one oocyte abnormal chromosome behavior was found during the first meiotic division, and in four oocytes during metaphase of the second meiotic division. These data suggest that chromosomal analysis of unfertilized oocytes in human IVF may improve the understanding human oocyte maturation and fertilization.

Lignans from the roots of Urtica dioica and their metabolites bind to human sex hormone binding globulin (SHBG).

PubMed

Schöttner, M; Gansser, D; Spiteller, G

1997-12-01

Polar extracts of the stinging nettle (Urtica dioica L.) roots contain the ligans (+)-neoolivil, (-)-secoisolariciresinol, dehydrodiconiferyl alcohol, isolariciresinol, pinoresinol, and 3,4-divanillyltetrahydrofuran. These compounds were either isolated from Urtica roots, or obtained semisynthetically. Their affinity to human sex hormone binding globulin (SHBG) was tested in an in vitro assay. In addition, the main intestinal transformation products of plant lignans in humans, enterodiol and enterolactone, together with enterofuran were checked for their activity. All lignans except (-)-pinoresinol developed a binding affinity to SHBG in the in vitro assay. The affinity of (-)-3,4-divanillyltetrahydrofuran was outstandingly high. These findings are discussed with respect to potential beneficial effects of plant lignans on benign prostatic hyperplasia (BPH).

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.

Phospholipase C-zeta deficiency as a cause for repetitive oocyte fertilization failure during ovarian stimulation for in vitro fertilization with ICSI: a case report.

PubMed

Chithiwala, Zahabiya H; Lee, Hoi Chang; Hill, David L; Jellerette-Nolan, Teru; Fissore, Rafael; Grow, Daniel; Dumesic, Daniel A

2015-09-01

The purpose of this study is to describe impaired oocyte fertilization from phospholipase C-zeta (PLC-ζ) deficiency in normal-appearing sperm that was successfully treated using calcium (Ca(2+)) ionophore with intracytoplasmic sperm injection (ICSI) of oocytes matured in vitro. An infertile couple undergoing in vitro fertilization (IVF) experienced failed oocyte fertilization following ICSI with normal-appearing sperm. A semen sample collected from the patient was used to assess the expression of sperm PLC- ζ protein by Western blot analysis and immunofluorescence and PLC-ζ bioactivity by an in vitro model of Ca(2+) release. A second IVF cycle was performed using Ca(2+) ionophore with ICSI to enhance Ca(2+)-induced oocyte activation of oocytes matured in vitro. Sperm PLC-ζ protein deficiency was demonstrated by Western blot analysis and immunofluorescence and confirmed by reduced PLC-ζ bioactivity using an in vitro model of Ca(2+) release. Nevertheless, with this sperm and supplementation of Ca(2+) ionophore following ICSI, fertilization of four of six oocytes matured in vitro was obtained. In addition, four embryos underwent cleavage and two of them reached the blastocyst stage. Transfer of these blastocysts into the uterus led to a single pregnancy and live birth. Deficiency of PLC-ζ in normal-appearing human sperm is associated with impaired Ca(2+)-dependent oocyte activation during ICSI. Under this condition, use of Ca(2+) ionophore following ICSI of oocytes matured in vitro improves embryo developmental competence, possibly through the activation of Ca(2+)-dependent mechanisms governing fertilization and preimplantation embryogenesis.

The presence of centrioles and centrosomes in ovarian mature cystic teratoma cells suggests human parthenotes developed in vitro can differentiate into mature cells without a sperm centriole.

PubMed

Lee, Bo Yon; Shim, Sang Woo; Kim, Young Sun; Kim, Seung Bo

2011-11-18

In most animals, somatic cell centrosomes are inherited from the centriole of the fertilizing spermatozoa. The oocyte centriole degenerates during oogenesis, and completely disappears in metaphase II. Therefore, the embryos generated by in vitro parthenogenesis are supposed to develop without any centrioles. Exceptional acentriolar and/or acentrosomal developments are possible in mice and in some experimental cells; however, in most animals, the full developmental potential of parthenogenetic cells in vitro and the fate of their centrioles/centrosomes are not clearly understood. To predict the future of in vitro human parthenogenesis, we explored the centrioles/centrosomes in ovarian mature cystic teratoma cells by immunofluorescent staining and transmission electron microscopy. We confirmed the presence of centrioles and centrosomes in these well-known parthenogenetic ovarian tumor cells. Our findings clearly demonstrate that, even without a sperm centriole, parthenotes that develop from activated oocytes can produce their own centrioles/centrosomes, and can even develop into the well-differentiated mature tissue. Copyright © 2011 Elsevier Inc. All rights reserved.

Investigations of oocyte in vitro maturation within a mouse model.

PubMed

Chin, Alexis Heng Boon; Chye, Ng Soon

2004-02-01

This study attempted to develop a 'less meiotically competent' murine model for oocyte in vitro maturation (IVM), which could more readily be extrapolated to human clinical assisted reproduction. Oocyte meiotic competence was drastically reduced upon shortening the standard duration of in vivo gonadotrophin stimulation from 48 h to 24 h, and by selecting only naked or partially naked germinal vesicle oocytes, instead of fully cumulus enclosed oocyte complexes. With such a less meiotically competent model, only porcine granulosa coculture significantly enhanced the oocyte maturation rate in vitro, whereas no significant enhancement was observed with macaque and murine granulosa coculture. Increased serum concentrations and the supplementation of gonadotrophins, follicular fluid and extracellular matrix gel within the culture medium did not enhance IVM under either cell-free or coculture conditions. Culture medium conditioned by porcine granulosa also enhanced the maturation rate, and this beneficial effect was not diminished upon freeze-thawing. Enhanced IVM in the presence of porcine granulosa coculture did not, however, translate into improved developmental competence, as assessed by in vitro fertilization and embryo culture to the blastocyst stage.

Guiding the evolution to catch the virus: An in silico study of affinity maturation against rapidly mutating antigen

NASA Astrophysics Data System (ADS)

Wang, Shenshen; Burton, Dennis; Kardar, Mehran; Chakraborty, Arup

2014-03-01

The immune system comprises an intricate and evolving collection of cells and molecules that enables a defense against pathogenic agents. Its workings present a rich source of physical problems that impact human health. One intriguing example is the process of affinity maturation (AM) through which an antibody (Ab)--a component of the host immune system--evolves to more efficiently bind an antigen (Ag)--a unique part of a foreign pathogen such as a virus. Sufficiently strong binding to the Ag enables recognition and neutralization. A major challenge is to contain a diversifying mixture of Ag variants, that arise in natural infection, from evading Ab neutralization. This entails a thorough understanding of AM against multiple Ag species and mutating Ag. During AM, Ab-encoding cells undergo cycles of mutation and selection, a process reminiscent of Darwinian evolution yet occurring in real time. We first cast affinity-dependent selection into an extreme value problem and show how the binding characteristics scale with Ag diversity. We then develop an agent-based residue-resolved computational model of AM which allows us to track the evolutionary trajectories of individual cells. This dynamic model not only reveals significant stochastic effects associated with the relatively small and highly dynamic population size, it also uncovers the markedly distinct maturation outcomes if designed Ag variants are presented in different temporal procedures. Insights thus obtained would guide rational design of vaccination protocols.

In vitro maturation of immature thymocytes into immunocompetent T cells in the absence of direct thymic influence.

PubMed

Irlé, C; Piguet, P F; Vassalli, P

1978-07-01

Peanut lectin (PNL) binds to a majority of mouse thymocytes (Thc) in suspension. By using cell affinity chromatography on a column of anti-PNL antibody, Thc populations at least 96 percent pure in PNL + or - cells, as judged by immunofluorescence, were obtained. PNL(+) cells are rich in Thy 1 and poor in H(2) antigens, cortisone sensitive, unresponsive to phytohemagglutinin (PHA), and immunologically incompetent, as judged by mixed lymphocyte reaction, popliteal lymph node graft-versus-host assay, and by testing helper activity in a primary in vitro antibody response to sheep erythrocytes; the converse is true of PNL(-) cells. Thus, PNL(+) and (-) cells appear to correspond to cortical and medullary Thc, respectively, as previously suggested. In culture, PNL(+) Thc show poor viability and a weak proliferative response to concanavalin A (Con A), except when supernate (SUP) of 24 h Con A stimulated lymph node lymphocyte cultures, or irradiated lymph node cells, are added, in which cases a strong proliferative response to the mitogen is observed. A variety of control experiments showed that the proliferating cells did not result from preferential stimulation of a few contaminating PNL(-) Thc present in the PNL(+) Thc cultures. The blasts resulting from PNL(+) Thc proliferation display mitogen-induced cytotoxicity, and give rise to a population of medium-sized lymphocytes, mostly PNL(-), poor in Thy 1 and rich in H(2) antigens, PHA responsive, and immunologically competent in the above-mentioned assays. Fresh PNL(+) Thc responded in mixed lymphocyte reaction in the presence of SUP (lectin depleted) and since incubation in SUP alone did not confer reactivity on PNL(+) Thc, it appears therefore that (a) immature Thc possess alloantigen and mitogen-specific surface receptors but lack the capacity to respond by proliferation to receptor triggering without the help of extracellular factor(s) released by mature lymphoid cells stimulated by mitogens (b) cell division is associated with the acquisition of immunological responsiveness, characteristic of mature T lymphocytes. The implications of these findings for the ontogenesis of thymus-derived lymphocytes, and for the possible traffic of Thc within and from the thymus, are discussed.

Effect of recombinant-LH and hCG in the absence of FSH on in vitro maturation (IVM) fertilization and early embryonic development of mouse germinal vesicle (GV)-stage oocytes.

PubMed

Dinopoulou, Vasiliki; Drakakis, Peter; Kefala, Stella; Kiapekou, Erasmia; Bletsa, Ritsa; Anagnostou, Elli; Kallianidis, Konstantinos; Loutradis, Dimitrios

2016-06-01

During in vitro maturation (IVM), intrinsic and extrinsic factors must co-operate properly in order to ensure cytoplasmic and nuclear maturation. We examined the possible effect of LH/hCG in the process of oocyte maturation in mice with the addition of recombinant LH (r-LH) and hCG in our IVM cultures of mouse germinal vesicle (GV)-stage oocytes. Moreover, the effects of these hormones on fertilization, early embryonic development and the expression of LH/hCG receptor were examined. Nuclear maturation of GV-stage oocytes was evaluated after culture in the presence of r-LH or hCG. Fertilization rates and embryonic development were assessed after 24h. Total RNA was isolated from oocytes of different stages of maturation and from zygotes and embryos of different stages of development in order to examine the expression of LH/hCG receptor, using RT-PCR. The in vitro nuclear maturation rate of GV-stage oocytes that received hCG was significantly higher compared to the control group. Early embryonic development was increased in the hCG and LH cultures of GV oocytes when LH was further added. The LH/hCG receptor was expressed in all stages of in vitro matured mouse oocytes and in every stage of early embryonic development. Addition of hCG in IVM cultures of mouse GV oocytes increased maturation rates significantly. LH, however, was more beneficial to early embryonic development than hCG. This suggests a promising new technique in basic science research or in clinical reproductive medicine. Copyright © 2016 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Estimation of the optimal timing of fertilization for embryo development of in vitro-matured bovine oocytes based on the times of nuclear maturation and sperm penetration.

PubMed

Koyama, Keisuke; Kang, Sung-Sik; Huang, Weiping; Yanagawa, Yojiro; Takahashi, Yoshiyuki; Nagano, Masashi

2014-05-01

The objective of this research was to estimate the optimal timing for fertilization to achieve proper embryonic development of in vitro-matured bovine oocytes. First, cumulus-oocyte complexes were subjected to in vitro maturation (IVM) for 14-22 hr. The timing when 50% of oocytes reached metaphase II stage was estimated to be 17.5 hr after IVM start. Next, using oocytes subjected to IVM for 12-30 hr, sperm penetration was examined after 4-18 hr of in vitro fertilization (IVF). A significant negative correlation between IVM duration and the timing when 50% of oocytes were penetrated by sperm after IVF start was observed (P<0.01). Finally, oocytes subjected to 12-30 hr of IVM were inseminated and cultured for 6 days to examine embryonic development. In the group with 22 hr of IVM, the percentages of cleaved embryos and blastocysts were the highest values in all groups. According to the regression equation describing the time from nuclear maturation to sperm penetration (x) and the percentage of blastocysts (y) (y=7.23x - 0.297x(2), P<0.01), the blastocyst rate peaked when sperm penetration occurred at 12.2 hr after achieving nuclear maturation. In conclusion, under the present IVM/IVF conditions, it was estimated that oocytes acquired their highest developmental competence at about 30 hr after IVM start, and thus, the optimal IVM duration was calculated to be about 21 hr.

Toxic effects and possible mechanisms of hydrogen sulfide and/or ammonia on porcine oocyte maturation in vitro.

PubMed

Yang, Lei-Lei; Zhao, Yong; Luo, Shi-Ming; Ma, Jun-Yu; Ge, Zhao-Jia; Shen, Wei; Yin, Shen

2018-03-15

Previous studies suggest that hydrogen sulfide (H 2 S) and ammonia (NH 3 ) are two major air pollutants which can cause damage to porcine health. However, the mechanisms underlying toxic effects of these compounds on porcine oocyte maturation are not clear. To clarify the mechanism, we evaluated the oocyte quality by detecting some events during oocytes maturation. In our study, porcine oocytes were cultured with different concentrations of Na 2 S and/or NH 4 Cl in vitro and the rate of the first polar body extrusion decreased significantly. Also, actin filament was seriously disrupted to damage the cytoskeleton which resulted in reduced rate of oocyte maturation. We explored the reactive oxygen species (ROS) generation and found that the ROS level was increased significantly after Na 2 S treatment but not after NH 4 Cl treatment. Moreover, early stage apoptosis rate was significantly increased and autophagy protein LC3 B expression level was higher in oocytes treated with Na 2 S and/or NH 4 Cl, which might be caused by ROS elevation. Additionally, exposure to Na 2 S and/or NH 4 Cl also caused ROS generation and early apoptosis in cumulus cells, which might further affect oocyte maturation in vitro. In summary, our data suggested that exposure to H 2 S and/or NH 3 decreased porcine oocyte maturation in vitro, which might be caused by actin disruption, ROS generation, early apoptosis and autophagy. Copyright © 2017 Elsevier B.V. All rights reserved.

Mature adipocyte-derived dedifferentiated fat cells can transdifferentiate into skeletal myocytes in vitro

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

Kazama, Tomohiko; Fujie, Masaki; Endo, Tuyoshi

2008-12-19

We have previously reported the establishment of preadipocyte cell lines, termed dedifferentiated fat (DFAT) cells, from mature adipocytes of various animals. DFAT cells possess long-term viability and can redifferentiate into adipocytes both in vivo and in vitro. Furthermore, DFAT cells can transdifferentiate into osteoblasts and chondrocytes under appropriate culture conditions. However, it is unclear whether DFAT cells are capable of transdifferentiating into skeletal myocytes, which is common in the mesodermal lineage. Here, we show that DFAT cells can be induced to transdifferentiate into skeletal myocytes in vitro. Myogenic induction of DFAT cells resulted in the expression of MyoD and myogenin,more » followed by cell fusion and formation of multinucleated cells expressing sarcomeric myosin heavy chain. These results indicate that DFAT cells derived from mature adipocytes can transdifferentiate into skeletal myocytes in vitro.« less

Follicle dynamics: visualization and analysis of follicle growth and maturation using murine ovarian tissue culture.

PubMed

Murase, Tomohiko; Iwase, Akira; Komatsu, Kouji; Bayasula; Nakamura, Tomoko; Osuka, Satoko; Takikawa, Sachiko; Goto, Maki; Kotani, Tomomi; Kikkawa, Fumitaka

2018-02-01

To visualize and analyze follicle development in ovarian tissue culture using physiological concentrations of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in order to establish an ovarian tissue culture system that enables efficient in vitro growth of follicles. Ovarian tissues from 4-week-old female ICR mice were sliced and cultured. Images of ovarian tissues in culture were obtained at 24-h or 30-min intervals by using a microscope. The area of each follicle observed in the ovarian tissue slices was tracked and analyzed in association with oocyte maturation. We were able to track the development of each follicle using this culture system. Follicle growth was associated with oocyte maturation. Meiotically matured oocytes (MII) were obtained from 33% of all follicles investigated. Approximately, a quarter of follicles (24%) did not grow and resulted in atresia. Follicle dynamics were successfully visualized and analyzed in murine ovarian tissue culture. We were able to obtain mature oocytes from the fully grown follicles in vitro. This culture system would be helpful for efficient in vitro culturing of ovarian tissues.

Cloning, heterologous expression and biochemical characterization of plastidial sn-glycerol-3-phosphate acyltransferase from Helianthus annuus.

PubMed

Payá-Milans, Miriam; Venegas-Calerón, Mónica; Salas, Joaquín J; Garcés, Rafael; Martínez-Force, Enrique

2015-03-01

The acyl-[acyl carrier protein]:sn-1-glycerol-3-phosphate acyltransferase (GPAT; E.C. 2.3.1.15) catalyzes the first step of glycerolipid assembly within the stroma of the chloroplast. In the present study, the sunflower (Helianthus annuus, L.) stromal GPAT was cloned, sequenced and characterized. We identified a single ORF of 1344base pairs that encoded a GPAT sharing strong sequence homology with the plastidial GPAT from Arabidopsis thaliana (ATS1, At1g32200). Gene expression studies showed that the highest transcript levels occurred in green tissues in which chloroplasts are abundant. The corresponding mature protein was heterologously overexpressed in Escherichia coli for purification and biochemical characterization. In vitro assays using radiolabelled acyl-ACPs and glycerol-3-phosphate as substrates revealed a strong preference for oleic versus palmitic acid, and weak activity towards stearic acid. The positional fatty acid composition of relevant chloroplast phospholipids from sunflower leaves did not reflect the in vitro GPAT specificity, suggesting a more complex scenario with mixed substrates at different concentrations, competition with other acyl-ACP consuming enzymatic reactions, etc. In summary, this study has confirmed the affinity of this enzyme which would partly explain the resistance to cold temperatures observed in sunflower plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders

PubMed Central

Cui, Naiwen; Zhang, Huidan; Schneider, Nils; Tao, Ye; Asahara, Haruichi; Sun, Zhiyi; Cai, Yamei; Koehler, Stephan A.; de Greef, Tom F. A.; Abbaspourrad, Alireza; Weitz, David A.; Chong, Shaorong

2016-01-01

Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (103–106). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution. PMID:26940078

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

Beta-lactam antibiotic-induced platelet dysfunction: Evidence for irreversible inhibition of platelet activation in vitro and in vivo after prolonged exposure to penicillin

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

Burroughs, S.F.; Johnson, G.J.

beta-Lactam antibiotics cause platelet dysfunction with bleeding complications. Previous in vitro studies documented reversible inhibition of agonist-receptor interaction. This mechanism is inadequate to explain the effect of beta-lactam antibiotics in vivo. Platelet function does not return to normal immediately after drug treatment, implying irreversible inhibition of platelet function. We report here evidence of irreversible platelet functional and biochemical abnormalities after in vitro and in vivo exposure to beta-lactam antibiotics. Irreversible binding of (14C)-penicillin (Pen) occurred in vitro. After 24 hours' in vitro incubation with 10 to 20 mmol/L Pen, or ex vivo after antibiotic treatment, irreversible functional impairment occurred; butmore » no irreversible inhibition of alpha 2 adrenergic receptors, measured with (3H)-yohimbine, or high-affinity thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors, measured with agonist (3H)-U46619 and antagonist (3H)-SQ29548, occurred. However, low-affinity platelet TXA2/PGH2 receptors were decreased 40% after Pen exposure in vitro or in vivo, indicating irreversible membrane alteration. Two postreceptor biochemical events were irreversibly inhibited in platelets incubated with Pen for 24 hours in vitro or ex vivo after antibiotic treatment. Thromboxane synthesis was inhibited 28.3% to 81.7%. Agonist-induced rises in cytosolic calcium ((Ca2+)i) were inhibited 40.1% to 67.5% in vitro and 26.6% to 52.2% ex vivo. Therefore, Pen binds to platelets after prolonged exposure, resulting in irreversible dysfunction attributable to inhibition of TXA2 synthesis and impairment of the rise in (Ca2+)i. The loss of low-affinity TXA2/PGH2 receptors suggests that the primary site of action of these drugs is on the platelet membrane.« less

Oocyte holding in the Iberian red deer (Cervus elaphus hispanicus): Effect of initial oocyte quality and epidermal growth factor addition on in vitro maturation.

PubMed

Macías-García, B; González-Fernández, L; Matilla, E; Hernández, N; Mijares, J; Sánchez-Margallo, F M

2018-02-01

Current in vitro embryo production protocols in the Iberian red deer (Cervus elaphus hispanicus) need to be optimized; oocyte harvesting in situ followed by overnight holding could reduce the human effort and shipping costs. In our work, post-mortem ovaries were retrieved, and the oocytes were harvested and allocated to G1 group (good quality) or G2 + G3 group (low quality). The oocytes were separately subjected to immediate in vitro maturation (IVM) or held overnight in a holding medium composed of 40% of TCM 199 with Earle's salts, 40% TCM 199 with Hanks' salts and 20% fetal bovine serum (FBS), at room temperature (16 hr). In vitro maturation was carried out in a basal medium supplemented or not with 50 ng/ml of epidermal growth factor (EGF). Our data showed that addition of EGF to the maturation medium increases the percentage of G1 oocytes reaching metaphase II (3.9% vs. 50%, basal vs. EGF; p < .001) and decreased their degeneration rate (69.9% vs. 22.2%, basal vs. EGF; p < .01) when oocytes were immediately matured. Overnight holding increased the meiotic competence of G1 oocytes (37.5% matured in basal medium) and EGF increased prophase arrest in G2 + G3 oocytes (16.1% vs. 38.8% in germinal vesicle [GV] stage in basal medium vs. EGF added medium; p < .05). Our data demonstrate that oocyte holding can be used in Iberian red deer oocytes. Interestingly, EGF addition increases the oocytes' meiotic competence in immediately matured oocytes but not after oocyte holding depending upon initial oocyte quality. © 2017 Blackwell Verlag GmbH.

Optimizing the conditions for in vitro maturation and artificial activation of sika deer (Cervus nippon hortulorum) oocytes.

PubMed

Yin, Y; Tang, L; Zhang, P; Kong, D; Wang, Z; Guan, J; Song, G; Tang, B; Li, Z

2013-02-01

With the goal of establishing experimental protocols for cloning sika deer, various conditions for in vitro maturation (IVM) and artificial activation of sika deer oocytes were examined. In vitro maturation was evaluated in seven different culture media. The highest rate of oocyte maturation was 75.4% in 10 μg/ml follicle-stimulating hormone (FSH), 1 μg/ml LH, 0.2 mm cysteamine and 50 ng/ml epidermal growth factor (EGF) after 24 h of IVM. The efficiency after 24 h of IVM did not differ significantly (p > 0.05) from that observed after 20 h. Cysteamine (0.2 mm) significantly increased the maturation rates after 20 h (from 59.1% to 67.2%, p < 0.05) and after 24 h (from 63.2% to 71.6%, p < 0.05) of IVM. The IVM rates of oocytes collected during the oestrous season (75.4%) and the anoestrous season (23.3%) were significantly different at 24 h. The 20 μg/ml FSH, 2 μg/ml LH, 0.4 mm cysteamine and 100 ng/ml EGF significantly increased the maturation rates (from 23.3% to 54.2%, p < 0.01) at 24 h during the anoestrous season. For the activation experiments, the most effective method was chemical activation [ionomycin + 6-dimethylaminopurine (6-DMAP)], which promoted the development of sika deer oocytes to the blastocyst stage (32.4%). Our results indicate that in vitro matured sika deer oocytes are good candidates for parthenogenetic activation and that chemical treatment is needed for relatively efficient activation of the oocytes. These optimized conditions for IVM and parthenogenetic activation may be useful for efforts to restore populations of the endangered sika deer using the somatic cell nuclear transfer technique. © 2012 Blackwell Verlag GmbH.

Aptamers and methods for their in vitro selection and uses thereof

DOEpatents

Doyle, Sharon A [Walnut Creek, CA; Murphy, Michael B [Severna Park, MD

2008-02-12

The present method is an improved in vitro selection protocol that relies on magnetic separations for DNA aptamer production that is relatively easy and scalable without the need for expensive robotics. The ability of aptamers selected by this method to recognize and bind their target protein with high affinity and specificity, and detail their uses in a number of assays is also described. Specific TTF1 and His6 aptamers were selected using the method described, and shown to be useful for enzyme-linked assays, Western blots, and affinity purification.

Aptamers and methods for their in vitro selection and uses thereof

DOEpatents

Doyle, Sharon A [Walnut Creek, CA; Murphy, Michael B [Severna Park, MD

2012-01-31

The present method is an improved in vitro selection protocol that relies on magnetic separations for DNA aptamer production that is relatively easy and scalable without the need for expensive robotics. The ability of aptamers selected by this method to recognize and bind their target protein with high affinity and specificity, and detail their uses in a number of assays is also described. Specific TTF1 and His6 aptamers were selected using the method described, and shown to be useful for enzyme-linked assays, Western blots, and affinity purification.

In vitro and in vivo evaluation of new radiolabeled neurotensin(8-13) analogues with high affinity for NT1 receptors.

PubMed

García-Garayoa, E; Allemann-Tannahill, L; Bläuenstein, P; Willmann, M; Carrel-Rémy, N; Tourwé, D; Iterbeke, K; Conrath, P; Schubiger, P A

2001-01-01

The potential utility of neurotensin (NT) in cancer diagnosis and therapy is limited by its rapid degradation. New stabilized analogues were synthesized, labeled with [99mTc] and screened in vitro and in vivo. High affinity and rapid internalization were obtained in binding assays. Despite their longer human plasma half-lives, a rapid degradation was observed with low concentrations as used in biodistribution tests. The tumor uptake rates were rather low but tumor/blood ratios increased according to the stability raise.

An in vitro Comparison of Microdialysis Relative Recovery of Met- and Leu-Enkephalin Using Cyclodextrins and Antibodies as Affinity Agents

PubMed Central

Fletcher, Heidi J.; Stenken, Julie A.

2008-01-01

Cyclodextrins and antibodies have been used as affinity agents to improve relative recovery during microdialysis sampling. Two neuropeptides, methionine-enkephalin (ME) and leucine-enkephalin (LE), were chosen to compare the use of cyclodextrins and antibodies as possible affinity agents for improving their relative recovery across polycarbonate and polyethersulfone membranes during in vitro sampling. Cyclodextrins (CD) including β-CD, 2-hydroxypropyl-β-cyclodextrin (2HPβ-CD), and γ-CD gave improvements of relative recovery for both peptides of less than 2-fold as compared to controls. Comparisons of relative recovery between tyrosine-glycine-glycine, tyrosine, and phenylalanine using different cyclodextrins in the perfusion fluid were also obtained. Inclusion of an antibody against met-enkephalin in the microdialysis perfusion fluid resulted in relative recovery increases of up to 2.5-fold. These results show that using antibodies as affinity agents during microdialysis sampling may be more effective agents to improve the relative recovery of these opioid neuropeptides. PMID:18558138

Liver condition of Holstein cows affects mitochondrial function and fertilization ability of oocytes

PubMed Central

TANAKA, Hiroshi; TAKEO, Shun; ABE, Takahito; KIN, Airi; SHIRASUNA, Koumei; KUWAYAMA, Takehito; IWATA, Hisataka

2016-01-01

The aim of the present study was to examine the fertilization ability and mitochondrial function of oocytes derived from cows with or without liver damage. Oocytes were collected from the ovaries of cows with damaged livers (DL) and those of cows with healthy livers (HL), subjected to in vitro maturation, and fertilized in vitro. A significantly high abnormal fertilization rate was observed for oocytes from DL cows compared to oocytes from HL cows. The time to dissolve the zona pellucida by protease before fertilization was similar between the two liver conditions, whereas after fertilization treatment this time was shorter for DL cows than for HL cows. The percentage of oocytes with equivalent cortical granule distributions underneath the membrane was greater for in vitro matured oocytes from HL cows, whereas an immature distribution pattern was observed for oocytes from DL cows. In addition, a greater percentage of oocytes derived from HL cows released cortical granules following fertilization compared with oocytes from DL cows. Mitochondrial function determined by ATP content and membrane potential were similar at the germinal vesicle stage, but post-in vitro maturation, the oocytes derived from HL cows showed higher values than DL cows. The mitochondrial DNA copy number in oocytes was similar between the two liver conditions for both the germinal vesicle and post-in vitro maturation oocytes. In conclusion, liver damage induces low fertilization, likely because of incomplete cortical granule distribution and release, and the maturation of oocytes from DL cows contain low-functioning mitochondria compared to their HL counterparts. PMID:26832309

Liver condition of Holstein cows affects mitochondrial function and fertilization ability of oocytes.

PubMed

Tanaka, Hiroshi; Takeo, Shun; Abe, Takahito; Kin, Airi; Shirasuna, Koumei; Kuwayama, Takehito; Iwata, Hisataka

2016-06-17

The aim of the present study was to examine the fertilization ability and mitochondrial function of oocytes derived from cows with or without liver damage. Oocytes were collected from the ovaries of cows with damaged livers (DL) and those of cows with healthy livers (HL), subjected to in vitro maturation, and fertilized in vitro. A significantly high abnormal fertilization rate was observed for oocytes from DL cows compared to oocytes from HL cows. The time to dissolve the zona pellucida by protease before fertilization was similar between the two liver conditions, whereas after fertilization treatment this time was shorter for DL cows than for HL cows. The percentage of oocytes with equivalent cortical granule distributions underneath the membrane was greater for in vitro matured oocytes from HL cows, whereas an immature distribution pattern was observed for oocytes from DL cows. In addition, a greater percentage of oocytes derived from HL cows released cortical granules following fertilization compared with oocytes from DL cows. Mitochondrial function determined by ATP content and membrane potential were similar at the germinal vesicle stage, but post-in vitro maturation, the oocytes derived from HL cows showed higher values than DL cows. The mitochondrial DNA copy number in oocytes was similar between the two liver conditions for both the germinal vesicle and post-in vitro maturation oocytes. In conclusion, liver damage induces low fertilization, likely because of incomplete cortical granule distribution and release, and the maturation of oocytes from DL cows contain low-functioning mitochondria compared to their HL counterparts.

The presence of centrioles and centrosomes in ovarian mature cystic teratoma cells suggests human parthenotes developed in vitro can differentiate into mature cells without a sperm centriole

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

Lee, Bo Yon, E-mail: boyonlee@gmail.com; Shim, Sang Woo; Kim, Young Sun

Highlights: Black-Right-Pointing-Pointer The sperm centriole is the progenitor of centrosomes in all somatic cells. Black-Right-Pointing-Pointer Centrioles and centrosomes exist in parthenogenetic ovarian teratoma cells. Black-Right-Pointing-Pointer Without a sperm centriole, parthenogenetic oocytes produce centrioles and centrosomes. Black-Right-Pointing-Pointer Parthenogenetic human oocytes can develop and differentiate into mature cells. -- Abstract: In most animals, somatic cell centrosomes are inherited from the centriole of the fertilizing spermatozoa. The oocyte centriole degenerates during oogenesis, and completely disappears in metaphase II. Therefore, the embryos generated by in vitro parthenogenesis are supposed to develop without any centrioles. Exceptional acentriolar and/or acentrosomal developments are possible in mice andmore » in some experimental cells; however, in most animals, the full developmental potential of parthenogenetic cells in vitro and the fate of their centrioles/centrosomes are not clearly understood. To predict the future of in vitro human parthenogenesis, we explored the centrioles/centrosomes in ovarian mature cystic teratoma cells by immunofluorescent staining and transmission electron microscopy. We confirmed the presence of centrioles and centrosomes in these well-known parthenogenetic ovarian tumor cells. Our findings clearly demonstrate that, even without a sperm centriole, parthenotes that develop from activated oocytes can produce their own centrioles/centrosomes, and can even develop into the well-differentiated mature tissue.« less

Plant regeneration from in vitro leaves of mature black cherry (Prunus serotina)

Treesearch

Xiaomei Liu; Paula M. Pijut

2008-01-01

A regeneration system was developed for Prunus serotina from a juvenile (F) and two mature genotypes (#3 and #4). Adventitious shoots regenerated from leaves of in vitro cultures on woody plant medium with thidiazuron (TDZ) and naphthaleneacetic acid (NAA). The best regeneration for genotype F (91.4%) was observed on medium with 9.08 µM TDZ...

Effect of open pulled straw (OPS) vitrification on the fertilisation rate and developmental competence of porcine oocytes.

PubMed

Varga, Erika; Gardón, J C; Papp, Agnes Bali

2006-03-01

Freezing technologies are very important to preserve gametes and embryos of animals with a good pedigree or those having high genetic value. The aim of this work was to compare immature and in vitro matured porcine oocytes regarding their morphology and ability to be fertilised after vitrification by the open pulled straw (OPS) method. In four experiments 830 oocytes were examined. To investigate the effect of cumulus cells on oocyte survival after OPS vitrification, both denuded and cumulus-enclosed oocytes were vitrified at the germinal vesicle (GV) stage, then after vitrification they were matured in vitro. Besides, in vitro matured oocytes surrounded with a cumulus and those without a cumulus were also vitrified. The survival of oocytes was evaluated by their morphology. After in vitro fertilisation the rates of oocytes penetrated by spermatozoa were compared. Our results suggest that the vitrification/warming procedure is the most effective in cumulus-enclosed oocytes (22.35 +/- 1.75%). There was no difference between the order of maturation and vitrification in cumulus-enclosed oocytes, which suggests the importance of cumulus cells in protecting the viability of oocytes during cryopreservation.

Impact of delipidated estrous sheep serum supplementation on in vitro maturation, cryotolerance and endoplasmic reticulum stress gene expression of sheep oocytes

PubMed Central

dos Santos Neto, Pedro C.; Cuadro, Federico; Bosolasco, Diego; Mulet, Ana P.; Crispo, Martina

2018-01-01

High lipid content of oocytes and embryos in domestic animals is one of the well-known factors associated with poor cryosurvival. Herein, we wanted to determine whether the use of delipidated estrous sheep serum during in vitro maturation (IVM) of ovine oocytes reduces the cytoplasmic lipid droplets content and improves embryo development and cryotolerance after vitrification. Cumulus oocytes complexes (COCs) were matured in vitro for 24 h in medium supplemented with whole or delipidated estrous sheep serum prior to vitrification. Neutral lipid present in lipid droplets of COCs, cleavage rate, embryo development rate on Day 6 and Day 8, and hatching rate on Day 8, were compared among experimental groups. Endoplasmic reticulum stress genes were evaluated in in vitro matured COCs under different lipid conditions prior to vitrification. The lipid droplets’ content (mean fluorescence intensity) of oocytes cultured with IVM media supplemented with delipidated serum was lower than COCs matured with whole serum (7.6 ± 1.7 vs. 22.8 ± 5.0 arbitrary units, respectively; P< 0.05). Despite IVM treatment, oocytes subjected to vitrification showed impaired competence compared with the non-vitrified groups (P<0.05). No significant differences in embryo production were observed in non-vitrified COCs after maturation in delipidated or whole serum (33.4±4.9 vs 31.9 ±4.2). COCs matured in delipidated serum and subjected to vitrification showed increased expression of ATF4, ATF6, GRP78, and CHOP10 genes (ER stress markers). Collectively, our results demonstrate that although supplementation of IVM medium with delipidated estrous sheep serum reduces the presence of cytoplasmic lipid droplets in oocytes after maturation, oocyte cryotolerance is not improved. Notably, the expression of genes associated with the unfolded protein response (UPR) was increased in COCs, with fewer lipid droplets subjected to vitrification, suggesting that oocyte cryopreservation is associated with ER stress and activation of adaptive responses. PMID:29912910

Human induced pluripotent stem cells can reach complete terminal maturation: in vivo and in vitro evidence in the erythropoietic differentiation model

PubMed Central

Kobari, Ladan; Yates, Frank; Oudrhiri, Noufissa; Francina, Alain; Kiger, Laurent; Mazurier, Christelle; Rouzbeh, Shaghayegh; El-Nemer, Wassim; Hebert, Nicolas; Giarratana, Marie-Catherine; François, Sabine; Chapel, Alain; Lapillonne, Hélène; Luton, Dominique; Bennaceur-Griscelli, Annelise; Douay, Luc

2012-01-01

Background Human induced pluripotent stem cells offer perspectives for cell therapy and research models for diseases. We applied this approach to the normal and pathological erythroid differentiation model by establishing induced pluripotent stem cells from normal and homozygous sickle cell disease donors. Design and Methods We addressed the question as to whether these cells can reach complete erythroid terminal maturation notably with a complete switch from fetal to adult hemoglobin. Sickle cell disease induced pluripotent stem cells were differentiated in vitro into red blood cells and characterized for their terminal maturation in terms of hemoglobin content, oxygen transport capacity, deformability, sickling and adherence. Nucleated erythroblast populations generated from normal and pathological induced pluripotent stem cells were then injected into non-obese diabetic severe combined immunodeficiency mice to follow the in vivo hemoglobin maturation. Results We observed that in vitro erythroid differentiation results in predominance of fetal hemoglobin which rescues the functionality of red blood cells in the pathological model of sickle cell disease. We observed, in vivo, the switch from fetal to adult hemoglobin after infusion of nucleated erythroid precursors derived from either normal or pathological induced pluripotent stem cells into mice. Conclusions These results demonstrate that human induced pluripotent stem cells: i) can achieve complete terminal erythroid maturation, in vitro in terms of nucleus expulsion and in vivo in terms of hemoglobin maturation; and ii) open the way to generation of functionally corrected red blood cells from sickle cell disease induced pluripotent stem cells, without any genetic modification or drug treatment. PMID:22733021

Identification and Characterization of BMS-955176, a Second-Generation HIV-1 Maturation Inhibitor with Improved Potency, Antiviral Spectrum, and Gag Polymorphic Coverage.

PubMed

Nowicka-Sans, Beata; Protack, Tricia; Lin, Zeyu; Li, Zhufang; Zhang, Sharon; Sun, Yongnian; Samanta, Himadri; Terry, Brian; Liu, Zheng; Chen, Yan; Sin, Ny; Sit, Sing-Yuen; Swidorski, Jacob J; Chen, Jie; Venables, Brian L; Healy, Matthew; Meanwell, Nicholas A; Cockett, Mark; Hanumegowda, Umesh; Regueiro-Ren, Alicia; Krystal, Mark; Dicker, Ira B

2016-07-01

BMS-955176 is a second-generation human immunodeficiency virus type 1 (HIV-1) maturation inhibitor (MI). A first-generation MI, bevirimat, showed clinical efficacy in early-phase studies, but ∼50% of subjects had viruses with reduced susceptibility associated with naturally occurring polymorphisms in Gag near the site of MI action. MI potency was optimized using a panel of engineered reporter viruses containing site-directed polymorphic changes in Gag that reduce susceptibility to bevirimat (including V362I, V370A/M/Δ, and T371A/Δ), leading incrementally to the identification of BMS-955176. BMS-955176 exhibits potent activity (50% effective concentration [EC50], 3.9 ± 3.4 nM [mean ± standard deviation]) toward a library (n = 87) of gag/pr recombinant viruses representing 96.5% of subtype B polymorphic Gag diversity near the CA/SP1 cleavage site. BMS-955176 exhibited a median EC50 of 21 nM toward a library of subtype B clinical isolates assayed in peripheral blood mononuclear cells (PBMCs). Potent activity was maintained against a panel of reverse transcriptase, protease, and integrase inhibitor-resistant viruses, with EC50s similar to those for the wild-type virus. A 5.4-fold reduction in EC50 occurred in the presence of 40% human serum plus 27 mg/ml of human serum albumin (HSA), which corresponded well to an in vitro measurement of 86% human serum binding. Time-of-addition and pseudotype reporter virus studies confirm a mechanism of action for the compound that occurs late in the virus replication cycle. BMS-955176 inhibits HIV-1 protease cleavage at the CA/SP1 junction within Gag in virus-like particles (VLPs) and in HIV-1-infected cells, and it binds reversibly and with high affinity to assembled Gag in purified HIV-1 VLPs. Finally, in vitro combination studies showed no antagonistic interactions with representative antiretrovirals (ARVs) of other mechanistic classes. In conclusion, BMS-955176 is a second-generation MI with potent in vitro anti-HIV-1 activity and a greatly improved preclinical profile compared to that of bevirimat. Copyright © 2016 Nowicka-Sans et al.

Cortical granule exocytosis in Bufo arenarum oocytes matured in vitro.

PubMed

Oterino, J; Sanchez Toranzo, G; Zelarayán, L; Valz-Gianinet, J N; Bühler, M I

2001-08-01

Denuded Bufo arenarum oocytes matured in vitro by progesterone treatment exhibited abnormal segmentation due to the penetration of more than one sperm. These oocytes were able to respond to activation stimuli and exhibited the external signs characteristic of activation. However, the prevention of polyspermy was not effective in these oocytes, which exhibited numerous sperm in their cytoplasm. The aim of this work was to analyse the cortical reaction in polyspermic Bufo arenarum oocytes matured in vitro. The result indicate that the cortical reaction of these oocytes seems to occur with a chronological sequence similar to that described for ovoposited oocytes of this species. In addition, when, 1 min after pricking, cortical granule exocytosis occurred, the oocytes became refractory to sperm entry, suggesting that they are able to establish a slow block to polyspermy.

Monoamine transporter and receptor interaction profiles in vitro predict reported human doses of novel psychoactive stimulants and psychedelics.

PubMed

Luethi, Dino; Liechti, Matthias E

2018-05-29

Pharmacological profiles of new psychoactive substances (NPSs) can be established rapidly in vitro and provide information on potential psychoactive effects in humans. The present study investigated whether specific in vitro monoamine transporter and receptor interactions can predict effective psychoactive doses in humans. We correlated previously assessed in vitro data of stimulants and psychedelics with human doses that are reported on the Internet and in books. For stimulants, dopamine and norepinephrine transporter inhibition potency was positively correlated with human doses, whereas serotonin transporter inhibition potency was inversely correlated with human doses. Serotonin 5-hydroxytryptamine-2A (5-HT2A) and 5-HT2C receptor affinity was significantly correlated with psychedelic doses, but 5-HT1A receptor affinity and 5-HT2A and 5-HT2B receptor activation potency were not. The rapid assessment of in vitro pharmacological profiles of NPSs can help to predict psychoactive doses and effects in humans and facilitate the appropriate scheduling of NPSs.

In-vitro maturation of round spermatids using co-culture on Vero cells.

PubMed

Cremades, N; Bernabeu, R; Barros, A; Sousa, M

1999-05-01

In an attempt to determine whether co-culture could promote sperm maturation, three patients with non-obstructive azoospermia, two with maturation arrest at the level of primary spermatocytes and one patient with <1% tubules showing complete spermatogenesis, and one patient with total globozoospermia, gave consent to experimentally co-culture round spermatids retrieved from the testicle on Vero cell monolayers. In all azoospermic patients elongating spermatids could be obtained from round spermatids. In one case of maturation arrest, of 37 round spermatids co-cultured for up to 5 days, 30% developed flagella, 46% matured to elongating and 19% to elongated spermatids, with one mature spermatozoon also obtained (3%). In the same patient, primary cultures of three round spermatids with flagella enabled development of one further mature spermatozoon. In the case with total globozoospermia, of six round spermatids co-cultured for up to 5 days, one mature spermatozoon was obtained, with a flagellum and normal head morphology. These preliminary findings suggest that it may be possible to overcome the round spermatid block, and even the triggering of morphological abnormalities arising at the spermiogenic level, by in-vitro maturation under special environmental conditions.

Influence of co-culture with denuded oocytes during in vitro maturation on fertilization and developmental competence of cumulus-enclosed porcine oocytes in a defined system.

PubMed

Appeltant, Ruth; Somfai, Tamás; Kikuchi, Kazuhiro; Maes, Dominiek; Van Soom, Ann

2016-04-01

Co-culture of cumulus-oocyte complexes (COCs) with denuded oocytes (DOs) during in vitro maturation (IVM) was reported to improve the developmental competence of oocytes via oocyte-secreted factors in cattle. The aim of the present study was to investigate if addition of DOs during IVM can improve in vitro fertilization (IVF) and in vitro culture (IVC) results for oocytes in a defined in vitro production system in pigs. The maturation medium was porcine oocyte medium supplemented with gonadotropins, dbcAMP and β-mercaptoethanol. Cumulus-oocyte complexes were matured without DOs or with DOs in different ratios (9 COC, 9 COC+16 DO and 9 COC+36 DO). Consequently; oocytes were subjected to IVF as intact COCs or after denudation to examine if DO addition during IVM would affect cumulus or oocyte properties. After fertilization, penetration and normal fertilization rates of zygotes were not different between all tested groups irrespective of denudation before IVF. When zygotes were cultured for 6 days, no difference could be observed between all treatment groups in cleavage rate, blastocyst rate and cell number per blastocyst. In conclusion, irrespective of the ratio, co-culture with DOs during IVM did not improve fertilization parameters and embryo development of cumulus-enclosed porcine oocytes in a defined system. © 2015 Japanese Society of Animal Science.

The maturation and germination of Phytophthora ramorum Chlamydospores

Treesearch

Aaron L. Smith; Everett M. Hansen

2008-01-01

Chlamydospores are a distinctive feature of Phytophthora ramorum. They are formed quickly in agar, and within colonized leaves. We followed their development and maturation in vitro and in vivo, and studied conditions affecting their germination. Cell walls of mature P. ramorum chlamydospores...

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.

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

Development and In Vitro Characterization of a Gemcitabine-loaded MUC4-targeted Immunoliposome Against Pancreatic Ductal Adenocarcinoma.

PubMed

Urey, Carlos; Hilmersson, Katarzyma Said; Andersson, Bodil; Ansari, Daniel; Andersson, Roland

2017-11-01

Pancreatic Ductal adeno-carcinoma (PDAC) is a devastating disease. Gemcitabine is the standard chemotherapeutic agent against PDAC but has only limited effectiveness. The aim of the study was to develop and study the targeting affinity and in vitro antiproliferative effect of a MUC4-targeted gemcitabine-loaded immuno-liposome for treatment of PDAC. Gemcitabine-loaded immunoliposomes were developed by grafting anti-MUC4 antibodies to the liposomal surface. Targeting affinity was compared in vitro between immunoliposomes and non-targeted liposomes and anti-proliferative effect was compared in vitro between free drug, non-targeted liposomal gemcitabine and MUC4-targeted immunoliposomal gemcitabine on a MUC4-positive pancreatic cancer cell line, Capan-1. Development of a MUC4-targeted immunoliposome was confirmed and characterized by immunoblots and size characterization. The MUC4-targeted immunoliposome showed a significantly higher targeting affinity compared to the non-targeted liposomes and also showed an improved antiproliferative effect compared to free and non-targeted liposomal drug. Successful development and characterization of a MUC4-targeted immunoliposome shows promising results for a targeted treatment and improved retention of gemcitabine for treatment of PDAC. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Analysis of the actions of the novel dopamine receptor-directed compounds (S)-OSU6162 and ACR16 at the D2 dopamine receptor

PubMed Central

Kara, Elodie; Lin, Hong; Svensson, Kjell; Johansson, Anette M; Strange, Philip G

2010-01-01

BACKGROUND AND PURPOSE The two phenylpiperidines, OSU6162 and ACR16, have been proposed as novel drugs for the treatment of brain disorders, including schizophrenia and Huntington's disease, because of their putative dopamine stabilizing effects. Here we evaluated the activities of these compounds in a range of assays for the D2 dopamine receptor in vitro. EXPERIMENTAL APPROACH The affinities of these compounds for the D2 dopamine receptor were evaluated in competition with [3H]spiperone and [3H]NPA. Agonist activity of these compounds was evaluated in terms of their ability to stimulate [35S]GTPγS binding. KEY RESULTS Both compounds had low affinities for inhibition of [3H]spiperone binding (pKi vs. [3H]spiperone, ACR16: <5, OSU6162: 5.36). Neither compound was able to stimulate [35S]GTPγS binding when assayed in the presence of Na+ ions, but if the Na+ ions were removed, both compounds were low-affinity, partial agonists (Emax relative to dopamine: ACR16: 10.2%, OSU6162:54.3%). Schild analysis of the effects of OSU6162 to inhibit dopamine-stimulated [35S]GTPγS binding indicated Schild slopes of ∼0.9, suggesting little deviation from competitive inhibition. OSU6162 was, however, able to accelerate [3H]NPA dissociation from D2 dopamine receptors, indicating some allosteric effects of this compound. CONCLUSIONS AND IMPLICATIONS The two phenylpiperidines were low-affinity, low-efficacy partial agonists at the D2 dopamine receptor in vitro, possibly exhibiting some allosteric effects. Comparing their in vitro and in vivo effects, the in vitro affinities were a reasonable guide to potencies in vivo. However, the lack of in vitro–in vivo correlation for agonist efficacy needs to be further addressed. PMID:20804495

Heat stress and antioxidant enzyme activity in bubaline ( Bubalus bubalis) oocytes during in vitro maturation

NASA Astrophysics Data System (ADS)

Waiz, Syma Ashraf; Raies-ul-Haq, Mohammad; Dhanda, Suman; Kumar, Anil; Goud, T. Sridhar; Chauhan, M. S.; Upadhyay, R. C.

2016-09-01

In vitro environments like heat stress usually increase the production of reactive oxygen species in bubaline oocytes which have been implicated as one of the major causes for reduced developmental competence. Oocytes during meiotic maturation are sensitive to oxidative stress, and heat stress accelerates cellular metabolism, resulting in the higher production of free radicals. Therefore, the aim of present work was to assess the impact of heat stress during meiotic maturation on bubaline cumulus-oocyte complexes (COC), denuded oocytes (DO), and cumulus cell mass in terms of their oxidative status. Accordingly, for control group, COC were matured at 38.5 °C for complete 24 h of meiotic maturation and heat stress of 40.5 and 41.5 °C was applied to COC during the first 12 h of maturation and then moved to 38.5 °C for rest of the 12 h. In another group, COC after maturation were denuded from the surrounding cumulus cells by manual pipetting. Results indicated that the production of reactive oxygen species (ROS), lipid peroxides, and nitric oxide (NO) was significantly ( P < 0.05) higher in the oocytes subjected to heat stress (40.5 and 41.5 °C) during meiotic maturation compared to the oocytes matured under standard in vitro culture conditions (38.5 °C). Also, the antioxidant enzymatic activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase were significantly ( P < 0.05) increased in all the treatment groups compared to the control group. Therefore, the present study clearly establishes that heat stress ensues oxidative stress in bubaline oocytes which triggers the induction of antioxidant enzymatic defense system for scavenging the ROS.

Pronuclear synchronization and nuclear morphology of mature and in vitro matured oocytes in the rat: an ultrastructural study.

PubMed

Cincik, M; Baykal, B; Zeteroglu, S; Onalan, G; Ceyhan, S T; Ergur, R

2005-01-01

The objective of this study was to evaluate synchronous and asynchronous pronucleus (PN) formation and the related patterns of juxtapositional nucleolus (n) formation in immature (prophase I [PI] and metaphase I [MI]) and mature (metaphase II [MII]) oocytes after fertilization, both ultrastructurally and at the level of light microscope. A single dose of 15 IU gonadotrophin was injected subcutaneously to twenty four 26-wk-old, female Wistar rats to induce ovulation. Human chorionic gonadotrophin (4 IU) was administered 40 h later, and after 4-6 h the ovaries were dissected, and the oocytes were aspirated. A total of 214 rat oocytes were classified according to a maturation index as follows: group I, 80 PI oocytes; group II, 50 MI oocytes; and group III, 84 MII oocytes. Immature oocytes were in vitro matured for 18-36 h. Spermatozoa were acquired by microepididymal sperm aspiration and processed using swim-up technique. Intracytoplasmic sperm injection was performed on mature oocytes after 2 h of incubation and on in vitro matured (IVM) oocytes 4 h after maturation. Pronuclear synchronization [both pronucleases (PNs) centrally located, equal sized, with equal numbers and sizes of juxtapositional nucleoli (Nn)] was observed in fertilized oocytes. Asynchronous PN formation (diversity between male and female PNs, related to dimensions, localization, and the number of Nn) in groups I, II, and III was found in 75, 86, and 47% of preembryos, respectively. There was a significant difference of synchronous pronuclear formation between mature and IVM oocytes (P < 0.05). In IVM oocytes, asynchronous PN formation is high, and juxtapositional pronucleolar patterns are observed to be low by transmission electron microscope (TEM).

Clinical benefit of metaphase I oocytes

PubMed Central

Vanhoutte, Leen; De Sutter, Petra; Van der Elst, Josiane; Dhont, Marc

2005-01-01

Background We studied the benefit of using in vitro matured metaphase I (MI) oocytes for ICSI in patients with a maximum of 6 mature metaphase II (MII) oocytes at retrieval. Methods In 2004, 187 ICSI cycles were selected in which maximum 6 MII oocytes and at least one MI oocyte were retrieved. MI oocytes were put in culture to mature until the moment of ICSI, which was performed between 2 to 11 hours after oocyte retrieval (day 0). In exceptional cases, when the patient did not have any mature oocyte at the scheduled time of ICSI, MI oocytes were left to mature overnight and were injected between 19 to 26 hours after retrieval (day 1). Embryos from MI oocytes were chosen for transfer only when no other good quality embryos from MII oocytes were available. Outcome parameters were time period of in vitro maturation (IVM), IVM and fertilization rates, embryo development, clinical pregnancy rates, implantation rates and total MI oocyte utilization rate. Results The overall IVM rate was 43%. IVM oocytes had lower fertilization rates compared to in vivo matured sibling oocytes (52% versus 68%, P < 0.05). The proportion of poor quality embryos was significantly higher in IVM derived oocytes. One pregnancy and live birth was obtained out of 13 transfers of embryos exclusively derived from IVM oocytes. This baby originated from an oocyte that was injected after 22 hrs of IVM. Conclusion Fertilization of in vitro matured MI oocytes can result in normal embryos and pregnancy, making IVM worthwhile, particularly when few MII oocytes are obtained at retrieval. PMID:16356175

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.

Evaluation of zona pellucida birefringence intensity during in vitro maturation of oocytes from stimulated cycles.

PubMed

Petersen, Claudia G; Vagnini, Laura D; Mauri, Ana L; Massaro, Fabiana C; Silva, Liliane F I; Cavagna, Mario; Baruffi, Ricardo L R; Oliveira, Joao B A; Franco, José G

2011-04-23

This study evaluated whether there is a relationship between the zona pellucida birefringence (ZP-BF) intensity and the nuclear (NM) and cytoplasmic (CM) in vitro maturation of human oocytes from stimulated cycles. The ZP-BF was evaluated under an inverted microscope with a polarizing optical system and was scored as high/positive (when the ZP image presented a uniform and intense birefringence) or low/negative (when the image presented moderate and heterogeneous birefringence). CM was analyzed by evaluating the distribution of cortical granules (CGs) throughout the ooplasm by immunofluorescence staining. CM was classified as: complete, when CG was localized in the periphery; incomplete, when oocytes presented a cluster of CGs in the center; or in transition, when oocytes had both in clusters throughout cytoplasm and distributed in a layer in the cytoplasm periphery Nuclear maturation: From a total of 83 germinal vesicle (GV) stage oocytes, 58 of oocytes (69.9%) reached NM at the metaphase II stage. From these 58 oocytes matured in vitro, the high/positively scoring ZP-BF was presented in 82.7% of oocytes at the GV stage, in 75.8% of oocytes when at the metaphase I, and in 82.7% when oocytes reached MII. No relationship was observed between NM and ZP-BF positive/negative scores (P = 0.55). These variables had a low Pearson's correlation coefficient (r = 0.081). Cytoplasmic maturation: A total of 85 in vitro-matured MII oocytes were fixed for CM evaluation. Forty-nine oocytes of them (57.6%) showed the complete CM, 30 (61.2%) presented a high/positively scoring ZP-BF and 19 (38.8%) had a low/negatively scoring ZP-BF. From 36 oocytes (42.3%) with incomplete CM, 18 (50%) presented a high/positively scoring ZPBF and 18 (50%) had a low/negatively scoring ZP-BF. No relationship was observed between CM and ZP-BF positive/negative scores (P = 0.42). These variables had a low Pearson's correlation coefficient (r = 0.11). The current study demonstrated an absence of relationship between ZP-BF high/positive or low/negative score and nuclear and cytoplasmic in vitro maturation of oocytes from stimulation cycles.

Effects of Vitrification on Outcomes of In Vivo-Mature, In Vitro-Mature and Immature Human Oocytes.

PubMed

Song, Wen-Yan; Peng, Zhao-Feng; Chen, Xue-Mei; Jin, Hai-Xia; Yao, Gui-Dong; Shi, Sen-Lin; Yang, Hong-Yi; Zhang, Xiang-Yang; Sun, Ying-Pu

2016-01-01

To observe the effects of vitrification on spindle, zona pellucida, embryonic aneuploidy and DNA injury in in vivo-maruted, in vitro-mature and immature human oocytes. Between January 2009 and February 2015, 223 immature oocytes from 450 infertile patients, and 31 in vivo-mature oocytes from 3 infertile couples were collected. Of the 223 immature oocytes, 113 were used for in vitro culture before vitrification. Some oocytes were randomly divided into in vivo-mature group (group A, n = 15), in vitro-mature group (group B, n = 88) and immature group (group C, n = 85), and then the oocytes with spindle in these three groups after freezing-thawing were selected to use for Polscope imaging, embryonic aneuploidy screening and embryo development evaluation. Other oocytes were randomly divided into group A (n = 16), group B (n = 25) and group C (n = 25) for detecting DNA injury. After thawing, spindle occurrence rate, spindle Retardance value, and cleavage rate were significantly higher in groups A and B than in group C (all P < 0.05), but there were no statistical differences in fertility rate, high-quality embryo rate, blastulation rate and aneuploidy rate amongst the three groups (all P > 0.05). Zona pellucida density (ZPD) was significantly lower in group A than in groups B and C both before and after vitrification (all P < 0.05). ZPD was significantly higher after thawing than before vitrification (all P < 0.05), but zona pellucida thickness (ZPT) was not significantly changed in all the three groups (all P > 0.05). Rate of comet cells was significantly lower in group A than in groups B and C (all P < 0.01). Comet tail was significantly longer in group C than in groups B and A (all P < 0.05). In vivo- and in vitro-mature human oocytes are more suitable to vitrification than immature human oocytes. Spindle Retardance value has more predictive value for embryonic development potential than ZPD and ZPT. © 2016 The Author(s) Published by S. Karger AG, Basel.

The effects of platelet lysate on maturation, fertilization and embryo development of NMRI mouse oocytes at germinal vesicle stage.

PubMed

Pazoki, Hassan; Eimani, Hussein; Farokhi, Farah; Shahverdi, Abdol-Hossein; Tahaei, Leila Sadat

2016-04-01

Improving in vitro maturation could increase the rate of pregnancy from oocytes matured in vitro. Consequently, patients will be prevented from using gonadotropin with its related side effects. In this study, the maturation medium was enriched by platelet lysate (PL), then maturation and subsequent developments were monitored. Oocytes at germinal vesicle stage with cumulus cells (cumulus-oocyte complex) and without cumulus cells (denuded oocytes) were obtained from mature female mice. The maturation medium was enriched by 5 and 10 % PL and 5 % PL + 5 % fetal bovine serum (FBS) as experimental groups; the control groups' media consisted of 5 and 10 % FBS. After 18 h, the matured oocytes were collected and, after fertilization, subsequent development was monitored. The rates of maturation, fertilization and 2-cell embryo development for the denuded oocyte groups in experimental media 5 % PL and 5 % PL + 5 % FBS were significantly higher than those of the control groups ( P < 0.05), while the results for the cumulus-oocyte complex groups were similar between the experimental groups and control groups. The results of this study indicated that platelet lysate could improve the maturation rate in the absence of granulosa cells compared to media with FBS. This extract also had positive effects on fertilization and embryo development.

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.

A fully human chimeric antigen receptor with potent activity against cancer cells but reduced risk for off-tumor toxicity

PubMed Central

Song, De-Gang; Ye, Qunrui; Poussin, Mathilde; Liu, Lin; Figini, Mariangela; Powell, Daniel J.

2015-01-01

Chimeric antigen receptors (CARs) can redirect T cells against antigen-expressing tumors in an HLA-independent manner. To date, various CARs have been constructed using mouse single chain antibody variable fragments (scFvs) of high affinity that are immunogenic in humans and have the potential to mediate “on-target” toxicity. Here, we developed and evaluated a fully human CAR comprised of the human C4 folate receptor-alpha (αFR)-specific scFv coupled to intracellular T cell signaling domains. Human T cells transduced to express the C4 CAR specifically secreted proinflammatory cytokine and exerted cytolytic functions when cultured with αFR-expressing tumors in vitro. Adoptive transfer of C4 CAR T cells mediated the regression of large, established human ovarian cancer in a xenogeneic mouse model. Relative to a murine MOv19 scFv-based αFR CAR, C4 CAR T cells mediated comparable cytotoxic tumor activity in vitro and in vivo but had lower affinity for αFR protein and exhibited reduced recognition of normal cells expressing low levels of αFR. Thus, T cells expressing a fully human CAR of intermediate affinity can efficiently kill antigen-expressing tumors in vitro and in vivo and may overcome issues of transgene immunogenicity and “on-target off-tumor” toxicity that plague trials utilizing CARs containing mouse-derived, high affinity scFvs. PMID:26101914

Granulocyte-macrophage colony stimulating factor (GM-CSF) enhances cumulus cell expansion in bovine oocytes

PubMed Central

2013-01-01

Background The objectives of the study were to characterize the expression of the α- and β-subunits of granulocyte-macrophage colony stimulating factor (GM-CSF) receptor in bovine cumulus cells and oocytes and to determine the effect of exogenous GM-CSF on cumulus cells expansion, oocyte maturation, IGF-2 transcript expression and subsequent competence for embryonic development. Methods Cumulus-oocyte complexes (COC) were obtained by aspirating follicles 3- to 8-mm in diameter with an 18 G needle connected to a vacuum pump at −50 mmHg. Samples of cumulus cells and oocytes were used to detect GM- CSF receptor by immunofluorescence. A dose–response experiment was performed to estimate the effect of GM-CSF on cumulus cell expansion and nuclear/cytoplasmic maturation. Also, the effect of GM-CSF on IGF-2 expression was evaluated in oocytes and cumulus cells after in vitro maturation by Q-PCR. Finally, a batch of COC was randomly assigned to in vitro maturation media consisting of: 1) synthetic oviductal fluid (SOF, n = 212); 2) synthetic oviductal fluid supplemented with 100 ng/ml of GM-CSF (SOF + GM-CSF, n = 224) or 3) tissue culture medium (TCM 199, n = 216) and then subsequently in vitro fertilized and cultured for 9 days. Results Immunoreactivity for both α and β GM-CSF receptors was localized in the cytoplasm of both cumulus cells and oocytes. Oocytes in vitro matured either with 10 or 100 ng/ml of GM-CSF presented a higher (P < 0.05) cumulus cells expansion than that of the control group (0 ng/ml of GM-CSF). GM-CSF did not affect the proportion of oocytes in metaphase II, cortical granules dispersion and IGF-2 expression. COC exposed to 100 ng/ml of GM-CSF during maturation did not display significant differences in terms of embryo cleavage rate (50.4% vs. 57.5%), blastocyst development at day 7 (31.9% vs. 28.7%) and at day 9 (17.4% vs. 17.9%) compared to untreated control (SOF alone, P = 0.2). Conclusions GM-CSF enhanced cumulus cell expansion of in vitro matured bovine COC. However, GM-CSF did not increase oocyte nuclear or cytoplasmic maturation rates, IGF-2 expression or subsequent embryonic development. PMID:23799974

Defective sperm head decondensation undermines the success of ICSI in the bovine.

PubMed

Águila, Luis; Felmer, Ricardo; Arias, María Elena; Navarrete, Felipe; Martin-Hidalgo, David; Lee, Hoi Chang; Visconti, Pablo; Fissore, Rafael

2017-09-01

The efficiency of intracytoplasmic sperm injection (ICSI) in the bovine is low compared to other species. It is unknown whether defective oocyte activation and/or sperm head decondensation limit the success of this technique in this species. To elucidate where the main obstacle lies, we used homologous and heterologous ICSI and parthenogenetic activation procedures. We also evaluated whether in vitro maturation negatively impacted the early stages of activation after ICSI. Here we showed that injected bovine sperm are resistant to nuclear decondensation by bovine oocytes and this is only partly overcome by exogenous activation. Remarkably, when we used heterologous ICSI, in vivo -matured mouse eggs were capable of mounting calcium oscillations and displaying normal PN formation following injection of bovine sperm, although in vitro -matured mouse oocytes were unable to do so. Together, our data demonstrate that bovine sperm are especially resistant to nuclear decondensation by in vitro -matured oocytes and this deficiency cannot be simply overcome by exogenous activation protocols, even by inducing physiological calcium oscillations. Therefore, the inability of a suboptimal ooplasmic environment to induce sperm head decondensation limits the success of ICSI in the bovine. Studies aimed to improve the cytoplasmic milieu of in vitro -matured oocytes and to replicate the molecular changes associated with in vivo capacitation and acrosome reaction will deepen our understanding of the mechanism of fertilization and improve the success of ICSI in this species. © 2017 Society for Reproduction and Fertility.

Tailoring in vitro evolution for protein affinity or stability

PubMed Central

Jermutus, Lutz; Honegger, Annemarie; Schwesinger, Falk; Hanes, Jozef; Plückthun, Andreas

2001-01-01

We describe a rapid and general technology working entirely in vitro to evolve either the affinity or the stability of ligand-binding proteins, depending on the chosen selection pressure. Tailored in vitro selection strategies based on ribosome display were combined with in vitro diversification by DNA shuffling to evolve either the off-rate or thermodynamic stability of single-chain Fv antibody fragments (scFvs). To demonstrate the potential of this method, we chose to optimize two proteins already possessing favorable properties. A scFv with an initial affinity of 1.1 nM (koff at 4°C of 10−4 s−1) was improved 30-fold by the use of off-rate selections over a period of several days. As a second example, a generic selection strategy for improved stability exploited the property of ribosome display that the conditions can be altered under which the folding of the displayed protein occurs. We used decreasing redox potentials in the selection step to select for molecules stable in the absence of disulfide bonds. They could be functionally expressed in the reducing cytoplasm, and, when allowed to form disulfides again, their stability had increased to 54 kJ/mol from an initial value of 24 kJ/mol. Sequencing revealed that the evolved mutant proteins had used different strategies of residue changes to adapt to the selection pressure. Therefore, by a combination of randomization and appropriate selection strategies, an in vitro evolution of protein properties in a predictable direction is possible. PMID:11134506

Effect of recombinant human follicle-stimulating hormone and luteinizing hormone on in vitro maturation of porcine oocytes evaluated by the subsequent in vitro development of embryos obtained by in vitro fertilization, intracytoplasmic sperm injection, or parthenogenetic activation.

PubMed

Silvestre, M A; Alfonso, J; García-Mengual, E; Salvador, I; Duque, C C; Molina, I

2007-05-01

The aim of this work was to study the effect of recombinant human (rh) FSH and LH on in vitro maturation of pig oocytes compared with a conventional hormonal supplement based on equine (PMSG) and human chorionic gonadotropins (hCG), as evaluated by the developmental ability of 3 types of pig embryos obtained by in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), or artificial activation (ATA). In Exp. 1, one cumulus-oocyte complex group (A group) was supplemented with rh-FSH and rh-LH (0.1 IU/mL each), and the other group (B group) was supplemented with PMSG and hCG (10 IU/mL each). No differences in nuclear maturation between the A and B groups were observed (68.5 vs. 71.4%, respectively). No differences were detected between hormonal treatments in the rates of cleavage or blastocyst formation of ATA, IVF, and ICSI embryos. Total cell number of the embryos was not significantly different in any experimental group (A: 31.1, 28.5, and 19.8 vs. B: 25.2, 25.5, and 20.6 for ATA, IVF, and ICSI embryos, respectively). In Exp. 2, the effects of different concentrations of rh-FSH and rh-LH (0.5, 0.1, or 0.05 IU/mL) in maturation medium on nuclear maturation and in vitro development of embryos obtained by IVF were studied. No effect of different hormonal concentrations on blastocyst formation rates was observed (8.5, 13.0, and 5.7%, respectively). Blastocyst cell number was not different in any experimental group. In conclusion, the results obtained here permit us to substitute PMSG and hCG with rh-FSH and rh-LH and to produce pig embryos obtained by IVF, ICSI, or ATA.

Selenium and vitamin E improve the in vitro maturation, fertilization and culture to blastocyst of porcine oocytes.

PubMed

Tareq, K M A; Akter, Quzi Sharmin; Khandoker, M A M Yahia; Tsujii, Hirotada

2012-01-01

Selenium (Se) and vitamin E (Vit-E), as integral parts of antioxidant systems, play important roles for sperm and embryos in vitro. In this study, the effects of Se and Vit-E on the maturation, in vitro fertilization and culture to blastocysts of porcine oocytes and accumulation of ammonia in the culture medium during different development stages were investigated. The maturation was performed in modified tissue culture medium (mTCM)-199 supplemented with 10% (v/v) porcine follicular fluid, the fertilization medium was modified Tyrode's albumin lactate pyruvate (mTALP), and the embryo culture medium was modified North Carolina State University (mNCSU)-23. Se in the form of sodium selenite (SS) and seleon-L-methionine (SeMet) and Vit-E at different concentrations were also used. The incorporation and oxidation of (14)C(U)-glucose were assessed with a liquid scintillation counter. In this study, SeMet and SeMet+Vit-E increased oocyte maturation, fertilization and incorporation and oxidation of (14)C(U)-glucose significantly (P<0.05) compared with the control and other treatments. In addition, embryo development, specifically in terms of the numbers of morulae and blastocysts, significantly increased (P<0.05) with SeMet and SeMet+Vit-E. In contrast, the accumulation of ammonia was reduced with SeMet and SeMet+Vit-E compared with other treatments. These findings indicate that SeMet and SeMet+Vit-E may play important roles in reducing the accumulation of ammonia and subsequently in increasing the rate of maturation of porcine oocytes and fertilization, as well as development of the blastocyst and utilization of glucose in in vitro maturation, fertilization and culture to blastocysts of porcine oocytes.

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.

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...

Differential sensitivity of immature and mature ventral mesencephalic neurons to rotenone induced neurotoxicity in vitro.

PubMed

Satish Bollimpelli, V; Kondapi, Anand K

2015-12-25

Rotenone induced neuronal toxicity in ventral mesencephalic (VM) dopaminergic (DA) neurons in culture is widely accepted as an important model for the investigation of Parkinson's disease (PD). However, little is known about developmental stage dependent toxic effects of rotenone on VM neurons in vitro. The objective of present study is to investigate the effect of rotenone on developing VM neurons at immature versus mature stages. Primary VM neurons were cultured in the absence of glial cells. Exposure of VM neurons to rotenone for 2 days induced cell death in both immature and mature neurons in a concentration-dependent manner, but to a greater extent in mature neurons. While rotenone-treated mature VM neurons showed α-synuclein aggregation and sensitivity to DA neurons, immature VM neurons exhibited only DA neuronal sensitivity but not α-synuclein aggregation. In addition, on rotenone treatment, enhancement of caspase-3 activity and reactive oxygen species (ROS) production were higher in mature VM neurons than in immature neurons. These results suggest that even though both mature and immature VM neurons are sensitive to rotenone, their manifestations differ from each other, with only mature VM neurons exhibiting Parkinsonian conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

Serotonergic ergoline derivatives.

PubMed

Mantegani, S; Brambilla, E; Caccia, C; Damiani, G; Fornaretto, M G; McArthur, R A; Varasi, M

1998-05-05

Novel classes of 13- and 14-tertbutyl-ergoline derivatives were prepared, and characterised in vitro for their affinity for adrenergic, dopaminergic and serotonergic binding sites. This study particularly examines the importance of the presence and the position of the tert-butyl group in conferring either significant 5-HT1A or 5-HT2 affinity and selectivity respectively.

Royal jelly may improve the metabolism of glucose and redox state of ovine oocytes matured in vitro and embryonic development following in vitro fertilization.

PubMed

Eshtiyaghi, Mahbobeh; Deldar, Hamid; Pirsaraei, Zarbakht Ansari; Shohreh, Bahram

2016-12-01

The aim of this study was to investigate the effect of different concentrations of royal jelly (RJ) on in vitro maturation (IVM), fertilization, cleavage, blastocyst rates, glutathione (GSH) content in ovine oocyte, mRNA abundance of antioxidant enzymes in both oocyte and cumulus, and glucose metabolism-related genes in cumulus cells. In vitro maturation of oocyte was performed in the presence of control (RJ 0 ), 2.5 (RJ 2.5 ), 5 (RJ 5 ), and 10 (RJ 10 ) mg/mL of RJ. Nuclear status, intracellular GSH content in oocytes, and mRNA abundance of selected genes were evaluated following 24 hours of IVM. Following the IVM, fertilization and embryo culture were carried out in all the groups and embryonic development was examined. The addition of 10-mg/mL RJ to maturation media not only yielded a higher number of oocytes at MII stage but also showed an increased level of intracellular GSH content than did RJ 2.5 and control groups. Fertilization, cleavage, and blastocyst rate were higher in the RJ 10 treatment group in comparison to the control one. In cumulus cells, the expression of PFKM, PFKL, and G6PDH were increased following the addition of RJ to the maturation media. Supplementation of 10-mg/mL RJ to IVM medium increased the GPx mRNA abundance in both oocyte and cumulus cells and SOD expression in the cumulus cells. The CAT mRNA abundance was not influenced by the addition of RJ to the maturation media in either oocyte or cumulus cells. It seems that the improvement of oocyte maturation and its subsequent development in RJ 10 group may be associated with amelioration of redox status in the oocytes and activation of glucose metabolic pathways in their surrounding cumulus cells. Copyright © 2016 Elsevier Inc. All rights reserved.

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 vitro growth and maturation of isolated caprine preantral follicles: Influence of insulin and FSH concentration, culture dish, coculture, and oocyte size on meiotic resumption.

PubMed

Silva, G M; Brito, I R; Sales, A D; Aguiar, F L N; Duarte, A B G; Araújo, V R; Vieira, L A; Magalhães-Padilha, D M; Lima, L F; Alves, B G; Silveira, L B R; Lo Turco, E G; Rodrigues, A P; Campello, C C; Wheeler, M B; Figueiredo, J R

2017-03-01

The aims of this study were: (1) to evaluate the effect of different insulin concentrations, alone or in combination with either a fixed FSH concentration or increasing FSH concentrations on the in vitro culture of isolated caprine preantral follicles and (2) to analyze the efficiency of two IVM media and maturation culture systems (with or without coculture with in vivo grown oocytes) on the meiosis resumption. Secondary follicles were cultured for 18 days in a basic medium supplemented with low- or high-insulin concentration alone or with a fixed FSH concentration or with increasing FSH concentrations. Oocytes grown in vivo or in vitro were matured alone or cocultured. The high-insulin concentration associated with fixed FSH treatment had higher meiotic resumption rate (P < 0.05) and was the only treatment capable of producing oocytes in metaphase II. The rates of germinal vesicle, germinal vesicle breakdown, metaphase I, metaphase II (MII), meiotic resumption, and oocyte diameter were similar between the maturation media. In conclusion, a basic medium supplemented with 10-μg/mL insulin and 100-μg/mL FSH throughout the culture period improved meiotic resumption rate and produced MII oocytes from caprine preantral follicles cultured in vitro. The MII rate was similar between in vivo and in vitro grown oocytes ≥110 μm. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

Functional and hypoglycemic properties of nopal cladodes (O. ficus-indica) at different maturity stages using in vitro and in vivo tests.

PubMed

Nuñez-López, María A; Paredes-López, Octavio; Reynoso-Camacho, Rosalía

2013-11-20

Nopal (Opuntia ficus-indica) cladodes are recommended for their therapeutic properties; their maturity stage may affect their biological properties. Cladodes of three maturity stages, from the same crop and location, were dehydrated and evaluated for some of their physicochemical and nutritional characteristics and antidiabetic properties. The flours of small and medium cladodes (SCF and MCF, respectively) had higher contents of dietary fiber, water absorption, swelling, and viscosity compared to those of the large cladode flour (LCF). Streptozotocin-induced diabetic rats, treated with MCF and SCF (doses of 50 mg/kg body weight), showed reduction of postprandial blood glucose on 46.0 and 23.6%, respectively (p < 0.05), in relation to the control; and LCF had no significant effect. In vitro, glucose diffusion tests showed similar ranking by the two former samples, whereas the latter was close to the control. Cladode maturity stages showed different fiber content and produced suspensions with differences in viscosity, which may affect in vitro and in vivo glucose responses.

Bioaccumulation of Ergovaline in Bovine Lateral Saphenous Veins in Vitro

USDA-ARS?s Scientific Manuscript database

Ergot alkaloids have been associated with vasoconstriction in grazing livestock afflicted with the fescue toxicosis syndrome. Previous in vitro investigations studying how ergot alkaloids cause the observed vasoconstriction have shown that ergovaline has a very distinct receptor affinity and sustain...

Cross species comparisons of chemical interaction with recombinant steroid receptors in vitro.

EPA Science Inventory

Typically, mammalian receptors are used for in vitro hazard identification and screening for endocrine disrupting compounds. There is concern, however, that differences may exist in the affinities of environmental compounds for steroid receptors from other vertebrate classes. S...

MUC1-specific CTLs are non-functional within a pancreatic tumor microenvironment.

PubMed

Mukherjee, P; Ginardi, A R; Madsen, C S; Tinder, T L; Jacobs, F; Parker, J; Agrawal, B; Longenecker, B M; Gendler, S J

2001-01-01

Pancreatic cancer is a highly aggressive, treatment refractory disease and is the fourth leading cause of death in the United States. In humans, 90% of pancreatic adenocarcinomas over-express altered forms of a tumor-associated antigen, MUC1 (an epithelial mucin glycoprotein), which is a target for immunotherapy. Using a clinically relevant mouse model of pancreas cancer that demonstrates peripheral and central tolerance to human MUC1 and develops spontaneous tumors of the pancreas, we have previously reported the presence of functionally active, low affinity, MUC1-specific precursor cytotoxic T cells (pCTLs). Hypothesis for this study is that MUC1-based immunization may enhance the low level MUC1-specific immunity that may lead to an effective anti-tumor response. Data demonstrate that MUC1 peptide-based immunization elicits mature MUC1-specific CTLs in the peripheral lymphoid organs. The mature CTLs secrete IFN-gamma and are cytolytic against MUC1-expressing tumor cells in vitro. However, active CTLs that infiltrate the pancreas tumor microenvironment become cytolytically anergic and are tolerized to MUC1 antigen, allowing the tumor to grow. We demonstrate that the CTL tolerance could be reversed at least in vitro with the use of anti-CD40 co-stimulation. The pancreas tumor cells secrete immunosuppressive cytokines, including IL-10 and TGF-beta that are partly responsible for the down-regulation of CTL activity. In addition, they down-regulate their MHC class I molecules to avoid immune recognition. CD4+ CD25+ T regulatory cells, which secrete IL-10, were also found in the tumor environment. Together these data indicate the use of several immune evasion mechanisms by tumor cells to evade CTL killing. Thus altering the tumor microenvironment to make it more conducive to CTL killing may be key in developing a successful anti-cancer immunotherapy.

Kangaroo IGF-II is structurally and functionally similar to the human [Ser29]-IGF-II variant.

PubMed

Yandell, C A; Francis, G L; Wheldrake, J F; Upton, Z

1999-06-01

Kangaroo IGF-II has been purified from western grey kangaroo (Macropus fuliginosus) serum and characterised in a number of in vitro assays. In addition, the complete cDNA sequence of mature IGF-II has been obtained by reverse-transcription polymerase chain reaction. Comparison of the kangaroo IGF-II cDNA sequence with known IGF-II sequences from other species revealed that it is very similar to the human variant, [Ser29]-hIGF-II. Both the variant and kangaroo IGF-II contain an insert of nine nucleotides that encode the amino acids Leu-Pro-Gly at the junction of the B and C domains of the mature protein. The deduced kangaroo IGF-II protein sequence also contains three other amino acid changes that are not observed in human IGF-II. These amino acid differences share similarities with the changes described in many of the IGF-IIs reported for non-mammalian species. Characterisation of human IGF-II, kangaroo IGF-II, chicken IGF-II and [Ser29]-hIGF-II in a number of in vitro assays revealed that all four proteins are functionally very similar. No significant differences were observed in the ability of the IGF-IIs to bind to the bovine IGF-II/cation-independent mannose 6-phosphate receptor or to stimulate protein synthesis in rat L6 myoblasts. However, differences were observed in their abilities to bind to IGF-binding proteins (IGFBPs) present in human serum. Kangaroo, chicken and [Ser29]-hIGF-II had lower apparent affinities for human IGFBPs than did human IGF-II. Thus, it appears that the major circulating form of IGF-II in the kangaroo and a minor form of IGF-II found in human serum are structurally and functionally very similar. This suggests that the splice site that generates both the variant and major form of human IGF-II must have evolved after the divergence of marsupials from placental mammals.

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.

Developmental potential of elongating and elongated spermatids obtained after in-vitro maturation of isolated round spermatids.

PubMed

Cremades, N; Sousa, M; Bernabeu, R; Barros, A

2001-09-01

Round spermatid injections are associated with disappointing clinical outcomes, and although these cells have been shown to mature into late spermatids in vitro, the developmental potential of such gametes remains to be demonstrated. Round spermatids were isolated from 12 testicle samples of patients with obstructive azoospermia, hypoplasia, complete maturation arrest, and incomplete Sertoli cell-only syndrome. They were cultured for 7 days at 32 degrees C, 5% CO(2)in air, in microdrops of Vero cell-conditioned medium containing 10% synthetic serum substitute. From the 238 round spermatids cultured, 25.2% attained the elongating and 5.5% the elongated spermatid stage (3-4 days per step). Relatively higher maturation rates were found in cases with obstructive azoospermia, but differences were significant only for elongated spermatids (9.3%). No differences were found in maturation rates between cases with non-obstructive azoospermia (4.3% of elongated spermatids). Experimental microinjections with elongating and elongated spermatids revealed a low fertilization rate (40.9%) but a normal blastocyst formation rate (60%). Late spermatids resulting from in-vitro culture of round spermatids in conditioned medium, either in controls in cases with a spermiogenetic block, appeared able to successfully fertilize the human oocyte and elicit normal embryo development.

Synthesis and binding affinity of new 1,4-disubstituted triazoles as potential dopamine D(3) receptor ligands.

PubMed

Insua, Ignacio; Alvarado, Mario; Masaguer, Christian F; Iglesias, Alba; Brea, José; Loza, María I; Carro, Laura

2013-10-15

A series of new 1,4-disubstituted triazoles was prepared from appropriate arylacetylenes and aminoalkylazides using click chemistry methodology. These compounds were evaluated as potential ligands on several subtypes of dopamine receptors in in vitro competition assays, showing high affinity for dopamine D3 receptors, lower affinity for D2 and D4, and no affinity for the D1 receptors. Compound 18 displayed the highest affinity at the D3 receptor with a Ki value of 2.7 nM, selectivity over D2 (70-fold) and D4 (200-fold), and behaviour as a competitive antagonist in the low nanomolar range. Copyright © 2013 Elsevier Ltd. All rights reserved.

Feed restriction and insulin-like growth factor-I (IGF-I) affect the oocyte maturation in matrinxã Brycon amazonicus.

PubMed

Montrezor, Luís Henrique; Urbinati, Elisabeth Criscuolo

2017-02-01

The feeding and nutrition of breeders are crucial aspects in the reproductive process. During the maturation period, metabolic changes occur aiming at mobilizing energy for growth and follicular development. The involvement of IGF-1 in metabolic and reproductive events is important. The aim of this work was to evaluate if alternate feed restriction and re-feeding have permissive effects on in vitro actions of IGF-1 on oocytes development of matrinxã. In vivo experiments were performed during vitellogenesis period. Females (n = 60) were fed with a commercial feed (2% of biomass) and they were divided into two treatments: fish receiving food daily (control - fed), and fish submitted to cycles of 3 days of feed restriction and 2 days of re-feeding (no-fed group). For the in vitro experiments, oocytes (n = 20) were obtained from the ovaries removed at the end of the in vivo experiment and were divided into four groups: fed -IGF-1; fed +IGF-1; no-fed -IGF-1 and no-fed +IGF-1. Fish under restriction had lower body weights, decreased plasma glucose, increased triglycerides levels, and their final maturation and mature oocyte were reduced and the atresic ones were in higher number. Moreover, IGF-1, in vitro, increased the percentage of mature oocytes in fed females and decreased the atresic ones. In no-fed females, IGF-1 increased the final maturation and mature oocytes and reduced the atresic ones. This study demonstrates the importance of the feeding management of female breeders of matrinxã during the vitellogenesis period.

Impaired response of mature adipocytes of diabetic mice to hypoxia

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

Hong, Seok Jong, E-mail: seok-hong@northwestern.edu; Jin, Da P.; Buck, Donald W.

2011-10-01

Adipose tissue contains various cells such as infiltrated monocytes/macrophages, endothelial cells, preadipocytes, and adipocytes. Adipocytes have an endocrine function by secreting adipokines such as interleukin (IL)-6, tumor necrosis factor (TNF)-{alpha}, leptin, and adiponectin. Dysregulation of adipokines in adipose tissues leads to a chronic low-grade inflammation which could result in atherosclerosis, hypertension, and type 2 diabetes. A sustained inflammatory state, which is characterized by prolonged persistence of macrophages and neutrophils, is found in diabetic wounds. In addition, subcutaneous adipocytes are enormously increased in amount clinically in type 2 diabetes. However, the function of subcutaneous adipocytes, which play an important role inmore » injured tissue subjected to hypoxia, has not been well characterized in vitro due to the difficulty of maintaining mature adipocytes in culture using conventional methods because of their buoyancy. In this study, we established a novel in vitro culture method of mature adipocytes by enclosing them in a hyaluronan (HA) based hydrogel to study their role in response to stress such as hypoxia. BrdU labeling and Ki67 immunostaining experiments showed that hydrogel enclosed mature adipocytes proliferate in vitro. Both mRNA and protein expression analyses for hypoxia regulated genes, such as vascular endothelial growth factor (VEGF) and heme oxygenase 1 (HO1), showed that mature adipocytes of wild type mice respond to hypoxia. In contrast, mature adipocytes of diabetic db/db and TallyHo mice did not efficiently respond to hypoxia. Our studies suggest that mature adipocytes are functionally active cells, and their abnormal function to hypoxia can be one of underlining mechanisms in type 2 diabetes.« less

Isolation of Latex Bead Phagosomes from Dictyostelium for in vitro Functional Assays.

PubMed

D'Souza, Ashwin; Sanghavi, Paulomi; Rai, Ashim; Pathak, Divya; Mallik, Roop

2016-12-05

We describe a protocol to purify latex bead phagosomes (LBPs) from Dictyostelium cells. These can be later used for various in vitro functional assays. For instance, we use these LBPs to understand the microtubule motor-driven transport on in vitro polymerized microtubules. Phagosomes are allowed to mature for defined periods inside cells before extraction for in vitro motility. These assays allow us to probe how lipids on the phagosome membrane recruit and organize motors, and also measure the motion and force generation resulting from underlying lipid-motor interactions. This provides a unique opportunity to interrogate native-like organelles using biophysical and biochemical assays, and understand the role of motor proteins in phagosome maturation and pathogen clearance.

Anti-Müllerian hormone is produced heterogeneously in primate preantral follicles and is a potential biomarker for follicle growth and oocyte maturation in vitro.

PubMed

Xu, Jing; Xu, Fuhua; Letaw, John H; Park, Byung S; Searles, Robert P; Ferguson, Betsy M

2016-12-01

The main goals of this study were to investigate the expression of anti-Müllerian hormone (AMH) and its receptor (AMHR2) during follicular development in primates, and to evaluate the potential of AMH as a biomarker for follicle growth and oocyte maturation in vitro. The mRNA and protein expression of AMH and AMHR2 were determined using isolated follicles and ovarian sections from rhesus macaques (n = 4) by real-time PCR and immunohistochemistry, respectively. Isolated secondary follicles were cultured individually. Follicle growth and media AMH concentrations were assessed by ELISA. The mRNA expression profiles, obtained from RNA sequencing, of in vitro- and in vivo-developed antral follicles were compared. Secondary follicles from additional animals (n = 35) were cultured. Follicle growth, oocyte maturation, and media AMH concentrations were evaluated for forecasting follicular development in vitro by AMH levels. AMH immunostaining was heterogeneous in the population of preantral follicles that were also stained for AMHR2. The mRNA expression profiles were comparable between in vivo- and in vitro-developed follicles. AMH levels produced by growing follicles were higher than those of nongrowing follicles in culture. With a cutoff value of 1.40 ng/ml, 85 % of nongrowing follicles could be identified while eliminating only 5 % of growing follicles. Growing follicles that generated metaphase II-stage oocytes secreted greater amounts of AMH than did those yielding immature germinal vesicle-stage oocytes. AMH, co-expressed with AMHR2, was produced heterogeneously by preantral follicles in macaques with levels correlated positively with follicle growth and oocyte maturation. AMH may serve as a biomarker for primate follicular development in vitro.

Ribonucleoprotein organization of eukaryotic RNA. XXXII. U2 small nuclear RNA precursors and their accurate 3' processing in vitro as ribonucleoprotein particles.

PubMed

Wieben, E D; Nenninger, J M; Pederson, T

1985-05-05

Biosynthetic precursors of U2 small nuclear RNA have been identified in cultured human cells by hybrid-selection of pulse-labeled RNA with cloned U2 DNA. These precursor molecules are one to approximately 16 nucleotides longer than mature U2 RNA and contain 2,2,7-trimethylguanosine "caps". The U2 RNA precursors are associated with proteins that react with a monoclonal antibody for antigens characteristic of small nuclear ribonucleoprotein particles. Like previously described precursors of U1 and U4 small nuclear RNAs, the pre-U2 RNAs are recovered in cytoplasmic fractions, although it is not known if this is their location in vivo. The precursors are processed to mature-size U2 RNA when cytoplasmic extracts are incubated in vitro at 37 degrees C. Mg2+ is required but ATP is not. The ribonucleoprotein structure of the pre-U2 RNA is maintained during the processing reaction in vitro, as are the 2,2,7-trimethylguanosine caps. The ribonucleoprotein organization is of major importance, as exogenous, protein-free U2 RNA precursors are degraded rapidly in the in vitro system. Two lines of evidence indicate that the conversion of U2 precursors to mature-size U2 RNA involves a 3' processing reaction. First, the reaction is unaffected by a large excess of mature U2 small nuclear RNP, whose 5' trimethylguanosine caps would be expected to compete for a 5' processing activity. Second, when pre-U2 RNA precursors are first stoichiometrically decorated with an antibody specific for 2,2,7-trimethylguanosine, the extent of subsequent processing in vitro is unaffected. These results provide the first demonstration of a eukaryotic RNA processing reaction in vitro occurring within a ribonucleoprotein particle.

Beneficial effect of resveratrol on bovine oocyte maturation and subsequent embryonic development after in vitro fertilization.

PubMed

Wang, Feng; Tian, XiuZhi; Zhang, Lu; He, ChangJiu; Ji, PengYun; Li, Yu; Tan, DunXian; Liu, GuoShi

2014-02-01

To analyze the potential beneficial effects and mechanisms of action of resveratrol on the maturation of bovine oocytes that were incubated in different concentrations of resveratrol (0.1, 1.0, or 10.0 μM) as germinal vesicle-stage oocytes. In vitro prospective study. University research laboratory. Animal models for human studies. In vitro culture in the presence of various concentrations of the antioxidant resveratrol. Parameters of hormone levels, oocyte nuclear maturation, cumulus expansion, levels of intracellular glutathione and reactive oxygen species, embryonic cleavage, blastocyst formation, gene expression associated with mature bovine oocytes and cumulus cells, and level of sirtuin 1 gene expression. Resveratrol statistically significantly increased progesterone secretion and decreased estradiol-17β secretion by cumulus cells. The elevated levels of progesterone activated the Mos/MEK/p42 mitogen-activated protein kinase (MAPK) cascade in the oocytes. At a concentration of 1.0 μM, resveratrol statistically significantly improved cumulus expansion, polar body formation, the (hatched) blastocyst rate, and the mean number of cells/blastocysts. Meanwhile, resveratrol statistically significantly reduced the level of reactive oxygen species (ROS) and increased the level of glutathione (GSH). For the first time, the expression of the sirtuin-1 gene was identified in granulosa cells, cumulus cells, oocytes, and blastocysts. Further studies revealed that resveratrol promoted sirtuin-1 gene expression. Resveratrol promoted bovine oocyte maturation and subsequent post-in vitro fertilization embryonic development by inducing progesterone secretion and an antioxidant effect, probably in a manner dependent on sirtuin-1. Copyright © 2014 American Society for Reproductive Medicine. All rights reserved.

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

Zukin, R.S.; Eghbali, M.; Olive, D.

{kappa} opioid receptors ({kappa} receptors) have been characterized in homogenates of guinea pig and rat brain under in vitro binding conditions. {kappa} receptors were labeled by using the tritiated prototypic {kappa} opioid ethylketocyclazocine under conditions in which {mu} and {delta} opioid binding was suppressed. In the case of guinea pig brain membranes, a single population of high-affinity {kappa} opioid receptor sites was observed. In contrast, in the case of rat brain, two populations of {kappa} sites were observed. To test the hypothesis that the high- and low-affinity {kappa} sites represent two distinct {kappa} receptor subtypes, a series of opioids weremore » tested for their abilities to compete for binding to the two sites. U-69,593 and Cambridge 20 selectively displaced the high-affinity {kappa} site in both guinea pig and rat tissue, but were inactive at the rat-brain low-affinity site. Other {kappa} opioid drugs competed for binding to both sites, but with different rank orders of potency. Quantitative light microscopy in vitro autoradiography was used to visualize the neuroanatomical pattern of {kappa} receptors in rat and guinea pig brain. The distribution patterns of the two {kappa} receptor subtypes of rat brain were clearly different. Collectively, these data provide direct evidence for the presence of two {kappa} receptor subtypes; the U-69,593-sensitive, high-affinity {kappa}{sub 1} site predominates in guinea pig brain, and the U-69,593-insensitive, low-affinity {kappa}{sub 2} site predominates in rat brain.« less

Exposure to cadmium during in vitro maturation at environmental nanomolar levels impairs oocyte fertilization through oxidative damage: A large animal model study.

PubMed

Martino, N A; Marzano, G; Mangiacotti, M; Miedico, O; Sardanelli, A M; Gnoni, A; Lacalandra, G M; Chiaravalle, A E; Ciani, E; Bogliolo, L; Minervini, F; Pizzi, F; Dell'Aquila, M E

2017-04-01

Cadmium is a highly toxic heavy metal with negative effects on oocyte fertilization. The aim of this study was to analyse whether cadmium-induced impairment of fertilization is caused by mitochondria dysfunction and oxidative stress in the cumulus-oocyte complex (COC). Preliminarily, 19 trace element levels were measured in ovaries from juvenile and adult ewes and age-related cadmium ovarian bioaccumulation at nanomolar concentrations was found. COCs from juvenile and adult ewes, exposed during in vitro maturation to 1nM or 100nM CdCl 2 , and subjected to in vitro fertilization showed significantly lower fertilization rates in exposed COCs compared with controls. In vitro matured exposed and control COCs underwent confocal microscopy analysis of mitochondria activity and reactive oxygen species (ROS) levels and lipid peroxidation (LPO) assay at cumulus cell and oocyte level. In both age groups, cadmium at nanomolar concentrations induced cumulus-oocyte mitochondria over-activity and oxidative damage which were related to impaired oocyte fertilization. Copyright © 2017 Elsevier Inc. All rights reserved.

Toxic effects of ethylene oxide residues on bovine embryos in vitro.

PubMed

Holyoak, G R; Wang, S; Liu, Y; Bunch, T D

1996-04-15

The potential of ethylene oxide (EtO) residues in exposed plastic tissue culture dishes to adversely affect bovine oocyte maturation, fertilization and subsequent embryonic development was monitored. In experiment 1, the effects of aeration time and aeration combined with washing of EtO-gassed culture dishes on the extent of residual toxicity were investigated. There was no cleavage in any treatment in which oocytes were matured and fertilized in dishes exposed to EtO. EtO residues caused functional degeneration of oocytes even when culture dishes were aerated for more than 12 days post EtO-exposure and repeatedly washed. In experiment 2, the residual toxicity of EtO gas on in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) were evaluated. Cleavage rate significantly decreased and post-cleavage development was retarded in ova maintained in dishes treated with EtO either during IVM or IVF. EtO residues may be more detrimental to spermatozoa than to oocytes which may have been the primary cause of fertilization failure during IVF.

Clonal mature adipocyte production of proliferative-competent daughter cells requires lipid export prior to cell division

USDA-ARS?s Scientific Manuscript database

Numerous in vitro observations have been published to show that mature adipocytes may resume proliferation and begin to populate the adipofibroblast fraction or form other cell types. In the present study, we evaluated clonal cultures of mature pig-derived adipocytes as they began to reestablish the...

Zoledronic acid modulates maturation of human monocyte-derived dendritic cells.

PubMed

Orsini, Giulia; Failli, Alessandra; Legitimo, Annalisa; Adinolfi, Barbara; Romanini, Antonella; Consolini, Rita

2011-12-01

Zoledronic acid (ZA) is a drug of the bisphosphonate class, which is widely used for the treatment of both osteoporosis and skeletal metastasis. Besides its main bone antiresorptive activity, ZA displays antitumor properties, by triggering the expansion and activation of γδ T-cells, which exert an antitumor effect through dendritic cells (DCs). Several studies have reported the interaction between ZA and γδ T-cells, but the potential immunoregulatory activity of this drug on DCs has scarcely been investigated. Therefore, in this paper, we evaluated the effects of a therapeutic dose of ZA on the in vitro generation and maturation of DCs derived from peripheral blood monocytes of healthy adult donors. We demonstrate that ZA treatment did not affect DC differentiation, but inhibited DC maturation on lipopolysaccharide activation, as shown by the impaired expression of maturation surface markers and reduced ability to induce allogeneic T-cell proliferation. Interestingly, IL-10 secretion by mature DCs was significantly lower in ZA-treated cells than in controls. We conclude that ZA exerts its immunological in vitro activity also by modulating the maturation of DCs.

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.

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

In vitro embryos production after oocytes treatment with forskolin.

PubMed

Paschoal, Daniela Martins; Maziero, Rosiára Rosária Dias; Sudano, Mateus José; Guastali, Midyan Daroz; Vergara, Luis Eduardo; Crocomo, Letícia Ferrari; Lima-Neto, João Ferreira de; Magalhães, Luis Carlos Oña; Monteiro, Bianca Andriolo; Rascado, Tatiana da Silva; Martins, Alício; Leal, Claudia Lima Verde; Landim-Alvarenga, Fernanda da Cruz

2016-04-01

The inhibition of nuclear maturation allows time for the oocyte to accumulate molecules that are important for embryonic development. Thus, the objective of this work was to evaluate the effect of blocking oocyte meiosis with the addition of forskolin, an efficient inhibitor of nuclear maturation, in in vitro maturation (IVM) medium. Forskolin was added to the IVM medium for 6 h at concentrations of 0.1 mM, 0.05 mM or 0.025 mM, then the oocytes were allowed to mature in drug-free medium for 18 h. The oocytes were assessed for the stage of nuclear maturation, the activity and distribution of mitochondria, oocyte ultrastructure, the number of viable cells and the apoptosis rate. After forskolin treatment, the oocytes were fertilized in vitro and cultured for 7 days. On day 7, the blastocyst rate, the ultrastructure, the number of intact cells and the apoptosis rate of the blastocysts were measured. No differences were observed for the stage of nuclear maturation of the oocyte, the mitochondrial activity and distribution, the blastocyst rate or total number of intact cells. However, a higher rate of apoptosis was observed in the blastocysts produced from oocytes blocked for 6 h with the higher concentration of forskolin (P < 0.05). We conclude that all the experimental groups reached the MII stage after the addition of forskolin and that the highest concentration of forskolin caused cellular degeneration without harming embryo production on the 7th day.

Occurrence of ovarian follicular dominance during stimulation for IVM impacts usable blastocyst yield

PubMed Central

Romero, Sergio; Pella, Ricardo; Escudero, Francisco; Pérez, Ygor; García, Mario; Orihuela, Patricia

2018-01-01

Objective To evaluate the influence of ovarian follicular dominance on the outcome of oocyte in-vitro maturation. Methods This retrospective cohort study included 21 patients with polycystic ovaries or polycystic ovary syndrome (Rotterdam criteria, 2004) subjected to 24 in-vitro maturation (IVM) cycles between October 2015 and January 2017. Patients undergoing IVM received minimal gonadotropin stimulation starting on day 2 or 3 of the cycle; ovum pick-up typically occurred on days 6 to 8. No hCG-trigger shot was given. Following 30h of IVM, mature oocytes were inseminated by ICSI and the resulting embryos cultured up to the blastocyst stage. Results Ovarian follicular dominance was observed in nine of the 24 IVM cycles. Oocyte IVM yielded an overall maturation rate of 69.3±23.8%, and no difference was observed when the groups with or without a dominant follicle were assessed independently. The rates of fertilization and usable blastocysts per fertilized oocyte, mature oocyte (Metaphase II) or cumulus-oocyte-complex were nearly three times higher (28.7±22.5%) in the group without ovarian follicular dominance. No differences were found in the clinical pregnancy rates attained by the individuals with or without a dominant follicle after 21 vitrified-warmed blastocyst transfer cycles. Conclusion Occurrence of ovarian follicular dominance during hormonal stimulation for in-vitro maturation negatively impacted embryological outcomes. Strategies devised to limit the appearance of ovarian follicular dominance must be further explored. PMID:29338139

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.

Biotechnology Challenges to In Vitro Maturation of Hepatic Stem Cells.

PubMed

Chen, Chen; Soto-Gutierrez, Alejandro; Baptista, Pedro M; Spee, Bart

2018-04-01

The incidence of liver disease is increasing globally. The only curative therapy for severe end-stage liver disease, liver transplantation, is limited by the shortage of organ donors. In vitro models of liver physiology have been developed and new technologies and approaches are progressing rapidly. Stem cells might be used as a source of liver tissue for development of models, therapies, and tissue-engineering applications. However, we have been unable to generate and maintain stable and mature adult liver cells ex vivo. We review factors that promote hepatocyte differentiation and maturation, including growth factors, transcription factors, microRNAs, small molecules, and the microenvironment. We discuss how the hepatic circulation, microbiome, and nutrition affect liver function, and the criteria for considering cells derived from stem cells to be fully mature hepatocytes. We explain the challenges to cell transplantation and consider future technologies for use in hepatic stem cell maturation, including 3-dimensional biofabrication and genome modification. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

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

Comparison of normal and abnormal fertilization of in vitro-matured human oocyte according to insemination method.

PubMed

Park, Ju Hee; Jee, Byung Chul; Kim, Seok Hyun

2016-04-01

Our purpose was to compare the normal fertilization rate, multi-pronuclei (PN) formation rate, and embryonic development of in vitro-matured oocytes between conventional insemination and intracytoplasmic sperm injection (ICSI). A total of 213 stimulated in vitro fertilization (IVF) cycles were selected, in which at least one immature oocyte was obtained (from 2010 to 2014). Immature oocytes were assigned to germinal vesicle (GV)-stage or metaphase I (MI)-stage oocyte groups. Cycles with obligatory ICSI due to male-factor infertility were excluded. Cycles were divided into two groups according to fertilization method: there were 97 cycles with conventional insemination and 116 cycles with ICSI. After in vitro maturation of 324 GV-stage oocytes and 341 MI-stage oocytes, the fertilization rate, multi-PN formation rate, and embryonic development were compared according to the fertilization method. The normal fertilization rate was similar in the conventional insemination and the ICSI both in GV-derived and MI-derived oocytes. Both fertilization methods resulted in a similar multi-PN formation rate in GV-derived oocytes; however, in MI-derived oocytes, the multi-PN formation rate was zero with ICSI and this was significantly lower than that with conventional insemination (9.6%, P = 0.001). In non-male-factor infertility, ICSI should be considered when MI oocytes are matured. © 2016 Japan Society of Obstetrics and Gynecology.

Noninvasive assays of in vitro matured human oocytes showed insignificant correlation with fertilization and embryo development.

PubMed

Ashourzadeh, Sareh; Khalili, Mohammad Ali; Omidi, Marjan; Mahani, Seyed Nooraldin Nematollahi; Kalantar, Seyed Mehdi; Aflatoonian, Abbas; Habibzadeh, Victoria

2015-08-01

Recently, the upgrading of in vitro maturation (IVM) of human oocytes as a promising strategy has emerged in assisted reproductive technology (ART). The goal was to evaluate the correlation of the in vitro matured oocytes selected on the basis of the zona pellucida (ZP) birefringence and meiotic spindles (MS) detection with fertilization and subsequent embryo development in ICSI program. A total of 168 immature oocytes [germinal vesicle (n = 140) and metaphase I (n = 28)] obtained from patients undergoing oocytes retrieval for ICSI. After in vitro culture for 24-40 h, 112 (67 %) oocytes reached to MII stage. Using a polarized microscopy, the presence of MS and ZP birefringence were assessed in matured oocytes, followed by ICSI performance. The rates of fertilization in oocytes with spindles (51.3 %) were similar to that of the oocytes without spindles (50.7 %; P = 1.00). Moreover, the fertilization rates in high birefringence (HB) oocytes was not statistically different than oocytes with low birefringence (LB) (P = 0.44). The findings also showed that 64.9 % of the fertilized oocytes developed to embryos, in which 33.3 % were derived from spindle-detected oocytes. Regarding the ZP birefringence, 35.5 % of the embryos were derived from HB oocytes. There were insignificant relationships between the MS detection and ZP birefringence score with the rates of fertilization and embryo development in IVM oocytes.

Sperm preparation through Sephadex™ filtration improves in vitro fertilization rate of buffalo oocytes.

PubMed

Husna, A U; Azam, A; Qadeer, S; Awan, M A; Nasreen, S; Shahzad, Q; Fouladi-Nashta, A; Khalid, M; Akhter, S

2018-04-01

Routinely, swim-up method is used to separate high-quality sperm; however, long processing time and close cell-to-cell contact during the centrifugation step are inevitable elements of oxidative stress to sperm. The objective was to evaluate Sephadex ™ and glass wool filtration to separate motile, intact and viable sperm for in vitro fertilization in buffalo. The cumulus-oocyte complexes (COCs) were collected from ovaries of slaughtered buffaloes by aspiration and matured for 24 hr in CO 2 incubator at 38.5°C and 5% CO 2 . Matured COCs were rinsed twice in fertilization TALP and placed in the pre-warmed fertilization medium without sperm. Cryopreserved buffalo semen was thawed at 37°C for 30 s and processed through Sephadex ™ , glass wool filtration and swim-up (control). Total and motile sperm recovery rates were assessed, resuspended in fertilization TALP and incubated for 15-20 min in CO 2 incubator. Samples prepared by each method were divided into two aliquots: one aliquot was studied for sperm quality (progressive motility, membrane integrity, viability, liveability), while the other was subjected to co-incubation with sets of 10-15 in vitro matured oocytes. Data on sperm quality were analysed by ANOVA, while in vitro fertilizing rates were compared by chi-squared test using SPSS-20. Least significant difference (LSD) test was used to compare treatment means. Glass wool filtration yielded higher total and motile sperm recovery rate, while Sephadex ™ filtration improved (p < .05) sperm quality (progressive motility, membrane integrity, viability, liveability). Sperm preparation through Sephadex filtration yielded higher in vitro fertilization rate in terms of cleavage rate compared to glass wool filtration and swim-up (control). In conclusion, cryopreserved Nili-Ravi buffalo sperm selected through Sephadex filtration showed improved quality and yielded better fertilization rates (cleavage rate) of in vitro matured/fertilized oocytes. Sephadex filtration could be a promising technique for use in in vitro fertilization in buffalo. © 2017 Blackwell Verlag GmbH.

The evolution of porcine embryo in vitro production.

PubMed

Grupen, Christopher G

2014-01-01

The in vitro production of porcine embryos has presented numerous challenges to researchers over the past four decades. Some of the problems encountered were specific to porcine gametes and embryos and needed the concerted efforts of many to overcome. Gradually, porcine embryo in vitro production systems became more reliable and acceptable rates of blastocyst formation were achieved. Despite the significant improvements, the problem of polyspermic fertilization has still not been adequately resolved and the embryo in vitro culture conditions are still considered to be suboptimal. Whereas early studies focused on increasing our understanding of the reproductive processes involved, the technology evolved to the point where in vitro-matured oocytes and in vitro-produced embryos could be used as research material for developing associated reproductive technologies, such as SCNT and embryo cryopreservation. Today, the in vitro procedures used to mature oocytes and culture embryos are integral to the production of transgenic pigs by SCNT. This review discusses the major achievements, advances, and knowledge gained from porcine embryo in vitro production studies and highlights the future research perspectives of this important technology. Copyright © 2014 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

Kinetics of nuclear maturation and effect of holding ovaries at room temperature on in vitro maturation of camel (Camelus dromedarius) oocytes.

PubMed

Wani, N A; Nowshari, M A

2005-07-01

Experiments were conducted to investigate kinetics of in vitro nuclear maturation and the effect of storing ovaries at room temperature on initial chromatin configuration and in vitro maturation of dromedary camel oocytes. Cumulus oocyte complexes (COCs) were collected from slaughterhouse ovaries and matured in vitro for 4-48h. At every 4h interval (starting from 0 to 48 h), groups of oocytes were fixed, stained and evaluated for the status of nuclear chromatin. Oocytes were categorized as germinal vesicle (GV), diakinesis (DK), metaphase-I (M-I), anaphase-I (A-I), metaphase-II (M-II) stage and those with degenerated, fragmented, activated or without a visible chromatin as others. At the start of culture, 74% (66/89) oocytes were at GV stage, 13% (12/89) at DK and 12% (11/89) were classified as others. Germinal vesicle breakdown started spontaneously in culture and at 20 h of culture 97% oocytes had already completed this process. After 8 and 16 h of maturation the highest proportion of oocytes (42%, 48/114 and 41%, 51/123) were at DK and M-I stage, respectively. The proportions of oocytes reaching M-II stage at 32 (42%, 50/118), 36 (45%, 47/104), 40 (49%, 57/117), 44 (52%, 103/198) and 48 h (46%, 55/120) of culture were not different from each other (P>0.05). The proportion of oocytes categorized as others, however, increased after 40 h of culture and was higher (P<0.05) at 48 h compared with other maturation periods. There was no difference (P>0.05) in the proportion of oocytes reaching M-II stage from the ovaries collected and stored in normal saline solution (NSS) at room temperature for 12h (43%, 64/148) and those collected in warm NSS (37 degrees C) and processed immediately after arrival in laboratory (49%, 57/117). However, low number of oocytes reached M-II stage from ovaries collected in warm NSS but stored at room temperature (29%, 37/128) compared with other two groups (P<0.05). It may be concluded that dromedary oocytes require 32-44h of in vitro culture to have an optimum number of oocytes in M-II stage. However, further studies are required to find out the most appropriate maturation period, which will result in the further development of these oocytes after IVF, ICSI, parthenogenetic activation or nuclear transfer. Ovaries can be collected and stored in normal saline solution at room temperature for 12h without any appreciable effect on the nuclear maturation of the oocytes.

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

Affinity Pulldown of Biotinylated RNA for Detection of Protein-RNA Complexes.

PubMed

Panda, Amaresh C; Martindale, Jennifer L; Gorospe, Myriam

2016-12-20

RNA-binding proteins (RBPs) have recently emerged as crucial players in the regulation of gene expression. The interactions of RBPs with target mRNAs control the levels of gene products by altering different regulatory steps, including pre-mRNA splicing and maturation, nuclear mRNA export, and mRNA stability and translation (Glisovic et al. , 2008). There are several methodologies available today to identify RNAs bound to specific RBPs; some detect only recombinant molecules in vitro , others detect recombinant and endogenous molecules, while others detect only endogenous molecules. Examples include systematic evolution of ligands by exponential enrichment (SELEX), biotinylated RNA pulldown assay, RNA immunoprecipitation (RIP) assay, electrophoretic mobility shift assay (EMSA), RNA footprinting analysis, and various UV crosslinking and immunoprecipitation (CLIP) methods such as CLIP, PAR-CLIP, and iCLIP (Popova et al. , 2015). Here, we describe a simple and informative method to study and identify the RNA region of interaction between an RBP and its target transcript (Panda et al. , 2014 and 2016). Its reproducibility and ease of use make this protocol a fast and useful method to identify interactions between RBPs and specific RNAs.

New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-affinity γ-Hydroxybutyrate (GHB) Binding Sites

PubMed Central

Vogensen, Stine B.; Marek, Aleš; Bay, Tina; Wellendorph, Petrine; Kehler, Jan; Bundgaard, Christoffer; Frølund, Bente; Pedersen, Martin H.F.; Clausen, Rasmus P.

2013-01-01

3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity and we demonstrate in vivo brain penetration. In vitro characterization of [3H]-1 binding shows high specificity to the high-affinity GHB binding sites. PMID:24053696

Rab10 Disruption Results in Delayed OPC Maturation.

PubMed

Zhang, Zhao-Huan; Zhao, Wei-Qian; Ma, Fan-Fei; Zhang, Hui; Xu, Xiao-Hui

2017-10-01

Oligodendrocyte precursor cell (OPC) maturation requires membrane addition for myelin sheath formation. Since the Rab system has been shown to contribute to membrane addition in other cell types, in this study, we explored the role of Rab in OPC maturation. SiRNA and shRNA techniques and conditional knockout mice provided in vitro and in vivo evidence that Rab10 is involved in OPC maturation and may affect myelination during OPC development.

Effect of in-utero diethylstilboestrol exposure on human oocyte quality and fertilization in a programme of in-vitro fertilization.

PubMed

Kerjean, A; Poirot, C; Epelboin, S; Jouannet, P

1999-06-01

Genital tract abnormalities and adverse pregnancy outcome are well known in women exposed in utero to diethylstilboestrol (DES). Data about adverse reproductive performance in women exposed to DES have been published, including controversial reports of menstrual dysfunction, poor responses after ovarian stimulation, oocyte maturation and fertilization abnormalities. We compared oocyte quality, in-vitro fertilization results and embryo quality for women exposed in utero to DES with a control group. Between 1989 and 1996, 56 DES-exposed women who had 125 in-vitro fertilization (IVF) attempts were retrospectively compared to a control group of 45 women with tubal disease, who underwent 73 IVF attempts. Couples suffering from male infertility were excluded. The parameters compared were oocyte quality (maturation abnormalities, immature oocyte, mature oocyte), fertilization and cleavage rate (per treated and metaphase II oocytes), and embryo quality (number and grade). We found no significant difference in oocyte maturational status, fertilization rates, cleavage rates, embryo quality and development between DES-exposed subjects and control subjects. These results suggest that in-utero exposure to DES has no significant influence on oocyte quality and fertilization ability as judged during IVF attempts.

Substrate specificity of the high-affinity glucose transport system of Pseudomonas aeruginosa.

PubMed

Wylie, J L; Worobec, E A

1993-07-01

Specificity of the high-affinity glucose transport system of Pseudomonas aeruginosa was examined. At a concentration of [14C]glucose near the Vmax of the system, inhibition by maltose, galactose, and xylose was detected. This inhibition is similar to that detected in earlier in vivo studies and correlates with the known specificity of OprB, a glucose-specific porin of P. aeruginosa. At a level of [14C]glucose 100 times lower, only unlabelled glucose inhibited uptake to any extent. This matches the known in vitro specificity of the periplasmic glucose binding protein. These findings were used to explain the discrepancy between earlier in vivo and in vitro results reported in the literature.

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

Ribosomal RNA maturation in Schizosaccharomyces pombe is dependent on a large ribonucleoprotein complex of the internal transcribed spacer 1.

PubMed

Lalev, A I; Abeyrathne, P D; Nazar, R N

2000-09-08

The interdependency of steps in the processing of pre-rRNA in Schizosaccharomyces pombe suggests that RNA processing, at least in part, acts as a quality control mechanism which helps assure that only functional RNA is incorporated into mature ribosomes. To determine further the role of the transcribed spacer regions in rRNA processing and to detect interactions which underlie the interdependencies, the ITS1 sequence was examined for its ability to form ribonucleoprotein complexes with cellular proteins. When incubated with protein extract, the spacer formed a specific large RNP. This complex was stable to fractionation by agarose or polyacrylamide gel electrophoresis. Modification exclusion analyses indicated that the proteins interact with a helical domain which is conserved in the internal transcribed spacers. Mutagenic analyses confirmed an interaction with this sequence and indicated that this domain is critical to the efficient maturation of the precursor RNA. The protein constituents, purified by affinity chromatography using the ITS1 sequence, retained an ability to form stable RNP. Protein analyses of gel purified complex, prepared with affinity-purified proteins, indicated at least 20 protein components ranging in size from 20-200 kDa. Peptide mapping by Maldi-Toff mass spectroscopy identified eight hypothetical RNA binding proteins which included four different RNA-binding motifs. Another protein was putatively identified as a pseudouridylate synthase. Additional RNA constituents were not detected. The significance of this complex with respect to rRNA maturation and interdependence in rRNA processing is discussed. Copyright 2000 Academic Press.

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.

Effects of Different Maturation Systems on Bovine Oocyte Quality, Plasma Membrane Phospholipid Composition and Resistance to Vitrification and Warming.

PubMed

Sprícigo, José F W; Diógenes, Mateus N; Leme, Ligiane O; Guimarães, Ana L; Muterlle, Carolle V; Silva, Bianca Damiani Marques; Solà-Oriol, David; Pivato, Ivo; Silva, Luciano Paulino; Dode, Margot A N

2015-01-01

The objective of this study was to evaluate the effects of different maturation systems on oocyte resistance after vitrification and on the phospholipid profile of the oocyte plasma membrane (PM). Four different maturation systems were tested: 1) in vitro maturation using immature oocytes aspirated from slaughterhouse ovaries (CONT; n = 136); 2) in vitro maturation using immature oocytes obtained by ovum pick-up (OPU) from unstimulated heifers (IMA; n = 433); 3) in vitro maturation using immature oocytes obtained by OPU from stimulated heifers (FSH; n = 444); and 4) in vivo maturation using oocytes obtained from heifers stimulated 24 hours prior by an injection of GnRH (MII; n = 658). A sample of matured oocytes from each fresh group was analyzed by matrix associated laser desorption-ionization (MALDI-TOF) to determine their PM composition. Then, half of the matured oocytes from each group were vitrified/warmed (CONT VIT, IMA VIT, FSH VIT and MII VIT), while the other half were used as fresh controls. Afterwards, the eight groups underwent IVF and IVC, and blastocyst development was assessed at D2, D7 and D8. A chi-square test was used to compare embryo development between the groups. Corresponding phospholipid ion intensity was expressed in arbitrary units, and following principal components analyses (PCA) the data were distributed on a 3D graph. Oocytes obtained from superstimulated animals showed a greater rate of developmental (P<0.05) at D7 (MII = 62.4±17.5% and FSH = 58.8±16.1%) compared to those obtained from unstimulated animals (CONT = 37.9±8.5% and IMA = 50.6±14.4%). However, the maturation system did not affect the resistance of oocytes to vitrification because the blastocyst rate at D7 was similar (P>0.05) for all groups (CONT VIT = 2.8±3.5%, IMA VIT = 2.9±4.0%, FSH VIT = 4.3±7.2% and MII VIT = 3.6±7.2%). MALDI-TOF revealed that oocytes from all maturation groups had similar phospholipid contents, except for 760.6 ([PC (34:1) + H]+), which was more highly expressed in MII compared to FSH (P<0.05). The results suggest that although maturation systems improve embryonic development, they do not change the PM composition nor the resistance of bovine oocytes to vitrification.

Effects of Different Maturation Systems on Bovine Oocyte Quality, Plasma Membrane Phospholipid Composition and Resistance to Vitrification and Warming

PubMed Central

Sprícigo, José F. W.; Diógenes, Mateus N.; Leme, Ligiane O.; Guimarães, Ana L.; Muterlle, Carolle V.; Silva, Bianca Damiani Marques; Solà-Oriol, David; Pivato, Ivo; Silva, Luciano Paulino; Dode, Margot A. N.

2015-01-01

The objective of this study was to evaluate the effects of different maturation systems on oocyte resistance after vitrification and on the phospholipid profile of the oocyte plasma membrane (PM). Four different maturation systems were tested: 1) in vitro maturation using immature oocytes aspirated from slaughterhouse ovaries (CONT; n = 136); 2) in vitro maturation using immature oocytes obtained by ovum pick-up (OPU) from unstimulated heifers (IMA; n = 433); 3) in vitro maturation using immature oocytes obtained by OPU from stimulated heifers (FSH; n = 444); and 4) in vivo maturation using oocytes obtained from heifers stimulated 24 hours prior by an injection of GnRH (MII; n = 658). A sample of matured oocytes from each fresh group was analyzed by matrix associated laser desorption-ionization (MALDI-TOF) to determine their PM composition. Then, half of the matured oocytes from each group were vitrified/warmed (CONT VIT, IMA VIT, FSH VIT and MII VIT), while the other half were used as fresh controls. Afterwards, the eight groups underwent IVF and IVC, and blastocyst development was assessed at D2, D7 and D8. A chi-square test was used to compare embryo development between the groups. Corresponding phospholipid ion intensity was expressed in arbitrary units, and following principal components analyses (PCA) the data were distributed on a 3D graph. Oocytes obtained from superstimulated animals showed a greater rate of developmental (P<0.05) at D7 (MII = 62.4±17.5% and FSH = 58.8±16.1%) compared to those obtained from unstimulated animals (CONT = 37.9±8.5% and IMA = 50.6±14.4%). However, the maturation system did not affect the resistance of oocytes to vitrification because the blastocyst rate at D7 was similar (P>0.05) for all groups (CONT VIT = 2.8±3.5%, IMA VIT = 2.9±4.0%, FSH VIT = 4.3±7.2% and MII VIT = 3.6±7.2%). MALDI-TOF revealed that oocytes from all maturation groups had similar phospholipid contents, except for 760.6 ([PC (34:1) + H]+), which was more highly expressed in MII compared to FSH (P<0.05). The results suggest that although maturation systems improve embryonic development, they do not change the PM composition nor the resistance of bovine oocytes to vitrification. PMID:26107169

IGF-I slightly improves nuclear maturation and cleavage rate of bovine oocytes exposed to acute heat shock in vitro.

PubMed

Meiyu, Qi; Liu, Di; Roth, Zvi

2015-08-01

An in vitro model of embryo production was used to examine the effects of insulin-like growth factor (IGF)-I on maturation and developmental competence of oocytes exposed to heat shock. Cumulus-oocyte complexes were matured at 38.5°C or exposed to acute heat shock (HS; 41.5°C), with or without 100 ng/ml IGF-I, for 22 h through in vitro maturation. The experimental groups were control (C), C + IGF-I, HS, and HS + IGF-I. Oocytes were fertilized at the end of maturation, and the proportion of cleaved embryos was recorded 44 h later. HS during maturation increased the proportion of TUNEL-positive oocytes (P < 0.05). HS did not have any effect on cortical granule translocation but impaired resumption of meiosis, expressed as a decreased proportion of oocytes with nuclei in metaphase I (P < 0.05) and metaphase II (MII; P < 0.05). HS decreased the proportion of oocytes that cleaved (P < 0.05), in particular those oocytes that further developed to 4-cell-stage embryos (P < 0.05). IGF-I alleviated, to some extent, the deleterious effects of HS on the oocytes as reflected by a reduced proportion of TUNEL-positive oocytes (P < 0.03). While not significant, IGF-I tended to increase the proportion of MII-stage oocytes (P < 0.08) and 4-cell-stage cleaved embryos (P < 0.06). Further examination is required to explore whether IGF-I also affects the developmental competence of oocytes exposed to HS.

Oxidative stress as a damage mechanism in porcine cumulus-oocyte complexes exposed to malathion during in vitro maturation.

PubMed

Flores, Diana; Souza, Verónica; Betancourt, Miguel; Teteltitla, Mario; González-Márquez, Humberto; Casas, Eduardo; Bonilla, Edmundo; Ramírez-Noguera, Patricia; Gutiérrez-Ruíz, María Concepción; Ducolomb, Yvonne

2017-06-01

Malathion is one of the most commonly used insecticides. Recent findings have demonstrated that it induces oxidative stress in somatic cells, but there are not enough studies that have demonstrated this effect in germ cells. Malathion impairs porcine oocyte viability and maturation, but studies have not shown how oxidative stress damages maturation and which biochemical mechanisms are affected in this process in cumulus-oocyte complexes (COCs). The aims of the present study were to determine the amount of oxidative stress produced by malathion in porcine COCs matured in vitro, to define how biochemical mechanisms affect this process, and determine whether trolox can attenuate oxidative damage. Sublethal concentrations 0, 750, and 1000 µM were used to evaluate antioxidant enzyme expressions, reactive oxygen species (ROS production), protein oxidation, and lipid peroxidation, among other oxidation products. COCs viability and oocyte maturation decreased in a concentration-dependent manner. Malathion increased Cu, Zn superoxide dismutase (SOD1), glutathione-S-transferase (GST), and glucose 6 phosphate dehydrogenase (G6PD) protein level and decreased glutathione peroxidase (GSH-Px) and catalase (CAT) protein level. Species reactives of oxygen (ROS), protein oxidation and Thiobarbituric acid reactive substances (TBARS) levels increased in COCs exposed to the insecticide, but when COCs were pre-treated with the trolox (50 µM) 30 min before and during malathion exposure, these parameters decreased down to control levels. This study showed that malathion has a detrimental effect on COCs during in vitro maturation, inducing oxidative stress, while trolox attenuated malathion toxicity by decreasing oxidative damage. © 2017 Wiley Periodicals, Inc.

Collecting near mature and immature orchid seeds for ex situ conservation: 'in vitro collecting' as a case study.

PubMed

Kendon, Jonathan P; Rajaovelona, Landy; Sandford, Helen; Fang, Rui; Bell, Jake; Sarasan, Viswambharan

2017-08-08

Lack of phenological information and efficient collecting methods are considered impediments for orchid seed collecting. This leads to opportunistic collecting as part of general seed collecting schedules that may last few weeks especially in remote areas. The study explored the feasibility of collecting near mature and immature seeds to support conservation action plans. Mature, near mature and immature seeds of orchids were collected from the wild in the Central Highlands of Madagascar (CHM). Seed capsules were collected in sterile culture medium in the wild, to prevent deterioration of seeds inside the capsule after collecting, later to be cultured under laboratory conditions. Seed capsules collected by the in vitro collecting (IVC) method were kept in very good condition for up to 4 weeks before germination under in vitro conditions. Significantly faster and higher germination rate (p < 0.001) than mature seeds was observed in a number of taxa collected during a 3 year-long study. In some taxa even immature seeds, with no apparent sign of testa covering the embryo, germinated following IVC where mature seeds failed to germinate. We propose that IVC method has potential to complement conventional seed collecting by increasing the germplasm that can be used in integrated conservation action plans. Improvements can be made in developing collections for taxa from biodiversity hotspots and remote areas where collecting requires considerable resources. This method can further be used on a wider selection of plants from different geographic areas and on embryo rescue programmes for economically important plants.

Effects of sorbitol on porcine oocyte maturation and embryo development in vitro.

PubMed

Lin, Tao; Zhang, Jin Yu; Diao, Yun Fei; Kang, Jung Won; Jin, Dong-Il

2015-04-01

In the present study, a porcine system was supplemented with sorbitol during in vitro maturation (IVM) or in vitro culture (IVC), and the effects of sorbitol on oocyte maturation and embryonic development following parthenogenetic activation were assessed. Porcine immature oocytes were treated with different concentrations of sorbitol during IVM, and the resultant metaphase II stage oocytes were activated and cultured in porcine zygote medium-3 (PZM-3) for 7 days. No significant difference was observed in cumulus expansion and the nuclear maturation between the control and sorbitol-treated groups, with the exception of the 100 mM group, which showed significantly decreased nuclear maturation and cumulus expansion. There was no significant difference in the intracellular reactive oxygen species (ROS) levels between oocytes matured with 10 or 20 mM sorbitol and control groups, but 50 and 100 mM groups had significantly higher ROS levels than other groups. The 20 mM group showed significant increases in intracellular glutathione and subsequent blastocyst formation rates following parthenogenetic activation compared with the other groups. During IVC, supplementation with sorbitol significantly reduced blastocyst formation and increased the apoptotic index compared with the control. The apoptotic index of blastocysts from the sorbitol-treated group for entire culture period was significantly higher than those of the partially sorbitol-exposed groups. Based on these findings, it can be concluded that the addition of a low concentration of sorbitol (20 mM) during IVM of porcine oocytes benefits subsequent blastocyst development and improves embryo quality, whereas sorbitol supplement during IVC has a negative effect on blastocyst formation.

Identification of 17,20beta,21-trihydroxy-4-pregnen-3-one as an oocyte maturation-inducing steroid in black porgy, Acanthopagrus schlegeli.

PubMed

Yueh, Wen-Shiun; Thomas, Peter; Chang, Ching-Fong

2005-02-01

The identity of the maturation-inducing steroid (MIS) in black porgy, Acanthopagrus schlegeli, a marine protandrous teleost, is unknown. Previous studies demonstrated that two teleost MISs, the progestins 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S) and 17,20beta-dihydroxy-4-pregnen-3-one (DHP) can induce maturation of black porgy oocytes in vitro. The purpose of the present study was to identify the major progestin produced during oocyte maturation (OM) in black porgy and investigate whether its secretion increases during this process. Females were injected twice with a LHRH analog to induce OM. Ovarian follicles undergoing OM were incubated in vitro with tritiated [3H]pregnenolone precursor and the tritiated products were extracted, purified, and identified by HPLC, TLC, acetylation, and recrystallization. Significant amounts of tritiated products were biosynthesized from [3H]pregnenolone that co-migrated with 20beta-S but not with DHP on HPLC and TLC. Similar TLC profiles were obtained with the tritiated products isolated from the HPLC/TLC 20beta-S fraction and standard 20beta-S after the acetylation reaction. The identity of the tritiated products as 20beta-S was confirmed by recrystallization. 20beta-S had a slightly higher potency than DHP in the inducing in vitro final oocyte maturation. Plasma 20beta-S concentrations increased significantly during the oocyte maturation after injection with a LHRH analog. The present data suggest that 20beta-S is the MIS in black porgy.

Influence of In Vitro and In Vivo Oxygen Modulation on β Cell Differentiation From Human Embryonic Stem Cells

PubMed Central

Cechin, Sirlene; Álvarez-Cubela, Silvia; Giraldo, Jaime A.; Molano, Ruth D.; Villate, Susana; Ricordi, Camillo; Pileggi, Antonello; Inverardi, Luca

2014-01-01

The possibility of using human embryonic stem (hES) cell-derived β cells as an alternative to cadaveric islets for the treatment of type 1 diabetes is now widely acknowledged. However, current differentiation methods consistently fail to generate meaningful numbers of mature, functional β cells. In order to address this issue, we set out to explore the role of oxygen modulation in the maturation of pancreatic progenitor (PP) cells differentiated from hES cells. We have previously determined that oxygenation is a powerful driver of murine PP differentiation along the endocrine lineage of the pancreas. We hypothesized that targeting physiological oxygen partial pressure (pO2) levels seen in mature islets would help the differentiation of PP cells along the β-cell lineage. This hypothesis was tested both in vivo (by exposing PP-transplanted immunodeficient mice to a daily hyperbaric oxygen regimen) and in vitro (by allowing PP cells to mature in a perfluorocarbon-based culture device designed to carefully adjust pO2 to a desired range). Our results show that oxygen modulation does indeed contribute to enhanced maturation of PP cells, as evidenced by improved engraftment, segregation of α and β cells, body weight maintenance, and rate of diabetes reversal in vivo, and by elevated expression of pancreatic endocrine makers, β-cell differentiation yield, and insulin production in vitro. Our studies confirm the importance of oxygen modulation as a key variable to consider in the design of β-cell differentiation protocols and open the door to future strategies for the transplantation of fully mature β cells. PMID:24375542

Glycopeptide Analogues of PSGL-1 Inhibit P-Selectin In Vitro and In Vivo

PubMed Central

Krishnamurthy, Venkata R; Sardar, Mohammed Y. R.; Yu, Ying; Song, Xuezheng; Haller, Carolyn; Dai, Erbin; Wang, Xiacong; Hanjaya-Putra, Donny; Sun, Lijun; Morikis, Vasilios; Simon, Scott I.; Woods, Robert; Cummings, Richard D.; Chaikof, Elliot L.

2015-01-01

Blockade of P-selectin/PSGL-1 interactions holds significant potential for treatment of disorders of innate immunity, thrombosis, and cancer. Current inhibitors remain limited due to low binding affinity or by the recognized disadvantages inherent to chronic administration of antibody therapeutics. Here we report an efficient approach for generating glycosulfopeptide mimics of N-terminal PSGL-1 through development of a stereoselective route for multi-gram scale synthesis of the C2 O-glycan building block and replacement of hydrolytically labile tyrosine sulfates with isosteric sulfonate analogs. Library screening afforded a compound of exceptional stability, GSnP-6, that binds to human P-selectin with nanomolar affinity (Kd ~ 22 nM). Molecular dynamics simulation defines the origin of this affinity in terms of a number of critical structural contributions. GSnP-6 potently blocks P-selectin/PSGL-1 interactions in vitro and in vivo and represents a promising candidate for the treatment of diseases driven by acute and chronic inflammation. PMID:25824568

Production of a soluble single-chain variable fragment antibody against okadaic acid and exploration of its specific binding.

PubMed

He, Kuo; Zhang, Xiuyuan; Wang, Lixia; Du, Xinjun; Wei, Dong

2016-06-15

Okadaic acid is a lipophilic marine algal toxin commonly responsible for diarrhetic shellfish poisoning (DSP). Outbreaks of DSP have been increasing and are of worldwide public health concern; therefore, there is a growing demand for more rapid, reliable, and economical analytical methods for the detection of this toxin. In this study, anti-okadaic acid single-chain variable fragment (scFv) genes were prepared by cloning heavy and light chain genes from hybridoma cells, followed by fusion of the chains via a linker peptide. An scFv-pLIP6/GN recombinant plasmid was constructed and transformed into Escherichia coli for expression, and the target scFv was identified with IC-CLEIA (chemiluminescent enzyme immunoassay). The IC15 was 0.012 ± 0.02 μg/L, and the IC50 was 0.25 ± 0.03 μg/L. The three-dimensional structure of the scFv was simulated with computer modeling, and okadaic acid was docked to the scFv model to obtain a putative structure of the binding complex. Two predicted critical amino acids, Ser32 and Thr187, were then mutated to verify this theoretical model. Both mutants exhibited significant loss of binding activity. These results help us to understand this specific scFv-antigen binding mechanism and provide guidance for affinity maturation of the antibody in vitro. The high-affinity scFv developed here also has potential for okadaic acid toxin detection. Copyright © 2016. Published by Elsevier Inc.

Targeting tumor cells via EGF receptors: selective toxicity of an HBEGF-toxin fusion protein.

PubMed

Chandler, L A; Sosnowski, B A; McDonald, J R; Price, J E; Aukerman, S L; Baird, A; Pierce, G F; Houston, L L

1998-09-25

Over-expression of the epidermal growth factor receptor (EGFR) is a hallmark of numerous solid tumors, thus providing a means of selectively targeting therapeutic agents. Heparin-binding epidermal growth factor (HBEGF) binds to EGFRs with high affinity and to heparan sulfate proteoglycans, resulting in increased mitogenic potential compared to other EGF family members. We have investigated the feasibility of using HBEGF to selectively deliver a cytotoxic protein into EGFR-expressing tumor cells. Recombinant fusion proteins consisting of mature human HBEGF fused to the plant ribosome-inactivating protein saporin (SAP) were expressed in Escherichia coli. Purified HBEGF-SAP chimeras inhibited protein synthesis in a cell-free assay and competed with EGF for binding to receptors on intact cells. A construct with a 22-amino-acid flexible linker (L22) between the HBEGF and SAP moieties exhibited an affinity for the EGFR that was comparable to that of HBEGF. The sensitivity to HBEGF-L22-SAP was determined for a variety of human tumor cell lines, including the 60 cell lines comprising the National Cancer Institute Anticancer Drug Screen. HBEGF-L22-SAP was cytotoxic in vitro to a variety of EGFR-bearing cell lines and inhibited growth of EGFR-over-expressing human breast carcinoma cells in vivo. In contrast, the fusion protein had no effect on small-cell lung carcinoma cells, which are EGFR-deficient. Our results demonstrate that fusion proteins composed of HBEGF and SAP exhibit targeting specificity and cytotoxicity that may be of therapeutic value in treating a variety of EGFR-bearing malignancies.

Generation of “LYmph Node Derived Antibody Libraries” (LYNDAL) for selecting fully human antibody fragments with therapeutic potential

PubMed Central

Diebolder, Philipp; Keller, Armin; Haase, Stephanie; Schlegelmilch, Anne; Kiefer, Jonathan D; Karimi, Tamana; Weber, Tobias; Moldenhauer, Gerhard; Kehm, Roland; Eis-Hübinger, Anna M; Jäger, Dirk; Federspil, Philippe A; Herold-Mende, Christel; Dyckhoff, Gerhard; Kontermann, Roland E; Arndt, Michaela AE; Krauss, Jürgen

2014-01-01

The development of efficient strategies for generating fully human monoclonal antibodies with unique functional properties that are exploitable for tailored therapeutic interventions remains a major challenge in the antibody technology field. Here, we present a methodology for recovering such antibodies from antigen-encountered human B cell repertoires. As the source for variable antibody genes, we cloned immunoglobulin G (IgG)-derived B cell repertoires from lymph nodes of 20 individuals undergoing surgery for head and neck cancer. Sequence analysis of unselected “LYmph Node Derived Antibody Libraries” (LYNDAL) revealed a naturally occurring distribution pattern of rearranged antibody sequences, representing all known variable gene families and most functional germline sequences. To demonstrate the feasibility for selecting antibodies with therapeutic potential from these repertoires, seven LYNDAL from donors with high serum titers against herpes simplex virus (HSV) were panned on recombinant glycoprotein B of HSV-1. Screening for specific binders delivered 34 single-chain variable fragments (scFvs) with unique sequences. Sequence analysis revealed extensive somatic hypermutation of enriched clones as a result of affinity maturation. Binding of scFvs to common glycoprotein B variants from HSV-1 and HSV-2 strains was highly specific, and the majority of analyzed antibody fragments bound to the target antigen with nanomolar affinity. From eight scFvs with HSV-neutralizing capacity in vitro, the most potent antibody neutralized 50% HSV-2 at 4.5 nM as a dimeric (scFv)2. We anticipate our approach to be useful for recovering fully human antibodies with therapeutic potential. PMID:24256717

Generation of “LYmph Node Derived Antibody Libraries” (LYNDAL) for selecting fully human antibody fragments with therapeutic potential.

PubMed

Diebolder, Philipp; Keller, Armin; Haase, Stephanie; Schlegelmilch, Anne; Kiefer, Jonathan D; Karimi, Tamana; Weber, Tobias; Moldenhauer, Gerhard; Kehm, Roland; Eis-Hübinger, Anna M; Jäger, Dirk; Federspil, Philippe A; Herold-Mende, Christel; Dyckhoff, Gerhard; Kontermann, Roland E; Arndt, Michaela A E; Krauss, Jürgen

2014-01-01

The development of efficient strategies for generating fully human monoclonal antibodies with unique functional properties that are exploitable for tailored therapeutic interventions remains a major challenge in the antibody technology field. Here, we present a methodology for recovering such antibodies from antigen-encountered human B cell repertoires. As the source for variable antibody genes, we cloned immunoglobulin G (IgG)-derived B cell repertoires from lymph nodes of 20 individuals undergoing surgery for head and neck cancer. Sequence analysis of unselected “LYmph Node Derived Antibody Libraries” (LYNDAL) revealed a naturally occurring distribution pattern of rearranged antibody sequences, representing all known variable gene families and most functional germline sequences. To demonstrate the feasibility for selecting antibodies with therapeutic potential from these repertoires, seven LYNDAL from donors with high serum titers against herpes simplex virus (HSV) were panned on recombinant glycoprotein B of HSV-1. Screening for specific binders delivered 34 single-chain variable fragments (scFvs) with unique sequences. Sequence analysis revealed extensive somatic hypermutation of enriched clones as a result of affinity maturation. Binding of scFvs to common glycoprotein B variants from HSV-1 and HSV-2 strains was highly specific, and the majority of analyzed antibody fragments bound to the target antigen with nanomolar affinity. From eight scFvs with HSV-neutralizing capacity in vitro,the most potent antibody neutralized 50% HSV-2 at 4.5 nM as a dimeric (scFv)2. We anticipate our approach to be useful for recovering fully human antibodies with therapeutic potential.

The pathogen-related yeast protein Pry1, a member of the CAP protein superfamily, is a fatty acid-binding protein

PubMed Central

Darwiche, Rabih; Mène-Saffrané, Laurent; Gfeller, David; Asojo, Oluwatoyin A.; Schneiter, Roger

2017-01-01

Members of the CAP superfamily (cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins), also known as SCP superfamily (sperm-coating proteins), have been implicated in many physiological processes, including immune defenses, venom toxicity, and sperm maturation. Their mode of action, however, remains poorly understood. Three proteins of the CAP superfamily, Pry1, -2, and -3 (pathogen related in yeast), are encoded in the Saccharomyces cerevisiae genome. We have shown previously that Pry1 binds cholesterol in vitro and that Pry function is required for sterol secretion in yeast cells, indicating that members of this superfamily may generally bind sterols or related small hydrophobic compounds. On the other hand, tablysin-15, a CAP protein from the horsefly Tabanus yao, has been shown to bind leukotrienes and free fatty acids in vitro. Therefore, here we assessed whether the yeast Pry1 protein binds fatty acids. Computational modeling and site-directed mutagenesis indicated that the mode of fatty acid binding is conserved between tablysin-15 and Pry1. Pry1 bound fatty acids with micromolar affinity in vitro, and its function was essential for fatty acid export in cells lacking the acyl-CoA synthetases Faa1 and Faa4. Fatty acid binding of Pry1 is independent of its capacity to bind sterols, and the two sterol- and fatty acid-binding sites are nonoverlapping. These results indicate that some CAP family members, such as Pry1, can bind different lipids, particularly sterols and fatty acids, at distinct binding sites, suggesting that the CAP domain may serve as a stable, secreted protein domain that can accommodate multiple ligand-binding sites. PMID:28365570

CAT-8015: A Second-generation Pseudomonas Exotoxin A-Based Immunotherapy Targeting CD22 -Expressing Hematological Malignancies

PubMed Central

Alderson, Ralph F.; Kreitman, Robert J.; Chen, Tianling; Yeung, Peter; Herbst, Ronald; Fox, Judy A.; Pastan, Ira

2009-01-01

Purpose To compare the in vitro and in vivo efficacy of CAT-8015, a second-generation recombinant immunotoxin composed of disulfide linked affinity matured VH and VL chains of the mouse anti-CD22 monoclonal antibody RFB4 fused to PE38, to the parental compound CAT-3888. Experimental Design The biological activity of CAT-8015 was examined in vitro using B cell tumor lines and in vivo in a JD38-based subcutaneous tumor model in NCr athymic mice. Pharmacokinetics and interspecies scaling of CAT-8015 were evaluated in mice, rats, and Cynomologus monkeys. The potential toxicity of CAT-8015 was assessed in monkeys in a toxicological study and compared to CAT-3888. Results The IC50s of CAT-8015 in vitro using the EHEB, MEC1, Daudi, CA46, and JD38 cell lines ranged from 0.3 - 8.6 ng/mL. Pharmacokinetic studies with CAT-8015 were conducted in mouse, rat and Cynomolgus monkey. The T1/2 was calculated to be 0.42, 0.61, and 0.79 hr and the Vss was 1.37, 5.57, and 140.3 mL in mouse, rat, and monkey, respectively. In vivo, when JD38 tumor-bearing animals were treated with CAT-8015 at doses ≥ 75 μg/kg at 48 hr intervals for a total of 3 doses, a rapid reduction in tumor volume and in some cases complete remission in tumor growth was observed. The comparative toxicological study showed comparable clinical and anatomical pathology changes for CAT-8015 and CAT-3888. Conclusions CAT-8015 is a CD22-targeting immunotoxin that, in preclinical studies, has greatly improved efficacy as compared to CAT-3888. PMID:19188153

Understanding mechanism of in vitro maturation, fertilization and culture of sheep embryoes through in silico analysis.

PubMed

Sreenivas, Dulam; Kaladhar, Dowluru Svgk; Samy, A Palni; Kumar, R Sangeeth

2012-01-01

Protein interations are presently required to understand the mechanisms of in vitro maturation, fertilization and culture of sheep embryoes through in silico analysis. The present work has been conducted on TCM-199 supplemented with epidermal growth factor (EGF), fetal bovine serum (FBS) or wheat peptones The maturation rate of oocyte was significantly higher in the FBS supplemented group when compared with BSA and wheat peptone supplemented groups. The in silico protein interaction studies has shown that the proteins EGFR (epidermal growth factor receptor), CCK (cholecystokinin)- a peptide hormone, Alb - a serum albumin, ESR- estrogen receptor 1, TGFA- transforming growth factor, STAT- signal transducer and FN1- fibronectin 1 has direct interaction and produces cell growth in in vitro culture. Alb is directly activates EGF and promotes MAPK3 that mediates diverse biological functions such as cell growth, adhesion and proliferation. Alb may also involve in stress response signalling and may be in cell cycle control.

The effect of glucocorticoids on ERK-1/2 phosphorylation during maturation of lamb oocytes and their subsequent fertilization and cleavage ability in vitro.

PubMed

González, Raquel; Ruiz-León, Yolanda; Gomendio, Montserrat; Roldan, Eduardo R S

2010-04-01

High levels of glucocorticoids may alter reproduction, but little is known about their direct actions on oocyte maturation, fertilization and subsequent development. Earlier work suggested negative effects of cortisol or dexamethasone on oocyte maturation but differences were noted between animal models. Both glucocorticoids reduce the p34(cdc2)-cyclin B1 complex but it is unknown if other signaling pathways important for meiosis progression are affected. In this study, using sheep oocytes as a model system, we assessed in vitro the effects of increasing concentration of glucocorticoids (0-250 microM) on oocyte maturation and underlying changes in the MAP kinase pathway, and the ability of oocytes to undergo fertilization and embryo development. Cortisol decreased oocyte maturation but only at the highest concentration, whereas dexamethasone had no effect. Fertilization and cleavage were not affected. On the other hand, both cortisol and dexamethasone inhibited ERK-1/2 activation in a concentration-dependent manner. It thus seems that oocytes can overcome deleterious effects of glucocorticoids during maturation despite the decrease in ERK-1/2 activity, but repercussions in vivo should be further explored. Copyright 2009 Elsevier Inc. All rights reserved.

Quercetin improves the in vitro development of porcine oocytes by decreasing reactive oxygen species levels

PubMed Central

Kang, Jung-Taek; Kwon, Dae-Kee; Park, Sol-Ji; Kim, Su-Jin; Moon, Joon-Ho; Koo, Ok-Jae; Jang, Goo

2013-01-01

Quercetin is a plant-derived flavonoid found in fruits or vegetables that has antioxidant properties and acts as a free radical scavenger. We investigated the effects of quercetin on porcine oocyte nuclear maturation and embryonic development after parthenogenetic activation. We then evaluated the antioxidant activities of quercetin by measuring reactive oxygen species (ROS) levels in matured oocytes. Immature oocytes were untreated or treated with 1, 10, and 50 µg/mL quercetin during in vitro maturation (IVM). Quercetin treatment did not improve oocyte nuclear maturation, but significantly higher blastocyst rates (p < 0.05) of parthenogenetically activated oocytes were achieved when the IVM medium was supplemented with an adequate concentration of quercetin (1 µg/mL). However, cleavage rates and blastocyst cell numbers were not affected. Oocytes treated with 1 or 10 µg/mL quercetin had significantly lower (p < 0.05) levels of ROS than the control and group treated with the highest concentration of quercetin (50 µg/mL). Moreover, this highest concentration was detrimental to oocyte nuclear maturation and blastocyst formation. Based on our findings, we concluded that exogenous quercetin reduces ROS levels during oocyte maturation and is beneficial for subsequent embryo development. PMID:23388446

Engineering epithelial-stromal interactions in vitro for toxicology assessment.

PubMed

Belair, David G; Abbott, Barbara D

2017-05-01

Crosstalk between epithelial and stromal cells drives the morphogenesis of ectodermal organs during development and promotes normal mature adult epithelial tissue homeostasis. Epithelial-stromal interactions (ESIs) have historically been examined using mammalian models and ex vivo tissue recombination. Although these approaches have elucidated signaling mechanisms underlying embryonic morphogenesis processes and adult mammalian epithelial tissue function, they are limited by the availability of tissue, low throughput, and human developmental or physiological relevance. In this review, we describe how bioengineered ESIs, using either human stem cells or co-cultures of human primary epithelial and stromal cells, have enabled the development of human in vitro epithelial tissue models that recapitulate the architecture, phenotype, and function of adult human epithelial tissues. We discuss how the strategies used to engineer mature epithelial tissue models in vitro could be extrapolated to instruct the design of organotypic culture models that can recapitulate the structure of embryonic ectodermal tissues and enable the in vitro assessment of events critical to organ/tissue morphogenesis. Given the importance of ESIs towards normal epithelial tissue development and function, such models present a unique opportunity for toxicological screening assays to incorporate ESIs to assess the impact of chemicals on mature and developing epidermal tissues. Published by Elsevier B.V.

Engineering epithelial-stromal interactions in vitro for toxicology assessment

PubMed Central

Belair, David G.; Abbott, Barbara D.

2018-01-01

Crosstalk between epithelial and stromal cells drives the morphogenesis of ectodermal organs during development and promotes normal mature adult epithelial tissue homeostasis. Epithelial-stromal interactions (ESIs) have historically been examined using mammalian models and ex vivo tissue recombination. Although these approaches have elucidated signaling mechanisms underlying embryonic morphogenesis processes and adult mammalian epithelial tissue function, they are limited by the availability of tissue, low throughput, and human developmental or physiological relevance. In this review, we describe how bioengineered ESIs, using either human stem cells or co-cultures of human primary epithelial and stromal cells, have enabled the development of human in vitro epithelial tissue models that recapitulate the architecture, phenotype, and function of adult human epithelial tissues. We discuss how the strategies used to engineer mature epithelial tissue models in vitro could be extrapolated to instruct the design of organotypic culture models that can recapitulate the structure of embryonic ectodermal tissues and enable the in vitro assessment of events critical to organ/tissue morphogenesis. Given the importance of ESIs towards normal epithelial tissue development and function, such models present a unique opportunity for toxicological screening assays to incorporate ESIs to assess the impact of chemicals on mature and developing epidermal tissues. PMID:28285100

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

Hellebrin and its aglycone form hellebrigenin display similar in vitro growth inhibitory effects in cancer cells and binding profiles to the alpha subunits of the Na+/K+-ATPase

PubMed Central

2013-01-01

Background Surface-expressed Na+/K+-ATPase (NaK) has been suggested to function as a non-canonical cardiotonic steroid-binding receptor that activates multiple signaling cascades, especially in cancer cells. By contrast, the current study establishes a clear correlation between the IC50in vitro growth inhibitory concentration in human cancer cells and the Ki for the inhibition of activity of purified human α1β1 NaK. Methods The in vitro growth inhibitory effects of seven cardiac glycosides including five cardenolides (ouabain, digoxin, digitoxin, gitoxin, uzarigenin-rhamnoside, and their respective aglycone forms) and two bufadienolides (gamabufotalin-rhamnoside and hellebrin, and their respective aglycone forms) were determined by means of the MTT colorimetric assay and hellebrigenin-induced cytotoxic effects were visualized by means of quantitative videomicroscopy. The binding affinity of ten of the 14 compounds under study was determined with respect to human α1β1, α2β1 and α3β1 NaK complexes. Lactate releases and oxygen consumption rates were also determined in cancer cells treated with these various cardiac glycosides. Results Although cardiotonic steroid aglycones usually display weaker binding affinity and in vitro anticancer activity than the corresponding glycoside, the current study demonstrates that the hellebrin / hellebrigenin pair is at odds with respect to this rule. In addition, while some cardiac steroid glycosides (e.g., digoxin), but not the aglycones, display a higher binding affinity for the α2β1 and α3β1 than for the α1β1 complex, both hellebrin and its aglycone hellebrigenin display ~2-fold higher binding affinity for α1β1 than for the α2β1 and α3β1 complexes. Finally, the current study highlights a common feature for all cardiotonic steroids analyzed here, namely a dramatic reduction in the oxygen consumption rate in cardenolide- and bufadienolide-treated cells, reflecting a direct impact on mitochondrial oxidative phosphorylation. Conclusions Altogether, these data show that the binding affinity of the bufadienolides and cardenolides under study is usually higher for the α2β1 and α3β1 than for the α1β1 NaK complex, excepted for hellebrin and its aglycone form, hellebrigenin, with hellebrigenin being as potent as hellebrin in inhibiting in vitro cancer cell growth. PMID:23621895

Synthesis and pharmacological evaluation of indole-based sigma receptor ligands

PubMed Central

Mésangeau, Christophe; Amata, Emanuele; Alsharif, Walid; Seminerio, Michael J.; Robson, Matthew J.; Matsumoto, Rae R.; Poupaert, Jacques H.; McCurdy, Christopher R.

2011-01-01

A series of novel indole-based analogues were prepared and their affinities for sigma receptors were determined using in vitro radioligand binding assays. The results of this study identified several compounds with nanomolar sigma-2 affinity and significant selectivity over sigma-1 receptors. In particular, 2-(4-(3-(4-fluorophenyl)indol-1-yl)butyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (9f) was found to display high affinity at sigma-2 receptors with good selectivity (σ-1/σ-2 = 395). The pharmacological binding profile for this compound was established with other relevant nonsigma sites. PMID:21899931

Bovine oocyte meiotic inhibition before in vitro maturation and its value to in vitro embryo production: does it improve developmental competence?

PubMed

Bilodeau-Goeseels, S

2012-08-01

The efficiency of bovine in vitro embryo production has remained low despite extensive effort to understand the effects of culture conditions, media composition and supplementation. As bovine oocytes resume meiosis spontaneously when cultured, it was hypothesized that preventing meiosis in vitro before in vitro maturation (IVM) and in vitro fertilization (IVF) would allow more oocytes to acquire developmental competence. This article reviews some of the factors involved in meiotic arrest as well as the effects of meiotic inhibition before IVM on bovine oocytes developmental competence following IVF. Follicular components and cAMP-elevating agents can delay or inhibit meiosis in various proportions of oocytes; however, few studies have examined their effects on development following IVM and IVF because they are not practical (follicular components) or have a transient effect on meiosis (cAMP-elevating agents). Protein synthesis or phosphorylation inhibition prevented meiosis in high percentages of oocytes; however, these non-specific inhibitions led to lower developmental competence compared with non-arrested oocytes. Maturation promoting factor (MPF) inhibition with specific inhibitors has been examined in several studies. Despite faster maturation following removal from inhibition and some structural damage to the oocytes, MPF inhibition generally led to blastocyst rates similar to control, non-arrested oocytes. Future work will involve evaluating the effects on arrested oocytes of molecules that can improve developmental competence in non-arrested oocytes. It is also anticipated that new IVM systems that take into consideration new knowledge of the mechanisms involved in the control of meiosis will be developed. Moreover, global gene expression analysis studies will also provide clues to the culture conditions required for optimal expression of developmental competence. © Her Majesty the Queen in Right of Canada 2011. Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada.

The Reproductive Toxicity of CdSe/ZnS Quantum Dots on the in vivo Ovarian Function and in vitro Fertilization

PubMed Central

Xu, Gaixia; Lin, Guimiao; Lin, Suxia; Wu, Na; Deng, Yueyue; Feng, Gang; Chen, Qiang; Qu, Junle; Chen, Danni; Chen, Siping; Niu, Hanben; Mei, Shujiang; Yong, Ken-Tye; Wang, Xiaomei

2016-01-01

Despite the usefulness of quantum dots (QDs) in biomedicine and optoelectronics, their toxicity risks remain a major obstacle for clinical usages. Hence, we studied the reproductive toxicity of CdSe/ZnS QDs on two aspects, (i) in vivo ovarian functions and (ii) in vitro fertilization process. The body weight, estrous cycles, biodistribution of QDs, and oocyte maturation are evaluated on female mice treated with QDs. The mRNA level of the follicle-stimulating hormone receptor (FSHr) and luteinizing hormone receptor (LHr) in ovaries are assayed. Then, the matured cumulus-oocyte-complexes are harvested to co-culture with in vitro capacitated sperms, and the in vitro fertilization is performed. The result revealed that QDs are found in the ovaries, but no changes are detected on the behavior and estrous cycle on the female mice. The mRNA downregulations of FSHr and LHr are observed and the number of matured oocytes has shown a significant decrease when the QDs dosage was above 1.0 pmol/day. Additionally, we found the presence of QDs has reduced the in vitro fertilization success rate. This study highly suggests that the exposure of CdSe/ZnS QDs to female mice can cause adverse effects to the ovary functions and such QDs may have limited applications in clinical usage. PMID:27876896

The Reproductive Toxicity of CdSe/ZnS Quantum Dots on the in vivo Ovarian Function and in vitro Fertilization.

PubMed

Xu, Gaixia; Lin, Guimiao; Lin, Suxia; Wu, Na; Deng, Yueyue; Feng, Gang; Chen, Qiang; Qu, Junle; Chen, Danni; Chen, Siping; Niu, Hanben; Mei, Shujiang; Yong, Ken-Tye; Wang, Xiaomei

2016-11-23

Despite the usefulness of quantum dots (QDs) in biomedicine and optoelectronics, their toxicity risks remain a major obstacle for clinical usages. Hence, we studied the reproductive toxicity of CdSe/ZnS QDs on two aspects, (i) in vivo ovarian functions and (ii) in vitro fertilization process. The body weight, estrous cycles, biodistribution of QDs, and oocyte maturation are evaluated on female mice treated with QDs. The mRNA level of the follicle-stimulating hormone receptor (FSHr) and luteinizing hormone receptor (LHr) in ovaries are assayed. Then, the matured cumulus-oocyte-complexes are harvested to co-culture with in vitro capacitated sperms, and the in vitro fertilization is performed. The result revealed that QDs are found in the ovaries, but no changes are detected on the behavior and estrous cycle on the female mice. The mRNA downregulations of FSHr and LHr are observed and the number of matured oocytes has shown a significant decrease when the QDs dosage was above 1.0 pmol/day. Additionally, we found the presence of QDs has reduced the in vitro fertilization success rate. This study highly suggests that the exposure of CdSe/ZnS QDs to female mice can cause adverse effects to the ovary functions and such QDs may have limited applications in clinical usage.

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.

Herbal preparation (HemoHIM) enhanced functional maturation of bone marrow-derived dendritic cells mediated toll-like receptor 4.

PubMed

Lee, Sung-Ju; Kim, Jong-Jin; Kang, Kyung-Yun; Hwang, Yun-Ho; Jeong, Gil-Yeon; Jo, Sung-kee; Jung, Uhee; Park, Hae-Ran; Yee, Sung-Tae

2016-02-19

HemoHIM, which is an herbal preparation of three edible herbs (Angelicam gigas Nakai, Cnidium offinale Makino, and Peaonia japonica Miyabe), is known to have various biological and immunological activities, but the modulatory effects of this preparation on dendritic cells (DCs)-mediated immune responses have not been examined previously. DCs are a unique group of white blood cells that initiate primary immune responses by capturing, processing, and presenting antigens to T cells. In the present study, we investigated the effect of HemoHIM on the functional and phenotypic maturation of murine bone marrow-derived dendritic cells (BMDCs) both in vitro and in vivo. The expression of co-stimulatory molecules (CD40, CD80, CD86, MHC I, and MHC II) and the production of cytokines (IL-1β, IL-6, IL-12p70, and TNF-α) were increased by HemoHIM in BMDCs. Furthermore, the antigen-uptake ability of BMDCs was decreased by HemoHIM, and the antigen-presenting ability of HemoHIM-treated mature BMDCs increased TLR4-dependent CD4(+) and CD8(+) T cell responses. Our findings demonstrated that HemoHIM induces TLR4-mediated BMDCs functional and phenotypic maturation through in vivo and in vitro. And our study showed the antigen-presenting ability that HemoHIM-treated mature BMDCs increase CD4(+) and CD8(+) T cell responses by in vitro. These results suggest that HemoHIM has the potential to mediate DC immune responses.

Differentiation and Characterization of Myeloid Cells

PubMed Central

Gupta, Dipti; Shah, Hetavi Parag; Malu, Krishnakumar; Berliner, Nancy; Gaines, Peter

2015-01-01

Recent molecular studies of myeloid differentiation have utilized several in vitro models of myelopoiesis, generated from either ex vivo differentiated bone marrow progenitors or induced immortalized myeloid cell lines. Ex vivo differentiation begins with an enriched population of bone marrow-derived hematopoietic stem cells generated by lineage depletion and/or positive selection for CD34+ antigen (human) or Sca-1+ (mouse) cells, which are then expanded and subsequently induced in vitro in a process that recapitulates normal myeloid development. Myeloid cell lines include two human leukemic cell lines, NB-4 and HL-60, which have been demonstrated to undergo retinoic acid–induced myeloid development, however, both cell lines exhibit defects in the upregulation of late-expressed neutrophil-specific genes. Multiple murine factor–dependent cell models of myelopoiesis are also available that express the full range of neutrophil maturation markers, including: 32Dcl3 cells, which undergo G-CSF-induced myeloid maturation, EML/EPRO cells, which develop into mature neutrophils in response to cytokines and retinoic acid, and ER-Hoxb8 cells, which undergo myeloid maturation upon removal of estradial in the maintenance medium. In this unit, the induction of myeloid maturation in each of these model systems is described, including their differentiation to either neutrophils or macrophages, if applicable. Commonly used techniques to test for myeloid characteristics of developing cells are also described, including flow cytometry and real time RT-PCR. Together, these assays provide a solid foundation for in vitro investigations of myeloid development with either human or mouse models. PMID:24510620

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

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

Cytotoxicity of seven bisphenol analogues compared to bisphenol A and relationships with membrane affinity data.

PubMed

Russo, Giacomo; Capuozzo, Antonella; Barbato, Francesco; Irace, Carlo; Santamaria, Rita; Grumetto, Lucia

2018-06-01

Bisphenol A (BPA) is a chemical used in numerous industrial applications. Due to its well ascertained toxicity as endocrine disruptor, industries have started to replace it with other bisphenols whose alleged greater safety is scarcely supported by literature studies. In this study, the toxicity of seven BPA analogues was evaluated using both in silico and in vitro techniques, as compared to BPA toxicity. Furthermore, their affinity indexes for phospholipids (i.e. phospholipophilicity) were determined by immobilized artificial membrane liquid chromatography (IAM-LC) and possible relationships with in vitro toxic activity were also investigated. The results on four different cell cultures yielded similar ranking of toxicity for the bisphenols considered, with IC 50 values confirming their poor acute toxicity. As compared to BPA, bisphenol AF, bisphenol B, bisphenol M, and bisphenol A diglycidyl ether resulted more toxic, while bisphenol S, bisphenol F and bisphenol E were found as the less toxic congeners. These results are partly consistent with the scale of phospholipid affinity showing that toxicity increases at increasing membrane affinity. Therefore, phospholipophilicity determination can be assumed as a useful preliminary tool to select less toxic congeners to surrogate BPA in industrial applications. Copyright © 2018 Elsevier Ltd. All rights reserved.

Cytokine refacing effect reduces granulocyte macrophage colony-stimulating factor susceptibility to antibody neutralization

PubMed Central

Heinzelman, Pete; Carlson, Sharon J.; Cox, George N.

2015-01-01

Crohn's Disease (CD) afflicts over half a million Americans with an annual economic impact exceeding $10 billion. Granulocyte macrophage colony-stimulating factor (GM-CSF) can increase patient immune responses against intestinal microbes that promote CD and has been effective for some patients in clinical trials. We have made important progress toward developing GM-CSF variants that could be more effective CD therapeutics by virtue of being less prone to neutralization by the endogenous GM-CSF autoantibodies that are highly expressed in CD patients. Yeast display engineering revealed mutations that increase GM-CSF variant binding affinity by up to ∼3-fold toward both GM-CSF receptor alpha and beta subunits in surface plasmon resonance experiments. Increased binding affinity did not reduce GM-CSF half-maximum effective concentration (EC50) values in conventional in vitro human leukocyte proliferation assays. Affinity-enhancing mutations did, however, promote a ‘refacing effect’ that imparted all five evaluated GM-CSF variants with increased in vitro bioactivity in the presence of GM-CSF-neutralizing polyclonal antisera. The most improved variant, H15L/R23L, was 6-fold more active than wild-type GM-CSF. Incorporation of additional known affinity-increasing mutations could augment the refacing effect and concomitant bioactivity improvements described here. PMID:25855658

Effects of polyunsaturated fatty acids and prostaglandins on oocyte maturation in a marine teleost, the European sea bass (Dicentrarchus labrax).

PubMed

Sorbera, L A; Asturiano, J F; Carrillo, M; Zanuy, S

2001-01-01

The effects of the polyunsaturated fatty acids (PUFAs), arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and prostaglandins (PGs) on oocyte maturation were investigated in a marine teleost, the sea bass (Dicentrarchus labrax). Follicle-enclosed postvitellogenic, preovulatory oocytes were cultured in vitro and maturation was verified by assessing volume increase, lipid droplet coalescence, yolk clarification, and germinal vesicle migration and breakdown. Human chorionic gonadotropin was administered as the maturation-inducing gonadotropin (GTH) and was capable of inducing maturation in a time- and dose-dependent manner. Free AA induced maturation in a dose- and time-dependent manner and enhanced GTH-induced maturation, while EPA, DHA, and oleic acid were ineffective. Maturation induced by GTH was significantly suppressed by a phospholipase A(2) blocker, suggesting that mobilization of AA was involved in GTH-induced maturation. Moreover, EPA and DHA exhibited a significant, dose-dependent attenuation of GTH-induced maturation. Maturation induced by GTH was inhibited in the presence of a cyclooxygenase inhibitor, indomethacin, and this inhibition was reversed by addition of AA, PGE(2), or PGF(2alpha). PGE(2) and PGF(2alpha) alone were both effective stimulators of maturation, while PGE(1) and PGE(3) were ineffective. The effect of PUFAs on oocyte maturation in vitro were corroborated with studies in vivo. Oocytes were obtained from females fed a commercial, PUFA-enriched diet (RD) and maturational behavior was compared with oocytes from females fed a natural diet (ND) with a higher EPA content and n-3:n-6 ratio. Although no significant difference was observed in the rate of spontaneous oocyte maturation, a higher percentage of GTH-induced maturation and lower percentage of atresia were observed in RD oocytes. Moreover, while basal PGE production from oocytes from both groups was the same, RD oocytes produced significantly higher levels of PGE in the presence of hCG. The results from this study provide evidence for the participation of AA metabolism in GTH-induced oocyte maturation, and suggest that other PUFAs and PGs may play important roles in the induction of maturation in a marine teleost.

Precision cancer immunotherapy: optimizing dendritic cell-based strategies to induce tumor antigen-specific T-cell responses against individual patient tumors.

PubMed

Osada, Takuya; Nagaoka, Koji; Takahara, Masashi; Yang, Xiao Yi; Liu, Cong-Xiao; Guo, Hongtao; Roy Choudhury, Kingshuk; Hobeika, Amy; Hartman, Zachary; Morse, Michael A; Lyerly, H Kim

2015-05-01

Most dendritic cell (DC)-based vaccines have loaded the DC with defined antigens, but loading with autologos tumor-derived antigens would generate DCs that activate personalized tumor-specific T-cell responses. We hypothesized that DC matured with an optimized combination of reagents and loaded with tumor-derived antigens using a clinically feasible electroporation strategy would induce potent antitumor immunity. We first studied the effects on DC maturation and antigen presentation of the addition of picibanil (OK432) to a combination of zoledronic acid, tumor necrosis factor-α, and prostaglandin E2. Using DC matured with the optimized combination, we tested 2 clinically feasible sources of autologous antigen for electroloading, total tumor mRNA or total tumor lysate, to determine which stimulated more potent antigen-specific T cells in vitro and activated more potent antitumor immunity in vivo. The combination of tumor necrosis factor-α/prostaglandin E2/zoledronic acid/OK432 generated DC with high expression of maturation markers and antigen-specific T-cell stimulatory function in vitro. Mature DC electroloaded with tumor-derived mRNA [mRNA electroporated dendritic cell (EPDC)] induced greater expansion of antigen-specific T cells in vitro than DC electroloaded with tumor lysate (lysate EPDC). In a therapeutic model of MC38-carcinoembryonic antigen colon cancer-bearing mice, vaccination with mRNA EPDC induced the most efficient anti-carcinoembryonic antigen cellular immune response, which significantly suppressed tumor growth. In conclusion, mature DC electroloaded with tumor-derived mRNA are a potent cancer vaccine, especially useful when specific tumor antigens for vaccination have not been identified, allowing autologous tumor, and if unavailable, allogeneic cell lines to be used as an unbiased source of antigen. Our data support clinical testing of this strategy.

A Single-Stranded DNA Aptamer That Selectively Binds to Staphylococcus aureus Enterotoxin B

PubMed Central

DeGrasse, Jeffrey A.

2012-01-01

The bacterium Staphylococcus aureus is a common foodborne pathogen capable of secreting a cocktail of small, stable, and strain-specific, staphylococcal enterotoxins (SEs). Staphylococcal food poisoning (SFP) results when improperly handled food contaminated with SEs is consumed. Gastrointestinal symptoms of SFP include emesis, diarrhea and severe abdominal pain, which manifest within hours of ingesting contaminated food. Immuno-affinity based methods directly detect, identify, and quantify several SEs within a food or clinical sample. However, the success of these assays depends upon the availability of a monoclonal antibody, the development of which is non-trivial and costly. The current scope of the available immuno-affinity based methods is limited to the classical SEs and does not encompass all of the known or emergent SEs. In contrast to antibodies, aptamers are short nucleic acids that exhibit high affinity and specificity for their targets without the high-costs and ethical concerns of animal husbandry. Further, researchers may choose to freely distribute aptamers and develop assays without the proprietary issues that increase the per-sample cost of immuno-affinity assays. This study describes a novel aptamer, selected in vitro, with affinity to staphylococcal enterotoxin B (SEB) that may be used in lieu of antibodies in SE detection assays. The aptamer, designated APTSEB1, successfully isolates SEB from a complex mixture of SEs with extremely high discrimination. This work sets the foundation for future aptamer and assay development towards the entire family of SEs. The rapid, robust, and low-cost identification and quantification of all of the SEs in S. aureus contaminated food is essential for food safety and epidemiological efforts. An in vitro generated library of SE aptamers could potentially allow for the comprehensive and cost-effective analysis of food samples that immuno-affinity assays currently cannot provide. PMID:22438927

A single-stranded DNA aptamer that selectively binds to Staphylococcus aureus enterotoxin B.

PubMed

DeGrasse, Jeffrey A

2012-01-01

The bacterium Staphylococcus aureus is a common foodborne pathogen capable of secreting a cocktail of small, stable, and strain-specific, staphylococcal enterotoxins (SEs). Staphylococcal food poisoning (SFP) results when improperly handled food contaminated with SEs is consumed. Gastrointestinal symptoms of SFP include emesis, diarrhea and severe abdominal pain, which manifest within hours of ingesting contaminated food. Immuno-affinity based methods directly detect, identify, and quantify several SEs within a food or clinical sample. However, the success of these assays depends upon the availability of a monoclonal antibody, the development of which is non-trivial and costly. The current scope of the available immuno-affinity based methods is limited to the classical SEs and does not encompass all of the known or emergent SEs. In contrast to antibodies, aptamers are short nucleic acids that exhibit high affinity and specificity for their targets without the high-costs and ethical concerns of animal husbandry. Further, researchers may choose to freely distribute aptamers and develop assays without the proprietary issues that increase the per-sample cost of immuno-affinity assays. This study describes a novel aptamer, selected in vitro, with affinity to staphylococcal enterotoxin B (SEB) that may be used in lieu of antibodies in SE detection assays. The aptamer, designated APT(SEB1), successfully isolates SEB from a complex mixture of SEs with extremely high discrimination. This work sets the foundation for future aptamer and assay development towards the entire family of SEs. The rapid, robust, and low-cost identification and quantification of all of the SEs in S. aureus contaminated food is essential for food safety and epidemiological efforts. An in vitro generated library of SE aptamers could potentially allow for the comprehensive and cost-effective analysis of food samples that immuno-affinity assays currently cannot provide.

The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy

PubMed Central

Honigberg, Lee A.; Smith, Ashley M.; Sirisawad, Mint; Verner, Erik; Loury, David; Chang, Betty; Li, Shyr; Pan, Zhengying; Thamm, Douglas H.; Miller, Richard A.; Buggy, Joseph J.

2010-01-01

Activation of the B-cell antigen receptor (BCR) signaling pathway contributes to the initiation and maintenance of B-cell malignancies and autoimmune diseases. The Bruton tyrosine kinase (Btk) is specifically required for BCR signaling as demonstrated by human and mouse mutations that disrupt Btk function and prevent B-cell maturation at steps that require a functional BCR pathway. Herein we describe a selective and irreversible Btk inhibitor, PCI-32765, that is currently under clinical development in patients with B-cell non-Hodgkin lymphoma. We have used this inhibitor to investigate the biologic effects of Btk inhibition on mature B-cell function and the progression of B cell-associated diseases in vivo. PCI-32765 blocked BCR signaling in human peripheral B cells at concentrations that did not affect T cell receptor signaling. In mice with collagen-induced arthritis, orally administered PCI-32765 reduced the level of circulating autoantibodies and completely suppressed disease. PCI-32765 also inhibited autoantibody production and the development of kidney disease in the MRL-Fas(lpr) lupus model. Occupancy of the Btk active site by PCI-32765 was monitored in vitro and in vivo using a fluorescent affinity probe for Btk. Active site occupancy of Btk was tightly correlated with the blockade of BCR signaling and in vivo efficacy. Finally, PCI-32765 induced objective clinical responses in dogs with spontaneous B-cell non-Hodgkin lymphoma. These findings support Btk inhibition as a therapeutic approach for the treatment of human diseases associated with activation of the BCR pathway. PMID:20615965

Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids.

PubMed

Seet, Christopher S; He, Chongbin; Bethune, Michael T; Li, Suwen; Chick, Brent; Gschweng, Eric H; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B; Baltimore, David; Crooks, Gay M; Montel-Hagen, Amélie

2017-05-01

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3 + TCR-αβ + single-positive CD8 + or CD4 + cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen-specific cytotoxicity and near-complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ-encoding loci. ATOs provide a robust tool for studying human T cell differentiation and for the future development of stem-cell-based engineered T cell therapies.

Generation of mature T cells from human hematopoietic stem/progenitor cells in artificial thymic organoids

PubMed Central

Seet, Christopher S.; He, Chongbin; Bethune, Michael T.; Li, Suwen; Chick, Brent; Gschweng, Eric H.; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B.; Baltimore, David; Crooks, Gay M.; Montel-Hagen, Amélie

2017-01-01

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3+TCRab+ single positive (SP) CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports highly efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naïve phenotypes, a diverse TCR repertoire, and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen specific cytotoxicity and near complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ loci. ATOs provide a robust tool for studying human T cell development and stem cell based approaches to engineered T cell therapies. PMID:28369043

CXCR4 engagement promotes dendritic cell survival and maturation

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

Kabashima, Kenji; Sugita, Kazunari; Shiraishi, Noriko

2007-10-05

It has been reported that human monocyte derived-dendritic cells (DCs) express CXCR4, responsible for chemotaxis to CXCL12. However, it remains unknown whether CXCR4 is involved in other functions of DCs. Initially, we found that CXCR4 was expressed on bone marrow-derived DCs (BMDCs). The addition of specific CXCR4 antagonist, 4-F-Benzoyl-TN14003, to the culture of mouse BMDCs decreased their number, especially the mature subset of them. The similar effect was found on the number of Langerhans cells (LCs) but not keratinocytes among epidermal cell suspensions. Since LCs are incapable of proliferating in vitro, these results indicate that CXCR4 engagement is important formore » not only maturation but also survival of DCs. Consistently, the dinitrobenzene sulfonic acid-induced, antigen-specific in vitro proliferation of previously sensitized lymph node cells was enhanced by CXCL12, and suppressed by CXCR4 antagonist. These findings suggest that CXCL12-CXCR4 engagement enhances DC maturation and survival to initiate acquired immune response.« less

Redesign of a cross-reactive antibody to dengue virus with broad-spectrum activity and increased in vivo potency

PubMed Central

Tharakaraman, Kannan; Robinson, Luke N.; Hatas, Andrew; Chen, Yi-Ling; Siyue, Liu; Raguram, S.; Sasisekharan, V.; Wogan, Gerald N.; Sasisekharan, Ram

2013-01-01

Affinity improvement of proteins, including antibodies, by computational chemistry broadly relies on physics-based energy functions coupled with refinement. However, achieving significant enhancement of binding affinity (>10-fold) remains a challenging exercise, particularly for cross-reactive antibodies. We describe here an empirical approach that captures key physicochemical features common to antigen–antibody interfaces to predict protein–protein interaction and mutations that confer increased affinity. We apply this approach to the design of affinity-enhancing mutations in 4E11, a potent cross-reactive neutralizing antibody to dengue virus (DV), without a crystal structure. Combination of predicted mutations led to a 450-fold improvement in affinity to serotype 4 of DV while preserving, or modestly increasing, affinity to serotypes 1–3 of DV. We show that increased affinity resulted in strong in vitro neutralizing activity to all four serotypes, and that the redesigned antibody has potent antiviral activity in a mouse model of DV challenge. Our findings demonstrate an empirical computational chemistry approach for improving protein–protein docking and engineering antibody affinity, which will help accelerate the development of clinically relevant antibodies. PMID:23569282

Digestion products of the PH20 hyaluronidase inhibit remyelination.

PubMed

Preston, Marnie; Gong, Xi; Su, Weiping; Matsumoto, Steven G; Banine, Fatima; Winkler, Clayton; Foster, Scott; Xing, Rubing; Struve, Jaime; Dean, Justin; Baggenstoss, Bruce; Weigel, Paul H; Montine, Thomas J; Back, Stephen A; Sherman, Larry S

2013-02-01

Oligodendrocyte progenitor cells (OPCs) recruited to demyelinating lesions often fail to mature into oligodendrocytes (OLs) that remyelinate spared axons. The glycosaminoglycan hyaluronan (HA) accumulates in demyelinating lesions and has been implicated in the failure of OPC maturation and remyelination. We tested the hypothesis that OPCs in demyelinating lesions express a specific hyaluronidase, and that digestion products of this enzyme inhibit OPC maturation. Mouse OPCs grown in vitro were analyzed for hyaluronidase expression and activity. Gain of function studies were used to define the hyaluronidases that blocked OPC maturation. Mouse and human demyelinating lesions were assessed for hyaluronidase expression. Digestion products from different hyaluronidases and a hyaluronidase inhibitor were tested for their effects on OPC maturation and functional remyelination in vivo. OPCs demonstrated hyaluronidase activity in vitro and expressed multiple hyaluronidases, including HYAL1, HYAL2, and PH20. HA digestion by PH20 but not other hyaluronidases inhibited OPC maturation into OLs. In contrast, inhibiting HA synthesis did not influence OPC maturation. PH20 expression was elevated in OPCs and reactive astrocytes in both rodent and human demyelinating lesions. HA digestion products generated by the PH20 hyaluronidase but not another hyaluronidase inhibited remyelination following lysolecithin-induced demyelination. Inhibition of hyaluronidase activity lead to increased OPC maturation and promoted increased conduction velocities through lesions. We determined that PH20 is elevated in demyelinating lesions and that increased PH20 expression is sufficient to inhibit OPC maturation and remyelination. Pharmacological inhibition of PH20 may therefore be an effective way to promote remyelination in multiple sclerosis and related conditions. Copyright © 2012 American Neurological Association.

Vitrification solution containing DMSO and EG can induce parthenogenetic activation of in vitro matured ovine oocytes and decrease sperm penetration.

PubMed

Tian, Shu-Jun; Yan, Chang-Liang; Yang, Hui-Xin; Zhou, Guang-Bin; Yang, Zhong-Qiang; Zhu, Shi-En

2007-10-01

This study was designed to examine the reduced incidence of normal fertilization in vitrified ovine oocytes. After in vitro maturation for 24 h, the oocytes were randomly allocated into three groups: (1) untreated (control), (2) exposed to vitrification solution (VS) without being plunged into liquid nitrogen (toxicity), or (3) vitrified by open-pulled straw method (vitrification). In experiment 1, the treated and control oocytes were matured for another 2 h, and the oocytes were then in vitro fertilized for 12 h to examine sperm penetration. The percentage of monospermy in toxicity group (29.3%) and vitrification group (28.2%) dramatically decreased compared to the control group (45.0%) (P<0.05). To find the mechanism that the VS decreased the monospermy, some treated and control oocytes were used to test the distribution of CG and the resistance of zona pellucida (ZP) to 0.1% pronase E immediately (IVM 24 h), after another 2 h of maturation (IVM 26 h), and after 12 h of in vitro fertilization (IVF 12 h) respectively. Others were used to examine female pronucleus formation after 12 h of culture in fertilization medium with the absence of sperm. The results showed that the percentage of CG completely release in the oocytes (IVM 24 and 26 h) of toxicity group (41.2% and 39.9%) and vitrification group (41.7% and 51.7%) was significantly higher than that of control group (7.1% and 18.4%) (P<0.05). The ZP digestion duration in the oocytes (IVM 26 h) of the toxicity group (435.6 s) and vitrification group (422.3 s) was longer than that of control group (381.6 s) (P<0.05). The percentage of female pronucleus formation in toxicity group (58.7%) and vitrification group (63.9%) was higher than that (8.2%) of control group (P<0.05). The data above demonstrated that the VS containing DMSO and EG could parthenogenetically activate in vitro matured ovine oocytes, resulting in ZP hardening and decreased sperm penetration.

Drug composition matters: the influence of carrier concentration on the radiochemical purity, hydroxyapatite affinity and in-vivo bone accumulation of the therapeutic radiopharmaceutical 188Rhenium-HEDP.

PubMed

Lange, R; de Klerk, J M H; Bloemendal, H J; Ramakers, R M; Beekman, F J; van der Westerlaken, M M L; Hendrikse, N H; Ter Heine, R

2015-05-01

(188)Rhenium-HEDP is an effective bone-targeting therapeutic radiopharmaceutical, for treatment of osteoblastic bone metastases. It is known that the presence of carrier (non-radioactive rhenium as ammonium perrhenate) in the reaction mixture during labeling is a prerequisite for adequate bone affinity, but little is known about the optimal carrier concentration. We investigated the influence of carrier concentration in the formulation on the radiochemical purity, in-vitro hydroxyapatite affinity and the in-vivo bone accumulation of (188)Rhenium-HEDP in mice. The carrier concentration influenced hydroxyapatite binding in-vitro as well as bone accumulation in-vivo. Variation in hydroxyapatite binding with various carrier concentrations seemed to be mainly driven by variation in radiochemical purity. The in-vivo bone accumulation appeared to be more complex: satisfactory radiochemical purity and hydroxyapatite affinity did not necessarily predict acceptable bio-distribution of (188)Rhenium-HEDP. For development of new bisphosphonate-based radiopharmaceuticals for clinical use, human administration should not be performed without previous animal bio-distribution experiments. Furthermore, our clinical formulation of (188)Rhenium-HEDP, containing 10 μmol carrier, showed excellent bone accumulation that was comparable to other bisphosphonate-based radiopharmaceuticals, with no apparent uptake in other organs. Radiochemical purity and in-vitro hydroxyapatite binding are not necessarily predictive of bone accumulation of (188)Rhenium-HEDP in-vivo. The formulation for (188)Rhenium-HEDP as developed by us for clinical use exhibits excellent bone uptake and variation in carrier concentration during preparation of this radiopharmaceutical should be avoided. Copyright © 2015 Elsevier Inc. All rights reserved.

Synthesis, Biodistribution and In vitro Evaluation of Brain Permeable High Affinity Type 2 Cannabinoid Receptor Agonists [11C]MA2 and [18F]MA3.

PubMed

Ahamed, Muneer; van Veghel, Daisy; Ullmer, Christoph; Van Laere, Koen; Verbruggen, Alfons; Bormans, Guy M

2016-01-01

The type 2 cannabinoid receptor (CB2) is a member of the endocannabinoid system and is known for its important role in (neuro)inflammation. A PET-imaging agent that allows in vivo visualization of CB2 expression may thus allow quantification of neuroinflammation. In this paper, we report the synthesis, radiosynthesis, biodistribution and in vitro evaluation of a carbon-11 ([ 11 C]MA2) and a fluorine-18 ([ 18 F]MA3) labeled analog of a highly potent N -arylamide oxadiazole CB2 agonist (EC 50 = 0.015 nM). MA2 and MA3 behaved as potent CB2 agonist (EC 50 : 3 nM and 0.1 nM, respectively) and their in vitro binding affinity for h CB2 was found to be 87 nM and 0.8 nM, respectively. Also MA3 (substituted with a fluoro ethyl group) was found to have higher binding affinity and EC 50 values when compared to the originally reported trifluoromethyl analog 12 . [ 11 C]MA2 and [ 18 F]MA3 were successfully synthesized with good radiochemical yield, high radiochemical purity and high specific activity. In mice, both tracers were efficiently cleared from blood and all major organs by the hepatobiliary pathway and importantly these compounds showed high brain uptake. In conclusion, [ 11 C]MA2 and [ 18 F]MA3 are shown to be high potent CB2 agonists with good brain uptake, these favorable characteristics makes them potential PET probes for in vivo imaging of brain CB2 receptors. However, in view of its higher affinity and selectivity, further detailed evaluation of MA3 as a PET tracer for CB2 is warranted.

Synthesis, Biodistribution and In vitro Evaluation of Brain Permeable High Affinity Type 2 Cannabinoid Receptor Agonists [11C]MA2 and [18F]MA3

PubMed Central

Ahamed, Muneer; van Veghel, Daisy; Ullmer, Christoph; Van Laere, Koen; Verbruggen, Alfons; Bormans, Guy M.

2016-01-01

The type 2 cannabinoid receptor (CB2) is a member of the endocannabinoid system and is known for its important role in (neuro)inflammation. A PET-imaging agent that allows in vivo visualization of CB2 expression may thus allow quantification of neuroinflammation. In this paper, we report the synthesis, radiosynthesis, biodistribution and in vitro evaluation of a carbon-11 ([11C]MA2) and a fluorine-18 ([18F]MA3) labeled analog of a highly potent N-arylamide oxadiazole CB2 agonist (EC50 = 0.015 nM). MA2 and MA3 behaved as potent CB2 agonist (EC50: 3 nM and 0.1 nM, respectively) and their in vitro binding affinity for hCB2 was found to be 87 nM and 0.8 nM, respectively. Also MA3 (substituted with a fluoro ethyl group) was found to have higher binding affinity and EC50 values when compared to the originally reported trifluoromethyl analog 12. [11C]MA2 and [18F]MA3 were successfully synthesized with good radiochemical yield, high radiochemical purity and high specific activity. In mice, both tracers were efficiently cleared from blood and all major organs by the hepatobiliary pathway and importantly these compounds showed high brain uptake. In conclusion, [11C]MA2 and [18F]MA3 are shown to be high potent CB2 agonists with good brain uptake, these favorable characteristics makes them potential PET probes for in vivo imaging of brain CB2 receptors. However, in view of its higher affinity and selectivity, further detailed evaluation of MA3 as a PET tracer for CB2 is warranted. PMID:27713686

Structure-5-HT/D2 Receptor Affinity Relationship in a New Group of 1-Arylpiperazynylalkyl Derivatives of 8-Dialkylamino-3,7-dimethyl-1H-purine-2,6(3H,7H)-dione.

PubMed

Żmudzki, Paweł; Satała, Grzegorz; Chłoń-Rzepa, Grażyna; Bojarski, Andrzej J; Kazek, Grzegorz; Siwek, Agata; Gryboś, Anna; Głuch-Lutwin, Monika; Wesołowska, Anna; Pawłowski, Maciej

2016-10-01

In our previous papers, we have reported that some 8-amino-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione derivatives possessed high affinity and displayed agonistic, partial agonistic, or antagonistic activity for serotonin 5-HT 1A and dopamine D 2 receptors. In order to examine further the influence of the substituent in the position 8 of the purine moiety and the influence of the xanthine core on the affinity for serotonin 5-HT 1A , 5-HT 2A , 5-HT 6 , 5-HT 7 , and dopamine D 2 receptors, two series of 1-arylpiperazynylalkyl derivatives of 8-amino-3,7-dimethyl-1H-purine-2,6(3H,7H)-dione were synthesized. All the final compounds were investigated in in vitro competition binding experiments for the serotonin 5-HT 1A , 5-HT 2A , 5-HT 6 , 5-HT 7 , and dopamine D 2 receptors. The structure-affinity relationships for this group of compounds were discussed. For selected compounds, the functional assays for the 5-HT 1A and D 2 receptors were carried out. The results of the assays indicated that these groups of derivatives possessed antagonistic activity for 5-HT 1A receptors and agonistic, partial agonistic, or antagonistic activity for D 2 receptors. In total, 26 new compounds were synthesized, 20 of which were tested in in vitro binding experiments and 5 were tested in in vitro functional assays. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

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.

Impact of vitrification on human oocytes before and after in vitro maturation: A systematic review and meta-analysis.

PubMed

Mohsenzadeh, Mehdi; Salehi-Abargouei, Amin; Tabibnejad, Nasim; Karimi-Zarchi, Mojgan; Khalili, Mohammad Ali

2018-05-21

There are controversies regarding in vitro maturation (IVM) procedure, the time of storing frozen oocytes and maturation stage of vitrified oocytes and its impact on oocytes fertilization capability. The aim of this systematic review and meta-analysis was to evaluate the impact of vitrification on human oocytes during IVM procedure. A systematic review with meta-analysis was undertaken. Main search terms were those related key words. We searched Medline, Embase, Scopus and ISI web of science to detect English-language studies. The final search was performed on 27 January 2018. The original articles which studied laboratory outcomes after vitrification of MII or GV oocytes before or after IVM were included. Exclusion criteria were animal trials and the studies that performed cryopreservation using slow-freeze method. Oocyte maturation, survival, fertilization and cleavage rates were assessed. Bias and quality assessments were applied. 2476 articles were screened and after duplicates removing together with application of inclusion and exclusion criteria, 14 studies assessed for eligibility. Finally 5 studies included for analysis. All studies compared laboratory outcomes between oocytes that vitrified at the GV stage and those which firstly matured in vitro, and then vitrified. Meta-analysis showed that vitrification of oocytes at GV stage had a negative impact on maturation rate (RR = 1.28, 95% CI: 0.96-1.70); but not on cleavage rate (RR = 1.07, 95% CI: 0.70-1.64); fertilization rate (RR = 0.99, 95% CI: 0.85-1.14) and survival rate(RR = 1.01, 95% CI: 0.96-1.06). In general, Based on our results, oocyte vitrification decreases the maturation rate. In addition, survival, fertilization as well as cleavage rates did not significantly differ between the oocytes vitrified before IVM versus oocytes vitrified after IVM. Copyright © 2018. Published by Elsevier B.V.

Pig oocytes with a large perivitelline space matured in vitro show greater developmental competence after parthenogenesis and somatic cell nuclear transfer.

PubMed

Lee, Joohyeong; You, Jinyoung; Lee, Geun-Shik; Hyun, Sang-Hwan; Lee, Eunsong

2013-09-01

The objective of this study was to examine the developmental competence of pig oocytes in relation to the size of the perivitelline space (PVS) of oocytes matured in vitro. Immature oocytes were matured in medium 199 or porcine zygote medium (PZM)-3 containing 108 or 61.6 mM NaCl. In vitro-matured (IVM) oocytes were examined for intracellular glutathione (GSH) level; cyclin-dependent kinase 1 (CDK1), proliferating cell nuclear antigen (PCNA), and extracellular signal-regulated kinase 2 (ERK2) mRNA levels; and developmental competence after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). IVM oocytes with a larger PVS had higher (P < 0.05) levels of intracellular GSH (1.00 pixels/oocyte vs. 0.57 pixels/oocyte) and blastocyst formation (54.3% vs. 37.3%) after PA than oocytes with a smaller PVS. Culturing oocytes for maturation in PZM-3 with reduced (61.6 mM) NaCl increased (P < 0.05) the size of the PVS (6.4 µm vs. 2.8 µm) compared to control oocytes that were matured in normal PZM-3 containing 108 mM NaCl. Moreover, oocytes with a larger PVS showed higher CDK1, PCNA, and ERK2 mRNA and intracellular GSH levels (1.6 pixels/oocyte vs. 1.2 pixels/oocyte) and increased blastocyst formation after PA (52.1% vs. 40.6%) and SCNT (31.8% vs. 18.2%) than control oocytes. Our results demonstrate that pig oocytes with a large PVS have greater developmental competence after PA and SCNT, which is attributed to improved cytoplasmic maturation based on the enhanced GSH level and transcription factor expression. Further, enlargement of the PVS by culturing in low-NaCl medium improves the developmental competence of pig oocytes. Copyright © 2013 Wiley Periodicals, Inc.

Combinatorial Polymer Matrices Enhance In Vitro Maturation of Human Induced Pluripotent Cell Cell-Derived Cardiomyocytes

PubMed Central

Chun, Young Wook; Balikov, Daniel A.; Feaster, Tromondae K.; Williams, Charles H.; Sheng, Calvin C.; Lee, Jung-Bok; Boire, Timothy C.; Neely, M. Diana; Bellan, Leon M.; Ess, Kevin C.; Bowman, Aaron B.; Sung, Hak-Joon; Hong, Charles C.

2015-01-01

Cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs) hold great promise for modeling human heart diseases. However, iPSC-CMs studied to date resemble immature embryonic myocytes and therefore do not adequately recapitulate native adult cardiomyocyte phenotypes. Since extracellular matrix plays an essential role in heart development and maturation in vivo, we sought to develop a synthetic culture matrix that could enhance functional maturation of iPSC-CMs in vitro. In this study, we employed a library of combinatorial polymers comprising of three functional subunits - poly-ε-caprolacton (PCL), polyethylene glycol (PEG), and carboxylated PCL (cPCL) - as synthetic substrates for culturing human iPSC-CMs. Of these, iPSC-CMs cultured on 4%PEG-96%PCL (each % indicates the corresponding molar ratio) exhibit the greatest contractility and mitochondrial function. These functional enhancements are associated with increased expression of cardiac myosin light chain-2v, cardiac troponin I and integrin alpha-7. Importantly, iPSC-CMs cultured on 4%PEG-95%PCL demonstrate troponin I (TnI) isoform switch from the fetal slow skeletal TnI (ssTnI) to the postnatal cardiac TnI (cTnI), the first report of such transition in vitro. Finally, culturing iPSC-CMs on 4%PEG-96%PCL also significantly increased expression of genes encoding intermediate filaments known to transduce integrin-mediated mechanical signals to the myofilaments. In summary, our study demonstrates that synthetic culture matrices engineered from combinatorial polymers can be utilized to promote in vitro maturation of human iPSC-CMs through the engagement of critical matrix-integrin interactions. PMID:26204225

Effect of a high fat diet on ovary morphology, in vitro development, in vitro fertilisation rate and oocyte quality in mice.

PubMed

Sohrabi, Maryam; Roushandeh, Amaneh Mohammadi; Alizadeh, Zohreh; Vahidinia, Aliasghar; Vahabian, Mehrangiz; Hosseini, Mahnaz

2015-10-01

The aim of this study was to determine the effect of a high-fat diet (HFD) on oocyte maturation and quality in a mouse model. Female BALB/c mice were allocated to one of the following groups: (a) control group (n = 40), which received a controlled diet; or (b) HFD group (n = 40), which received an HFD for 12 weeks. Sections of the ovary were examined histologically. The number of follicles and corpora lutea were counted. In vitro maturation and in vitro fertilisation (IVF) were assessed in germinal vesicle (GV) and metaphase II (MII) oocytes, respectively. The expression of bone morphogenetic protein 15 (BMP15) and leptin receptor genes in GV and MII oocytes was evaluated using reverse transcription real-time polymerase chain reactions. In the HFD group, there was a decreased number of primordial and Graafian follicles, as well as corpora lutea (p < 0.05). The rate of oocyte development to the MII stage was also reduced (p < 0.001). Cumulus expansion was observed more frequently in the control group than the HFD group (p < 0.05). The IVF rate in the HFD group was lower than that in the control group (p < 0.05). In the HFD group, BMP15 and leptin receptor genes were upregulated in the GV stage (p > 0.05) and MII stage (p < 0.05), compared to the control group. An HFD reduces folliculogenesis in the primordial and Graafian stages, in vitro maturation and in vitro fertilisation rates, as well as oocyte quality in mice.

Developmental competence of mature yak vitrified-warmed oocytes is enhanced by IGF-I via modulation of CIRP during in vitro maturation.

PubMed

Pan, Yangyang; Cui, Yan; He, Honghong; Baloch, Abdul Rasheed; Fan, Jiangfeng; Xu, Gengquan; He, Junfeng; Yang, Kun; Li, Guyue; Yu, Sijiu

2015-12-01

The objective of this study was to investigate whether developmental competence of mature vitrified-warmed yak (Bos grunniens) oocytes can be enhanced by supplemented insulin-like growth factor I (IGF-1) during in vitro maturation (IVM), and its relationship with the expression of cold-inducible RNA-binding protein (CIRP). In experiment 1, immature yak oocytes were divided into four groups, and IVM supplemented with 0, 50, 100 and 200 ng/mL IGF-1 was evaluated; the mRNA and protein expression levels of CIRP in mature oocytes in the four groups were evaluated using quantitative real-time PCR and western blotting analyses. In experiment 2, the mature yak oocytes in the four groups were cryopreserved using the Cryotop (CT) method, followed by chemical activation and in vitro culture for two days and eight days to determine cleavage, blastocyst rates, and total cell number in the blastocysts. Mature yak oocytes without vitrification served as a control group. The outcomes were as following: (1) the expression of CIRP in the matured oocytes was up-regulated in the IGF-1 groups and was highest expression was observed in the 100 ng/mL IGF-1 treatment group. (2) In the vitrified-warmed groups, the rates of cleavage and blastocyst were also highest in the 100 ng/mL IGF-1 treatment group (81.04 ± 1.06%% and 32.16 ± 1.01%), which were close to the rates observed in groups without vitrification (83.25 ± 0.85% and 32.54 ± 0.34%). The rates of cleavage and blastocyst in the other vitrified-warmed groups were 70.92 ± 1.32% and 27.33 ± 1.31% (0 ng/mL); 72.73 ± 0.74% and 29.41 ± 0.84% (50 ng/mL); 72.43 ± 0.61% and 27.61 ± 0.59% (200 ng/mL), respectively. There was no significant difference in the total cell number per blastocysts between the vitrified-warmed groups and group without vitrification. Thus, we conclude that the enhancement in developmental competence of mature yak vitrified-warmed oocytes after the addition of IGF-1 during IVM might result from the regulation of CIRP expression in mature yak oocytes prior to vitrification. Copyright © 2015 Elsevier Inc. All rights reserved.

Effect of collection-maturation interval time and pregnancy status of donor mares on oocyte developmental competence in horse cloning.

PubMed

Gambini, A; Andrés, G; Jarazo, J; Javier, J; Karlanian, F; Florencia, K; De Stéfano, A; Salamone, D F

2014-02-01

The current limitations for obtaining ovaries from slaughterhouses and the low efficiency of in vivo follicular aspiration necessitate a complete understanding of the variables that affect oocyte developmental competence in the equine. For this reason, we assessed the effect on equine oocyte meiotic competence and the subsequent in vitro cloned embryo development of 1) the time interval between ovary collection and the onset of oocyte in vitro maturation (collection-maturation interval time) and 2) the pregnancy status of the donor mares. To define the collection-maturation interval time, collected oocytes were classified according to the slaughtering time and the pregnancy status of the mare. Maturation rate was recorded and some matured oocytes of each group were used to reconstruct zona free cloned embryos. Nuclear maturation rates were lower when the collection-maturation interval time exceeded 10 h as compared to 4 h (32/83 vs. 76/136, respectively; P = 0.0128) and when the donor mare was pregnant as compared to nonpregnant (53/146 vs. 177/329, respectively; P = 0.0004). Low rates of cleaved embryos were observed when the collection-maturation interval time exceeded 10 h as compared to 6 to 10 h (11/27 vs. 33/44, respectively; P = 0.0056), but the pregnancy status of donor mares did not affect cloned equine blastocyst development (3/49 vs. 1/27 for blastocyst rates of nonpregnant and pregnant groups, respectively; P = 1.00). These results indicate that, to apply assisted reproductive technologies in horses, oocytes should be harvested within approximately 10 h after ovary collection. Also, even though ovaries from pregnant mares are a potential source of oocytes, they should be processed at the end of the collection routine due to the lower collection and maturation rate in this group.

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.

Fertilisation in the horse and paracrine signalling in the oviduct.

PubMed

Goudet, Ghylène

2011-01-01

The mammalian oviduct plays a crucial role in the preparation of gametes for fertilisation (transport and final maturation) and fertilisation itself. An increasing number of studies offers a comprehensive overview of the functions of the oviduct and its secretions, but this topic has had limited investigation in the horse. Limited data are available on the final oocyte maturation in the equine oviduct. However, in vitro and in vivo systems have been established to analyse the influence of equine oviduct epithelial cells (OEC) during maturation on the potential of oocytes for fertilisation and development. Most studies focus on the role of the oviduct in equine sperm function, such as spermatozoa transport, attachment to oviduct epithelium, viability, motility and capacitation. Moreover, some possible candidate molecules for sperm-oviducal interactions have been identified in the horse. Finally, the low efficiency of conventional in vitro fertilisation and the in vivo fertilisation of equine oocytes transferred into the oviduct of an inseminated mare predicted an influence of oviduct in equine fertilisation. Actually, in vivo and in vitro experiments demonstrated a role of the oviduct in equine fertilisation. Moreover, recent studies showed a beneficial effect of homologous and heterologous OEC on equine in vitro fertilisation, and some candidate molecules have been studied.

Positive effects of Forskolin (stimulator of lipolysis) treatment on cryosurvival of in vitro matured porcine oocytes.

PubMed

Fu, Xiang-Wei; Wu, Guo-Quan; Li, Jun-Jie; Hou, Yun-Peng; Zhou, Guang-Bin; Lun-Suo; Wang, Yan-Ping; Zhu, Shi-En

2011-01-15

In order to examine its effect on oocyte lipid content and cryosurvival, Forskolin was added to the medium for in vitro maturation of porcine oocytes. Treatments were control (IVM without Forskolin during the 42 h incubation period), addition of 10 μM Forskolin for the entire 42 h (0-42) and addition of 10 μM Forskolin between 24 and 42 h only (24-42). In Experiment 1, treatments did not differ significantly in cleavage rate, but the blastocyst formation rate was lower in the 0-42 group than for control and 24-42 group oocytes (17, 32 and 40%, respectively; P < 0.05). It was shown in Experiment 2 that Forskolin treatment from 0-42 h and from 24-42 h significantly reduced lipid content of oocytes compared to that of control cells (65 and 99 vs. 140 μm(2) intensity of fluorescence, respectively; P < 0.05). In Experiment 3, the percentage of oocyte survival after cryopreservation and thawing was significantly higher in both Forskolin treatment groups than in control oocytes (72% for 0-42, 65% for 24-42 and 52% for control; P < 0.05). However, Forskolin treatment did not increase cleavage rates of vitrified in vitro matured porcine oocytes (Control group 28%, 0-42 h group 0%, 24-42 h group 26.67%). Addition of Forskolin affected the nuclear maturation of porcine oocytes. The percentage of PBE (polar body extrusion) were significantly reduced in the 0-42 h group (0-42 h group 42.00 ± 2.08 vs. Control group 79.70 ± 2.82 and 24-42 h group 70.60 ± 2.83; P < 0.05). The 24-42 h group showed similar nuclear status to that of the Control group. We propose that delipation engendered by incubation with 10 μM Forskolin during 24-42 hours of maturation increased cryosurvival of in vitro-maturated porcine oocytes and that attendant chemical lipolysis did not impair their further development as it may have done in oocytes incubated with Forskolin for the full 42 h. Copyright © 2011 Elsevier Inc. All rights reserved.

Lactobacillus acidophilus binds to MUC3 component of cultured intestinal epithelial cells with highest affinity.

PubMed

Das, Jugal Kishore; Mahapatra, Rajani Kanta; Patro, Shubhransu; Goswami, Chandan; Suar, Mrutyunjay

2016-04-01

Lactobacillus strains have been shown to adhere to the mucosal components of intestinal epithelial cells. However, established in vitro adhesion assays have several drawbacks in assessing the adhesion of new Lactobacillus strains. The present study aimed to compare the adhesion of four different Lactobacillus strains and select the most adherent microbe, based on in silico approach supported by in vitro results. The mucus-binding proteins in Lactobacillus acidophilus, L. plantarum, L. brevis and L. fermentum were identified and their capacities to interact with intestinal mucin were compared by molecular docking analysis. Lactobacillus acidophilus had the maximal affinity of binding to mucin with predicted free energy of -6.066 kcal mol(-1) Further, in vitro experimental assay of adhesion was performed to validate the in silico results. The adhesion of L. acidophilus to mucous secreting colon epithelial HT-29 MTX cells was highest at 12%, and it formed biofilm with maximum depth (Z = 84 μm). Lactobacillus acidophilus was determined to be the most adherent strain in the study. All the Lactobacillus strains tested in this study, displayed maximum affinity of binding to MUC3 component of mucus as compared to other gastrointestinal mucins. These findings may have importance in the design of probiotics and health care management. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Developmental competence of frozen-thawed yak (Bos grunniens) oocytes followed by in vitro maturation and fertilization.

PubMed

Niu, Hui-Ran; Zi, Xiang-Dong; Xiao, Xiao; Xiong, Xian-Rong; Zhong, Jin-Cheng; Li, Jian; Wang, Li; Wang, Yong

2014-02-01

In the present study, we examined the ability of immature germinal vesicle (GV) and subjected to in vitro matured (MII) yak oocytes to survive after cryopreservation as well as their subsequent development following in vitro maturation and fertilization. Both GV and MII oocytes were cryopreserved by using two different vitrification solutions (VS); VS-I contained 10% ethylene glycol (EG) and 10% dimethylsulfoxide (DMSO) in TCM-199 + 20% (v/v) fetal calf serum (FCS) whereas VS-II contained 40% EG + 18% Ficoll + 0.5 M sucrose in TCM-199 + 20% FCS. The percentage of oocytes found to be morphologically normal was greater (P < 0.01) in VS-I group than in VS-II group. Rates of cleavage (30.6-42.2%) and blastocyst formation (2.9-8.9%) did not differ among groups, but were lower than in unfrozen control (55.7% and 25.4%, P < 0.01). These results show that a combination of EG and DMSO or EG, Ficoll and sucrose can be used to cryopreserve yak oocytes in French straws. Copyright © 2014 Elsevier Inc. All rights reserved.

Agrobacterium-medicated transformation of mature Prunus serotina (black cherry) and regeneration of trangenic shoots

Treesearch

Xiaomei Liu; Paula Pijut

2010-01-01

A protocol for Agrobacterium-mediated transformation was developed for in vitro leaf explants of an elite, mature Prunus serotina tree. Agrobacterium tumefaciens strain EHA105 harboring an RNAi plasmid with the black cherry AGAMOUS (AG) gene was used. Bacteria were induced...

Kisspeptin as a promising oocyte maturation trigger for in vitro fertilisation in humans.

PubMed

Kasum, Miro; Franulić, Daniela; Čehić, Ermin; Orešković, Slavko; Lila, Albert; Ejubović, Emina

2017-08-01

The aim of this review is to analyse the effectiveness of exogenous kisspeptin administration as a novel alternative of triggering oocyte maturation, instead of currently used triggers such as human chorionic gonadotropin (hCG) or gonadotropin releasing hormone (GnRH) agonist, in women undergoing in vitro fertilisation (IVF) treatment. Kisspeptin has been considered a master regulator of two modes of GnRH and hence gonadotropin secretion, pulses and surges. Administration of kisspeptin-10 and kisspeptin-54 induces the luteinising hormone (LH) surge required for egg maturation and ovulation in animal investigations and LH release during the preovulatory phase of the menstrual cycle and hypothalamic amenorrhoea in humans. Exogenous kisspeptin-54 has been successfully administered as a promising method of triggering oocyte maturation, following ovarian stimulation with gonadotropins and GnRH antagonists in women undergoing IVF, due to its efficacy considering achieved pregnancy rates compared to hCG and GnRH agonists. Also, its safety in patients at high risk of developing ovarian hyperstimulation syndrome is noteworthy. Nevertheless, further studies would be desirable to establish the optimal trigger of egg maturation and to improve the reproductive outcome for women undergoing IVF treatment.

Impact of supplementary royal jelly on in vitro maturation of sheep oocytes: genes involved in apoptosis and embryonic development.

PubMed

Valiollahpoor Amiri, Mohammad; Deldar, Hamid; Ansari Pirsaraei, Zarbakht

2016-01-01

Optimizing culture conditions lead to the improvement of oocyte developmental competence and additives with anti-oxidative activity in culture media improved embryonic development. Royal jelly (RJ) is a product from the cephalic glands of nurse bees that has considerable health effects. The aim of this study was to investigate the effect of different concentrations of RJ on the maturation, cleavage, and blastocyst rates and gene expression in the oocyte and cumulus cells during in vitro maturation (IVM) of sheep oocyte. IVM of oocyte was performed in the presence of control (RJ0), 2.5 (RJ2.5), 5 (RJ5), 10 (RJ10), 20 (RJ20), and 40 (RJ40) mg/mL of RJ. Following the maturation period, parthenogenetic activation was carried out in two treatment groups (RJ0 and RJ10) and embryonic development was examined three and eight days thereafter. Moreover, the relative expression of BCL2 and BAX in oocyte as well as BCL2, BAX, HAS2, PTGS2, and STAR in cumulus cells were assessed. The results indicated that the addition of 10 mg/mL of RJ (90 ± 4.51%) to the maturation medium linearly increased the oocyte maturation rate compared to the control group (57 ± 2.42%), then it remained constant to the RJ40 (93 ± 3.10%) group. The higher RJ concentrations were associated with increased (p < 0.01) cleavage (53.3 ± 1.55% to 82.3 ± 2.82%) and blastocyst rate (15.5 ± 1.16% to 33.8 ± 3.09%) from the RJ0 to the RJ10 group. The relative mRNA expression of BCL2 and BAX in the oocyte was higher at RJ10. In cumulus cells, the expression of BCL2 was not affected, but that of BAX decreased, and expression of HAS2, PTGS2, and STAR were increased following the addition of RJ to the maturation media. In conclusion, the addition of 10 mg/mL of RJ to maturation medium improved blastocyst formation and decreased the apoptotic incidence in sheep cumulus cells and the oocyte during the in vitro development.

17,20β,21-Trihydroxy-4-pregnen-3-one biosynthesis and 20β-hydroxysteroid dehydrogenase expression during final oocyte maturation in the protandrous yellowfin porgy, Acanthopagrus latus.

PubMed

Jeng, Shan-Ru; Yueh, Wen-Shiun; Lee, Yan-Horn; Yen, Hsiu-Fang; Chang, Ching-Fong

2012-04-01

The purpose of this study was to investigate the physiological maturation-inducing steroid (MIS) in the marine protandrous yellowfin porgy (Acanthopagrus latus). Female fish were injected with 2 doses of LHRH analog (10 and 40 μg per kg). Ovarian tissue was obtained at 6 h intervals for in vitro analysis of oocyte maturation. The most effective steroids for inducing in vitro maturation (germinal vesicle breakdown and GVBD) in cultured oocytes were 17,20β-dihydroxy-4-pregnen-3-one (17,20βP) and 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S). 17,20βP was less potent than 20βS in inducing oocyte maturation. At higher concentrations, 11-deoxycortisol, 17α-hydroxy-progesterone, and 20β-21-dihydroxy-4-pregnen-3-one also significantly induced oocyte maturation. A tritiated precursor [(3)H]-pregnenolone, was cultured in vitro together with the maturing ovarian tissue. The tritiated metabolites were purified and identified by solvent extraction, HPLC, TLC, acetylation reaction and recrystallization. HPLC, TLC and recrystallization analysis showed that significant levels of tritiated 11-deoxycortisol (a precursor of 20β-S) and 20β-S, but not 17,20βP, were biosynthesized from [(3)H]-pregnenolone. Similar TLC profiles were obtained from the tritiated products that were isolated from the HPLC/TLC 20β-S fraction and standard 20β-S after the acetylation reaction. Constant specific radioactivity of tritiated 11-deoxycortisol and 20β-S but not 17,20βP by recrystallization was obtained in the tritiated metabolites isolated from HPLC and TLC fractions. The expression of 20β-hydroxysteroid dehydrogenase (20β-HSD) mRNA (a key enzyme that converts 11-deoxycortisol to 20β-S) was significantly increased in maturing ovarian tissue. This study provides the first evidence that 20β-S is converted from 11-deoxycortisol and is the possible MIS in yellowfin porgy. Copyright © 2012. Published by Elsevier Inc.

Reproductive Performance of Holstein Dairy Cows Grazing in Dry-summer Subtropical Climatic Conditions: Effect of Heat Stress and Heat Shock on Meiotic Competence and In vitro Fertilization

PubMed Central

Pavani, Krishna; Carvalhais, Isabel; Faheem, Marwa; Chaveiro, Antonio; Reis, Francisco Vieira; da Silva, Fernando Moreira

2015-01-01

The present study was designed to evaluate how environmental factors in a dry-summer subtropical climate in Terceira-Azores (situated in the North Atlantic Ocean: 38° 43′ N 27° 12′ W) can affect dairy cow (Holstein) fertility, as well as seasonal influence on in vitro oocytes maturation and embryos development. Impact of heat shock (HS) effects on in vitro oocyte’s maturation and further embryo development after in vitro fertilization (IVF) was also evaluated. For such purpose the result of the first artificial insemination (AI) performed 60 to 90 days after calving of 6,300 cows were recorded for one year. In parallel, climatic data was obtained at different elevation points (n = 5) from 0 to 1,000 m and grazing points from 0 to 500 m, in Terceira island, and the temperature humidity index (THI) was calculated. For in vitro experiments, oocytes (n = 706) were collected weekly during all year, for meiotic maturation and IVF. Further, to evaluate HS effect, 891 oocytes were collected in the cold moths (December, January, February and March) and divided in three groups treated to HS for 24 h during in vitro maturation at: C (Control = 38.5°C), HS1 (39.5°C) and HS2 (40.5°C). Oocytes from each group were used for meiotic assessment and IVF. Cleavage, morula and blastocyst development were evaluated respectively on day 2, 6, and 9 after IVF. A negative correlation between cow’s conception rate (CR) and THI in grazing points (−91.3%; p<0.001) was observed. Mean THI in warmer months (June, July, August and September) was 71.7±0.7 and the CR (40.2±1.5%) while in cold months THI was 62.8±0.2 and CR was 63.8±0.4%. A similar impact was obtained with in vitro results in which nuclear maturation rate (NMR) ranged from 78.4% (±8.0) to 44.3% (±8.1), while embryos development ranged from 53.8% (±5.8) to 36.3% (±3.3) in cold and warmer months respectively. In vitro HS results showed a significant decline (p<0.05) on NMR of oocytes for every 1°C rising temperature (78.4±8.0, 21.7±3.1 and 8.9±2.2, respectively for C, HS1, and HS2). Similar results were observed in cleavage rate and embryo development, showing a clear correlation (96.9 p<0.05) between NMR and embryo development with respect to temperatures. Results clearly demonstrated that, up to a THI of 70.6, a decrease in the CR occurs in first AI after calving; this impairment was confirmed with in vitro results. PMID:25656191

Reproductive Performance of Holstein Dairy Cows Grazing in Dry-summer Subtropical Climatic Conditions: Effect of Heat Stress and Heat Shock on Meiotic Competence and In vitro Fertilization.

PubMed

Pavani, Krishna; Carvalhais, Isabel; Faheem, Marwa; Chaveiro, Antonio; Reis, Francisco Vieira; da Silva, Fernando Moreira

2015-03-01

The present study was designed to evaluate how environmental factors in a dry-summer subtropical climate in Terceira-Azores (situated in the North Atlantic Ocean: 38° 43' N 27° 12' W) can affect dairy cow (Holstein) fertility, as well as seasonal influence on in vitro oocytes maturation and embryos development. Impact of heat shock (HS) effects on in vitro oocyte's maturation and further embryo development after in vitro fertilization (IVF) was also evaluated. For such purpose the result of the first artificial insemination (AI) performed 60 to 90 days after calving of 6,300 cows were recorded for one year. In parallel, climatic data was obtained at different elevation points (n = 5) from 0 to 1,000 m and grazing points from 0 to 500 m, in Terceira island, and the temperature humidity index (THI) was calculated. For in vitro experiments, oocytes (n = 706) were collected weekly during all year, for meiotic maturation and IVF. Further, to evaluate HS effect, 891 oocytes were collected in the cold moths (December, January, February and March) and divided in three groups treated to HS for 24 h during in vitro maturation at: C (Control = 38.5°C), HS1 (39.5°C) and HS2 (40.5°C). Oocytes from each group were used for meiotic assessment and IVF. Cleavage, morula and blastocyst development were evaluated respectively on day 2, 6, and 9 after IVF. A negative correlation between cow's conception rate (CR) and THI in grazing points (-91.3%; p<0.001) was observed. Mean THI in warmer months (June, July, August and September) was 71.7±0.7 and the CR (40.2±1.5%) while in cold months THI was 62.8±0.2 and CR was 63.8±0.4%. A similar impact was obtained with in vitro results in which nuclear maturation rate (NMR) ranged from 78.4% (±8.0) to 44.3% (±8.1), while embryos development ranged from 53.8% (±5.8) to 36.3% (±3.3) in cold and warmer months respectively. In vitro HS results showed a significant decline (p<0.05) on NMR of oocytes for every 1°C rising temperature (78.4±8.0, 21.7±3.1 and 8.9±2.2, respectively for C, HS1, and HS2). Similar results were observed in cleavage rate and embryo development, showing a clear correlation (96.9 p<0.05) between NMR and embryo development with respect to temperatures. Results clearly demonstrated that, up to a THI of 70.6, a decrease in the CR occurs in first AI after calving; this impairment was confirmed with in vitro results.

[Peroxynitrite effect on the haemoglobin oxygen affinity in vitro in presence of different partial pressure of carbon dioxide].

PubMed

Stepuro, T L; Zinchuk, V V

2011-08-01

Peroxynitrite (ONOO-) besides its toxic possesses regulatory action that includes the modulation of oxygen binding properties of blood. The aim of this work was to estimate ONOO- effect on the haemoglobin oxygen affinity (HOA) in vitro in presence of different partial pressure of carbon dioxide (CO2). The ONOO- presence in venous blood in conditions of hypercapnia induced oxyhaemoglobin dissociation curve shift leftward while in hypocapnic conditions the result of a different character was obtained. The revealed effect of ONOO- is realized, possibly, through various modifications ofhaemoglobin whose formation is dependent on the CO2 pressure. The ONOO- influences the HOA in different manner that can be important in regulation of blood oxygenation in lungs and maintenance of oxygen consumption in tissues.

Sericin Accelerates the Production of Hyaluronan and Decreases the Incidence of Polyspermy Fertilization in Bovine Oocytes During In Vitro Maturation

PubMed Central

HOSOE, Misa; YOSHIDA, Nao; HASHIYADA, Yutaka; TERAMOTO, Hidetoshi; TAKAHASHI, Toru; NIIMURA, Sueo

2014-01-01

Fetal bovine serum (FBS) has been widely used as a supplement in the maturation medium of bovine oocytes in vitro. However, serum contains many undefined factors and is potentially infectious to humans and animals. As a serum replacement, we evaluated the feasibility of using the silk protein, sericin, derived from the cocoons of silkworm. To examine the rates of oocyte maturation and fertilization, cumulus-oocyte complexes were cultured in TCM-199 supplemented with 0.01%, 0.05%, 0.1% or 0.15% sericin or 5% FBS. The sizes of the perivitelline space that might relate to polyspermy, the expressions of Has2 and CD44 mRNA, the amount of hyaluronan (hyaluronic acid: HA) contained in the oocytes and the rates of blastocyst formation following insemination were then compared between the oocytes cultured with 0.05% sericin and 5% FBS, because the polyspermy rates in oocytes cultured with 0.05% sericin were significantly lower than in those cultured with 5% FBS. After in vitro maturation (IVM), the mean size of the perivitelline space was significantly greater in oocytes cultured with sericin than in those cultured with FBS, although the rates of nuclear maturation, fertilization and blastocyst formation of oocytes under both IVM conditions were not significantly different. The expression of HAS2 and CD44 mRNA and the amount of HA in the denuded oocytes cultured with 0.05% sericin were significantly greater than in those cultured with FBS. These results indicate the feasibility of sericin as an alternative protein supplement for IVM in bovine oocytes. PMID:24748396

Sericin accelerates the production of hyaluronan and decreases the incidence of polyspermy fertilization in bovine oocytes during in vitro maturation.

PubMed

Hosoe, Misa; Yoshida, Nao; Hashiyada, Yutaka; Teramoto, Hidetoshi; Takahashi, Toru; Niimura, Sueo

2014-01-01

Fetal bovine serum (FBS) has been widely used as a supplement in the maturation medium of bovine oocytes in vitro. However, serum contains many undefined factors and is potentially infectious to humans and animals. As a serum replacement, we evaluated the feasibility of using the silk protein, sericin, derived from the cocoons of silkworm. To examine the rates of oocyte maturation and fertilization, cumulus-oocyte complexes were cultured in TCM-199 supplemented with 0.01%, 0.05%, 0.1% or 0.15% sericin or 5% FBS. The sizes of the perivitelline space that might relate to polyspermy, the expressions of Has2 and CD44 mRNA, the amount of hyaluronan (hyaluronic acid: HA) contained in the oocytes and the rates of blastocyst formation following insemination were then compared between the oocytes cultured with 0.05% sericin and 5% FBS, because the polyspermy rates in oocytes cultured with 0.05% sericin were significantly lower than in those cultured with 5% FBS. After in vitro maturation (IVM), the mean size of the perivitelline space was significantly greater in oocytes cultured with sericin than in those cultured with FBS, although the rates of nuclear maturation, fertilization and blastocyst formation of oocytes under both IVM conditions were not significantly different. The expression of HAS2 and CD44 mRNA and the amount of HA in the denuded oocytes cultured with 0.05% sericin were significantly greater than in those cultured with FBS. These results indicate the feasibility of sericin as an alternative protein supplement for IVM in bovine oocytes.

Selective inhibition of polymorphonuclear neutrophil activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

PubMed

Ackermann, M F; Gasiewicz, T A; Lamm, K R; Germolec, D R; Luster, M I

1989-12-01

Although the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), via its interaction with the Ah receptor, is an extremely potent carcinogen and immunosuppressive agent in experimental animals, its possible actions on polymorphonuclear (PMN) function have not been determined. In addition to their importance against infectious organisms, PMNs have been implicated in antitumor resistance. The present studies examined the effects of in vivo exposure to TCDD on PMN function in B6C3F1 (TCDD sensitive, presence of high affinity Ah receptor) and DBA/2N (TCDD resistant at low doses, defective Ah receptor) mice. Animals received a single oral exposure of 5 or 10 micrograms/kg of TCDD and PMNs were obtained 5 days later from the peritoneal cavity following elicitation with sodium caseinate. TCDD reduced the cytolytic and cytostatic activity of PMA-activated PMNs in B6C3F1, but not in DBA/2N mice, suggesting that this response segregates with the Ah locus. Furthermore, TCDD was found to bind specifically to PMNs from Ah-responsive mice. Neither the production of superoxide and hydrogen peroxide nor degranulation, the latter measured by beta-glucuronidase release, was impaired. Supernatants recovered from PMN cell cultures of TCDD-sensitive mice, but not from resistant DBA/2N mice, showed reduced killing capacity for actinomycin D-treated L929 tumor cells, while their ability to bind to tumor cells was not altered. These data suggest that TCDD interferes with PMN-mediated tumor cell killing by altering the production or secretion of a cytolytic factor. Examination of bone marrow stem cells revealed that granulocytic but not monocytic colonies were reduced after TCDD exposure in vivo and in vitro. Although mature PMNs had detectable levels of Ah receptor, exposure in vitro of these cells to TCDD had no effect on antitumor activity. Thus, it is possible that TCDD may affect PMNs at the level of hematopoiesis, via a direct interaction with granulocyte precursor cells, or modulate PMNs at different stages of maturation.

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.

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

Naturally Engineered Maturation of Cardiomyocytes

PubMed Central

Scuderi, Gaetano J.; Butcher, Jonathan

2017-01-01

Ischemic heart disease remains one of the most prominent causes of mortalities worldwide with heart transplantation being the gold-standard treatment option. However, due to the major limitations associated with heart transplants, such as an inadequate supply and heart rejection, there remains a significant clinical need for a viable cardiac regenerative therapy to restore native myocardial function. Over the course of the previous several decades, researchers have made prominent advances in the field of cardiac regeneration with the creation of in vitro human pluripotent stem cell-derived cardiomyocyte tissue engineered constructs. However, these engineered constructs exhibit a functionally immature, disorganized, fetal-like phenotype that is not equivalent physiologically to native adult cardiac tissue. Due to this major limitation, many recent studies have investigated approaches to improve pluripotent stem cell-derived cardiomyocyte maturation to close this large functionality gap between engineered and native cardiac tissue. This review integrates the natural developmental mechanisms of cardiomyocyte structural and functional maturation. The variety of ways researchers have attempted to improve cardiomyocyte maturation in vitro by mimicking natural development, known as natural engineering, is readily discussed. The main focus of this review involves the synergistic role of electrical and mechanical stimulation, extracellular matrix interactions, and non-cardiomyocyte interactions in facilitating cardiomyocyte maturation. Overall, even with these current natural engineering approaches, pluripotent stem cell-derived cardiomyocytes within three-dimensional engineered heart tissue still remain mostly within the early to late fetal stages of cardiomyocyte maturity. Therefore, although the end goal is to achieve adult phenotypic maturity, more emphasis must be placed on elucidating how the in vivo fetal microenvironment drives cardiomyocyte maturation. This information can then be utilized to develop natural engineering approaches that can emulate this fetal microenvironment and thus make prominent progress in pluripotent stem cell-derived maturity toward a more clinically relevant model for cardiac regeneration. PMID:28529939

The influence of N-acetyl-L-cysteine on damage of porcine oocyte exposed to zearalenone in vitro

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

Lai, Fang-Nong; Institute of Reproductive Sciences, Qingdao Agricultural University, Qingdao 266109; Ma, Jun-Yu

Zearalenone (ZEA), one of the mycotoxins produced by Fusarium fungi, impacts porcine reproduction by interfering with the estrogen signaling pathway. Previous studies have shown that ZEA inhibits porcine oocyte maturation through the formation of aberrant spindle. To explore the effect of ZEA on porcine oocyte meiotic maturation, the extent of both nuclear and cytoplasmic maturation was examined in this study. Compared with control group, presence of ZEA (3 μM) during oocyte maturation, significantly inhibited the polar body extrusions from 71% to 51%, and significantly increased intracellular reactive oxygen species (ROS) level (12.01 vs. 5.89). Intracellular glutathione (GSH) content in ZEAmore » treatment group was lower than in the control group (1.08 pmol/oocyte vs. 0.18 pmol/oocyte), and cortical granules of cortical area distributed oocytes were reduced (88% vs. 62%). ZEA decreases cumulus expansion in both morphology and mRNA level (HAS2, PTX3, TNFAIP6 and CX43). Addition of N-acetyl-L-cysteine (NAC) to the oocyte maturation media reversed the ZEA-induced inhibition of polar body extrusion (from 69% to 81%), up-regulated ROS (from 7.9 to 6.5), down-regulated GSH content (from 0.16 to 0.82 pmol/oocyte) and recovered cumulus cells expansion in morphology and mRNA level. It is concluded that ZEA affects both oocyte nucleus and cytoplasmic maturation during in vitro maturation, and NAC can reverse these damages to some extent. - Highlights: • ZEA significantly inhibited the polar body extrusions during oocyte maturation. • ZEA significantly increased intracellular ROS level and reduced GSH content. • ZEA disturbed cortical granules of cortical area distributed oocytes. • NAC reversed the ZEA-induced inhibition of oocyte maturation.« less

Parthenogenic Blastocysts Derived from Cumulus-Free In Vitro Matured Human Oocytes

PubMed Central

McElroy, Sohyun L.; Byrne, James A.; Chavez, Shawn L.; Behr, Barry; Hsueh, Aaron J.; Westphal, Lynn M.; Reijo Pera, Renee A.

2010-01-01

Background Approximately 20% of oocytes are classified as immature and discarded following intracytoplasmic sperm injection (ICSI) procedures. These oocytes are obtained from gonadotropin-stimulated patients, and are routinely removed from the cumulus cells which normally would mature the oocytes. Given the ready access to these human oocytes, they represent a potential resource for both clinical and basic science application. However culture conditions for the maturation of cumulus-free oocytes have not been optimized. We aimed to improve maturation conditions for cumulus-free oocytes via culture with ovarian paracrine/autocrine factors identified by single cell analysis. Methodology/Principal Finding Immature human oocytes were matured in vitro via supplementation with ovarian paracrine/autocrine factors that were selected based on expression of ligands in the cumulus cells and their corresponding receptors in oocytes. Matured oocytes were artificially activated to assess developmental competence. Gene expression profiles of parthenotes were compared to IVF/ICSI embryos at morula and blastocyst stages. Following incubation in medium supplemented with ovarian factors (BDNF, IGF-I, estradiol, GDNF, FGF2 and leptin), a greater percentage of oocytes demonstrated nuclear maturation and subsequently, underwent parthenogenesis relative to control. Similarly, cytoplasmic maturation was also improved as indicated by development to blastocyst stage. Parthenogenic blastocysts exhibited mRNA expression profiles similar to those of blastocysts obtained after IVF/ICSI with the exception for MKLP2 and PEG1. Conclusions/Significance Human cumulus-free oocytes from hormone-stimulated cycles are capable of developing to blastocysts when cultured with ovarian factor supplementation. Our improved IVM culture conditions may be used for obtaining mature oocytes for clinical purposes and/or for derivation of embryonic stem cells following parthenogenesis or nuclear transfer. PMID:20539753

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.

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.

Up-regulation of angiotensin II receptors by in vitro differentiation of murine N1E-115 neuroblastoma cells.

PubMed

Reagan, L P; Ye, X H; Mir, R; DePalo, L R; Fluharty, S J

1990-12-01

In vitro differentiation of murine neuroblastoma N1E-115 cells induced by low serum (0.5%) and dimethyl sulfoxide (1.5%) increased the uptake of 45Ca2+ as well as basal and forskolin-stimulated adenylate cyclase activity. Associated with these biochemical indices of differentiation was an increase in the density of binding sites for the angiotensin II (Ang II) receptor agonist 125I-[Sar1]-Ang II and the antagonist 125I-[Sar1,Ile8]-Ang II (125I-SARILE). This up-regulation was apparent within 24 hr and was maximal at 72 hr. Other manipulations that independently increased intracellular cAMP or Ca2+ levels produced a qualitatively similar up-regulation of Ang II receptors. In vitro differentiation did not diminish the specificity of these receptors for Ang-II related peptides. Sarcosine-substituted Ang II receptor antagonists such as [Sar1,Gly8]-Ang II, [Sar1,Thr8]-Ang II, or SARILE itself competed for 125I-SARILE in a monophasic fashion, whereas the competition displayed by the agonists Ang II, angiotensin III, and Crinia-Ang II for 125I-SARILE-labeled sites was biphasic, consisting of distinct high and low affinity components. Moreover, in vitro differentiation predominantly increased the density of high affinity sites for angiotensin III and Crinia-Ang II, but the lower affinity site for Ang II, and in all three cases the majority of this increased binding was insensitive to guanine nucleotides. Collectively, these results demonstrate that the expression of Ang II receptors on neuron-like cells is regulated by the biochemical events accompanying differentiation and suggest that the biphasic nature of the binding of some angiotensin agonists may be indicative of multiple receptor subtypes.

Estrogen Receptor-β Agonist Diarylpropionitrile: Biological Activities of R- and S-Enantiomers on Behavior and Hormonal Response to Stress

PubMed Central

Weiser, Michael J.; Wu, T. John; Handa, Robert J.

2009-01-01

Estrogens have been shown to have positive and negative effects on anxiety and depressive-like behaviors, perhaps explained by the existence of two distinct estrogen receptor (ER) systems, ERα and ERβ. The ERβ agonist, diarylpropionitrile (DPN) has been shown to have anxiolytic properties in rats. DPN exists as a racemic mixture of two enantiomers, R-DPN and S-DPN. In this study, we compared R-DPN and S-DPN for their in vitro binding affinity, ability to activate transcription in vitro at an estrogen response element, and in vivo endocrine and behavioral responses. In vitro binding studies using recombinant rat ERβ revealed that S-DPN has a severalfold greater relative binding affinity for ERβ than does R-DPN. Furthermore, cotransfection of N-38 immortalized hypothalamic cells with an estrogen response element-luc reporter and ERβ revealed that S-DPN is a potent activator of transcription in vitro, whereas R-DPN is not. Subsequently, we examined anxiety-like behaviors using the open-field test and elevated plus maze or depressive-like behaviors, using the forced swim test. Ovariectomized young adult female Sprague Dawley rats treated with racemic DPN, S-DPN, and the ERβ agonist, WAY-200070, showed significantly decreased anxiety-like behaviors in both the open-field and elevated plus maze and significantly less depressive-like behaviors in the forced swim test compared with vehicle-, R-DPN-, or propylpyrazoletriol (ERα agonist)-treated animals. In concordance with the relative binding affinity and transcriptional potency, these results demonstrate that the S-enantiomer is the biologically active form of DPN. These studies also indicate that estrogen's positive effects on mood, including its anxiolytic and antidepressive actions, are due to its actions at ERβ. PMID:19074580

A Simplified Method for Three-Dimensional (3-D) Ovarian Tissue Culture Yielding Oocytes Competent to Produce Full-Term Offspring in Mice

PubMed Central

Higuchi, Carolyn M.; Maeda, Yuuki; Horiuchi, Toshitaka; Yamazaki, Yukiko

2015-01-01

In vitro growth of follicles is a promising technology to generate large quantities of competent oocytes from immature follicles and could expand the potential of assisted reproductive technologies (ART). Isolated follicle culture is currently the primary method used to develop and mature follicles in vitro. However, this procedure typically requires complicated, time-consuming procedures, as well as destruction of the normal ovarian microenvironment. Here we describe a simplified 3-D ovarian culture system that can be used to mature multilayered secondary follicles into antral follicles, generating developmentally competent oocytes in vitro. Ovaries recovered from mice at 14 days of age were cut into 8 pieces and placed onto a thick Matrigel drop (3-D culture) for 10 days of culture. As a control, ovarian pieces were cultured on a membrane filter without any Matrigel drop (Membrane culture). We also evaluated the effect of activin A treatment on follicle growth within the ovarian pieces with or without Matrigel support. Thus we tested four different culture conditions: C (Membrane/activin-), A (Membrane/activin+), M (Matrigel/activin-), and M+A (Matrigel/activin+). We found that the cultured follicles and oocytes steadily increased in size regardless of the culture condition used. However, antral cavity formation occurred only in the follicles grown in the 3-D culture system (M, M+A). Following ovarian tissue culture, full-grown GV oocytes were isolated from the larger follicles to evaluate their developmental competence by subjecting them to in vitro maturation (IVM) and in vitro fertilization (IVF). Maturation and fertilization rates were higher using oocytes grown in 3-D culture (M, M+A) than with those grown in membrane culture (C, A). In particular, activin A treatment further improved 3-D culture (M+A) success. Following IVF, two-cell embryos were transferred to recipients to generate full-term offspring. In summary, this simple and easy 3-D ovarian culture system using a Matrigel drop and activin A supplementation (M+A) provides optimal and convenient conditions to support growth of developmentally competent oocytes in vitro. PMID:26571501

Moloney leukemia virus immortalizes B lymphocytes in vitro.

PubMed Central

Runnels, J; Serunian, L; Thursby, M; Rosenberg, N

1991-01-01

An in vitro culture system in which Moloney murine leukemia virus induces immortalization of mature B lymphocytes has been developed. The cell lines derived in this way are nontumorigenic, and virus production is not required to sustain them. This system provides a new in vitro model with which to study the stepwise process of transformation by retroviruses lacking oncogenes. Images PMID:1895405

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

Bovine in vitro fertilization: in vitro oocyte maturation and sperm capacitation with heparin.

PubMed

Parrish, John J

2014-01-01

As a result of research in the 1980s on in vitro maturation, sperm capacitation, and in vitro fertilization, the bovine is now one of the important models for development. Further, the current production of bovine embryos in vitro rivals that of in vivo embryo production for commercial applications. Researchers of today may be unaware of why decisions were made in the procedures. This review addresses the state of the art at the time of the work by Parrish et al. (Bovine in vitro fertilization with frozen thawed semen. Theriogenology 1986;25:591-600), and how later work would explain success or failure of competing procedures. Important was the use of frozen semen and heparin capacitation, because this allowed future researchers/practitioners to change sperm numbers and capacitation conditions to adjust for variations among bulls. The large numbers of citation of the original work stand the testament of time in the repeatability and success of the procedures. The work was done within the environment of the N.L. First laboratory and the unique interactions with a large number of talented graduate students, postdoctoral researchers, and technicians. Copyright © 2014 Elsevier Inc. All rights reserved.

In Silico and In Vitro Analysis of Bacoside A Aglycones and Its Derivatives as the Constituents Responsible for the Cognitive Effects of Bacopa monnieri

PubMed Central

Ramasamy, Seetha; Chin, Sek Peng; Sukumaran, Sri Devi; Buckle, Michael James Christopher; Kiew, Lik Voon; Chung, Lip Yong

2015-01-01

Bacopa monnieri has been used in Ayurvedic medicine to improve memory and cognition. The active constituent responsible for its pharmacological effects is bacoside A, a mixture of dammarane-type triterpenoid saponins containing sugar chains linked to a steroid aglycone skeleton. Triterpenoid saponins have been reported to be transformed in vivo to metabolites that give better biological activity and pharmacokinetic characteristics. Thus, the activities of the parent compounds (bacosides), aglycones (jujubogenin and pseudojujubogenin) and their derivatives (ebelin lactone and bacogenin A1) were compared using a combination of in silico and in vitro screening methods. The compounds were docked into 5-HT1A, 5-HT2A, D1, D2, M1 receptors and acetylcholinesterase (AChE) using AutoDock and their central nervous system (CNS) drug-like properties were determined using Discovery Studio molecular properties and ADMET descriptors. The compounds were screened in vitro using radioligand receptor binding and AChE inhibition assays. In silico studies showed that the parent bacosides were not able to dock into the chosen CNS targets and had poor molecular properties as a CNS drug. In contrast, the aglycones and their derivatives showed better binding affinity and good CNS drug-like properties, were well absorbed through the intestines and had good blood brain barrier (BBB) penetration. Among the compounds tested in vitro, ebelin lactone showed binding affinity towards M1 (Ki = 0.45 μM) and 5-HT2A (4.21 μM) receptors. Bacoside A and bacopaside X (9.06 μM) showed binding affinity towards the D1 receptor. None of the compounds showed any inhibitory activity against AChE. Since the stimulation of M1 and 5-HT2A receptors has been implicated in memory and cognition and ebelin lactone was shown to have the strongest binding energy, highest BBB penetration and binding affinity towards M1 and 5-HT2A receptors, we suggest that B. monnieri constituents may be transformed in vivo to the active form before exerting their pharmacological activity. PMID:25965066

In Silico and In Vitro Analysis of Bacoside A Aglycones and Its Derivatives as the Constituents Responsible for the Cognitive Effects of Bacopa monnieri.

PubMed

Ramasamy, Seetha; Chin, Sek Peng; Sukumaran, Sri Devi; Buckle, Michael James Christopher; Kiew, Lik Voon; Chung, Lip Yong

2015-01-01

Bacopa monnieri has been used in Ayurvedic medicine to improve memory and cognition. The active constituent responsible for its pharmacological effects is bacoside A, a mixture of dammarane-type triterpenoid saponins containing sugar chains linked to a steroid aglycone skeleton. Triterpenoid saponins have been reported to be transformed in vivo to metabolites that give better biological activity and pharmacokinetic characteristics. Thus, the activities of the parent compounds (bacosides), aglycones (jujubogenin and pseudojujubogenin) and their derivatives (ebelin lactone and bacogenin A1) were compared using a combination of in silico and in vitro screening methods. The compounds were docked into 5-HT1A, 5-HT2A, D1, D2, M1 receptors and acetylcholinesterase (AChE) using AutoDock and their central nervous system (CNS) drug-like properties were determined using Discovery Studio molecular properties and ADMET descriptors. The compounds were screened in vitro using radioligand receptor binding and AChE inhibition assays. In silico studies showed that the parent bacosides were not able to dock into the chosen CNS targets and had poor molecular properties as a CNS drug. In contrast, the aglycones and their derivatives showed better binding affinity and good CNS drug-like properties, were well absorbed through the intestines and had good blood brain barrier (BBB) penetration. Among the compounds tested in vitro, ebelin lactone showed binding affinity towards M1 (Ki = 0.45 μM) and 5-HT2A (4.21 μM) receptors. Bacoside A and bacopaside X (9.06 μM) showed binding affinity towards the D1 receptor. None of the compounds showed any inhibitory activity against AChE. Since the stimulation of M1 and 5-HT2A receptors has been implicated in memory and cognition and ebelin lactone was shown to have the strongest binding energy, highest BBB penetration and binding affinity towards M1 and 5-HT2A receptors, we suggest that B. monnieri constituents may be transformed in vivo to the active form before exerting their pharmacological activity.

Cadexomer iodine provides superior efficacy against bacterial wound biofilms in vitro and in vivo.

PubMed

Fitzgerald, Daniel J; Renick, Paul J; Forrest, Emma C; Tetens, Shannon P; Earnest, David N; McMillan, Jillian; Kiedaisch, Brett M; Shi, Lei; Roche, Eric D

2017-01-01

Examination of clinical samples indicates bacterial biofilms are present in the majority of chronic wounds, and substantial evidence suggests biofilms contribute significantly to delayed healing. Bacteria in biofilms are highly tolerant of antimicrobials, and little data exist to guide the choice of anti-biofilm wound therapy. Cadexomer iodine (CI) was recently reported to have superior efficacy compared to diverse wound dressings against Pseudomonas aeruginosa biofilms in an ex vivo model. In the current study, the strong performance of CI vs. P. aeruginosa biofilm was confirmed using colony and colony drip-flow in vitro wound biofilm models. Similar in vitro efficacy of CI was also demonstrated against mature Staphylococcus aureus biofilms using the same models. Additionally, the rapid kill of mature S. aureus and P. aeruginosa colony biofilms was visualized by confocal microscopy using Live/Dead fluorescent stains. Superior in vitro efficacy of CI vs. staphylococcal biofilms was further demonstrated against methicillin-resistant S. aureus (MRSA) using multiple biofilm models with log reduction, Live/Dead, and metabolic endpoints. Comparator antimicrobial dressings, including silver-based dressings used throughout and other active agents used in individual models, elucidated only limited effects against the mature biofilms. Given the promising in vitro activity, CI was tested in an established mouse model of MRSA wound biofilm. CI had significantly greater impact on MRSA biofilm in mouse wounds than silver dressings or mupirocin based on Gram-stained histology sections and quantitative microbiology from biopsy samples (>4 log reduction in CFU/g vs. 0.7-1.6, p < 0.0001). The superior efficacy for CI in these in vitro and in vivo models suggests CI topical products may represent a better choice to address established bacterial biofilm in chronic wounds. © 2016 by the Wound Healing Society.

Occurrence of ovarian follicular dominance during stimulation for IVM impacts usable blastocyst yield.

PubMed

Romero, Sergio; Pella, Ricardo; Escudero, Francisco; Pérez, Ygor; García, Mario; Orihuela, Patricia

2018-03-01

To evaluate the influence of ovarian follicular dominance on the outcome of oocyte in-vitro maturation. This retrospective cohort study included 21 patients with polycystic ovaries or polycystic ovary syndrome (Rotterdam criteria, 2004) subjected to 24 invitro maturation (IVM) cycles between October 2015 and January 2017. Patients undergoing IVM received minimal gonadotropin stimulation starting on day 2 or 3 of the cycle; ovum pick-up typically occurred on days 6 to 8. No hCG-trigger shot was given. Following 30h of IVM, mature oocytes were inseminated by ICSI and the resulting embryos cultured up to the blastocyst stage. Ovarian follicular dominance was observed in nine of the 24 IVM cycles. Oocyte IVM yielded an overall maturation rate of 69.3±23.8%, and no difference was observed when the groups with or without a dominant follicle were assessed independently. The rates of fertilization and usable blastocysts per fertilized oocyte, mature oocyte (Metaphase II) or cumulus-oocyte-complex were nearly three times higher (28.7±22.5%) in the group without ovarian follicular dominance. No differences were found in the clinical pregnancy rates attained by the individuals with or without a dominant follicle after 21 vitrified-warmed blastocyst transfer cycles. Occurrence of ovarian follicular dominance during hormonal stimulation for in-vitro maturation negatively impacted embryological outcomes. Strategies devised to limit the appearance of ovarian follicular dominance must be further explored.

Radiosynthesis and in vitro evaluation of 2-(N-alkyl-N-1'-11C-propyl)amino-5-hydroxytetralin analogs as high affinity agonists for dopamine D-2 receptors.

PubMed

Shi, B; Narayanan, T K; Yang, Z Y; Christian, B T; Mukherjee, J

1999-10-01

We have developed radiotracers based on agonists that may potentially allow the in vivo assessment of the high affinity (HA) state of the dopamine D-2 receptors. The population of HA state, which is likely the functional state of the receptor, may be altered in certain diseases. We carried out radiosyntheses and evaluated the binding affinities, lipophilicity, and in vitro autoradiographic binding characteristics of three dopamine D-2 receptor agonists: (+/-)-2-(N,N-dipropyl)amino-5-hydroxytetralin (5-OH-DPAT), (+/-)-2-(N-phenethyl-N-propyl)amino-5-hydroxytetralin (PPHT), and (+/-)-2-(N-cyclohexylethyl-N-propyl)amino-5-hydroxytetralin (ZYY-339). In 3H-spiperone assays using rat striata, ZYY-339 exhibited subnanomolar affinity for D-2 receptor sites (IC50 = 0.010 nM), PPHT was somewhat weaker (IC50 = 0.65 nM), and 5-OH-DPAT exhibited the weakest affinity (IC50 = 2.5 nM) of the three compounds. Radiosynthesis of these derivatives, 2-(N-propyl-N-1'-11C-propyl)amino-5-hydroxytetralin (11C-5-OH-DPAT), 2-(N-phenethyl-N-1'-11C-propyl)amino-5-hydroxytetralin (11C-PPHT), and 2-(N-cyclohexylethyl-N-1'-11C-propyl)amino-5-hydroxytetralin (11C-ZYY-339) was achieved by first synthesizing 11C-1-propionyl chloride and subsequent coupling with the appropriate secondary amine precursor to form the respective amide, which was then reduced to provide the desired tertiary amine products. The final products were obtained by reverse-phase high performance liquid chromatography (HPLC) purification in radiochemical yields of 5-10% after 60-75 min from the end of 11CO2 trapping and with specific activities in the range of 250-1,000 Ci/mmol. In vitro autoradiographs in rat brain slices with 11C-5-OH-DPAT, 11C-PPHT, and 11C-ZYY-339 revealed selective binding of the three radiotracers to the dopamine D-2 receptors in the striata.

The fertilization ability and developmental competence of bovine oocytes grown in vitro

PubMed Central

MAKITA, Miho; UEDA, Mayuko; MIYANO, Takashi

2016-01-01

In vitro growth culture systems for oocytes are being developed in several mammalian species. In these growth culture systems, in vitro grown oocytes usually have lower blastocyst formation than in vivo grown oocytes after in vitro fertilization. Furthermore, there have been a few reports that investigated the fertilization ability of in vitro grown oocytes in large animals. The purpose of this study was to investigate the fertilization process and developmental competence of bovine oocytes grown in vitro. Oocyte-granulosa cell complexes collected from bovine early antral follicles (0.4−0.7 mm in diameter) were cultured for growth with 17β-estradiol and androstenedione for 14 days and matured in vitro. These oocytes were then inseminated for 6 or 12 h, and further cultured for development up to 8 days in vitro. After growth culture, oocytes grew from 95 µm to around 120 µm and acquired maturation competence (79%). Although fertilization rates of in vitro grown oocytes were low after 6 h of insemination, 34% of in vitro grown oocytes fertilized normally after 12 h of insemination, having two polar bodies and two pronuclei with a sperm tail, and 22% of these oocytes developed into blastocysts after 8 days of culture. The fertilization and blastocyst formation rates were similar to those of in vivo grown oocytes. In addition, blastocyst cell numbers were also similar between in vitro and in vivo grown oocytes. In conclusion, in vitro grown bovine oocytes are similar to in vivo grown oocytes in fertilization ability and can develop into blastocysts. PMID:27151093

The fertilization ability and developmental competence of bovine oocytes grown in vitro.

PubMed

Makita, Miho; Ueda, Mayuko; Miyano, Takashi

2016-08-25

In vitro growth culture systems for oocytes are being developed in several mammalian species. In these growth culture systems, in vitro grown oocytes usually have lower blastocyst formation than in vivo grown oocytes after in vitro fertilization. Furthermore, there have been a few reports that investigated the fertilization ability of in vitro grown oocytes in large animals. The purpose of this study was to investigate the fertilization process and developmental competence of bovine oocytes grown in vitro. Oocyte-granulosa cell complexes collected from bovine early antral follicles (0.4-0.7 mm in diameter) were cultured for growth with 17β-estradiol and androstenedione for 14 days and matured in vitro. These oocytes were then inseminated for 6 or 12 h, and further cultured for development up to 8 days in vitro. After growth culture, oocytes grew from 95 µm to around 120 µm and acquired maturation competence (79%). Although fertilization rates of in vitro grown oocytes were low after 6 h of insemination, 34% of in vitro grown oocytes fertilized normally after 12 h of insemination, having two polar bodies and two pronuclei with a sperm tail, and 22% of these oocytes developed into blastocysts after 8 days of culture. The fertilization and blastocyst formation rates were similar to those of in vivo grown oocytes. In addition, blastocyst cell numbers were also similar between in vitro and in vivo grown oocytes. In conclusion, in vitro grown bovine oocytes are similar to in vivo grown oocytes in fertilization ability and can develop into blastocysts.

A bioengineered niche promotes in vivo engraftment and maturation of pluripotent stem cell derived human lung organoids.

PubMed

Dye, Briana R; Dedhia, Priya H; Miller, Alyssa J; Nagy, Melinda S; White, Eric S; Shea, Lonnie D; Spence, Jason R

2016-09-28

Human pluripotent stem cell (hPSC) derived tissues often remain developmentally immature in vitro, and become more adult-like in their structure, cellular diversity and function following transplantation into immunocompromised mice. Previously we have demonstrated that hPSC-derived human lung organoids (HLOs) resembled human fetal lung tissue in vitro (Dye et al., 2015). Here we show that HLOs required a bioartificial microporous poly(lactide-co-glycolide) (PLG) scaffold niche for successful engraftment, long-term survival, and maturation of lung epithelium in vivo. Analysis of scaffold-grown transplanted tissue showed airway-like tissue with enhanced epithelial structure and organization compared to HLOs grown in vitro. By further comparing in vitro and in vivo grown HLOs with fetal and adult human lung tissue, we found that in vivo transplanted HLOs had improved cellular differentiation of secretory lineages that is reflective of differences between fetal and adult tissue, resulting in airway-like structures that were remarkably similar to the native adult human lung.

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.

Detection of Variation in Long-Term Micropropagated Mature Pistachio via DNA-Based Molecular Markers.

PubMed

Akdemir, Hülya; Suzerer, Veysel; Tilkat, Engin; Onay, Ahmet; Çiftçi, Yelda Ozden

2016-12-01

Determination of genetic stability of in vitro-grown plantlets is needed for safe and large-scale production of mature trees. In this study, genetic variation of long-term micropropagated mature pistachio developed through direct shoot bud regeneration using apical buds (protocol A) and in vitro-derived leaves (protocol B) was assessed via DNA-based molecular markers. Randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), and amplified fragment length polymorphism (AFLP) were employed, and the obtained PIC values from RAPD (0.226), ISSR (0.220), and AFLP (0.241) showed that micropropagation of pistachio for different periods of time resulted in "reasonable polymorphism" among donor plant and its 18 clones. Mantel's test showed a consistence polymorphism level between marker systems based on similarity matrices. In conclusion, this is the first study on occurrence of genetic variability in long-term micropropagated mature pistachio plantlets. The obtained results clearly indicated that different marker approaches used in this study are reliable for assessing tissue culture-induced variations in long-term cultured pistachio plantlets.

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.

Effects of anticonvulsants in vivo on high affinity choline uptake in vitro in mouse hippocampal synaptosomes.

PubMed Central

Miller, J. A.; Richter, J. A.

1985-01-01

The effects of several anticonvulsant drugs on sodium-dependent high affinity choline uptake (HACU) in mouse hippocampal synaptosomes was investigated. HACU was measured in vitro after in vivo administration of the drug to mice. HACU was inhibited by drugs which have in common the ability to facilitate gamma-aminobutyric acid (GABA) transmission, pentobarbitone, phenobarbitone, barbitone, diazepam, chloridiazepoxide, and valproic acid. Dose-response relationships were determined for these drugs and the drugs' potencies at inhibiting HACU correlated well with their anticonvulsant potencies. Clonazepam, ethosuximide, carbamazepine, and barbituric acid had no effect on HACU in the doses used while phenytoin and trimethadione stimulated HACU. These results suggest that certain anticonvulsants may elicit a part of their anticonvulsant activity by modulating cholinergic neurones. This effect may be mediated through a GABA mechanism. PMID:3978310

Development of dopamine receptor radiopharmaceuticals for the study of neurological and psychiatric disorders

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

Dr. Jogeshwar Mukherjee

Our goals in this grant application are directed towards the development of radiotracers that may allow the study of the high-affinity state (functional state) of the dopamine receptors. There have been numerous reports on the presence of two inter-convertible states of these (G-protein coupled) receptors in vitro. However, there is no report that establishes the presence of these separate affinity states in vivo. We have made efforts in this direction in order to provide such direct in vivo evidence about the presence of the high affinity state. This understanding of the functional state of the receptors is of critical significancemore » in our overall diagnosis and treatment of diseases that implicate the G-protein coupled receptors. Four specific aims have been listed in the grant application: (1). Design and syntheses of agonists (2). Radiosyntheses of agonists (3). In vitro pharmacology of agonists (4). In vivo distribution and pharmacology of labeled derivatives. We have accomplished the syntheses and radiosyntheses of three agonist radiotracers labeled with carbon-11. In vitro and in vivo pharmacological experiments have been accomplished in rats and preliminary PET studies in non-human primates have been carried out. Various accomplishments during the funded years, briefly outlined in this document, have been disseminated by several publications in various journals and presentations in national and international meetings (Society of Nuclear Medicine, Society for Neuroscience and International Symposium on Radiopharmaceutical Chemistry).« less

A Unique Human Chorionic Gonadotropin Antagonist Suppresses Ovarian Hyperstimulation Syndrome in Rats

PubMed Central

Vardhana, Pratibhasri A.; Julius, Martin A.; Pollak, Susan V.; Lustbader, Evan G.; Trousdale, Rhonda K.; Lustbader, Joyce W.

2009-01-01

Ovarian hyperstimulation syndrome (OHSS) is a complication of in vitro fertilization associated with physiological changes after hCG administration to induce final oocyte maturation. It presents as widespread increases in vascular permeability and, in rare cases, results in cycle cancellation, multi-organ dysfunction, and pregnancy termination. These physiological changes are due primarily to activation of the vascular endothelial growth factor (VEGF) system in response to exogenous human chorionic gonadotropin (hCG). An hCG antagonist (hCG-Ant) could attenuate these effects by competitively binding to the LH/CG receptor, thereby blocking LH activity in vivo. We expressed a form of hCG that lacks three of its four N-linked glycosylation sites and tested its efficacy as an antagonist. The hCG-Ant binds the LH receptor with an affinity similar to native hCG and inhibits cAMP response in vitro. In a rat model for ovarian stimulation, hCG-Ant dramatically reduces ovulation and steroid hormone production. In a well-established rat OHSS model, vascular permeability and vascular endothelial growth factor (VEGF) expression are dramatically reduced after hCG-Ant treatment. Finally, hCG-Ant does not appear to alter blastocyst development when given after hCG in mice. These studies demonstrate that removing specific glycosylation sites on native hCG can produce an hCG-Ant that is capable of binding without activating the LH receptor and blocking the actions of hCG. Thus hCG-Ant will be investigated as a potential therapy for OHSS. PMID:19443574

Engineering hyperthermophilic archaeon Pyrococcus furiosus to overproduce its cytoplasmic [NiFe]-hydrogenase.

PubMed

Chandrayan, Sanjeev K; McTernan, Patrick M; Hopkins, R Christopher; Sun, Junsong; Jenney, Francis E; Adams, Michael W W

2012-01-27

The cytoplasmic hydrogenase (SHI) of the hyperthermophilic archaeon Pyrococcus furiosus is an NADP(H)-dependent heterotetrameric enzyme that contains a nickel-iron catalytic site, flavin, and six iron-sulfur clusters. It has potential utility in a range of bioenergy systems in vitro, but a major obstacle in its use is generating sufficient amounts. We have engineered P. furiosus to overproduce SHI utilizing a recently developed genetic system. In the overexpression (OE-SHI) strain, transcription of the four-gene SHI operon was under the control of a strong constitutive promoter, and a Strep-tag II was added to the N terminus of one subunit. OE-SHI and wild-type P. furiosus strains had similar rates of growth and H(2) production on maltose. Strain OE-SHI had a 20-fold higher transcription of the polycistronic hydrogenase mRNA encoding SHI, and the specific activity of the cytoplasmic hydrogenase was ∼10-fold higher when compared with the wild-type strain, although the expression levels of genes encoding processing and maturation of SHI were the same in both strains. Overexpressed SHI was purified by a single affinity chromatography step using the Strep-tag II, and it and the native form had comparable activities and physical properties. Based on protein yield per gram of cells (wet weight), the OE-SHI strain yields a 100-fold higher amount of hydrogenase when compared with the highest homologous [NiFe]-hydrogenase system previously reported (from Synechocystis). This new P. furiosus system will allow further engineering of SHI and provide hydrogenase for efficient in vitro biohydrogen production.

Engineering Hyperthermophilic Archaeon Pyrococcus furiosus to Overproduce Its Cytoplasmic [NiFe]-Hydrogenase*

PubMed Central

Chandrayan, Sanjeev K.; McTernan, Patrick M.; Hopkins, R. Christopher; Sun, Junsong; Jenney, Francis E.; Adams, Michael W. W.

2012-01-01

The cytoplasmic hydrogenase (SHI) of the hyperthermophilic archaeon Pyrococcus furiosus is an NADP(H)-dependent heterotetrameric enzyme that contains a nickel-iron catalytic site, flavin, and six iron-sulfur clusters. It has potential utility in a range of bioenergy systems in vitro, but a major obstacle in its use is generating sufficient amounts. We have engineered P. furiosus to overproduce SHI utilizing a recently developed genetic system. In the overexpression (OE-SHI) strain, transcription of the four-gene SHI operon was under the control of a strong constitutive promoter, and a Strep-tag II was added to the N terminus of one subunit. OE-SHI and wild-type P. furiosus strains had similar rates of growth and H2 production on maltose. Strain OE-SHI had a 20-fold higher transcription of the polycistronic hydrogenase mRNA encoding SHI, and the specific activity of the cytoplasmic hydrogenase was ∼10-fold higher when compared with the wild-type strain, although the expression levels of genes encoding processing and maturation of SHI were the same in both strains. Overexpressed SHI was purified by a single affinity chromatography step using the Strep-tag II, and it and the native form had comparable activities and physical properties. Based on protein yield per gram of cells (wet weight), the OE-SHI strain yields a 100-fold higher amount of hydrogenase when compared with the highest homologous [NiFe]-hydrogenase system previously reported (from Synechocystis). This new P. furiosus system will allow further engineering of SHI and provide hydrogenase for efficient in vitro biohydrogen production. PMID:22157005

Resveratrol Improves the Mitochondrial Function and Fertilization Outcome of Bovine Oocytes

PubMed Central

TAKEO, Shun; SATO, Daichi; KIMURA, Koji; MONJI, Yasunori; KUWAYAMA, Takehito; KAWAHARA-MIKI, Ryoka; IWATA, Hisataka

2013-01-01

The aim of the present study was to address the effect of resveratrol-mediated upregulation of sirtuin 1 (SIRT1) during oocyte maturation on mitochondrial function, the developmental ability of oocytes and on mechanisms responsible for blockage of polyspermic fertilization. Oocytes collected from slaughterhouse-derived ovaries were cultured in TCM-199 medium supplemented with 10% FCS and 0 or 20 µM resveratrol (Res). We examined the effect of Res on SIRT1 expression in in vitro-matured oocytes (Exp 1); fertilization and developmental ability (Exp 2); mitochondrial DNA copy number (Mt number), ATP content and mitochondrial membrane potential in matured oocytes (Exp 3); and the time required for proteinase to dissolve the zona pellucida following in vitro fertilization (as a marker of zona pellucida hardening), as well as on the distribution of cortical granules before and after fertilization (Exp 4). In Exp 1, the 20 µM Res treatment upregulated protein expression of SIRT1 in oocytes. In Exp 2, Res treatment improved the ratio of normal fertilization and the total cell number of blastocysts. In Exp 3, Res treatment significantly increased the ATP content in matured oocytes. Additionally, Res increased the overall Mt number and mitochondrial membrane potential, but the effect was donor-dependent. In Exp 4, Res-induced zona hardening improved the distribution and exocytosis of cortical granules after in vitro fertilization. In conclusion, Res improved the quality of oocytes by improving mitochondrial quantity and quality. In addition, Res added to the maturation medium enhanced SIRT1 protein expression in oocytes and improved fertilization via reinforcement of the mechanisms responsible for blockage of polyspermic fertilization. PMID:24390595

Pore-formation by adenylate cyclase toxoid activates dendritic cells to prime CD8+ and CD4+ T cells.

PubMed

Svedova, Martina; Masin, Jiri; Fiser, Radovan; Cerny, Ondrej; Tomala, Jakub; Freudenberg, Marina; Tuckova, Ludmila; Kovar, Marek; Dadaglio, Gilles; Adkins, Irena; Sebo, Peter

2016-04-01

The adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis is a bi-functional leukotoxin. It penetrates myeloid phagocytes expressing the complement receptor 3 and delivers into their cytosol its N-terminal adenylate cyclase enzyme domain (~400 residues). In parallel, ~1300 residue-long RTX hemolysin moiety of CyaA forms cation-selective pores and permeabilizes target cell membrane for efflux of cytosolic potassium ions. The non-enzymatic CyaA-AC(-) toxoid, has repeatedly been successfully exploited as an antigen delivery tool for stimulation of adaptive T-cell immune responses. We show that the pore-forming activity confers on the CyaA-AC(-) toxoid a capacity to trigger Toll-like receptor and inflammasome signaling-independent maturation of CD11b-expressing dendritic cells (DC). The DC maturation-inducing potency of mutant toxoid variants in vitro reflected their specifically enhanced or reduced pore-forming activity and K(+) efflux. The toxoid-induced in vitro phenotypic maturation of DC involved the activity of mitogen activated protein kinases p38 and JNK and comprised increased expression of maturation markers, interleukin 6, chemokines KC and LIX and granulocyte-colony-stimulating factor secretion, prostaglandin E2 production and enhancement of chemotactic migration of DC. Moreover, i.v. injected toxoids induced maturation of splenic DC in function of their cell-permeabilizing capacity. Similarly, the capacity of DC to stimulate CD8(+) and CD4(+) T-cell responses in vitro and in vivo was dependent on the pore-forming activity of CyaA-AC(-). This reveals a novel self-adjuvanting capacity of the CyaA-AC(-) toxoid that is currently under clinical evaluation as a tool for delivery of immunotherapeutic anti-cancer CD8(+) T-cell vaccines into DC.

Use of antigen-primed dendritic cells for inducing antitumor immune responses in vitro in patients with non-small cell lung cancer

PubMed Central

Obleukhova, Irina; Kiryishina, Nataliya; Falaleeva, Svetlana; Lopatnikova, Julia; Kurilin, Vasiliy; Kozlov, Vadim; Vitsin, Aleksander; Cherkasov, Andrey; Kulikova, Ekaterina; Sennikov, Sergey

2018-01-01

Cancer is associated with a reduction in immature and mature circulating dendritic cells (DCs), and with an impaired migratory capacity, compared with healthy donors. Therefore, modern approaches to the in vitro generation of DCs loaded with tumor antigens and their use for inducing antitumor immune responses in vivo are being investigated. The purpose of the present study was to investigate the phenotypic and functional characteristics of peripheral blood DC subsets in patients with non-small cell lung cancer (NSCLC), and the development of an antitumor cytotoxic response by mononuclear cells (MNCs) from patients using in vitro generated antigen-primed DCs. Heparinized peripheral venous blood samples were obtained from 10 healthy donors and 20 patients with a histologically verified diagnosis of NSCLC. The ability of antigen-activated DCs to stimulate the activity of MNCs against autologous tumor cells was evaluated using a cytotoxic test. Peripheral blood DC subsets from patients with NSCLC were identified to be decreased and to exhibit an impaired ability to mature, compared with healthy donors. Furthermore, DCs generated from MNCs from patients with NSCLC were able to stimulate a specific cytotoxic response when loaded with autologous tumor lysates or RNA and matured, in vitro. A perforin and granzyme B-dependent mode of cytotoxicity was primarily induced. The ability of DCs loaded with tumor antigens to increase the cytotoxic activity of MNCs against NSCLC cells in vitro indicates the effective induction and co-stimulation of T lymphocytes by the generated DCs. PMID:29399182

Quality of common marmoset (Callithrix jacchus) oocytes collected after ovarian stimulation.

PubMed

Kanda, Akifumi; Nobukiyo, Asako; Yoshioka, Miyuki; Hatakeyama, Teruhiko; Sotomaru, Yusuke

2018-01-15

The common marmoset (Callithrix jacchus) is an experimental animal that is considered suitable for the creation of next-generation human disease models. It has recently been used in the reproductive technology field. Oocytes can be effectively collected from female marmosets via ovarian stimulation with injections of follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG). The oocytes, collected about 28 h after the hCG injection, include both premature oocytes and postmature (in vivo matured; IVO) oocytes, and the premature oocytes can be matured by in vitro culture (in vitro matured; IVM). Although IVM and IVO oocytes are equivalent in appearance at the MII stage, it remains unclear whether there are differences in their properties. Therefore, we investigated their in vitro fertilization and developmental capacities and cytoskeletal statuses. Our findings revealed that the IVM and IVO oocytes had similar fertilization rates but that no IVO oocytes could develop to the blastocyst stage. Additionally, IVO oocytes showed abnormal cytoskeletal formation. It is concluded that IVM oocytes maintain normal function, whereas IVO oocytes would be affected by aging and other factors when they remain for a long time in the ovary. Copyright © 2017 Elsevier Inc. All rights reserved.

An in vitro study of functional maturation of murine thymus cells.

PubMed

Chakravarty, A K

1977-05-26

Critical time of onset of thymus cell functions in ontogeny was studied in vitro. Collaborative function in an antibody response and ability to induce a graft-versus-host (GvH) response by murine thymocytes from different stages of ontogeny were investigated. Thymocytes from as early as 16-day mouse embryos were capable of collaborating in the antibody response to sheep-erythrocyte-antigen in vitro following 24 h of pretreatment with concanavalin A (con A). By contrast, maturation of thymus cell function as measured by competence to induce a graft-versus-host reaction, was first manifested by newborn thymus cells, and pretreatment with con A did not facilitate the maturation of this thymus cell function. Experiments to understand the effect of con A on the expression of cell surface antigens have also been reported. Con A-treated thymus cells of different ontogenic stages tested were less susceptible to killing by anti-theta serum than nontreated thymus cells; reverse was true with anti-H-2 serum. The significance of the differential susceptibility of con A-treated thymus cells to anti-sera treatment and the finding that mouse thymocytes can provide helper function as early as the 16th day of gestation have been discussed.

Supplementation with nanomolar concentrations of verbascoside during in vitro maturation improves embryo development by protecting the oocyte against oxidative stress: a large animal model study.

PubMed

Martino, Nicola Antonio; Ariu, Federica; Bebbere, Daniela; Uranio, Manuel Filioli; Chirico, Adriana; Marzano, Giuseppina; Sardanelli, Anna Maria; Cardinali, Angela; Minervini, Fiorenza; Bogliolo, Luisa; Dell'Aquila, Maria Elena

2016-10-01

The effects of verbascoside (VB), added at nanomolar concentrations during in vitro maturation (IVM) of juvenile sheep oocytes, on in vitro embryo development and its mechanisms of action at the oocyte level were analyzed. Developmental rates, after IVM in the presence/absence of VB (1nM for 24h; 1nM for 2h; 10nM for 2h), were evaluated. The bioenergetic/oxidative status of oocytes matured after IVM in the presence/absence of 1nM VB for 24h was assessed by confocal analysis of mitochondria and reactive oxygen species (ROS), lipid peroxidation (LPO) assay, and quantitative PCR of bioenergy/redox-related genes. The addition of 1nM VB during 24h IVM significantly increased blastocyst formation and quality. Verbascoside reduced oocyte ROS and LPO and increased mitochondria/ROS colocalization while keeping mitochondria activity and gene expression unchanged. In conclusion, supplementation with nanomolar concentrations of VB during IVM, in the juvenile sheep model, promotes embryo development by protecting the oocyte against oxidative stress. Copyright © 2016 Elsevier Inc. All rights reserved.

Hydrostatic pressure affects in vitro maturation of oocytes and follicles and increases granulosa cell death.

PubMed

Rashidi, Zahra; Azadbakht, Mehri; Amini, Ali; Karimi, Isac

2014-01-01

This study examines the effects of hydrostatic pressure on in vitro maturation (IVM) of oocytes derived from in vitro grown follicles. In this experimental study, preantral follicles were isolated from 12-day-old female NMRI mice. Each follicle was cultured individually in Alpha Minimal Essential Medium (α-MEM) under mineral oil for 12 days. Then, follicles were induced for IVM and divided into two groups, control and experiment. In the experiment group follicles were subjected to 20 mmHg pressure for 30 minutes and cultured for 24-48 hours. We assessed for viability and IVM of the oocytes. The percentage of apoptosis in cumulus cells was determined by the TUNEL assay. A comparison between groups was made using the student's t test. The percentage of metaphase II oocytes (MII) increased in hydrostatic pressuretreated follicles compared to controls (p<0.05). Cumulus cell viability reduced in hydrostatic pressure-treated follicles compared to controls (p<0.05). Exposure of follicles to pressure increased apoptosis in cumulus cells compared to controls (p<0.05). Hydrostatic pressure, by inducing apoptosis in cumulus cells, participates in the cumulus oocyte coupled relationship with oocyte maturation.

Production, Nutritional Quality and In vitro Methane Production from Andropogon gayanus Grass Harvested at Different Maturities and Preserved as Hay or Silage

PubMed Central

Ribeiro, G. O.; Teixeira, A. M.; Velasco, F. O.; Faria Júnior, W. G.; Pereira, L. G. R.; Chaves, A. V.; Gonçalves, L. C.; McAllister, T. A.

2014-01-01

Andropogon gayanus is an important grass due to its high biomass production, drought tolerance and favorable growth on low fertility acidic soils. Currently, there is little research on the impact of growth stage on the nutritional quality or the degree of CH4 production that may arise from this forage during ruminal fermentation. The objectives of this study were to determine the effects of regrowth stage of A. gayanus on its chemical composition, in vitro production of gas and CH4, as well as in vitro dry matter (DM) digestibility when grown under tropical Brazilian conditions and conserved as hay or as silage. The nutritional value of A. gayanus grass declined with increasing maturity; however digestible DM yield linearly increased. After 112 d of regrowth, A. gayanus produced higher quality silage (higher lactate and lower pH and butyrate content) and higher DM yield. However, the low levels of crude protein at this time would make protein supplementation a necessity for proper rumen fermentation. No differences in CH4 kinetic parameters were found with advancing maturity or preservation method (hay or silage). PMID:25049959

In vitro antiprotozoal activity of the leaves of Artemisia ludoviciana.

PubMed

Said Fernández, Salvador; Ramos Guerra, Monica Celina; Mata Cárdenas, Benito David; Vargas Villarreal, Javier; Villarreal Treviño, Licet

2005-07-01

The inhabitants of Northeast of Mexico use an infusion of leaves from Artemisia ludoviciana as an antidiarrheal remedy. The aqueous, methanol, acetone and hexane leaf extracts from mature plants were found to be active in vitro against the parasitic protozoa Entamoeba histolytica and Giardia lamblia.

Development of an intracanal mature Enterococcus faecalis biofilm and its susceptibility to some antimicrobial intracanal medications; an in vitro study

PubMed Central

Saber, Shehab El-Din Mohamed; El-Hady, Soha A.

2012-01-01

Objectives: To develop a mature biofilm of Enterococcus faecalis inside the root canal system and to test its susceptibility to some antimicrobial medications in vitro. Methods: Single rooted premolars were mechanically enlarged, sterilized, and then infected with a clinical isolate of E. faecalis. Biofilm formation and maturation was monitored using SEM. Biofilm bacteria were exposed to Amoxicillin+clavulanate, Ciprofloxacin, Clindamycin, Doxycycline, and calcium hydroxide as intracanal medications for 1 week. Finally bacterial samples were collected, and colony-forming units were enumerated. Results: SEM examination confirmed the formation of a mature biofilm at the end of the incubation period. All the chemotherapeutic agents used were significantly better than Calcium hydroxide in elimination of biofilm bacteria. The antimicrobial effect of Amoxicillin + clavulanate, Ciprofloxacin and Clindamycin was significantly better than Doxycycline (P=.05). However the difference in the antimicrobial effectiveness among them was statistically non-significant (P=.05). Conclusions: The method used for bacterial biofilm development and maturation is reliable and can be used to assess the anti bacterial potential of endodontic materials. Also, the local application of antibacterial agents can be beneficial in resistant cases of apical periodontitis but only after careful culture and sensitivity testing to choose the appropriate agent for the existing flora. PMID:22229006

Effect of oxygen tension and serum during IVM on developmental competence of bovine oocytes.

PubMed

Pereira, Michele M; Machado, Marco A; Costa, Fernanda Q; Serapiao, Raquel V; Viana, Joao H M; Camargo, Luiz S A

2010-01-01

With an aim to improve the in vitro production of bovine embryos, the present study investigated the effect of serum and oxygen tension during IVM on oocyte developmental competence. Four experimental groups were evaluated: G1, 10% oestrus cow serum (OCS) with 20% O(2); G2, 0.1% polyvinyl alcohol (PVA) with 20% O(2); G3, 10% OCS with 5% O(2); and G4, 0.1% PVA with 5% O(2). The proportion of MII oocytes, blastocyst rates and total cell number were not affected (P > 0.05) when the OCS was replaced with PVA under 5% O(2), whereas a higher (P < 0.05) blastocyst rate and total cell number were found with OCS compared with PVA under 20% O(2). The apoptosis index was lower in blastocysts from oocytes matured with PVA under 5% O(2) (G4) compared with other groups (G1, G2 and G3), but no differences (P > 0.05) were found in maturation and blastocyst rates. Significant differences were found in the amount of specific transcripts in oocytes matured under different conditions. In conclusion maturation with PVA and 5% O(2) provides an efficient in vitro culture condition for the maturation of bovine oocytes.

The vascular plant-pathogenic bacterium Ralstonia solanacearum produces biofilms required for its virulence on the surfaces of tomato cells adjacent to intercellular spaces.

PubMed

Mori, Yuka; Inoue, Kanako; Ikeda, Kenichi; Nakayashiki, Hitoshi; Higashimoto, Chikaki; Ohnishi, Kouhei; Kiba, Akinori; Hikichi, Yasufumi

2016-08-01

The mechanism of colonization of intercellular spaces by the soil-borne and vascular plant-pathogenic bacterium Ralstonia solanacearum strain OE1-1 after invasion into host plants remains unclear. To analyse the behaviour of OE1-1 cells in intercellular spaces, tomato leaves with the lower epidermis layers excised after infiltration with OE1-1 were observed under a scanning electron microscope. OE1-1 cells formed microcolonies on the surfaces of tomato cells adjacent to intercellular spaces, and then aggregated surrounded by an extracellular matrix, forming mature biofilm structures. Furthermore, OE1-1 cells produced mushroom-type biofilms when incubated in fluids of apoplasts including intercellular spaces, but not xylem fluids from tomato plants. This is the first report of biofilm formation by R. solanacearum on host plant cells after invasion into intercellular spaces and mushroom-type biofilms produced by R. solanacearum in vitro. Sugar application led to enhanced biofilm formation by OE1-1. Mutation of lecM encoding a lectin, RS-IIL, which reportedly exhibits affinity for these sugars, led to a significant decrease in biofilm formation. Colonization in intercellular spaces was significantly decreased in the lecM mutant, leading to a loss of virulence on tomato plants. Complementation of the lecM mutant with native lecM resulted in the recovery of mushroom-type biofilms and virulence on tomato plants. Together, our findings indicate that OE1-1 produces mature biofilms on the surfaces of tomato cells after invasion into intercellular spaces. RS-IIL may contribute to biofilm formation by OE1-1, which is required for OE1-1 virulence. © 2015 BSPP AND JOHN WILEY & SONS LTD.

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.

In vitro erythrocyte membrane stabilization properties of Carica papaya L. leaf extracts

PubMed Central

Ranasinghe, Priyanga; Ranasinghe, Pathmasiri; Abeysekera, W. P. Kaushalya M.; Premakumara, G. A. Sirimal; Perera, Yashasvi S.; Gurugama, Padmalal; Gunatilake, Saman B.

2012-01-01

Background: Carica papaya L. fruit juice and leaf extracts are known to have many beneficial medical properties. Recent reports have claimed possible beneficial effects of C. papaya L. leaf juice in treating patients with dengue viral infections. This study aims to evaluate the membrane stabilization potential of C. papaya L. leaf extracts using an in vitro hemolytic assay. Materials and Methods: The study was conducted in between June and August 2010. Two milliliters of blood from healthy volunteers and patients with serologically confirmed current dengue infection were freshly collected and used in the assays. Fresh papaya leaves at three different maturity stages (immature, partly matured, and matured) were cleaned with distilled water, crushed, and the juice was extracted with 10 ml of cold distilled water. Freshly prepared cold water extracts of papaya leaves (1 ml containing 30 μl of papaya leaf extracts, 20 μl from 40% erythrocytes suspension, and 950 μl of phosphate buffered saline) were used in the heat-induced and hypotonic-induced hemolytic assays. In dose response experiments, six different concentrations (9.375, 18.75, 37.5, 75, 150, and 300 μg/ml) of freeze dried extracts of the partly matured leaves were used. Membrane stabilization properties were investigated with heat-induced and hypotonicity-induced hemolysis assays. Results: Extracts of papaya leaves of all three maturity levels showed a significant reduction in heat-induced hemolysis compared to controls (P < 0.05). Papaya leaf extracts of all three maturity levels showed more than 25% inhibition at a concentration of 37.5 μg/ml. The highest inhibition of heat-induced hemolysis was observed at 37.5 μg/ml. Inhibition activity of different maturity levels was not significantly (P < 0.05) different from one another. Heat-induced hemolysis inhibition activity did not demonstrate a linear dose response relationship. At 37.5 μg/ml concentration of the extract, a marked inhibition of hypotonicity-induced hemolysis was observed. Conclusion: C. papaya L. leaf extracts showed a significant inhibition of hemolysis in vitro and could have a potential therapeutic effect on disease processes causing destabilization of biological membranes. PMID:23225962

In vitro erythrocyte membrane stabilization properties of Carica papaya L. leaf extracts.

PubMed

Ranasinghe, Priyanga; Ranasinghe, Pathmasiri; Abeysekera, W P Kaushalya M; Premakumara, G A Sirimal; Perera, Yashasvi S; Gurugama, Padmalal; Gunatilake, Saman B

2012-10-01

Carica papaya L. fruit juice and leaf extracts are known to have many beneficial medical properties. Recent reports have claimed possible beneficial effects of C. papaya L. leaf juice in treating patients with dengue viral infections. This study aims to evaluate the membrane stabilization potential of C. papaya L. leaf extracts using an in vitro hemolytic assay. The study was conducted in between June and August 2010. Two milliliters of blood from healthy volunteers and patients with serologically confirmed current dengue infection were freshly collected and used in the assays. Fresh papaya leaves at three different maturity stages (immature, partly matured, and matured) were cleaned with distilled water, crushed, and the juice was extracted with 10 ml of cold distilled water. Freshly prepared cold water extracts of papaya leaves (1 ml containing 30 μl of papaya leaf extracts, 20 μl from 40% erythrocytes suspension, and 950 μl of phosphate buffered saline) were used in the heat-induced and hypotonic-induced hemolytic assays. In dose response experiments, six different concentrations (9.375, 18.75, 37.5, 75, 150, and 300 μg/ml) of freeze dried extracts of the partly matured leaves were used. Membrane stabilization properties were investigated with heat-induced and hypotonicity-induced hemolysis assays. Extracts of papaya leaves of all three maturity levels showed a significant reduction in heat-induced hemolysis compared to controls (P < 0.05). Papaya leaf extracts of all three maturity levels showed more than 25% inhibition at a concentration of 37.5 μg/ml. The highest inhibition of heat-induced hemolysis was observed at 37.5 μg/ml. Inhibition activity of different maturity levels was not significantly (P < 0.05) different from one another. Heat-induced hemolysis inhibition activity did not demonstrate a linear dose response relationship. At 37.5 μg/ml concentration of the extract, a marked inhibition of hypotonicity-induced hemolysis was observed. C. papaya L. leaf extracts showed a significant inhibition of hemolysis in vitro and could have a potential therapeutic effect on disease processes causing destabilization of biological membranes.

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

Effect of different shipping temperatures (∼22 °C vs. ∼7 °C) and holding media on blastocyst development after overnight holding of immature equine cumulus-oocyte complexes.

PubMed

Diaw, Mouhamadou; Salgado, Renato M; Canesin, Heloísa S; Gridley, Nell; Hinrichs, Katrin

2018-04-15

Intracytoplasmic sperm injection (ICSI) is an important tool for equine embryo production in both clinical and research settings. In clinical ICSI programs, immature equine cumulus-oocyte complexes (COCs) are often collected at the mare's location and shipped to the ICSI laboratory. To simplify shipment and aid scheduling of subsequent procedures, COCs can be held overnight at room temperature (∼22 °C) before placement into maturation culture, with no detrimental effect on meiotic or developmental competence. A recent study indicated that it might be possible to hold COCs overnight at cold (∼4 °C) temperatures. If so, this might allow longer holding periods that would ease shipping requirements. In this study, we compared oocyte maturation rates, as well as cleavage and blastocyst rates after ICSI, for COCs held at either room or cold temperatures overnight before the onset of in vitro maturation. In Exp. 1, COCs were shipped overnight in a commercial embryo holding medium, ViGRO (Vg), in insulated containers designed to hold at either room temperature (RT, ∼22 °C) or cold temperatures (Cold, ∼7 °C). Subsequent rates of in vitro maturation, cleavage and blastocyst formation were significantly higher in the RT treatment (39%, 90% and 41%, respectively) than in the Cold treatment (23%, 60% and 17%, respectively, P < .05). In Exp. 2, we compared Vg medium with a second commercial embryo holding medium, SYNGRO (Sy). There was no significant difference between Vg and Sy groups in any evaluated parameter within either RT or Cold treatments. Within each medium group and for both media combined, the rates of in vitro maturation, cleavage and blastocyst formation were significantly higher in the RT treatment (42%, 81% and 42%, respectively for the combined media) than in the Cold treatment (29%, 54% and 10%, respectively for the combined media, P < .05). We conclude that shipment of immature equine COCs at cold temperatures (∼7 °C) is detrimental to subsequent in vitro maturation and embryo production. Copyright © 2018 Elsevier Inc. All rights reserved.

Retrovirus-specific differences in matrix and nucleocapsid protein-nucleic acid interactions: implications for genomic RNA packaging.

PubMed

Sun, Meng; Grigsby, Iwen F; Gorelick, Robert J; Mansky, Louis M; Musier-Forsyth, Karin

2014-01-01

Retroviral RNA encapsidation involves a recognition event between genomic RNA (gRNA) and one or more domains in Gag. In HIV-1, the nucleocapsid (NC) domain is involved in gRNA packaging and displays robust nucleic acid (NA) binding and chaperone functions. In comparison, NC of human T-cell leukemia virus type 1 (HTLV-1), a deltaretrovirus, displays weaker NA binding and chaperone activity. Mutation of conserved charged residues in the deltaretrovirus bovine leukemia virus (BLV) matrix (MA) and NC domains affects virus replication and gRNA packaging efficiency. Based on these observations, we hypothesized that the MA domain may generally contribute to NA binding and genome encapsidation in deltaretroviruses. Here, we examined the interaction between HTLV-2 and HIV-1 MA proteins and various NAs in vitro. HTLV-2 MA displays higher NA binding affinity and better chaperone activity than HIV-1 MA. HTLV-2 MA also binds NAs with higher affinity than HTLV-2 NC and displays more robust chaperone function. Mutation of two basic residues in HTLV-2 MA α-helix II, previously implicated in BLV gRNA packaging, reduces NA binding affinity. HTLV-2 MA binds with high affinity and specificity to RNA derived from the putative packaging signal of HTLV-2 relative to nonspecific NA. Furthermore, an HIV-1 MA triple mutant designed to mimic the basic character of HTLV-2 MA α-helix II dramatically improves binding affinity and chaperone activity of HIV-1 MA in vitro and restores RNA packaging to a ΔNC HIV-1 variant in cell-based assays. Taken together, these results are consistent with a role for deltaretrovirus MA proteins in viral RNA packaging.

The tau positron-emission tomography tracer AV-1451 binds with similar affinities to tau fibrils and monoamine oxidases.

PubMed

Vermeiren, Céline; Motte, Philippe; Viot, Delphine; Mairet-Coello, Georges; Courade, Jean-Philippe; Citron, Martin; Mercier, Joël; Hannestad, Jonas; Gillard, Michel

2018-02-01

Lilly/Avid's AV-1451 is one of the most advanced tau PET tracers in the clinic. Although results obtained in Alzheimer's disease patients are compelling, discrimination of tracer uptake in healthy individuals and patients with supranuclear palsy (PSP) is less clear as there is substantial overlap of signal in multiple brain regions. Moreover, accurate quantification of [ 18 F]AV-1451 uptake in Alzheimer's disease may not be possible. The aim of the present study was to characterize the in vitro binding of AV-1451 to understand and identify potential off-target binding that could explain the poor discrimination observed in PSP patients. [ 3 H]AV-1451 and AV-1451 were characterized in in vitro binding assays using recombinant and native proteins/tissues from postmortem samples of controls and Alzheimer's disease and PSP patients. [ 3 H]AV-1451 binds to multiple sites with nanomolar affinities in brain homogenates and to tau fibrils isolated from Alzheimer's disease or PSP patients. [ 3 H]AV-1451 also binds with similarly high affinities in brain homogenates devoid of tau pathology. This unexpected binding was demonstrated to be because of nanomolar affinities of [ 3 H]AV-1451 for monoamine oxidase A and B enzymes. High affinity of AV-1451 for monoamine oxidase proteins may limit its utility as a tau PET tracer in PSP and Alzheimer's disease because of high levels of monoamine oxidase expression in brain regions also affected by tau deposition, especially if monoamine oxidase levels change over time or with a treatment intervention. © 2017 International Parkinson and Movement Disorder Society. © 2017 International Parkinson and Movement Disorder Society.

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

Human bone marrow-derived mesenchymal cells differentiate and mature into endocrine pancreatic lineage in vivo.

PubMed

Phadnis, Smruti M; Joglekar, Mugdha V; Dalvi, Maithili P; Muthyala, Sudhakar; Nair, Prabha D; Ghaskadbi, Surendra M; Bhonde, Ramesh R; Hardikar, Anandwardhan A

2011-03-01

The scarcity of human islets for transplantation remains a major limitation of cell replacement therapy for diabetes. Bone marrow-derived progenitor cells are of interest because they can be isolated, expanded and offered for such therapy under autologous/allogeneic settings. We characterized and compared human bone marrow-derived mesenchymal cells (hBMC) obtained from (second trimester), young (1-24 years) and adult (34-81 years) donors. We propose a novel protocol that involves assessment of paracrine factors from regenerating pancreas in differentiation and maturation of hBMC into endocrine pancreatic lineage in vivo. We observed that donor age was inversely related to growth potential of hBMC. Following in vitro expansion and exposure to specific growth factors involved in pancreatic development, hBMC migrated and formed islet-like cell aggregates (ICA). ICA show increased abundance of pancreatic transcription factors (Ngn3, Brn4, Nkx6.1, Pax6 and Isl1). Although efficient differentiation was not achieved in vitro, we observed significant maturation and secretion of human c-peptide (insulin) upon transplantation into pancreactomized and Streptozotocin (STZ)-induced diabetic mice. Transplanted ICA responded to glucose and maintained normoglycemia in diabetic mice. Our data demonstrate that hBMC have tremendous in vitro expansion potential and can be differentiated into multiple lineages, including the endocrine pancreatic lineage. Paracrine factors secreted from regenerating pancreas help in efficient differentiation and maturation of hBMC, possibly via recruiting chromatin modulators, to generate glucose-responsive insulin-secreting cells.

Replacement of serum with sericin in in vitro maturation and culture media: Effects on embryonic developmental competence of Sanjabi sheep embryo during breeding season.

PubMed

Hajarian, H; Aghaz, F; Karami Shabankareh, H

2017-04-01

Sericin is a water-soluble component of silk and has been used as a biomaterial due to its antibacterial and ultraviolet radiation-resistant properties. This study was designed to evaluate the effect of sericin supplementation, as a serum replacement, in maturation and culture media on the meiotic competence of oocytes or in vitro culture of ovine embryos. In experiment 1, oocytes were matured in the presence of 10% fetal ovine serum (FOS), 0.1% polyvinyl alcohol (PVA) and different concentrations of sericin (0.1, 0.5, 1 and 2.5%), for 24 h. The addition of 0.5% sericin to maturation medium increased the rates of maturation to metaphase II of oocytes compared with those in cultures with 0.1% PVA. Following fertilization, blastocyst development was higher for oocytes matured with 0.5% of sericin compared with 0.1% PVA. However, the rates of nuclear maturation of oocytes and blastocyst development under FOS and 0.5% sericin were not significantly different. In experiment 2, presumptive zygotes were cultured in the presence of 10% FOS, 0.1% PVA and different concentrations of sericin (0.1, 0.5, 1 and 2.5%), for 7-8 days. The addition of 0.5% sericin to culture medium increased the blastocyst rate compared with those in cultures without sericin or addition of 0.1% PVA and 10% FOS. These results indicate the feasibility of sericin as an alternative protein supplement for IVM and IVC in ovine oocytes and zygotes. Copyright © 2016. Published by Elsevier Inc.

Fatty Acid Synthesis and Oxidation in Cumulus Cells Support Oocyte Maturation in Bovine

PubMed Central

Sanchez-Lazo, Laura; Brisard, Daphné; Elis, Sébastien; Maillard, Virginie; Uzbekov, Rustem; Labas, Valérie; Desmarchais, Alice; Papillier, Pascal; Monget, Philippe

2014-01-01

Oocyte meiotic maturation requires energy from various substrates including glucose, amino acids, and lipids. Mitochondrial fatty acid (FA) β-oxidation (FAO) in the oocyte is required for meiotic maturation, which is accompanied by differential expression of numerous genes involved in FAs metabolism in surrounding cumulus cells (CCs) in vivo. The objective was to elucidate components involved in FAs metabolism in CCs during oocyte maturation. Twenty-seven genes related to lipogenesis, lipolysis, FA transport, and FAO were chosen from comparative transcriptome analysis of bovine CCs before and after maturation in vivo. Using real-time PCR, 22 were significantly upregulated at different times of in vitro maturation (IVM) in relation to oocyte meiosis progression from germinal vesicle breakdown to metaphase-II. Proteins FA synthase, acetyl-coenzyme-A carboxylase, carnitine palmitoyltransferase, perilipin 2, and FA binding protein 3 were detected by Western blot and immunolocalized to CCs and oocyte cytoplasm, with FA binding protein 3 concentrated around oocyte chromatin. By mass spectrometry, CCs lipid profiling was shown to be different before and after IVM. FAO inhibitors etomoxir and mildronate dose-dependently decreased the oocyte maturation rate in vitro. In terms of viability, cumulus enclosed oocytes were more sensitive to etomoxir than denuded oocytes. In CCs, etomoxir (150μM) led to downregulation of lipogenesis genes and upregulated lipolysis and FAO genes. Moreover, the number of lipid droplets decreased, whereas several lipid species were more abundant compared with nontreated CCs after IVM. In conclusion, FAs metabolism in CCs is important to maintain metabolic homeostasis and may influence meiosis progression and survival of enclosed oocytes. PMID:25058602

Induction of human dendritic cell maturation using transfection with RNA encoding a dominant positive toll-like receptor 4.

PubMed

Cisco, Robin M; Abdel-Wahab, Zeinab; Dannull, Jens; Nair, Smita; Tyler, Douglas S; Gilboa, Eli; Vieweg, Johannes; Daaka, Yehia; Pruitt, Scott K

2004-06-01

Maturation of dendritic cells (DC) is critical for the induction of Ag-specific immunity. Ag-loaded DC matured with LPS, which mediates its effects by binding to Toll-like receptor 4 (TLR4), induce Ag-specific CTL in vitro and in vivo in animal models. However, clinical use of LPS is limited due to potential toxicity. Therefore, we sought to mimic the maturation-inducing effects of LPS on DC by stimulating TLR4-mediated signaling in the absence of exogenous LPS. We developed a constitutively active TLR4 (caTLR4) and demonstrated that transfection of human DC with RNA encoding caTLR4 led to IL-12 and TNF-alpha secretion. Transfection with caTLR4 RNA also induced a mature DC phenotype. Functionally, transfection of DC with caTLR4 RNA enhanced allostimulation of CD4(+) T cells. DC transfected with RNA encoding the MART (Melan-A/MART-1) melanoma Ag were then used to stimulate T cells in vitro. Cotransfection of these DC with caTLR4 RNA enhanced the generation of MART-specific CTL. This CTL activity was superior to that seen when DC maturation was induced using either LPS or a standard mixture of cytokines (TNF-alpha, IL-6, IL-1beta, and PGE(2)). We conclude that transfection of DC with RNA encoding a functional signaling protein, such as caTLR4, may provide a new tool for studying TLR signaling in DC and may be a promising approach for the induction of DC maturation for tumor immunotherapy.

The anti-tumor efficacy of 3C23K, a glyco-engineered humanized anti-MISRII antibody, in an ovarian cancer model is mainly mediated by engagement of immune effector cells

PubMed Central

Estupina, Pauline; Fontayne, Alexandre; Barret, Jean-Marc; Kersual, Nathalie; Dubreuil, Olivier; Le Blay, Marion; Pichard, Alexandre; Jarlier, Marta; Pugnière, Martine; Chauvin, Maëva; Chardès, Thierry; Pouget, Jean-Pierre; Deshayes, Emmanuel; Rossignol, Alexis; Abache, Toufik; de Romeuf, Christophe; Terrier, Aurélie; Verhaeghe, Lucie; Gaucher, Christine; Prost, Jean-François

2017-01-01

Ovarian cancer is the leading cause of death in women with gynecological cancers and despite recent advances, new and more efficient therapies are crucially needed. Müllerian Inhibiting Substance type II Receptor (MISRII, also named AMHRII) is expressed in most ovarian cancer subtypes and is a novel potential target for ovarian cancer immunotherapy. We previously developed and tested 12G4, the first murine monoclonal antibody (MAb) against human MISRII. Here, we report the humanization, affinity maturation and glyco-engineering steps of 12G4 to generate the Fc-optimized 3C23K MAb, and the evaluation of its in vivo anti-tumor activity. The epitopes of 3C23K and 12G4 were strictly identical and 3C23K affinity for MISRII was enhanced by a factor of about 14 (KD = 5.5 × 10−11 M vs 7.9 × 10−10 M), while the use of the EMABling® platform allowed the production of a low-fucosylated 3C23K antibody with a 30-fold KD improvement of its affinity to FcγRIIIa. In COV434-MISRII tumor-bearing mice, 3C23K reduced tumor growth more efficiently than 12G4 and its combination with carboplatin was more efficient than each monotherapy with a mean tumor size of 500, 1100 and 100 mm3 at the end of treatment with 3C23K (10 mg/kg, Q3-4D12), carboplatin (60 mg/kg, Q7D4) and 3C23K+carboplatin, respectively. Conversely, 3C23K-FcKO, a 3C23K form without affinity for the FcγRIIIa receptor, did not display any anti-tumor effect in vivo. These results strongly suggested that 3C23K mechanisms of action are mainly Fc-related. In vitro, antibody-dependent cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP) were induced by 3C23K, as demonstrated with human effector cells. Using human NK cells, 50% of the maximal lysis was obtained with a 46-fold lower concentration of low-fucosylated 3C23K (2.9 ng/ml) than of 3C23K expressed in CHO cells (133.35 ng/ml). As 3C23K induced strong ADCC with human PBMC but almost none with murine PBMC, antibody-dependent cell phagocytosis (ADCP) was then investigated. 3C23K-dependent (100 ng/ml) ADCP was more active with murine than human macrophages (only 10% of living target cells vs. about 25%). These in vitro results suggest that the reduced ADCC with murine effectors could be partially balanced by ADCP activity in in vivo experiments. Taken together, these preclinical data indicate that 3C23K is a new promising therapeutic candidate for ovarian cancer immunotherapy and justify its recent introduction in a phase I clinical trial. PMID:28427157

Fluorescent and Lanthanide Labeling for Ligand Screens, Assays, and Imaging

PubMed Central

Josan, Jatinder S.; De Silva, Channa R.; Yoo, Byunghee; Lynch, Ronald M.; Pagel, Mark D.; Vagner, Josef; Hruby, Victor J.

2012-01-01

The use of fluorescent (or luminescent) and metal contrast agents in high-throughput screens, in vitro assays, and molecular imaging procedures has rapidly expanded in recent years. Here we describe the development and utility of high-affinity ligands for cancer theranostics and other in vitro screening studies. In this context, we also illustrate the syntheses and use of heteromultivalent ligands as targeted imaging agents. PMID:21318902

Inhibition of cytochrome P450 3A by acetoxylated analogues of resveratrol in in vitro and in silico models

NASA Astrophysics Data System (ADS)

Basheer, Loai; Schultz, Keren; Kerem, Zohar

2016-08-01

Many dietary compounds, including resveratrol, are potent inhibitors of CYP3A4. Here we examined the potential to predict inhibition capacity of dietary polyphenolics using an in silico and in vitro approaches and synthetic model compounds. Mono, di, and tri-acetoxy resveratrol were synthesized, a cell line of human intestine origin and microsomes from rat liver served to determine their in vitro inhibition of CYP3A4, and compared to that of resveratrol. Docking simulation served to predict the affinity of the synthetic model compounds to the enzyme. Modelling of the enzyme’s binding site revealed three types of interaction: hydrophobic, electrostatic and H-bonding. The simulation revealed that each of the examined acetylations of resveratrol led to the loss of important interactions of all types. Tri-acetoxy resveratrol was the weakest inhibitor in vitro despite being the more lipophilic and having the highest affinity for the binding site. The simulation demonstrated exclusion of all interactions between tri-acetoxy resveratrol and the heme due to distal binding, highlighting the complexity of the CYP3A4 binding site, which may allow simultaneous accommodation of two molecules. Finally, the use of computational modelling may serve as a quick predictive tool to identify potential harmful interactions between dietary compounds and prescribed drugs.

Improving cattle nutrition on the Great Plains with shrubs and fecal seeding of fourwing saltbush

USDA-ARS?s Scientific Manuscript database

Two in vitro trials were conducted for estimates of dietary percentage of fourwing saltbush (Atriplex canescens; FS) or winterfat (Krascheninnikovia lanata; WF) to improve diet digestibility when cattle graze mature cool-season grass. Three in vitro trials were conducted to estimate the percentage ...

Surfactant-free Colloidal Particles with Specific Binding Affinity

PubMed Central

2017-01-01

Colloidal particles with specific binding affinity are essential for in vivo and in vitro biosensing, targeted drug delivery, and micrometer-scale self-assembly. Key to these techniques are surface functionalizations that provide high affinities to specific target molecules. For stabilization in physiological environments, current particle coating methods rely on adsorbed surfactants. However, spontaneous desorption of these surfactants typically has an undesirable influence on lipid membranes. To address this issue and create particles for targeting molecules in lipid membranes, we present here a surfactant-free coating method that combines high binding affinity with stability at physiological conditions. After activating charge-stabilized polystyrene microparticles with EDC/Sulfo-NHS, we first coat the particles with a specific protein and subsequently covalently attach a dense layer of poly(ethyelene) glycol. This polymer layer provides colloidal stability at physiological conditions as well as antiadhesive properties, while the protein coating provides the specific affinity to the targeted molecule. We show that NeutrAvidin-functionalized particles bind specifically to biotinylated membranes and that Concanavalin A-functionalized particles bind specifically to the glycocortex of Dictyostelium discoideum cells. The affinity of the particles changes with protein density, which can be tuned during the coating procedure. The generic and surfactant-free coating method reported here transfers the high affinity and specificity of a protein onto colloidal polystyrene microparticles. PMID:28847149

Poly(hydroxyethyl methacrylate) based affinity membranes for in vitro removal of anti-dsDNA antibodies from SLE plasma.

PubMed

Uzun, Lokman; Yavuz, Handan; Osman, Bilgen; Celik, Hamdi; Denizli, Adil

2010-07-01

The preparation of polymeric membrane using affinity technology for application in blood filtration devices is described here. DNA attached poly(hydroxyethyl methacrylate) (PHEMA) based microporous affinity membrane was prepared for selective removal of anti-dsDNA antibodies from systemic lupus erythematosus (SLE) patient plasma in in vitro. In order to further increase blood-compatibility of affinity membrane, aminoacid based comonomer N-methacryloyl-L-alanine (MAAL) was included in the polymerization recipe. PHEMAAL membrane was produced by a photopolymerization technique and then characterized by swelling tests and scanning electron microscope (SEM) studies. Blood-compatibility tests were also performed. The water swelling ratio of PHEMAAL membrane increased significantly (133.2%) compared with PHEMA (58%). PHEMAAL membrane has large pores around in the range of 5-10 microm. All the clotting times increased when compared with PHEMA membrane. Loss of platelets and leukocytes was very low. DNA loading was 7.8 mg/g. There was a very low anti-dsDNA-antibody adsorption onto the plain PHEMAAL membrane, about 78 IU/g. The PHEMAAL-DNA membrane adsorbed anti-dsDNA-antibody in the range of 10-68 x 10(3)IU/g from SLE plasma. Anti-dsDNA-antibody concentration decreased significantly from 875 to 144 IU/ml with the time. Anti-dsDNA-antibodies could be repeatedly adsorbed and eluted without noticeable loss in the anti-dsDNA-antibody adsorption amount. (c) 2010 Elsevier B.V. All rights reserved.

Relative binding affinity of carboxylate-, phosphonate-, and bisphosphonate-functionalized gold nanoparticles targeted to damaged bone tissue

NASA Astrophysics Data System (ADS)

Ross, Ryan D.; Cole, Lisa E.; Roeder, Ryan K.

2012-10-01

Functionalized Au NPs have received considerable recent interest for targeting and labeling cells and tissues. Damaged bone tissue can be targeted by functionalizing Au NPs with molecules exhibiting affinity for calcium. Therefore, the relative binding affinity of Au NPs surface functionalized with either carboxylate ( l-glutamic acid), phosphonate (2-aminoethylphosphonic acid), or bisphosphonate (alendronate) was investigated for targeted labeling of damaged bone tissue in vitro. Targeted labeling of damaged bone tissue was qualitatively verified by visual observation and backscattered electron microscopy, and quantitatively measured by the surface density of Au NPs using field-emission scanning electron microscopy. The surface density of functionalized Au NPs was significantly greater within damaged tissue compared to undamaged tissue for each functional group. Bisphosphonate-functionalized Au NPs exhibited a greater surface density labeling damaged tissue compared to glutamic acid- and phosphonic acid-functionalized Au NPs, which was consistent with the results of previous work comparing the binding affinity of the same functionalized Au NPs to synthetic hydroxyapatite crystals. Targeted labeling was enabled not only by the functional groups but also by the colloidal stability in solution. Functionalized Au NPs were stabilized by the presence of the functional groups, and were shown to remain well dispersed in ionic (phosphate buffered saline) and serum (fetal bovine serum) solutions for up to 1 week. Therefore, the results of this study suggest that bisphosphonate-functionalized Au NPs have potential for targeted delivery to damaged bone tissue in vitro and provide motivation for in vivo investigation.

Mass Spectrometric Determination of ILPR G-quadruplex Binding Sites in Insulin and IGF-2

PubMed Central

Xiao, JunFeng

2009-01-01

The insulin-linked polymorphic region (ILPR) of the human insulin gene promoter region forms G-quadruplex structures in vitro. Previous studies show that insulin and insulin-like growth factor-2 (IGF-2) exhibit high affinity binding in vitro to 2-repeat sequences of ILPR variants a and h, but negligible binding to variant i. Two-repeat sequences of variants a and h form intramolecular G-quadruplex structures that are not evidenced for variant i. Here we report on the use of protein digestion combined with affinity capture and MALDI-MS detection to pinpoint ILPR binding sites in insulin and IGF-2. Peptides captured by ILPR variants a and h were sequenced by MALDI-MS/MS, LC-MS and in silico digestion. On-bead digestion of insulin-ILPR variant a complexes supported the conclusions. The results indicate that the sequence VCG(N)RGF is generally present in the captured peptides and is likely involved in the affinity binding interactions of the proteins with the ILPR G-quadruplexes. The significance of arginine in the interactions was studied by comparing the affinities of synthesized peptides VCGERGF and VCGEAGF with ILPR variant a. Peptides from other regions of the proteins that are connected through disulfide linkages were also detected in some capture experiments. Identification of binding sites could facilitate design of DNA binding ligands for capture and detection of insulin and IGF-2. The interactions may have biological significance as well. PMID:19747845

Production of blastocysts following in vitro maturation and fertilization of dromedary camel oocytes vitrified at the germinal vesicle stage.

PubMed

Fathi, Mohamed; Moawad, Adel R; Badr, Magdy R

2018-01-01

Cryopreservation of oocytes would serve as an alternative to overcome the limited availability of dromedary camel oocytes and facilitate improvements in IVP techniques in this species. Our goal was to develop a protocol for the vitrification of camel oocytes at the germinal vesicle (GV) stage using different cryoprotectant combinations: 20% EG and 20% DMSO (VS1), 25% EG plus 25% DMSO (VS2) or 25% EG and 25% glycerol (VS3) and various cryo-carriers; straws or open pulled-straw (OPS) or solid surface vitrification (SSV); and Cryotop. Viable oocytes were cultured in vitro for 30 h. Matured oocytes were fertilized with epididymal spermatozoa and then cultured in vitro in modified KSOMaa medium for 7 days. Survival and nuclear maturation rates were significantly lower (P ≤ 0.05) in oocytes exposed to VS3 (44.8% and 34.0%, respectively) than those exposed to VS1 (68.2% and 48.0%, respectively) and VS2 (79.3% and 56.9%, respectively). Although recovery rates were significantly lower (P ≤ 0.05) in SSV and Cryotop vitrified oocytes (66.9% to 71.1%) than those vitrified by straws with VS1 or VS2 solutions (86.3% to 91.0%), survival rates were higher in the SSV and Cryotop groups (90.7% to 94.8%) than in the straw and OPS groups (68.2% to 86.5%). Among vitrified groups, maturation and fertilization rates were the highest in the Cryotop-VS2 group (51.8% and 39.2%, respectively). These values were comparable to those seen in the controls (59.2% and 44.6%, respectively). Cleavage (22.5% to 27.9%), morula (13.2% to 14.5%), and blastocyst (6.4% to 8.5%) rates were significantly higher (P ≤ 0.05) in SSV and Cryotop groups than in straws. No significant differences were observed in these parameters between the Cryotop and control groups. We report for the first time that dromedary oocytes vitrified at the GV-stage have the ability to be matured, fertilized and subsequently develop in vitro to produce blastocysts at frequencies comparable to those obtained using fresh oocytes.

Effects of recipient oocyte age and interval from fusion to activation on development of buffalo (Bubalus bubalis) nuclear transfer embryos derived from fetal fibroblasts.

PubMed

Lu, F; Jiang, J; Li, N; Zhang, S; Sun, H; Luo, C; Wei, Y; Shi, D

2011-09-15

The objective was to investigate the effect of recipient oocyte age and the interval from activation to fusion on developmental competence of buffalo nuclear transfer (NT) embryos. Buffalo oocytes matured in vitro for 22 h were enucleated by micromanipulation under the spindle view system, and a fetal fibroblast (pretreated with 0.1 μg/mL aphidicolin for 24 h, followed by culture for 48 h in 0.5% fetal bovine serum) was introduced into the enucleated oocyte, followed by electrofusion. Both oocytes and NT embryos were activated by exposure to 5 μM ionomycin for 5 min, followed by culture in 2 mM 6-dimethyl-aminopurine for 3 h. When oocytes matured in vitro for 28, 29, 30, 31, or 32 h were activated, more oocytes matured in vitro for 30 h developed into blastocysts in comparison with oocytes matured in vitro for 32 h (31.3 vs 19.9%, P < 0.05). When electrofusion was induced 27 h after the onset of oocyte maturation, the cleavage rate (78.0%) was higher than that of electrofusion induced at 28 h (67.2%, P < 0.05), and the blastocyst yield (18.1%) was higher (P < 0.05) than that of electrofusion induced at 25 or 26 h (7.4 and 8.5%, respectively). A higher proportion of NT embryos activated at 3 h after electrofusion developed to the blastocyst stage (18.6%) in comparison with NT embryos activated at 1 h (6.0%), 2 h (8.3%), or 4 h (10.6%) after fusion (P < 0.05). No recipient was pregnant 60 d after transfer of blastocysts developed from NT embryos activated at 1 h (0/8), 2 h (0/10), or 4 h (0/9) after fusion. However, 3 of 16 recipients were pregnant following transfer of blastocysts developed from the NT embryos activated at 3 h after fusion, and two of these recipients maintained pregnancy to term. We concluded that the developmental potential of buffalo NT embryos was related to recipient oocyte age and the interval from fusion to activation. Copyright © 2011 Elsevier Inc. All rights reserved.

Effect of recombinant bovine somatotropin (rbST) on cytoplasmic maturation of bovine oocytes and their developmental competence in vitro.

PubMed

Kuzmina, Tatjana I; Alm, Hannelore; Denisenko, Vitaly; Tuchscherer, Armin; Kanitz, Wilhelm; Torner, Helmut

2007-04-01

The objectives of this study were to evaluate the effects of recombinant bovine somatotropin (rbST) on the nuclear and cytoplasmic maturation of bovine oocytes and their further developmental competence to blastocysts in vitro. We analyzed the mitochondrial activity and concentration of intracellular stored calcium ([Ca(2+)](is)) in matured oocytes and the morphology and chromatin status of produced embryos after in vitro fertilization. Cumulus-oocyte complexes were incubated in TCM 199 containing 10% fetal calf serum (control medium 1: CM 1) or 10% estrus cow serum (control medium 2: CM 2). The culture medium of the treatment groups was modified by supplementation of the control medium with 10 ng/ml rbST (CM 1A and CM 2A), 10(6)/ml granulosa cells (CM 1B and CM 2B), or 10 ng/ml rbST plus 10(6)/ml granulosa cells (CM 1C and CM 2C). No differences were observed in the percentages of oocytes reaching metaphase II between the groups. However, the proportion of blastocysts was highest in treatment groups CM 1C and CM 2C (P<0.05). The type of serum did not alter the positive effect of rbST on the developmental competence of embryos. The fluorescence intensity of metabolically active mitochondria measured by intensity per oocyte (Em 570) after MitoTracker CMTM Ros Orange labeling was significantly increased in oocytes matured in the presence of 10 ng/ml rbST and granulosa cells (309.21 vs. 119.97 microA; P<0.01). In parallel, the concentration of [Ca(2+)](is) in oocytes, determined using fluorophore chlortetracycline, was significantly decreased (0.85 +/- 0.02 vs. 0.97 +/- 0.03 AU; P<0.05). Based on these results, we concluded that rbST, in interaction with granulosa cells stimulates the oxidative activity of ooplasmic mitochondria and decreases the content of [Ca(2+)](is) in oocytes. These facts support the hypothesis that somatotropin influences the developmental competence of bovine oocytes during maturation in vitro, and this effect can be modulated by granulosa cells.

Birth after human chorionic gonadotropin-primed oocyte in vitro maturation and fertilization with testicular sperm in a normo-ovulatory patient.

PubMed

González-Ortega, Claudia; Piña-Aguilar, Raul Eduardo; Cancino-Villareal, Patricia; Gutiérrez-Gutiérrez, Antonio Martin

2016-01-01

In this report, we present a case of in vitro maturation (IVM) with surgical retrieved testicular sperm in a normo-ovulatory female. Human chorionic gonadotropin-primed IVM, testicular biopsy for sperm retrieval and intracytoplasmic sperm injection with fresh sperm were performed. Fourteen cumulus-oocyte complexes were obtained in germinal vesicle or metaphase I stage, eight oocytes reached metaphase II, seven presumptive zygotes were obtained, and three cleavage stages embryos in day 2 were transferred producing a singleton pregnancy. A single healthy newborn was obtained. Our results suggest that IVM may be an alternative for in vitro fertilization in normo-ovulatory women even if surgical retrieval of sperm is needed. Further research is required to depict contributing factors to the success of IVM in indications different from polycystic ovaries syndrome and the role of male gamete.

Micropropagation of annatto (Bixa orellana L.) from mature tree and assessment of genetic fidelity of micropropagated plants with RAPD markers.

PubMed

Siril, E A; Joseph, Nisha

2013-01-01

An in vitro propagation technique based on axillary bud proliferation was developed for the first time to mature annatto (Bixa orellana L.) tree. Nodal segments cultured on Murashige and Skoog (MS) medium supplemented with 1.0 μM benzyl adenine (BA) and tender coconut water (10 %) showed significantly high (P < 0.05) explant response (67.0 %), development of elongated shoots (3.36), shoot buds (8.9) and shoot elongation (3.53 cm). Cytokinins like zeatin, isopentenyl adenine (2-iP), kinetin, or thidiazuron (TDZ) were inferior to BA to induce multiple shoots. Seasonal variations significantly affected the in vitro response of nodal explants. In vitro rooting experiments have showed 55.6 % rooting on MS medium containing 15 μM indole-3-butyric acid (IBA). Alternatively, in vitro raised shoots were rooted (61.1 %) ex vitro, by 10 mM indole-3-butyric acid (IBA) for 30 s. The results of the RAPD marker system revealed the genetic stability among the micropropagated plants. The present protocol in brief, can be used for the clonal propagation of the superior genotype and preservation of germplasm.

Utility of color Doppler indices of dominant follicular blood flow for prediction of clinical factors in in vitro fertilization-embryo transfer cycles.

PubMed

Ozaki, T; Hata, K; Xie, H; Takahashi, K; Miyazaki, K

2002-12-01

To investigate the relationship between color Doppler indices of dominant follicular blood flow and clinical factors in in vitro fertilization-embryo transfer cycles. This was a prospective study involving 26 patients completing a total of 33 in vitro fertilization cycles. Dominant follicular blood flow indices, peak systolic velocities, the resistance index and the pulsatility index were evaluated using transvaginal color Doppler. The indices were compared to the clinical outcomes of in vitro fertilization-embryo transfer. There was a significant correlation between dominant follicular peak systolic velocities and the number of oocytes retrieved, as well as the number of mature oocytes obtained. There was no significant correlation between dominant follicular resistance index or pulsatility index and the number of follicles > 10 mm in diameter, the number of oocytes retrieved or the number of mature oocytes. There were no significant differences between dominant follicular peak systolic velocities, resistance index or pulsatility index, and fertilization rate or the ratio of good quality embryos. However, significant differences were found between the number of oocytes retrieved, as well as the number of mature oocytes for those patients in which the peak systolic velocity was below 25 cm/s. Doppler assessment of dominant follicle blood flow alone is useful for predicting the number of retrievable oocytes. However, morphological quality of the embryo produced or the pregnancy rate cannot be predicted by this method.

Expression stability of two housekeeping genes (18S rRNA and G3PDH) during in vitro maturation of follicular oocytes in buffalo (Bubalus bubalis).

PubMed

Aswal, Ajay Pal Singh; Raghav, Sarvesh; De, Sachinandan; Thakur, Manish; Goswami, Surender Lal; Datta, Tirtha Kumar

2008-01-15

The present study was undertaken to evaluate the expression stability of two housekeeping genes (HKGs), 18S rRNA and G3PDH during in vitro maturation (IVM) of oocytes in buffalo, which qualifies their use as internal controls for valid qRT-PCR estimation of other oocyte transcripts. A semi quantitative RT-PCR system was used with optimised qRT-PCR parameters at exponential PCR cycle for evaluation of temporal expression pattern of these genes over 24 h of IVM. 18S rRNA was found more stable in its expression pattern than G3PDH.

Sperm chromatin maturity and integrity correlated to zygote development in ICSI program.

PubMed

Asmarinah; Syauqy, Ahmad; Umar, Liya Agustin; Lestari, Silvia Werdhy; Mansyur, Eliza; Hestiantoro, Andon; Paradowszka-Dogan, Agnieszka

2016-10-01

This study aimed to evaluate sperm chromatin maturity and integrity of that injected into good-quality oocytes in an in vitro fertilization-intra cytoplasmic sperm injection (IVF-ICSI) program. A cut-off value of sperm chromatin maturity and integrity was developed as a function of their correlation to the zygote development, i.e., embryo formation and cleavage rate. The study assessed sperm chromatin maturity using aniline blue (AB) staining, whereas toluidine blue (TB) staining was used to assess sperm chromatin integrity. Ejaculates from 59 patients undergoing ICSI and 46 fertile normozoospermic donors for determination of normal values of sperm chromatin status were used in this study. Embryo formation and cleavage rates were observed for the period of 3 days after ICSI. There was a significant difference in the percentage of sperm with mature chromatin between ejaculate from ICSI patients and fertile donor (p=0.020); while there was no significant difference in sperm chromatin integrity of both samples (p=0.120). There was no significant correlation between sperm chromatin maturity and either embryo formation or cleavage rate; as well as sperm chromatin integrity to both parameters of zygote development (p>0.05). Furthermore, we found that the cut-off value of sperm chromatin maturity and integrity of the fertile normozoospermic ejaculates were 87.2% and 80.2%, respectively. Using the cut-offs, we found that low sperm chromatin maturity at the level of <87% correlated significantly with the cleavage rate of the zygote (p=0.022; r=0.371); whereas poor sperm chromatin integrity at the level of <80% correlated with embryo formation (p=0.048; r=0,485). In conclusion, this study showed that poor maturity and integrity of sperm chromatin (AB<87% and TB<80%, respectively), could affect zygote development following ICSI. AB: aniline blue; CMA3: chromomycin A3; ICSI: intra cytoplasmic sperm injection; IVF: in vitro fertilization; PBS: phosphate buffer saline; SPSS: Statistical Package for Social Science; TB: toluidine blue; WHO: World Health Organization.

Temporal and Embryonic Lineage-Dependent Regulation of Human Vascular SMC Development by NOTCH3

PubMed Central

Granata, Alessandra; Bernard, William G.; Zhao, Ning; Mccafferty, John; Lilly, Brenda

2015-01-01

Vascular smooth muscle cells (SMCs), which arise from multiple embryonic progenitors, have unique lineage-specific properties and this diversity may contribute to spatial patterns of vascular diseases. We developed in vitro methods to generate distinct vascular SMC subtypes from human pluripotent stem cells, allowing us to explore their intrinsic differences and the mechanisms involved in SMC development. Since Notch signaling is thought to be one of the several key regulators of SMC differentiation and function, we profiled the expression of Notch receptors, ligands, and downstream elements during the development of origin-specific SMC subtypes. NOTCH3 expression in our in vitro model varied in a lineage- and developmental stage-specific manner so that the highest expression in mature SMCs was in those derived from paraxial mesoderm (PM). This pattern was consistent with the high expression level of NOTCH3 observed in the 8–9 week human fetal descending aorta, which is populated by SMCs of PM origin. Silencing NOTCH3 in mature SMCs in vitro reduced SMC markers in cells of PM origin preferentially. Conversely, during early development, NOTCH3 was highly expressed in vitro in SMCs of neuroectoderm (NE) origin. Inhibition of NOTCH3 in early development resulted in a significant downregulation of specific SMC markers exclusively in the NE lineage. Corresponding to this prediction, the Notch3-null mouse showed reduced expression of Acta2 in the neural crest-derived SMCs of the aortic arch. Thus, Notch3 signaling emerges as one of the key regulators of vascular SMC differentiation and maturation in vitro and in vivo in a lineage- and temporal-dependent manner. PMID:25539150

Perinatal maturation of the mouse respiratory rhythm-generator: in vivo and in vitro studies.

PubMed

Viemari, Jean-Charles; Burnet, Henri; Bévengut, Michelle; Hilaire, Gérard

2003-03-01

In vivo (plethysmography) and in vitro (en bloc preparations) experiments were performed from embryonic day 16 (E16) to postnatal day 9 (P9) in order to analyse the perinatal maturation of the respiratory rhythm-generator in mice. At E16, delivered foetuses did not ventilate and survive but at E18 they breathed at about 110 cycles/min with respiratory cycles of variable individual duration. From E18 to P0-P2, the respiratory cycles stabilised without changes in the breathing parameters. However, these increased several-fold during the next days. Hypoxia increased breathing frequency from E18-P5 and only significantly affected ventilation from P3 onwards. At E16, in vitro medullary preparations (pons resection) produced rhythmic phrenic bursts at a low frequency (about 5 cycles/min) with variable cycle duration. At E18, their frequency doubled but cycle duration remained variable. After birth, the frequency did not change although cycle duration stabilised. At E18 and P0-P2, the in vitro frequency decreased by around 50% under hypoxia, increased by 40-50% under noradrenaline or substance P and was permanently depressed by the pontine A5 areas. At E16 however, hypoxia had no effects, both noradrenaline and substance P drastically increased the frequency and area A5 inhibition was not expressed at this time. At E18 and P0-P2, electrical stimulation and electrolytic lesion of the rostral ventrolateral medulla affected the in vitro rhythm but failed to induce convincing effects at E16. Thus, a major maturational step in respiratory rhythmogenesis occurs between E16-E18, in agreement with the concept of multiple rhythmogenic mechanisms.

Early in vitro fertilization improves development of bovine ova heat stressed during in vitro maturation.

PubMed

Schrock, G E; Saxton, A M; Schrick, F N; Edwards, J L

2007-09-01

The objectives were to examine the development of embryos derived from control (38.5 degrees C) or heat-stressed ova [41.0 degrees C during the first 12 h of in vitro maturation (hIVM)] when in vitro fertilization (IVF) was performed at 16, 18, 20, 24, or 30 hIVM. Effects of heat stress in compromising ovum development depended on when IVF was performed (in vitro maturation temperature x IVF time interaction). When IVF was performed at 24 or 30 hIVM, fewer heat-stressed ova developed to the blastocyst stage compared with the respective controls. In contrast, when IVF was performed at 16, 18, or 20 hIVM, more heat-stressed ova developed to the blastocyst stage compared with the respective controls. Performing IVF earlier than usual was beneficial, because the ability of heat-stressed ova to develop to the blastocyst stage was improved when IVF was performed at 18 or 20 vs. 24 hIVM. Blastocyst stage and quality were equivalent to non-heat-stressed controls regardless of IVF time. Control ova undergoing IVF at 20, 24, 30, or 32 hIVM and heat-stressed ova undergoing IVF at 16, 18, 20, or 24 hIVM were compared for blastocyst development by multisource regression. Although linear and quadratic slopes were similar, heat stress reduced the peak and shifted the developmental response of ova by 7.3 h. In other words, obtaining optimal blastocyst development from heat-stressed ova would depend on performing IVF at 19.5 hIVM compared with 26.7 hIVM for non-heat-stressed controls. Heat-induced reductions in peak blastocyst development significantly reduced the window of time available to perform IVF and obtain > or = 20% blastocyst development. In summary, results support an effect of heat stress to hasten developmentally important events during oocyte maturation. The inability of earlier IVF to fully restore the development of heat-stressed ova to that of non-heat-stressed controls highlights the importance of further study.

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.

[High non-specific binding of the beta(1) -selective radioligand 2-(125)I-ICI-H].

PubMed

Riemann, B; Law, M P; Kopka, K; Wagner, St; Luthra, S; Pike, V W; Neumann, J; Kirchhefer, U; Schmitz, W; Schober, O; Schäfers, M

2003-08-01

As results of cardiac biopsies suggest, myocardial beta(1) -adrenoceptor density is reduced in patients with chronic heart failure. However, changes in cardiac beta(2)-adrenoceptors vary. With suitable radiopharmaceuticals single photon emission computed tomography (SPECT) and positron emission tomography (PET) offer the opportunity to assess beta-adrenoceptors non-invasively. Among the novel racemic analogues of the established beta(1)-selective adrenoceptor antagonist ICI 89.406 the iodinated 2-I-ICI-H showed high affinity and selectivity to beta(1)-adrenoceptors in murine ventricular membranes. The aim of this study was its evaluation as a putative sub-type selective beta(1)-adrenergic radioligand in cardiac imaging. Competition studies in vitro and in vivo were used to investigate the kinetics of 2-I-ICI-H binding to cardiac beta-adrenoceptors in mice and rats. In addition, the radiosynthesis of 2-(125)I-ICI-H from the silylated precursor 2-SiMe(3)-ICI-H was established. The specific activity was 80 GBq/ micro mol, the radiochemical yield ranged from 70 to 80%. The unlabelled compound 2-I-ICI-H showed high beta(1)-selectivity and -affinity in the in vitro competition studies. In vivo biodistribution studies apparently showed low affinity to cardiac beta-adrenoceptors. The radiolabelled counterpart 2-(125)I-ICI-H showed a high degree of non-specific binding in vitro and no specific binding to cardiac beta(1)-adrenoceptors in vivo. Because of its high non-specific binding 2-(125)I-ICI-H is no suitable radiotracer for imaging in vivo.

Synthesis and Nicotinic Acetylcholine Receptor In Vitro and In Vivo Pharmacological Properties of 2'-Fluoro-3'-(substituted phenyl)deschloroepibatidine Analogues of 2'-Fluoro-3'-(4-nitrophenyl)deschloroepibatidine (4-Nitro-PFEB or RTI-7527-102)

PubMed Central

Ondachi, Pauline; Castro, Ana; Luetje, Charles W.; Damaj, M. Imad; Mascarella, S. Wayne; Navarro, Hernán A.; Carroll, F. Ivy

2012-01-01

Herein, we report the synthesis and nicotinic acetylcholine receptor (nAChR) in vitro and in vivo pharmacological properties of 2'-fluoro-3'-(substituted phenyl)deschloroepibatidines 5b–g, analogues of 3'-(4-nitrophenyl) compound 5a. All compounds had high affinity for the α4β2-nAChR and low affinity for α7-nAChR. Initial electrophysiological studies showed that all analogues were antagonists at α4β2-, α3β4-, and α7-nAChRs. The 4-carbamoylphenyl analogue 5g was highly selective for α4β2-nAChR over α3β4- and α7-nAChRs. All the analogues were antagonists of nicotine-induced antinociception in the tail-flick test. Molecular modeling docking studies using agonist-bound form of the X-ray crystal structure of the acetylcholine binding protein suggested several different binding modes for epibatidine, varenicline, and 5a–5g. In particular, a unique binding mode for 5g was suggested by these docking simulations. The high binding affinity, in vitro efficacy, and selectivity of 5g for α4β2-nAChR combined with its nAChR functional antagonist properties suggest that 5g will be a valuable pharmacological tool for studying the nAChR and may have potential as a pharmacotherapy for addiction and other CNS disorders. PMID:22742586

A bioengineered niche promotes in vivo engraftment and maturation of pluripotent stem cell derived human lung organoids

PubMed Central

Dye, Briana R; Dedhia, Priya H; Miller, Alyssa J; Nagy, Melinda S; White, Eric S; Shea, Lonnie D; Spence, Jason R

2016-01-01

Human pluripotent stem cell (hPSC) derived tissues often remain developmentally immature in vitro, and become more adult-like in their structure, cellular diversity and function following transplantation into immunocompromised mice. Previously we have demonstrated that hPSC-derived human lung organoids (HLOs) resembled human fetal lung tissue in vitro (Dye et al., 2015). Here we show that HLOs required a bioartificial microporous poly(lactide-co-glycolide) (PLG) scaffold niche for successful engraftment, long-term survival, and maturation of lung epithelium in vivo. Analysis of scaffold-grown transplanted tissue showed airway-like tissue with enhanced epithelial structure and organization compared to HLOs grown in vitro. By further comparing in vitro and in vivo grown HLOs with fetal and adult human lung tissue, we found that in vivo transplanted HLOs had improved cellular differentiation of secretory lineages that is reflective of differences between fetal and adult tissue, resulting in airway-like structures that were remarkably similar to the native adult human lung. DOI: http://dx.doi.org/10.7554/eLife.19732.001 PMID:27677847

Solving the puzzle of pluripotent stem cell-derived cardiomyocyte maturation: piece by piece.

PubMed

Lundy, David J; Lee, Desy S; Hsieh, Patrick C H

2017-03-01

There is a growing need for in vitro models which can serve as platforms for drug screening and basic research. Human adult cardiomyocytes cannot be readily obtained or cultured, and so pluripotent stem cell-derived cardiomyocytes appear to be an attractive option. Unfortunately, these cells are structurally and functionally immature-more comparable to foetal cardiomyocytes than adult. A recent study by Ruan et al ., provides new insights into accelerating the maturation process and takes us a step closer to solving the puzzle of pluripotent stem cell-derived cardiomyocyte maturation.

New analogues of oxotremorine and oxotremorine-M: estimation of their in vitro affinity and efficacy at muscarinic receptor subtypes.

PubMed

Barocelli, E; Ballabeni, V; Bertoni, S; Dallanoce, C; De Amici, M; De Micheli, C; Impicciatore, M

2000-06-30

Two subsets of tertiary amines (1a-6a) and methiodides (1b-6b) with a structural resemblance to oxotremorine and oxotremorine-M were tested at rabbit vas deferens (M1), guinea pig left atrium (M2), guinea pig ileum and urinary bladder (M3) muscarinic receptor subtypes. The pharmacological profile of the derivatives under study has been discussed by evaluating their potency, affinity and efficacy as well as the regional differences in muscarinic receptor occupancy.

Current status of in vitro embryo production in sheep and goats.

PubMed

Paramio, M-T; Izquierdo, D

2014-10-01

Sheep and goat production is an important economic activity in Spain with an increasing interest in milk production. Multiovulation and Embryo Transfer (MOET) and In vitro Embryo Production (IVEP) are assisted reproductive technologies aimed at increasing the genetic diffusion of females. In vitro embryo production is a multi-step methodology comprising the following procedures: (i) In vitro Maturation (IVM) of oocytes recovered directly from the follicles, (ii) In vitro Fertilization (IVF) or co-incubation of capacitated spermatozoa with in vitro matured oocytes and (iii) In vitro culture (IVC) of zygotes up to the blastocyst stage. In vitro embryo production from oocytes recovered from prepubertal females is called JIVET (Juvenile in vitro Embryo Transfer) and allows shortened generation intervals and increased genetic gain. Embryo production together with embryo cryoconservation would allow large-scale embryo marketing, a pathogen-free genetic movement and easier and cheaper germplasm commercial transactions. Commercial Embryo activity in small ruminants is low compared to cows in the European Union (data from the European Embryo Transfer Association) and in the world (data from the International Embryo Transfer Association). There is less IVEP research in small ruminants compared to other livestock species. The aim of this review was to provide an overview of the current status of IVEP of small ruminant with an emphasis on (i) description of the main methodologies currently used for IVM, IVF and IVC of embryos (ii) comparing procedures and outputs from JIVET and IVEP of adult females and (iii) the future research perspectives of this technology. © 2014 Blackwell Verlag GmbH.

Comparison of Quinn's Advantage fertilization medium and tissue culture medium 199 for in vitro maturation of oocytes.

PubMed

Lin, Yu-Hung; Hwang, Jiann-Loung; Huang, Lee-Wen; Seow, Kok-Min; Hsieh, Bih-Chwen; Tzeng, Chii-Ruey

2014-03-01

The purpose of the study was to compare the Quinn's Advantage fertilization medium (Q1) and the tissue culture medium 199 (TCM199) for in vitro maturation (IVM) of oocytes and ammonium production during IVM. The immature murine oocytes were randomly added into Q1 and TCM199. Ammonium concentrations were measured at the start and after 18 hours of IVM, and the mature oocytes were fertilized and cultured into blastocysts. The blastocysts were then stained for inner cell mass (ICM) and trophectoderm. The maturation rate was higher in Q1 than in TCM199 (85.7% vs. 76.6%, p = 0.024). The fertilization and blastocyst rates were slightly higher in Q1, but not significant. Differential staining of the blastocysts showed slightly higher ICM ratio in the blastocysts derived from Q1. Mean ammonium concentrations in Q1 and TCM199 at Time 0 were 184.9 and 339.2 μg/dL, respectively (p = 0.05), and after 18 hours of IVM were 268.7 and 443.6 μg/dL, respectively (p = 0.045). Addition of ammonium chloride into Q1 adversely affects IVM. Q1 is superior to TCM199 in terms of oocyte maturation, which may be due to lower ammonium concentration. Copyright © 2014. Published by Elsevier B.V.

In-vitro maturation of germinal vesicle and metaphase I eggs prior to cryopreservation optimizes reproductive potential in patients undergoing fertility preservation.

PubMed

Lee, Joseph A; Sekhon, Lucky; Grunfeld, Lawrence; Copperman, Alan B

2014-06-01

To evaluate current and previous findings related to a timely implementation of in-vitro maturation (IVM) of germinal vesicle, metaphase I and metaphase II oocytes with an optimal cryopreservation to determine whether IVM should be attempted prior to (fresh IVM) or IVM after cryopreservation (postthaw IVM). Mitochondrion, chromatin and spindle formation in both groups were interpreted from referenced studies to establish best management of all oocytes. The postthaw survival of germinal vesicle, metaphase I, fresh IVM-metaphase II and control metaphase II oocytes did not differ significantly [83.3% (n=9), 86.7% (n=12), 83% (n=57) and 86% (n=68), respectively]. Overall, combined survival and maturation were significantly higher (P<0.05) in the fresh IVM group at 63.8% (44 of 69) compared with the postthaw IVM group at 33.3% (nine of 27). Conservation of retrieved immature oocytes after vaginal oocyte retrieval has become a major concern for patients, as they strive to maximize the reproductive viability of all oocytes obtained during treatment. Oocyte cryopreservation is important for patients at risk of ovarian cancer, elective fertility preservation and potentially for ovum donation. The superior maturation rate of germinal vesicle and metaphase I oocytes in the fresh IVM vs. postthaw groups provides strong impetus to mature oocytes to the metaphase II stage prior to cryopreservation.

No Amelioration of Uromodulin Maturation and Trafficking Defect by Sodium 4-Phenylbutyrate in Vivo

PubMed Central

Kemter, Elisabeth; Sklenak, Stefanie; Rathkolb, Birgit; Hrabě de Angelis, Martin; Wolf, Eckhard; Aigner, Bernhard; Wanke, Ruediger

2014-01-01

Uromodulin (UMOD)-associated kidney disease (UAKD) belongs to the hereditary progressive ER storage diseases caused by maturation defects of mutant UMOD protein. Current treatments of UAKD patients are symptomatic and cannot prevent disease progression. Two in vitro studies reported a positive effect of the chemical chaperone sodium 4-phenylbutyrate (4-PBA) on mutant UMOD maturation. Thus, 4-PBA was suggested as a potential treatment for UAKD. This study evaluated the effects of 4-PBA in two mouse models of UAKD. In contrast to previous in vitro studies, treatment with 4-PBA did not increase HSP70 expression or improve maturation and trafficking of mutant UMOD in vivo. Kidney function of UAKD mice was actually deteriorated by 4-PBA treatment. In transfected tubular epithelial cells, 4-PBA did not improve maturation but increased the expression level of both mutant and wild-type UMOD protein. Activation of NF-κB pathway in thick ascending limb of Henle's loop cells of UAKD mice was detected by increased abundance of RelB and phospho-IκB kinase α/β, an indirect activator of NF-κB. Furthermore, the abundance of NF-κB1 p105/p50, NF-κB2 p100/p52, and TRAF2 was increased in UAKD. NF-κB activation was identified as a novel disease mechanism of UAKD and might be a target for therapeutic intervention. PMID:24567330

Ethylene-associated phase change from juvenile to mature phenotype of daylily (Hemerocallis) in vitro

NASA Technical Reports Server (NTRS)

Smith, D. L.; Kelly, K.; Krikorian, A. D.

1989-01-01

Hemerocallis plantlets maintained in vitro for extended periods of time in tightly closed culture vessels frequently show a phenotype, albeit on a miniaturized scale, typical of more mature, field-grown plants. The positive relationship of elevated ethylene in the headspace of such vessels to the phase shift from juvenile to mature form is established. Rigorous restriction in air exchange with the external environment by means of silicone grease seals hastens the phase change and improves uniformity of response. Although some plantlets may take longer to accumulate enough ethylene in sealed jars to undergo change, added ethylene and ethylene-releasing agents promote it. Ethylene adsorbants (e.g. mercuric perchlorate) block the shift of juvenile to mature form. Critical ambient ethylene level for the shift is ca 1 microliter l-1. Levels up to 1000 microliters l-1 do not hasten the response but are not toxic. The phase change is fully reversible when air exchange permits ethylene to drop below 1 microliter l-1. At least 1 microliter l-1 ethylene is required to sustain the mature phenotype. The ethylene synthesis inhibitor aminoethoxyvinylglycine (AVG) prevents the phase change, while the ethylene biosynthesis intermediate 1-aminocyclopropanecarboxylic acid (ACC) improves it. KOH, as a CO2 absorbent, does not prevent the phase change. Histology sections demonstrate subtle changes in the form of shoot tips of plantlets undergoing phase change.

Zinc supplementation of vitrification medium improves in vitro maturation and fertilization of oocytes derived from vitrified-warmed mouse ovaries.

PubMed

Geravandi, Shirin; Azadbakht, Mehri; Pourmoradi, Mahsa; Nowrouzi, Fatemeh

2017-02-01

Oocyte cryopreservation is an approach for fertility preservation for normal women and cancer patients facing chemo and radiotherapy. The present study evaluated the effect of adding zinc chloride to the vitrification medium used for whole mouse ovaries and then assessing the in vitro maturation and fertilization of oocytes when they were subsequently extracted from these vitrified ovarian tissues. Four vitrification solutions with 0, 100,150 and 200 μg/dl zinc (V0, V1, V2 and V3 respectively) were compared. The viability of oocytes isolated from ovaries vitrified-warmed in the highest concentration of zinc (V3) was significantly higher after 24 than in the control V0 group (72.99 vs 85.97). Progression to the MII stage, fertilization and cleavage by 48 h was also higher in the V3 than V0 control group (35.55 vs 44.73), (47.67 vs 63.74), (28.72 vs 43.03) (P < 0.05) respectively. These results indicate that supplementation of vitrification medium for intact ovaries with zinc can improve the oocyte viability and in vitro maturation-fertilization rate. Copyright © 2017 Elsevier Inc. All rights reserved.

Engineered human skin substitutes undergo large-scale genomic reprogramming and normal skin-like maturation after transplantation to athymic mice.

PubMed

Klingenberg, Jennifer M; McFarland, Kevin L; Friedman, Aaron J; Boyce, Steven T; Aronow, Bruce J; Supp, Dorothy M

2010-02-01

Bioengineered skin substitutes can facilitate wound closure in severely burned patients, but deficiencies limit their outcomes compared with native skin autografts. To identify gene programs associated with their in vivo capabilities and limitations, we extended previous gene expression profile analyses to now compare engineered skin after in vivo grafting with both in vitro maturation and normal human skin. Cultured skin substitutes were grafted on full-thickness wounds in athymic mice, and biopsy samples for microarray analyses were collected at multiple in vitro and in vivo time points. Over 10,000 transcripts exhibited large-scale expression pattern differences during in vitro and in vivo maturation. Using hierarchical clustering, 11 different expression profile clusters were partitioned on the basis of differential sample type and temporal stage-specific activation or repression. Analyses show that the wound environment exerts a massive influence on gene expression in skin substitutes. For example, in vivo-healed skin substitutes gained the expression of many native skin-expressed genes, including those associated with epidermal barrier and multiple categories of cell-cell and cell-basement membrane adhesion. In contrast, immunological, trichogenic, and endothelial gene programs were largely lacking. These analyses suggest important areas for guiding further improvement of engineered skin for both increased homology with native skin and enhanced wound healing.

In vitro steroid-induced meiosis in Rhinella arenarum oocytes: role of pre-MPF activation.

PubMed

Arias Torres, Ana Josefina; Bühler, Marta Inés; Zelarayán, Liliana Isabel

2016-04-01

In this work we showed the relationship between seasonal periods and the response of R. arenarum follicles and oocytes to different steroids. Using in vitro germinal vesicle breakdown (GVBD) assays, we demonstrated that P4 is the main steroid capable of inducing maturation in R. arenarum oocytes and follicles. In the second part of this work we showed that androgens can activate pre-maturation promoting factors (pre-MPFs) such as P4, by cytoplasm microinjection experiments. The results indicated that the steroids assayed induced oocyte and follicle maturation in a dose- and time-dependent manner. In oocytes, P4 was the most efficient steroid as a maturation inducer (EC50 of the reproductive period, 6 nM, EC50 of the non-reproductive period ≅ 30 nM). Androgens (DHEA, dehydroepiandrosterone; T, testosterone; and AD, androstenedione) were less efficient maturation inducers than P4 (EC50 reproductive period ≅ 50, 120 and 600 nM respectively). Similar results were obtained with intact follicles in both seasonal periods. Although the response of follicles to the different androgens was variable, in no case was it above the above the response induced by P4. Independently of the season, oocytes and follicles incubated in P4, P5 and T underwent GVBD after 6-10 h while oocytes and follicles incubated in DHEA and AD matured more slowly. Furthermore, we demonstrated that microinjection of mature cytoplasm from androgen-treated oocytes is sufficient to promote GVBD in immature recipient oocytes (DHEA, 57 ± 12%; AD, 60 ± 8%; T, 56 ± 13%). Thus, androgens such as DHEA, T and AD are as competent as P4 to activate pre-MPF.

Thymocyte Maturation Is Regulated by the Activity of the Helix-Loop-Helix Protein, E47

PubMed Central

Bain, Gretchen; Quong, Melanie W.; Soloff, Rachel S.; Hedrick, Stephen M.; Murre, Cornelis

1999-01-01

The E2A proteins, E12 and E47, are required for progression through multiple developmental pathways, including early B and T lymphopoiesis. Here, we provide in vitro and in vivo evidence demonstrating that E47 activity regulates double-positive thymocyte maturation. In the absence of E47 activity, positive selection of both major histocompatibility complex (MHC) class I– and class II–restricted T cell receptors (TCRs) is perturbed. Additionally, development of CD8 lineage T cells in an MHC class I–restricted TCR transgenic background is sensitive to the dosage of E47. Mice deficient for E47 display an increase in production of mature CD4 and CD8 lineage T cells. Furthermore, ectopic expression of an E2A inhibitor helix-loop-helix protein, Id3, promotes the in vitro differentiation of an immature T cell line. These results demonstrate that E2A functions as a regulator of thymocyte positive selection. PMID:10587351

Monitoring sinew contraction during formation of tissue-engineered fibrin-based ligament constructs.

PubMed

Paxton, Jennifer Z; Wudebwe, Uchena N G; Wang, Anqi; Woods, Daniel; Grover, Liam M

2012-08-01

The ability to study the gross morphological changes occurring during tissue formation is vital to producing tissue-engineered structures of clinically relevant dimensions in vitro. Here, we have used nondestructive methods of digital imaging and optical coherence tomography to monitor the early-stage formation and subsequent maturation of fibrin-based tissue-engineered ligament constructs. In addition, the effect of supplementation with essential promoters of collagen synthesis, ascorbic acid (AA) and proline (P), has been assessed. Contraction of the cell-seeded fibrin gel occurs unevenly within the first 5 days of culture around two fixed anchor points before forming a longitudinal ligament-like construct. AA+P supplementation accelerates gel contraction in the maturation phase of development, producing ligament-like constructs with a higher collagen content and distinct morphology to that of unsupplemented constructs. These studies highlight the importance of being able to control the methods of tissue formation and maturation in vitro to enable the production of tissue-engineered constructs with suitable replacement tissue characteristics for repair of clinical soft-tissue injuries.

Fibrocyte measurement in peripheral blood correlates with number of cultured mature fibrocytes in vitro and is a potential biomarker for interstitial lung disease in Rheumatoid Arthritis.

PubMed

Just, Søren Andreas; Lindegaard, Hanne; Hejbøl, Eva Kildall; Davidsen, Jesper Rømhild; Bjerring, Niels; Hansen, Søren Werner Karlskov; Schrøder, Henrik Daa; Hansen, Inger Marie Jensen; Barington, Torben; Nielsen, Christian

2017-07-18

Interstitial lung disease (ILD) can be a severe extra-articular disease manifestation in Rheumatoid Arthritis (RA). A potential role of fibrocytes in RA associated ILD (RA-ILD) has not previously been described. We present a modified faster method for measuring circulating fibrocytes, without intracellular staining. The results are compared to the traditional culture method, where the number of monocytes that differentiate into mature fibrocytes in vitro are counted. The results are following compared to disease activity in patients with severe asthma, ILD, RA (without diagnosed ILD) and RA with verified ILD (RA-ILD). CD45 + CD34 + CD11b + (7-AAD - CD3 - CD19 - CD294 - ) cells were isolated by cell sorting and stained for pro-collagen type 1. Thirty-nine patients (10 RA, 9 ILD and 10 with severe asthma, 10 with RA-ILD) and 10 healthy controls (HC) were included. Current medication, disease activity, pulmonary function test and radiographic data were collected. Circulating fibrocytes were quantified by flow cytometry. Peripheral blood mononuclear cells were isolated and cultured for 5 days and the numbers of mature fibrocytes were counted. 90.2% (mean, SD = 1.5%) of the sorted cells were pro-collagen type 1 positive and thereby fulfilled the criteria for being circulating fibrocytes. The ILD and RA-ILD groups had increased levels of circulating fibrocytes compared to HC (p < 0.05). Levels of circulating fibrocytes correlated overall to number of monocytes that subsequently in vitro differentiated to mature fibrocytes (r = 0.81, p < 0.001). RA patients with pathologically reduced diffusion capacity for carbon monoxide adjusted for hemoglobin (DLCO c ) in both the RA and in the combined RA + RA-ILD group, had significantly higher levels of both circulating and number of cultured mature fibrocytes (both p < 0.05). In both groups, the level of circulating fibrocytes and number of mature fibrocytes in culture also correlated to a reduction in DLCO c (r = -0.61 an r = -0.58 both p < 0.05). We presented a fast and valid method for measuring circulating fibrocytes using flow cytometry on lysed peripheral blood. Further, we showed for the first time, that the level of circulating fibrocytes correlated with the number of peripheral blood mononuclear cells, that differentiated into mature fibrocytes in vitro. Reduced DLCO c was correlated with high levels of circulating and mature fibrocytes in RA, which have not been reported previously. In such, this study suggests that fibrocytes may exhibit an important role in the pathogenesis of RA-ILD, which requires further clarification in future studies. ClinicalTrials.gov : NCT02711657 , registered 13/3-2016, retrospectively registered.

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

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

Birth of Healthy Offspring following ICSI in In Vitro-Matured Common Marmoset (Callithrix jacchus) Oocytes

PubMed Central

Takahashi, Tsukasa; Hanazawa, Kisaburo; Inoue, Takashi; Sato, Kenya; Sedohara, Ayako; Okahara, Junko; Suemizu, Hiroshi; Yagihashi, Chie; Yamamoto, Masafumi; Eto, Tomoo; Konno, Yusuke; Okano, Hideyuki; Suematsu, Makoto; Sasaki, Erika

2014-01-01

Intracytoplasmic sperm injection (ICSI), an important method used to treat male subfertility, is applied in the transgenic technology of sperm-mediated gene transfer. However, no study has described successful generation of offspring using ICSI in the common marmoset, a small non-human primate used as a model for biomedical translational research. In this study, we investigated blastocyst development and the subsequent live offspring stages of marmoset oocytes matured in vitro and fertilized by ICSI. To investigate the optimal timing of performing ICSI, corrected immature oocytes were matured in vitro and ICSI was performed at various time points (1–2 h, 2–4 h, 4–6 h, 6–8 h, and 8–10 h after extrusion of the first polar body (PB)). Matured oocytes were then divided randomly into two groups: one was used for in vitro fertilization (IVF) and the other for ICSI. To investigate in vivo development of embryos followed by ICSI, 6-cell- to 8-cell-stage embryos and blastocysts were nonsurgically transferred into recipient marmosets. Although no significant differences were observed in the fertilization rate of blastocysts among ICSI timing after the first PB extrusion, the blastocyst rate at 1–2 h was lowest among groups at 2–4 h, 4–6 h, 6–8 h, and 8–10 h. Comparing ICSI to IVF, the fertilization rates obtained in ICSI were higher than in IVF (p>0.05). No significant difference was noted in the cleaved blastocyst rate between ICSI and IVF. Following the transfer of 37 ICSI blastocysts, 4 of 20 recipients became pregnant, while with the transfer of 21 6-cell- to 8-cell-stage ICSI embryos, 3 of 8 recipients became pregnant. Four healthy offspring were produced and grew normally. These are the first marmoset offspring produced by ICSI, making it an effective fertilization method for marmosets. PMID:24751978

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

Discovery of 5-substituted tetrahydronaphthalen-2yl-methyl with N-phenyl-N-(piperidin-4-yl)propionamide derivatives as potent opioid receptor ligands.

PubMed

Deekonda, Srinivas; Wugalter, Lauren; Kulkarni, Vinod; Rankin, David; Largent-Milnes, Tally M; Davis, Peg; Bassirirad, Neemah M; Lai, Josephine; Vanderah, Todd W; Porreca, Frank; Hruby, Victor J

2015-09-15

A new series of novel opioid ligands have been designed and synthesized based on the 4-anilidopiperidine scaffold containing a 5-substituted tetrahydronaphthalen-2yl)methyl group with different N-phenyl-N-(piperidin-4-yl)propionamide derivatives to study the biological effects of these substituents on μ and δ opioid receptor interactions. Recently our group reported novel 4-anilidopiperidine analogues, in which several aromatic ring-contained amino acids were conjugated with N-phenyl-N-(piperidin-4-yl)propionamide and examined their biological activities at the μ and δ opioid receptors. In continuation of our efforts in these novel 4-anilidopiperidine analogues, we took a peptidomimetic approach in the present design, in which we substituted aromatic amino acids with tetrahydronaphthalen-2yl methyl moiety with amino, amide and hydroxyl substitutions at the 5th position. In in vitro assays these ligands, showed very good binding affinity and highly selective toward the μ opioid receptor. Among these, the lead ligand 20 showed excellent binding affinity (2 nM) and 5000 fold selectivity toward the μ opioid receptor, as well as functional selectivity in GPI assays (55.20 ± 4.30 nM) and weak or no agonist activities in MVD assays. Based on the in vitro bioassay results the lead compound 20 was chosen for in vivo assessment for efficacy in naïve rats after intrathecal administration. Compound 20 was not significantly effective in alleviating acute pain. This discrepancy between high in vitro binding affinity, moderate in vitro activity, and low in vivo activity may reflect differences in pharmacodynamics (i.e., engaging signaling pathways) or pharmacokinetics (i.e., metabolic stability). In sum, our data suggest that further optimization of this compound 20 is required to enhance in vivo activity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Interfering effects of bisphenol A on in vitro growth of preantral follicles and maturation of oocyes.

PubMed

Wang, Xiyan; Jiang, Shi-Wen; Wang, Liguo; Sun, Yanmei; Xu, Fangyan; He, Hai; Wang, Shuo; Zhang, Zhenghong; Pan, Xiaoyan

2018-06-26

In order to investigate the effects and mechanism of Bisphenol A (BPA) on the growth of preantral follicles and the maturation of oocytes in vitro, preantral follicles were harvested from mouse ovaries and in vitro cultured for 11 days with different concentrations of BPA (0, 4.5 and 45 μM) for calculating the percentages of antral follicles, denuded oocytes, degenerative oocytes and the maturation rate of oocytes, besides measuring the diameter of follicles and the thickness of cumulus cell layers. The contents of estradiol (E 2 ) in the culture media on Day 4, 8 and 10 were detected by ELISA. The estrogen receptor (ER) expression, spindle morphology and chromosome distribution in oocytes on Day 10 and 11 were observed by immunofluorescence. Western blotting was used to detect the expressions of growth differentiation factor 9 (GDF-9), bone morphogenetic protein-15 (BMP-15), phosphorylated extracellular signal-regulated kinase 1 (p-Erk1) and phosphorylated Ca 2 + /calmodulin-dependent protein kinase II (p-CaMKII) in the oocytes. Compared with control, BPA (45 μM) significantly reduced percentages of antral follicles (9.25% vs. 91.17%, P < 0.05) and the maturation rate of oocytes (7.61% vs. 79.83%, P < 0.05), but increased the percentages of denuded oocytes (30.29% vs. 3.36%, P < 0.05) and degenerative oocytes (45.70% vs. 2.45%, P < 0.05). The diameter of follicles and the thickness of the cumulus cell layers were decreased significantly (P < 0.05). Moreover, BPA (45 μM) significantly decreased E 2 contents in the culture medium on Day 8 and 10 (P < .05) and the expressions of ER, GDF-9 and BMP-15 in oocytes (P < 0.05). Furthermore, BPA (4.5 and 45 μM) treatment resulted in the abnormal spindle morphology and chromosome distribution, and the decreased expressions of p-Erk1 and p-CaMKII in the MII oocytes. Together, these results clearly demonstrated BPA retarded the preantral follicle growth in vitro through interfering with the synthesis and secretion of E 2 and reducing the expressions of ER, GDF-9 and BMP-15, and led to the abnormal meioses of oocytes through reducing p-Erk1 and p-CaMKII expressions in the preantral follicles, which will help us to further unsderstand the mechanism of BPA exposure retarding in vitro growth of preantral follicles and maturation of oocyes. Copyright © 2018. Published by Elsevier B.V.

Computational design of cyclic peptides for the customized oriented immobilization of globular proteins.

PubMed

Soler, Miguel A; Rodriguez, Alex; Russo, Anna; Adedeji, Abimbola Feyisara; Dongmo Foumthuim, Cedrix J; Cantarutti, Cristina; Ambrosetti, Elena; Casalis, Loredana; Corazza, Alessandra; Scoles, Giacinto; Marasco, Daniela; Laio, Alessandro; Fortuna, Sara

2017-01-25

The oriented immobilization of proteins, key for the development of novel responsive biomaterials, relies on the availability of effective probes. These are generally provided by standard approaches based on in vivo maturation and in vitro selection of antibodies and/or aptamers. These techniques can suffer technical problems when a non-immunogenic epitope needs to be targeted. Here we propose a strategy to circumvent this issue by in silico design. In our method molecular binders, in the form of cyclic peptides, are computationally evolved by stochastically exploring their sequence and structure space to identify high-affinity peptides for a chosen epitope of a target globular protein: here a solvent-exposed site of β2-microglobulin (β2m). Designed sequences were screened by explicit solvent molecular dynamics simulations (MD) followed by experimental validation. Five candidates gave dose-response surface plasmon resonance signals with dissociation constants in the micromolar range. One of them was further analyzed by means of isothermal titration calorimetry, nuclear magnetic resonance, and 250 ns of MD. Atomic-force microscopy imaging showed that this peptide is able to immobilize β2m on a gold surface. In short, we have shown by a variety of experimental techniques that it is possible to capture a protein through an epitope of choice by computational design.

Tramadol, an Opioid Receptor Agonist: An Inhibitor of Growth, Morphogenesis, and Biofilm Formation in the Human Pathogen, Candida albicans.

PubMed

Kathwate, Gunderao Hanumantrao; Karuppayil, S Mohan

2016-12-01

Tramadol is a synthetic, centrally acting low-affinity agonist of μ-opioid receptors in humans. It is used as an analgesic and is shown to have local anesthetic action. In this study, we have tried to explore its anti-Candida potential. Minimum inhibitory concentration (MIC50) and minimum fungicidal concentration (MFC) values were established. MIC50 ranged from 2 to 4 mg/mL, whereas MFC was recorded at 8 mg/mL. Also, the effect of tramadol on germ tube formation, adhesion, and biofilms in Candida albicans was studied. Tramadol impaired in vitro growth of C. albicans. A time-dependent killing assay showed that it kills C. albicans within 24 h of exposure. Tramadol has strong activity against Candida virulence factors such as yeast-to-hyphal form switching and adhesion. C. albicans biofilms, which are notoriously resistant to many antifungals, were sensitive to tramadol. At 8 mg/mL of tramadol, 82% of early stage biofilms and 52.88% of matured biofilms were inhibited. Although our results show that the antifungal effect of tramadol requires concentrations that can be achieved only locally, they may provide potential candidates for development of novel antifungal drugs.

Spore coat protein synthesis in cell-free systems from sporulating cells of Bacillus subtilis.

PubMed

Nakayama, T; Munoz, L E; Sadaie, Y; Doi, R H

1978-09-01

Cell-free systems for protein synthesis were prepared from Bacillus subtilis 168 cells at several stages of sporulation. Immunological methods were used to determine whether spore coat protein could be synthesized in the cell-free systems prepared from sporulating cells. Spore coat protein synthesis first occurred in extracts from stage t2 cells. The proportion of spore coat protein to total proteins synthesized in the cell-free systems was 2.4 and 3.9% at stages t2 and t4, respectively. The sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis patterns of immunoprecipitates from the cell-free systems showed the complete synthesis of an apparent spore coat protein precursor (molecular weight, 25,000). A polypeptide of this weight was previously identified in studies in vivo (L.E. Munoz, Y. Sadaie, and R.H. Doi, J. Biol. Chem., in press). The synthesis in vitro of polysome-associated nascent spore coat polypeptides with varying molecular weights up to 23,000 was also detected. These results indicate that the spore coat protein may be synthesized as a precursor protein. The removal of proteases in the crude extracts by treatment with hemoglobin-Sepharose affinity techniques may be preventing the conversion of the large 25,000-dalton precursor to the 12,500-dalton mature spore coat protein.

The use of in vitro transcription to probe regulatory functions of viral protein domains.

PubMed

Loewenstein, Paul M; Song, Chao-Zhong; Green, Maurice

2007-01-01

Adenoviruses (Ads), like other DNA tumor viruses, have evolved specific regulatory genes that facilitate virus replication by controlling the transcription of other viral genes as well as that of key cellular genes. In this regard, the E1A transcription unit contains multiple protein domains that can transcriptionally activate or repress cellular genes involved in the regulation of cell proliferation and cell differentiation. Studies using in vitro transcription have provided a basis for a molecular understanding of the interaction of viral regulatory proteins with the transcriptional machinery of the cell and continue to inform our understanding of transcription regulation. This chapter provides examples of the use of in vitro transcription to analyze transcriptional activation and transcriptional repression by purified, recombinant Ad E1A protein domains and single amino acid substitution mutants as well as the use of protein-affinity chromatography to identify host cell transcription factors involved in viral transcriptional regulation. A detailed description is provided of the methodology to prepare nuclear transcription extract, to prepare biologically active protein domains, to prepare affinity depleted transcription extracts, and to analyze transcription by primer extension and by run-off assay using naked DNA templates.

New Coumarin Derivatives as Potent Selective COX-2 Inhibitors: Synthesis, Anti-Inflammatory, QSAR, and Molecular Modeling Studies.

PubMed

Dawood, Dina H; Batran, Rasha Z; Farghaly, Thoraya A; Khedr, Mohammed A; Abdulla, Mohamed M

2015-12-01

Two new series of coumarin derivatives incorporating thiazoline and thiazolidinone moieties were designed, synthesized, and investigated in vivo for their anti-inflammatory activities using the carrageenan-induced rat paw edema model and in vitro for their inhibitory activities against the human cyclooxygenase (COX)-1 and COX-2 isoforms. Most of the synthesized compounds demonstrated exceptionally high in vivo anti-inflammatory activity and displayed superior GI safety profiles (0-7% ulceration) as compared to indomethacin. All the bioactive compounds showed in vitro high affinity and selectivity toward the COX-2 isoenzyme, compared to the reference celecoxib with IC50 values ranging from 0.31 to 0.78 μM. The ethyl thiosemicarbazone 2b, thiazoline derivatives 3a, 3b, 5b, 6a, and 7f, and the thiazolidinone compounds 8b and 9a showed the highest in vivo and in vitro anti-inflammatory activities with remarkable COX-2 selectivity. Quantitative structure-activity relationship study (QSAR) was done and resulted in a highly predictive power R(2) (0.908). A molecular docking study revealed a relationship between the docking affinity and the biological results. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Malathion-induced inhibition of human plasma cholinesterase studied by the fluorescence spectroscopy method

NASA Astrophysics Data System (ADS)

Pavelkić, V. M.; Krinulović, K. S.; Savić, J. Z.; Ilić, M. A.

2008-05-01

The in vitro effect of technical grade malathion was assessed via the kinetic parameters of human plasma butyrylcholinesterase (BChE) using N-methylindoxyl acetate as a substrate for BChE. An inhibitor kinetics study demonstrated the existence of a biphasic inhibition curve, indicating high-and low-affinity binding sites of malathion. The IC 50 values as calculated from the experimental inhibition curves were 1.33 × 10-9 and 1.48 × 10-5 M for the high-and low-affinity binding sites, respectively; Hill’s analysis gave 1.29 × 10-9 and 1.38 × 10-6 M. The Cornish-Bowden plots and their secondary plots indicated that the nature of inhibition was of mixed type with the predominant competitive character of both affinity binding sites.

Binding of perlecan to transthyretin in vitro.

PubMed Central

Smeland, S; Kolset, S O; Lyon, M; Norum, K R; Blomhoff, R

1997-01-01

Transthyretin is one of two specific proteins involved in the transport of thyroid hormones in plasma; it possesses two binding sites for serum retinol-binding protein. In the present study we demonstrate that transthyretin also interacts in vitro with [35S]sulphate-labelled material from the medium of HepG2 cells. By using the same strategy as for purifying serum retinol-binding protein, [35S]sulphate-labelled medium was specifically eluted from a transthyretin-affinity column. Ion-exchange chromatography showed that the material was highly polyanionic, and its size and alkali susceptibility suggested that it was a proteoglycan. Structural analyses with chondroitinase ABC lyase and nitrous acid revealed that approx. 20% was chondroitin sulphate and 80% heparan sulphate. Immunoprecipitation showed that the [35S]sulphate-labelled material contained perlecan. Further analysis by binding studies revealed specific and saturable binding of 125I-transthyretin to perlecan-enriched Matrigel. Because inhibition of sulphation by treating HepG2 cells with sodium chlorate increased the affinity of the perlecan for transthyretin, and [3H]heparin was not retained by the transthyretin affinity column, the binding is probably mediated by the core protein and is not a protein-glycosaminoglycan interaction. Because perlecan is released from transthyretin in water, the binding might be due to hydrophobic interactions. PMID:9307034

Interaction between DNA and Drugs Having Protonable Basic Groups: Characterization through Affinity Constants, Drug Release Kinetics, and Conformational Changes

PubMed Central

Alarcón, Liliana P.; Baena, Yolima; Manzo, Rubén H.

2017-01-01

This paper reports the in vitro characterization of the interaction between the phosphate groups of DNA and the protonated species of drugs with basic groups through the determination of the affinity constants, the reversibility of the interaction, and the effect on the secondary structure of the macromolecule. Affinity constants of the counterionic condensation DNA–drug were in the order of 106. The negative electrokinetic potential of DNA decreased with the increase of the proportion of loading drugs. The drugs were slowly released from the DNA–drug complexes and had release kinetics consistent with the high degree of counterionic condensation. The circular dichroism profile of DNA was not modified by complexation with atenolol, lidocaine, or timolol, but was significantly altered by the more lipophilic drugs benzydamine and propranolol, revealing modifications in the secondary structure of the DNA. The in vitro characterization of such interactions provides a physicochemical basis that would contribute to identify the effects of this kind of drugs in cellular cultures, as well as side effects observed under their clinical use. Moreover, this methodology could also be projected to the fields of intracellular DNA transfection and the use of DNA as a carrier of active drugs. PMID:28054999

Comparison of ex vivo harvested and in vitro cultured materials from Echinococcus granulosus by measuring expression levels of five genes putatively involved in the development and maturation of adult worms.

PubMed

Dezaki, Ebrahim Saedi; Yaghoubi, Mohammad Mehdi; Spiliotis, Markus; Boubaker, Ghalia; Taheri, Elham; Almani, Pooya Ghaseminejad; Tohidi, Farideh; Harandi, Majid Fasihi; Gottstein, Bruno

2016-11-01

Parts of the natural life cycle of Echinococcus granulosus can be retraced in vitro such as the development of protoscoleces into semiadult worms with three or more proglottids, or the redifferentiation of in vitro cultured protoscoleces into metacestode-like cystic structures. Most in vitro generated samples share-at the microscopical level-high similarities with those naturally grown, but developmental differences have also been documented, such as missing egg production in in vitro grown adults or unusual bladder/vesicle formation in protoscoleces cultured into the metacestode direction. The aim of the present study was to explore how far different in vitro generated stage-specific materials/structures match the natural situation on the transcriptome level, based on testing five exemplarily chosen different genes: the frizzled receptor eg-fz4 (posterior marker), the FGF receptor-like factor eg-fgfrl (anterior association), the cell differentiation protein eg-rcd1 (part of the CCR4-NOT complex, a key regulator of eukaryotic gene expression), the rapidly accelerated fibrosarcoma serin/threonin kinase eg-braf (part of the MAPK pathway involved, e.g., in EGF signaling) and the co-smad eg-smadD (downstream factor of TGFβ/BMP2/activin signaling). These genes-tested via qPCR-were selected such as to allow a discussion on their potential role in the development of E. granulosus into the adult stage. Thus, testing took place with three ex vivo isolated samples, namely (i) egg-containing adult worms, (ii) invaginated protoscoleces, and (iii) protoscolex-free germinal layer tissue. Respective data were compared (a) with in vitro generated metacestode-like microcysts developed from protoscolices, and (b) different development stages of protoscoleces in vitro cultured toward adult maturation. As a finding, only eg-smadD and partially eg-fz4 showed high expression similarities between ex vivo harvested and in vitro cultured E. granulosus, thus suggesting a putative role in adult maturation. Conclusively, the fact of using "only" five genes did not allow answering the question if ex vivo and in vitro materials are similar on the transcriptome level. Another experimental restriction arises from different growth conditions of the in vitro cultured materials, and comparing these to the ex vivo harvested ones. Future experiments may solve the problems by using fully standardized E. granulosus sample collection and fully standardized culture conditions.

Variations of DNA methylation in Eucalyptus urophylla×Eucalyptus grandis shoot tips and apical meristems of different physiological ages.

PubMed

Mankessi, François; Saya, Aubin R; Favreau, Bénédicte; Doulbeau, Sylvie; Conéjéro, Geneviève; Lartaud, Marc; Verdeil, Jean-Luc; Monteuuis, Olivier

2011-10-01

Global DNA methylation was assessed by high-performance liquid chromatography (HPLC) for the first time in Eucalyptus urophylla×Eucalyptus grandis shoot tips comparing three outdoor and one in vitro sources of related genotypes differing in their physiological age. The DNA methylation levels found were consistent with those reported for other Angiosperms using the same HPLC technology. Notwithstanding noticeable time-related fluctuations within each source of plant material, methylation rate was overall higher for the mature clone (13.7%) than for the rejuvenated line of the same clone (12.6%) and for the juvenile offspring seedlings (11.8%). The in vitro microshoots of the mature clone were less methylated (11.3%) than the other outdoor origins, but the difference with the juvenile seedlings was not significant. Immunofluorescence investigations on shoot apices established that the mature source could be distinguished from the rejuvenated and juvenile origins by a higher density of cells with methylated nuclei in leaf primordia. Shoot apical meristems (SAMs) from the mature clone also showed a greater proportion and more methylated cells than SAMs from the rejuvenated and juvenile origins. The nuclei of these latter were characterized by fewer and more dispersed labeled spots than for the mature source. Our findings establish that physiological ageing induced quantitative and qualitative variations of DNA methylation at shoot tip, SAM and even cellular levels. Overall this DNA methylation increased with maturation and conversely decreased with rejuvenation to reach the lower scores and to show the immunolabeling patterns that characterized juvenile material nuclei. Copyright © Physiologia Plantarum 2011.

Influence of bone affinity on the skeletal distribution of fluorescently labeled bisphosphonates in vivo.

PubMed

Roelofs, Anke J; Stewart, Charlotte A; Sun, Shuting; Błażewska, Katarzyna M; Kashemirov, Boris A; McKenna, Charles E; Russell, R Graham G; Rogers, Michael J; Lundy, Mark W; Ebetino, Frank H; Coxon, Fraser P

2012-04-01

Bisphosphonates are widely used antiresorptive drugs that bind to calcium. It has become evident that these drugs have differing affinities for bone mineral; however, it is unclear whether such differences affect their distribution on mineral surfaces. In this study, fluorescent conjugates of risedronate, and its lower-affinity analogues deoxy-risedronate and 3-PEHPC, were used to compare the localization of compounds with differing mineral affinities in vivo. Binding to dentine in vitro confirmed differences in mineral binding between compounds, which was influenced predominantly by the characteristics of the parent compound but also by the choice of fluorescent tag. In growing rats, all compounds preferentially bound to forming endocortical as opposed to resorbing periosteal surfaces in cortical bone, 1 day after administration. At resorbing surfaces, lower-affinity compounds showed preferential binding to resorption lacunae, whereas the highest-affinity compound showed more uniform labeling. At forming surfaces, penetration into the mineralizing osteoid was found to inversely correlate with mineral affinity. These differences in distribution at resorbing and forming surfaces were not observed at quiescent surfaces. Lower-affinity compounds also showed a relatively higher degree of labeling of osteocyte lacunar walls and labeled lacunae deeper within cortical bone, indicating increased penetration of the osteocyte canalicular network. Similar differences in mineralizing surface and osteocyte network penetration between high- and low-affinity compounds were evident 7 days after administration, with fluorescent conjugates at forming surfaces buried under a new layer of bone. Fluorescent compounds were incorporated into these areas of newly formed bone, indicating that "recycling" had occurred, albeit at very low levels. Taken together, these findings indicate that the bone mineral affinity of bisphosphonates is likely to influence their distribution within the skeleton. Copyright © 2012 American Society for Bone and Mineral Research.

Production of lion (Panthera leo) blastocysts after in vitro maturation of oocytes and intracytoplasmic sperm injection.

PubMed

Fernandez-Gonzalez, Lorena; Hribal, Romy; Stagegaard, Julia; Zahmel, Jennifer; Jewgenow, Katarina

2015-04-01

Assisted reproductive techniques are becoming widely applied to the breeding of endangered species, but establishing reliable protocols for the production of embryos in vitro is challenging because of the scarcity of sample material. In our study, we applied an assisted reproductive technique protocol for IVM and intracytoplasmic sperm injection (ICSI), developed in the domestic cat, to oocytes retrieved from ovaries of four 2-year-old lionesses (Panthera leo) eight hours postmortem. In total, 68 cumulus-oocyte complexes of good quality were randomly distributed and cultured for 32 to 34 hours in two different maturation culture media, consisting of Medium 199 with Earle's salts, 3 mg/mL BSA, 0.1 mg/mL cysteine, 1.4 mg/mL sodium pyruvate, 0.6 mg/mL sodium lactate, 0.15 mg/mL l-glutamine, and 0.055 mg/mL gentamicin. Hormonal supplementation of IVM_1 was 0.02 IU/mL FSH and 0.05 IU/mL LH; IVM_2 consisted of 1.64 IU/mL FSH, 1.06 IU/mL LH, and 1 μg/mL 17ß-estradiol. Differences in hormonal supplementation did not produce significant differences in oocyte maturation rates, which were 39.4% in IVM_1 and 34.3% in IVM_2. Matured oocytes were microinjected with homologous frozen-thawed spermatozoa, and subsequent cleavage rates were 30.8% and 58.3%, respectively. Half of the embryos derived from oocytes matured in IVM_1 developed into blastocysts, whereas only 28.6% of embryos from oocytes matured in IVM_2 reached the blastocyst stage. Morula stages were present from Day 6 onward, and blastocyst stages from Day 9 on, indicating a slower developmental speed in comparison with domestic cats. This is the first report of in vitro-produced blastocysts using ICSI in the lion, and the results report that IVM and ICSI can be successfully performed with cumulus-oocyte complexes retrieved from ovaries after eight hours of shipping, obtaining competent embryos in culture. Copyright © 2015 Elsevier Inc. All rights reserved.

Inhibition of cytochrome P450 3A by acetoxylated analogues of resveratrol in in vitro and in silico models

PubMed Central

Basheer, Loai; Schultz, Keren; Kerem, Zohar

2016-01-01

Many dietary compounds, including resveratrol, are potent inhibitors of CYP3A4. Here we examined the potential to predict inhibition capacity of dietary polyphenolics using an in silico and in vitro approaches and synthetic model compounds. Mono, di, and tri-acetoxy resveratrol were synthesized, a cell line of human intestine origin and microsomes from rat liver served to determine their in vitro inhibition of CYP3A4, and compared to that of resveratrol. Docking simulation served to predict the affinity of the synthetic model compounds to the enzyme. Modelling of the enzyme’s binding site revealed three types of interaction: hydrophobic, electrostatic and H-bonding. The simulation revealed that each of the examined acetylations of resveratrol led to the loss of important interactions of all types. Tri-acetoxy resveratrol was the weakest inhibitor in vitro despite being the more lipophilic and having the highest affinity for the binding site. The simulation demonstrated exclusion of all interactions between tri-acetoxy resveratrol and the heme due to distal binding, highlighting the complexity of the CYP3A4 binding site, which may allow simultaneous accommodation of two molecules. Finally, the use of computational modelling may serve as a quick predictive tool to identify potential harmful interactions between dietary compounds and prescribed drugs. PMID:27530542

Screening for Natural Inhibitors of Topoisomerases I from Rhamnus davurica by Affinity Ultrafiltration and High-Performance Liquid Chromatography–Mass Spectrometry

PubMed Central

Chen, Guilin; Guo, Mingquan

2017-01-01

Topoisomerase I (Topo I) catalyzes topological interconversion of duplex DNA during DNA replication and transcription, and has been deemed as important antineoplastic targets. In this study, the fraction R.d-60 from ethyl acetate extracts of Rhamnus davurica showed higher inhibitory rates against SGC-7901 and HT-29 compared with the R.d-30 fraction in vitro. However, the specific active components of R.d-60 fraction remain elusive. To this end, a method based on bio-affinity ultrafiltration and high performance liquid chromatography/electrospray mass spectrometry (HPLC- ESI-MS/MS) was developed to rapidly screen and identify the Topo I inhibitors in this fraction. The enrichment factors (EFs) were calculated to evaluate the binding affinities between the bioactive constituents and Topo I. As a result, eight ligands were identified and six of which with higher EFs showed more potential antitumor activity. Furthermore, antiproliferative assays in vitro (IC50 values) with two representative candidates (apigenin, quercetin) against SGC-7901, HT-29 and Hep G2 cells were conducted and further validated. Finally, the structure-activity relationships revealed that flavones contain a C2-C3 double bond of C ring exhibited higher bio-affinities to Topo I than those without it. This integrated method combining Topo I ultrafiltration with HPLC-MS/MS proved to be very efficient in rapid screening and identification of potential Topo I inhibitors from the complex extracts of medicinal plants, and could be further explored as a valuable high-throughput screening platform in the early drug discovery stage. PMID:28919906

Fusion of a Novel Genetically Engineered Chitosan Affinity Protein and Green Fluorescent Protein for Specific Detection of Chitosan In Vitro and In Situ

PubMed Central

Nampally, Malathi; Moerschbacher, Bruno Maria

2012-01-01

Chitin is the second most abundant polysaccharide, present, e.g., in insect and arthropod exoskeletons and fungal cell walls. In some species or under specific conditions, chitin appears to be enzymatically de-N-acetylated to chitosan—e.g., when pathogenic fungi invade their host tissues. Here, the deacetylation of chitin is assumed to represent a pathogenicity mechanism protecting the fungus from the host's chitin-driven immune response. While highly specific chitin binding lectins are well known and easily available, this is not the case for chitosan-specific probes. This is partly due to the poor antigenicity of chitosan so that producing high-affinity, specific antibodies is difficult. Also, lectins with specificity to chitosan have been described but are not commercially available, and our attempts to reproduce the findings were not successful. We have, therefore, generated a fusion protein between a chitosanase inactivated by site-directed mutagenesis, the green fluorescent protein (GFP), and StrepII, as well as His6 tags for purification and detection. The recombinant chitosan affinity protein (CAP) expressed in Escherichia coli was shown to specifically bind to chitosan, but not to chitin, and the affinity increased with decreasing degree of acetylation. In vitro, CAP detection was possible either based on GFP fluorescence or using Strep-Tactin conjugates or anti-His5 antibodies. CAP fluorescence microscopy revealed binding to the chitosan exposing endophytic infection structures of the wheat stem rust fungus, but not the chitin exposing ectophytic infection structures, verifying its suitability for in situ chitosan staining. PMID:22367086

In vitro propagation, ex vitro rooting and leaf micromorphology of Bauhinia racemosa Lam.: a leguminous tree with medicinal values.

PubMed

Sharma, Udit; Kataria, Vinod; Shekhawat, N S

2017-10-01

A micropropagation system for Bauhinia racemosa Lam. was developed involving axillary shoot proliferation and ex vitro rooting using nodal explants obtained from mature tree. MS medium with 3.0 mg l -1 BA (6-benzyladenine) was optimum for shoot bud induction. For shoot multiplication, mother explants were transferred repeatedly on medium containing low concentration of BA (0.75 mg l -1 ). Number of shoots was increased up to two passages and decreased thereafter. Shoot multiplication was further enhanced on MS medium containing 0.25 mg l -1 each of BA and Kin (Kinetin) with 0.1 mg l -1 of NAA (α-naphthalene acetic acid). Addition of 0.004 mg l -1 TDZ (thidiazuron) increased the rate of shoot multiplication and 21.81 ± 1.26 shoots per culture vessel were obtained. In vitro regenerated shoots were rooted under ex vitro conditions treated with 400 mg l -1 IBA (indole-3-butyric acid) for 7 min on sterile soilrite. After successful hardening in greenhouse, ex vitro rooted plants were transferred to the field conditions with ≈85% of survival rate. Micromorphological changes were observed on leaf surface i.e. development of vein density and trichomes and stomatal appearance, when plants were subjected to environmental conditions. This is the first report on in vitro regeneration of B. racemosa from mature tree.

Effect of high and low antral follicle count in pubertal beef heifers on in vitro fertilization (IVF)

USDA-ARS?s Scientific Manuscript database

Pubertal heifers can be classified between those with high (= 25) and low (= 15) antral follicle counts (AFC). The objective of this study was to determine oocyte development and maturation (e.g., fertility) in an in vitro fertilization (IVF) system for high and low AFC heifers. From a pool of 120...

In vitro fertilization (IVF) from low or high antral follicle count pubertal beef heifers using semi-defined culture conditions

USDA-ARS?s Scientific Manuscript database

Antral follicle counts (AFC) vary among pubertal beef heifers. Our objective was to compare the in vitro maturation and fertilization of oocytes collected from low and high AFC heifers. Previously we reported results using serum-based IVF media and in this study report results using semi-defined m...

NifX and NifEN exchange NifB cofactor and the VK-cluster, a newly isolated intermediate of the iron-molybdenum cofactor biosynthetic pathway.

PubMed

Hernandez, Jose A; Igarashi, Robert Y; Soboh, Basem; Curatti, Leonardo; Dean, Dennis R; Ludden, Paul W; Rubio, Luis M

2007-01-01

The iron-molybdenum cofactor of nitrogenase (FeMo-co) is synthesized in a multistep process catalysed by several Nif proteins and is finally inserted into a pre-synthesized apo-dinitrogenase to generate mature dinitrogenase protein. The NifEN complex serves as scaffold for some steps of this synthesis, while NifX belongs to a family of small proteins that bind either FeMo-co precursors or FeMo-co during cofactor synthesis. In this work, the binding of FeMo-co precursors and their transfer between purified Azotobacter vinelandii NifX and NifEN proteins was studied to shed light on the role of NifX on FeMo-co synthesis. Purified NifX binds NifB cofactor (NifB-co), a precursor to FeMo-co, with high affinity and is able to transfer it to the NifEN complex. In addition, NifEN and NifX exchange another [Fe-S] cluster that serves as a FeMo-co precursor, and we have designated it as the VK-cluster. In contrast to NifB-co, the VK-cluster is electronic paramagnetic resonance (EPR)-active in the reduced and the oxidized states. The NifX/VK-cluster complex is unable to support in vitro FeMo-co synthesis in the absence of NifEN because further processing of the VK-cluster into FeMo-co requires the simultaneous activities of NifEN and NifH. Our in vitro studies suggest that the role of NifX in vivo is to serve as transient reservoir of FeMo-co precursors and thus help control their flux during FeMo-co synthesis.

Kaplan-Meier Meets Chemical Kinetics: Intrinsic Rate of SOD1 Amyloidogenesis Decreased by Subset of ALS Mutations and Cannot Fully Explain Age of Disease Onset.

PubMed

Abdolvahabi, Alireza; Shi, Yunhua; Rasouli, Sanaz; Croom, Corbin M; Aliyan, Amir; Martí, Angel A; Shaw, Bryan F

2017-06-21

Over 150 mutations in SOD1 (superoxide dismutase-1) cause amyotrophic lateral sclerosis (ALS), presumably by accelerating SOD1 amyloidogenesis. Like many nucleation processes, SOD1 fibrillization is stochastic (in vitro), which inhibits the determination of aggregation rates (and obscures whether rates correlate with patient phenotypes). Here, we diverged from classical chemical kinetics and used Kaplan-Meier estimators to quantify the probability of apo-SOD1 fibrillization (in vitro) from ∼10 3 replicate amyloid assays of wild-type (WT) SOD1 and nine ALS variants. The probability of apo-SOD1 fibrillization (expressed as a Hazard ratio) is increased by certain ALS-linked SOD1 mutations but is decreased or remains unchanged by other mutations. Despite this diversity, Hazard ratios of fibrillization correlated linearly with (and for three mutants, approximately equaled) Hazard ratios of patient survival (R 2 = 0.67; Pearson's r = 0.82). No correlation exists between Hazard ratios of fibrillization and age of initial onset of ALS (R 2 = 0.09). Thus, Hazard ratios of fibrillization might explain rates of disease progression but not onset. Classical kinetic metrics of fibrillization, i.e., mean lag time and propagation rate, did not correlate as strongly with phenotype (and ALS mutations did not uniformly accelerate mean rate of nucleation or propagation). A strong correlation was found, however, between mean ThT fluorescence at lag time and patient survival (R 2 = 0.93); oligomers of SOD1 with weaker fluorescence correlated with shorter survival. This study suggests that SOD1 mutations trigger ALS by altering a property of SOD1 or its oligomers other than the intrinsic rate of amyloid nucleation (e.g., oligomer stability; rates of intercellular propagation; affinity for membrane surfaces; and maturation rate).

Accurate and sensitive quantification of protein-DNA binding affinity.

PubMed

Rastogi, Chaitanya; Rube, H Tomas; Kribelbauer, Judith F; Crocker, Justin; Loker, Ryan E; Martini, Gabriella D; Laptenko, Oleg; Freed-Pastor, William A; Prives, Carol; Stern, David L; Mann, Richard S; Bussemaker, Harmen J

2018-04-17

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes. Copyright © 2018 the Author(s). Published by PNAS.

Accurate and sensitive quantification of protein-DNA binding affinity

PubMed Central

Rastogi, Chaitanya; Rube, H. Tomas; Kribelbauer, Judith F.; Crocker, Justin; Loker, Ryan E.; Martini, Gabriella D.; Laptenko, Oleg; Freed-Pastor, William A.; Prives, Carol; Stern, David L.; Mann, Richard S.; Bussemaker, Harmen J.

2018-01-01

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes. PMID:29610332

Complementary DNA display selection of high-affinity peptides binding the vacuolating toxin (VacA) of Helicobacter pylori.

PubMed

Hayakawa, Yumiko; Matsuno, Mitsuhiro; Tanaka, Makoto; Wada, Akihiro; Kitamura, Koichiro; Takei, Osamu; Sasaki, Ryuzo; Mizukami, Tamio; Hasegawa, Makoto

2015-09-01

Artificial peptides designed for molecular recognition of a bacterial toxin have been developed. Vacuolating cytotoxin A protein (VacA) is a major virulence factor of Helicobacter pylori, a gram-negative microaerophilic bacterium inhabiting the upper gastrointestinal tract, particularly the stomach. This study attempted to identify specific peptide sequences with high affinity for VacA using systematic directed evolution in vitro, a cDNA display method. A surface plasmon resonance-based biosensor and fluorescence correlation spectroscopy to examine binding of peptides with VacA identified a peptide (GRVNQRL) with high affinity. Cyclization of the peptide by attaching cysteine residues to both termini improved its binding affinity to VacA, with a dissociation constant (Kd ) of 58 nm. This study describes a new strategy for the development of artificial functional peptides, which are promising materials in biochemical analyses and medical applications. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

One-pot synthesis and sigma receptor binding studies of novel spirocyclic-2,6-diketopiperazine derivatives.

PubMed

Ghandi, Mehdi; Sherafat, Fatemeh; Sadeghzadeh, Masoud; Alirezapour, Behrouz

2016-06-01

New spirocyclic-2,6-diketopiperazine derivatives containing benzylpiperidine and cycloalkane moieties were synthesized by a one-pot two-step sequential Ugi/intramolecular N-amidation process in moderate to good yields. The in vitro ligand-binding profile studies performed on the sigma-1 and sigma-2 receptors revealed that the σ1 affinities and subtype selectivities of three spirocyclic piperidine derivatives are generally comparable to those of spirocycloalkane analogues. Compared to the low σ1 affinities obtained for cycloalkyl-substituted spirocyclic-2,6-diketopiperazines with n=2, those with n=1 proved to have optimal fitting with σ2 subtype by exhibiting higher affinities. Moreover, the best binding affinity and subtype selectivity was identified for compound 3c with Kiσ1=5.9±0.5nM and Kiσ2=563±21nM as well as 95-fold σ1/σ2 selectivity ratio, respectively. Copyright © 2016. Published by Elsevier Ltd.

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.

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.

Overaccumulation of the chloroplast antisense RNA AS5 is correlated with decreased abundance of 5S rRNA in vivo and inefficient 5S rRNA maturation in vitro

PubMed Central

Sharwood, Robert E.; Hotto, Amber M.; Bollenbach, Thomas J.; Stern, David B.

2011-01-01

Post-transcriptional regulation in the chloroplast is exerted by nucleus-encoded ribonucleases and RNA-binding proteins. One of these ribonucleases is RNR1, a 3′-to-5′ exoribonuclease of the RNase II family. We have previously shown that Arabidopsis rnr1-null mutants exhibit specific abnormalities in the expression of the rRNA operon, including the accumulation of precursor 23S, 16S, and 4.5S species and a concomitant decrease in the mature species. 5S rRNA transcripts, however, accumulate to a very low level in both precursor and mature forms, suggesting that they are unstable in the rnr1 background. Here we demonstrate that rnr1 plants overaccumulate an antisense RNA, AS5, that is complementary to the 5S rRNA, its intergenic spacer, and the downstream trnR gene, which encodes tRNAArg, raising the possibility that AS5 destabilizes 5S rRNA or its precursor and/or blocks rRNA maturation. To investigate this, we used an in vitro system that supports 5S rRNA and trnR processing. We show that AS5 inhibits 5S rRNA maturation from a 5S-trnR precursor, and shorter versions of AS5 demonstrate that inhibition requires intergenic sequences. To test whether the sense and antisense RNAs form double-stranded regions in vitro, treatment with the single-strand-specific mung bean nuclease was used. These results suggest that 5S–AS5 duplexes interfere with a sense-strand secondary structure near the endonucleolytic cleavage site downstream from the 5S rRNA coding region. We hypothesize that these duplexes are degraded by a dsRNA-specific ribonuclease in vivo, contributing to the 5S rRNA deficiency observed in rnr1. PMID:21148395

Regeneration of Acer caudatifolium Hayata plantlets from juvenile explants.

PubMed

Durkovic, J

2003-07-01

Juvenile and fully mature Acer caudatifolium Hayata explants were assayed for their organogenic capacity. A protocol for multiple shoot culture formation and in vitro plant regeneration was developed for juvenile axillary bud cultures. Mature explants failed in shoot regeneration. Shoot multiplication was achieved by releasing apical dominance of the single elongated shoot on woody plant medium (WPM) supplemented with 0.7 mg l(-1) 6-benzylaminopurine and 0.05 mg l(-1) alpha-naphthaleneacetic acid. The highest rooting percentage was recorded on half-strength WPM containing 1.0 mg l(-1) indole-3-butyric acid. Regenerated plantlets were successfully hardened to ex vitro conditions and continued to grow after transfer to soil. No morphological aberrations were observed in the regenerates.

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.

Pharmacological characterization of the cloned kappa opioid receptor as a kappa 1b subtype.

PubMed

Lai, J; Ma, S W; Zhu, R H; Rothman, R B; Lentes, K U; Porreca, F

1994-10-27

Substantial pharmacological evidence in vitro and in vivo has suggested the existence of subtypes of the kappa opioid receptor. Quantitative radioligand binding techniques resolved the presence of two high affinity binding sites for the kappa 1 ligand [3H]U69,593 in mouse brain membranes, termed kappa 1a and kappa 1b, respectively. Whereas the kappa 1a site has high affinity for fedotozine and oxymorphindole and low affinity for bremazocine and alpha-neoendorphin, site kappa 1b has high affinity for bremazocine and alpha-neoendorphin and low affinity for fedotozine and oxymorphindole. CI-977 and U69,593 bind equally well at both sites. To determine the relationship between these kappa 1 receptor subtypes and the recently cloned mouse kappa 1 receptor (KOR), we examined [3H]U69,593 binding to the KOR in stably transfected cells (KORCHN-8). Competition of [3H]U69,593 binding to the KOR by bremazocine, alpha-neoendorphin, fedotozine and oxymorphindole resolved a single class of binding sites at which these agents had binding affinities similar to that of the kappa 1b site present in mouse brain. These results suggest that the cloned KOR corresponds to the kappa 1 site in mouse brain defined as kappa 1b.

Cryopreservation of in vitro matured oocytes in addition to ovarian tissue freezing for fertility preservation in paediatric female cancer patients before and after cancer therapy.

PubMed

Abir, R; Ben-Aharon, I; Garor, R; Yaniv, I; Ash, S; Stemmer, S M; Ben-Haroush, A; Freud, E; Kravarusic, D; Sapir, O; Fisch, B

2016-04-01

Is a protocol that combines in vitro maturation of germinal vesicle-stage oocytes and their vitrification with freezing of cortical ovarian tissue feasible for use in fertility preservation for both chemotherapy-naive paediatric patients as well as patients after initiation of cancer therapy? Follicle-containing ovarian tissue as well as oocytes that can undergo maturation in vitro can be obtained from paediatric patients (including prepubertal girls) both before and after cancer therapy. Anticancer therapy reduces the number of follicles/oocytes but this effect is less severe in young patients, particularly the paediatric age group. Autotransplantation of ovarian tissue has yielded to date 60 live births, including one from tissue that was cryostored in adolescence. However, it is assumed that autografting cryopreserved-thawed ovarian cortical tissue poses a risk of reseeding the malignancy. Immature oocytes can be collected from very young girls without hormonal stimulation and then matured in vitro and vitrified. We have previously shown that there is no difference in the number of ovarian cortical follicles between paediatric patients before and after chemotherapy. A prospective study was conducted in a cohort of 42 paediatric females with cancer (before and after therapy initiation) who underwent fertility preservation procedures in 2007-2014 at a single tertiary medical centre. The study group included girls and adolescent females with cancer: 22 before and 20 after chemotherapy. Following partial or complete oophorectomy, immature oocytes were either aspirated manually ex vivo from visible small antral follicles or filtered from spent media. Oocytes were incubated in oocyte maturation medium, and those that matured at 24 or 48 h were vitrified. Ovarian cortical tissue was cut and prepared for slow-gradual cryopreservation. Anti-Mullerian hormone (AMH) levels were measured in serum before and after oophorectomy. Ovarian tissue was successfully collected from 78.7% of the 42 patients. Oocytes were obtained from 20 patients before chemotherapy and 13 after chemotherapy. The youngest patients from whom oocytes were retrieved were aged 2 years (two atretic follicles) and 3 years. Of the 395 oocytes collected, ∼30% were atretic (29.6% in the pre-chemotherapy group, 37% in the post-chemotherapy group). One hundred twenty-one oocytes (31%) were matured in vitro and vitrified: 67.8% from patients before chemotherapy, the rest after chemotherapy. Mature oocytes suitable for vitrification were obtained from 16/20 patients before chemotherapy and from 12/13 patients after chemotherapy (maturation rate, 32 and 26.4%, respectively). There were significant correlations of the number of vitrified oocytes with patient age (more matured oocytes with older age) (P = 0.001) and with pre-oophorectomy AMH levels (P = 0.038 pre-chemotherapy group, P = 0.029 post-chemotherapy group). Oocytes suitable for vitrification were obtained both by manual aspiration of antral follicles (45%) and from rinse solutions after dissection. There were significantly more matured oocytes in the pre-chemotherapy group from aspiration than in the post-chemotherapy group after both aspiration (P < 0.033) and retrieval from rinsing fluids (P < 0.044). The number of pre-antral follicles per histological section did not differ in the pre- versus post-chemotherapy. AMH levels dropped by approximately 50% after ovarian removal in both groups, with a significant correlation between pre- and post-oophorectomy levels (P = 0.002 pre-chemotherapy group, P = 0.001 post-chemotherapy group). There were no patients between 5 years and 10 years old in the post-chemotherapy group, which might have affected some results and correlations. Oocytes from patients soon after chemotherapy might be damaged, and caution is advised when using them for fertility-restoration purposes. The viability, development capability and fertilization potential of oocytes from paediatric patients, especially prepubertal and after chemotherapy, are unknown, in particular oocytes recovered from the media after the tissue dissection step. Although more oocytes were collected and matured from chemotherapy-naïve paediatric patients, ovarian tissue and immature oocytes were also retrieved from young girls in whom cancer therapy has already been initiated. Our centre has established a protocol for potential maximal fertility preservation in paediatric female patients with cancer. Vitrified-in vitro-matured oocytes may serve as an important gamete source in paediatric female patients with cancer because the risk of reseeding the disease is avoided. Further studies are needed on the fertility-restoring potential of oocytes from paediatric and prepubertal patients, especially after exposure to chemotherapy. The study was conducted as part of the routine procedures for fertility preservation at our IVF unit. No funding outside of the IVF laboratory was received. Funding for the AMH measurements was obtained by a research grant from the Israel Science Foundation (to B.-A.I., ISF 13-1873). None of the authors have competing interests. N/A. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Potential mechanisms of development-dependent adverse effects of the herbicide paraquat in 3D rat brain cell cultures.

PubMed

Sandström, J; Broyer, A; Zoia, D; Schilt, C; Greggio, C; Fournier, M; Do, K Q; Monnet-Tschudi, F

2017-05-01

Exposure to environmental toxicants during vulnerable windows of brain development is suspected to raise the prevalence for neurological dysfunctions at later stages in life. Differentiation processes and changes in morphology, as well as a lack of physiological barriers, might be reasons that render a developing brain more susceptible to neurotoxicants than an adult. However, also the intrinsic capacity of cells to combat toxicant induced cellular stress might differ between the immature- and mature brain. In order to study whether this intrinsic protection capacity differs between immature and maturated brain cells we chose to study the maturation-dependent adverse effects of the known neurotoxicant Paraquat Dichloride (PQ) in 3D rat brain cell cultures. This in vitro system consists of the major brain cell types - neurons, astrocytes, oligodendrocytes and microglia - and over the time in vitro cultured cells undergo differentiation and maturation into a tissue-like organization. PQ was applied repeatedly over ten days in the sub-micromolar range, and effects were evaluated on neurons and glial cells. We observed that despite a higher PQ-uptake in mature cultures, PQ-induced adverse effects on glutamatergic-, GABAergic- and dopaminergic neurons, as assessed by gene expression and enzymatic activity, were more pronounced in immature cultures. This was associated with a stronger astrogliosis in immature- as compared to mature cultures, as well as perturbations of the glutathione-mediated defense against oxidative stress. Furthermore we observed evidence of microglial activation only in mature cultures, whereas immature cultures appeared to down-regulate markers for neuroprotective M2-microglial phenotype upon PQ-exposure. Taken together our results indicate that immature brain cell cultures have less intrinsic capacity to cope with cellular stress elicited by PQ as compared to mature cells. This may render immature brain cells more susceptible to the adverse effects of PQ. Copyright © 2017 Elsevier B.V. All rights reserved.

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

[18F]F15599, a novel 5-HT1A receptor agonist, as a radioligand for PET neuroimaging.

PubMed

Lemoine, Laëtitia; Verdurand, Mathieu; Vacher, Bernard; Blanc, Elodie; Le Bars, Didier; Newman-Tancredi, Adrian; Zimmer, Luc

2010-03-01

The serotonin-1A (5-HT(1A)) receptor is implicated in the pathophysiology of major neuropsychiatric disorders. Thus, the functional imaging of 5-HT(1A) receptors by positron emission tomography (PET) may contribute to the understanding of its role in those pathologies and their therapeutics. These receptors exist in high- and low-affinity states and it is proposed that agonists bind preferentially to the high-affinity state of the receptor and therefore could provide a measure of the functional 5-HT(1A) receptors. Since all clinical PET 5-HT(1A) radiopharmaceuticals are antagonists, it is of great interest to develop a( 18)F labelled agonist. F15599 (3-chloro-4-fluorophenyl-(4-fluoro-4{[(5-methyl-pyrimidin-2-ylmethyl)-amino]-methyl}-piperidin-1-yl)-methanone) is a novel ligand with high affinity and selectivity for 5-HT(1A) receptors and is currently tested as an antidepressant. In pharmacological tests in rat, it exhibits preferential agonist activity at post-synaptic 5-HT(1A) receptors in cortical brain regions. Here, its nitro-precursor was synthesised and radiolabelled via a fluoronucleophilic substitution. Radiopharmacological evaluations included in vitro and ex vivo autoradiography in rat brain and PET scans on rats and cats. Results were compared with simultaneous studies using [(18)F]MPPF, a validated 5-HT(1A) antagonist radiopharmaceutical. The chemical and radiochemical purities of [(18)F]F15599 were >98%. In vitro [(18)F]F15599 binding was consistent with the known 5-HT(1A) receptors distribution (hippocampus, dorsal raphe nucleus, and notably cortical areas) and addition of Gpp(NH)p inhibited [(18)F]F15599 binding, consistent with a specific binding to G protein-coupled receptors. In vitro binding of [(18)F]F15599 was blocked by WAY100635 and 8-OH-DPAT, respectively, prototypical 5-HT(1A) antagonist and agonist. The ex vivo and in vivo studies demonstrated that the radiotracer readily entered the rat and the cat brain and generated few brain radioactive metabolites. Remarkably, in microPET studies, [(18)F]F15599 notably displayed a pattern of brain labelling that did not correlate with in vitro observations. Thus, in cat, the highest binding was observed in dorsal raphe and cingulate cortex with little binding in other cortical regions and none in hippocampus. In vivo binding was abolished by WAY100635, indicating specific labelling of 5-HT(1A) receptors. [(18)F]F15599 is a radiofluorinated agonist presenting interesting characteristics for probing in vitro and in vivo the high-affinity states of the 5-HT(1A) receptors. Its differential labelling of 5-HT(1A) receptors in vitro and in vivo may result from its reported preferential interaction with receptors coupled to specific G-protein subtypes.

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

Generation of human adipose stem cells through dedifferentiation of mature adipocytes in ceiling cultures.

PubMed

Lessard, Julie; Côté, Julie Anne; Lapointe, Marc; Pelletier, Mélissa; Nadeau, Mélanie; Marceau, Simon; Biertho, Laurent; Tchernof, André

2015-03-07

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Changes in chemical components and cytotoxicity at different maturity stages of Pleurotus eryngii fruiting body.

PubMed

Cui, Fengjie; Li, Yunhong; Yang, Yan; Sun, Wenjing; Wu, Di; Ping, Lifeng

2014-12-31

The present study investigated the changes of the chemical components and cytotoxicity potency at 5 developmental stages of Pleurotus eryngii fruiting body. The carbohydrate and protein contents increased along the maturity of fruiting body while fat content decreased. By comparison, the polysaccharide-protein fractions had the highest antiproliferative effect on SGC-7901 and HepG-2 cells in vitro and increasing activity with growing maturity of P. eryngii fruiting body.The maturation process increased the protein content and acid property through the enhanced relative abundance of Asp, Thr, and Glu in polysaccharide-protein fractions. Further purification and electrophoresis identified that the polysaccharide-protein PEG-1with three subunits possibly was the target cytotoxical component. Our findings proved that mature fruiting body of P. eryngii containing these polysaccharide-proteins possessed highly nutritional values and therapeutical benefits.

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.

Generation of functional podocytes from human induced pluripotent stem cells.

PubMed

Ciampi, Osele; Iacone, Roberto; Longaretti, Lorena; Benedetti, Valentina; Graf, Martin; Magnone, Maria Chiara; Patsch, Christoph; Xinaris, Christodoulos; Remuzzi, Giuseppe; Benigni, Ariela; Tomasoni, Susanna

2016-07-01

Generating human podocytes in vitro could offer a unique opportunity to study human diseases. Here, we describe a simple and efficient protocol for obtaining functional podocytes in vitro from human induced pluripotent stem cells. Cells were exposed to a three-step protocol, which induced their differentiation into intermediate mesoderm, then into nephron progenitors and, finally, into mature podocytes. After differentiation, cells expressed the main podocyte markers, such as synaptopodin, WT1, α-Actinin-4, P-cadherin and nephrin at the protein and mRNA level, and showed the low proliferation rate typical of mature podocytes. Exposure to Angiotensin II significantly decreased the expression of podocyte genes and cells underwent cytoskeleton rearrangement. Cells were able to internalize albumin and self-assembled into chimeric 3D structures in combination with dissociated embryonic mouse kidney cells. Overall, these findings demonstrate the establishment of a robust protocol that, mimicking developmental stages, makes it possible to derive functional podocytes in vitro. Copyright © 2016. Published by Elsevier B.V.

HIGH INCIDENCE OF POLYSPERMIC FERTILIZATION IN BOVINE OOCYTES MATURED IN VITRO AFTER CRYOTOP VITRIFICATION.

PubMed

Hwang, In-Sul; Kwon, Dae-Jin; Im, Gi-Sun; Tashima, Kazuya; Hochi, Shinichi; Hwang, Seongsoo

2016-01-01

Vitrification with the Cryotop device is the most promising technique for oocyte cryopreservation, but the high post-warming morphological survival of bovine oocytes does not guarantee high developmental competence after in vitro fertilization (IVF). This study was designed to examine achievement of normal fertilization in bovine oocytes vitrified-warmed with the Cryotop device. Oocytes were matured in vitro and vitrified-warmed after complete removal of the cumulus layers. Distribution of cortical granules (CGs) was assessed by Lens culinaris agglutinin (LCA) lectin staining. Ten hours after IVF, presumptive zygotes were analyzed for pronuclear formation. Day-8 blastocysts were harvested and stained with Hoechst-33342 for total cell counting. Both yield and mean cell number of the blastocysts were impaired by Cryotop vitrification. Incidence of polyspermic fertilization was three-times higher in vitrified oocytes compared to fresh oocytes. No difference in CG distribution was found between vitrified and fresh oocytes. Polyspermic fertilization induced in vitrified-warmed bovine oocytes may be one of the possible causes responsible for their low developmental potential.

In vitro pathogenicity assay for the ergot fungus Claviceps purpurea.

PubMed

Scheffer, Jan; Tudzynski, Paul

2006-04-01

The pathogenic development of the biotrophic ergot fungus Claviceps purpurea is strictly limited to the ovary of grasses. Early colonization stages occur within a defined spatio-temporal course of events, including the directed growth to the vascular tissue for nutrient supply. To characterize mutant strains with putative defects in pathogenicity, the close observation of the infection pathway is therefore indispensable. Here, we describe the establishment of a new pathogenicity assay, based on the in vitro cultivation of isolated rye ovaries. The pathogenic development of a wild-type strain of C. purpurea was compared with the infection of mature rye flowers on whole plants. Up to the sixth day post inoculation, the route of infection within the isolated ovaries was maintained and temporally equal to that seen in mature flowers. Therefore, the in vitro pathogenicity assay is an effective alternative to the whole-plant infection tests, and suitable for detailed infection studies and screening high numbers of mutants for defects in early pathogenesis.

Increasing the cAMP concentration during in vitro maturation of pig oocytes improves cumulus maturation and subsequent fertilization in vitro.

PubMed

Appeltant, R; Beek, J; Vandenberghe, L; Maes, D; Van Soom, A

2015-02-01

Porcine IVF faces various problems such as incomplete cytoplasmic maturation of the oocyte and polyspermy. Previous studies proved the importance of cAMP in regulating nuclear and cytoplasmic maturation of oocytes. This study investigated the effect of the cAMP-modulating agents 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cAMP sodium salt (dbcAMP) on several parameters during in vitro production of porcine embryos. First, we wanted to see if oocyte collection in IBMX could meiotically arrest oocytes and, as such, improve synchronization of nuclear and cytoplasmic maturation. To this end, cumulus-oocyte complexes (COCs) were collected from gilts in HEPES-buffered Tyrode balanced salt solution medium with 0.5-mM IBMX or without IBMX. At the end of oocyte collection, the effect of IBMX on chromatin configuration was evaluated. However, no differences could be observed in nuclear configuration between IBMX- and IBMX+ oocytes (P > 0.05). Second, we added dbcAMP during IVM to improve cytoplasmic maturation and evaluated cumulus expansion (lack of adhesion), a disintegrin and metalloproteinase with thrombospondin-like repeats (ADAMTS-1) levels in cumulus cells, fertilization, and blastocyst rates. Cumulus-oocyte complexes were matured in modified North Carolina State University medium 37 with or without 1-mM dbcAMP. Frozen-thawed, epididymal, boar spermatozoa were used for IVF. After IVF, presumed zygotes were cultured for 7 days in North Carolina State University medium 23. Penetration rate decreased in dbcAMP+ (57.3%) compared with dbcAMP- (67.8%), but the polyspermy rate also decreased (43.3% vs. 53.4%, respectively) leading to an increased normal fertilization rate (56.7% vs. 46.6%, respectively; P < 0.05). Only 7.2% of the COCs showed adhesion in dbcAMP+ which was lower than 15.7% in dbcAMP- (P < 0.05) probably because of an upregulation of the ADAMTS-1 protein by dbcAMP. When the adherent oocytes were removed during maturation, no difference could be detected between the blastocyst rate of dbcAMP- and dbcAMP+ (17.1% and 21.0% on Day 7, respectively; P > 0.05). In conclusion, the use of IBMX during collection did not cause a meiotic arrest. Using dbcAMP during IVM caused a greater normal fertilization rate, a lower rate of adherent COCs during IVM, higher levels of ADAMTS-1 in cumulus cells, and an equal blastocyst rate after screening out adherent COCs. These findings contribute to a better understanding of cAMP involvement in porcine oocyte maturation and provide a basis to develop an improved system with less polyspermy and higher blastocyst rates. Copyright © 2015 Elsevier Inc. All rights reserved.

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.

Are cannabidiol and Δ(9) -tetrahydrocannabivarin negative modulators of the endocannabinoid system? A systematic review.

PubMed

McPartland, John M; Duncan, Marnie; Di Marzo, Vincenzo; Pertwee, Roger G

2015-02-01

Based upon evidence that the therapeutic properties of Cannabis preparations are not solely dependent upon the presence of Δ(9) -tetrahydrocannabinol (THC), pharmacological studies have been recently carried out with other plant cannabinoids (phytocannabinoids), particularly cannabidiol (CBD) and Δ(9) -tetrahydrocannabivarin (THCV). Results from some of these studies have fostered the view that CBD and THCV modulate the effects of THC via direct blockade of cannabinoid CB1 receptors, thus behaving like first-generation CB1 receptor inverse agonists, such as rimonabant. Here, we review in vitro and ex vivo mechanistic studies of CBD and THCV, and synthesize data from these studies in a meta-analysis. Synthesized data regarding mechanisms are then used to interpret results from recent pre-clinical animal studies and clinical trials. The evidence indicates that CBD and THCV are not rimonabant-like in their action and thus appear very unlikely to produce unwanted CNS effects. They exhibit markedly disparate pharmacological profiles particularly at CB1 receptors: CBD is a very low-affinity CB1 ligand that can nevertheless affect CB1 receptor activity in vivo in an indirect manner, while THCV is a high-affinity CB1 receptor ligand and potent antagonist in vitro and yet only occasionally produces effects in vivo resulting from CB1 receptor antagonism. THCV has also high affinity for CB2 receptors and signals as a partial agonist, differing from both CBD and rimonabant. These cannabinoids illustrate how in vitro mechanistic studies do not always predict in vivo pharmacology and underlie the necessity of testing compounds in vivo before drawing any conclusion on their functional activity at a given target. © 2014 The British Pharmacological Society.

Are cannabidiol and Δ9-tetrahydrocannabivarin negative modulators of the endocannabinoid system? A systematic review

PubMed Central

McPartland, John M; Duncan, Marnie; Di Marzo, Vincenzo; Pertwee, Roger G

2015-01-01

Based upon evidence that the therapeutic properties of Cannabis preparations are not solely dependent upon the presence of Δ9-tetrahydrocannabinol (THC), pharmacological studies have been recently carried out with other plant cannabinoids (phytocannabinoids), particularly cannabidiol (CBD) and Δ9-tetrahydrocannabivarin (THCV). Results from some of these studies have fostered the view that CBD and THCV modulate the effects of THC via direct blockade of cannabinoid CB1 receptors, thus behaving like first-generation CB1 receptor inverse agonists, such as rimonabant. Here, we review in vitro and ex vivo mechanistic studies of CBD and THCV, and synthesize data from these studies in a meta-analysis. Synthesized data regarding mechanisms are then used to interpret results from recent pre-clinical animal studies and clinical trials. The evidence indicates that CBD and THCV are not rimonabant-like in their action and thus appear very unlikely to produce unwanted CNS effects. They exhibit markedly disparate pharmacological profiles particularly at CB1 receptors: CBD is a very low-affinity CB1 ligand that can nevertheless affect CB1 receptor activity in vivo in an indirect manner, while THCV is a high-affinity CB1 receptor ligand and potent antagonist in vitro and yet only occasionally produces effects in vivo resulting from CB1 receptor antagonism. THCV has also high affinity for CB2 receptors and signals as a partial agonist, differing from both CBD and rimonabant. These cannabinoids illustrate how in vitro mechanistic studies do not always predict in vivo pharmacology and underlie the necessity of testing compounds in vivo before drawing any conclusion on their functional activity at a given target. PMID:25257544

In Vitro and In Vivo Characterization of a Fully Felinized Therapeutic Anti-Nerve Growth Factor Monoclonal Antibody for the Treatment of Pain in Cats.

PubMed

Gearing, D P; Huebner, M; Virtue, E R; Knight, K; Hansen, P; Lascelles, B D X; Gearing, R P; Drew, A C

2016-07-01

Limited options are available for the treatment of pain in cats. Monoclonal antibodies (mAbs) that neutralize nerve growth factor (NGF) have demonstrated analgesic capacity in rodent models, people with osteoarthritis, and dogs with degenerative joint disease. This study describes the design and characterization of a fully felinized anti-NGF monoclonal antibody. In vitro potency, pharmacokinetics, and the ability of the antibody to treat pain in a self-resolving, acute inflammation model were investigated in cats. Thirty-eight cats at a research colony at Charles River Laboratories, Ireland. Felinized anti-NGF mAb, NV-02, was produced using a complementary DNA (cDNA)-based method (PETization). Purified NV-02 was tested for affinity, potency, and immunoreactivity in vitro, then for safety and plasma pharmacokinetic distribution in vivo, and analgesic efficacy in a model of kaolin-induced inflammatory pain. Anti-NGF mAb, NV-02 neutralized NGF with high affinity and potency and did not bind complement. NV-02-administered SC had a plasma half-life of 7-15 days and was well tolerated at dosages up to 28 mg/kg. A dosage of 2 mg/kg NV-02 SC significantly decreased signs of lameness on day 2 (P = .0027), day 3 (P = .016), day 4, (P = .0063), day 5 (P = .0085), day 6 (P = .0014), and day 7 (P = .0034) after induction of inflammation. The high affinity, long plasma half-life, safety, and analgesic efficacy of felinized anti-NGF mAb (NV-02) support further investigation of the analgesic potential of this antibody in the cat. Copyright © 2016 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

[Evaluation on a fast weight reduction model in vitro].

PubMed

Li, Songtao; Li, Ying; Wen, Ying; Sun, Changhao

2010-03-01

To establish a fast and effective model in vitro for screening weight-reducing drugs and taking preliminary evaluation of the model. Mature adipocytes of SD rat induced by oleic acid were used to establish a obesity model in vitro. Isoprel, genistein, caffeine were selected as positive agents and curcumine as negative agent to evaluate the obesity model. Lipolysis of adipocytes was stimulated significantly by isoprel, genistein and caffeine rather than curcumine. This model could be used efficiently for screening weight-losing drugs.

Rapamycin treatment during in vitro maturation of oocytes improves embryonic development after parthenogenesis and somatic cell nuclear transfer in pigs.

PubMed

Lee, Joohyeong; Park, Jong-Im; Yun, Jung Im; Lee, Yongjin; Yong, Hwanyul; Lee, Seung Tae; Park, Choon-Keun; Hyun, Sang-Hwan; Lee, Geun-Shik; Lee, Eunsong

2015-01-01

This study was conducted to investigate the effects of rapamycin treatment during in vitro maturation (IVM) on oocyte maturation and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Morphologically good (MGCOCs) and poor oocytes (MPCOCs) were untreated or treated with 1 nM rapamycin during 0-22 h, 22-42 h, or 0-42 h of IVM. Rapamycin had no significant effects on nuclear maturation and blastocyst formation after PA of MGCOCs. Blastocyst formation after PA was significantly increased by rapamycin treatment during 22-42 h and 0-42 h (46.6% and 46.5%, respectively) relative to the control (33.3%) and 0-22 h groups (38.6%) in MPCOCs. In SCNT, blastocyst formation tended to increase in MPCOCs treated with rapamycin during 0-42 h of IVM relative to untreated oocytes (20.3% vs. 14.3%, 0.05 < p < 0.1), while no improvement was observed in MGCOCs. Gene expression analysis revealed that transcript abundance of Beclin 1 and microtubule-associated protein 1 light chain 3 mRNAs was significantly increased in MPCOCs by rapamycin relative to the control. Our results demonstrated that autophagy induction by rapamycin during IVM improved developmental competence of oocytes derived from MPCOCs.

The influence of powdered coconut water (ACP-318®) in in vitro maturation of canine oocytes.

PubMed

Silva, A E F; Cavalcante, L F; Rodrigues, B A; Rodrigues, J L

2010-12-01

The objective of this study was to determine the influence of powdered coconut water (ACP-318(®)) diluted in high glucose (11.0 mM) TCM199 in the achievement of nuclear in vitro maturation (IVM) of canine oocytes. Cumulus oocyte complexes (COCs) (n = 632) were randomly allocated into three experimental groups named as group 1 (control group), group 2 (5% powdered coconut water) and group 3 (10% powdered coconut water). The percentage of meiotic resumption (MR) (GVBD to MII) was 39.1% (81/207), 50.2% (108/215) and 46.6% (98/210) for groups 1, 2 and 3 respectively (p < 0.05). There were no differences in MR rates among groups 2 and 3. The medium with ACP-318(®) slightly enhanced the nuclear maturation of canine oocytes when a comparison was established with rates of maturation exhibited by oocytes in the experimental group 1 without ACP-318(®) (p < 0.05). The results suggest that oocytes' nuclear morphology integrity and meiosis achievement were positively influenced when exposed to high glucose TCM199 supplemented with 5% powdered coconut water. Further investigation must be performed for a better understanding of powdered coconut water influence in cellular events during IVM of dog oocytes. © 2009 Blackwell Verlag GmbH.

Hanging drop monoculture for selection of optimal antioxidants during in vitro maturation of porcine oocytes.

PubMed

Ishikawa, S; Machida, R; Hiraga, K; Hiradate, Y; Suda, Y; Tanemura, K

2014-04-01

We analysed the effect of three antioxidants that have different functional mechanisms on the in vitro maturation (IVM) of porcine oocytes. Single oocyte monoculture using the hanging drop (HD) system has some advantages such as improving analysis efficiency brought by the smaller number of samples than the number of oocytes cultured in one drop. Direct effects of ligands on single oocytes could also be detected without considering the effects of paracrine factors from other oocytes. After 22 h of pre-culture, denuded oocytes were cultured for 22 h with 0.01 and 0.1 μg/ml of L-carnitine (LC), lactoferrin (LF) or sulforaphane (SF) in the presence/non-presence of oxidant stress induced by H2O2 supplementation to evaluate the reducing effects against oxidative stress on nuclear maturation. As a result, compared with LC and SF, LF showed effective reduction in oxidative stress at a lower concentration (0.01 μg/ml), suggesting that LF is a more effective antioxidant in porcine oocyte IVM. Additionally, LF also increased maturation rate even in culture without H2O2. Our results clearly suggest that the HD monoculture system is useful for screening the substances that affect porcine oocyte culture. © 2014 Blackwell Verlag GmbH.

Rapamycin treatment during in vitro maturation of oocytes improves embryonic development after parthenogenesis and somatic cell nuclear transfer in pigs

PubMed Central

Lee, Joohyeong; Park, Jong-Im; Yun, Jung Im; Lee, Yongjin; Yong, Hwanyul; Lee, Seung Tae; Park, Choon-Keun; Hyun, Sang-Hwan; Lee, Geun-Shik

2015-01-01

This study was conducted to investigate the effects of rapamycin treatment during in vitro maturation (IVM) on oocyte maturation and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Morphologically good (MGCOCs) and poor oocytes (MPCOCs) were untreated or treated with 1 nM rapamycin during 0-22 h, 22-42 h, or 0-42 h of IVM. Rapamycin had no significant effects on nuclear maturation and blastocyst formation after PA of MGCOCs. Blastocyst formation after PA was significantly increased by rapamycin treatment during 22-42 h and 0-42 h (46.6% and 46.5%, respectively) relative to the control (33.3%) and 0-22 h groups (38.6%) in MPCOCs. In SCNT, blastocyst formation tended to increase in MPCOCs treated with rapamycin during 0-42 h of IVM relative to untreated oocytes (20.3% vs. 14.3%, 0.05 < p < 0.1), while no improvement was observed in MGCOCs. Gene expression analysis revealed that transcript abundance of Beclin 1 and microtubule-associated protein 1 light chain 3 mRNAs was significantly increased in MPCOCs by rapamycin relative to the control. Our results demonstrated that autophagy induction by rapamycin during IVM improved developmental competence of oocytes derived from MPCOCs. PMID:25797293

Stimulating CTL Towards HER2/neu Overexpressing Breast Cancer

DTIC Science & Technology

2000-10-01

cytotoxicity possibly due to the peptides poor solubility and poor binding affinity. To gain further insight into the factors that govern CTL activity, we...662 peptide. Objective: Complete by 12/96. Methods: A soluble recombinant form of HLA-A2 is folded in vitro in the presence of j2m and HN654-662. The...decided to substitute the first position from isoleucine to lysine to improve solubility of the peptide library. The library was used in our in vitro

No amelioration of uromodulin maturation and trafficking defect by sodium 4-phenylbutyrate in vivo: studies in mouse models of uromodulin-associated kidney disease.

PubMed

Kemter, Elisabeth; Sklenak, Stefanie; Rathkolb, Birgit; Hrabě de Angelis, Martin; Wolf, Eckhard; Aigner, Bernhard; Wanke, Ruediger

2014-04-11

Uromodulin (UMOD)-associated kidney disease (UAKD) belongs to the hereditary progressive ER storage diseases caused by maturation defects of mutant UMOD protein. Current treatments of UAKD patients are symptomatic and cannot prevent disease progression. Two in vitro studies reported a positive effect of the chemical chaperone sodium 4-phenylbutyrate (4-PBA) on mutant UMOD maturation. Thus, 4-PBA was suggested as a potential treatment for UAKD. This study evaluated the effects of 4-PBA in two mouse models of UAKD. In contrast to previous in vitro studies, treatment with 4-PBA did not increase HSP70 expression or improve maturation and trafficking of mutant UMOD in vivo. Kidney function of UAKD mice was actually deteriorated by 4-PBA treatment. In transfected tubular epithelial cells, 4-PBA did not improve maturation but increased the expression level of both mutant and wild-type UMOD protein. Activation of NF-κB pathway in thick ascending limb of Henle's loop cells of UAKD mice was detected by increased abundance of RelB and phospho-IκB kinase α/β, an indirect activator of NF-κB. Furthermore, the abundance of NF-κB1 p105/p50, NF-κB2 p100/p52, and TRAF2 was increased in UAKD. NF-κB activation was identified as a novel disease mechanism of UAKD and might be a target for therapeutic intervention.

Effects of dendritic cells from hepatitis B virus transgenic mice-stimulated autologous lymphocytes on hepatitis B virus replication: a study on the impact of specific sensitized effector cells on in vitro virus replication.

PubMed

Shen, Zhong-Yang; Zheng, Wei-Ping; Liu, Tao; Yang, Yang; Song, Hong-Li

2015-03-01

The objective of this study was to explore the effects of dendritic cells (DCs) from hepatitis B virus (HBV) transgenic mice-stimulated autologous lymphocytes on in vitro HBV replication. DCs from HBV transgenic mice were induced to maturity by lipopolysaccharide, followed by incubation with hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) in vitro. Mature DCs and autologous lymphocytes were co-stimulated to form specific sensitized immune effector cells (IEC), which were then co-cultured with the human hepatoma cell line HepG2.2.15. Changes in morphology and activity of hepatocytes were then observed, as well as analysis of changes in liver enzyme, and HBV DNA and inflammatory cytokine levels in the culture supernatant. Intracellular HBV DNA and covalently closed circular DNA (cccDNA) concentration were measured by real-time polymerase chain reaction. Co-stimulation by mature DCs and IEC showed no impact on the morphology and liver enzyme expression level of HepG2.2.15 cells, but the supernatant HBV DNA and intracellular HBV DNA and cccDNA levels decreased significantly compared with those cells co-cultured with immature DCs. Secretion of inflammatory cytokines in the supernatant showed that when HBV DNA was highly expressed, the concentration of IFN-γ and IL-2 decreased, while IL-10 increased. Contrastingly, when HBV DNA had low expression, the concentration of IFN-γ and IL-2 increased and IL-10 decreased. Co-stimulation of HBV-related antigen-induced mature DCs and autologous lymphocytes showed inhibitory effects on ex vivo HBV replication, and cytokines were suggested to mediate this effect.

Somatic cell nuclear transfer in horses.

PubMed

Galli, Cesare; Lagutina, Irina; Duchi, Roberto; Colleoni, Silvia; Lazzari, Giovanna

2008-07-01

The cloning of equids was achieved in 2003, several years after the birth of Dolly the sheep and also after the cloning of numerous other laboratory and farm animal species. The delay was because of the limited development in the horse of more classical-assisted reproductive techniques required for successful cloning, such as oocyte maturation and in vitro embryo production. When these technologies were developed, the application of cloning also became possible and cloned horse offspring were obtained. This review summarizes the main technical procedures that are required for cloning equids and the present status of this technique. The first step is competent oocyte maturation, this is followed by oocyte enucleation and reconstruction, using either zona-enclosed or zona-free oocytes, by efficient activation to allow high cleavage rates and finally by a suitable in vitro embryo culture technique. Cloning of the first equid, a mule, was achieved using an in vivo-matured oocytes and immediate transfer of the reconstructed embryo, i.e. at the one cell stage, to the recipient oviduct. In contrast, the first horse offspring was obtained using a complete in vitro procedure from oocyte maturation to embryo culture to the blastocyst stage, followed by non-surgical transfer. Later studies on equine cloning report high efficiency relative to that for other species. Cloned equid offspring reported to date appear to be normal and those that have reached puberty have been confirmed to be fertile. In summary, horse cloning is now a reproducible technique that offers the opportunity to preserve valuable genetics and notably to generate copies of castrated champions and therefore, offspring from those champions that would be impossible to obtain otherwise.

Decreased pregnancy and live birth rates after vitrification of in vitro matured oocytes.

PubMed

Cohen, Yoni; St-Onge-St-Hilaire, Alexandra; Tannus, Samer; Younes, Grace; Dahan, Michael H; Buckett, William; Son, Weon-Young

2018-06-04

To assess effects on fertilization rate, embryo quality, pregnancy, and live birth rates of vitrification and warming of oocytes that matured in vitro (vIVM) compared to fresh in vitro maturation (fIVM) cycles. A retrospective cohort study conducted at a university hospital-affiliated IVF unit. Fifty-six cycles of vIVM cycles and 263 fIVM in women diagnosed with polycystic ovarian syndrome (PCOS) ovaries were included in the analysis. The study group included PCOS patients who failed ovulation induction with intrauterine insemination and were offered IVM cycle followed by oocyte vitrification and warming. The embryological aspects and clinical outcomes were compared to those of controls undergoing fresh IVM cycles during the same period. The main outcome measure was live birth rate. One thousand seventy oocytes were collected from 56 patients and underwent vitrification and warming. In the control group, 4781 oocytes were collected from 219 patients who had undergone a fresh IVM cycle. Oocyte maturation rates were similar between the groups (mean ± SD: 0.7 ± 0.2 vs. 0.6 ± 0.2, for vIVM and fIVM, respectively). Survival rate after warming was 59.8%. Fertilization and embryo cleavage rates per oocyte were significantly lower in the vIVM group. Clinical pregnancy (10.7 vs. 36.1%) and live birth rates (8.9 vs. 25.9%) per cycle were significantly lower in the vIVM group than those in the fIVM group (P = 0.005 and P < 0.001, respectively). Five healthy babies were born in the vIVM group. The reproductive potential of vitrified IVM oocytes is impaired. This injury likely occurs through vitrification and warming.

Brucella discriminates between mouse dendritic cell subsets upon in vitro infection.

PubMed

Papadopoulos, Alexia; Gagnaire, Aurélie; Degos, Clara; de Chastellier, Chantal; Gorvel, Jean-Pierre

2016-01-01

Brucella is a Gram-negative bacterium responsible for brucellosis, a worldwide re-emerging zoonosis. Brucella has been shown to infect and replicate within Granulocyte macrophage colony-stimulating factor (GMCSF) in vitro grown bone marrow-derived dendritic cells (BMDC). In this cell model, Brucella can efficiently control BMDC maturation. However, it has been shown that Brucella infection in vivo induces spleen dendritic cells (DC) migration and maturation. As DCs form a complex network composed by several subpopulations, differences observed may be due to different interactions between Brucella and DC subsets. Here, we compare Brucella interaction with several in vitro BMDC models. The present study shows that Brucella is capable of replicating in all the BMDC models tested with a high infection rate at early time points in GMCSF-IL15 DCs and Flt3l DCs. GMCSF-IL15 DCs and Flt3l DCs are more activated than the other studied DC models and consequently intracellular bacteria are not efficiently targeted to the ER replicative niche. Interestingly, GMCSF-DC and GMCSF-Flt3l DC response to infection is comparable. However, the key difference between these 2 models concerns IL10 secretion by GMCSF DCs observed at 48 h post-infection. IL10 secretion can explain the weak secretion of IL12p70 and TNFα in the GMCSF-DC model and the low level of maturation observed when compared to GMCSF-IL15 DCs and Flt3l DCs. These models provide good tools to understand how Brucella induce DC maturation in vivo and may lead to new therapeutic design using DCs as cellular vaccines capable of enhancing immune response against pathogens.

N-octanoylated ghrelin peptide inhibits bovine oocyte meiotic resumption.

PubMed

Xu, X L; Bai, J H; Feng, T; Xiao, L L; Song, Y Q; Xiao, Y X; Liu, Y

2018-07-01

Studies have shown that ghrelin plays an important role in the mammalian reproductive system, including the central, gonadal levels, and also during in vitro maturation of oocytes; however, the functions of ghrelin in bovine oocyte meiosis require further investigation. We aimed to evaluate the effects of an n-octanoylated ghrelin peptide on oocyte meiotic resumption and the developmental competence of mature oocytes in vitro. design: The expression of GHRL (encoding ghrelin) mRNA and its receptor (the growth hormone secretagogue receptor, GHSR) in the cumulus-oocyte complex (COCs), denuded oocytes (DOs), and cumulus cells (CCs) was assessed using quantitative real-time reverse transcription PCR (qRT-PCR), and the effects of the n-octanoylated ghrelin peptide on meiotic resumption were studied at four different doses (0, 10, 50, and 100 ng/mL) in a 6 h culture system. qRT-PCR analysis showed that GHRL and GHSR mRNAs were expressed in all tested samples; however, GHRL was predominantly expressed in DOs, and GHSR was predominantly expressed in CCs. Germinal vesicle breakdown was inhibited significantly by 50 ng/mL ghrelin compared with that in the negative control (P < 0.05). Further studies showed that n-octanoylated ghrelin increased the levels of cAMP and cGMP in the CCs and DOs, which inhibited the meiotic resumption of bovine oocytes. And the inhibitory role in the developmental competence of mature oocytes were also included, ghrelin could significantly improve the cleavage rate (P < 0.05) and blastocyst rate (P < 0.05). N-octanoylated ghrelin maintained bovine oocytes meiotic arrest and further improved their developmental competence; therefore, n-octanoylated ghrelin could be considered as a potential pharmaceutical inhibitor of meiosis for the in vitro maturation of bovine oocytes. Copyright © 2018 Elsevier Inc. All rights reserved.

Peritoneal fluid of women with endometriosis reduces SOD1 in bovine oocytes in vitro maturation.

PubMed

Malvezzi, Helena; Da Broi, Michele Gomes; Meola, Juliana; Rosa-E-Silva, Júlio César; Ferriani, Rui Alberto; Navarro, Paula Andrea

2018-06-01

Studies have demonstrated oxidative stress in peritoneal fluid (PF) from women with endometriosis and the importance of enzymatic antioxidant machinery to avoid oocyte oxidative damage. Considering that PF constantly surrounds the ovaries and has direct contact with the oocyte at ovulation, we wonder if PF from women with endometriosis may affect antioxidant enzyme gene expression. Thus, the present study aims to evaluate the PF impact from infertile women with minimal and mild endometriosis and from fertile control women without endometriosis on SOD1, CAT, GSR gene's expression in experimental bovine oocytes matured in vitro. Samples of PF were obtained from women who underwent videolaparoscopy-7 infertile with EI/II and 7 fertile without endometriosis. Immature bovine oocytes underwent in vitro maturation in the absence of PF and in the presence of three concentrations (1, 5 and 10%) of PF from fertile and from infertile women with EI/II. After 22 to 24 h of IVM, oocytes were denuded and stored for analysis of SOD1, CAT and GSR by real-time polymerase chain reaction. Oocyte SOD1 expression was significantly lower in the 10% endometriosis group (0.67 ± 0.32) when compared with no-peritoneal fluid (1.05 ± 0.24, p < 0.008) and 10% control groups (1.06 ± 0.22, p < 0.006). These findings raise the possibility of a deleterious influence of PF from women with EI/II on the oocyte, not only after ovulation but also during the maturation process, which could contribute to worsening oocyte quality, being one of the mechanisms related to infertility in patients with endometriosis.

Fibroblast growth factor 10 enhances bovine oocyte maturation and developmental competence in vitro.

PubMed

Zhang, Kun; Hansen, Peter J; Ealy, Alan D

2010-12-01

The ability of oocytes to resume meiosis, become fertilized, and generate viable pregnancies is controlled during folliculogenesis by several endocrine and paracrine factors. The aim of this work is to determine whether fibroblast growth factor 10 (FGF10) is an oocyte competent factor. Transcripts for each of the four FGF receptor types (FGFR) were present in cumulus and oocytes after their extraction from the follicles. FGFR1 transcripts predominated in cumulus cells whereas FGFR2 was most abundant in oocytes. Exposing the cumulus-oocyte complexes to FGF10 during in vitro maturation did not affect cleavage rates, but increases (P<0.05) in the percentage of embryos at the 8-16-cell stage on day 3 and at the blastocyst stage on day 7, which were evident in FGF10-supplemented oocytes. The progression of oocytes through meiosis and cumulus expansion was increased (P<0.05) by FGF10. The importance of the endogenous sources of FGFs was examined by adding anti-FGF10 IgG during oocyte maturation. Blocking endogenous FGF10 activity decreased (P<0.05) the percentage of oocytes developing into blastocysts and limited (P<0.05) cumulus expansion. Expression profiles of putative cumulus and oocyte competency markers were examined for their involvement in FGF10-mediated responses. FGF10 influenced the expression of CTSB and SPRY2 in cumulus cells and BMP15 in oocytes. In summary, this work provides new insight into the importance of FGFRs and locally derived FGF10 during oocyte maturation in cattle. Its subsequent impact on in vitro embryo development implicates it as a noteworthy oocyte competent factor.

Automated On-tip Affinity Capture Coupled with Mass Spectrometry to Characterize Intact Antibody-Drug Conjugates from Blood

NASA Astrophysics Data System (ADS)

Li, Ke Sherry; Chu, Phillip Y.; Fourie-O'Donohue, Aimee; Srikumar, Neha; Kozak, Katherine R.; Liu, Yichin; Tran, John C.

2018-05-01

Antibody-drug conjugates (ADCs) present unique challenges for ligand-binding assays primarily due to the dynamic changes of the drug-to-antibody ratio (DAR) distribution in vivo and in vitro. Here, an automated on-tip affinity capture platform with subsequent mass spectrometry analysis was developed to accurately characterize the DAR distribution of ADCs from biological matrices. A variety of elution buffers were tested to offer optimal recovery, with trastuzumab serving as a surrogate to the ADCs. High assay repeatability (CV 3%) was achieved for trastuzumab antibody when captured below the maximal binding capacity of 7.5 μg. Efficient on-tip deglycosylation was also demonstrated in 1 h followed by affinity capture. Moreover, this tip-based platform affords higher throughput for DAR characterization when compared with a well-characterized bead-based method.

DNA aptamers for the detection of Haemophilus influenzae type b by cell SELEX.

PubMed

Bitaraf, F S; Rasooli, I; Mousavi Gargari, S L

2016-03-01

Haemophilus influenzae type b (Hib) causes acute bacterial meningitis (ABM) in children, with a mortality rate of about 3-6 % of the affected patients. ABM can lead to death during a period of hours to several days and, hence, rapid and early detection of the infection is crucial. Aptamers, the short single-stranded DNA or RNA with high affinity to target molecules, are selected by a high-flux screening technique known as in vitro screening and systematic evolution of ligands by exponential enrichment technology (SELEX). In this study, whole-cell SELEX was applied for the selection of target-specific aptamers with high affinity to Hib. ssDNA aptamers prepared by lambda exonuclease were incubated with the target cells (Hib). The aptameric binding rate to Hib was characterized for binding affinity after seven SELEX rounds by flow cytometry. The aptamers with higher binding affinity were cloned. Four of 68 aptamer clones were selected for sequencing. The dissociation constant (Kd) of the high-affinity aptamer clones 45 and 63 were 47.10 and 28.46 pM, respectively. These aptamers did not bind to other bacterial species, including the seven meningitis-causing bacteria. They showed distinct affinity to various H. influenzae strains only. These aptamers showed the highest affinity to Hib and the lowest affinity to H. influenzae type c and to other meningitis-causing bacteria. Clone 63 could detect Hib in patients' cerebrospinal fluid (CSF) samples at 60 colony-forming units (CFU)/mL. The results indicate applicability of the aptamers for rapid and early detection of infections brought about by Hib.

Evaluation of new antimicrobial agents on Bacillus spp. strains: docking affinity and in vitro inhibition of glutamate-racemase.

PubMed

Tamay-Cach, Feliciano; Correa-Basurto, José; Villa-Tanaca, Lourdes; Mancilla-Percino, Teresa; Juárez-Montiel, Margarita; Trujillo-Ferrara, José G

2013-10-01

Three glutamic acid derivatives, two boron-containing and one imide-containing compound, were synthesized and tested for antimicrobial activity targeting glutamate-racemase. Antimicrobial effect was evaluated over Bacillus spp. Docking analysis shown that the test compounds bind near the active site of racemase isoforms, suggesting an allosteric effect. The boron derivatives had greater affinity than the imide derivative. In vitro assays shown good antimicrobial activity for the boron-containing compounds, and no effectiveness for the imide-containing compounds. The minimum inhibitory concentration of tetracycline, used as standard, was lower than that of the boron-containing derivatives. However, it seems that the boron-containing derivatives are more selective for bacteria. Experimental evidence suggests that the boron-containing derivatives act by inhibiting the racemase enzyme. Therefore, these test compounds probably impede the formation of the bacterial cell wall. Thus, the boron-containing glutamic acid derivatives should certainly be of interest for future studies as antimicrobial agents for Bacillus spp.

Active site-directed double mutants of dihydrofolate reductase.

PubMed

Ercikan-Abali, E A; Mineishi, S; Tong, Y; Nakahara, S; Waltham, M C; Banerjee, D; Chen, W; Sadelain, M; Bertino, J R

1996-09-15

Variants of dihydrofolate reductase (DHFR), which confer resistance to antifolates, are used as dominant selectable markers in vitro and in vivo and may be useful in the context of gene therapy. To identify improved mutant human DHFRs with increased catalytic efficiency and decreased binding to methotrexate, we constructed by site-directed mutagenesis four variants with substitutions at both Leu22 and Phe31 (i.e., Phe22-Ser31, Tyr22-Ser31, Phe22-Gly31, and Tyr22-Gly31). Antifolate resistance has been observed previously when individual changes are made at these active-site residues. Substrate and antifolate binding properties of these "double" mutants revealed that each have greatly diminished affinity for antifolates (> 10,000-fold) yet only slightly reduced substrate affinity. Comparison of in vitro measured properties with those of single-residue variants indicates that double mutants are indeed significantly superior. This was verified for one of the double mutants that provided high-level methotrexate resistance following retrovirus-mediated gene transfer in NIH3T3 cells.

Creation of Novel Cores for β-Secretase (BACE-1) Inhibitors: A Multiparameter Lead Generation Strategy

PubMed Central

2014-01-01

In order to find optimal core structures as starting points for lead optimization, a multiparameter lead generation workflow was designed with the goal of finding BACE-1 inhibitors as a treatment for Alzheimer’s disease. De novo design of core fragments was connected with three predictive in silico models addressing target affinity, permeability, and hERG activity, in order to guide synthesis. Taking advantage of an additive SAR, the prioritized cores were decorated with a few, well-characterized substituents from known BACE-1 inhibitors in order to allow for core-to-core comparisons. Prediction methods and analyses of how physicochemical properties of the core structures correlate to in vitro data are described. The syntheses and in vitro data of the test compounds are reported in a separate paper by Ginman et al. [J. Med. Chem.2013, 56, 4181–4205]. The affinity predictions are described in detail by Roos et al. [J. Chem. Inf.2014, DOI: 10.1021/ci400374z]. PMID:24900855

Creation of Novel Cores for β-Secretase (BACE-1) Inhibitors: A Multiparameter Lead Generation Strategy.

PubMed

Viklund, Jenny; Kolmodin, Karin; Nordvall, Gunnar; Swahn, Britt-Marie; Svensson, Mats; Gravenfors, Ylva; Rahm, Fredrik

2014-04-10

In order to find optimal core structures as starting points for lead optimization, a multiparameter lead generation workflow was designed with the goal of finding BACE-1 inhibitors as a treatment for Alzheimer's disease. De novo design of core fragments was connected with three predictive in silico models addressing target affinity, permeability, and hERG activity, in order to guide synthesis. Taking advantage of an additive SAR, the prioritized cores were decorated with a few, well-characterized substituents from known BACE-1 inhibitors in order to allow for core-to-core comparisons. Prediction methods and analyses of how physicochemical properties of the core structures correlate to in vitro data are described. The syntheses and in vitro data of the test compounds are reported in a separate paper by Ginman et al. [J. Med. Chem. 2013, 56, 4181-4205]. The affinity predictions are described in detail by Roos et al. [J. Chem. Inf. 2014, DOI: 10.1021/ci400374z].

Low-Dose Urinary Human Chorionic Gonadotropin Is Effective for Oocyte Maturation in In Vitro Fertilization/ Intracytoplasmic Sperm Injection Cycles Independent of Body Mass Index

PubMed Central

R. Hoyos, Luis; Khan, Sana; Dai, Jing; Singh, Manvinder; P. Diamond, Michael; E. Puscheck, Elizabeth; O. Awonuga, Awoniyi

2017-01-01

Background: Currently, there is no agreement on the optimal urinary derived human chorionic gonadotropin (u-hCG) dose requirement for initiating final oocyte maturation prior to oocyte collection in in vitro fertilization (IVF), but doses that range from 2500- 15000 IU have been used. We intended to determine whether low dose u-hCG was effective for oocyte maturation in IVF/intracytoplasmic sperm injection (ICSI) cycles independent of body mass index (BMI). Materials and Methods: We retrospectively evaluated a cohort of 295 women who underwent their first IVF/ICSI cycles between January 2003 and December 2010 at the Division of Reproductive Endocrinology and Infertility, Wayne State University, Detroit, MI, USA. Treatment cycles were divided into 3 groups based on BMI (kg/ m2): <25 (n=136), 25- <30 (n=84), and ≥30 (n=75) women. Patients received 5000, 10000 or 15000 IU u-hCG for final maturation prior to oocyte collection. The primary outcome was clinical pregnancy rates (CPRs) and secondary outcome was live birth rates (LBRs). Results: Only maternal age negatively impacted (P<0.001) CPR [odds ratio (OR=0.85, confidence interval (CI: 0.79-0.91)] and LBR (OR=0.84, CI: 0.78-0.90). Conclusion: Administration of lower dose u-hCG was effective for oocyte maturation in IVF and did not affect the CPRs and LBRs irrespective of BMI. Women’s BMI need not be taken into consideration in choosing the appropriate dose of u-hCG for final oocyte maturation prior to oocyte collection in IVF. Only maternal age at the time of IVF negatively influenced CPRs and LBRs in this study. PMID:28367299

Interfering of the Reelin/ApoER2/PSD95 Signaling Axis Reactivates Dendritogenesis of Mature Hippocampal Neurons.

PubMed

Ampuero, Estibaliz; Jury, Nur; Härtel, Steffen; Marzolo, María-Paz; van Zundert, Brigitte

2017-05-01

Reelin, an extracellular glycoprotein secreted in embryonic and adult brain, participates in neuronal migration and neuronal plasticity. Extensive evidence shows that reelin via activation of the ApoER2 and VLDLR receptors promotes dendrite and spine formation during early development. Further evidence suggests that reelin signaling is needed to maintain a stable architecture in mature neurons, but, direct evidence is lacking. During activity-dependent maturation of the neuronal circuitry, the synaptic protein PSD95 is inserted into the postsynaptic membrane to induce structural refinement and stability of spines and dendrites. Given that ApoER2 interacts with PSD95, we tested if reelin signaling interference in adult neurons reactivates the dendritic architecture. Unlike findings in developing cultures, the presently obtained in vitro and in vivo data show, for the first time, that reelin signaling interference robustly increase dendritogenesis and reduce spine density in mature hippocampal neurons. In particular, the expression of a mutant ApoER2 form (ApoER2-tailless), which is unable to interact with PSD95 and hence cannot transduce reelin signaling, resulted in robust dendritogenesis in mature hippocampal neurons in vitro. These results indicate that reelin/ApoER2/PSD95 signaling is important for neuronal structure maintenance in mature neurons. Mechanistically, obtained immunofluorescent data indicate that reelin signaling impairment reduced synaptic PSD95 levels, consequently leading to synaptic re-insertion of NR2B-NMDARs. Our findings underscore the importance of reelin in maintaining adult network stability and reveal a new mode for reactivating dendritogenesis in neurological disorders where dendritic arbor complexity is limited, such as in depression, Alzheimer's disease, and stroke. J. Cell. Physiol. 232: 1187-1199, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Direct influence of titanium and zirconia particles on the morphology and functionality of mature human osteoclasts.

PubMed

Pasold, Juliane; Markhoff, Jana; Tillmann, Jenny; Krogull, Martin; Pisowocki, Phillip; Bader, Rainer

2017-09-01

Within the last ten years of biomedical implants, the focus is increasingly on bioceramics, specifically on zirconia (ZrO 2 ). Hence, we analyzed the impact of ZrO 2 particles in comparison to titanium particles on mature human osteoclasts (OCs) as little is known about the direct effect of wear particles on mature OCs and their role in the osteolytic process during aseptic endoprosthesis loosening. Changes in cell morphology and functionality of OCs incubated with particles in different concentrations were investigated in vitro. OCs tend to be enlarged after three days of cultivation with both types of particles, especially with high concentrations of ZrO 2 , suggesting increased cell fusion. Further, we identified significantly increased expression of OC specific and bone matrix related genes: VNR, RANK, TRAP, and CTSK pointing on a direct stimulatory particle effect on the functionality of mature OCs. In completion, we quantified the bone resorption activity of particle treated mature OCs but could not detect a significant difference in bone resorption compared to OCs cultivated without particles. However, we could identify significantly higher gene expression of MMP-1 in particle treated OCs compared to untreated control OCs after three days of incubation. We also detected an impaired production of the tissue inhibitor of metalloproteinase, especially for OCs treated with high ZrO 2 concentrations. In conclusion, our in vitro data show that abrasion particles could have a direct influence on mature OCs and therefore could promote increased OC-mediated bone resorption during aseptic loosening of total joint replacements. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2608-2615, 2017. © 2017 Wiley Periodicals, Inc.

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.

Live Birth after Rescue In vitro Maturation-intracytoplasmic Sperm Injection in Type 1 Diabetes, Polycystic Ovary Syndrome Patient Using Clomiphene-antagonist Protocol.

PubMed

Sankari, Samundi; Elanchezhian, M; Selvamani, Divya; Nagarajan, M; Gopikrishnan, D

2018-01-01

Infertility in patients with polycystic ovary syndrome (PCOS) associated with diabetes leads to challenging situations seeking alternative treatments. In vitro maturation (IVM) followed by intracytoplasmic sperm injection (ICSI) could overcome the challenges with promising pregnancies in such patients. In the treatment of a 32-year-old diabetic woman who also had PCOS, single immature oocyte was retrieved. Rescue IVM followed by ICSI yielded a grade 1 day 3 embryo which on transferring resulted in pregnancy and a healthy infant was delivered. Rescue IVM-ICSI could help in achieving pregnancy and live birth. Stimulation involving clomiphene and gonadotropin-releasing hormone antagonist is an effective and patient-friendly protocol for women with PCOS and diabetes and also for poor responders.

Effects of cilostamide and/or forskolin on the meiotic resumption and development competence of growing ovine oocytes selected by brilliant cresyl blue staining.

PubMed

Azari-Dolatabad, Nima; Rahmani, H R; Hajian, M; Ostadhosseini, S; Hosseini, S M; Nasr-Esfahani, M H

2016-05-01

The relevance of low developmental competence of in vitro-matured oocyte to the incomplete/delayed cytoplasmic maturation, and the heterogeneity of retrieved oocytes is well established in several species. A short phase of prematuration culture was used to allow better oocyte cytoplasmic maturation. The preselection of growing and fully grown oocytes has been proposed to improve developmental competency. This study investigated the effects of phosphodiesterase type 3-specific inhibitor, cilostamide, and adenylate cyclase activator, forskolin, on the resumption of meiosis and developmental competence of growing ovine oocytes selected by brilliant cresyl blue (BCB) staining. Results indicate that cilostamide, forskolin, and their combination significantly (P < 0.05) increased the percentage of growing (BCB-) oocytes maintained at the germinal vesicle stage. However, only forskolin significantly (P < 0.05) increased the yield and quality of blastocysts derived from BCB- oocytes compared with non-BCB-treated oocytes. We conclude that a short prematuration culture with forskolin may improve the in vitro developmental competency of growing oocytes in ovine. Copyright © 2016 Elsevier Inc. All rights reserved.

Characterization of New Cationic N,N-Dimethyl[70]fulleropyrrolidinium Iodide Derivatives as Potent HIV-1 Maturation Inhibitors.

PubMed

Castro, Edison; Martinez, Zachary S; Seong, Chang-Soo; Cabrera-Espinoza, Andrea; Ruiz, Mauro; Hernandez Garcia, Andrea; Valdez, Federico; Llano, Manuel; Echegoyen, Luis

2016-12-22

HIV-1 maturation can be impaired by altering protease (PR) activity, the structure of the Gag-Pol substrate, or the molecular interactions of viral structural proteins. Here we report the synthesis and characterization of new cationic N,N-dimethyl[70]fulleropyrrolidinium iodide derivatives that inhibit more than 99% of HIV-1 infectivity at low micromolar concentrations. Analysis of the HIV-1 life cycle indicated that these compounds inhibit viral maturation by impairing Gag and Gag-Pol processing. Importantly, fullerene derivatives 2a-c did not inhibit in vitro PR activity and strongly interacted with HIV immature capsid protein in pull-down experiments. Furthermore, these compounds potently blocked infectivity of viruses harboring mutant PR that are resistant to multiple PR inhibitors or mutant Gag proteins that confer resistance to the maturation inhibitor Bevirimat. Collectively, our studies indicate fullerene derivatives 2a-c as potent and novel HIV-1 maturation inhibitors.

Effect of human milk and colostrum on Entamoeba histolytica.

PubMed

Akisu, Ciler; Aksoy, Umit; Cetin, Hasan; Ustun, Sebnem; Akisu, Mete

2004-03-01

Many defense factors of the mother's colostrum or milk protect infants from intestinal, respiratory and systemic infections. In the present study, we investigated the effect of colostrum and mature human milk on E. histolytica parasites in vitro. Samples of human milk were collected from 5 healthy lactating mothers. The medium with human milk at concentrations of 2%, 5% and 10% was obtained. The lethal effect of E. histolytica on the medium supplemented with different concentrations of both colostrum and mature human milk was significant during the first 30 min. We also detected that the results of colostrum and mature human milk were similar. No statistically significant differences were found between same concentrations of colostrum and mature human milk at the same times. Colostrum and mature human milk have significant lethal effect on E. histolytica and protect against its infection in breast fed children.

Mature brain-derived neurotrophic factor and its receptor TrkB are upregulated in human glioma tissues.

PubMed

Xiong, Jing; Zhou, L I; Lim, Yoon; Yang, Miao; Zhu, Yu-Hong; Li, Zhi-Wei; Fu, Deng-Li; Zhou, Xin-Fu

2015-07-01

There are two forms of brain-derived neurotrophic factor (BDNF), precursor of BDNF (proBDNF) and mature BDNF, which each exert opposing effects through two different transmembrane receptor signaling systems, consisting of p75 neurotrophin receptor (p75NTR) and tyrosine receptor kinase B (TrkB). Previous studies have demonstrated that proBDNF promotes cell death and inhibits the growth and migration of C6 glioma cells through p75NTR in vitro , while mature BDNF has opposite effects on C6 glioma cells. It is hypothesized that mature BDNF is essential in the development of malignancy in gliomas. However, histological data obtained in previous studies were unable distinguish mature BDNF from proBDNF due to the lack of specific antibodies. The present study investigated the expression of mature BDNF using a specific sheep monoclonal anti-mature BDNF antibody in 42 human glioma tissues of different grades and 10 control tissues. The correlation between mature BDNF and TrkB was analyzed. Mature BDNF expression was significantly increased in high-grade gliomas, and was positively correlated with the malignancy of the tumor and TrkB receptor expression. The present data have demonstrated that increased levels of mature BDNF contribute markedly to the development of malignancy of human gliomas through the primary BDNF receptor TrkB.

Biochemical and pharmacological properties of SR 49059, a new, potent, nonpeptide antagonist of rat and human vasopressin V1a receptors.

PubMed

Serradeil-Le Gal, C; Wagnon, J; Garcia, C; Lacour, C; Guiraudou, P; Christophe, B; Villanova, G; Nisato, D; Maffrand, J P; Le Fur, G

1993-07-01

SR 49059, a new potent and selective orally active, nonpeptide vasopressin (AVP) antagonist has been characterized in several in vitro and in vivo models. SR 49059 showed high affinity for V1a receptors from rat liver (Ki = 1.6 +/- 0.2) and human platelets, adrenals, and myometrium (Ki ranging from 1.1 to 6.3 nM). The previously described nonpeptide V1 antagonist, OPC-21268, was almost inactive in human tissues at concentrations up to 100 microM. SR 49059 exhibited much lower affinity (two orders of magnitude or more) for AVP V2 (bovine and human), V1b (human), and oxytocin (rat and human) receptors and had no measurable affinity for a great number of other receptors. In vitro, AVP-induced contraction of rat caudal artery was competitively antagonized by SR 49059 (pA2 = 9.42). Furthermore, SR 49059 inhibited AVP-induced human platelet aggregation with an IC50 value of 3.7 +/- 0.4 nM, while OPC-21268 was inactive up to 20 microM. In vivo, SR 49059 inhibited the pressor response to exogenous AVP in pithed rats (intravenous) and in conscious normotensive rats (intravenous and per os) with a long duration of action (> 8 h at 10 mg/kg p.o). In all the biological assays used, SR 49059 was devoid of any intrinsic agonistic activity. Thus, SR 49059 is the most potent and selective nonpeptide AVP V1a antagonist described so far, with marked affinity, selectivity, and efficacy toward both animal and human receptors. With this original profile, SR 49059 constitutes a powerful tool for exploring the therapeutical usefulness of a selective V1a antagonist.

Binding affinity and decontamination of dermal decontamination gel to model chemical warfare agent simulants.

PubMed

Cao, Yachao; Elmahdy, Akram; Zhu, Hanjiang; Hui, Xiaoying; Maibach, Howard

2018-05-01

Six chemical warfare agent simulants (trimethyl phosphate, dimethyl adipate, 2-chloroethyl methyl sulfide, diethyl adipate, chloroethyl phenyl sulfide and diethyl sebacate) were studied in in vitro human skin to explore relationship between dermal penetration/absorption and the mechanisms of simulant partitioning between stratum corneum (SC) and water as well as between dermal decontamination gel (DDGel) and water. Both binding affinity to and decontamination of simulants using DDGel were studied. Partition coefficients of six simulants between SC and water (Log P SC/w ) and between DDGel and water (Log P DDGel/w ) were determined. Results showed that DDGel has a similar or higher binding affinity to each simulant compared to SC. The relationship between Log P octanol/water and Log P SC/w as well as between Log P octanol/water and Log P DDGel/w demonstrated that partition coefficient of simulants correlated to their lipophilicity or hydrophilicity. Decontamination efficiency results with DDGel for these simulants were consistent with binding affinity results. Amounts of percentage dose of chemicals in DDGel of trimethyl phosphate, dimethyl adipate, 2-chloroethyl methyl sulfide, diethyl adipate, chloroethyl phenyl sulfide and diethyl sebacate were determined to be 61.15, 85.67, 75.91, 53.53, 89.89 and 76.58, with corresponding amounts absorbed in skin of 0.96, 0.65, 1.68, 0.72, 0.57 and 1.38, respectively. In vitro skin decontamination experiments coupled with a dermal absorption study demonstrated that DDGel can efficiently remove chemicals from skin surface, back-extract from the SC, and significantly reduced chemical penetration into skin or systemic absorption for all six simulants tested. Therefore, DDGel offers a great potential as a NextGen skin Decon platform technology for both military and civilian use. Copyright © 2018 John Wiley & Sons, Ltd.

New approaches for the reliable in vitro assessment of binding affinity based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics.

PubMed

Zeilinger, Markus; Pichler, Florian; Nics, Lukas; Wadsak, Wolfgang; Spreitzer, Helmut; Hacker, Marcus; Mitterhauser, Markus

2017-12-01

Resolving the kinetic mechanisms of biomolecular interactions have become increasingly important in early-phase drug development. Since traditional in vitro methods belong to dose-dependent assessments, binding kinetics is usually overlooked. The present study aimed at the establishment of two novel experimental approaches for the assessment of binding affinity of both, radiolabelled and non-labelled compounds targeting the A 3 R, based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics. A novel time-resolved competition assay was developed and applied to determine the K i of eight different A 3 R antagonists, using CHO-K1 cells stably expressing the hA 3 R. In addition, a new kinetic real-time cell-binding approach was established to quantify the rate constants k on and k off , as well as the dedicated K d of the A 3 R agonist [ 125 I]-AB-MECA. Furthermore, lipophilicity measurements were conducted to control influences due to physicochemical properties of the used compounds. Two novel real-time cell-binding approaches were successfully developed and established. Both experimental procedures were found to visualize the kinetic binding characteristics with high spatial and temporal resolution, resulting in reliable affinity values, which are in good agreement with values previously reported with traditional methods. Taking into account the lipophilicity of the A 3 R antagonists, no influences on the experimental performance and the resulting affinity were investigated. Both kinetic binding approaches comprise tracer administration and subsequent binding to living cells, expressing the dedicated target protein. Therefore, the experiments resemble better the true in vivo physiological conditions and provide important markers of cellular feedback and biological response.

Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies

PubMed Central

Schlothauer, Tilman; Rueger, Petra; Stracke, Jan Olaf; Hertenberger, Hubert; Fingas, Felix; Kling, Lothar; Emrich, Thomas; Drabner, Georg; Seeber, Stefan; Auer, Johannes; Koch, Stefan; Papadimitriou, Apollon

2013-01-01

The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity. PMID:23765230

Transcriptional regulators TRIM28, SETDB1, and TP53 are aberrantly expressed in porcine embryos produced by in vitro fertilization in comparison to in vivo- and somatic-cell nuclear transfer-derived embryos

PubMed Central

Hamm, Jennifer; Tessanne, Kim; Murphy, Clifton N; Prather, Randall S

2014-01-01

In vitro embryo production is important for research in animal reproduction, embryo transfer, transgenics, and cloning. Yet, in vitro-fertilized (IVF) embryos are generally developmentally delayed and are inferior to in vivo-derived (IVV) embryos; this discrepancy is likely a result of aberrant gene expression. Transcription of three genes implicated to be important in normal preimplantation embryo development, TRIM28, SETDB1, and TP53, was determined by quanitative PCR in IVF, somatic-cell nuclear transfer (SCNT), parthenogenetic, and IVV porcine oocytes and embryos. There was no difference in TRIM28 or SETDB1 abundance between oocytes matured in vitro versus in vivo (P > 0.05), whereas TP53 levels were higher in in vitro-matured oocytes. TRIM28 increased from metaphase-II oocytes to the 4-cell and blastocyst stages in IVF embryos, whereas IVV embryos showed a reduction in TRIM28 abundance from maturation throughout development. The relative abundance of TP53 increased by the blastocyst stage in all treatment groups, but was higher in IVF embryos compared to IVV and SCNT embryos. In contrast, SETDB1 transcript levels decreased from the 2-cell to blastocyst stage in all treatments. For each gene analyzed, SCNT embryos of both hard-to-clone and easy-to-clone cell lines were more comparable to IVV than IVF embryos. Knockdown of TRIM28 also had no effect on blastocyst development or expression of SETDB1 or TP53. Thus, TRIM28, SETDB1, and TP53 are dynamically expressed in porcine oocytes and embryos. Furthermore, TRIM28 and TP53 abundances in IVV and SCNT embryos are similar, but different from quantities in IVF embryos. Mol. Reprod. Dev. 81: 552–556, 2014. © 2014 The Authors. Published by Wiley Periodicals, Inc. PMID:24659575

Life-span of in vitro differentiated Plasmodium falciparum gametocytes.

PubMed

Gebru, Tamirat; Lalremruata, Albert; Kremsner, Peter G; Mordmüller, Benjamin; Held, Jana

2017-08-11

The sexual stages (gametocytes) of Plasmodium falciparum do not directly contribute to the pathology of malaria but are essential for transmission of the parasite from the human host to the mosquito. Mature gametocytes circulate in infected human blood for several days and their circulation time has been modelled mathematically from data of previous in vivo studies. This is the first time that longevity of gametocytes is studied experimentally in vitro. The in vitro longevity of P. falciparum gametocytes of 1 clinical isolate and 2 laboratory strains was assessed by three different methods: microscopy, flow cytometry and reverse transcription quantitative real-time PCR (RT-qPCR). Additionally, the rate of gametocytogenesis of the used P. falciparum strains was compared. The maximum in vitro lifespan of P. falciparum gametocytes reached almost 2 months (49 days by flow cytometry, 46 days by microscopy, and at least 52 days by RT-qPCR) from the starting day of gametocyte culture to death of last parasite in the tested strains with an average 50% survival rate of 6.5, 2.6 and 3.5 days, respectively. Peak gametocytaemia was observed on average 19 days after initiation of gametocyte culture followed by a steady decline due to natural decay of the parasites. The rate of gametocytogenesis was highest in the NF54 strain. Plasmodium falciparum mature gametocytes can survive up to 16-32 days (at least 14 days for mature male gametocytes) in vitro in absence of the influence of host factors. This confirms experimentally a previous modelling estimate that used molecular tools for gametocyte detection in treated patients. The survival time might reflect the time the parasite can be transmitted to the mosquito after clearance of asexual parasites. These results underline the importance of efficient transmission blocking agents in the fight against malaria.

The roles of two O-donor ligands in the Fe2+-binding and H2O2-sensing by the Fe2+-dependent H2O2 sensor PerR.

PubMed

Ji, Chang-Jun; Yang, Yoon-Mo; Kim, Jung-Hoon; Ryu, Su-Hyun; Youn, Hwan; Lee, Jin-Won

2018-05-10

PerR is a metal-dependent peroxide sensing transcription factor which controls the expression of genes involved in peroxide resistance. The function of Bacillus subtilis PerR is mainly dictated by the regulatory metal ion (Fe 2+ or Mn 2+ ) coordinated by three N-donor ligands (His37, His91, and His93) and two O-donor ligands (Asp85 and Asp104). While H 2 O 2 sensing by PerR is mediated by Fe 2+ -dependent oxidation of N-donor ligand (either His37 or His91), one of the O-donor ligands (Asp104), but not Asp85, has been proposed as the key residue that regulates the sensitivity of PerR to H 2 O 2 . Here we systematically investigated the relative roles of two O-donor ligands of PerR in metal-binding affinity and H 2 O 2 sensitivity in vivo and in vitro. Consistent with the previous report, in vitro the D104E-PerR could not sense low levels of H 2 O 2 in the presence of excess Fe 2+ sufficient for the formation of the Fe 2+ -bound D104E-PerR. However, the expression of PerR-regulated reporter fusion was not repressed by D104E-PerR in the presence of Fe 2+ , suggesting that Fe 2+ is not an effective corepressor for this mutant protein in vivo. Furthermore, in vitro metal titration assays indicate that D104E-PerR has a significantly reduced affinity for Fe 2+ , but not for Mn 2+ , when compared to wild type PerR. These data indicate that the type of O-donor ligand (Asp vs. Glu) at position 104 is an important determinant in providing high Fe 2+ -binding affinity required for the sensing of the physiologically relevant Fe 2+ -levels, in addition to its role in rendering PerR highly sensitive to physiological levels of H 2 O 2 . In comparison, the D85E-PerR did not show a perturbed change in Fe 2+ -binding affinity, however, it displayed a slightly decreased sensitivity to H 2 O 2 both in vivo and in vitro, suggesting that the type of O-donor ligand (Asp vs. Glu) at position 85 may be important for the fine-tuning of H 2 O 2 sensitivity. Copyright © 2018 Elsevier Inc. All rights reserved.

Differential affinities of MinD and MinE to anionic phospholipid influence Min Patterning dynamics in vitro

PubMed Central

Vecchiarelli, Anthony G.; Li, Min; Mizuuchi, Michiyo; Mizuuchi, Kiyoshi

2014-01-01

The E. coli Min system forms a cell-pole-to-cell-pole oscillator that positions the divisome at mid-cell. The MinD ATPase binds the membrane and recruits the cell division inhibitor MinC. MinE interacts with and releases MinD (and MinC) from the membrane. The chase of MinD by MinE creates the in vivo oscillator that maintains a low level of the division inhibitor at mid-cell. In vitro reconstitution and visualization of Min proteins on a supported lipid bilayer has provided significant advances in understanding Min patterns in vivo. Here we studied the effects of flow, lipid composition, and salt concentration on Min patterning. Flow and no-flow conditions both supported Min protein patterns with somewhat different characteristics. Without flow, MinD and MinE formed spiraling waves. MinD and, to a greater extent MinE, have stronger affinities for anionic phospholipid. MinD-independent binding of MinE to anionic lipid resulted in slower and narrower waves. MinE binding to the bilayer was also more susceptible to changes in ionic strength than MinD. We find that modulating protein diffusion with flow, or membrane binding affinities with changes in lipid composition or salt concentration, can differentially affect the retention time of MinD and MinE, leading to spatiotemporal changes in Min patterning. PMID:24930948

Imaging of Cerebral Amyloid Angiopathy with Bivalent 99mTc-Hydroxamamide Complexes

NASA Astrophysics Data System (ADS)

Iikuni, Shimpei; Ono, Masahiro; Watanabe, Hiroyuki; Matsumura, Kenji; Yoshimura, Masashi; Kimura, Hiroyuki; Ishibashi-Ueda, Hatsue; Okamoto, Yoko; Ihara, Masafumi; Saji, Hideo

2016-05-01

Cerebral amyloid angiopathy (CAA), characterized by the deposition of amyloid aggregates in the walls of cerebral vasculature, is a major factor in intracerebral hemorrhage and vascular cognitive impairment and is also associated closely with Alzheimer’s disease (AD). We previously reported 99mTc-hydroxamamide (99mTc-Ham) complexes with a bivalent amyloid ligand showing high binding affinity for β-amyloid peptide (Aβ(1-42)) aggregates present frequently in the form in AD. In this article, we applied them to CAA-specific imaging probes, and evaluated their utility for CAA-specific imaging. In vitro inhibition assay using Aβ(1-40) aggregates deposited mainly in CAA and a brain uptake study were performed for 99mTc-Ham complexes, and all 99mTc-Ham complexes with an amyloid ligand showed binding affinity for Aβ(1-40) aggregates and very low brain uptake. In vitro autoradiography of human CAA brain sections and ex vivo autoradiography of Tg2576 mice were carried out for bivalent 99mTc-Ham complexes ([99mTc]SB2A and [99mTc]BT2B), and they displayed excellent labeling of Aβ depositions in human CAA brain sections and high affinity and selectivity to CAA in transgenic mice. These results may offer new possibilities for the development of clinically useful CAA-specific imaging probes based on the 99mTc-Ham complex.

Imaging of Cerebral Amyloid Angiopathy with Bivalent (99m)Tc-Hydroxamamide Complexes.

PubMed

Iikuni, Shimpei; Ono, Masahiro; Watanabe, Hiroyuki; Matsumura, Kenji; Yoshimura, Masashi; Kimura, Hiroyuki; Ishibashi-Ueda, Hatsue; Okamoto, Yoko; Ihara, Masafumi; Saji, Hideo

2016-05-16

Cerebral amyloid angiopathy (CAA), characterized by the deposition of amyloid aggregates in the walls of cerebral vasculature, is a major factor in intracerebral hemorrhage and vascular cognitive impairment and is also associated closely with Alzheimer's disease (AD). We previously reported (99m)Tc-hydroxamamide ((99m)Tc-Ham) complexes with a bivalent amyloid ligand showing high binding affinity for β-amyloid peptide (Aβ(1-42)) aggregates present frequently in the form in AD. In this article, we applied them to CAA-specific imaging probes, and evaluated their utility for CAA-specific imaging. In vitro inhibition assay using Aβ(1-40) aggregates deposited mainly in CAA and a brain uptake study were performed for (99m)Tc-Ham complexes, and all (99m)Tc-Ham complexes with an amyloid ligand showed binding affinity for Aβ(1-40) aggregates and very low brain uptake. In vitro autoradiography of human CAA brain sections and ex vivo autoradiography of Tg2576 mice were carried out for bivalent (99m)Tc-Ham complexes ([(99m)Tc]SB2A and [(99m)Tc]BT2B), and they displayed excellent labeling of Aβ depositions in human CAA brain sections and high affinity and selectivity to CAA in transgenic mice. These results may offer new possibilities for the development of clinically useful CAA-specific imaging probes based on the (99m)Tc-Ham complex.

Adipogenesis of human adipose-derived stem cells within three-dimensional hollow fiber-based bioreactors.

PubMed

Gerlach, Jörg C; Lin, Yen-Chih; Brayfield, Candace A; Minteer, Danielle M; Li, Han; Rubin, J Peter; Marra, Kacey G

2012-01-01

To further differentiate adipose-derived stem cells (ASCs) into mature adipocytes and create three-dimensional (3D) adipose tissue in vitro, we applied multicompartment hollow fiber-based bioreactor technology with decentral mass exchange for more physiological substrate gradients and integral oxygenation. We hypothesize that a dynamic 3D perfusion in such a bioreactor will result in longer-term culture of human adipocytes in vitro, thus providing metabolically active tissue serving as a diagnostic model for screening drugs to treat diabetes. ASCs were isolated from discarded human abdominal subcutaneous adipose tissue and then inoculated into dynamic 3D culture bioreactors to undergo adipogenic differentiation. Insulin-stimulated glucose uptake from the medium was assessed with and without TNF-alpha. 3D adipose tissue was generated in the 3D-bioreactors. Immunohistochemical staining indicated that 3D-bioreactor culture displayed multiple mature adipocyte markers with more unilocular morphologies as compared with two-dimensional (2D) cultures. Results of real-time polymerase chain reaction showed 3D-bioreactor treatment had more efficient differentiation in fatty acid-binding protein 4 expression. Repeated insulin stimulation resulted in increased glucose uptake, with a return to baseline between testing. Importantly, TNF-alpha inhibited glucose uptake, an indication of the metabolic activity of the tissue. 3D bioreactors allow more mature adipocyte differentiation of ASCs compared with traditional 2D culture and generate adipose tissue in vitro for up to 2 months. Reproducible metabolic activity of the adipose tissue in the bioreactor was demonstrated, which is potentially useful for drug discovery. We present here, to the best of our knowledge for the first time, the development of a coherent 3D high density fat-like tissue consisting of unilocular structure from primary adipose stem cells in vitro.

Differential regulation of immature articular cartilage compressive moduli and Poisson's ratios by in vitro stimulation with IGF-1 and TGF-beta1.

PubMed

Williams, Gregory M; Dills, Kristin J; Flores, Christian R; Stender, Michael E; Stewart, Kevin M; Nelson, Lauren M; Chen, Albert C; Masuda, Koichi; Hazelwood, Scott J; Klisch, Stephen M; Sah, Robert L

2010-09-17

Mechanisms of articular cartilage growth and maturation have been elucidated by studying composition-function dynamics during in vivo development and in vitro culture with stimuli such as insulin-like growth factor-1 (IGF-1) and transforming growth factor-beta 1 (TGF-beta1). This study tested the hypothesis that IGF-1 and TGF-beta1 regulate immature cartilage compressive moduli and Poisson's ratios in a manner consistent with known effects on tensile properties. Bovine calf articular cartilage from superficial-articular (S) and middle-growth (M) regions were analyzed fresh or following culture in medium with IGF-1 or TGF-beta1. Mechanical properties in confined (CC) and unconfined (UCC) compression, cartilage matrix composition, and explant size were assessed. Culture with IGF-1 resulted in softening in CC and UCC, increased Poisson's ratios, substantially increased tissue volume, and accumulation of glycosaminoglycan (GAG) and collagen (COL). Culture with TGF-beta1 promoted maturational changes in the S layer, including stiffening in CC and UCC and increased concentrations of GAG, COL, and pyridinoline crosslinks (PYR), but little growth. Culture of M layer explants with TGF-beta1 was nearly homeostatic. Across treatment groups, compressive moduli in CC and UCC were positively related to GAG, COL, and PYR concentrations, while Poisson's ratios were negatively related to concentrations of these matrix components. Thus, IGF-1 and TGF-beta1 differentially regulate the compressive mechanical properties and size of immature articular cartilage in vitro. Prescribing tissue growth, maturation, or homeostasis by controlling the in vitro biochemical environment with such growth factors may have applications in cartilage repair and tissue engineering.

Insulin resistance does not affect early embryo development but lowers implantation rate in in vitro maturation-in vitro fertilization-embryo transfer cycle.

PubMed

Chang, Eun M; Han, Ji E; Seok, Hyun H; Lee, Dong R; Yoon, Tae K; Lee, Woo S

2013-07-01

Although extensive evidence indicates the hyperinsulinemia directly contributes to reproductive dysfunction in polycystic ovarian syndrome (PCOS), influence of insulin resistance (IR) on assisted reproductive technology outcomes is poorly understood. In this study we aimed to evaluate the effects of IR on in vitro maturation-in vitro fertilization-embryo transfer (IVM-IVF-ET) in patients with PCOS. Prospective observational study. Women with PCOS (n = 115) commencing IVM. IR (n = 51) and non-IR (n = 64) women with PCOS ready to commence an IVM cycle were recruited. IR was diagnosed using the glucose tolerance test (GTT) and homeostasis model assessment (HOMA) index. Patients with an abnormal GTT and/or HOMA index >2·4 were considered IR. Patients underwent 115 cycles of unstimulated hCG-primed IVM. Maturation, fertilization, cleavage rates, the number of good-quality embryo, and blastocyst formation rates were not significantly different between groups. However, implantation (11·6% vs 28·7%, P = 0·001, respectively), clinical pregnancy (23·5% vs 53·1%, P = 0·002, respectively), and ongoing pregnancy rates (21·6% vs 46·9%. P = 0·006, respectively) were significantly decreased in the IR group. The negative effect of IR on pregnancy outcomes remained after controlling for age, body mass index (BMI) and lipid profiles (OR 4·928, 95% CI 1·735-13·991, P = 0·003). Pregnancy rate after IVM is impaired in IR patients with PCOS. Oocyte development and embryo quality are not affected, suggesting that the effects of hyperinsulinemia on endometrial function and implantation process underlie the decreased pregnancy rate. © 2012 John Wiley & Sons Ltd.

Adipogenesis of Human Adipose-Derived Stem Cells Within Three-Dimensional Hollow Fiber-Based Bioreactors

PubMed Central

Gerlach, Jörg C.; Lin, Yen-Chih; Brayfield, Candace A.; Minteer, Danielle M.; Li, Han; Rubin, J. Peter

2012-01-01

To further differentiate adipose-derived stem cells (ASCs) into mature adipocytes and create three-dimensional (3D) adipose tissue in vitro, we applied multicompartment hollow fiber-based bioreactor technology with decentral mass exchange for more physiological substrate gradients and integral oxygenation. We hypothesize that a dynamic 3D perfusion in such a bioreactor will result in longer-term culture of human adipocytes in vitro, thus providing metabolically active tissue serving as a diagnostic model for screening drugs to treat diabetes. ASCs were isolated from discarded human abdominal subcutaneous adipose tissue and then inoculated into dynamic 3D culture bioreactors to undergo adipogenic differentiation. Insulin-stimulated glucose uptake from the medium was assessed with and without TNF-alpha. 3D adipose tissue was generated in the 3D-bioreactors. Immunohistochemical staining indicated that 3D-bioreactor culture displayed multiple mature adipocyte markers with more unilocular morphologies as compared with two-dimensional (2D) cultures. Results of real-time polymerase chain reaction showed 3D-bioreactor treatment had more efficient differentiation in fatty acid-binding protein 4 expression. Repeated insulin stimulation resulted in increased glucose uptake, with a return to baseline between testing. Importantly, TNF-alpha inhibited glucose uptake, an indication of the metabolic activity of the tissue. 3D bioreactors allow more mature adipocyte differentiation of ASCs compared with traditional 2D culture and generate adipose tissue in vitro for up to 2 months. Reproducible metabolic activity of the adipose tissue in the bioreactor was demonstrated, which is potentially useful for drug discovery. We present here, to the best of our knowledge for the first time, the development of a coherent 3D high density fat-like tissue consisting of unilocular structure from primary adipose stem cells in vitro. PMID:21902468