A unique polysaccharide purified from Hericium erinaceus mycelium prevents oxidative stress induced by H2O2 in human gastric mucosa epithelium cell.

PubMed

Wang, Mingxing; Kanako, Nakajima; Zhang, Yanqiu; Xiao, Xulang; Gao, Qipin; Tetsuya, Konishi

2017-01-01

Hericium erinaceus (HE) has been used both as a traditional Chinese medicine and home remedy for treatment of gastric and duodenal ulcers and gastritis. EP-1, a purified polysaccharide isolated from HE mycelium, has recently been identified as the active component responsible for HE anti-gastritis activity. Because oxidative stress has been implicated as a pathogenic cause of gastritis and gastric ulcers, EP-1 antioxidant properties were systematically examined in vitro using the human gastric mucosal epithelial cell line, GES-1. Results showed that EP-1 possessed higher oxygen radical absorbance capacity (ORAC) and 2-3 times higher ability to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH), superoxide and hydroxyl radicals than a hot water extract of commercially available HE fruiting body. A crude mycelial polysaccharide (CMPS) extract of HE, from which EP-1 was purified, showed slightly stronger radical scavenging activity and ORAC than EP-1, with the exception of DPPH-scavenging activity. Antioxidant activities of these extracts were further studied using hydrogen peroxide (H2O2)-abused GES-1 cells; EP-1 dose-dependently preserved cell viability of abused cells as assessed via MTT assay. Moreover, FACS analysis revealed that EP-1 prevented H2O2-induced apoptotic cell death by inhibiting activation of apoptotic cellular signals within mitochondria-dependent apoptotic pathways. CMPS also prevented H2O2-induced oxidative stress, but to a lesser degree than did EP-1, even though CMPS exhibited comparable or stronger in vitro antioxidant activity than did EP-1.

The nucleotide composition of microbial genomes indicates differential patterns of selection on core and accessory genomes.

PubMed

Bohlin, Jon; Eldholm, Vegard; Pettersson, John H O; Brynildsrud, Ola; Snipen, Lars

2017-02-10

The core genome consists of genes shared by the vast majority of a species and is therefore assumed to have been subjected to substantially stronger purifying selection than the more mobile elements of the genome, also known as the accessory genome. Here we examine intragenic base composition differences in core genomes and corresponding accessory genomes in 36 species, represented by the genomes of 731 bacterial strains, to assess the impact of selective forces on base composition in microbes. We also explore, in turn, how these results compare with findings for whole genome intragenic regions. We found that GC content in coding regions is significantly higher in core genomes than accessory genomes and whole genomes. Likewise, GC content variation within coding regions was significantly lower in core genomes than in accessory genomes and whole genomes. Relative entropy in coding regions, measured as the difference between observed and expected trinucleotide frequencies estimated from mononucleotide frequencies, was significantly higher in the core genomes than in accessory and whole genomes. Relative entropy was positively associated with coding region GC content within the accessory genomes, but not within the corresponding coding regions of core or whole genomes. The higher intragenic GC content and relative entropy, as well as the lower GC content variation, observed in the core genomes is most likely associated with selective constraints. It is unclear whether the positive association between GC content and relative entropy in the more mobile accessory genomes constitutes signatures of selection or selective neutral processes.

Evolution of CCL11: genetic characterization in lagomorphs and evidence of positive and purifying selection in mammals.

PubMed

Neves, Fabiana; Abrantes, Joana; Esteves, Pedro J

2016-07-01

The interactions between chemokines and their receptors are crucial for differentiation and activation of inflammatory cells. CC chemokine ligand 11 (CCL11) binds to CCR3 and to CCR5 that in leporids underwent gene conversion with CCR2. Here, we genetically characterized CCL11 in lagomorphs (leporids and pikas). All lagomorphs have a potentially functional CCL11, and the Pygmy rabbit has a mutation in the stop codon that leads to a longer protein. Other mammals also have mutations at the stop codon that result in proteins with different lengths. By employing maximum likelihood methods, we observed that, in mammals, CCL11 exhibits both signatures of purifying and positive selection. Signatures of purifying selection were detected in sites important for receptor binding and activation. Of the three sites detected as under positive selection, two were located close to the stop codon. Our results suggest that CCL11 is functional in all lagomorphs, and that the signatures of purifying and positive selection in mammalian CCL11 probably reflect the protein's biological roles. © The Author(s) 2016.

Purification of carbon nanotubes via selective heating

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

Rogers, John A.; Wilson, William L.; Jin, Sung Hun

The present invention provides methods for purifying a layer of carbon nanotubes comprising providing a precursor layer of substantially aligned carbon nanotubes supported by a substrate, wherein the precursor layer comprises a mixture of first carbon nanotubes and second carbon nanotubes; selectively heating the first carbon nanotubes; and separating the first carbon nanotubes from the second carbon nanotubes, thereby generating a purified layer of carbon nanotubes. Devices benefiting from enhanced electrical properties enabled by the purified layer of carbon nanotubes are also described.

A Strain of Bacillus sphaericus Causes Slower Development of Resistance in Culex quinquefasciatus

PubMed Central

Pei, Guofeng; Oliveira, Cláudia M. F.; Yuan, Zhiming; Nielsen-LeRoux, Christina; Silva-Filha, Maria Helena; Yan, Jianpin; Regis, Lêda

2002-01-01

Two field-collected Culex quinquefasciatus colonies were subjected to selection pressure by three strains of Bacillus sphaericus, C3-41, 2362, and IAB59, under laboratory conditions. After 13 and 18 generations of exposure to high concentrations of C3-41 and IAB59, a field-collected low-level-resistant colony developed >144,000- and 46.3-fold resistance to strains C3-41 and IAB59, respectively. A field-collected susceptible colony was selected with 2362 and IAB59 for 46 and 12 generations and attained >162,000- and 5.7-fold resistance to the two agents, respectively. The pattern of resistance evolution in mosquitoes depended on continuous selection pressure, and the stronger the selection pressure, the more quickly resistance developed. The resistant colonies obtained after selection with B. sphaericus C3-41 and 2362 showed very high levels of cross-resistance to B. sphaericus 2362 and C3-41, respectively, but they displayed only low-level cross-resistance to IAB59. On the other hand, the IAB59-selected colonies had high cross-resistance to both strains C3-41 and 2362. Additionally, the slower evolution of resistance against strain IAB59 may be explained by the presence of another larvicidal factor. This is in agreement with the nontoxicity of the cloned and purified binary toxin (Bin1) of IAB59 for 2362-resistant larvae. We also verified that all the B. sphaericus-selected colonies showed no cross-resistance to Bacillus thuringiensis subsp. israelensis, suggesting that it would be a promising alternative in managing resistance to B. sphaericus in C. quinquefasciatus larvae. PMID:12039761

Development of Cell-SELEX Technology and Its Application in Cancer Diagnosis and Therapy.

PubMed

Chen, Man; Yu, Yuanyuan; Jiang, Feng; Zhou, Junwei; Li, Yongshu; Liang, Chao; Dang, Lei; Lu, Aiping; Zhang, Ge

2016-12-10

SELEX (systematic evolution of ligands by exponential enrichment) is a process involving the progressive isolation of high selective ssDNA/RNA from a combinatorial single-stranded oligonucleotide library through repeated rounds of binding, partitioning and amplification. SELEX-derived single-stranded DNA/RNA molecules, called aptamers, are selected against a wide range of targets, including purified proteins, live cells, tissues, microorganisms, small molecules and so on. With the development of SELEX technology over the last two decades, various modified SELEX processes have been arisen. A majority of aptamers are selected against purified proteins through traditional SELEX. Unfortunately, more and more evidence showed aptamers selected against purified membrane proteins failed to recognize their targets in live cells. Cell-SELEX could develop aptamers against a particular target cell line to discriminate this cell line from others. Therefore, cell-SELEX has been widely used to select aptamers for the application of both diagnosis and therapy of various diseases, especially for cancer. In this review, the advantages and limitations of cell-SELEX and SELEX against purified protein will be compared. Various modified cell-SELEX techniques will be summarized, and application of cell-SELEX in cancer diagnosis and therapy will be discussed.

Development of Cell-SELEX Technology and Its Application in Cancer Diagnosis and Therapy

PubMed Central

Chen, Man; Yu, Yuanyuan; Jiang, Feng; Zhou, Junwei; Li, Yongshu; Liang, Chao; Dang, Lei; Lu, Aiping; Zhang, Ge

2016-01-01

SELEX (systematic evolution of ligands by exponential enrichment) is a process involving the progressive isolation of high selective ssDNA/RNA from a combinatorial single-stranded oligonucleotide library through repeated rounds of binding, partitioning and amplification. SELEX-derived single-stranded DNA/RNA molecules, called aptamers, are selected against a wide range of targets, including purified proteins, live cells, tissues, microorganisms, small molecules and so on. With the development of SELEX technology over the last two decades, various modified SELEX processes have been arisen. A majority of aptamers are selected against purified proteins through traditional SELEX. Unfortunately, more and more evidence showed aptamers selected against purified membrane proteins failed to recognize their targets in live cells. Cell-SELEX could develop aptamers against a particular target cell line to discriminate this cell line from others. Therefore, cell-SELEX has been widely used to select aptamers for the application of both diagnosis and therapy of various diseases, especially for cancer. In this review, the advantages and limitations of cell-SELEX and SELEX against purified protein will be compared. Various modified cell-SELEX techniques will be summarized, and application of cell-SELEX in cancer diagnosis and therapy will be discussed. PMID:27973403

Overproduction, purification, and ATPase activity of the Escherichia coli RuvB protein involved in DNA repair.

PubMed Central

Iwasaki, H; Shiba, T; Makino, K; Nakata, A; Shinagawa, H

1989-01-01

The ruvA and ruvB genes of Escherichia coli constitute an operon which belongs to the SOS regulon. Genetic evidence suggests that the products of the ruv operon are involved in DNA repair and recombination. To begin biochemical characterization of these proteins, we developed a plasmid system that overproduced RuvB protein to 20% of total cell protein. Starting from the overproducing system, we purified RuvB protein. The purified RuvB protein behaved like a monomer in gel filtration chromatography and had an apparent relative molecular mass of 38 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which agrees with the value predicted from the DNA sequence. The amino acid sequence of the amino-terminal region of the purified protein was analyzed, and the sequence agreed with the one deduced from the DNA sequence. Since the deduced sequence of RuvB protein contained the consensus sequence for ATP-binding proteins, we examined the ATP-binding and ATPase activities of the purified RuvB protein. RuvB protein had a stronger affinity to ADP than to ATP and weak ATPase activity. The results suggest that the weak ATPase activity of RuvB protein is at least partly due to end product inhibition by ADP. Images PMID:2529252

Antihemorrhagin in the blood serum of king cobra (Ophiophagus hannah): purification and characterization.

PubMed

Chanhome, Lawan; Khow, Orawan; Omori-Satoh, Tamotsu; Sitprija, Visith

2003-06-01

King cobra (Ophiophagus hannah) serum was found to possess antihemorrhagic activity against king cobra hemorrhagin. The activity was stronger than that in commercial king cobra antivenom. An antihemorrhagin has been purified by ion exchange chromatography, affinity chromatography and gel filtration with a 22-fold purification and an overall yield of 12% of the total antihemorrhagic activity contained in crude serum. The purified antihemorrhagin was homogeneous in disc-PAGE and SDS-PAGE. Its apparent molecular weight determined by SDS-PAGE was 120 kDa. The antihemorrhagin was also active against other hemorrhagic snake venoms obtained in Thailand and Japan such as Calloselasma rhodostoma, Trimeresurus albolabris, Trimeresurus macrops and Trimeresurus flavoviridis (Japanese Habu). It inhibited the proteolytic activity of king cobra venom. It is an acid- and thermolabile protein and does not form precipitin lines against king cobra venom.

Anticoagulant and antithrombotic evaluation of native fucosylated chondroitin sulfates and their derivatives as selective inhibitors of intrinsic factor Xase.

PubMed

Wu, Mingyi; Wen, Dandan; Gao, Na; Xiao, Chuang; Yang, Lian; Xu, Li; Lian, Wu; Peng, Wenlie; Jiang, Jianmin; Zhao, Jinhua

2015-03-06

Fucosylated chondroitin sulfate (FCS), a structurally unusual glycosaminoglycan, has distinct anticoagulant properties, and is an especially strong inhibitor of the intrinsic factor Xase (anti-Xase). To obtain a highly selective inhibitor of human Xase, we purified six native FCSs with various sulfation patterns, prepared a series of FCS derivatives, and then elucidated the relationship between the structures and the anticoagulant activities of FCSs. FCSs 1-3 containing higher Fuc2S4S exhibit stronger AT-dependent anti-IIa activities, whereas 4-6 containing more Fuc3S4S produce potent HCII-dependent anti-IIa activities. Saccharides containing a minimum of 6-8 trisaccharide units, free carboxyl groups, and full fucosylation of GlcA may be required for potent anti-Xase activity, and approximately six trisaccharide units and partial fucosylation of GlcA may contribute to potent HCII-dependent activity. Decreasing of the molecular weights markedly reduces their AT-dependent anti-IIa activities, and even eliminates human platelet and factor XII activation. Furthermore, in vitro and in vivo studies suggested that fractions of 6-12 kDa may be very promising compounds as putative selective intrinsic Xase inhibitors with antithrombotic action, but without the consequences of major bleeding and factor XII activation. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Homage to Bateman: sex roles predict sex differences in sexual selection.

PubMed

Fritzsche, Karoline; Arnqvis, Göran

2013-07-01

Classic sex role theory predicts that sexual selection should be stronger in males in taxa showing conventional sex roles and stronger in females in role reversed mating systems. To test this very central prediction and to assess the utility of different measures of sexual selection, we estimated sexual selection in both sexes in four seed beetle species with divergent sex roles using a novel experimental design. We found that sexual selection was sizeable in females and the strength of sexual selection was similar in females and males in role-reversed species. Sexual selection was overall significantly stronger in males than in females and residual selection formed a substantial component of net selection in both sexes. Furthermore, sexual selection in females was stronger in role-reversed species compared to species with conventional sex roles. Variance-based measures of sexual selection (the Bateman gradient and selection opportunities) were better predictors of sexual dimorphism in reproductive behavior and morphology across species compared to trait-based measures (selection differentials). Our results highlight the importance of using assays that incorporate components of fitness manifested after mating. We suggest that the Bateman gradient is generally the most informative measure of the strength of sexual selection in comparisons across sexes and/or species. © 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.

Toxicological Profiling of Highly Purified Single-Walled Carbon Nanotubes with Different Lengths in the Rodent Lung and Escherichia Coli.

PubMed

Wang, Xiang; Lee, Jae-Hyeok; Li, Ruibin; Liao, Yu-Pei; Kang, Joohoon; Chang, Chong Hyun; Guiney, Linda M; Mirshafiee, Vahid; Li, Linjiang; Lu, Jianqin; Xia, Tian; Hersam, Mark C; Nel, André E

2018-06-01

Carbon nanotubes (CNTs) exhibit a number of physicochemical properties that contribute to adverse biological outcomes. However, it is difficult to define the independent contribution of individual properties without purified materials. A library of highly purified single-walled carbon nanotubes (SWCNTs) of different lengths is prepared from the same base material by density gradient ultracentrifugation, designated as short (318 nm), medium (789 nm), and long (1215 nm) SWCNTs. In vitro screening shows length-dependent interleukin-1β (IL-1β) production, in order of long > medium > short. However, there are no differences in transforming growth factor-β1 production in BEAS-2B cells. Oropharyngeal aspiration shows that all the SWCNTs induce profibrogenic effects in mouse lung at 21 d postexposure, but there are no differences between tube lengths. In contrast, these SWCNTs demonstrate length-dependent antibacterial effects on Escherichia coli, with the long SWCNT exerting stronger effects than the medium or short tubes. These effects are reduced by Pluronic F108 coating or supplementing with glucose. The data show length-dependent effects on proinflammatory response in macrophage cell line and antibacterial effects, but not on collagen deposition in the lung. These data demonstrate that over the length scale tested, the biological response to highly purified SWCNTs is dependent on the complexity of the nano/bio interface. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A study on biological activity of marine fungi from different habitats in coastal regions.

PubMed

Zhou, Songlin; Wang, Min; Feng, Qi; Lin, Yingying; Zhao, Huange

2016-01-01

In recent years, marine fungi have become an important source of active marine natural products. Former researches are limited in habitats selection of fungi with bioactive compounds. In this paper were to measure antibacterial and antitumor cell activity for secondary metabolites of marine fungi, which were isolated from different habitats in coastal regions. 195 strains of marine fungi were isolated and purified from three different habitats. They biologically active experiment results showed that fungi isolation from the mangrove habitats had stronger antibacterial activity than others, and the stains isolated from the estuarial habitats had the least antibacterial activity. However, the strains separated from beach habitats strongly inhibited tumor cell proliferation in vitro, and fungi of mangrove forest habitats had the weakest activity of inhibiting tumor. Meanwhile, 195 fungal strains belonged to 46 families, 84 genera, 142 species and also showed 137 different types of activity combinations by analyzing the inhibitory activity of the metabolites fungi for 4 strains of pathogenic bacteria and B-16 cells. The study investigated the biological activity of marine fungi isolated from different habitats in Haikou coastal regions. The results help us to understand bioactive metabolites of marine fungi from different habitats, and how to selected biological activity fungi from various marine habitats effectively.

Position specific variation in the rate of evolution in transcription factor binding sites

PubMed Central

Moses, Alan M; Chiang, Derek Y; Kellis, Manolis; Lander, Eric S; Eisen, Michael B

2003-01-01

Background The binding sites of sequence specific transcription factors are an important and relatively well-understood class of functional non-coding DNAs. Although a wide variety of experimental and computational methods have been developed to characterize transcription factor binding sites, they remain difficult to identify. Comparison of non-coding DNA from related species has shown considerable promise in identifying these functional non-coding sequences, even though relatively little is known about their evolution. Results Here we analyse the genome sequences of the budding yeasts Saccharomyces cerevisiae, S. bayanus, S. paradoxus and S. mikatae to study the evolution of transcription factor binding sites. As expected, we find that both experimentally characterized and computationally predicted binding sites evolve slower than surrounding sequence, consistent with the hypothesis that they are under purifying selection. We also observe position-specific variation in the rate of evolution within binding sites. We find that the position-specific rate of evolution is positively correlated with degeneracy among binding sites within S. cerevisiae. We test theoretical predictions for the rate of evolution at positions where the base frequencies deviate from background due to purifying selection and find reasonable agreement with the observed rates of evolution. Finally, we show how the evolutionary characteristics of real binding motifs can be used to distinguish them from artefacts of computational motif finding algorithms. Conclusion As has been observed for protein sequences, the rate of evolution in transcription factor binding sites varies with position, suggesting that some regions are under stronger functional constraint than others. This variation likely reflects the varying importance of different positions in the formation of the protein-DNA complex. The characterization of the pattern of evolution in known binding sites will likely contribute to the effective use of comparative sequence data in the identification of transcription factor binding sites and is an important step toward understanding the evolution of functional non-coding DNA. PMID:12946282

Production and Evaluation of a Purified Adenovirus Group-Specific (Hexon) Antigen for Use in the Diagnostic Complement Fixation Test

PubMed Central

Dowdle, W. R.; Lambriex, M.; Hierholzer, J. C.

1971-01-01

A simple procedure for the production of large volumes of purified adenovirus group-specific complement-fixing (CF) (hexon) antigen by selective adsorption to and elution from CaHPO4 is described. Results of immunodiffusion tests, electrophoresis, electron microscopy, and tests for hemagglutination and infectivity indicate that the purified antigen consisted of a single virus component (hexon). The purified product contained little host materials. Unlike the crude virus harvest usually employed for serodiagnostic CF tests, the purified antigen demonstrated no anticomplementary activity and did not develop such activity during storage. The purified antigen was equal to or slightly more sensitive than crude virus harvests for serodiagnosis of adenovirus infections. Images PMID:4325021

Genomic Signatures of Selective Pressures and Introgression from Archaic Hominins at Human Innate Immunity Genes

PubMed Central

Deschamps, Matthieu; Laval, Guillaume; Fagny, Maud; Itan, Yuval; Abel, Laurent; Casanova, Jean-Laurent; Patin, Etienne; Quintana-Murci, Lluis

2016-01-01

Human genes governing innate immunity provide a valuable tool for the study of the selective pressure imposed by microorganisms on host genomes. A comprehensive, genome-wide study of how selective constraints and adaptations have driven the evolution of innate immunity genes is missing. Using full-genome sequence variation from the 1000 Genomes Project, we first show that innate immunity genes have globally evolved under stronger purifying selection than the remainder of protein-coding genes. We identify a gene set under the strongest selective constraints, mutations in which are likely to predispose individuals to life-threatening disease, as illustrated by STAT1 and TRAF3. We then evaluate the occurrence of local adaptation and detect 57 high-scoring signals of positive selection at innate immunity genes, variation in which has been associated with susceptibility to common infectious or autoimmune diseases. Furthermore, we show that most adaptations targeting coding variation have occurred in the last 6,000–13,000 years, the period at which populations shifted from hunting and gathering to farming. Finally, we show that innate immunity genes present higher Neandertal introgression than the remainder of the coding genome. Notably, among the genes presenting the highest Neandertal ancestry, we find the TLR6-TLR1-TLR10 cluster, which also contains functional adaptive variation in Europeans. This study identifies highly constrained genes that fulfill essential, non-redundant functions in host survival and reveals others that are more permissive to change—containing variation acquired from archaic hominins or adaptive variants in specific populations—improving our understanding of the relative biological importance of innate immunity pathways in natural conditions. PMID:26748513

Cylindrospermopsin inhibits growth and modulates protease activity in the aquatic plants Lemna minor L. and Wolffia arrhiza (L.) Horkel.

PubMed

Jámbrik, Katalin; Máthé, C; Vasas, G; Bácsi, I; Surányi, G; Gonda, S; Borbély, G; M-Hamvas, Márta

2010-01-01

The toxic effects of cylindrospermopsin (cyanobacterial toxin) on animals have been examined extensively, but little research has focused on their effects on plants. In this study cylindrospermopsin (CYN) caused alterations of growth, soluble protein content and protease enzyme activity were studied on two aquatic plants Lemna minor and Wolffia arrhiza in short-term (5 days) experiments. For the treatments we used CYN containing crude extracts of Aphanizomenon ovalisporum (BGSD-423) and purified CYN as well. The maximal inhibitory effects on fresh weight of L. minor and W. arrhiza caused by crude extract were 60% and 54%, respectively, while the maximum inhibitory effects were 30% and 43% in the case of purified CYN at 20 μg ml(-1) CYN content of culture medium. In CYN-treated plants the concentration of soluble protein showed mild increases, especially in W. arrhiza. Protease isoenzyme activity gels showed significant alterations of enzyme activities under the influence of CYN. Several isoenzymes were far more active and new ones appeared in CYN-treated plants. Treatments with cyanobacterial crude extract caused stronger effects than the purified cyanobacterial toxins used in equivalent CYN concentrations.

Structural Characterization and Evaluation of the Antioxidant Activity of Phenolic Compounds from Astragalus taipaishanensis and Their Structure-Activity Relationship

NASA Astrophysics Data System (ADS)

Pu, Wenjun; Wang, Dongmei; Zhou, Dan

2015-09-01

Eight phenolic compounds were isolated using bio-guided isolation and purified from the roots of Astragalus taipaishanensis Y. C. Ho et S. B. Ho (A. taipaishanensis) for the first time. Their structures were elucidated by ESI-MS, HR-ESI-MS, 1D-NMR and 2D-NMR as 7,2‧-dihydroxy-3‧,4‧-dimethoxy isoflavan (1), formononetin (2), isoliquiritigenin (3), quercetin (4), kaempferol (5), ononin (6), p-hydroxybenzoic acid (7) and vanillic acid (8). Six flavonoids (compounds 1-6) exhibited stronger antioxidant activities (determined by DPPH, ABTS, FRAP and lipid peroxidation inhibition assays) than those of BHA and TBHQ and also demonstrated noticeable protective effects (particularly quercetin and kaempferol) on Escherichia coli under oxidative stress. Additionally, the chemical constituents compared with those of Astragalus membranaceus and the structure-activity relationship of the isolated compounds were both analyzed. The results clearly demonstrated that A. taipaishanensis has the potential to be selected as an alternative medicinal and food plant that can be utilized in health food products, functional tea and pharmaceutical products.

[Studies on antigencity of human immunodeficiency virus type 1 (HIV-1) external glycoprotein as well as its expression in Pichia pastoris].

PubMed

Zhao, Li-Hui; Yu, Xiang-Hui; Jiang, Chun-Lai; Wu, Yong-Ge; Shen, Jia-Cong; Kong, Wei

2007-05-01

Based on the computer simulation, we analyzed hydrophobicity, potential epitope of recombined subtypes HIV-1 Env protein (851 amino acids) from Guangxi in China. Compared with conservative peptides of other subtypes in env protein, three sequences (469-511aa, 538-674aa, 700-734aa) were selected to recombine into a chimeric gene that codes three conservative epitope peptides with stronger antigencity, and was constructed in the yeast expression plasmid pPICZB. Chimeric proteins were expressed in Pichia pastoris under the induction of methanol, and were analyzed by SDS-PAGE and Westernblot. The results showed that fusion proteins of three-segment antigen were expressed in Pichia pastoris and that specific protein band at the site of 40kD was target protein, which is interacted with HIV-1 serum. The target proteins were purified by metal Ni-sepharose 4B, and were demonstrated to possess good antigenic specificity from the data of ELISA. This chimeric antigen may be used as research and developed into HIV diagnostic reagents.

Evidence of pervasive biologically functional secondary structures within the genomes of eukaryotic single-stranded DNA viruses.

PubMed

Muhire, Brejnev Muhizi; Golden, Michael; Murrell, Ben; Lefeuvre, Pierre; Lett, Jean-Michel; Gray, Alistair; Poon, Art Y F; Ngandu, Nobubelo Kwanele; Semegni, Yves; Tanov, Emil Pavlov; Monjane, Adérito Luis; Harkins, Gordon William; Varsani, Arvind; Shepherd, Dionne Natalie; Martin, Darren Patrick

2014-02-01

Single-stranded DNA (ssDNA) viruses have genomes that are potentially capable of forming complex secondary structures through Watson-Crick base pairing between their constituent nucleotides. A few of the structural elements formed by such base pairings are, in fact, known to have important functions during the replication of many ssDNA viruses. Unknown, however, are (i) whether numerous additional ssDNA virus genomic structural elements predicted to exist by computational DNA folding methods actually exist and (ii) whether those structures that do exist have any biological relevance. We therefore computationally inferred lists of the most evolutionarily conserved structures within a diverse selection of animal- and plant-infecting ssDNA viruses drawn from the families Circoviridae, Anelloviridae, Parvoviridae, Nanoviridae, and Geminiviridae and analyzed these for evidence of natural selection favoring the maintenance of these structures. While we find evidence that is consistent with purifying selection being stronger at nucleotide sites that are predicted to be base paired than at sites predicted to be unpaired, we also find strong associations between sites that are predicted to pair with one another and site pairs that are apparently coevolving in a complementary fashion. Collectively, these results indicate that natural selection actively preserves much of the pervasive secondary structure that is evident within eukaryote-infecting ssDNA virus genomes and, therefore, that much of this structure is biologically functional. Lastly, we provide examples of various highly conserved but completely uncharacterized structural elements that likely have important functions within some of the ssDNA virus genomes analyzed here.

Evidence of Pervasive Biologically Functional Secondary Structures within the Genomes of Eukaryotic Single-Stranded DNA Viruses

PubMed Central

Muhire, Brejnev Muhizi; Golden, Michael; Murrell, Ben; Lefeuvre, Pierre; Lett, Jean-Michel; Gray, Alistair; Poon, Art Y. F.; Ngandu, Nobubelo Kwanele; Semegni, Yves; Tanov, Emil Pavlov; Monjane, Adérito Luis; Harkins, Gordon William; Varsani, Arvind; Shepherd, Dionne Natalie

2014-01-01

Single-stranded DNA (ssDNA) viruses have genomes that are potentially capable of forming complex secondary structures through Watson-Crick base pairing between their constituent nucleotides. A few of the structural elements formed by such base pairings are, in fact, known to have important functions during the replication of many ssDNA viruses. Unknown, however, are (i) whether numerous additional ssDNA virus genomic structural elements predicted to exist by computational DNA folding methods actually exist and (ii) whether those structures that do exist have any biological relevance. We therefore computationally inferred lists of the most evolutionarily conserved structures within a diverse selection of animal- and plant-infecting ssDNA viruses drawn from the families Circoviridae, Anelloviridae, Parvoviridae, Nanoviridae, and Geminiviridae and analyzed these for evidence of natural selection favoring the maintenance of these structures. While we find evidence that is consistent with purifying selection being stronger at nucleotide sites that are predicted to be base paired than at sites predicted to be unpaired, we also find strong associations between sites that are predicted to pair with one another and site pairs that are apparently coevolving in a complementary fashion. Collectively, these results indicate that natural selection actively preserves much of the pervasive secondary structure that is evident within eukaryote-infecting ssDNA virus genomes and, therefore, that much of this structure is biologically functional. Lastly, we provide examples of various highly conserved but completely uncharacterized structural elements that likely have important functions within some of the ssDNA virus genomes analyzed here. PMID:24284329

Natural selection reduced diversity on human y chromosomes.

PubMed

Wilson Sayres, Melissa A; Lohmueller, Kirk E; Nielsen, Rasmus

2014-01-01

The human Y chromosome exhibits surprisingly low levels of genetic diversity. This could result from neutral processes if the effective population size of males is reduced relative to females due to a higher variance in the number of offspring from males than from females. Alternatively, selection acting on new mutations, and affecting linked neutral sites, could reduce variability on the Y chromosome. Here, using genome-wide analyses of X, Y, autosomal and mitochondrial DNA, in combination with extensive population genetic simulations, we show that low observed Y chromosome variability is not consistent with a purely neutral model. Instead, we show that models of purifying selection are consistent with observed Y diversity. Further, the number of sites estimated to be under purifying selection greatly exceeds the number of Y-linked coding sites, suggesting the importance of the highly repetitive ampliconic regions. While we show that purifying selection removing deleterious mutations can explain the low diversity on the Y chromosome, we cannot exclude the possibility that positive selection acting on beneficial mutations could have also reduced diversity in linked neutral regions, and may have contributed to lowering human Y chromosome diversity. Because the functional significance of the ampliconic regions is poorly understood, our findings should motivate future research in this area.

Natural Selection Reduced Diversity on Human Y Chromosomes

PubMed Central

Wilson Sayres, Melissa A.; Lohmueller, Kirk E.; Nielsen, Rasmus

2014-01-01

The human Y chromosome exhibits surprisingly low levels of genetic diversity. This could result from neutral processes if the effective population size of males is reduced relative to females due to a higher variance in the number of offspring from males than from females. Alternatively, selection acting on new mutations, and affecting linked neutral sites, could reduce variability on the Y chromosome. Here, using genome-wide analyses of X, Y, autosomal and mitochondrial DNA, in combination with extensive population genetic simulations, we show that low observed Y chromosome variability is not consistent with a purely neutral model. Instead, we show that models of purifying selection are consistent with observed Y diversity. Further, the number of sites estimated to be under purifying selection greatly exceeds the number of Y-linked coding sites, suggesting the importance of the highly repetitive ampliconic regions. While we show that purifying selection removing deleterious mutations can explain the low diversity on the Y chromosome, we cannot exclude the possibility that positive selection acting on beneficial mutations could have also reduced diversity in linked neutral regions, and may have contributed to lowering human Y chromosome diversity. Because the functional significance of the ampliconic regions is poorly understood, our findings should motivate future research in this area. PMID:24415951

Induction of Pro-Angiogenic Factors by Pregnancy-Specific Glycoproteins and Studies on Receptor Usage

DTIC Science & Technology

2008-01-01

expression vector. The purified bacoluvirus DNA, containing PSG11, was purified and used to transfect Spodoptera frugiperda (Sf-9) cells (Invitrogen) to...proteins in Spodoptera frugiperda cells using biotin acceptor peptides. Anal Biochem, 1998. 262(2): p. 122-8. 175. Hirt, B., Selective extraction of

Dissociative absorption: An empirically unique, clinically relevant, dissociative factor.

PubMed

Soffer-Dudek, Nirit; Lassri, Dana; Soffer-Dudek, Nir; Shahar, Golan

2015-11-01

Research of dissociative absorption has raised two questions: (a) Is absorption a unique dissociative factor within a three-factor structure, or a part of one general dissociative factor? Even when three factors are found, the specificity of the absorption factor is questionable. (b) Is absorption implicated in psychopathology? Although commonly viewed as "non-clinical" dissociation, absorption was recently hypothesized to be specifically associated with obsessive-compulsive symptoms. To address these questions, we conducted exploratory and confirmatory factor analyses on 679 undergraduates. Analyses supported the three-factor model, and a "purified" absorption scale was extracted from the original inclusive absorption factor. The purified scale predicted several psychopathology scales. As hypothesized, absorption was a stronger predictor of obsessive-compulsive symptoms than of general psychopathology. In addition, absorption was the only dissociative scale that longitudinally predicted obsessive-compulsive symptoms. We conclude that absorption is a unique and clinically relevant dissociative tendency that is particularly meaningful to obsessive-compulsive symptoms. Copyright © 2015 Elsevier Inc. All rights reserved.

Phylogeny reconstruction in the Caesalpinieae grade (Leguminosae) based on duplicated copies of the sucrose synthase gene and plastid markers.

PubMed

Manzanilla, Vincent; Bruneau, Anne

2012-10-01

The Caesalpinieae grade (Leguminosae) forms a morphologically and ecologically diverse group of mostly tropical tree species with a complex evolutionary history. This grade comprises several distinct lineages, but the exact delimitation of the group relative to subfamily Mimosoideae and other members of subfamily Caesalpinioideae, as well as phylogenetic relationships among the lineages are uncertain. With the aim of better resolving phylogenetic relationships within the Caesalpinieae grade, we investigated the utility of several nuclear markers developed from genomic studies in the Papilionoideae. We cloned and sequenced the low copy nuclear gene sucrose synthase (SUSY) and combined the data with plastid trnL and matK sequences. SUSY has two paralogs in the Caesalpinieae grade and in the Mimosoideae, but occurs as a single copy in all other legumes tested. Bayesian and maximum likelihood phylogenetic analyses suggest the two nuclear markers are congruent with plastid DNA data. The Caesalpinieae grade is divided into four well-supported clades (Cassia, Caesalpinia, Tachigali and Peltophorum clades), a poorly supported clade of Dimorphandra Group genera, and two paraphyletic groups, one with other Dimorphandra Group genera and the other comprising genera previously recognized as the Umtiza clade. A selection analysis of the paralogs, using selection models from PAML, suggests that SUSY genes are subjected to a purifying selection. One of the SUSY paralogs, under slightly stronger positive selection, may be undergoing subfunctionalization. The low copy SUSY gene is useful for phylogeny reconstruction in the Caesalpinieae despite the presence of duplicate copies. This study confirms that the Caesalpinieae grade is an artificial group, and highlights the need for further analyses of lineages at the base of the Mimosoideae. Copyright © 2012 Elsevier Inc. All rights reserved.

Discovering functional DNA elements using population genomic information: a proof of concept using human mtDNA.

PubMed

Schrider, Daniel R; Kern, Andrew D

2014-06-09

Identifying the complete set of functional elements within the human genome would be a windfall for multiple areas of biological research including medicine, molecular biology, and evolution. Complete knowledge of function would aid in the prioritization of loci when searching for the genetic bases of disease or adaptive phenotypes. Because mutations that disrupt function are disfavored by natural selection, purifying selection leaves a detectable signature within functional elements; accordingly, this signal has been exploited for over a decade through the use of genomic comparisons of distantly related species. While this is so, the functional complement of the genome changes extensively across time and between lineages; therefore, evidence of the current action of purifying selection in humans is essential. Because the removal of deleterious mutations by natural selection also reduces within-species genetic diversity within functional loci, dense population genetic data have the potential to reveal genomic elements that are currently functional. Here, we assess the potential of this approach by examining an ultradeep sample of human mitochondrial genomes (n = 16,411). We show that the high density of polymorphism in this data set precisely delineates regions experiencing purifying selection. Furthermore, we show that the number of segregating alleles at a site is strongly correlated with its divergence across species after accounting for known mutational biases in human mitochondrial DNA (ρ = 0.51; P < 2.2 × 10(-16)). These two measures track one another at a remarkably fine scale across many loci-a correlation that is purely the result of natural selection. Our results demonstrate that genetic variation has the potential to reveal with surprising precision which regions in the genome are currently performing important functions and likely to have deleterious fitness effects when mutated. As more complete human genomes are sequenced, similar power to reveal purifying selection may be achievable in the human nuclear genome. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Selfish drive can trump function when animal mitochondrial genomes compete.

PubMed

Ma, Hansong; O'Farrell, Patrick H

2016-07-01

Mitochondrial genomes compete for transmission from mother to progeny. We explored this competition by introducing a second genome into Drosophila melanogaster to follow transmission. Competitions between closely related genomes favored those functional in electron transport, resulting in a host-beneficial purifying selection. In contrast, matchups between distantly related genomes often favored those with negligible, negative or lethal consequences, indicating selfish selection. Exhibiting powerful selfish selection, a genome carrying a detrimental mutation displaced a complementing genome, leading to population death after several generations. In a different pairing, opposing selfish and purifying selection counterbalanced to give stable transmission of two genomes. Sequencing of recombinant mitochondrial genomes showed that the noncoding region, containing origins of replication, governs selfish transmission. Uniparental inheritance prevents encounters between distantly related genomes. Nonetheless, in each maternal lineage, constant competition among sibling genomes selects for super-replicators. We suggest that this relentless competition drives positive selection, promoting change in the sequences influencing transmission.

Selfish drive can trump function when animal mitochondrial genomes compete

PubMed Central

Ma, Hansong; O’Farrell, Patrick H.

2016-01-01

Mitochondrial genomes compete for transmission from mother to progeny. We explored this competition by introducing a second genome into Drosophila melanogaster to follow transmission. Competitions between closely related genomes favored those functional in electron transport, resulting in a host-beneficial purifying selection1. Contrastingly, matchups between distant genomes often favored those with negligible, negative or lethal consequences, indicating selfish selection. Exhibiting powerful selfish selection, a genome carrying a detrimental mutation displaced a complementing genome leading to population death after several generations. In a different pairing, opposing selfish and purifying selection counterbalanced to give stable transmission of two genomes. Sequencing of recombinant mitochondrial genomes revealed that the non-coding region, containing origins of replication, governs selfish transmission. Uniparental inheritance prevents encounters between distantly related genomes. Nonetheless, within each maternal lineage, constant competition among sibling genomes selects for super-replicators. We suggest that this relentless competition drives positive selection promoting change in the sequences influencing transmission. PMID:27270106

Outer membrane defect and stronger biofilm formation caused by inactivation of a gene encoding for heptosyltransferase I in Cronobacter sakazakii ATCC BAA-894.

PubMed

Wang, L; Hu, X; Tao, G; Wang, X

2012-05-01

To investigate the role of lipopolysaccharide (LPS) structure in the stability of outer membrane and the ability of biofilm formation in Cronobacter sakazakii. A C. sakazakii mutant strain LWW02 was constructed by inactivating the gene ESA_04107 encoding for heptosyltransferase I. LPS were purified from LWW02, and changes in their structure were confirmed by thin-layer chromatography and electrospray ionization mass spectrometry. Comparing with the wild-type strain BAA-894, slower growth, higher membrane permeability, higher surface hydrophobicity, stronger ability of autoaggregation and biofilm formation were observed for the mutant strain LWW02. The gene ESA_04107 encodes heptosyltransferase I in C. sakazakii ATCC BAA-894. The cleavage of LPS in C. sakazakii could cause its outer membrane defects and increase its ability to form biofilms. The study is important for understanding the pathogenic mechanism and efficient control of C. sakazakii. © 2012 The Authors. Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

Increased Growth Inhibitory Effects on Human Cancer Cells and Anti-Inflammatory Potency of Shogaols from Zingiber officinale Relative to Gingerols

PubMed Central

Sang, Shengmin; Hong, Jungil; Wu, Hou; Liu, Jing; Yang, Chung S.; Pan, Min-Hsiung; Badmaev, Vadimir; Ho, Chi-Tang

2009-01-01

Ginger, the rhizome of the plant Zingiber officinale, has received extensive attention due to its antioxidant, anti-inflammatory, and anti-tumor activities. Most researchers have considered gingerols as the active principles and have paid little attention to shogaols, the dehydration products of corresponding gingerols during storage or thermal processing. In this study, we have purified and identified eight major components including three major gingerols and corresponding shogaols from ginger extract and compared their anti-carcinogenic and anti-inflammatory activities. Our results showed that shogaols ([6]-, [8]-, and [10]-) had much stronger growth inhibitory effects than gingerols ([6]-, [8]-, and [10]-) on H-1299 human lung cancer cells and HCT-116 human colon cancer cells, especially when comparing [6]-shogaol with [6]-gingerol (IC50: ~8 µM vs. ~150 µM). In addition, we found that [6]-shogaol had much stronger inhibitory effects on arachidonic acid release and nitric oxide (NO) synthesis than [6]-gingerol. PMID:19877681

Increased growth inhibitory effects on human cancer cells and anti-inflammatory potency of shogaols from Zingiber officinale relative to gingerols.

PubMed

Sang, Shengmin; Hong, Jungil; Wu, Hou; Liu, Jing; Yang, Chung S; Pan, Min-Hsiung; Badmaev, Vladimir; Ho, Chi-Tang

2009-11-25

Ginger, the rhizome of the plant Zingiber officinale , has received extensive attention because of its antioxidant, anti-inflammatory, and antitumor activities. Most researchers have considered gingerols as the active principles and have paid little attention to shogaols, the dehydration products of corresponding gingerols during storage or thermal processing. In this study, we have purified and identified eight major components, including three major gingerols and corresponding shogaols, from ginger extract and compared their anticarcinogenic and anti-inflammatory activities. Our results showed that shogaols ([6], [8], and [10]) had much stronger growth inhibitory effects than gingerols ([6], [8], and [10]) on H-1299 human lung cancer cells and HCT-116 human colon cancer cells, especially when comparing [6]-shogaol with [6]-gingerol (IC50 of approximately 8 versus approximately 150 microM). In addition, we found that [6]-shogaol had much stronger inhibitory effects on arachidonic acid release and nitric oxide (NO) synthesis than [6]-gingerol.

Comparative analysis of complete mitochondrial genomes suggests that relaxed purifying selection is driving high nonsynonymous evolutionary rate of the NADH2 gene in whitefish (Coregonus ssp.).

PubMed

Jacobsen, Magnus W; da Fonseca, Rute R; Bernatchez, Louis; Hansen, Michael M

2016-02-01

Several studies have recently reported evidence for positive selection acting on the mitochondrial genome (mitogenome), emphasizing its potential role in adaptive divergence and speciation. In this study we searched 107 full mitogenomes of recently diverged species and lineages of whitefish (Coregonus ssp.) for signals of positive selection. These salmonids show several distinct morphological and ecological differences that may be associated with energetics and therefore potentially positive selection at the mitogenome level. We found that purifying selection and genetic drift were the predominant evolutionary forces acting on the analyzed mitogenomes. However, the NADH dehydrogenase 2 gene (ND2) showed a highly elevated dN/dS ratio compared to the other mitochondrial genes, which was significantly higher in whitefish compared to other salmonids. We therefore further examined nonsynonymous evolution in ND2 by (i) mapping amino acid changes to a protein model structure which showed that they were located away from key functional residues of the protein, (ii) locating them in the sequences of other species of fish (Salmonidae, Anguillidae, Scombridae and Percidae) only to find pronounced overlap of nonsynonymous regions. We thus conclude that relaxed purifying selection is driving the evolution of ND2 by affecting mostly regions that have lower functional relevance. Copyright © 2015 Elsevier Inc. All rights reserved.

Evolutionary Strategies of Viruses, Bacteria and Archaea in Hydrothermal Vent Ecosystems Revealed through Metagenomics

PubMed Central

Anderson, Rika E.; Sogin, Mitchell L.; Baross, John A.

2014-01-01

The deep-sea hydrothermal vent habitat hosts a diverse community of archaea and bacteria that withstand extreme fluctuations in environmental conditions. Abundant viruses in these systems, a high proportion of which are lysogenic, must also withstand these environmental extremes. Here, we explore the evolutionary strategies of both microorganisms and viruses in hydrothermal systems through comparative analysis of a cellular and viral metagenome, collected by size fractionation of high temperature fluids from a diffuse flow hydrothermal vent. We detected a high enrichment of mobile elements and proviruses in the cellular fraction relative to microorganisms in other environments. We observed a relatively high abundance of genes related to energy metabolism as well as cofactors and vitamins in the viral fraction compared to the cellular fraction, which suggest encoding of auxiliary metabolic genes on viral genomes. Moreover, the observation of stronger purifying selection in the viral versus cellular gene pool suggests viral strategies that promote prolonged host integration. Our results demonstrate that there is great potential for hydrothermal vent viruses to integrate into hosts, facilitate horizontal gene transfer, and express or transfer genes that manipulate the hosts’ functional capabilities. PMID:25279954

Frequent associations between CTL and T-Helper epitopes in HIV-1 genomes and implications for multi-epitope vaccine designs

PubMed Central

2010-01-01

Background Epitope vaccines have been suggested as a strategy to counteract viral escape and development of drug resistance. Multiple studies have shown that Cytotoxic T-Lymphocyte (CTL) and T-Helper (Th) epitopes can generate strong immune responses in Human Immunodeficiency Virus (HIV-1). However, not much is known about the relationship among different types of HIV epitopes, particularly those epitopes that can be considered potential candidates for inclusion in the multi-epitope vaccines. Results In this study we used association rule mining to examine relationship between different types of epitopes (CTL, Th and antibody epitopes) from nine protein-coding HIV-1 genes to identify strong associations as potent multi-epitope vaccine candidates. Our results revealed 137 association rules that were consistently present in the majority of reference and non-reference HIV-1 genomes and included epitopes of two different types (CTL and Th) from three different genes (Gag, Pol and Nef). These rules involved 14 non-overlapping epitope regions that frequently co-occurred despite high mutation and recombination rates, including in genomes of circulating recombinant forms. These epitope regions were also highly conserved at both the amino acid and nucleotide levels indicating strong purifying selection driven by functional and/or structural constraints and hence, the diminished likelihood of successful escape mutations. Conclusions Our results provide a comprehensive systematic survey of CTL, Th and Ab epitopes that are both highly conserved and co-occur together among all subtypes of HIV-1, including circulating recombinant forms. Several co-occurring epitope combinations were identified as potent candidates for inclusion in multi-epitope vaccines, including epitopes that are immuno-responsive to different arms of the host immune machinery and can enable stronger and more efficient immune responses, similar to responses achieved with adjuvant therapies. Signature of strong purifying selection acting at the nucleotide level of the associated epitopes indicates that these regions are functionally critical, although the exact reasons behind such sequence conservation remain to be elucidated. PMID:20696039

Testing whether Metazoan Tyrosine Loss Was Driven by Selection against Promiscuous Phosphorylation

PubMed Central

Pandya, Siddharth; Struck, Travis J.; Mannakee, Brian K.; Paniscus, Mary; Gutenkunst, Ryan N.

2015-01-01

Protein tyrosine phosphorylation is a key regulatory modification in metazoans, and the corresponding kinase enzymes have diversified dramatically. This diversification is correlated with a genome-wide reduction in protein tyrosine content, and it was recently suggested that this reduction was driven by selection to avoid promiscuous phosphorylation that might be deleterious. We tested three predictions of this intriguing hypothesis. 1) Selection should be stronger on residues that are more likely to be phosphorylated due to local solvent accessibility or structural disorder. 2) Selection should be stronger on proteins that are more likely to be promiscuously phosphorylated because they are abundant. We tested these predictions by comparing distributions of tyrosine within and among human and yeast orthologous proteins. 3) Selection should be stronger against mutations that create tyrosine versus remove tyrosine. We tested this prediction using human population genomic variation data. We found that all three predicted effects are modest for tyrosine when compared with the other amino acids, suggesting that selection against deleterious phosphorylation was not dominant in driving metazoan tyrosine loss. PMID:25312910

Purified glycosaminoglycans from cooked haddock may enhance Fe uptake via endocytosis in a Caco-2 cell culture model

USDA-ARS?s Scientific Manuscript database

This study aims to understand the enhancing effect of glycosaminoglycans (GAGs), such as chondroitin/dermatan structures, on Fe uptake to Caco-2 cells. High sulfated GAGs were selectively purified from cooked haddock. An in vitro digestion/Caco-2 cell culture model was used to evaluate Fe uptake (ce...

Purified ryanodine receptor from rabbit skeletal muscle is the calcium- release channel of sarcoplasmic reticulum

PubMed Central

1988-01-01

The ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was purified as a single 450,000-dalton polypeptide from CHAPS- solubilized triads using immunoaffinity chromatography. The purified receptor had a [3H]ryanodine-binding capacity (Bmax) of 490 pmol/mg and a binding affinity (Kd) of 7.0 nM. Using planar bilayer recording techniques, we show that the purified receptor forms cationic channels selective for divalent ions. Ryanodine receptor channels were identical to the Ca-release channels described in native sarcoplasmic reticulum using the same techniques. In the present work, four criteria were used to establish this identity: (a) activation of channels by micromolar Ca and millimolar ATP and inhibition by micromolar ruthenium red, (b) a main channel conductance of 110 +/- 10 pS in 54 mM trans Ca, (c) a long- term open state of lower unitary conductance induced by ryanodine concentrations as low as 20 nM, and (d) a permeability ratio PCa/PTris approximately equal to 14. In addition, we show that the purified ryanodine receptor channel displays a saturable conductance in both monovalent and divalent cation solutions (gamma max for K and Ca = 1 nS and 172 pS, respectively). In the absence of Ca, channels had a broad selectivity for monovalent cations, but in the presence of Ca, they were selectively permeable to Ca against K by a permeability ratio PCa/PK approximately equal to 6. Receptor channels displayed several equivalent conductance levels, which suggest an oligomeric pore structure. We conclude that the 450,000-dalton polypeptide ryanodine receptor is the Ca-release channel of the sarcoplasmic reticulum and is the target site of ruthenium red and ryanodine. PMID:2459298

Lichen acids may be used as a potential drug for cancer therapy; by inhibiting mitochondrial thioredoxin reductase purified from rat lung.

PubMed

Ozgencli, Ilknur; Budak, Harun; Ciftci, Mehmet; Anar, Mustafa

2018-05-24

Thioredoxin reductase (E.C 1.6.4.5.; TrxR) is a widely distributed flavoprotein that catalyzes the NADPH-dependent reduction of thioredoxin (Trx) in many cellular events such as DNA synthesis, DNA repair, angiogenesis, antioxidative defense, and regulating apoptosis. Although TrxR is indispensible in protecting cells against oxidative stress, the overexpression of TrxR is seen in many aggressive tumors. Therefore, targeted inhibition of TrxR has been accepted as a new approach for chemotherapy. In this study, in vitro inhibition effect of the lichen acids (diffractaic, evernic, lobaric, lecanoric, and vulpinic acid) on mitochondrial TrxR purified from rat lung was investigated. It was the first time the enzyme was purified from rat lungs by using 2', 5'-ADP Sepharose 4B affinity chromatography. The purity of the enzyme was checked with SDS-PAGE. In vitro inhibition effect of the lichen acids was investigated spectrophotometrically. To emphasize the importance of the obtained data, the commercial anticancer drugs cisplatin and doxorubicin were used as positive controls. Molecular mass of the enzyme was calculated as approximately 52.4 kDa. The enzyme was purified with a 63.6% yield, 208.3 fold, and 0.5 EU/mg proteins specific activity. The IC50 values of five lichen acids were significantly lower than IC50 values of anticancer drugs. All of the lichen acids, especially lecanoric and vulpinic acid, exhibited much stronger inhibitory effect on TrxR than the anticancer drugs cisplatin and doxorubicin. These lichen acids have pharmacological potential as effective natural antioxidants, antimicrobials, and anticancer agents. . Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Quercetin and quercetin 3-O-glycosides from Bauhinia longifolia (Bong.) Steud. show anti-Mayaro virus activity.

PubMed

dos Santos, Alda E; Kuster, Ricardo M; Yamamoto, Kristie A; Salles, Tiago S; Campos, Renata; de Meneses, Marcelo D F; Soares, Márcia R; Ferreira, Davis

2014-03-28

The arthropod-borne Mayaro virus (MAYV) causes 'Mayaro fever', a disease of medical significance, primarily affecting individuals in permanent contact with forested areas in tropical South America. Recently, MAYV has attracted attention due to its likely urbanization. Currently, there are no licensed drugs against most mosquito-transmitted viruses. Here, we investigated the in vitro anti-MAYV activity of the flavonoids quercetin and its derivatives from the Brazilian shrub Bauhinia longifolia (Bong.) Steud. Flavonoids were purified by chromatographic fractionation from leaf extracts of B. longifolia and chemically identified as quercetin and quercetin glycosides using spectroscopic techniques. Cytotoxicity of purified flavonoids and of EtOAc- and n-BuOH-containing flavonoid mixtures was measured by the dye-uptake assay while their antiviral activity was evaluated by a virus yield inhibition assay. The following flavonoids were purified from B. longifolia leaves: non-glycosylated quercetin and its glycosides guaijaverin, quercitrin, isoquercitrin, and hyperin. EtOAc and n-BuOH fractions containing these flavonoids demonstrated the highest antiviral activity of all tested substances, while quercetin had the highest antiviral activity amongst purified flavonoids. Quercetin, EtOAc, or n-BuOH fractions inhibited MAYV production by more than 90% at 25 μg/mL, displaying a stronger antiviral effect than the licensed antiviral ribavirin. A mixture of the isomers isoquercitrin and hyperin had a modest antiviral effect (IC90 = 104.9), while guaijaverin and quercitrin did not show significant antiviral activity. B. longifolia is a good source of flavonoids with anti-Mayaro virus activity. This is the first report of the activity of quercetin and its derivatives against an alphavirus.

Quercetin and quercetin 3-O-glycosides from Bauhinia longifolia (Bong.) Steud. show anti-Mayaro virus activity

PubMed Central

2014-01-01

Background The arthropod-borne Mayaro virus (MAYV) causes ‘Mayaro fever’, a disease of medical significance, primarily affecting individuals in permanent contact with forested areas in tropical South America. Recently, MAYV has attracted attention due to its likely urbanization. Currently, there are no licensed drugs against most mosquito-transmitted viruses. Here, we investigated the in vitro anti-MAYV activity of the flavonoids quercetin and its derivatives from the Brazilian shrub Bauhinia longifolia (Bong.) Steud. Methods Flavonoids were purified by chromatographic fractionation from leaf extracts of B. longifolia and chemically identified as quercetin and quercetin glycosides using spectroscopic techniques. Cytotoxicity of purified flavonoids and of EtOAc- and n-BuOH-containing flavonoid mixtures was measured by the dye-uptake assay while their antiviral activity was evaluated by a virus yield inhibition assay. Results The following flavonoids were purified from B. longifolia leaves: non-glycosylated quercetin and its glycosides guaijaverin, quercitrin, isoquercitrin, and hyperin. EtOAc and n-BuOH fractions containing these flavonoids demonstrated the highest antiviral activity of all tested substances, while quercetin had the highest antiviral activity amongst purified flavonoids. Quercetin, EtOAc, or n-BuOH fractions inhibited MAYV production by more than 90% at 25 μg/mL, displaying a stronger antiviral effect than the licensed antiviral ribavirin. A mixture of the isomers isoquercitrin and hyperin had a modest antiviral effect (IC90 = 104.9), while guaijaverin and quercitrin did not show significant antiviral activity. Conclusions B. longifolia is a good source of flavonoids with anti-Mayaro virus activity. This is the first report of the activity of quercetin and its derivatives against an alphavirus. PMID:24678592

INVESTIGATIONS ON THE ANTIGENICITY OF SNAKE VENOMS

DTIC Science & Technology

The chemical composition of viperotoxin, the neurotoxic protein isolated from the venom of Vipera palestinae has been determined. Viperotoxin is...carboxy-terminal position of the viperotoxin chain. Vipera palestinae hemorrhagin has been purified and isolated. Further tests have established that...active sites, or selective blocking of a part of one active center having two distinct biological activities. Purified neurotoxin of V. palestinae

Methods for purifying carbon materials

DOEpatents

Dailly, Anne [Pasadena, CA; Ahn, Channing [Pasadena, CA; Yazami, Rachid [Los Angeles, CA; Fultz, Brent T [Pasadena, CA

2009-05-26

Methods of purifying samples are provided that are capable of removing carbonaceous and noncarbonaceous impurities from a sample containing a carbon material having a selected structure. Purification methods are provided for removing residual metal catalyst particles enclosed in multilayer carbonaceous impurities in samples generate by catalytic synthesis methods. Purification methods are provided wherein carbonaceous impurities in a sample are at least partially exfoliated, thereby facilitating subsequent removal of carbonaceous and noncarbonaceous impurities from the sample. Methods of purifying carbon nanotube-containing samples are provided wherein an intercalant is added to the sample and subsequently reacted with an exfoliation initiator to achieve exfoliation of carbonaceous impurities.

Selection by pollinators on floral traits in generalized Trollius ranunculoides (Ranunculaceae) along altitudinal gradients.

PubMed

Zhao, Zhi-Gang; Wang, Yi-Ke

2015-01-01

Abundance and visitation of pollinator assemblages tend to decrease with altitude, leading to an increase in pollen limitation. Thus increased competition for pollinators may generate stronger selection on attractive traits of flowers at high elevations and cause floral adaptive evolution. Few studies have related geographically variable selection from pollinators and intraspecific floral differentiation. We investigated the variation of Trollius ranunculoides flowers and its pollinators along an altitudinal gradient on the eastern Qinghai-Tibet Plateau, and measured phenotypic selection by pollinators on floral traits across populations. The results showed significant decline of visitation rate of bees along altitudinal gradients, while flies was unchanged. When fitness is estimated by the visitation rate rather than the seed number per plant, phenotypic selection on the sepal length and width shows a significant correlation between the selection strength and the altitude, with stronger selection at higher altitudes. However, significant decreases in the sepal length and width of T. ranunculoides along the altitudinal gradient did not correspond to stronger selection of pollinators. In contrast to the pollinator visitation, mean annual precipitation negatively affected the sepal length and width, and contributed more to geographical variation in measured floral traits than the visitation rate of pollinators. Therefore, the sepal size may have been influenced by conflicting selection pressures from biotic and abiotic selective agents. This study supports the hypothesis that lower pollinator availability at high altitude can intensify selection on flower attractive traits, but abiotic selection is preventing a response to selection from pollinators.

Selection by Pollinators on Floral Traits in Generalized Trollius ranunculoides (Ranunculaceae) along Altitudinal Gradients

PubMed Central

Zhao, Zhi-Gang; Wang, Yi-Ke

2015-01-01

Abundance and visitation of pollinator assemblages tend to decrease with altitude, leading to an increase in pollen limitation. Thus increased competition for pollinators may generate stronger selection on attractive traits of flowers at high elevations and cause floral adaptive evolution. Few studies have related geographically variable selection from pollinators and intraspecific floral differentiation. We investigated the variation of Trollius ranunculoides flowers and its pollinators along an altitudinal gradient on the eastern Qinghai-Tibet Plateau, and measured phenotypic selection by pollinators on floral traits across populations. The results showed significant decline of visitation rate of bees along altitudinal gradients, while flies was unchanged. When fitness is estimated by the visitation rate rather than the seed number per plant, phenotypic selection on the sepal length and width shows a significant correlation between the selection strength and the altitude, with stronger selection at higher altitudes. However, significant decreases in the sepal length and width of T. ranunculoides along the altitudinal gradient did not correspond to stronger selection of pollinators. In contrast to the pollinator visitation, mean annual precipitation negatively affected the sepal length and width, and contributed more to geographical variation in measured floral traits than the visitation rate of pollinators. Therefore, the sepal size may have been influenced by conflicting selection pressures from biotic and abiotic selective agents. This study supports the hypothesis that lower pollinator availability at high altitude can intensify selection on flower attractive traits, but abiotic selection is preventing a response to selection from pollinators. PMID:25692295

Massive expansion and differential evolution of small heat shock proteins with wheat (Triticum aestivum L.) polyploidization.

PubMed

Wang, Xiaoming; Wang, Ruochen; Ma, Chuang; Shi, Xue; Liu, Zhenshan; Wang, Zhonghua; Sun, Qixin; Cao, Jun; Xu, Shengbao

2017-05-31

Wheat (Triticum aestivum), one of the world's most important crops, is facing unprecedented challenges due to global warming. To evaluate the gene resources for heat adaptation in hexaploid wheat, small heat shock proteins (sHSPs), the key plant heat protection genes, were comprehensively analysed in wheat and related species. We found that the sHSPs of hexaploid wheat were massively expanded in A and B subgenomes with intrachromosomal duplications during polyploidization. These expanded sHSPs were under similar purifying selection and kept the expressional patterns with the original copies. Generally, a strong purifying selection acted on the α-crystallin domain (ACD) and theoretically constrain conserved function. Meanwhile, weaker purifying selection and strong positive selection acted on the N-terminal region, which conferred sHSP flexibility, allowing adjustments to a wider range of substrates in response to genomic and environmental changes. Notably, in CI, CV, ER, MI and MII subfamilies, gene duplications, expression variations and functional divergence occurred before wheat polyploidization. Our results indicate the massive expansion of active sHSPs in hexaploid wheat may also provide more raw materials for evolving functional novelties and generating genetic diversity to face future global climate changes, and highlight the expansion of stress response genes with wheat polyploidization.

Mitogenome Sequencing in the Genus Camelus Reveals Evidence for Purifying Selection and Long-term Divergence between Wild and Domestic Bactrian Camels.

PubMed

Mohandesan, Elmira; Fitak, Robert R; Corander, Jukka; Yadamsuren, Adiya; Chuluunbat, Battsetseg; Abdelhadi, Omer; Raziq, Abdul; Nagy, Peter; Stalder, Gabrielle; Walzer, Chris; Faye, Bernard; Burger, Pamela A

2017-08-30

The genus Camelus is an interesting model to study adaptive evolution in the mitochondrial genome, as the three extant Old World camel species inhabit hot and low-altitude as well as cold and high-altitude deserts. We sequenced 24 camel mitogenomes and combined them with three previously published sequences to study the role of natural selection under different environmental pressure, and to advance our understanding of the evolutionary history of the genus Camelus. We confirmed the heterogeneity of divergence across different components of the electron transport system. Lineage-specific analysis of mitochondrial protein evolution revealed a significant effect of purifying selection in the concatenated protein-coding genes in domestic Bactrian camels. The estimated dN/dS < 1 in the concatenated protein-coding genes suggested purifying selection as driving force for shaping mitogenome diversity in camels. Additional analyses of the functional divergence in amino acid changes between species-specific lineages indicated fixed substitutions in various genes, with radical effects on the physicochemical properties of the protein products. The evolutionary time estimates revealed a divergence between domestic and wild Bactrian camels around 1.1 [0.58-1.8] million years ago (mya). This has major implications for the conservation and management of the critically endangered wild species, Camelus ferus.

Selection is stronger in early-versus-late stages of divergence in a Neotropical livebearing fish.

PubMed

Ingley, Spencer J; Johnson, Jerald B

2016-03-01

How selection acts to drive trait evolution at different stages of divergence is of fundamental importance in our understanding of the origins of biodiversity. Yet, most studies have focused on a single point along an evolutionary trajectory. Here, we provide a case study evaluating the strength of divergent selection acting on life-history traits at early-versus-late stages of divergence in Brachyrhaphis fishes. We find that the difference in selection is stronger in the early-diverged population than the late-diverged population, and that trait differences acquired early are maintained over time. © 2016 The Author(s).

Duplication and population dynamics shape historic patterns of selection and genetic variation at the major histocompatibility complex in rodents

PubMed Central

Winternitz, Jamie C; Wares, John P

2013-01-01

Genetic variation at the major histocompatibility complex (MHC) is vitally important for wildlife populations to respond to pathogen threats. As natural populations can fluctuate greatly in size, a key issue concerns how population cycles and bottlenecks that could reduce genetic diversity will influence MHC genes. Using 454 sequencing, we characterized genetic diversity at the DRB Class II locus in montane voles (Microtus montanus), a North American rodent that regularly undergoes high-amplitude fluctuations in population size. We tested for evidence of historic balancing selection, recombination, and gene duplication to identify mechanisms maintaining allelic diversity. Counter to our expectations, we found strong evidence of purifying selection acting on the DRB locus in montane voles. We speculate that the interplay between population fluctuations and gene duplication might be responsible for the weak evidence of historic balancing selection and strong evidence of purifying selection detected. To further explore this idea, we conducted a phylogenetically controlled comparative analysis across 16 rodent species with varying demographic histories and MHC duplication events (based on the maximum number of alleles detected per individual). On the basis of phylogenetic generalized linear model-averaging, we found evidence that the estimated number of duplicated loci was positively related to allelic diversity and, surprisingly, to the strength of purifying selection at the DRB locus. Our analyses also revealed that species that had undergone population bottlenecks had lower allelic richness than stable species. This study highlights the need to consider demographic history and genetic structure alongside patterns of natural selection to understand resulting patterns of genetic variation at the MHC. PMID:23789067

Immune Selection In Vitro Reveals Human Immunodeficiency Virus Type 1 Nef Sequence Motifs Important for Its Immune Evasion Function In Vivo

PubMed Central

Lee, Patricia; Ng, Hwee L.; Yang, Otto O.

2012-01-01

Human immunodeficiency virus type 1 (HIV-1) Nef downregulates major histocompatibility complex class I (MHC-I), impairing the clearance of infected cells by CD8+ cytotoxic T lymphocytes (CTLs). While sequence motifs mediating this function have been determined by in vitro mutagenesis studies of laboratory-adapted HIV-1 molecular clones, it is unclear whether the highly variable Nef sequences of primary isolates in vivo rely on the same sequence motifs. To address this issue, nef quasispecies from nine chronically HIV-1-infected persons were examined for sequence evolution and altered MHC-I downregulatory function under Gag-specific CTL immune pressure in vitro. This selection resulted in decreased nef diversity and strong purifying selection. Site-by-site analysis identified 13 codons undergoing purifying selection and 1 undergoing positive selection. Of the former, only 6 have been reported to have roles in Nef function, including 4 associated with MHC-I downregulation. Functional testing of naturally occurring in vivo polymorphisms at the 7 sites with no previously known functional role revealed 3 mutations (A84D, Y135F, and G140R) that ablated MHC-I downregulation and 3 (N52A, S169I, and V180E) that partially impaired MHC-I downregulation. Globally, the CTL pressure in vitro selected functional Nef from the in vivo quasispecies mixtures that predominately lacked MHC-I downregulatory function at the baseline. Overall, these data demonstrate that CTL pressure exerts a strong purifying selective pressure for MHC-I downregulation and identifies novel functional motifs present in Nef sequences in vivo. PMID:22553319

Pseudomonas aeruginosa arylsulfatase: a purified enzyme for the mild hydrolysis of steroid sulfates.

PubMed

Stevenson, Bradley J; Waller, Christopher C; Ma, Paul; Li, Kunkun; Cawley, Adam T; Ollis, David L; McLeod, Malcolm D

2015-10-01

The hydrolysis of sulfate ester conjugates is frequently required prior to analysis for a range of analytical techniques including gas chromatography-mass spectrometry (GC-MS). Sulfate hydrolysis may be achieved with commercial crude arylsulfatase enzyme preparations such as that derived from Helix pomatia but these contain additional enzyme activities such as glucuronidase, oxidase, and reductase that make them unsuitable for many analytical applications. Strong acid can also be used to hydrolyze sulfate esters but this can lead to analyte degradation or increased matrix interference. In this work, the heterologously expressed and purified arylsulfatase from Pseudomonas aeruginosa is shown to promote the mild enzyme-catalyzed hydrolysis of a range of steroid sulfates. The substrate scope of this P. aeruginosa arylsulfatase hydrolysis is compared with commercial crude enzyme preparations such as that derived from H. pomatia. A detailed kinetic comparison is reported for selected examples. Hydrolysis in a urine matrix is demonstrated for dehydroepiandrosterone 3-sulfate and epiandrosterone 3-sulfate. The purified P. aeruginosa arylsulfatase contains only sulfatase activity allowing for the selective hydrolysis of sulfate esters in the presence of glucuronide conjugates as demonstrated in the short three-step chemoenzymatic synthesis of 5α-androstane-3β,17β-diol 17-glucuronide (ADG, 1) from epiandrosterone 3-sulfate. The P. aeruginosa arylsulfatase is readily expressed and purified (0.9 g per L of culture) and thus provides a new and selective method for the hydrolysis of steroid sulfate esters in analytical sample preparation. Copyright © 2015 John Wiley & Sons, Ltd.

Reconstitution and functional comparison of purified GlpF and AqpZ, the glycerol and water channels from Escherichia coli.

PubMed

Borgnia, M J; Agre, P

2001-02-27

A large family of membrane channel proteins selective for transport of water (aquaporins) or water plus glycerol (aquaglyceroporins) has been found in diverse life forms. Escherichia coli has two members of this family-a water channel, AqpZ, and a glycerol facilitator, GlpF. Despite having similar primary amino acid sequences and predicted structures, the oligomeric state and solute selectivity of AqpZ and GlpF are disputed. Here we report biochemical and functional characterizations of affinity-purified GlpF and compare it to AqpZ. Histidine-tagged (His-GlpF) and hemagglutinin-tagged (HA-GlpF) polypeptides encoded by a bicistronic construct were expressed in bacteria. HA-GlpF and His-GlpF appear to form oligomers during Ni-nitrilotriacetate affinity purification. Sucrose gradient sedimentation analyses showed that the oligomeric state of octyl glucoside-solubilized GlpF varies: low ionic strength favors subunit dissociation, whereas Mg(2+) stabilizes tetrameric assembly. Reconstitution of affinity-purified GlpF into proteoliposomes increases glycerol permeability more than 100-fold and water permeability up to 10-fold compared with control liposomes. Glycerol and water permeability of GlpF both occur with low Arrhenius activation energies and are reversibly inhibited by HgCl(2). Our studies demonstrate that, unlike AqpZ, a water-selective stable tetramer, purified GlpF exists in multiple oligomeric forms under nondenaturing conditions and is highly permeable to glycerol but less well permeated by water.

Detection of a lysosomal carboxypeptidase and a lysosomal dipeptidase in highly-purified dipeptidyl aminopeptidase I /cathepsin C/ and the elimination of their activities from preparations used to sequence peptides.

NASA Technical Reports Server (NTRS)

Mcdonald, J. K.; Zeitman, B. B.; Ellis, S.

1972-01-01

Description of the properties of a carboxypeptidase, termed 'catheptic carboxypeptidase C,' and a dipeptidase, termed 'Ser-Met dipeptidase.' Both were found in highly purified DAP I from either beef spleen or rat liver. Methods are described for the removal or selective inactivation of these contaminating enzymes.

HDL-associated dehydroepiandrosterone fatty acyl esters: enhancement of vasodilatory effect of HDL.

PubMed

Paatela, Hanna; Mervaala, Eero; Deb, Somdatta; Wähälä, Kristiina; Tikkanen, Matti J

2009-10-01

Dehydroepiandrosterone (DHEA) and high-density lipoprotein (HDL) are both vascular relaxants. In the circulation, HDL transports DHEA fatty acyl esters (DHEA-FAEs), which are naturally occurring lipophilic derivatives of DHEA. We studied in isolated rat mesenteric arteries whether HDL-associated DHEA-FAE improves the vasodilatory effect of HDL. To prepare DHEA-FAE-enriched HDL, we incubated DHEA with human plasma. After incubation, HDL was isolated, purified, and added in cumulative doses (0.1-125 microg/ml) to noradrenaline-precontracted rat arterial rings. DHEA-FAE-enriched HDL caused a dose-dependent relaxation (maximal 43+/-4%), which was significantly stronger than the effect of HDL from the control incubation without addition of DHEA (25+/-2%, p<0.001). When plasma incubation of DHEA was carried out in the presence of lecithin:cholesterol acyltransferase (LCAT) inhibitor, the relaxation response to HDL (25+/-3%) did not differ from the control HDL (p=0.98). Pretreatment of the arterial rings with nitric oxide synthase (NOS) antagonist impaired the relaxation response to DHEA-FAE-enriched HDL (43+/-4% vs. 30+/-3%, p=0.008). Similar experiments were performed with 17beta-estradiol (E(2)). Compared to control HDL, E(2)-FAE-enriched HDL induced slightly but non-significantly stronger relaxation. DHEA-FAE-enriched HDL was a stronger vasodilator than native HDL, and vascular relaxation was in part mediated by NOS, suggesting that DHEA-FAE may improve HDL's antiatherogenic function.

Mitigating Mitochondrial Genome Erosion Without Recombination.

PubMed

Radzvilavicius, Arunas L; Kokko, Hanna; Christie, Joshua R

2017-11-01

Mitochondria are ATP-producing organelles of bacterial ancestry that played a key role in the origin and early evolution of complex eukaryotic cells. Most modern eukaryotes transmit mitochondrial genes uniparentally, often without recombination among genetically divergent organelles. While this asymmetric inheritance maintains the efficacy of purifying selection at the level of the cell, the absence of recombination could also make the genome susceptible to Muller's ratchet. How mitochondria escape this irreversible defect accumulation is a fundamental unsolved question. Occasional paternal leakage could in principle promote recombination, but it would also compromise the purifying selection benefits of uniparental inheritance. We assess this tradeoff using a stochastic population-genetic model. In the absence of recombination, uniparental inheritance of freely-segregating genomes mitigates mutational erosion, while paternal leakage exacerbates the ratchet effect. Mitochondrial fusion-fission cycles ensure independent genome segregation, improving purifying selection. Paternal leakage provides opportunity for recombination to slow down the mutation accumulation, but always at a cost of increased steady-state mutation load. Our findings indicate that random segregation of mitochondrial genomes under uniparental inheritance can effectively combat the mutational meltdown, and that homologous recombination under paternal leakage might not be needed. Copyright © 2017 by the Genetics Society of America.

Pleistocene divergence across a mountain range and the influence of selection on mitogenome evolution in threatened Australian freshwater cod species.

PubMed

Harrisson, K; Pavlova, A; Gan, H M; Lee, Y P; Austin, C M; Sunnucks, P

2016-06-01

Climatic differences across a taxon's range may be associated with specific bioenergetic demands and may result in genetics-based metabolic adaptation, particularly in aquatic ectothermic organisms that rely on heat exchange with the environment to regulate key physiological processes. Extending down the east coast of Australia, the Great Dividing Range (GDR) has a strong influence on climate and the evolutionary history of freshwater fish species. Despite the GDR acting as a strong contemporary barrier to fish movement, many species, and species with shared ancestries, are found on both sides of the GDR, indicative of historical dispersal events. We sequenced complete mitogenomes from the four extant species of the freshwater cod genus Maccullochella, two of which occur on the semi-arid, inland side of the GDR, and two on the mesic coastal side. We constructed a dated phylogeny and explored the relative influences of purifying and positive selection in the evolution of mitogenome divergence among species. Results supported mid- to late-Pleistocene divergence of Maccullochella across the GDR (220-710 thousand years ago), bringing forward previously reported dates. Against a background of pervasive purifying selection, we detected potentially functionally relevant fixed amino acid differences across the GDR. Although many amino acid differences between inland and coastal species may have become fixed under relaxed purifying selection in coastal environments rather than positive selection, there was evidence of episodic positive selection acting on specific codons in the Mary River coastal lineage, which has consistently experienced the warmest and least extreme climate in the genus.

Pleistocene divergence across a mountain range and the influence of selection on mitogenome evolution in threatened Australian freshwater cod species

PubMed Central

Harrisson, K; Pavlova, A; Gan, H M; Lee, Y P; Austin, C M; Sunnucks, P

2016-01-01

Climatic differences across a taxon's range may be associated with specific bioenergetic demands and may result in genetics-based metabolic adaptation, particularly in aquatic ectothermic organisms that rely on heat exchange with the environment to regulate key physiological processes. Extending down the east coast of Australia, the Great Dividing Range (GDR) has a strong influence on climate and the evolutionary history of freshwater fish species. Despite the GDR acting as a strong contemporary barrier to fish movement, many species, and species with shared ancestries, are found on both sides of the GDR, indicative of historical dispersal events. We sequenced complete mitogenomes from the four extant species of the freshwater cod genus Maccullochella, two of which occur on the semi-arid, inland side of the GDR, and two on the mesic coastal side. We constructed a dated phylogeny and explored the relative influences of purifying and positive selection in the evolution of mitogenome divergence among species. Results supported mid- to late-Pleistocene divergence of Maccullochella across the GDR (220–710 thousand years ago), bringing forward previously reported dates. Against a background of pervasive purifying selection, we detected potentially functionally relevant fixed amino acid differences across the GDR. Although many amino acid differences between inland and coastal species may have become fixed under relaxed purifying selection in coastal environments rather than positive selection, there was evidence of episodic positive selection acting on specific codons in the Mary River coastal lineage, which has consistently experienced the warmest and least extreme climate in the genus. PMID:26883183

Antibacterial, antifungal and anticoagulant activities of chicken PLA2 group V expressed in Pichia pastoris.

PubMed

Karray, Aida; Bou Ali, Madiha; Kharrat, Nedia; Gargouri, Youssef; Bezzine, Sofiane

2018-03-01

Secretory class V phospholipase A2 (PLA2-V) has been shown to be involved in inflammatory processes in cellular studies, but the biochemical and physical properties of this important enzyme have been unclear. As a first step towards understanding the structure, function and regulation of this PLA2, we report the expression and characterization of PLA2-V from chicken (ChPLA2-V). The ChPLA2-V cDNA was synthesized from chicken heart polyA mRNA by RT-PCR, and an expression construct containing the PLA2 was established. After expression in Pichia pastoris cells, the active enzyme was purified. The purified ChPLA2-V protein was biochemically and physiologically characterized. The recombinant ChPLA2-V has an absolute requirement for Ca 2+ for enzymatic activity. The optimum pH for this enzyme is pH 8.5 in Tris-HCl buffer with phosphatidylcholine as substrate. ChPLA2-V was found to display potent Gram-positive and Gram-negative bactericidal activity and antifungal activity in vitro. The purified enzyme ChPLA2-V with much stronger anticoagulant activity compared with the intestinal and pancreatic chicken PLA2-V was approximately 10 times more active. Chicken group V PLA2, like mammal one, may be considered as a future therapeutic agents against fungal and bacterial infections and as an anticoagulant agent. Copyright © 2017. Published by Elsevier B.V.

Nucleotide substitutions in dengue virus serotypes from Asian and American countries: insights into intracodon recombination and purifying selection

PubMed Central

2013-01-01

Background Dengue virus (DENV) infection represents a significant public health problem in many subtropical and tropical countries. Although genetically closely related, the four serotypes of DENV differ in antigenicity for which cross protection among serotypes is limited. It is also believed that both multi-serotype infection as well as the evolution of viral antigenicity may have confounding effects in increased dengue epidemics. Numerous studies have been performed that investigated genetic diversity of DENV, but the precise mechanism(s) of dengue virus evolution are not well understood. Results We investigated genome-wide genetic diversity and nucleotide substitution patterns in the four serotypes among samples collected from different countries in Asia and Central and South America and sequenced as part of the Genome Sequencing Center for Infectious Diseases at the Broad Institute. We applied bioinformatics, statistical and coalescent simulation methods to investigate diversity of codon sequences of DENV samples representing the four serotypes. We show that fixation of nucleotide substitutions is more prominent among the inter-continental isolates (Asian and American) of serotypes 1, 2 and 3 compared to serotype 4 isolates (South and Central America) and are distributed in a non-random manner among the genes encoded by the virus. Nearly one third of the negatively selected sites are associated with fixed mutation sites within serotypes. Our results further show that of all the sites showing evidence of recombination, the majority (~84%) correspond to sites under purifying selection in the four serotypes. The analysis further shows that genetic recombination occurs within specific codons, albeit with low frequency (< 5% of all recombination sites) throughout the DENV genome of the four serotypes and reveals significant enrichment (p < 0.05) among sites under purifying selection in the virus. Conclusion The study provides the first evidence for intracodon recombination in DENV and suggests that within codons, genetic recombination has a significant role in maintaining extensive purifying selection of DENV in natural populations. Our study also suggests that fixation of beneficial mutations may lead to virus evolution via translational selection of specific sites in the DENV genome. PMID:23410119

"Orphan" retrogenes in the human genome.

PubMed

Ciomborowska, Joanna; Rosikiewicz, Wojciech; Szklarczyk, Damian; Makałowski, Wojciech; Makałowska, Izabela

2013-02-01

Gene duplicates generated via retroposition were long thought to be pseudogenized and consequently decayed. However, a significant number of these genes escaped their evolutionary destiny and evolved into functional genes. Despite multiple studies, the number of functional retrogenes in human and other genomes remains unclear. We performed a comparative analysis of human, chicken, and worm genomes to identify "orphan" retrogenes, that is, retrogenes that have replaced their progenitors. We located 25 such candidates in the human genome. All of these genes were previously known, and the majority has been intensively studied. Despite this, they have never been recognized as retrogenes. Analysis revealed that the phenomenon of replacing parental genes with their retrocopies has been taking place over the entire span of animal evolution. This process was often species specific and contributed to interspecies differences. Surprisingly, these retrogenes, which should evolve in a more relaxed mode, are subject to a very strong purifying selection, which is, on average, two and a half times stronger than other human genes. Also, for retrogenes, they do not show a typical overall tendency for a testis-specific expression. Notably, seven of them are associated with human diseases. Recognizing them as "orphan" retrocopies, which have different regulatory machinery than their parents, is important for any disease studies in model organisms, especially when discoveries made in one species are transferred to humans.

Sexual selection predicts advancement of avian spring migration in response to climate change.

PubMed

Spottiswoode, Claire N; Tøttrup, Anders P; Coppack, Timothy

2006-12-22

Global warming has led to earlier spring arrival of migratory birds, but the extent of this advancement varies greatly among species, and it remains uncertain to what degree these changes are phenotypically plastic responses or microevolutionary adaptations to changing environmental conditions. We suggest that sexual selection could help to understand this variation, since early spring arrival of males is favoured by female choice. Climate change could weaken the strength of natural selection opposing sexual selection for early migration, which would predict greatest advancement in species with stronger female choice. We test this hypothesis comparatively by investigating the degree of long-term change in spring passage at two ringing stations in northern Europe in relation to a synthetic estimate of the strength of female choice, composed of degree of extra-pair paternity, relative testes size and degree of sexually dichromatic plumage colouration. We found that species with a stronger index of sexual selection have indeed advanced their date of spring passage to a greater extent. This relationship was stronger for the changes in the median passage date of the whole population than for changes in the timing of first-arriving individuals, suggesting that selection has not only acted on protandrous males. These results suggest that sexual selection may have an impact on the responses of organisms to climate change, and knowledge of a species' mating system might help to inform attempts at predicting these.

Arrayed antibody library technology for therapeutic biologic discovery.

PubMed

Bentley, Cornelia A; Bazirgan, Omar A; Graziano, James J; Holmes, Evan M; Smider, Vaughn V

2013-03-15

Traditional immunization and display antibody discovery methods rely on competitive selection amongst a pool of antibodies to identify a lead. While this approach has led to many successful therapeutic antibodies, targets have been limited to proteins which are easily purified. In addition, selection driven discovery has produced a narrow range of antibody functionalities focused on high affinity antagonism. We review the current progress in developing arrayed protein libraries for screening-based, rather than selection-based, discovery. These single molecule per microtiter well libraries have been screened in multiplex formats against both purified antigens and directly against targets expressed on the cell surface. This facilitates the discovery of antibodies against therapeutically interesting targets (GPCRs, ion channels, and other multispanning membrane proteins) and epitopes that have been considered poorly accessible to conventional discovery methods. Copyright © 2013. Published by Elsevier Inc.

High mobility high efficiency organic films based on pure organic materials

DOEpatents

Salzman, Rhonda F [Ann Arbor, MI; Forrest, Stephen R [Ann Arbor, MI

2009-01-27

A method of purifying small molecule organic material, performed as a series of operations beginning with a first sample of the organic small molecule material. The first step is to purify the organic small molecule material by thermal gradient sublimation. The second step is to test the purity of at least one sample from the purified organic small molecule material by spectroscopy. The third step is to repeat the first through third steps on the purified small molecule material if the spectroscopic testing reveals any peaks exceeding a threshold percentage of a magnitude of a characteristic peak of a target organic small molecule. The steps are performed at least twice. The threshold percentage is at most 10%. Preferably the threshold percentage is 5% and more preferably 2%. The threshold percentage may be selected based on the spectra of past samples that achieved target performance characteristics in finished devices.

Experimental evolution of a sexually selected display in yeast

PubMed Central

Rogers, David W.; Greig, Duncan

2008-01-01

The fundamental principle underlying sexual selection theory is that an allele conferring an advantage in the competition for mates will spread through a population. Remarkably, this has never been demonstrated empirically. We have developed an experimental system using yeast for testing genetic models of sexual selection. Yeast signal to potential partners by producing an attractive pheromone; stronger signallers are preferred as mates. We tested the effect of high and low levels of sexual selection on the evolution of a gene determining the strength of this signal. Under high sexual selection, an allele encoding a stronger signal was able to invade a population of weak signallers, and we observed a corresponding increase in the amount of pheromone produced. By contrast, the strong signalling allele failed to invade under low sexual selection. Our results demonstrate, for the first time, the spread of a sexually selected allele through a population, confirming the central assumption of sexual selection theory. Our yeast system is a powerful tool for investigating the genetics of sexual selection. PMID:18842545

Photovoltaic performance of TiO2 electrode adsorbed with gardenia yellow purified by nonionic polymeric sorbent in dye-sensitized solar cells.

PubMed

Kwon, Oh Oun; Kim, Eui Jin; Lee, Jae Hyeok; Kim, Tae Young; Park, Kyung Hee; Kim, Sang Yook; Suh, Hwa Jin; Lee, Hyo Jung; Lee, Jae Wook

2015-02-05

To improve the photovoltaic conversion efficiency in dye-sensitized solar cells (DSSCs), TiO2 electrode adsorbed with gardenia yellow purified by nonionic polymeric sorbent was successfully formulated on nanoporous TiO2 surface. Adsorption and desorption properties of crude gardenia yellow solution on a macroporous resin, XAD-1600, were investigated to purify gardenia yellow because of its strong adsorption and desorption abilities as well as high selectivity. To this end, adsorption equilibrium and kinetic data were measured and fitted using adsorption isotherms and kinetic models. Adsorption and desorption breakthrough curves in a column packed with XAD-1600 resin was obtained to optimize the separation process of gardenia yellow. The photovoltaic performance of the photo-electrode adsorbed with the crude and purified gardenia yellow in DSSCs was compared from current-voltage measurements. The results showed that the photovoltaic conversion efficiency was highly dependent on how to separate and purify gardenia yellow as a photosensitizer. Copyright © 2014 Elsevier B.V. All rights reserved.

Origin of a function by tandem gene duplication limits the evolutionary capability of its sister copy.

PubMed

Hasselmann, Martin; Lechner, Sarah; Schulte, Christina; Beye, Martin

2010-07-27

The most remarkable outcome of a gene duplication event is the evolution of a novel function. Little information exists on how the rise of a novel function affects the evolution of its paralogous sister gene copy, however. We studied the evolution of the feminizer (fem) gene from which the gene complementary sex determiner (csd) recently derived by tandem duplication within the honey bee (Apis) lineage. Previous studies showed that fem retained its sex determination function, whereas the rise of csd established a new primary signal of sex determination. We observed a specific reduction of nonsynonymous to synonymous substitution ratios in Apis to non-Apis fem. We found a contrasting pattern at two other genetically linked genes, suggesting that hitchhiking effects to csd, the locus under balancing selection, is not the cause of this evolutionary pattern. We also excluded higher synonymous substitution rates by relative rate testing. These results imply that stronger purifying selection is operating at the fem gene in the presence of csd. We propose that csd's new function interferes with the function of Fem protein, resulting in molecular constraints and limited evolvability of fem in the Apis lineage. Elevated silent nucleotide polymorphism in fem relative to the genome-wide average suggests that genetic linkage to the csd gene maintained more nucleotide variation in today's population. Our findings provide evidence that csd functionally and genetically interferes with fem, suggesting that a newly evolved gene and its functions can limit the evolutionary capability of other genes in the genome.

Essentiality, conservation, evolutionary pressure and codon bias in bacterial genomes.

PubMed

Dilucca, Maddalena; Cimini, Giulio; Giansanti, Andrea

2018-07-15

Essential genes constitute the core of genes which cannot be mutated too much nor lost along the evolutionary history of a species. Natural selection is expected to be stricter on essential genes and on conserved (highly shared) genes, than on genes that are either nonessential or peculiar to a single or a few species. In order to further assess this expectation, we study here how essentiality of a gene is connected with its degree of conservation among several unrelated bacterial species, each one characterised by its own codon usage bias. Confirming previous results on E. coli, we show the existence of a universal exponential relation between gene essentiality and conservation in bacteria. Moreover, we show that, within each bacterial genome, there are at least two groups of functionally distinct genes, characterised by different levels of conservation and codon bias: i) a core of essential genes, mainly related to cellular information processing; ii) a set of less conserved nonessential genes with prevalent functions related to metabolism. In particular, the genes in the first group are more retained among species, are subject to a stronger purifying conservative selection and display a more limited repertoire of synonymous codons. The core of essential genes is close to the minimal bacterial genome, which is in the focus of recent studies in synthetic biology, though we confirm that orthologs of genes that are essential in one species are not necessarily essential in other species. We also list a set of highly shared genes which, reasonably, could constitute a reservoir of targets for new anti-microbial drugs. Copyright © 2018 Elsevier B.V. All rights reserved.

Hemizygosity Enhances Purifying Selection: Lack of Fast-Z Evolution in Two Satyrine Butterflies.

PubMed

Rousselle, Marjolaine; Faivre, Nicolas; Ballenghien, Marion; Galtier, Nicolas; Nabholz, Benoit

2016-10-23

The fixation probability of a recessive beneficial mutation is increased on the X or Z chromosome, relative to autosomes, because recessive alleles carried by X or Z are exposed to selection in the heterogametic sex. This leads to an increased dN/dS ratio on sex chromosomes relative to autosomes, a pattern called the "fast-X" or "fast-Z" effect. Besides positive selection, the strength of genetic drift and the efficacy of purifying selection, which affect the rate of molecular evolution, might differ between sex chromosomes and autosomes. Disentangling the complex effects of these distinct forces requires the genome-wide analysis of polymorphism, divergence and gene expression data in a variety of taxa. Here we study the influence of hemizygosity of the Z chromosome in Maniola jurtina and Pyronia tithonus, two species of butterflies (Lepidoptera, Nymphalidae, Satyrinae). Using transcriptome data, we compare the strength of positive and negative selection between Z and autosomes accounting for sex-specific gene expression. We show that M. jurtina and P. tithonus do not experience a faster, but rather a slightly slower evolutionary rate on the Z than on autosomes. Our analysis failed to detect a significant difference in adaptive evolutionary rate between Z and autosomes, but comparison of male-biased, unbiased and female-biased Z-linked genes revealed an increased efficacy of purifying selection against recessive deleterious mutations in female-biased Z-linked genes. This probably contributes to the lack of fast-Z evolution of satyrines. We suggest that the effect of hemizygosity on the fate of recessive deleterious mutations should be taken into account when interpreting patterns of molecular evolution in sex chromosomes vs. autosomes. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Background diet and fat type alters plasma lipoprotein response but not aortic cholesterol accumulation in F1B Golden Syrian hamsters.

PubMed

Dillard, Alice; Matthan, Nirupa R; Spartano, Nicole L; Butkowski, Ann E; Lichtenstein, Alice H

2013-12-01

Dietary modification alters plasma lipoprotein profiles and atherosclerotic lesion progression in humans and some animal models. Variability in response to diet induced atherosclerosis has been reported in hamsters. Assessed was the interaction between background diet composition and dietary fat type on aortic cholesterol accumulation, lipoprotein profiles, hepatic lipids and selected genes. F1B Golden Syrian hamsters (20/group) were fed (12 weeks) semi-purified or non-purified diets containing either 10 % (w/w) coconut oil or safflower oil and 0.15 % (w/w) cholesterol. The non-purified diets relative to semi-purified diets resulted in significantly higher TC (72 % [percent difference] and 38 %, coconut oil and safflower oil, respectively) and nHDL-C (84 and 61 %, coconut oil and safflower oil, respectively), and lower HDL-C (-47 and -45 %, coconut oil and safflower oil, respectively) concentrations. Plasma triacylglycerol concentrations in the hamsters fed the non-purified coconut oil-supplemented diets were three- to fourfold higher than non-purified safflower oil-supplemented, and both semi-purified diets. With the exception of HDL-C, a significant effect of fat type was observed in TC, nHDL-C and triacylglycerol (all P < 0.05) concentrations. Regardless of diet induced differences in lipoprotein profiles, there was no significant effect on aortic cholesterol accumulation. There was an inverse relationship between plasma nHDL-C and triacylglycerol, and hepatic cholesteryl ester content (P < 0.001). Diet induced differences in hepatic gene transcription (LDL receptor, apoB-100, microsomal transfer protein) were not reflected in protein concentrations. Although hamsters fed non-purified and/or saturated fatty acid-supplemented diets had more atherogenic lipoprotein profiles compared to hamsters fed semi-purified and/or polyunsaturated fatty acid-supplemented diets these differences were not reflected in aortic cholesterol accumulation.

The stabilisation of purified, reconstituted P-glycoprotein by freeze drying with disaccharides.

PubMed

Heikal, Adam; Box, Karl; Rothnie, Alice; Storm, Janet; Callaghan, Richard; Allen, Marcus

2009-02-01

The drug efflux pump P-glycoprotein (P-gp) (ABCB1) confers multidrug resistance, a major cause of failure in the chemotherapy of tumours, exacerbated by a shortage of potent and selective inhibitors. A high throughput assay using purified P-gp to screen and characterise potential inhibitors would greatly accelerate their development. However, long-term stability of purified reconstituted ABCB1 can only be reliably achieved with storage at -80 degrees C. For example, at 20 degrees C, the activity of ABCB1 was abrogated with a half-life of <1 day. The aim of this investigation was to stabilise purified, reconstituted ABCB1 to enable storage at higher temperatures and thereby enable design of a high throughput assay system. The ABCB1 purification procedure was optimised to allow successful freeze drying by substitution of glycerol with the disaccharides trehalose or maltose. Addition of disaccharides resulted in ATPase activity being retained immediately following lyophilisation with no significant difference between the two disaccharides. However, during storage trehalose preserved ATPase activity for several months regardless of the temperature (e.g. 60% retention at 150 days), whereas ATPase activity in maltose purified P-gp was affected by both storage time and temperature. The data provide an effective mechanism for the production of resilient purified, reconstituted ABCB1.

Agelenopsis aperta venom and FTX, a purified toxin, inhibit acetylcholine release in Torpedo synaptosomes.

PubMed

Moulian, N; Gaudry-Talarmain, Y M

1993-06-01

The presence of P-type calcium channels in synaptosomes prepared from electric organ of Torpedo marmorata was investigated by using the venom of Agelenopsis aperta, a toxin purified from it, FTX, and its synthetic analog. We analysed the action of these agents on acetylcholine release which was continuously followed using a chemiluminescent assay. Agelenopsis aperta venom, FTX and synthetic FTX inhibit acetylcholine release from synaptosomes induced by a presynaptic membrane depolarization with 60 mM KCl. A stronger inhibition of acetylcholine release was observed with the venom than with FTX (70 and 50%, respectively). Another way of triggering acetylcholine release from Torpedo synaptosomes is to insert in the presynaptic membrane a calcium ionophore A23187 which allows the bypass of the natural calcium channels. The venom of Agelenopsis aperta inhibits A23187-evoked acetylcholine release. Purified and synthetic FTX does not possess this property, suggesting that this inhibition of acetylcholine release was due to other toxins of the venom. Another type of pharmacological sensitivity of Torpedo calcium channels was also demonstrated using omega-conotoxin GVIA. At a concentration of 20 microM, this toxin was able to inhibit about 35% of KCl-evoked acetylcholine release. When FTX + omega-conotoxin GVIA were applied together, the inhibitory effect on KCl-evoked acetylcholine release was not significantly increased in comparison with the one observed with FTX alone. In conclusion, we examined the effect of different agents on acetylcholine release from Torpedo marmorata electric organ synaptosomes; acetylcholine release was elicited with KCl depolarization and followed continuously with a chemiluminescent assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Selection Gradients, the Opportunity for Selection, and the Coefficient of Determination

PubMed Central

Moorad, Jacob A.; Wade, Michael J.

2013-01-01

We derive the relationship between R2 (the coefficient of determination), selection gradients, and the opportunity for selection for univariate and multivariate cases. Our main result is to show that the portion of the opportunity for selection that is caused by variation for any trait is equal to the product of its selection gradient and its selection differential. This relationship is a corollary of the first and second fundamental theorems of natural selection, and it permits one to investigate the portions of the total opportunity for selection that are involved in directional selection, stabilizing (and diversifying) selection, and correlational selection, which is important to morphological integration. It also allows one to determine the fraction of fitness variation not explained by variation in measured phenotypes and therefore attributable to random (or, at least, unknown) influences. We apply our methods to a human data set to show how sex-specific mating success as a component of fitness variance can be decoupled from that owing to prereproductive mortality. By quantifying linear sources of sexual selection and quadratic sources of sexual selection, we illustrate that the former is stronger in males, while the latter is stronger in females. PMID:23448880

Selection gradients, the opportunity for selection, and the coefficient of determination.

PubMed

Moorad, Jacob A; Wade, Michael J

2013-03-01

Abstract We derive the relationship between R(2) (the coefficient of determination), selection gradients, and the opportunity for selection for univariate and multivariate cases. Our main result is to show that the portion of the opportunity for selection that is caused by variation for any trait is equal to the product of its selection gradient and its selection differential. This relationship is a corollary of the first and second fundamental theorems of natural selection, and it permits one to investigate the portions of the total opportunity for selection that are involved in directional selection, stabilizing (and diversifying) selection, and correlational selection, which is important to morphological integration. It also allows one to determine the fraction of fitness variation not explained by variation in measured phenotypes and therefore attributable to random (or, at least, unknown) influences. We apply our methods to a human data set to show how sex-specific mating success as a component of fitness variance can be decoupled from that owing to prereproductive mortality. By quantifying linear sources of sexual selection and quadratic sources of sexual selection, we illustrate that the former is stronger in males, while the latter is stronger in females.

Electrochemical ion separation in molten salts

DOEpatents

Spoerke, Erik David; Ihlefeld, Jon; Waldrip, Karen; Wheeler, Jill S.; Brown-Shaklee, Harlan James; Small, Leo J.; Wheeler, David R.

2017-12-19

A purification method that uses ion-selective ceramics to electrochemically filter waste products from a molten salt. The electrochemical method uses ion-conducting ceramics that are selective for the molten salt cations desired in the final purified melt, and selective against any contaminant ions. The method can be integrated into a slightly modified version of the electrochemical framework currently used in pyroprocessing of nuclear wastes.

Mutation Accumulation, Soft Selection and the Middle-Class Neighborhood

PubMed Central

Moorad, Jacob A.; Hall, David W.

2009-01-01

The “middle-class neighborhood” is a breeding design intended to allow new mutations to accumulate by lessening the effects of purifying selection through the elimination of among-line fitness variation. We show that this design effectively applies soft selection to the experimental population, potentially causing biased estimates of mutational effects if social effects contribute to fitness. PMID:19448272

Phylogeny and evolution of Newcastle disease virus genotypes isolated in Asia during 2008-2011.

PubMed

Ebrahimi, Mohammad Majid; Shahsavandi, Shahla; Moazenijula, Gholamreza; Shamsara, Mahdi

2012-08-01

The full-length fusion (F) genes of 51 Newcastle disease (ND) strains isolated from chickens in Asia during the period 2008-2011 were genetically analyzed. Phylogenetic analysis showed that genotype VII of NDV still predominant in the domestic poultry of Asia. The sub-genotype VIIb circulated in the Iran and Indian sub-continent countries, whereas VIId sub-genotype existed in Far East countries. The non-synonymous to synonymous substitutions ratio was calculated 0.27 for VIId sub-genotype and 0.51 for VIIb sub-genotype indicates purifying/stabilizing selection which resulted in a low evolution rate in F gene of VIIb sub-genotype. There is evidence of localized positive selection when comparing these sub-genotypes protein sequences. Five codons in F gene of ND viruses had a posterior probability >90% using the Bayesian method, indicating these sites were under positive selection. To identify sites under positive selection; amino acid substitution classified depends on their radicalism and neutrality. The results indicate that although most positions were under purifying selection and can be eliminated, a few positions located in sub-genotype specific regions were subject to positive selection.

New Instrumentation for Phase Partitioning

NASA Technical Reports Server (NTRS)

Harris, J. M.

1985-01-01

Cells and molecules can be purified by partitioning between the two immiscible liquid phases formed by aqueous solutions of poly/ethylene glycol and dextran. Such purification can be more selective, higher yielding, and less destructive to sensitive biological materials than other available techniques. Earth's gravitational field is a hindering factor as it causes sedimentation of particles to be purified and shear-induced particle randomization. The present proposal is directed toward developing new instrumentation for performing phase partitioning both on Earth and in microgravity.

Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteins.

PubMed Central

Elliott, J; Blanchard, S G; Wu, W; Miller, J; Strader, C D; Hartig, P; Moore, H P; Racs, J; Raftery, M A

1980-01-01

A rapid methof for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from Torpedo californica electroplax is described. The major step in this purification involves sucrose-density-gradient centrifugation in a reorienting rotor. Further purification of these membranes can be achieved by selective extraction of proteins by use of alkaline pH or by treatment with solutions of lithium di-idosalicylate. The alkali-treated membranes retain functional characteristics of the untreated membranes and in addition contain essentially only the four polypeptides (mol.wts. 40000, 50000, 60000 and 65000) characteristic of the receptor purified by affinity chromatography. Dissolution of the purified membranes or of the alkali-treated purified membranes in sodium cholate solution followed by sucrose-density-gradient centrifugation in the same detergent solution yields solubilized receptor preparations comparable with the most highly purified protein obtained by affinity-chromatographic procedures. Images Fig. 1. Fig. 2. Fig. 3. Fig. 5. Fig. 7. PLATE 1 PMID:7387629

The effects of a parenting prime on sex differences in mate selection criteria.

PubMed

Millar, Murray G; Ostlund, Nelse M

2006-11-01

This study tested an evolutionary hypothesis that the mere prospect of caring for a child will increase sex differences in human mate selection criteria. That is, women would adopt a stronger preference for socially dominant men when parenting had been primed and men would adopt a stronger preference for physically attractive women when parenting had been primed. Male and female university students were randomly assigned to be exposed to a parenting prime or a nonparenting prime. Following the priming procedure, participants rated the romantic appeal of a target person of the opposite sex. Exposure to the parenting prime, the target's social dominance, and the target's physical attractiveness were orthogonally manipulated. As predicted, women adopted a stronger mate preference for social dominance when parenting was at the forefront of the mind. Contrary to predictions, the parenting prime had no effect on men's mate preference for physical attractiveness.

Are Forensic Experts Already Biased before Adversarial Legal Parties Hire Them?

PubMed Central

2016-01-01

This survey of 206 forensic psychologists tested the “filtering” effects of preexisting expert attitudes in adversarial proceedings. Results confirmed the hypothesis that evaluator attitudes toward capital punishment influence willingness to accept capital case referrals from particular adversarial parties. Stronger death penalty opposition was associated with higher willingness to conduct evaluations for the defense and higher likelihood of rejecting referrals from all sources. Conversely, stronger support was associated with higher willingness to be involved in capital cases generally, regardless of referral source. The findings raise the specter of skewed evaluator involvement in capital evaluations, where evaluators willing to do capital casework may have stronger capital punishment support than evaluators who opt out, and evaluators with strong opposition may work selectively for the defense. The results may provide a partial explanation for the “allegiance effect” in adversarial legal settings such that preexisting attitudes may contribute to partisan participation through a self-selection process. PMID:27124416

The activity of thymidine phosphorylase obtained from human uterine leiomyomas and studied in the presence of pyrimidine derivatives.

PubMed

Miszczak-Zaborska, E; Woźniak, K

1997-01-01

Partially purified samples of thymidine phosphorylase were obtained from four preparations of human uterine leiomyomas and uteri using the method of Yoshimura et al. (1990), Biochim. Biophys. Acta 1034, 107-113. Among the studied twelve pyrimidine derivatives, 5-bromouracil, 5-nitrouracil, 5-fluorouracil, 6-aminouracil, 4, 6-dihydroxy-5-nitropyrimidine are competitive inhibitors, while allyloxymethylthymine is an uncompetitive inhibitor of thymidine phosphorylase activity, 6-benzyl-2-thiouracil inhibits the activity of the enzyme in a mixed way. The most potent inhibitor of the thymidine phosphorylase activity is 5-bromouracil and uracil the weakest one. Stronger inhibition of these compounds on the activity of thymidine phosphorylase was found in uterine leiomyomas than in uteri.

High-throughput protein concentration and buffer exchange: comparison of ultrafiltration and ammonium sulfate precipitation.

PubMed

Moore, Priscilla A; Kery, Vladimir

2009-01-01

High-throughput protein purification is a complex, multi-step process. There are several technical challenges in the course of this process that are not experienced when purifying a single protein. Among the most challenging are the high-throughput protein concentration and buffer exchange, which are not only labor-intensive but can also result in significant losses of purified proteins. We describe two methods of high-throughput protein concentration and buffer exchange: one using ammonium sulfate precipitation and one using micro-concentrating devices based on membrane ultrafiltration. We evaluated the efficiency of both methods on a set of 18 randomly selected purified proteins from Shewanella oneidensis. While both methods provide similar yield and efficiency, the ammonium sulfate precipitation is much less labor intensive and time consuming than the ultrafiltration.

Evolutionary Diversifaction of Aminopeptidase N in Lepidoptera by Conserved Clade-specific Amino Acid Residues

PubMed Central

Hughes, Austin L.

2015-01-01

Members of the aminopepidase N (APN) gene family of the insect order Lepidoptera (moths and butterflies) bind the naturally insecticidal Cry toxins produced by the bacterium Bacillus thuringiensis. Phylogenetic analysis of amino acid sequences of seven lepidopteran APN classes provided strong support for the hypothesis that lepidopteran APN2 class arose by gene duplication prior to the most recent common ancestor of Lepidoptera and Diptera. The Cry toxin-binding region (BR) of lepidopteran and dipteran APNs was subject to stronger purifying selection within APN classes than was the remainder of the molecule, reflecting conservation of catalytic site and adjoining residues within the BR. Of lepidopteran APN classes, APN2, APN6, and APN8 showed the strongest evidence of functional specialization, both in expression patterns and in the occurrence of conserved derived amino acid residues. The latter three APN classes also shared a convergently evolved conserved residue close to the catalytic site. APN8 showed a particularly strong tendency towards class-specific conserved residues, including one of the catalytic site residues in the BR and ten others in close vicinity to the catalytic site residues. The occurrence of class-specific sequences along with the conservation of enzymatic function is consistent with the hypothesis that the presence of Cry toxins in the environment has been a factor shaping the evolution of this multi-gene family. PMID:24675701

Selection of pecan shell based activated carbons for removal of organic and inorganic impurities from simulated well-water

USDA-ARS?s Scientific Manuscript database

Activated carbons are a byproduct from pyrolysis and have value as a purifying agent. The effectiveness of activated carbons is dependent on feedstock selection and pyrolysis conditions that modify its surface properties. Therefore, pecan shell-based activated carbons (PSACs) were prepared by soakin...

Automated high throughput microscale antibody purification workflows for accelerating antibody discovery

PubMed Central

Luan, Peng; Lee, Sophia; Paluch, Maciej; Kansopon, Joe; Viajar, Sharon; Begum, Zahira; Chiang, Nancy; Nakamura, Gerald; Hass, Philip E.; Wong, Athena W.; Lazar, Greg A.

2018-01-01

ABSTRACT To rapidly find “best-in-class” antibody therapeutics, it has become essential to develop high throughput (HTP) processes that allow rapid assessment of antibodies for functional and molecular properties. Consequently, it is critical to have access to sufficient amounts of high quality antibody, to carry out accurate and quantitative characterization. We have developed automated workflows using liquid handling systems to conduct affinity-based purification either in batch or tip column mode. Here, we demonstrate the capability to purify >2000 antibodies per day from microscale (1 mL) cultures. Our optimized, automated process for human IgG1 purification using MabSelect SuRe resin achieves ∼70% recovery over a wide range of antibody loads, up to 500 µg. This HTP process works well for hybridoma-derived antibodies that can be purified by MabSelect SuRe resin. For rat IgG2a, which is often encountered in hybridoma cultures and is challenging to purify via an HTP process, we established automated purification with GammaBind Plus resin. Using these HTP purification processes, we can efficiently recover sufficient amounts of antibodies from mammalian transient or hybridoma cultures with quality comparable to conventional column purification. PMID:29494273

Variability of selected nutrients and contaminants monitored in rodent diets: A 6-year study

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

Oller, W.L.; Kendall, D.C.; Greenman, D.L.

1989-01-01

The results are given from monitoring a commercial closed-formula cereal-based rodent diet (Purina 5010), two open-formula cereal-based diets (NIH-31 and NIH-07), and one purified diet (AIN-76) for selected nutrients and contaminants. The observed concentrations of nutrients (protein, fat, vitamin A, and thiamine) approximated the manufacturer specifications for closed-formula cereal diet, while the average concentrations of nutrients found in the open-formula cereal diets were well above the nominal concentrations. Nominal concentrations for these open-formula diets tended to be close to the minimum values that were observed. Except for protein levels, greater variation in nutrient concentrations was found in the purified dietmore » than in the cereal diets. Contaminants were generally much lower in the purified diet than in the cereal diets, but the variation of contaminants was about equal in the two types of diets. Open- and closed-formula cereal diets appear to be very similar to each other in the degree of variation of nutrients and contaminants. Cadmium, lead, and selenium are the constituents of greatest concern in assuring the quality of the rodent diets that were evaluated.« less

Divergent selection for fiber length and bundle strength and correlated responses in cotton

USDA-ARS?s Scientific Manuscript database

Cotton breeders must develop cultivars to meet the demand for longer, stronger, and more uniform fibers. In the current study, two cycles of divergent selection for fiber upper-half mean length (UHML) and bundle strength (Str) were conducted within five diverse parental combinations selected based o...

The acetylcholinesterase inhibitor BW284c51 is a potent blocker of Torpedo nicotinic AchRs incorporated into the Xenopus oocyte membrane

PubMed Central

Olivera-Bravo, Silvia; Ivorra, Isabel; Morales, Andrés

2004-01-01

This work was aimed to determine if 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284c51), the most selective acetylcholinesterase inhibitor (AchEI), affects the nicotinic acetylcholine (Ach) receptor (AchR) function. Purified Torpedo nicotinic AchRs were injected into Xenopus laevis oocytes and BW284c51 effects on Ach- and carbamylcholine (Cch)-elicited currents were assessed using the voltage-clamp technique. BW284c51 (up to 1 mM) did not evoke any change in the oocyte membrane conductance. When BW284c51 (10 pM–100 μM) and Ach were coapplied, Ach-evoked currents (IAch) were reversibly inhibited in a concentration-dependent manner (Hill coefficient, 1; IC50, 0.2–0.5 μM for 0.1–1000 μM Ach). Cch-elicited currents showed a similar inhibition by BW284c51. IAch blockade by BW284c51 showed a strong voltage dependence, being only apparent at hyperpolarising potentials. BW284c51 also enhanced IAch desensitisation. BW284c51 changed the Ach concentration-dependence curve of Torpedo AchR response from two-site to single-site kinetics, without noticeably affecting the EC50 value. The BW284c51 blocking effect was highly selective for nicotinic over muscarinic receptors. BW284c51 inhibition potency was stronger than that of tacrine, and similar to that of d-tubocurarine (d-TC). Coapplication of BW284c51 with either tacrine or d-TC revealed synergistic inhibitory effects. Our results indicate that BW284c51 antagonises nicotinic AchRs in a noncompetitive way by blocking the receptor channel, and possibly by other, yet unknown, mechanisms. Therefore, besides acting as a selective AchEI, BW284c51 constitutes a powerful and reversible blocker of nicotinic AchRs that might be used as a valuable tool for understanding their function. PMID:15644872

Selective enrichment and desalting of hydrophilic peptides using graphene oxide.

PubMed

Jiang, Miao; Qi, Linyu; Liu, Peiru; Wang, Zijun; Duan, Zhigui; Wang, Ying; Liu, Zhonghua; Chen, Ping

2016-08-01

The wide variety and low abundance of peptides in tissue brought great difficulties to the separation and identification of peptides, which is not in favor of the development of peptidomics. RP-HPLC, which could purify small molecules based on their hydrophobicity, has been widely used in the separation and enrichment of peptide due to its fast, good reproducibility and high resolution. However, RP-HPLC requires the instrument and expensive C18 column and its sample capacity is also limited. Recently, graphene oxide has been applied to the adsorption of amino acids. However, the enrichment efficiency and selectivity of graphene oxide for peptides remain unclear. In this study, the adsorption efficiency and selectivity of graphene oxide and RP-C18 matrix were compared on trypsinized α-actin and also on tissue extracts from pituitary gland and hippocampus. For α-actin, there exhibit similar elution peaks for total trypsinized products and those adsorpted by GO and C18 matrix. But peptides adsorbed by GO showed the higher hydrophilic peaks than which adsorbed by C18 matrix. The resulted RP-HPLC profile showed that most of peptides enriched by graphene oxide were eluted at low concentration of organic solvent, while peptides adsorbed by RP-C18 matrix were mostly eluted at relatively high concentration. Moreover, mass spectrometry analysis suggested that, in pituitary sample, there were 495 peptides enriched by graphene oxide, 447 peptides enriched by RP-C18 matrix while in hippocampus sample 333 and 243 peptides respectively. The GRAVY value analysis suggested that the graphene oxide has a stronger adsorption for highly hydrophilic peptides compared to the RP-C18 matrix. Furthermore, the combination of these two methods could notably increase the number of identification peptides but also the number of predicted protein precursors. Our study provided a new thought to the role of graphene oxide during the enrichment of peptides from tissue which should be useful for peptidomics study. Copyright © 2016 Elsevier B.V. All rights reserved.

[Construction, expression and characterization of the fusion gene of super-antigen SEA and single chain Fv of the ND-1 monoclonal antibody against human colorectal cancer].

PubMed

Chen, Hang; Li, Li; Fang, Jin

2012-04-01

To construct and express the recombinant ND-1-scFv/SEA, a fusion protein of superantigen (staphylococcal enterotoxinA, SEA) and single-chain variable fragment of monoclonal antibody ND-1 against human clolorectal carcinoma, and to enhance the targeted killing effect of SEA. The expression of the fusion protein was induced in E.coli M15 by IPTG. Ni-NTA resin affinity chromatography was used to separate and purify the expressed product. The specific binding activity of the purified ND-1-scFv/SEA protein was examined by indirect immunofluorescence assay and the targeted-cytotoxicity was determined using MTT assay. The expressing vector of fusion gene ND-1scFv/SEA was constructed successfully. ND-1-scFv/SEA protein retained a high binding affinity to antigen-positive human colorectal cancer cell CCL-187 and had a stronger capability to activate PBMC and kill the target cells compared to SEA alone, with a killing rate of 91% at 4 μg/mL. ND-1-scFv/SEA fusion protein could specifically target colorectal cancer cell, enhance the activity of kill tumor cell and has potential applications in the targeted therapy of colorectal cancer.

Purification and complete amino acid sequence of a new type of sweet protein taste-modifying activity, curculin.

PubMed

Yamashita, H; Theerasilp, S; Aiuchi, T; Nakaya, K; Nakamura, Y; Kurihara, Y

1990-09-15

A new taste-modifying protein named curculin was extracted with 0.5 M NaCl from the fruits of Curculigo latifolia and purified by ammonium sulfate fractionation, CM-Sepharose ion-exchange chromatography, and gel filtration. Purified curculin thus obtained gave a single band having a Mr of 12,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 8 M urea. The molecular weight determined by low-angle laser light scattering was 27,800. These results suggest that native curculin is a dimer of a 12,000-Da polypeptide. The complete amino acid sequence of curculin was determined by automatic Edman degradation. Curculin consists of 114 residues. Curculin itself elicits a sweet taste. After curculin, water elicits a sweet taste, and sour substances induce a stronger sense of sweetness. No protein with both sweet-tasting and taste-modifying activities has ever been found. There are five sets of tripeptides common to miraculin (a taste-modifying protein), six sets of tripeptides common to thaumatin (a sweet protein), and two sets of tripeptides common to monellin (a sweet protein). Anti-miraculin serum was not immunologically reactive with curculin. The mechanism of the taste-modifying action of curculin is discussed.

Expression, purification, and antibacterial activity of bovine lactoferrampin-lactoferricin in Pichia pastoris.

PubMed

Tang, Xiang-Shan; Tang, Zhi-Ru; Wang, Sheng-Ping; Feng, Ze-Meng; Zhou, Dong; Li, Tie-Jun; Yin, Yu-Long

2012-02-01

Bovine lactoferrampin (LFA) and bovine lactoferricin (LFC) are two antimicrobial peptides located in the N(1) domain of bovine lactoferrin. The bactericidal activity of the fused peptide LFA-LFC is stronger than that of either LFA or LFC. The high cost of peptide production from either native digestion or chemical synthesis limits the clinical application of antimicrobial peptides. The expression of recombinant peptides in yeast may be an effective alternative. In the current study, the expression, purification, and antibacterial activity of LFA-LFC using the Pichia pastoris expression system are reported. The linearized expression vector pPICZaA-LFA-LFC was transformed into P. pastoris KM71 by electroporation, and positive colonies harboring the target genes were screened out and used for fermentation. The recombinant LFA-LFC peptide was purified via two-step column chromatography and identified by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The results indicate that P. pastoris is a suitable system for secreting LFA-LFC. The fermentation supernate and the purified LFA-LFC show high antimicrobial activities. The current study is the first to report on the expression and purification of LFA-LFC in P. pastoris and may have potential practical applications in microbial peptide production.

Evolutionary characterization of hemagglutinin gene of H9N2 influenza viruses isolated from Asia.

PubMed

Shahsavandi, Shahla; Salmanian, Ali-Hatef; Ghorashi, Seyed Ali; Masoudi, Shahin; Ebrahimi, Mohammad Majid

2012-08-01

The full length hemagglutinin (HA) genes of 287 H9N2 AI strains isolated from chickens in Asia during the period 1994-2009 were genetically analyzed. Phylogenetic analysis showed that G1-like viruses circulated in the Middle East and Indian sub-continent countries, whereas other sublineages existed in Far East countries. It also revealed G1-like viruses with an average 96.7% identity clustered into two subgroups largely based on their time of isolation. The Ka/Ks ratio was calculated 0.34 for subgroup 1 and 0.57 for subgroup 2 indicates purifying/stabilizing selection, but despite this there is evidence of localized positive selection when comparing the subgroups 1 and 2 protein sequences. Five sites in HA H9N2 viruses had a posterior probability >0.5 using the Bayesian method, indicating these sites were under positive selection. These sites were found to be associated with the globular head region of HA. To identify sites under positive selection; amino acid substitution classified depends on their radicalism and neutrality. The results indicate that, although most positions in HAs were under purifying selection and can be eliminated, a few positions located in the antigenic regions and receptor binding sites were subject to positive selection. Copyright © 2011 Elsevier Ltd. All rights reserved.

Incubation period and immune function: A comparative field study among coexisting birds

USGS Publications Warehouse

Palacios, M.G.; Martin, T.E.

2006-01-01

Developmental periods are integral components of life history strategies that can have important fitness consequences and vary enormously among organisms. However, the selection pressures and mechanisms causing variation in length of developmental periods are poorly understood. Particularly puzzling are prolonged developmental periods, because their selective advantage is unclear. Here we tested the hypotheses that immune function is stronger in species that are attacked at a higher rate by parasites and that prolonged embryonic development allows the development of this stronger immune system. Through a comparative field study among 12 coexisting passerine bird species, we show that species with higher blood parasite prevalence mounted stronger cellular immune responses than species with lower prevalence. These results provide support for the hypothesis that species facing greater selection pressure from parasites invest more in immune function. However, species with longer incubation periods mounted weaker cellular immune responses than species with shorter periods. Therefore, cellular immune responses do not support the hypothesis that longer development time enhances immunocompentence. Future studies should assess other components of the immune system and test alternative causes of variation in incubation periods among bird species. ?? Springer-Verlag 2005.

Darwinian sex roles confirmed across the animal kingdom

PubMed Central

Janicke, Tim; Häderer, Ines K.; Lajeunesse, Marc J.; Anthes, Nils

2016-01-01

Since Darwin’s conception of sexual selection theory, scientists have struggled to identify the evolutionary forces underlying the pervasive differences between male and female behavior, morphology, and physiology. The Darwin-Bateman paradigm predicts that anisogamy imposes stronger sexual selection on males, which, in turn, drives the evolution of conventional sex roles in terms of female-biased parental care and male-biased sexual dimorphism. Although this paradigm forms the cornerstone of modern sexual selection theory, it still remains untested across the animal tree of life. This lack of evidence has promoted the rise of alternative hypotheses arguing that sex differences are entirely driven by environmental factors or chance. We demonstrate that, across the animal kingdom, sexual selection, as captured by standard Bateman metrics, is indeed stronger in males than in females and that it is evolutionarily tied to sex biases in parental care and sexual dimorphism. Our findings provide the first comprehensive evidence that Darwin’s concept of conventional sex roles is accurate and refute recent criticism of sexual selection theory. PMID:26933680

Darwinian sex roles confirmed across the animal kingdom.

PubMed

Janicke, Tim; Häderer, Ines K; Lajeunesse, Marc J; Anthes, Nils

2016-02-01

Since Darwin's conception of sexual selection theory, scientists have struggled to identify the evolutionary forces underlying the pervasive differences between male and female behavior, morphology, and physiology. The Darwin-Bateman paradigm predicts that anisogamy imposes stronger sexual selection on males, which, in turn, drives the evolution of conventional sex roles in terms of female-biased parental care and male-biased sexual dimorphism. Although this paradigm forms the cornerstone of modern sexual selection theory, it still remains untested across the animal tree of life. This lack of evidence has promoted the rise of alternative hypotheses arguing that sex differences are entirely driven by environmental factors or chance. We demonstrate that, across the animal kingdom, sexual selection, as captured by standard Bateman metrics, is indeed stronger in males than in females and that it is evolutionarily tied to sex biases in parental care and sexual dimorphism. Our findings provide the first comprehensive evidence that Darwin's concept of conventional sex roles is accurate and refute recent criticism of sexual selection theory.

Process for removal of water and silicon mu-oxides from chlorosilanes

DOEpatents

Tom, Glenn M.; McManus, James V.

1992-03-10

A scavenger composition having utility for removal of water and silicon mu-oxide impurities from chlorosilanes, such scavenger composition comprising: (a) a support; and (b) associated with the support, one or more compound(s) selected from the group consisting of compounds of the formula: R.sub.a-x MCl.sub.x wherein: M is a metal selected from the group consisting of the monovalent metals lithium, sodium, and potassium; the divalent metals magnesium, strontium, barium, and calcium; and the trivalent metal aluminum; R is alkyl; a is a number equal to the valency of metal M; and x is a number having a value of from 0 to a, inclusive; and wherein said compound(s) of the formula R.sub.a-x MCl.sub.x have been activated for impurity-removal service by a reaction scheme selected from those of the group consisting of: (i) reaction of such compound(s) with hydrogen chloride to form a first reaction product therefrom, followed by reaction of the first reaction product with a chlorosilane of the formula: SiH.sub.4-y Cl.sub.y, wherein y is a number having a value of from 1 to 3, inclusive; and (ii) reaction of such compound(s) with a chlorosilane of the formula: SiH.sub.4-y Cl.sub.y wherein y is a number having a value of 1 to 3, inclusive. A corresponding method of making the scavenger composition, and of purifying a chlorosilane which contains oxygen and silicon mu-oxide impurities, likewise are disclosed, together with a purifier apparatus, in which a bed of the scavenger composition is disposed. The composition, purification process, and purifier apparatus of the invention have utility in purifying gaseous chlorosilanes which are employed in the semiconductor industry as silicon source reagents for forming epitaxial silicon layers.

Composition, process, and apparatus, for removal of water and silicon mu-oxides from chlorosilanes

DOEpatents

Tom, Glenn M.; McManus, James V.

1991-10-15

A scavenger composition having utility for removal of water and silicon mu-oxide impurities from chlorosilanes, such scavenger composition comprising: (a) a support; and (b) associated with the support, one or more compound(s) selected from the group consisting of compounds of the formula: R.sub.a-x MCl.sub.x wherein: M is a metal selected from the group consisting of the monovalent metals lithium, sodium, and potassium; the divalent metals magnesium, strontium, barium, and calcium; and the trivalent metal aluminum; R is alkyl; a is a number equal to the valency of metal M; and x is a number having a value from 0 to a, inclusive; and wherein said compound(s) of the formula R.sub.a-x MCl.sub.x have been activated for impurity-removal service by a reaction scheme selected from those of the group consisting of: (i) reaction of such compound(s) with hydrogen chloride to form a first reaction product therefrom, followed by reaction of the first reaction product with a chlorosilane of the formula: SiH.sub.4"y Cl.sub.y, wherein y is a number having a value of from 1 to 3, inclusive; and (ii) reaction of such compound(s) with a chlorosilane of the formula: SiH.sub.4-y Cl.sub.y wherein y is a number having a value of 1 to 3, inclusive. A corresponding method of making the scavenger composition, and of purifying a chlorosilane which contains oxygen and silicon mu-oxide impurities, likewise are disclosed, together with a purifier apparatus, in which a bed of the scavenger composition is disposed. The composition, purification process, and purifier apparatus of the invention have utility in purifying gaseous chlorosilanes which are employed in the semiconductor industry as silicon source reagents for forming epitaxial silicon layers.

Systems, compositions, and methods for fluid purification

DOEpatents

Ho, W.S. Winston; Verweij, Hendrik; Shqau, Krenar; Ramasubranian, Kartik

2015-12-22

Disclosed herein are membranes comprising a substrate, a support layer, and a selective layer. In some embodiments the membrane may further comprise a permeable layer. Methods of forming membranes are also disclosed comprising forming a support layer on a substrate, removing adsorbed species from the support layer, preparing a solution containing inorganic materials of a selective layer, contacting the support layer with the solution, drying the membrane, and exposing the membrane to rapid thermal processing. Also disclosed are methods of fluid purification comprising providing a membrane having a feed side and a permeable side, passing a fluid mixture across the feed side of the membrane, providing a driving force for transmembrane permeation, removing from the permeate side a permeate stream enriched in a purified fluid, and withdrawing from the feed side a fluid that is depleted in a purified fluid.

Purified phenolics from hydrothermal treatments of biomass: ability to protect sunflower bulk oil and model food emulsions from oxidation.

PubMed

Conde, Enma; Moure, Andrés; Domínguez, Herminia; Gordon, Michael H; Parajó, Juan Carlos

2011-09-14

The phenolic fractions released during hydrothermal treatment of selected feedstocks (corn cobs, eucalypt wood chips, almond shells, chestnut burs, and white grape pomace) were selectively recovered by extraction with ethyl acetate and washed with ethanol/water solutions. The crude extracts were purified by a relatively simple adsorption technique using a commercial polymeric, nonionic resin. Utilization of 96% ethanol as eluting agent resulted in 47.0-72.6% phenolic desorption, yielding refined products containing 49-60% w/w phenolics (corresponding to 30-58% enrichment with respect to the crude extracts). The refined extracts produced from grape pomace and from chestnut burs were suitable for protecting bulk oil and oil-in-water and water-in-oil emulsions. A synergistic action with bovine serum albumin in the emulsions was observed.

Bovicin HC5 inhibits wasteful amino acid degradation by mixed ruminal bacteria in vitro.

PubMed

Lima, Janaína R; Ribon, Andréa de O Barros; Russell, James B; Mantovani, Hilário C

2009-03-01

Streptococcus bovis HC5 produces a broad spectrum lantibiotic (bovicin HC5) that inhibits pure cultures of hyper ammonia-producing bacteria (HAB). Experiments were preformed to see if: (1) S. bovis HC5 cells could inhibit the deamination of amino acids by mixed ruminal bacteria taken directly from a cow, (2) semi-purified bovicin was as effective as S. bovis HC5 cells, and 3) semi-purified and the feed additive monensin were affecting the same types of ammonia-producing ruminal bacteria. Because purified and semi-purified bovicin HC5 was as effective as S. bovis HC5 cells, it appeared that bovicin HC5 was penetrating the cell membranes of HAB before it could be degraded by peptidases and proteinases. Mixed ruminal bacteria that were successively transferred and enriched nine times with trypticase did not become significantly more resistant to either bovicin HC5 (50 AU mL(-1)) or monensin (5 microM), and amplified rDNA restriction analysis indicated that bovicin HC5 and monensin appeared to be selecting against the same types of bacteria.

Purifying Selection Maintains Dosage-Sensitive Genes during Degeneration of the Threespine Stickleback Y Chromosome

PubMed Central

White, Michael A.; Kitano, Jun; Peichel, Catherine L.

2015-01-01

Sex chromosomes are subject to unique evolutionary forces that cause suppression of recombination, leading to sequence degeneration and the formation of heteromorphic chromosome pairs (i.e., XY or ZW). Although progress has been made in characterizing the outcomes of these evolutionary processes on vertebrate sex chromosomes, it is still unclear how recombination suppression and sequence divergence typically occur and how gene dosage imbalances are resolved in the heterogametic sex. The threespine stickleback fish (Gasterosteus aculeatus) is a powerful model system to explore vertebrate sex chromosome evolution, as it possesses an XY sex chromosome pair at relatively early stages of differentiation. Using a combination of whole-genome and transcriptome sequencing, we characterized sequence evolution and gene expression across the sex chromosomes. We uncovered two distinct evolutionary strata that correspond with known structural rearrangements on the Y chromosome. In the oldest stratum, only a handful of genes remain, and these genes are under strong purifying selection. By comparing sex-linked gene expression with expression of autosomal orthologs in an outgroup, we show that dosage compensation has not evolved in threespine sticklebacks through upregulation of the X chromosome in males. Instead, in the oldest stratum, the genes that still possess a Y chromosome allele are enriched for genes predicted to be dosage sensitive in mammals and yeast. Our results suggest that dosage imbalances may have been avoided at haploinsufficient genes by retaining function of the Y chromosome allele through strong purifying selection. PMID:25818858

Cellular immune response in MDR-TB patients to different protein expression of MDR and susceptible Mycobacterium tuberculosis: Rv0147, a novel MDR-TB biomarker.

PubMed

Hadizadeh Tasbiti, Alireza; Yari, Shamsi; Siadat, Seyed Davar; Tabarsi, Payam; Saeedfar, Kayvan; Yari, Fatemeh

2018-02-01

Tuberculosis (TB) is a crucial public health problem with prevalence of multidrug resistant (MDR) rising. An accurate TB biomarker is urgently needed to monitor the response to treatment in patients with MDR tuberculosis. To analyze interaction between selected MDR-TB purified protein and immune cells, dendritic cells from MDR-TB patients and healthy subjects were stimulated by 55KDa protein fractions (Rv0147). The purified proteins identified by proteomic techniques (two-dimensional gel electrophoresis, mass spectrometry) and peptide sequences are known to bind a MHC class I alleles which are extracted from the Immune Epitope Database and Analysis Resource database ( www.iedb.org ). T cells were isolated from PBMC by negative selection and cells were cultured in RPMI-1640 at 37 °C and 5% CO 2 . Cell culture was assayed for cytokine IL-10 and INF-γ by ELISA. We found that INF-γ production was significantly (335 ± 35.5 pg/ml, P ˂ 0.05) upregulated after protein candidate (Rv0147) stimulation by dendritic cells from MDR-TB patients, whereas IL-10 production was greatly reduced compared with production in healthy subjects (212 ± 9.94 pg/ml, P ˂ 0.05). In fact, the purified protein, Rv0147, stimulated dendritic cells from MDR-TB patients, failed to produce IL-10 and directly stimulates INF-γ production by T cells. These results suggest that the purified protein, Rv0147, may stimulate Th1 type protective cytokine response in MDR-TB patients but not in normal subjects. The production of INF-γ but not IL-10 in the presence of purified protein, Rv0147, may be shifted to Th1 responses in MDR-TB patients and supports its potential as protein vaccine candidates against TB.

Biopotency of serine protease inhibitors from cowpea (Vigna unguiculata) seeds on digestive proteases and the development of Spodoptera littoralis (Boisduval).

PubMed

Abd El-latif, Ashraf Oukasha

2015-05-01

Serine protease inhibitors (PIs) have been described in many plant species and are universal throughout the plant kingdom, where trypsin inhibitors is the most common type. In the present study, trypsin and chymotrypsin inhibitory activity was detected in the seed flour extracts of 13 selected cultivars/accessions of cowpea. Two cowpea cultivars, Cream7 and Buff, were found to have higher trypsin and chymotrypsin inhibitory potential compared to other tested cultivars for which they have been selected for further purification studies using ammonium sulfate fractionation and DEAE-Sephadex A-25 column. Cream7-purified proteins showed two bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) corresponding to molecular mass of 17.10 and 14.90 kDa, while the purified protein from Buff cultivar showed a single band corresponding mass of 16.50 kDa. The purified inhibitors were stable at temperature below 60°C and were active at wide range of pH from 2 to 12. The kinetic analysis revealed noncompetitive type of inhibition for both inhibitors against both enzymes. The inhibitor constant (Ki ) values suggested high affinity between inhibitors and enzymes. Purified inhibitors were found to have deep and negative effects on the mean larval weight, larval mortality, pupation, and mean pupal weight of Spodoptera littoralis, where Buff PI was more effective than Cream7 PI. It may be concluded that cowpea PI gene(s) could be potential insect control protein for future studies in developing insect-resistant transgenic plants. © 2014 Wiley Periodicals, Inc.

Mutants of feline immunodeficiency virus resistant to 2',3'-dideoxy-2',3'-didehydrothymidine.

PubMed Central

Zhu, Y Q; Remington, K M; North, T W

1996-01-01

We selected mutants of feline immunodeficiency virus (FIV) that are resistant to 2',3'-dideoxy-2',3'-didehydrothymidine (d4T). Two mutants were selected in cultured cells with a stepwise increase in d4T concentration, resulting in mutants able to replicate in 100 microM d4T. These mutants were three- to sixfold more resistant to d4T than wild-type FIV. They were also cross-resistant to 3'-azido-3'-deoxythymidine (AZT), 3'-fluoro-2',3'-dideoxythymidine, 2',3'-dideoxycytidine, 2',3'-dideoxyinosine, and 9-(2-phosphonylmethoxyethyl)adenine, and they were highly resistant to phosphonoformic acid (PFA). Plaque-purified mutants were isolated from each of the mutant populations. The mutant phenotype was stable, because both of the plaque-purified mutants remained d4T resistant even after three passages in the absence of d4T. One of the plaque-purified mutants, designated D4R-3c, was further characterized. Compared with wild-type reverse transcriptase (RT), RT purified from D4R-3c was 3-fold resistant to inhibition by the 5'-triphosphate of d4T, 10-fold resistant to inhibition by the 5'-triphosphate of AZT, and 6-fold resistant to PFA. D4R-3c had a single point mutation in the RT-encoding region of the pol gene at position 2474, resulting in a Val to Ile mutation at codon 47 of the FIV RT. The role of this mutation in d4T resistance was confirmed by site-directed mutagenesis. PMID:8878567

Purification of diverse hemoglobins by metal salt precipitation.

PubMed

Zimmerman, Devon; Dienes, Jack; Abdulmalik, Osheiza; Elmer, Jacob J

2016-09-01

Although donated blood is the preferred material for transfusion, its limited availability and stringent storage requirements have motivated the development of blood substitutes. The giant extracellular hemoglobin (aka erythrocruorin) of the earthworm Lumbricus terrestris (LtEc) has shown promise as a blood substitute, but an efficient purification method for LtEc must be developed to meet the potential large demand for blood substitutes. In this work, an optimized purification process that uses divalent and trivalent metal salts to selectively precipitate human, earthworm, and bloodworm hemoglobin (HbA, LtEc, and GdHb, respectively) from crude solutions was developed. Although several metal ions were able to selectively precipitate LtEc, Zn(2+) and Ni(2+) provided the lowest heme oxidation and highest overall yield of LtEc. In contrast, Zn(2+) was the only metal ion that completely precipitated HbA and GdHb. Polyacrylamide gel electrophoresis (PAGE) analysis shows that metal precipitation removes several impurities to provide highly pure hemoglobin samples. Heme oxidation levels were relatively low for Zn(2+)-purified HbA and LtEc (2.4±1.3% and 5.3±2.1%, respectively), but slightly higher for Ni(2+)-purified LtEc (8.4±1.2%). The oxygen affinity and cooperativity of the precipitated samples are also identical to samples purified with tangential flow filtration (TFF) alone, indicating the metal precipitation does not significantly affect the function of the hemoglobins. Overall, these results show that hemoglobins from several different species can be highly purified using a combination of metal (Zn(2+)) precipitation and tangential flow filtration. Copyright © 2015 Elsevier Inc. All rights reserved.

Selective adsorption of benzhydroxamic acid on fluorite rendering selective separation of fluorite/calcite

NASA Astrophysics Data System (ADS)

Jiang, Wei; Gao, Zhiyong; Khoso, Sultan Ahmed; Gao, Jiande; Sun, Wei; Pu, Wei; Hu, Yuehua

2018-03-01

Fluorite, a chief source of fluorine in the nature, usually coexists with calcite mineral in ore deposits. Worldwide, flotation techniques with a selective collector and/or a selective depressant are commonly preferred for the separation of fluorite from calcite. In the present study, an attempt was made to use benzhydroxamic acid (BHA) as a collector for the selective separation of fluorite from calcite without using any depressant. Results obtained from the flotation experiments for single mineral and mixed binary minerals revealed that the BHA has a good selective collecting ability for the fluorite when 50 mg/L of BHA was used at pH of 9. The results from the zeta potential and X-ray photoelectron spectroscopy (XPS) indicated that the BHA easily chemisorbs onto the fluorite as compared to calcite. Crystal chemistry calculations showed the larger Ca density and the higher Ca activity on fluorite surface mainly account for the selective adsorption of BHA on fluorite, leading to the selective separation of fluorite from calcite. Moreover, a stronger hydrogen bonding with BHA and the weaker electrostatic repulsion with BHA- also contribute to the stronger interaction of BHA species with fluorite surface.

Silymarin versus Silibinin: Differential Antioxidant and Neuroprotective Effects against H2O2-induced Oxidative Stress in PC12 Cells.

PubMed

Jiang, Hui-Hui; Yan, Fa-Shun; Shen, Liang; Ji, Hong-Fang

2016-05-01

The present study assessed comparatively the antioxidant activities of silymarin and its major active component silibinin and their neuroprotective effects against hydrogen peroxide (H2O2)-induced oxidative stress in rat pheochromocytoma PC12 cells. It was found that despite newly prepared silymarin and silibinin solution possessing comparable superoxide anion (O2*-)-scavenging activities, with time the activity of silymarin lowered slightly, but that of silibinin decreased dramatically. Both silymarin and silibinin suppressed H2O2-induced oxidative stress and apoptosis, and the neuroprotective effect of silymarin was overall relatively stronger than that of silibinin. The findings provided clues for future studies on therapeutic potentials of the whole silymarin or purified silibinin for neurodegenerative diseases.

Composition and antioxidant activities of four polysaccharides extracted from Herba Lophatheri.

PubMed

Ge, Qing; Mao, Jian-wei; Guo, Xiao-qing; Zhou, Yi-feng; Gong, Jing-yan; Mao, Shuang-rong

2013-09-01

Four polysaccharides (BLF80-A, BLF80-B, BLF80-C and BLF80-D) were isolated by hot-water extraction and purified from the leaves of Herba Lophatheri by DEAE-Sepharose fast flow. Their chemical and physical characteristics were determined and antioxidant activities were investigated on the basis of DPPH radical assay, hydroxyl radical assay and superoxide radical assay. The results showed that four polysaccharides exhibited antioxidant activities in a concentration-dependent manner, and the higher molecular weight, the stronger antioxidant activities of polysaccharides. Besides, the monosaccharide compositions of polysaccharides also influence their antioxidant activities. BLP80-D showed the strongest scavenging ability, followed by BLP80-C, BLP80-B and BLP80-A. Copyright © 2013 Elsevier B.V. All rights reserved.

Strong Purifying Selection at Synonymous Sites in D. melanogaster

PubMed Central

Lawrie, David S.; Messer, Philipp W.; Hershberg, Ruth; Petrov, Dmitri A.

2013-01-01

Synonymous sites are generally assumed to be subject to weak selective constraint. For this reason, they are often neglected as a possible source of important functional variation. We use site frequency spectra from deep population sequencing data to show that, contrary to this expectation, 22% of four-fold synonymous (4D) sites in Drosophila melanogaster evolve under very strong selective constraint while few, if any, appear to be under weak constraint. Linking polymorphism with divergence data, we further find that the fraction of synonymous sites exposed to strong purifying selection is higher for those positions that show slower evolution on the Drosophila phylogeny. The function underlying the inferred strong constraint appears to be separate from splicing enhancers, nucleosome positioning, and the translational optimization generating canonical codon bias. The fraction of synonymous sites under strong constraint within a gene correlates well with gene expression, particularly in the mid-late embryo, pupae, and adult developmental stages. Genes enriched in strongly constrained synonymous sites tend to be particularly functionally important and are often involved in key developmental pathways. Given that the observed widespread constraint acting on synonymous sites is likely not limited to Drosophila, the role of synonymous sites in genetic disease and adaptation should be reevaluated. PMID:23737754

Evolution of the Male-Determining Gene SRY Within the Cat Family Felidae

PubMed Central

King, V.; Goodfellow, P. N.; Wilkerson, A. J. Pearks; Johnson, W. E.; O'Brien, S. J.; Pecon-Slattery, J.

2007-01-01

In most placental mammals, SRY is a single-copy gene located on the Y chromosome and is the trigger for male sex determination during embryonic development. Here, we present comparative genomic analyses of SRY (705 bp) along with the adjacent noncoding 5′ flank (997 bp) and 3′ flank (948 bp) in 36 species of the cat family Felidae. Phylogenetic analyses indicate that the noncoding genomic flanks and SRY closely track species divergence. However, several inconsistencies are observed in SRY. Overall, the gene exhibits purifying selection to maintain function (ω = 0.815) yet SRY is under positive selection in two of the eight felid lineages. SRY has low numbers of nucleotide substitutions, yet most encode amino acid changes between species, and four different species have significantly altered SRY due to insertion/deletions. Moreover, fixation of nonsynonymous substitutions between sister taxa is not consistent and may occur rapidly, as in the case of domestic cat, or not at all over long periods of time, as observed within the Panthera lineage. The former resembles positive selection during speciation, and the latter purifying selection to maintain function. Thus, SRY evolution in cats likely reflects the different phylogeographic histories, selection pressures, and patterns of speciation in modern felids. PMID:17277366

Positive selection in the SLC11A1 gene in the family Equidae.

PubMed

Bayerova, Zuzana; Janova, Eva; Matiasovic, Jan; Orlando, Ludovic; Horin, Petr

2016-05-01

Immunity-related genes are a suitable model for studying effects of selection at the genomic level. Some of them are highly conserved due to functional constraints and purifying selection, while others are variable and change quickly to cope with the variation of pathogens. The SLC11A1 gene encodes a transporter protein mediating antimicrobial activity of macrophages. Little is known about the patterns of selection shaping this gene during evolution. Although it is a typical evolutionarily conserved gene, functionally important polymorphisms associated with various diseases were identified in humans and other species. We analyzed the genomic organization, genetic variation, and evolution of the SLC11A1 gene in the family Equidae to identify patterns of selection within this important gene. Nucleotide SLC11A1 sequences were shown to be highly conserved in ten equid species, with more than 97 % sequence identity across the family. Single nucleotide polymorphisms (SNPs) were found in the coding and noncoding regions of the gene. Seven codon sites were identified to be under strong purifying selection. Codons located in three regions, including the glycosylated extracellular loop, were shown to be under diversifying selection. A 3-bp indel resulting in a deletion of the amino acid 321 in the predicted protein was observed in all horses, while it has been maintained in all other equid species. This codon comprised in an N-glycosylation site was found to be under positive selection. Interspecific variation in the presence of predicted N-glycosylation sites was observed.

Quantification of quercetin glycosides in 6 onion cultivars and comparisons of hydrolysis-HPLC and spectrophotometric methods in measuring total quercetin concentrations.

PubMed

Yoo, Kil Sun; Lee, Eun Jin; Patil, Bhimanagouda S

2010-03-01

This study was performed to purify and quantify quercetin glycosides (QG) and aglycone (free) quercetin (Q) in 6 selected onion cultivars and to compare analytical approaches based on high-performance liquid chromatography (HPLC) and spectrophotometry for the quantification of total quercetin (TQ) concentrations. Individual mono- and di-glycoside Q compounds were purified using a semipreparative HPLC and identified by comparing spectral data and by confirming corresponding peaks of QG and Q after incomplete enzyme-hydrolysis. Purified QG were quantified as Q by enzyme-hydrolysis/HPLC. TQ concentrations obtained from 20 onion bulbs with enzyme-hydrolysis/HPLC, no-hydrolysis/HPLC, and a spectrophotometric method without prior hydrolysis were significantly correlated (r(2)= 0.99) and were about 15% higher, identical, or 10% less than those concentrations by a standard acid-hydrolysis/HPLC method, respectively. During enzyme-hydrolysis of onion extracts, progressive reduction of the QG and formation of the corresponding mono-glycosides and Q were monitored using an analytical HPLC. TQ ranged from 83 to 330 microg/g F.W. in 6 selected cultivars of long-day or short-day onions. Q3,4'G and Q4'G were the 2 major compounds and comprised approximately between 94% and 97% of TQ in onions.

Anticancer activity of bacteriophage T4 and its mutant HAP1 in mouse experimental tumour models.

PubMed

Dabrowska, Krystyna; Opolski, Adam; Wietrzyk, Joanna; Switala-Jelen, Kinga; Godlewska, Joanna; Boratynski, Janusz; Syper, Danuta; Weber-Dabrowska, Beata; Gorski, Andrzej

2004-01-01

Previously, we have shown the ability of the bacteriophage T4 and its substrain HAP1 (selected for a higher affinity to melanoma cells) to reveal antimetastatic activity in a mouse melanoma model. Here, we investigated the potential phage anticancer activity in primary tumour models. Mice were inoculated subcutaneously with B16 or LLC cells (collected from in vitro culture). Bacteriophages T4 and HAP1 were injected intraperitoneally daily (8 x 10(8)pfu/mouse, except the experiment concerning the dose-dependence). Treatment with purified preparations of bacteriophage T4 resulted in significant reduction of tumour size, the effect being dose-dependent. HAP1 was more effective than T4 and its activity was also dose-dependent. Parallel experiments with non-purified bacteriophage lysates resulted in significant stimulation of tumour growth. These data suggest that purified bacteriophages may inhibit tumour growth, a phenomenon with potentially important clinical implications in oncology.

[Study on extraction and purification technology of Hubei ophiopogon saponins].

PubMed

Lin, Yun-Han; Li, Chong-Ming; Li, Xiao-Dong; Xiang, Yang; Zhang, Ya-Qin; Zhang, Xiao-Cun; Liu, Xia

2013-05-01

To explore the extraction and purification technology of total saponins from the effective parts of Liriope spicata. Orthogonal design was used. Macroporous resin was selected to separate and purify total saponin from the effective parts of Liriope spicata. The process validation was conducted. The total saponins was determined by Ultraviolet Spectrophotometry. The optimal extraction conditions were as follows: 10 times the amount of ethanol (70%) for each occasion and hot reflux (3 x 2 h). Total saponins was purified by D101 macroporous resin. The concentration of eluting ethanol was 50% - 70%. Ethanol (70%) was selected as the best eluent. The result of process validation was consistent with the study. The process is simple and stable enough to significantly improve the extraction rate of the effective parts. The study can provide reference for the research and production of effective parts of traditional Chinese medicine such as Liriope spicata.

Host-Parasite Interactions and Purifying Selection in a Microsporidian Parasite of Honey Bees

PubMed Central

Huang, Qiang; Chen, Yan Ping; Wang, Rui Wu; Cheng, Shang; Evans, Jay D.

2016-01-01

To clarify the mechanisms of Nosema ceranae parasitism, we deep-sequenced both honey bee host and parasite mRNAs throughout a complete 6-day infection cycle. By time-series analysis, 1122 parasite genes were significantly differently expressed during the reproduction cycle, clustering into 4 expression patterns. We found reactive mitochondrial oxygen species modulator 1 of the host to be significantly down regulated during the entire infection period. Our data support the hypothesis that apoptosis of honey bee cells was suppressed during infection. We further analyzed genome-wide genetic diversity of this parasite by comparing samples collected from the same site in 2007 and 2013. The number of SNP positions per gene and the proportion of non-synonymous substitutions per gene were significantly reduced over this time period, suggesting purifying selection on the parasite genome and supporting the hypothesis that a subset of N. ceranae strains might be dominating infection. PMID:26840596

Host-Parasite Interactions and Purifying Selection in a Microsporidian Parasite of Honey Bees.

PubMed

Huang, Qiang; Chen, Yan Ping; Wang, Rui Wu; Cheng, Shang; Evans, Jay D

2016-01-01

To clarify the mechanisms of Nosema ceranae parasitism, we deep-sequenced both honey bee host and parasite mRNAs throughout a complete 6-day infection cycle. By time-series analysis, 1122 parasite genes were significantly differently expressed during the reproduction cycle, clustering into 4 expression patterns. We found reactive mitochondrial oxygen species modulator 1 of the host to be significantly down regulated during the entire infection period. Our data support the hypothesis that apoptosis of honey bee cells was suppressed during infection. We further analyzed genome-wide genetic diversity of this parasite by comparing samples collected from the same site in 2007 and 2013. The number of SNP positions per gene and the proportion of non-synonymous substitutions per gene were significantly reduced over this time period, suggesting purifying selection on the parasite genome and supporting the hypothesis that a subset of N. ceranae strains might be dominating infection.

Role of laccase from Coriolus versicolor MTCC-138 in selective oxidation of aromatic methyl group.

PubMed

Chaurasia, Pankaj Kumar; Singh, Sunil Kumar; Bharati, Shashi Lata

2014-01-01

Now a day, laccases are the most promising enzymes in the area of biotechnology and synthesis. One of the best applications of laccases is the selective oxidation of aromatic methyl group to aldehyde group. Such transformations are valuable because it is difficult to stop the reaction at aldehyde stage. Chemical methods used for such biotransformations areexpensive and give poor yields. But, the laccase-catalyzed biotransformations of such type are non-expensive and yield is excellent. Authors have used crude laccase obtained from the liquid culture growth medium of fungal strain Coriolus versicolor MTCC-138 for the biotransformations of toluene, 3-nitrotoluene, and 4-chlorotoluene to benzaldehyde, 3-nitrobenzaldehyde, and 4-chlorobenzaldehyde, respectively, instead of purified laccase because purification process requires much time and cost. This communication reports that crude laccase can also be used in the place of purified laccase as effective biocatalyst.

Effects of Simulated Hypogravity and Diet on Estrous Cycling in Rats

NASA Technical Reports Server (NTRS)

Tou, Janet C.; Grindeland, Richard E.; Baer, Lisa A.; Wade, Charles E.

2003-01-01

Environmental factors can disrupt ovulatory cycles. The study objective was to determine the effect of diet and simulated hypogravity on rat estrous cycles. Age 50 d Sprague-Dawley rats were randomly assigned to he fed either a purified or chow diet. Only normal cycling rats were used. Experimental rats (n=9-10/group) were kept as ambulatory controls (AC) or subjected to 40 d simulated hypogravity using a disuse atrophy hindlimb suspension (HLS) model. There was no effect on estrous cycles of AC fed either diet. At day 18, HLS rats fed either diet, had lengthened estrous cycles due to prolonged diestrus. HLS rats fed purified diet also had reduced time in estrus. Plasma estradiol was reduced in HLS rats fed purified diet but there was no effect on progesterone. This may have occurred because blood was collected from rats in estrus. Urinary progesterone collected during initial HLS was elevated in rats fed purified diet. In AC, corticosterone was elevated in chow vs purified diet fed rats. Differences were particularly striking following the application of a stressor with HLS/chow-fed rats displaying an enhanced stress response. Results emphasize the importance of diet selection when measuring endocrine-sensitive endpoints. HLS is a useful model for investigating the effects of environment on reproduction and providing insight about the impact extreme environment such as spaceflight on female reproductive health.

Molecular and functional characterization of FcγRIIIb receptor-ligand interaction: implications for neutrophil mediated immune mechanisms in malaria.

PubMed

Simtong, Piyapong; Romphruk, Amornrat V; Traum, Annalena; Burg-Roderfeld, Monika; Bein, Gregor; Jakubowski, Konstantin; Dominik, Andreas; Theisen, Michael; Kana, Ikhlaq Hussain; Sachs, Ulrich J; Santoso, Sentot

2018-05-21

The Fcγ receptor IIIb (FcγRIIIb) is a low-affinity receptor of IgG and is essential in neutrophil mediated effector functions. Different allelic forms of FcγRIIIb carrying human neutrophil antigen (HNA-1a, -1b, -1c and -1d) have been identified. Here, we have generated stable transfected HEK293 cell lines expressing HNA-1aa, -1bb, and -1bc. Of these, cells expressing HNA-1bc interacted significantly stronger (2.277 versus 0.743) with human IgG than cells expressing the HNA-1aa or -1bb alloforms. The higher affinity of IgG towards the HNA-1c alloform was confirmed using neutrophils derived from German blood donors. Neutrophils from HNA-1abc phenotyped individuals bound IgG significantly stronger (1.825 versus 0.903) than neutrophils from HNA-1ab typed individuals. These findings were confirmed by the SPR analysis demonstrating that recombinant HNA-1bc had a higher affinity (KD 7.24 x 10 -6 M) than recombinant HNA-1bb (KD 1.15 x 10 -5 M) against normal IgG. Finally, we demonstrated that Plasmodium falciparum merozoites opsonized with human IgG affinity purified against P. falciparum Glutamate rich protein (GLURP) enhanced stronger ROS emission in neutrophils obtained from HNA-1abc donors compared to neutrophils from HNA-1ab donors. Collectively, these results indicate that the amino acid substitution Ala 78 Asp resulting in the HNA-1c allotype leads to higher affinity towards human IgG, enhancement of neutrophil activation and possibly effective clearance of malaria by intracellular ROS. Copyright © 2018 American Society for Microbiology.

Selection and characterization of a mutant of feline immunodeficiency virus resistant to 2',3'-dideoxycytidine.

PubMed Central

Medlin, H K; Zhu, Y Q; Remington, K M; Phillips, T R; North, T W

1996-01-01

We have selected and plaque purified a mutant of feline immunodeficiency virus (FIV) that is resistant to 2',3'-dideoxycytidine (ddC). This mutant was selected in cultured cells in the continuous presence of 25 microM ddC. The mutant, designated DCR-5c, was fourfold resistant to ddC, threefold resistant to 2',3'-dideoxyinosine, and more than fourfold resistant to phosphonoformic acid. DCR-5c displayed little or no resistance to (-)-beta-2',3'-dideoxy-3'-thiacytidine, 3'-azido-3'-deoxythymidine, or 9-(2-phosphonylmethoxyethyl) adenine. Reverse transcriptase purified from DCR-5c was less susceptible to inhibition by ddCTP, phosphonoformic acid, ddATP, or azido-dTTP than the wild-type FIV reverse transcriptase. Sequence analysis of DCR-5c revealed a single base change (G to C at nucleotide 2342) in the reverse transcriptase-encoding region of FIV. This mutation results in substitution of His for Asp at codon 3 of FIV reverse transcriptase. The role of this mutation in ddC resistance was confirmed by site-directed mutagenesis. PMID:8849258

Method for refining contaminated iridium

DOEpatents

Heshmatpour, B.; Heestand, R.L.

1982-08-31

Contaminated iridium is refined by alloying it with an alloying agent selected from the group consisting of manganese and an alloy of manganese and copper, and then dissolving the alloying agent from the formed alloy to provide a purified iridium powder.

The population genomics of rhesus macaques (Macaca mulatta) based on whole-genome sequences

PubMed Central

Xue, Cheng; Raveendran, Muthuswamy; Harris, R. Alan; Fawcett, Gloria L.; Liu, Xiaoming; White, Simon; Dahdouli, Mahmoud; Rio Deiros, David; Below, Jennifer E.; Salerno, William; Cox, Laura; Fan, Guoping; Ferguson, Betsy; Horvath, Julie; Johnson, Zach; Kanthaswamy, Sree; Kubisch, H. Michael; Liu, Dahai; Platt, Michael; Smith, David G.; Sun, Binghua; Vallender, Eric J.; Wang, Feng; Wiseman, Roger W.; Chen, Rui; Muzny, Donna M.; Gibbs, Richard A.; Yu, Fuli; Rogers, Jeffrey

2016-01-01

Rhesus macaques (Macaca mulatta) are the most widely used nonhuman primate in biomedical research, have the largest natural geographic distribution of any nonhuman primate, and have been the focus of much evolutionary and behavioral investigation. Consequently, rhesus macaques are one of the most thoroughly studied nonhuman primate species. However, little is known about genome-wide genetic variation in this species. A detailed understanding of extant genomic variation among rhesus macaques has implications for the use of this species as a model for studies of human health and disease, as well as for evolutionary population genomics. Whole-genome sequencing analysis of 133 rhesus macaques revealed more than 43.7 million single-nucleotide variants, including thousands predicted to alter protein sequences, transcript splicing, and transcription factor binding sites. Rhesus macaques exhibit 2.5-fold higher overall nucleotide diversity and slightly elevated putative functional variation compared with humans. This functional variation in macaques provides opportunities for analyses of coding and noncoding variation, and its cellular consequences. Despite modestly higher levels of nonsynonymous variation in the macaques, the estimated distribution of fitness effects and the ratio of nonsynonymous to synonymous variants suggest that purifying selection has had stronger effects in rhesus macaques than in humans. Demographic reconstructions indicate this species has experienced a consistently large but fluctuating population size. Overall, the results presented here provide new insights into the population genomics of nonhuman primates and expand genomic information directly relevant to primate models of human disease. PMID:27934697

Increased burden of deleterious variants in essential genes in autism spectrum disorder.

PubMed

Ji, Xiao; Kember, Rachel L; Brown, Christopher D; Bućan, Maja

2016-12-27

Autism spectrum disorder (ASD) is a heterogeneous, highly heritable neurodevelopmental syndrome characterized by impaired social interaction, communication, and repetitive behavior. It is estimated that hundreds of genes contribute to ASD. We asked if genes with a strong effect on survival and fitness contribute to ASD risk. Human orthologs of genes with an essential role in pre- and postnatal development in the mouse [essential genes (EGs)] are enriched for disease genes and under strong purifying selection relative to human orthologs of mouse genes with a known nonlethal phenotype [nonessential genes (NEGs)]. This intolerance to deleterious mutations, commonly observed haploinsufficiency, and the importance of EGs in development suggest a possible cumulative effect of deleterious variants in EGs on complex neurodevelopmental disorders. With a comprehensive catalog of 3,915 mammalian EGs, we provide compelling evidence for a stronger contribution of EGs to ASD risk compared with NEGs. By examining the exonic de novo and inherited variants from 1,781 ASD quartet families, we show a significantly higher burden of damaging mutations in EGs in ASD probands compared with their non-ASD siblings. The analysis of EGs in the developing brain identified clusters of coexpressed EGs implicated in ASD. Finally, we suggest a high-priority list of 29 EGs with potential ASD risk as targets for future functional and behavioral studies. Overall, we show that large-scale studies of gene function in model organisms provide a powerful approach for prioritization of genes and pathogenic variants identified by sequencing studies of human disease.

Increased burden of deleterious variants in essential genes in autism spectrum disorder

PubMed Central

Kember, Rachel L.; Brown, Christopher D.; Bućan, Maja

2016-01-01

Autism spectrum disorder (ASD) is a heterogeneous, highly heritable neurodevelopmental syndrome characterized by impaired social interaction, communication, and repetitive behavior. It is estimated that hundreds of genes contribute to ASD. We asked if genes with a strong effect on survival and fitness contribute to ASD risk. Human orthologs of genes with an essential role in pre- and postnatal development in the mouse [essential genes (EGs)] are enriched for disease genes and under strong purifying selection relative to human orthologs of mouse genes with a known nonlethal phenotype [nonessential genes (NEGs)]. This intolerance to deleterious mutations, commonly observed haploinsufficiency, and the importance of EGs in development suggest a possible cumulative effect of deleterious variants in EGs on complex neurodevelopmental disorders. With a comprehensive catalog of 3,915 mammalian EGs, we provide compelling evidence for a stronger contribution of EGs to ASD risk compared with NEGs. By examining the exonic de novo and inherited variants from 1,781 ASD quartet families, we show a significantly higher burden of damaging mutations in EGs in ASD probands compared with their non-ASD siblings. The analysis of EGs in the developing brain identified clusters of coexpressed EGs implicated in ASD. Finally, we suggest a high-priority list of 29 EGs with potential ASD risk as targets for future functional and behavioral studies. Overall, we show that large-scale studies of gene function in model organisms provide a powerful approach for prioritization of genes and pathogenic variants identified by sequencing studies of human disease. PMID:27956632

Extreme primary and secondary protein structure variability in the chimeric male-transmitted cytochrome c oxidase subunit II protein in freshwater mussels: Evidence for an elevated amino acid substitution rate in the face of domain-specific purifying selection

PubMed Central

2008-01-01

Background Freshwater unionoidean bivalves, and species representing two marine bivalve orders (Mytiloida and Veneroida), exhibit a mode of mtDNA inheritance involving distinct maternal (F) and paternal (M) transmission routes concomitant with highly divergent gender-associated mtDNA genomes. Additionally, male unionoidean bivalves have a ~550 bp 3' coding extension to the cox2 gene (Mcox2e), that is apparently absent from all other metazoan taxa. Results Our molecular sequence analyses of MCOX2e indicate that both the primary and secondary structures of the MCOX2e region are evolving much faster than other regions of the F and M COX2-COX1 gene junction. The near N-terminus ~2/3 of the MCOX2e region contains an interspecifically variable number of predicted transmembrane helices (TMH) and interhelical loops (IHL) whereas the C-terminus ~1/3 is relatively conserved and hydrophilic while containing conserved functional motifs. MCOX2e displays an overall pattern of purifying selection that leads to the preservation of TMH/IHL and C-terminus tail sub-regions. However, 14 amino acid positions in the MCOX2e TMH/IHL sub-region might be targeted by diversifying selection, each representing a site where there exists interspecific variation for the constituent amino acids residing in a TMH or IHL. Conclusion Our results indicate that Mcox2e is unique to unionoidean bivalves, likely the result of a single insertion event that took place over 65 MYA and that MCOX2e is functional. The predicted TMH number, length and position variability likely stems from substitution-based processes rather than the typically implicated insertion/deletion events. MCOX2e has relatively high rates of primary and secondary structure evolution, with some amino acid residues potentially subjected to site-specific positive selection, yet an overall pattern of purifying selection leading to the preservation of the TMH/IHL and hydrophilic C-terminus tail subregions. The more conserved C-terminus tail (relative to the TMH/IHL sub-region of MCOX2e) is likely biologically active because it contains functional motifs. The rapid evolution of primary and secondary structure in MCOX2e, combined with the action of both positive and purifying selection, provide supporting evidence for the hypothesis that MCOX2e has a novel reproductive function within unionoidean bivalves. All tolled, our data indicate that unionoidean bivalve MCOX2 is the first reported chimeric animal mtDNA-encoded protein. PMID:18513440

Identification of Conflicting Selective Effects on Highly Expressed Genes

PubMed Central

Higgs, Paul G.; Hao, Weilong; Golding, G. Brian

2007-01-01

Many different selective effects on DNA and proteins influence the frequency of codons and amino acids in coding sequences. Selection is often stronger on highly expressed genes. Hence, by comparing high- and low-expression genes it is possible to distinguish the factors that are selected by evolution. It has been proposed that highly expressed genes should (i) preferentially use codons matching abundant tRNAs (translational efficiency), (ii) preferentially use amino acids with low cost of synthesis, (iii) be under stronger selection to maintain the required amino acid content, and (iv) be selected for translational robustness. These effects act simultaneously and can be contradictory. We develop a model that combines these factors, and use Akaike’s Information Criterion for model selection. We consider pairs of paralogues that arose by whole-genome duplication in Saccharmyces cerevisiae. A codon-based model is used that includes asymmetric effects due to selection on highly expressed genes. The largest effect is translational efficiency, which is found to strongly influence synonymous, but not non-synonymous rates. Minimization of the cost of amino acid synthesis is implicated. However, when a more general measure of selection for amino acid usage is used, the cost minimization effect becomes redundant. Small effects that we attribute to selection for translational robustness can be identified as an improvement in the model fit on top of the effects of translational efficiency and amino acid usage. PMID:19430600

Selecting Capabilities for Quality of Life Measurement

ERIC Educational Resources Information Center

Robeyns, Ingrid

2005-01-01

The capability approach advocates that interpersonal comparisons be made in the space of functionings and capabilities. However, Amartya Sen has not specified which capabilities should be selected as the relevant ones. This has provoked two types of criticism. The stronger critique is Martha Nussbaum's claim that Sen should endorse one specific…

Diversity at Issue in Teacher Selection

ERIC Educational Resources Information Center

Sawchuk, Stephen

2013-01-01

Slowly but surely, a growing number of states are eyeing policies to select academically stronger individuals for their teaching programs as one avenue to improve the quality of new teachers. Underneath the attention such plans are attracting, though, run deep-seated fears about their potential consequences--particularly whether they will result…

Apparatus for purifying arsine, phosphine, ammonia, and inert gases to remove Lewis acid and oxidant impurities therefrom

DOEpatents

Tom, Glenn M.; Brown, Duncan W.

1991-01-08

An apparatus for purifying a gaseous mixture comprising arsine, phosphine, ammonia, and/or inert gases, to remove Lewis acid and/or oxidant impurities therefrom, comprising a vessel containing a bed of a scavenger, the scavenger including a support having associated therewith an anion which is effective to remove such impurities, such anion being selected from one or more members of the group consisting of: (i) carbanions whose corresponding protonated compounds have a pK.sub.a value of from about 22 to about 36; and (ii) anions formed by reaction of such carbanions with the primary component of the mixture.

Ozone removal capability of a welding fume respirator containing activated charcoal.

PubMed

Johnston, A R; Dyrud, J F; Shih, Y T

1989-09-01

Development of air purifying respirators for protection against ozone has been slowed by concerns about oxidation of charcoal and other available sorbents. The suitability of a charcoal sorbent for low concentrations of ozone was evaluated as a part of the development of a half-mask air purifying respirator designed for welding fumes and ozone. Testing of the respirator confirmed that charcoal can be a suitable sorbent for low levels of ozone. Where the respirator is properly selected, fit tested, and worn, respirator use against welding fumes and ozone at concentrations not exceeding 10 times the permissible exposure limit had been recommended.

Selection of Human Antibody Fragments which Bind Novel Breast Tumor Antigens

DTIC Science & Technology

1996-09-01

OmniGene cycler. The products were gel C6.5 must occur rarely in the repertoire, since none purified, isolated from the gel using DEAE membranes, of 92...C6VHCDR3A, C6VHCDR3B, C6VHCDR3C, and minute) using a Hybaid OmniGene cycler. To introduce C6VHCDR3D. a Notl restriction site at the 3’ end of the scFv gene...34C for 20 30 sec, 55 *C for 30 sec and 72°C for 30 sec) using a Hybaid OmniGene cycler. The products were gel purified, isolated from the gel using

Technical adequacy of bisulfite sequencing and pyrosequencing for detection of mitochondrial DNA methylation: Sources and avoidance of false-positive detection.

PubMed

Owa, Chie; Poulin, Matthew; Yan, Liying; Shioda, Toshi

2018-01-01

The existence of cytosine methylation in mammalian mitochondrial DNA (mtDNA) is a controversial subject. Because detection of DNA methylation depends on resistance of 5'-modified cytosines to bisulfite-catalyzed conversion to uracil, examined parameters that affect technical adequacy of mtDNA methylation analysis. Negative control amplicons (NCAs) devoid of cytosine methylation were amplified to cover the entire human or mouse mtDNA by long-range PCR. When the pyrosequencing template amplicons were gel-purified after bisulfite conversion, bisulfite pyrosequencing of NCAs did not detect significant levels of bisulfite-resistant cytosines (brCs) at ND1 (7 CpG sites) or CYTB (8 CpG sites) genes (CI95 = 0%-0.94%); without gel-purification, significant false-positive brCs were detected from NCAs (CI95 = 4.2%-6.8%). Bisulfite pyrosequencing of highly purified, linearized mtDNA isolated from human iPS cells or mouse liver detected significant brCs (~30%) in human ND1 gene when the sequencing primer was not selective in bisulfite-converted and unconverted templates. However, repeated experiments using a sequencing primer selective in bisulfite-converted templates almost completely (< 0.8%) suppressed brC detection, supporting the false-positive nature of brCs detected using the non-selective primer. Bisulfite-seq deep sequencing of linearized, gel-purified human mtDNA detected 9.4%-14.8% brCs for 9 CpG sites in ND1 gene. However, because all these brCs were associated with adjacent non-CpG brCs showing the same degrees of bisulfite resistance, DNA methylation in this mtDNA-encoded gene was not confirmed. Without linearization, data generated by bisulfite pyrosequencing or deep sequencing of purified mtDNA templates did not pass the quality control criteria. Shotgun bisulfite sequencing of human mtDNA detected extremely low levels of CpG methylation (<0.65%) over non-CpG methylation (<0.55%). Taken together, our study demonstrates that adequacy of mtDNA methylation analysis using methods dependent on bisulfite conversion needs to be established for each experiment, taking effects of incomplete bisulfite conversion and template impurity or topology into consideration.

Assessment of hemolytic activity, enzyme production and bacteriocin characterization of Bacillus subtilis LR1 isolated from the gastrointestinal tract of fish.

PubMed

Banerjee, Goutam; Nandi, Ankita; Ray, Arun Kumar

2017-01-01

In the present investigation, probiotic potential (antagonistic activity, enzyme production, hemolytic activity, biosafety, antibiotic sensitivity and bile tolerance level) of Bacillus subtilis LR1 was evaluated. Bacteriocin produced by the bacterial strain B. subtilis LR1 isolated from the gastrointestinal tract of Labeo rohita was purified and characterized. The molecular weight of the purified bacteriocin was ~50 kDa in 12 % Native PAGE and showed inhibitory activity against four fish pathogens such as Bacillus mycoides, Aeromonas salmonicida, Pseudomonas fluorescens and Aeromonas hydrophila. The purified bacteriocin was maximally active at temperature 40 °C and pH 7.0, while none of the tested surfactants affect the bacteriocin activity. Extracellular enzyme activity of the selected bacterial strain was also evaluated. Amylase activity was estimated to be highest (38.23 ± 1.15 µg of maltose liberated mg -1  protein ml -1 of culture filtrate) followed by cellulase and protease activity. The selected bacterium was sensitive to most of the antibiotics used in this experiment, can tolerate 0.25 % bile salt and non-hemolytic in nature. Finally, the efficiency of the proposed probiotic candidate was evaluated in in vivo condition. It was detected that the bacterial strain can effectively reduce bacterial pathogenicity in Indian major carps.

Development of a Novel Human scFv Against EGFR L2 Domain by Phage Display Technology.

PubMed

Rahbarnia, Leila; Farajnia, Safar; Babaei, Hossein; Majidi, Jafar; Veisi, Kamal; Khosroshahi, Shiva Ahdi; Tanomand, Asghar

2017-01-01

Epidermal growth factor receptor (EGFR) as a transmembrane tyrosine kinase receptor frequently overexpresses in tumors with epithelial origin. The L2 domain from extracellular part of EGFR is involved in ligand binding and the blockage of this domain prevents activation of related signaling pathways. This study was aimed to develop a novel human scFv against EGFR L2 domain as a promising target for cancer therapy. The L2 recombinant protein was purified and used for panning a human scFv phage library (Tomlinson I). In this study, a novel screening strategy was applied to select clones with high binding and enrichment of rare specific phage clones of the L2 protein. After five biopanning rounds several specific clones were isolated which among them one phage clone with high binding was purified for further analysis. The specific interaction of selected clone against target antigen was confirmed by ELISA and western blotting. Immunofluorescence staining showed that purified scFv binds to A431 cells surface, displaying EGFR surface receptor. In the present study, we isolated for the first time a novel human scFv against EGFR L2 domain. This study can be the groundwork for developing more effective diagnostic and therapeutic agents against EGFR overexpressing cancers using this novel human anti-L2 ScFv. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Molecular population genetics of the insulin/TOR signal transduction pathway: a network-level analysis in Drosophila melanogaster.

PubMed

Alvarez-Ponce, David; Guirao-Rico, Sara; Orengo, Dorcas J; Segarra, Carmen; Rozas, Julio; Aguadé, Montserrat

2012-01-01

The IT-insulin/target of rapamycin (TOR)-signal transduction pathway is a relatively well-characterized pathway that plays a central role in fundamental biological processes. Network-level analyses of DNA divergence in Drosophila and vertebrates have revealed a clear gradient in the levels of purifying selection along this pathway, with the downstream genes being the most constrained. Remarkably, this feature does not result from factors known to affect selective constraint such as gene expression, codon bias, protein length, and connectivity. The present work aims to establish whether the selective constraint gradient detected along the IT pathway at the between-species level can also be observed at a shorter time scale. With this purpose, we have surveyed DNA polymorphism in Drosophila melanogaster and divergence from D. simulans along the IT pathway. Our network-level analysis shows that DNA polymorphism exhibits the same polarity in the strength of purifying selection as previously detected at the divergence level. This equivalent feature detected both within species and between closely and distantly related species points to the action of a general mechanism, whose action is neither organism specific nor evolutionary time dependent. The detected polarity would be, therefore, intrinsic to the IT pathway architecture and function.

Efficient production of artificially designed gelatins with a Bacillus brevis system.

PubMed

Kajino, T; Takahashi, H; Hirai, M; Yamada, Y

2000-01-01

Artificially designed gelatins comprising tandemly repeated 30-amino-acid peptide units derived from human alphaI collagen were successfully produced with a Bacillus brevis system. The DNA encoding the peptide unit was synthesized by taking into consideration the codon usage of the host cells, but no clones having a tandemly repeated gene were obtained through the above-mentioned strategy. Minirepeat genes could be selected in vivo from a mixture of every possible sequence encoding an artificial gelatin by randomly ligating the mixed sequence unit and transforming it into Escherichia coli. Larger repeat genes constructed by connecting minirepeat genes obtained by in vivo selection were also stable in the expression host cells. Gelatins derived from the eight-unit and six-unit repeat genes were extracellularly produced at the level of 0.5 g/liter and easily purified by ammonium sulfate fractionation and anion-exchange chromatography. The purified artificial gelatins had the predicted N-terminal sequences and amino acid compositions and a solgel property similar to that of the native gelatin. These results suggest that the selection of a repeat unit sequence stable in an expression host is a shortcut for the efficient production of repetitive proteins and that it can conveniently be achieved by the in vivo selection method. This study revealed the possible industrial application of artificially designed repetitive proteins.

Cell selection and characterization of a novel human endothelial cell specific nanobody.

PubMed

Ahmadvand, Davoud; Rasaee, Mohammad J; Rahbarizadeh, Fatemeh; Kontermann, Roland E; Sheikholislami, Farzaneh

2009-05-01

Antibody-based targeting of angiogenesis and vascular targeting therapy of cancer are extremely attractive conceptually and open new important diagnostic and therapeutic opportunities. Compelling evidence suggests that CD105 represents an ideal target for anti-angiogenic therapy and its presence in solid tumor vasculature has prognostic value. Camelids produce functional antibodies devoid of light chains and constant heavy chain domain (CH1). Nanobodies, the antigen-binding fragments of such heavy chain antibodies, are therefore comprised in one single domain. The aim of this study was to explore the possibilities of using anti-endoglin nanobody as an angiogenesis inhibitor. The anti-CD105 nanobody (AR-86a) was isolated from immune library by selections on purified antigens and target cells. Immunocytochemistry and FACS analysis showed that the purified nanobody reacted specifically with human umbilical vein endothelial cells (HUVECs) but not with other cell lines such as MDA-MB-453, Mel III, T-47D, MCF-7, AGO and HT 29. Further, selected nanobody potently inhibited proliferation of human endothelial cells and formation of capillary-like structures. This selected high affinity anti-endoglin nanobody may offer high specificity towards tumors with reduced side effects, and may be less likely to elicit drug resistance compared to conventional therapy.

A selective glucose sensor: the cooperative effect of monoboronic acid-modified poly(amidoamine) dendrimers.

PubMed

Tsai, Ching-Hua; Tang, Yi-Hsuan; Chen, Hui-Ting; Yao, Yi-Wen; Chien, Tun-Cheng; Kao, Chai-Lin

2018-05-01

Selective glucose binding was identified through five generations of monoboronic acid-functionalized PAMAM dendrimers. The best selectivity obtained when using G3 dendrimers (1b) generated 71.1, 94.9, and 1309 times stronger binding than when using galactose, fructose, and lactose, respectively. Further experiments using dendrimer analogues and glucose derivatives suggested that two nearby monoboronic acids cooperatively bound one glucose.

Parent-progeny sequencing indicates higher mutation rates in heterozygotes.

PubMed

Yang, Sihai; Wang, Long; Huang, Ju; Zhang, Xiaohui; Yuan, Yang; Chen, Jian-Qun; Hurst, Laurence D; Tian, Dacheng

2015-07-23

Mutation rates vary within genomes, but the causes of this remain unclear. As many prior inferences rely on methods that assume an absence of selection, potentially leading to artefactual results, we call mutation events directly using a parent-offspring sequencing strategy focusing on Arabidopsis and using rice and honey bee for replication. Here we show that mutation rates are higher in heterozygotes and in proximity to crossover events. A correlation between recombination rate and intraspecific diversity is in part owing to a higher mutation rate in domains of high recombination/diversity. Implicating diversity per se as a cause, we find an ∼3.5-fold higher mutation rate in heterozygotes than in homozygotes, with mutations occurring in closer proximity to heterozygous sites than expected by chance. In a genome that is a patchwork of heterozygous and homozygous domains, mutations occur disproportionately more often in the heterozygous domains. If segregating mutations predispose to a higher local mutation rate, clusters of genes dominantly under purifying selection (more commonly homozygous) and under balancing selection (more commonly heterozygous), might have low and high mutation rates, respectively. Our results are consistent with this, there being a ten times higher mutation rate in pathogen resistance genes, expected to be under positive or balancing selection. Consequently, we do not necessarily need to evoke extremely weak selection on the mutation rate to explain why mutational hot and cold spots might correspond to regions under positive/balancing and purifying selection, respectively.

Evolutionary dynamics of Rh2 opsins in birds demonstrate an episode of accelerated evolution in the New World warblers (Setophaga)

PubMed Central

Price, Trevor D.

2015-01-01

Low rates of sequence evolution associated with purifying selection can be interrupted by episodic changes in selective regimes. Visual pigments are a unique system in which we can investigate the functional consequences of genetic changes, therefore connecting genotype to phenotype in the context of natural and sexual selection pressures. We study the RH2 and RH1 visual pigments (opsins) across 22 bird species belonging to two ecologically convergent clades, the New World warblers (Parulidae) and Old World warblers (Phylloscopidae), and evaluate rates of evolution in these clades along with data from 21 additional species. We demonstrate generally slow evolution of these opsins: both Rh1 and Rh2 are highly conserved across Old World and New World warblers. However, Rh2 underwent a burst of evolution within the New World genus Setophaga, where it accumulated substitutions at 6 amino acid sites across the species we studied. Evolutionary analyses revealed a significant increase in dN/dS in Setophaga, implying relatively strong selective pressures to overcome long-standing purifying selection. We studied the effects of each substitution on spectral tuning and found they do not cause large spectral shifts. Thus substitutions may reflect other aspects of opsin function, such as those affecting photosensitivity and/or dark-light adaptation. Although it is unclear what these alterations mean for color perception, we suggest that rapid evolution is linked to sexual selection, given the exceptional plumage colour diversification in Setophaga. PMID:25827331

MC1R diversity in Northern Island Melanesia has not been constrained by strong purifying selection and cannot explain pigmentation phenotype variation in the region.

PubMed

Norton, Heather L; Werren, Elizabeth; Friedlaender, Jonathan

2015-10-19

Variation in human skin pigmentation evolved in response to the selective pressure of ultra-violet radiation (UVR). Selection to maintain darker skin in high UVR environments is expected to constrain pigmentation phenotype and variation in pigmentation loci. Consistent with this hypothesis, the gene MC1R exhibits reduced diversity in African populations from high UVR regions compared to low-UVR non-African populations. However, MC1R diversity in non-African populations that have evolved under high-UVR conditions is not well characterized. In order to test the hypothesis that MC1R variation has been constrained in Melanesians the coding region of the MC1R gene was sequenced in 188 individuals from Northern Island Melanesia. The role of purifying selection was assessed using a modified McDonald Kreitman's test. Pairwise FST was calculated between Melanesian populations and populations from the 1000 Genomes Project. The SNP rs2228479 was genotyped in a larger sample (n = 635) of Melanesians and tested for associations with skin and hair pigmentation. We observe three nonsynonymous and two synonymous mutations. A modified McDonald Kreitman's test failed to detect a significant signal of purifying selection. Pairwise FST values calculated between the four islands sampled here indicate little regional substructure in MC1R. When compared to African, European, East and South Asian populations, Melanesians do not exhibit reduced population divergence (measured as FST) or a high proportion of haplotype sharing with Africans, as one might expect if ancestral haplotypes were conserved across high UVR populations in and out of Africa. The only common nonsynonymous polymorphism observed, rs2228479, is not significantly associated with skin or hair pigmentation in a larger sample of Melanesians. The pattern of sequence diversity here does not support a model of strong selective constraint on MC1R in Northern Island Melanesia This absence of strong constraint, as well as the recent population history of the region, may explain the observed frequencies of the derived rs2228479 allele. These results emphasize the complex genetic architecture of pigmentation phenotypes, which are controlled by multiple, possibly interacting loci. They also highlight the role that population history can play in influencing phenotypic diversity in the absence of strong natural selection.

Human scFv antibodies (Afribumabs) against Africanized bee venom: Advances in melittin recognition.

PubMed

Pessenda, Gabriela; Silva, Luciano C; Campos, Lucas B; Pacello, Elenice M; Pucca, Manuela B; Martinez, Edson Z; Barbosa, José E

2016-03-15

Africanized Apis mellifera bees, also known as killer bees, have an exceptional defensive instinct, characterized by mass attacks that may cause envenomation or death. From the years 2000-2013, 77,066 bee accidents occurred in Brazil. Bee venom comprises several substances, including melittin and phospholipase A2 (PLA2). Due to the lack of antivenom for bee envenomation, this study aimed to produce human monoclonal antibody fragments (single chain fragment variable; scFv), by using phage display technology. These fragments targeted melittin and PLA2, the two major components of bee venom, to minimize their toxic effects in cases of mass envenomation. Two phage antibody selections were performed using purified melittin. As the commercial melittin is contaminated with PLA2, phages specific to PLA2 were also obtained during one of the selections. Specific clones for melittin and PLA2 were selected for the production of soluble scFvs, named here Afribumabs: prefix: afrib- (from Africanized bee); stem/suffix: -umab (fully human antibody). Afribumabs 1 and 2 were tested in in vitro and in vivo assays to assess their ability to inhibit the toxic actions of purified melittin, PLA2, and crude bee venom. Afribumabs reduced hemolysis caused by purified melittin and PLA2 and by crude venom in vitro and reduced edema formation in the paws of mice and prolonged the survival of venom-injected animals in vivo. These results demonstrate that Afribumabs may contribute to the production of the first non-heterologous antivenom treatment against bee envenomation. Such a treatment may overcome some of the difficulties associated with conventional immunotherapy techniques. Copyright © 2016 Elsevier Ltd. All rights reserved.

Expression and purification of recombinant human serum albumin from selectively terminable transgenic rice.

PubMed

Zhang, Qing; Yu, Hui; Zhang, Feng-zhen; Shen, Zhi-cheng

2013-10-01

Human serum albumin (HSA) is widely utilized for medical purposes and biochemical research. Transgenic rice has proved to be an attractive bioreactor for mass production of recombinant HSA (rHSA). However, transgene spread is a major environmental and food safety concern for transgenic rice expressing proteins of medical value. This study aimed to develop a selectively terminable transgenic rice line expressing HSA in rice seeds, and a simple process for recovery and purification of rHSA for economical manufacture. An HSA expression cassette was inserted into a T-DNA vector encoding an RNA interference (RNAi) cassette suppressing the CYP81A6 gene. This gene detoxifies the herbicide bentazon and is linked to the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) cassette which confers glyphosate tolerance. ANX Sepharose Fast Flow (ANX FF) anion exchange chromatography coupled with Butyl Sepharose High Performance (Butyl HP) hydrophobic interaction chromatography was used to purify rHSA. A transgenic rice line, HSA-84, was obtained with stable expression of rHSA of up to 0.72% of the total dry weight of the dehusked rice seeds. This line also demonstrated high sensitivity to bentazon, and thus could be killed selectively by a spray of bentazon. A two-step chromatography purification scheme was established to purify the rHSA from rice seeds to a purity of 99% with a recovery of 62.4%. Results from mass spectrometry and N-terminus sequencing suggested that the purified rHSA was identical to natural plasma-derived HSA. This study provides an alternative strategy for large-scale production of HSA with a built-in transgene safety control mechanism.

Solubilization conditions for bovine heart mitochondrial membranes allow selective purification of large quantities of respiratory complexes I, III, and V.

PubMed

Shimada, Satoru; Maeda, Shintaro; Hikita, Masahide; Mieda-Higa, Kaoru; Uene, Shigefumi; Nariai, Yukiko; Shinzawa-Itoh, Kyoko

2018-04-24

Ascertaining the structure and functions of mitochondrial respiratory chain complexes is essential to understanding the biological mechanisms of energy conversion; therefore, numerous studies have examined these complexes. A fundamental part of that research involves devising a method for purifying samples with good reproducibility; the samples obtained need to be stable and their constituents need to retain the same structure and functions they possess when in mitochondrial membranes. Submitochondrial bovine heart particles were isolated using differential centrifugation to adjust to a membrane concentration of 46.0% (w/v) or 31.5% (w/v) based on weight. After 0.7% (w/v) deoxycholic acid, 0.4% (w/v) decyl maltoside, and 7.2% (w/v) potassium chloride were added to the mitochondrial membranes, those membranes were solubilized. At a membrane concentration of 46%, complex V was selectively solubilized, whereas at a concentration of 31.5% (w/v), complexes I and III were solubilized. Two steps-sucrose density gradient centrifugation and anion-exchange chromatography on a POROS HQ 20 μm column-enabled selective purification of samples that retained their structure and functions. These two steps enabled complexes I, III, and V to be purified in two days with a high yield. Complexes I, III, and V were stabilized with n-decyl-β-D-maltoside. A total of 200 mg-300 mg of those complexes from one bovine heart (1.1 kg muscle) was purified with good reproducibility, and the complexes retained the same functions they possessed while in mitochondrial membranes. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Expression and purification of native and functional influenza A virus matrix 2 proton selective ion channel.

PubMed

Desuzinges Mandon, Elodie; Traversier, Aurélien; Champagne, Anne; Benier, Lorraine; Audebert, Stéphane; Balme, Sébastien; Dejean, Emmanuel; Rosa Calatrava, Manuel; Jawhari, Anass

2017-03-01

Influenza A virus displays one of the highest infection rates of all human viruses and therefore represents a severe human health threat associated with an important economical challenge. Influenza matrix protein 2 (M2) is a membrane protein of the viral envelope that forms a proton selective ion channel. Here we report the expression and native isolation of full length active M2 without mutations or fusions. The ability of the influenza virus to efficiently infect MDCK cells was used to express native M2 protein. Using a Calixarene detergents/surfactants based approach; we were able to solubilize most of M2 from the plasma membrane and purify it. The tetrameric form of native M2 was maintained during the protein preparation. Mass spectrometry shows that M2 was phosphorylated in its cytoplasmic tail (serine 64) and newly identifies an acetylation of the highly conserved Lysine 60. ELISA shows that solubilized and purified M2 was specifically recognized by M2 antibody MAB65 and was able to displace the antibody from M2 MDCK membranes. Using a bilayer voltage clamp measurement assay, we demonstrate a pH dependent proton selective ion channel activity. The addition of the M2 ion channel blocker amantadine allows a total inhibition of the channel activity, illustrating therefore the specificity of purified M2 activity. Taken together, this work shows the production and isolation of a tetrameric and functional native M2 ion channel that will pave the way to structural and functional characterization of native M2, conformational antibody development, small molecules compounds screening towards vaccine treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

Characterization of polysaccharides with marked inhibitory effect on lipid accumulation in Pleurotus eryngii.

PubMed

Chen, Jingjing; Yong, Yangyang; Xing, Meichun; Gu, Yifan; Zhang, Zhao; Zhang, Shizhu; Lu, Ling

2013-09-12

Mushrooms have a great potential for the production of useful bioactive metabolites. To explore the bioactive compounds from edible mushrooms for interfering with the development of macrophage-derived foam cells, which is recognized as the hallmark of early atherosclerosis, eight types of mushrooms polysaccharides had been selected to be tested. Consequently, different mushrooms polysaccharides displayed diverse component profiles. Of polysaccharides that we tested, the Pleurotus eryngii polysaccharide had the strongest inhibitory effect on lipid accumulation. Furthermore, through fractionation of DEAE-52 and Sephadex G-100, the polysaccharide from P. eryngii had been successfully purified and identified. By the analysis of IR, GC, and HPLC, the purified polysaccharide was estimated to be 30-38 kDa for the average molecular weight with the monosaccharide composition mainly composed of D-types of mannose, glucose and galactose. Findings presented in this report firstly provide direct evidence, which links the purified polysaccharide moiety with the biological function in foam-cell model. Copyright © 2013 Elsevier Ltd. All rights reserved.

Human proinsulin C-peptide from a precursor overexpressed in Pichia pastoris.

PubMed

Huang, Yang-Bin; Li, Jiang; Gao, Xin; Sun, Jiu-Ru; Lu, Yi; Feng, Tao; Fei, Jian; Cui, Da-Fu; Xia, Qi-Chang; Ren, Jun; Zhang, You-Shang

2006-08-01

In this article we report the production of human proinsulin C-peptide with 31 amino acid residues from a precursor overexpressed in Pichia pastoris. A C-peptide precursor expression plasmid containing nine C-peptide genes in tandem was constructed and used to transform P. pastoris. Transformants with a high copy number of the C-peptide precursor gene integrated into the chromosome of P. pastoris were selected. In high-density fermentation in a 300 liter fermentor using a simple culture medium composed mainly of salt and methanol, the C-peptide precursor was overexpressed to a level of 2.28 g per liter. A simple procedure was established to purify the expression product from the culture medium. The purified C-peptide precursor was converted into C-peptide by trypsin and carboxypeptidase B joint digestion. The yield of C-peptide with a purity of 96% was 730 mg per liter of culture. The purified C-peptide was characterized by mass spectrometry, N- and C-terminal amino acid sequencing, and sodium dodecylsulfate-polyacrylamide gel electrophoresis.

Dorsolateral neostriatum contribution to incentive salience: Opioid or dopamine stimulation makes one reward cue more motivationally attractive than another

PubMed Central

DiFeliceantonio, Alexandra G.; Berridge, Kent C.

2016-01-01

Pavlovian cues for rewards can become attractive incentives: approached and ‘wanted’ as the rewards themselves. The motivational attractiveness of a previously learned cue is not fixed, but can be dynamically amplified during re-encounter by simultaneous activation of brain limbic circuitry. Here we report that opioid or dopamine microinjections in the dorsolateral quadrant of the neostriatum (DLS) of rats selectively amplify attraction toward a previously learned Pavlovian cue in an individualized fashion, at the expense of a competing cue. In an autoshaping (sign-tracking vs goal-tracking) paradigm, microinjection of the mu opioid receptor agonist (DAMGO) or dopamine indirect agonist (amphetamine) in DLS of sign-tracker individuals selectively enhanced their sign-tracking attraction toward the reward-predictive lever cue. By contrast, DAMGO or amphetamine in DLS of goal-trackers selectively enhanced prepotent attraction toward the reward-proximal cue of sucrose dish. Amphetamine also enhanced goal-tracking in some sign-tracker individuals (if they ever defected to the dish even once). That DLS enhancement of cue attraction was due to stronger motivation, not stronger habits was suggested by: 1) sign-trackers flexibly followed their cue to a new location when the lever was suddenly moved after DLS DAMGO microinjection, and 2) DAMGO in DLS also made sign-trackers work harder on a new instrumental nose-poke response required to earn presentations of their Pavlovian lever cue (instrumental conditioned reinforcement). Altogether, our results suggest that DLS circuitry can enhance the incentive salience of a Pavlovian reward cue, selectively making that cue a stronger motivational magnet. PMID:26924040

How does the foraging behavior of large herbivores cause different associational plant defenses?

PubMed Central

Huang, Yue; Wang, Ling; Wang, Deli; Zeng, De-Hui; Liu, Chen

2016-01-01

The attractant-decoy hypothesis predicts that focal plants can defend against herbivory by neighboring with preferred plant species when herbivores make decisions at the plant species scale. The repellent-plant hypothesis assumes that focal plants will gain protection by associating with nonpreferred neighbors when herbivores are selective at the patch scale. However, herbivores usually make foraging decisions at these scales simultaneously. The net outcomes of the focal plant vulnerability could depend on the spatial scale at which the magnitude of selectivity by the herbivores is stronger. We quantified and compared the within- and between-patch overall selectivity index (OSI) of sheep to examine the relationships between associational plant effects and herbivore foraging selectivity. We found that the sheep OSI was stronger at the within- than the between-patch scale, but focal plant vulnerability followed both hypotheses. Focal plants defended herbivory with preferred neighbors when the OSI difference between the two scales was large. Focal plants gained protection with nonpreferred neighbors when the OSI difference was narrowed. Therefore, the difference in selectivity by the herbivores between the relevant scales results in different associational plant defenses. Our study suggests important implications for understanding plant-herbivore interactions and grassland management. PMID:26847834

Efficient syntheses of polyamine and polyamine amide voltage-sensitive calcium channel blockers: FTX-3.3 and sFTX-3.3.

PubMed

Moya, E; Blagbrough, I S

1996-02-01

Efficient syntheses of FTX-3.3 and sFTX-3.3, voltage-sensitive calcium channel blockers are described. These modified polyamines were prepared from selectively protected polyamines and purified on a practical scale.

Size-selective separation of polydisperse gold nanoparticles in supercritical ethane.

PubMed

Williams, Dylan P; Satherley, John

2009-04-09

The aim of this study was to use supercritical ethane to selectively disperse alkanethiol-stabilized gold nanoparticles of one size from a polydisperse sample in order to recover a monodisperse fraction of the nanoparticles. A disperse sample of metal nanoparticles with diameters in the range of 1-5 nm was prepared using established techniques then further purified by Soxhlet extraction. The purified sample was subjected to supercritical ethane at a temperature of 318 K in the pressure range 50-276 bar. Particles were characterized by UV-vis absorption spectroscopy, TEM, and MALDI-TOF mass spectroscopy. The results show that with increasing pressure the dispersibility of the nanoparticles increases, this effect is most pronounced for smaller nanoparticles. At the highest pressure investigated a sample of the particles was effectively stripped of all the smaller particles leaving a monodisperse sample. The relationship between dispersibility and supercritical fluid density for two different size samples of alkanethiol-stabilized gold nanoparticles was considered using the Chrastil chemical equilibrium model.

Determination of zearalenone and its metabolites in endometrial cancer by coupled separation techniques.

PubMed

Gadzała-Kopciuch, Renata; Cendrowski, Krzysztof; Cesarz, Anna; Kiełbasa, Paweł; Buszewski, Bogusław

2011-10-01

This study presents a selective method of isolation of zearalenone (ZON) and its metabolite, α-zearalenol (α-ZOL), in neoplastically changed human tissue by accelerated solvent and ultrasonic extractions using a mixture of acetonitrile/water (84/16% v/v) as the extraction solvent. Extraction effectiveness was determined through the selection of parameters (composition of the solvent mixture, temperature, pressure, number of cycles) with tissue contamination at the level of nanograms per gram. The produced acetonitrile/water extracts were purified, and analytes were enriched in columns packed with homemade molecularly imprinted polymers. Purified extracts were determined by liquid chromatography (LC) coupled with different detection systems (diode array detection--DAD and mass spectrometry--MS) involving the Ascentis RP-Amide as a stationary phase and gradient elution. The combination of UE-MISPE-LC (ultrasonic extraction--molecularly imprinted solid-phase extraction--liquid chromatography) produced high (R≈95-98%) and repeatable (RSD<3%) recovery values for ZON and α-ZOL. © The Author(s) 2011. This article is published with open access at Springerlink.com

Commercial Milk Enzyme-Linked Immunosorbent Assay (ELISA) Kit Reactivities to Purified Milk Proteins and Milk-Derived Ingredients.

PubMed

Ivens, Katherine O; Baumert, Joseph L; Taylor, Steve L

2016-07-01

Numerous commercial enzyme-linked immunosorbent assay (ELISA) kits exist to quantitatively detect bovine milk residues in foods. Milk contains many proteins that can serve as ELISA targets including caseins (α-, β-, or κ-casein) and whey proteins (α-lactalbumin or β-lactoglobulin). Nine commercially-available milk ELISA kits were selected to compare the specificity and sensitivity with 5 purified milk proteins and 3 milk-derived ingredients. All of the milk kits were capable of quantifying nonfat dry milk (NFDM), but did not necessarily detect all individual protein fractions. While milk-derived ingredients were detected by the kits, their quantitation may be inaccurate due to the use of different calibrators, reference materials, and antibodies in kit development. The establishment of a standard reference material for the calibration of milk ELISA kits is increasingly important. The appropriate selection and understanding of milk ELISA kits for food analysis is critical to accurate quantification of milk residues and informed risk management decisions. © 2016 Institute of Food Technologists®

Crude and purified proteasome activity assays are affected by type of microplate.

PubMed

Cui, Ziyou; Gilda, Jennifer E; Gomes, Aldrin V

2014-02-01

Measurement of proteasome activity is fast becoming a commonly used assay in many laboratories. The most common method to measure proteasome activity involves measuring the release of fluorescent tags from peptide substrates in black microplates. Comparisons of black plates used for measuring fluorescence with different properties show that the microplate properties significantly affect the measured activities of the proteasome. The microplate that gave the highest reading of trypsin-like activity of the purified 20S proteasome gave the lowest reading of chymotrypsin-like activity of the 20S proteasome. Plates with medium binding surfaces from two different companies showed an approximately 2-fold difference in caspase-like activity for purified 20S proteasomes. Even standard curves generated using free 7-amino-4-methylcoumarin (AMC) were affected by the microplate used. As such, significantly different proteasome activities, as measured in nmol AMC released/mg/min, were obtained for purified 20S proteasomes as well as crude heart and liver samples when using different microplates. The naturally occurring molecule betulinic acid activated the chymotrypsin-like proteasome activity in three different plates but did not affect the proteasome activity in the nonbinding surface microplate. These findings suggest that the type of proteasome activity being measured and sample type are important when selecting a microplate. Copyright © 2013 Elsevier Inc. All rights reserved.

Growth inhibitory and proapoptotic effects of l-asparaginase from Fusarium culmorum ASP-87 on human leukemia cells (Jurkat).

PubMed

Meghavarnam, Anil K; Salah, Maryam; Sreepriya, Meenakshisundaram; Janakiraman, Savitha

2017-06-01

The objective of this study was to evaluate the anticancer properties of l-asparaginase purified from fungal isolate Fusarium culmorum ASP-87 against human T-cell leukemia cell line (Jurkat). The growth inhibitory and proapoptotic effects of purified l-asparaginase on Jurkat cell lines were investigated by determining its influence on cell viability, colony formation, DNA fragmentation, and cell cycle progression. The results revealed that purified l-asparaginase showed significant decrease in cell survival with IC 50 value of 90 μg/mL (9 IU/mL). The enzyme inhibited colony formation and showed characteristic laddering pattern on agarose gel thereby confirming the induction of apoptosis. Further, cell cycle analysis revealed that the enzyme induced apoptotic cell death by arresting the growth of cells at G 2 -M phase. However, the enzyme did not elicit any toxic effects on human erythrocytes. l-asparaginase purified from F. culmorum ASP-87 showed significant and selective cytotoxic and apoptotic effects on human T-cell leukemic cells in dose-dependent manner. Results of the study give leads for the anticancer effects of fungal l-asparaginase and its potential usefulness in the chemotherapy of leukemia. © 2016 Société Française de Pharmacologie et de Thérapeutique.

[Cloning, expression and identification of functional fragment rC3B of human complement C3 in E. Coli].

PubMed

Gan, Hui; Zhou, Yong; Sun, Ping; Zhu, Xiao-Xia; Wang, Quan-Li; Zhan, Lin-Sheng

2007-08-01

This study was purposed to verify the binding part of human complement C3 to complement receptor III (CRIII) in monocytes, the peptide rC3B, including the binding-site, was expressed, purified and identified. rC3B, the binding part of human complement C3 to CRIII, was selected by computer-aided modeling and summarizing researches published. Then, rC3B gene fragment was amplified by PCR, and cloned into prokaryotic vector pQE30a. The fusion protein rC3B was expressed in E.coli M15 and purified by Ni(2+)-chelating affinity chromatography. The activity of rC3B was identified by Western blot and adherence assay with monocytes. The results showed that rC3B fragment was obtained, and a prokaryotic expression vector pQE30-rC3B was constructed. rC3B was efficiently expressed and purified. In Western blot, the target protein showed the activity of binding with C3 antibody, while the purified protein showed the activity of adherence with monocytes. It is concluded that the recombinant C3B was obtained and identified, and this study lay the basis for the further functional analysis of C3.

Photocatalytic air purifiers for indoor air: European standard and pilot room experiments.

PubMed

Costarramone, N; Cantau, C; Desauziers, V; Pécheyran, C; Pigot, T; Lacombe, S

2017-05-01

At the European level (CEN/TC386), some efforts are currently devoted to new standards for comparing the efficiency of commercial photocatalytic material/devices in various application fields. Concerning prototype or commercial indoor photocatalytic air purifiers designed for volatile organic compounds (VOC) abatement, the methodology is based on a laboratory airtight chamber. The photocatalytic function is demonstrated by the mineralization of a mixture of five VOCs. Experimental data were obtained for four selected commercial devices and three commercial materials: drop of VOC concentration, but also identification of secondary species (with special attention to formaldehyde), mineralization rates, and Clean Air Delivery Rate (CADR). With two efficient air purifiers, these laboratory experiments were compared to the results in two experimental rooms (35-40 m 3 ) where air pollution was introduced through wooden floor and furniture. The systems' ageing was also studied. The safety of the commercial products was also assessed by the determination of nanoparticle release. Standardized tests are useful to rank photocatalytic air purifiers and passive materials and to discard inefficient ones. A good correlation between the standard experiments and the experimental room experiments was found, even if in the latter case, the concentration of lower weight VOCs drops less quickly than that of heavier VOCs.

Overproduction and partial purification of the Norrie disease gene product, norrin, from a recombinant baculovirus.

PubMed

Shastry, Barkur S; Trese, Michael T

2003-12-05

Abnormal vascularization of the peripheral retina and retinal detachment are common clinical characteristics of Norrie disease (ND), familial exudative vitreoretinopathy, Coats' disease, and retinopathy of prematurity. Although little is known about the molecular basis of these diseases, studies have shown that all of these diseases are associated with mutations in the ND gene. In spite of this, little is known about norrin, its molecular mechanism of action, and its functional relationship with the development of abnormal retinal vasculature. To obtain a large quantity of norrin for structural and functional studies, we have overproduced it in insect cells. For this purpose, a cDNA fragment (869 bp) was isolated from a human retinal cDNA library by amplification and was cloned into an expression vector. The purified plasmid was co-transfected with wild-type linearized Bac-N-Blue DNA into S. frugiperda Sf21 insect cells. The recombinant virus plaques were purified and clones were selected based on the level of recombinant protein expressed in Sf21 cells infected with a purified recombinant virus. From these, a high-titer stock was generated and subsequently used to prepare a fused protein on a large scale. The protein was partially purified by the process of immobilized metal affinity chromatography and the use of ion exchange chromatography

High level of microsynteny and purifying selection affect the evolution of WRKY family in Gramineae.

PubMed

Jin, Jing; Kong, Jingjing; Qiu, Jianle; Zhu, Huasheng; Peng, Yuancheng; Jiang, Haiyang

2016-01-01

The WRKY gene family, which encodes proteins in the regulation processes of diverse developmental stages, is one of the largest families of transcription factors in higher plants. In this study, by searching for interspecies gene colinearity (microsynteny) and dating the age distributions of duplicated genes, we found 35 chromosomal segments of subgroup I genes of WRKY family (WRKY I) in four Gramineae species (Brachypodium, rice, sorghum, and maize) formed eight orthologous groups. After a stepwise gene-by-gene reciprocal comparison of all the protein sequences in the WRKY I gene flanking areas, highly conserved regions of microsynteny were found in the four Gramineae species. Most gene pairs showed conserved orientation within syntenic genome regions. Furthermore, tandem duplication events played the leading role in gene expansion. Eventually, environmental selection pressure analysis indicated strong purifying selection for the WRKY I genes in Gramineae, which may have been followed by gene loss and rearrangement. The results presented in this study provide basic information of Gramineae WRKY I genes and form the foundation for future functional studies of these genes. High level of microsynteny in the four grass species provides further evidence that a large-scale genome duplication event predated speciation.

Calcium Channels: Structure and Function (Annals of the New York Academy of Sciences. Volume 560)

DTIC Science & Technology

1989-06-26

many protease inhibitors were used , we believe that proteolysis was a problem. We therefore modified our purification protocol and have obtained a...recover activity by selective combination of fractions were unsuccessful. Chemical Cross-Linking of [ 25 Jo-CgTX Cross-linking of w-CgTX derivatives using ... using the planar bilayer recording technique and by comparing ligand-dependent gating, ionic selectivity , and pharmacology of purified ryanodine

Selective Activation of Transcription by a Novel CCAAT Binding Factor

NASA Astrophysics Data System (ADS)

Maity, Sankar N.; Golumbek, Paul T.; Karsenty, Gerard; de Crombrugghe, Benoit

1988-07-01

A novel CCAAT binding factor (CBF) composed of two different subunits has been extensively purified from rat liver. Both subunits are needed for specific binding to DNA. Addition of this purified protein to nuclear extracts of NIH 3T3 fibroblasts stimulates transcription from several promoters including the α 2(I) collagen, the α 1(I) collagen, the Rous sarcoma virus long terminal repeat (RSV-LTR), and the adenovirus major late promoter. Point mutations in the CCAAT motif that show either no binding or a decreased binding of CBF likewise abolish or reduce activation of transcription by CBF. Activation of transcription requires, therefore, the specific binding of CBF to its recognition sites.

Osvaldo and Isis retrotransposons as markers of the Drosophila buzzatii colonisation in Australia.

PubMed

Guerreiro, María Pilar García; Fontdevila, Antonio

2011-04-24

Transposable elements (TEs) constitute an important source of genetic variability owing to their jumping and regulatory properties, and are considered to drive species evolution. Several factors that are able to induce TE transposition in genomes have been documented (for example environmental stress and inter- and intra-specific crosses) but in many instances the reasons for TE mobilisation have yet to be elucidated. Colonising populations constitute an ideal model for studying TE behaviour and distribution as they are exposed to different environmental and new demographic conditions. In this study, the distribution of two TEs, Osvaldo and Isis, was examined in two colonising populations of D. buzzatii from Australia. Comparing Osvaldo copy numbers between Australian and Old World (reported in previous studies) colonisations provides a valuable tool for elucidating the colonisation process and the effect of new conditions encountered by colonisers on TEs. The chromosomal distributions of Osvaldo and Isis retrotransposons in two colonising populations of D. buzzatii from Australia revealed sites of high insertion frequency (>10%) and low frequency sites. Comparisons between Osvaldo insertion profiles in colonising populations from the Old World and Australia demonstrate a tendency towards a higher number of highly occupied sites with higher insertion frequency in the Old World than in Australian populations. Tests concerning selection against deleterious TE insertions indicate that Isis is more controlled by purifying selection than Osvaldo. The distribution of both elements on chromosomal arms follows a Poisson distribution and there are non-significant positive correlations between highly occupied sites and chromosomal inversions. The occupancy profile of Osvaldo and Isis retrotransposons is characterised by the existence of high and low insertion frequency sites in the populations. These results demonstrate that Australian D. buzzatii populations were subjected to a founder effect during the colonisation process. Moreover, there are more sites with high insertion frequency in the Old World colonisation than in the Australian colonisation, indicating a probable stronger bottleneck effect in Australia. The results suggest that selection does not seem to play a major role, compared to demography, in the distribution of transposable elements in the Australian populations.

Osvaldo and Isis retrotransposons as markers of the Drosophila buzzatii colonisation in Australia

PubMed Central

2011-01-01

Background Transposable elements (TEs) constitute an important source of genetic variability owing to their jumping and regulatory properties, and are considered to drive species evolution. Several factors that are able to induce TE transposition in genomes have been documented (for example environmental stress and inter- and intra-specific crosses) but in many instances the reasons for TE mobilisation have yet to be elucidated. Colonising populations constitute an ideal model for studying TE behaviour and distribution as they are exposed to different environmental and new demographic conditions. In this study, the distribution of two TEs, Osvaldo and Isis, was examined in two colonising populations of D. buzzatii from Australia. Comparing Osvaldo copy numbers between Australian and Old World (reported in previous studies) colonisations provides a valuable tool for elucidating the colonisation process and the effect of new conditions encountered by colonisers on TEs. Results The chromosomal distributions of Osvaldo and Isis retrotransposons in two colonising populations of D. buzzatii from Australia revealed sites of high insertion frequency (>10%) and low frequency sites. Comparisons between Osvaldo insertion profiles in colonising populations from the Old World and Australia demonstrate a tendency towards a higher number of highly occupied sites with higher insertion frequency in the Old World than in Australian populations. Tests concerning selection against deleterious TE insertions indicate that Isis is more controlled by purifying selection than Osvaldo. The distribution of both elements on chromosomal arms follows a Poisson distribution and there are non-significant positive correlations between highly occupied sites and chromosomal inversions. Conclusions The occupancy profile of Osvaldo and Isis retrotransposons is characterised by the existence of high and low insertion frequency sites in the populations. These results demonstrate that Australian D. buzzatii populations were subjected to a founder effect during the colonisation process. Moreover, there are more sites with high insertion frequency in the Old World colonisation than in the Australian colonisation, indicating a probable stronger bottleneck effect in Australia. The results suggest that selection does not seem to play a major role, compared to demography, in the distribution of transposable elements in the Australian populations. PMID:21513573

Positive and purifying selection in mitochondrial genomes of a bird with mitonuclear discordance.

PubMed

Morales, Hernán E; Pavlova, Alexandra; Joseph, Leo; Sunnucks, Paul

2015-06-01

Diversifying selection on metabolic pathways can reduce intraspecific gene flow and promote population divergence. An opportunity to explore this arises from mitonuclear discordance observed in an Australian bird Eopsaltria australis. Across >1500 km, nuclear differentiation is low and latitudinally structured by isolation by distance, whereas two highly divergent, parapatric mitochondrial lineages (>6.6% in ND2) show a discordant longitudinal geographic pattern and experience different climates. Vicariance, incomplete lineage sorting and sex-biased dispersal were shown earlier to be unlikely drivers of the mitonuclear discordance; instead, natural selection on a female-linked trait was the preferred hypothesis. Accordingly, here we tested for signals of positive, divergent selection on mitochondrial genes in E. australis. We used codon models and physicochemical profiles of amino acid replacements to analyse complete mitochondrial genomes of the two mitochondrial lineages in E. australis, its sister species Eopsaltria griseogularis, and outgroups. We found evidence of positive selection on at least five amino acids, encoded by genes of two oxidative phosphorylation pathway complexes NADH dehydrogenase (ND4 and ND4L) and cytochrome bc1 (cyt-b) against a background of widespread purifying selection on all mitochondrial genes. Three of these amino acid replacements were fixed in ND4 of the geographically most widespread E. australis lineage. The other two replacements were fixed in ND4L and cyt-b of the geographically more restricted E. australis lineage. We discuss whether this selection may reflect local environmental adaptation, a by-product of other selective processes, or genetic incompatibilities, and propose how these hypotheses can be tested in future. © 2015 John Wiley & Sons Ltd.

40 CFR 1065.307 - Linearity verification.

Code of Federal Regulations, 2011 CFR

2011-07-01

... different flow rates. Use a gravimetric reference measurement (such as a scale, balance, or mass comparator... the gas-division system to divide the span gas with purified air or nitrogen. Select gas divisions... verification for gravimetric PM balances, use external calibration weights that that meet the requirements in...

A rapid and sensitive dot-blot hybridization assay for the detection of citrus exocortis viroid in Citrus medica with digoxigenin-labelled RNA probes.

PubMed

Fonseca, M E; Marcellino, L H; Gander, E

1996-04-05

A rapid and sensitive dot-blot hybridization assay using in vitro-transcribed digoxigenin-labelled RNA probes (riboprobes) was developed aiming at detection of citrus exocortis viroid (CEVd) in crude sap of infected Citrus medica plants. The protocol includes a very quick and simple preparation of RNA extracts from samples using a denaturation step with formaldehyde. From our results, the employment of this step is highly recommended because the hybridization signals in formaldehyde-denatured samples were significantly stronger when compared with that of extracts without formaldehyde treatment. The assay was found to be sensitive enough to detect 0.1 ng of purified CEVd RNA and was able to detect viroid in 0.2 mg of symptomatic Citrus medica leaves. The use of riboprobes also allowed hybridization under high temperature conditions, avoiding non-specific background.

Soya Saponins Induce Enteritis in Atlantic Salmon (Salmo salar L.).

PubMed

Krogdahl, Åshild; Gajardo, Karina; Kortner, Trond M; Penn, Michael; Gu, Min; Berge, Gerd Marit; Bakke, Anne Marie

2015-04-22

Soybean meal-induced enteritis (SBMIE) is a well-described condition in the distal intestine of salmonids, and saponins have been implicated as the causal agent. However, the question remains whether saponins alone cause SBMIE. Moreover, the dose-response relationship has not been described. In a 10 week feeding trial with Atlantic salmon, a highly purified (95%) soya saponin preparation was supplemented (0, 2, 4, 6, or 10 g/kg) to two basal diets, one containing fishmeal as the major protein source (FM) and the other 25% lupin meal (LP). Saponins caused dose-dependent increases in the severity of inflammation independent of the basal diet, with concomitant alterations in digestive functions and immunological marker expression. Thus, saponins induced inflammation whether the diet contained other legume components or not. However, responses were often the same or stronger in fish fed the corresponding saponin-supplemented LP diets despite lower saponin exposure, suggesting potentiation by other legume component(s).

RELAX: detecting relaxed selection in a phylogenetic framework.

PubMed

Wertheim, Joel O; Murrell, Ben; Smith, Martin D; Kosakovsky Pond, Sergei L; Scheffler, Konrad

2015-03-01

Relaxation of selective strength, manifested as a reduction in the efficiency or intensity of natural selection, can drive evolutionary innovation and presage lineage extinction or loss of function. Mechanisms through which selection can be relaxed range from the removal of an existing selective constraint to a reduction in effective population size. Standard methods for estimating the strength and extent of purifying or positive selection from molecular sequence data are not suitable for detecting relaxed selection, because they lack power and can mistake an increase in the intensity of positive selection for relaxation of both purifying and positive selection. Here, we present a general hypothesis testing framework (RELAX) for detecting relaxed selection in a codon-based phylogenetic framework. Given two subsets of branches in a phylogeny, RELAX can determine whether selective strength was relaxed or intensified in one of these subsets relative to the other. We establish the validity of our test via simulations and show that it can distinguish between increased positive selection and a relaxation of selective strength. We also demonstrate the power of RELAX in a variety of biological scenarios where relaxation of selection has been hypothesized or demonstrated previously. We find that obligate and facultative γ-proteobacteria endosymbionts of insects are under relaxed selection compared with their free-living relatives and obligate endosymbionts are under relaxed selection compared with facultative endosymbionts. Selective strength is also relaxed in asexual Daphnia pulex lineages, compared with sexual lineages. Endogenous, nonfunctional, bornavirus-like elements are found to be under relaxed selection compared with exogenous Borna viruses. Finally, selection on the short-wavelength sensitive, SWS1, opsin genes in echolocating and nonecholocating bats is relaxed only in lineages in which this gene underwent pseudogenization; however, selection on the functional medium/long-wavelength sensitive opsin, M/LWS1, is found to be relaxed in all echolocating bats compared with nonecholocating bats. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Sequence polymorphism in an insect RNA virus field population: A snapshot from a single point in space and time reveals stochastic differences among and within individual hosts

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

Stenger, Drake C., E-mail: drake.stenger@ars.usda.

Population structure of Homalodisca coagulata Virus-1 (HoCV-1) among and within field-collected insects sampled from a single point in space and time was examined. Polymorphism in complete consensus sequences among single-insect isolates was dominated by synonymous substitutions. The mutant spectrum of the C2 helicase region within each single-insect isolate was unique and dominated by nonsynonymous singletons. Bootstrapping was used to correct the within-isolate nonsynonymous:synonymous arithmetic ratio (N:S) for RT-PCR error, yielding an N:S value ~one log-unit greater than that of consensus sequences. Probability of all possible single-base substitutions for the C2 region predicted N:S values within 95% confidence limits of themore » corrected within-isolate N:S when the only constraint imposed was viral polymerase error bias for transitions over transversions. These results indicate that bottlenecks coupled with strong negative/purifying selection drive consensus sequences toward neutral sequence space, and that most polymorphism within single-insect isolates is composed of newly-minted mutations sampled prior to selection. -- Highlights: •Sampling protocol minimized differential selection/history among isolates. •Polymorphism among consensus sequences dominated by negative/purifying selection. •Within-isolate N:S ratio corrected for RT-PCR error by bootstrapping. •Within-isolate mutant spectrum dominated by new mutations yet to undergo selection.« less

A monoclonal antibody recognizes undifferentiation-specific carbohydrate moieties expressed on cell surface of the human dental pulp cells.

PubMed

Kang, Kyung-Jung; Ko, Seon-Yle; Ryu, Chun-Jeih; Jang, Young-Joo

2017-05-01

Human dental pulp cells are obtained from dental pulp tissue, and have the ability to form dentin and a pulp-like complex. Although adult stem cells have been identified from the primary culture by using specific cell surface markers, the identity of surface markers for the purification of stem cells within the dental pulp population are still unclear. Previously, we had constructed monoclonal antibodies against the undifferentiated cell-specific surface markers of human dental pulp cells (hDPCs) by performing decoy immunization. Among them, a monoclonal antibody against the cell surface antigen of the undifferentiated hDPCs (named UPSA-1) was purified and its heavy and light chain consensus regions were analyzed. The cell surface binding affinity of UPSA-1 mAb on the undifferentiated hDPCs was stronger than that on the differentiated cells. When tunicamycin was applied to hDPSCs during culture, the cell surface binding affinity of the antibody was dramatically decreased, and dentinogenic differentiation was reduced. The purified UPSA-1 antigen band resulting from immunoprecipitation disappeared or shifted down on the SDS-PAGE by deglycosylation. These data suggested that glycosylation on the cell surface might be a marker of an undifferentiated state, and that UPSA-1 mAb might be useful for identifying the carbohydrate moiety on the cell surface of undifferentiated pulp cells. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Composition analysis and antioxidant properties of black garlic extract.

PubMed

Lu, Xiaoming; Li, Ningyang; Qiao, Xuguang; Qiu, Zhichang; Liu, Pengli

2017-04-01

Black garlic produced from fresh garlic under controlled high temperature and humidity has strong antioxidant properties. To determine these compounds, five fractions (from F1 to F5) were separated and purified by elution with chloroform:methanol at different ratios (8:1, 6:1, 4:1, 2:1, and 0:1; v/v). The antioxidant activity of each fraction was analyzed. The results showed that F3 and F4 had higher phenolic contents and stronger 2,2-diphenyl-2-picrylhydrazyl radical scavenging activity than the others. Seven purified individual components were further separated using semipreparation high-performance liquid chromatography from these two intensely antioxidant fractions (F3 and F4), their structures were elucidated by high-performance liquid chromatography coupled to diode array detection, electrospray ionization, mass spectrometry, 1 H nuclear magnetic resonance, and 13 C nuclear magnetic resonance spectrometry. Three compounds including adenosine, uridine, and 2-acetylpyrrole were first identified in black garlic, except for 5-hydroxymethylfurfural, (1S, 3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, and (1R, 3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid. The cellular antioxidant activities of uridine, adenosine, carboline alkaloids, 5-hydroxymethylfurfural, and ethyl acetate extracts were consistent with the results of in vitro experimental antioxidant properties. The results provide useful information for understanding the health benefits of black garlic products. Copyright © 2016. Published by Elsevier B.V.

Development of a water purifier for radioactive cesium removal from contaminated natural water by radiation-induced graft polymerization

NASA Astrophysics Data System (ADS)

Seko, Noriaki; Hoshina, Hiroyuki; Kasai, Noboru; Shibata, Takuya; Saiki, Seiichi; Ueki, Yuji

2018-02-01

Six years after the Fukushima-nuclear accident, the dissolved radioactive cesium (Cs) is now hardly detected in environmental natural waters. These natural waters are directly used as source of drinking and domestic waters in disaster-stricken areas in Fukushima. However, the possibility that some radioactive Cs adsorbed on soil or leaves will contaminate these natural waters during heavy rains or typhoon is always present. In order for the returning residents to live with peace of mind, it is important to demonstrate the safety of the domestic waters that they will use for their daily life. For this purpose, we have synthesized a material for selective removal of radioactive Cs by introducing ammonium 12-molybdophosphate (AMP) onto polyethylene nonwoven fabric through radiation-induced emulsion graft polymerization technique. Water purifiers filled with the grafted Cs adsorbent were installed in selected houses in Fukushima. The capability of the grafted adsorbent to remove Cs from domestic waters was evaluated for a whole year. The results showed that the tap water filtered through the developed water purifier contained no radioactive Cs, signifying the very effective adsorption performance of the developed grafted adsorbent. From several demonstrations, we have commercialized the water purifier named "KranCsair®". Furthermore, we have also developed a method for the mass production of the grafted nonwoven fabric. Using a 30 L grafting reactor, it was possible to produce the grafted nonwoven fabric with a suitable range of degree of grafting. When an irradiated roll of nonwoven trunk fabric with a length of 10 m and a width of 30 cm was set in the reactor filled with glycidyl methacrylate (GMA), AMP, Tween 80 monomer emulsion solution at 40 °C for 1 h, the difference of Dgs in the length and the width on roll of fabrics was negligible.

Selective recruitment of nuclear factors to productively replicating herpes simplex virus genomes.

PubMed

Dembowski, Jill A; DeLuca, Neal A

2015-05-01

Much of the HSV-1 life cycle is carried out in the cell nucleus, including the expression, replication, repair, and packaging of viral genomes. Viral proteins, as well as cellular factors, play essential roles in these processes. Isolation of proteins on nascent DNA (iPOND) was developed to label and purify cellular replication forks. We adapted aspects of this method to label viral genomes to both image, and purify replicating HSV-1 genomes for the identification of associated proteins. Many viral and cellular factors were enriched on viral genomes, including factors that mediate DNA replication, repair, chromatin remodeling, transcription, and RNA processing. As infection proceeded, packaging and structural components were enriched to a greater extent. Among the more abundant proteins that copurified with genomes were the viral transcription factor ICP4 and the replication protein ICP8. Furthermore, all seven viral replication proteins were enriched on viral genomes, along with cellular PCNA and topoisomerases, while other cellular replication proteins were not detected. The chromatin-remodeling complexes present on viral genomes included the INO80, SWI/SNF, NURD, and FACT complexes, which may prevent chromatinization of the genome. Consistent with this conclusion, histones were not readily recovered with purified viral genomes, and imaging studies revealed an underrepresentation of histones on viral genomes. RNA polymerase II, the mediator complex, TFIID, TFIIH, and several other transcriptional activators and repressors were also affinity purified with viral DNA. The presence of INO80, NURD, SWI/SNF, mediator, TFIID, and TFIIH components is consistent with previous studies in which these complexes copurified with ICP4. Therefore, ICP4 is likely involved in the recruitment of these key cellular chromatin remodeling and transcription factors to viral genomes. Taken together, iPOND is a valuable method for the study of viral genome dynamics during infection and provides a comprehensive view of how HSV-1 selectively utilizes cellular resources.

Anti-CDR3 Therapy for B-Cell Malignancies

DTIC Science & Technology

2013-10-01

1-5 in the report). The "Tomlinson" human antibody phage library will be used to pan for antibodies that bind these target CDR3s and not the parent...selection of phage to a test antigen. Successful expression will lead directly to the selection of CDR3-specific antibody-encoding phage : from which we... phage that bind to the purified candidate antibody and not to irrelevant antibodies. Phage are from single chain Fv library. Sequence phage that

Different evolutionary patterns of SNPs between domains and unassigned regions in human protein-coding sequences.

PubMed

Pang, Erli; Wu, Xiaomei; Lin, Kui

2016-06-01

Protein evolution plays an important role in the evolution of each genome. Because of their functional nature, in general, most of their parts or sites are differently constrained selectively, particularly by purifying selection. Most previous studies on protein evolution considered individual proteins in their entirety or compared protein-coding sequences with non-coding sequences. Less attention has been paid to the evolution of different parts within each protein of a given genome. To this end, based on PfamA annotation of all human proteins, each protein sequence can be split into two parts: domains or unassigned regions. Using this rationale, single nucleotide polymorphisms (SNPs) in protein-coding sequences from the 1000 Genomes Project were mapped according to two classifications: SNPs occurring within protein domains and those within unassigned regions. With these classifications, we found: the density of synonymous SNPs within domains is significantly greater than that of synonymous SNPs within unassigned regions; however, the density of non-synonymous SNPs shows the opposite pattern. We also found there are signatures of purifying selection on both the domain and unassigned regions. Furthermore, the selective strength on domains is significantly greater than that on unassigned regions. In addition, among all of the human protein sequences, there are 117 PfamA domains in which no SNPs are found. Our results highlight an important aspect of protein domains and may contribute to our understanding of protein evolution.

Purifying Selection on Exonic Splice Enhancers in Intronless Genes

PubMed Central

Savisaar, Rosina; Hurst, Laurence D.

2016-01-01

Exonic splice enhancers (ESEs) are short nucleotide motifs, enriched near exon ends, that enhance the recognition of the splice site and thus promote splicing. Are intronless genes under selection to avoid these motifs so as not to attract the splicing machinery to an mRNA that should not be spliced, thereby preventing the production of an aberrant transcript? Consistent with this possibility, we find that ESEs in putative recent retrocopies are at a higher density and evolving faster than those in other intronless genes, suggesting that they are being lost. Moreover, intronless genes are less dense in putative ESEs than intron-containing ones. However, this latter difference is likely due to the skewed base composition of intronless sequences, a skew that is in line with the general GC richness of few exon genes. Indeed, after controlling for such biases, we find that both intronless and intron-containing genes are denser in ESEs than expected by chance. Importantly, nucleotide-controlled analysis of evolutionary rates at synonymous sites in ESEs indicates that the ESEs in intronless genes are under purifying selection in both human and mouse. We conclude that on the loss of introns, some but not all, ESE motifs are lost, the remainder having functions beyond a role in splice promotion. These results have implications for the design of intronless transgenes and for understanding the causes of selection on synonymous sites. PMID:26802218

Theory of prokaryotic genome evolution.

PubMed

Sela, Itamar; Wolf, Yuri I; Koonin, Eugene V

2016-10-11

Bacteria and archaea typically possess small genomes that are tightly packed with protein-coding genes. The compactness of prokaryotic genomes is commonly perceived as evidence of adaptive genome streamlining caused by strong purifying selection in large microbial populations. In such populations, even the small cost incurred by nonfunctional DNA because of extra energy and time expenditure is thought to be sufficient for this extra genetic material to be eliminated by selection. However, contrary to the predictions of this model, there exists a consistent, positive correlation between the strength of selection at the protein sequence level, measured as the ratio of nonsynonymous to synonymous substitution rates, and microbial genome size. Here, by fitting the genome size distributions in multiple groups of prokaryotes to predictions of mathematical models of population evolution, we show that only models in which acquisition of additional genes is, on average, slightly beneficial yield a good fit to genomic data. These results suggest that the number of genes in prokaryotic genomes reflects the equilibrium between the benefit of additional genes that diminishes as the genome grows and deletion bias (i.e., the rate of deletion of genetic material being slightly greater than the rate of acquisition). Thus, new genes acquired by microbial genomes, on average, appear to be adaptive. The tight spacing of protein-coding genes likely results from a combination of the deletion bias and purifying selection that efficiently eliminates nonfunctional, noncoding sequences.

Dorsolateral neostriatum contribution to incentive salience: opioid or dopamine stimulation makes one reward cue more motivationally attractive than another.

PubMed

DiFeliceantonio, Alexandra G; Berridge, Kent C

2016-05-01

Pavlovian cues for rewards can become attractive incentives: approached and 'wanted' as the rewards themselves. The motivational attractiveness of a previously learned cue is not fixed, but can be dynamically amplified during re-encounter by simultaneous activation of brain limbic circuitry. Here it was reported that opioid or dopamine microinjections in the dorsolateral quadrant of the neostriatum (DLS) of rats selectively amplify attraction toward a previously learned Pavlovian cue in an individualized fashion, at the expense of a competing cue. In an autoshaping (sign-tracking vs. goal-tracking) paradigm, microinjection of the mu opioid receptor agonist (DAMGO) or dopamine indirect agonist (amphetamine) in the DLS of sign-tracker individuals selectively enhanced their sign-tracking attraction toward the reward-predictive lever cue. By contrast, DAMGO or amphetamine in the DLS of goal-trackers selectively enhanced prepotent attraction toward the reward-proximal cue of sucrose dish. Amphetamine also enhanced goal-tracking in some sign-tracker individuals (if they ever defected to the dish even once). That DLS enhancement of cue attraction was due to stronger motivation, not stronger habits, was suggested by: (i) sign-trackers flexibly followed their cue to a new location when the lever was suddenly moved after DLS DAMGO microinjection; and (ii) DAMGO in the DLS also made sign-trackers work harder on a new instrumental nose-poke response required to earn presentations of their Pavlovian lever cue (instrumental conditioned reinforcement). Altogether, the current results suggest that DLS circuitry can enhance the incentive salience of a Pavlovian reward cue, selectively making that cue a stronger motivational magnet. © 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

40 CFR 1065.307 - Linearity verification.

Code of Federal Regulations, 2010 CFR

2010-07-01

... different flow rates. Use a gravimetric reference measurement (such as a scale, balance, or mass comparator... the gas-division system to divide the span gas with purified air or nitrogen. Select gas divisions... PM balance, m max refers to the typical mass of a PM filter. (ii) For linearity verification of...

Host-parasite interactions and purifying selection in a microsporidian parasite of honey bees

USDA-ARS?s Scientific Manuscript database

Nosema ceranae is a microsporidian parasite that infects honey bee mid-gut epithelial cells. Infection can impair honey bee cognitive function, shorten lifespan, suppress the innate immune response and inhibit the apoptosis of infected gut cells. However, the virulence factors of this parasite are s...

Method for removal of phosgene from boron trichloride

DOEpatents

Freund, Samuel M.

1983-01-01

Selective ultraviolet photolysis using an unfiltered mercury arc lamp has been used to substantially reduce the phosgene impurity in a mixture of boron trichloride and phosgene. Infrared spectrophotometric analysis of the sample before and after irradiation shows that is is possible to highly purify commercially available boron trichloride with this method.

PURIFICATION AND PARTIAL CHARACTERIZATION OF AN ACID PHOSPHATASE FROM SPIRODELA OLIGORRHIZA AND ITS AFFINITY FOR SELECTED ORGANOPHOSPHATE PESTICIDES

EPA Science Inventory

An acid phosphatase from the aquatic plant Spirodela oligorrhiza (duckweed) was isolated by fast protein liquid chromatography (FPLC) and partially characterized. The enzyme was purified 1871-fold with a total yield of 40%. SDS-PAGE electrophoresis of the pure acid phosphatase ...

Determining the Effect of Natural Selection on Linked Neutral Divergence across Species

PubMed Central

Phung, Tanya N.; Lohmueller, Kirk E.

2016-01-01

A major goal in evolutionary biology is to understand how natural selection has shaped patterns of genetic variation across genomes. Studies in a variety of species have shown that neutral genetic diversity (intra-species differences) has been reduced at sites linked to those under direct selection. However, the effect of linked selection on neutral sequence divergence (inter-species differences) remains ambiguous. While empirical studies have reported correlations between divergence and recombination, which is interpreted as evidence for natural selection reducing linked neutral divergence, theory argues otherwise, especially for species that have diverged long ago. Here we address these outstanding issues by examining whether natural selection can affect divergence between both closely and distantly related species. We show that neutral divergence between closely related species (e.g. human-primate) is negatively correlated with functional content and positively correlated with human recombination rate. We also find that neutral divergence between distantly related species (e.g. human-rodent) is negatively correlated with functional content and positively correlated with estimates of background selection from primates. These patterns persist after accounting for the confounding factors of hypermutable CpG sites, GC content, and biased gene conversion. Coalescent models indicate that even when the contribution of ancestral polymorphism to divergence is small, background selection in the ancestral population can still explain a large proportion of the variance in divergence across the genome, generating the observed correlations. Our findings reveal that, contrary to previous intuition, natural selection can indirectly affect linked neutral divergence between both closely and distantly related species. Though we cannot formally exclude the possibility that the direct effects of purifying selection drive some of these patterns, such a scenario would be possible only if more of the genome is under purifying selection than currently believed. Our work has implications for understanding the evolution of genomes and interpreting patterns of genetic variation. PMID:27508305

Determining the Effect of Natural Selection on Linked Neutral Divergence across Species.

PubMed

Phung, Tanya N; Huber, Christian D; Lohmueller, Kirk E

2016-08-01

A major goal in evolutionary biology is to understand how natural selection has shaped patterns of genetic variation across genomes. Studies in a variety of species have shown that neutral genetic diversity (intra-species differences) has been reduced at sites linked to those under direct selection. However, the effect of linked selection on neutral sequence divergence (inter-species differences) remains ambiguous. While empirical studies have reported correlations between divergence and recombination, which is interpreted as evidence for natural selection reducing linked neutral divergence, theory argues otherwise, especially for species that have diverged long ago. Here we address these outstanding issues by examining whether natural selection can affect divergence between both closely and distantly related species. We show that neutral divergence between closely related species (e.g. human-primate) is negatively correlated with functional content and positively correlated with human recombination rate. We also find that neutral divergence between distantly related species (e.g. human-rodent) is negatively correlated with functional content and positively correlated with estimates of background selection from primates. These patterns persist after accounting for the confounding factors of hypermutable CpG sites, GC content, and biased gene conversion. Coalescent models indicate that even when the contribution of ancestral polymorphism to divergence is small, background selection in the ancestral population can still explain a large proportion of the variance in divergence across the genome, generating the observed correlations. Our findings reveal that, contrary to previous intuition, natural selection can indirectly affect linked neutral divergence between both closely and distantly related species. Though we cannot formally exclude the possibility that the direct effects of purifying selection drive some of these patterns, such a scenario would be possible only if more of the genome is under purifying selection than currently believed. Our work has implications for understanding the evolution of genomes and interpreting patterns of genetic variation.

The Rise and Fall of an Evolutionary Innovation: Contrasting Strategies of Venom Evolution in Ancient and Young Animals

PubMed Central

Sunagar, Kartik; Moran, Yehu

2015-01-01

Animal venoms are theorized to evolve under the significant influence of positive Darwinian selection in a chemical arms race scenario, where the evolution of venom resistance in prey and the invention of potent venom in the secreting animal exert reciprocal selection pressures. Venom research to date has mainly focused on evolutionarily younger lineages, such as snakes and cone snails, while mostly neglecting ancient clades (e.g., cnidarians, coleoids, spiders and centipedes). By examining genome, venom-gland transcriptome and sequences from the public repositories, we report the molecular evolutionary regimes of several centipede and spider toxin families, which surprisingly accumulated low-levels of sequence variations, despite their long evolutionary histories. Molecular evolutionary assessment of over 3500 nucleotide sequences from 85 toxin families spanning the breadth of the animal kingdom has unraveled a contrasting evolutionary strategy employed by ancient and evolutionarily young clades. We show that the venoms of ancient lineages remarkably evolve under the heavy constraints of negative selection, while toxin families in lineages that originated relatively recently rapidly diversify under the influence of positive selection. We propose that animal venoms mostly employ a ‘two-speed’ mode of evolution, where the major influence of diversifying selection accompanies the earlier stages of ecological specialization (e.g., diet and range expansion) in the evolutionary history of the species–the period of expansion, resulting in the rapid diversification of the venom arsenal, followed by longer periods of purifying selection that preserve the potent toxin pharmacopeia–the period of purification and fixation. However, species in the period of purification may re-enter the period of expansion upon experiencing a major shift in ecology or environment. Thus, we highlight for the first time the significant roles of purifying and episodic selections in shaping animal venoms. PMID:26492532

The interaction between the DRD4 VNTR polymorphism and perceived peer drinking norms in adolescent alcohol use and misuse

PubMed Central

Park, Aesoon; Kim, Jueun; Zaso, Michelle J.; Glatt, Stephen J.; Sher, Kenneth J.; Scott-Sheldon, Lori A. J.; Eckert, Tanya L.; Vanable, Peter A.; Carey, Kate B.; Ewart, Craig K.; Carey, Michael P.

2015-01-01

Peer drinking norms are arguably one of the strongest correlates of adolescent drinking. Prospective studies indicate that adolescents tend to select peers based on drinking (peer-selection) and their peers’ drinking is associated with changes in adolescent drinking over time (peer socialization). The present study investigated whether the peer selection and socialization processes in adolescent drinking differed as a function of the DRD4 VNTR genotype in two independent prospective datasets. The first sample was 174 high school students drawn from a 2-wave 6-month prospective study. The second sample was 237 college students drawn from a 3-wave annual prospective study. Multigroup cross-lagged panel analyses of the high school student sample indicated stronger socialization via peer drinking norms among carriers, whereas analyses of the college student sample indicated stronger drinking-based peer selection in the junior year among carriers, compared to non-carriers. Although replication and meta-analytic synthesis are needed, these findings suggest in part genetically determined peer-selection (carriers of the DRD4 7-repeat allele tend to associate with peers who have more favorable attitudes toward drinking and greater alcohol use) and peer socialization (carriers’ subsequent drinking behaviors are more strongly associated with their peer drinking norms) may differ across adolescent developmental stages. PMID:26902782

A Novel Point Mutation at Position 156 of Reverse Transcriptase from Feline Immunodeficiency Virus Confers Resistance to the Combination of (−)-β-2′,3′-Dideoxy-3′-Thiacytidine and 3′-Azido-3′-Deoxythymidine

PubMed Central

Smith, Robert A.; Remington, Kathryn M.; Preston, Bradley D.; Schinazi, Raymond F.; North, Thomas W.

1998-01-01

Mutants of feline immunodeficiency virus (FIV) resistant to (−)-β-2′,3′-dideoxy-3′-thiacytidine (3TC) were selected by culturing virus in the presence of increasing stepwise concentrations of 3TC. Two plaque-purified variants were isolated from the original mutant population, and both of these mutants were resistant to 3TC. Surprisingly, these mutants were also phenotypically resistant to 3′-azido-3′-deoxythymidine (AZT) and to the combination of 3TC and AZT. Purified reverse transcriptase (RT) from one of these plaque-purified mutants was resistant to the 5′-triphosphates of 3TC and AZT. DNA sequence analysis of the RT-encoding region of the pol gene amplified from the plaque-purified mutants revealed a Pro-to-Ser mutation at position 156 of RT. A site-directed mutant of FIV engineered to contain this Pro-156-Ser mutation was resistant to 3TC, AZT, and the combination of 3TC and AZT, confirming the role of the Pro-156-Ser mutation in the resistance of FIV to these two nucleoside analogs. This represents the first report of a lentiviral mutant resistant to the combination of AZT and 3TC due to a single, unique point mutation. PMID:9499094

Isolation of friedelin from black condensate of cork.

PubMed

Pires, Ricardo A; Aroso, Ivo; Silva, Susana P; Mano, João F; Reis, Rui L

2011-11-01

Black condensates (BC) are wastes of the insulation corkboard industry that contain several valuable chemicals, including friedelin, a terpene exhibiting biological activity. Herein, we report a straightforward procedure to extract friedelin from BC. Using this procedure, we were able to extract friedelin with yields between 0.4% and 2.9% and to further purify it obtaining purities from 77.0% to 99.3% (HPLC). The initial BC (2 batches), extracted raw product and purified friedelin were analyzed using FTIR. The extraction yields and purities were found to be directly related to the intensity of the carbonyl vibration at 1713 cm(-1) in the FTIR spectrum of the used BC batch. Therefore, these spectra can be used to screen and select BC batches suitable for friedelin extraction.

Expression, purification, and characterization of an enzymatically active truncated human rho-kinase I (ROCK I) domain expressed in Sf-9 insect cells.

PubMed

Khandekar, Sanjay S; Yi, Tracey; Dul, Ed; Wright, Lois L; Chen, Susan; Scott, Gilbert F; Smith, Gary K; Lee, Dennis; Hu, Erding; Kirkpatrick, Robert B

2006-01-01

Rho Kinase I (ROCK I) is a serine/threonine kinase that is involved in diverse cellular signaling. To further understand the physiological role of ROCK I and to identify and develop potent and selective inhibitors of ROCK I, we have overexpressed and purified a constitutively active dimeric human ROCK I (3-543) kinase domain using the Sf9-baculovirus expression system. In addition, using a limited proteolysis technique, we have identified a minimal functional subdomain of ROCK I that can be used in crystallization studies. The availability of multimilligram amounts of purified and well characterized functional human ROCK I kinase domains will be useful in screening and structural studies.

Preparative isolation and purification of seven main antioxidants from Eucommia ulmoides Oliv. (Du-zhong) leaves using HSCCC guided by DPPH-HPLC experiment.

PubMed

Dai, Xingping; Huang, Qiong; Zhou, Boting; Gong, Zhicheng; Liu, Zhaoqian; Shi, Shuyun

2013-08-15

Seven antioxidants were purified from Eucommia ulmoides Oliv. leaves using HSCCC guided by DPPH-HPLC experiment. HSCCC was successfully used to separate target antioxidants by three runs with different solvent systems after D101 column chromatography fractionation. Ethyl acetate-n-butanol-water (1:2:3, v/v/v) was selected as the optimum solvent system to purify geniposidic acid. Ethyl acetate-ethanol-water (4:1:5, v/v/v) was used to isolate caffeic acid, chlorogenic acid and ferulic acid. While three flavonoids, quercetin-3-O-sambubioside, rutin and isoquercitrin were purified by petroleum ether-ethyl acetate-methanol-water (1:5:1:5, v/v/v/v). The structures were identified by MS and NMR. Antioxidant activities were assessed, and compounds 2-7 showed strong antioxidant activities. This is the first report about separation of antioxidants from E. ulmoides leaves by HSCCC. The results indicated that the combinative methods using DPPH-HPLC and HSCCC could be widely applied for screening and isolation of antioxidants from complex extracts. Copyright © 2013 Elsevier Ltd. All rights reserved.

Lectins discriminate between pathogenic and nonpathogenic South American trypanosomes

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

de Miranda Santos, I.K.; Pereira, M.E.

1984-09-01

Cell surface carbohydrates of Trypanosoma cruzi, Trypanosoma rangeli, and Trypanosoma conorhini were analyzed by a micro-agglutination assay employing 27 highly purified lectins and by binding assays using various /sup 125/I-labeled lectins. The following seven lectins discriminated between the trypanosomes: 1) tomato lectin (an N-acetyl-D-glucosamine-binding protein), both in purified form and as crude tomato juice; 2) Bauhinea purpurea and Sophora japonica lectins (both N-acetyl-D-galactosamine-binding proteins), which selectively agglutinated T. cruzi; 3) Vicia villosa (an N-acetyl-D-galactosamine-binding protein) which was specific for T. rangeli; 4) peanut lectin (a D-galactose-binding protein) both in purified form and as crude saline extract; and 5) Ulex europaeusmore » and Lotus tetragonolobus (both L-fucose-binding proteins) lectins which reacted only with T. conorhini. Binding studies with 125I-labeled lectins were performed to find whether unagglutinated cells of the three different species of trypanosomes might have receptors for these lectins, in which case absence of agglutination could be due to a peculiar arrangement of the receptors. These assays essentially confirmed the agglutination experiments.« less

Studies on the selective lysis and purification of Trypanosoma cruzi

PubMed Central

1975-01-01

The mechanism by which culture forms of Trypanosoma cruzi are lysed by normal mammalian sera was examined. Lysis is restricted to the epimastigote form of the organism and is not dependent on the presence of agglutinins. Lysis is a complement-dependent process, the activity being generated by the alternate pathway. The selective lysis by serum was exploited to purify viable trypomastigotes by means of centrifugation in an albumin column. Essentially pure trypomastigote populations are now being employed in cell culture experiments. PMID:807672

In vitro study of histamine and histamine receptor ligands influence on the adhesion of purified human eosinophils to endothelium.

PubMed

Grosicki, Marek; Wójcik, Tomasz; Chlopicki, Stefan; Kieć-Kononowicz, Katarzyna

2016-04-15

It is a well-known fact that histamine is involved in eosinophil-dependent inflammatory responses including cellular chemotaxis and migration. Nevertheless, the relative role of histamine receptors in the mechanisms of eosinophils adhesion to endothelial cells is not known. Therefore the aim of presented study was to examine the effect of selective histamine receptors ligands on eosinophils adhesion to endothelium. For that purpose the highly purified human eosinophils have been isolated from the peripheral blood. The viability and functional integrity of isolated eosinophils have been validated in several tests. Histamine as well as 4-methylhistamine (selective H4 agonist) in concentration-dependent manner significantly increased number of eosinophils that adhere to endothelium. Among the selective histamine receptors antagonist or H1 inverse agonist only JNJ7777120 (histamine H4 antagonist) and thioperamide (dual histamine H3/H4 antagonist) had direct effect on eosinophils adhesion to endothelial cells. Antagonists of H1 (diphenhydramine, mepyramine) H2 (ranitidine and famotidine) and H3 (pitolisant) histamine receptors were ineffective. To the best of our knowledge, this is the first study to demonstrate that histamine receptor H4 plays a dominant role in histamine-induced eosinophils adhesion to endothelium. Copyright © 2016 Elsevier B.V. All rights reserved.

The NS3 proteins of global strains of bluetongue virus evolve into regional topotypes through negative (purifying) selection.

PubMed

Balasuriya, U B R; Nadler, S A; Wilson, W C; Pritchard, L I; Smythe, A B; Savini, G; Monaco, F; De Santis, P; Zhang, N; Tabachnick, W J; Maclachlan, N J

2008-01-01

Comparison of the deduced amino acid sequences of the genes (S10) encoding the NS3 protein of 137 strains of bluetongue virus (BTV) from Africa, the Americas, Asia, Australia and the Mediterranean Basin showed limited variation. Common to all NS3 sequences were potential glycosylation sites at amino acid residues 63 and 150 and a cysteine at residue 137, whereas a cysteine at residue 181 was not conserved. The PPXY and PS/TAP late-domain motifs were conserved in all but three of the viruses. Phylogenetic analyses of these same sequences yielded two principal clades that grouped the viruses irrespective of their serotype or year of isolation (1900-2003). All viruses from Asia and Australia were grouped in one clade, whereas those from the other regions were present in both clades. Each clade segregated into distinct subclades that included viruses from single or multiple regions, and the S10 genes of some field viruses were identical to those of live-attenuated BTV vaccines. There was no evidence of positive selection on the S10 gene as assessed by reconstruction of ancestral codon states on the phylogeny, rather the functional constraints of the NS3 protein are expressed through substantial negative (purifying) selection.

Purification of bioactive phenolics from Phanerochaete chysosporium biomass extract on selected macroporous resins

NASA Astrophysics Data System (ADS)

Idris, Z. M.; Dzahir, M. I. H. M.; Jamal, P.; Barkat, A. A.; Xian, R. L. W.

2017-06-01

In this study, two different types of macroporous resins known as XAD-7HP and HP-20 were evaluated for the adsorption and desorption properties against bioactive phenolics extracted from Phanerochaete chrysosporium. From the previous static sorption studies, it was found that the adsorption capacity for both resins had has no significant difference. Then, the kinetic adsorption data were analyzed with both pseudo-first-order and pseudo-second-order equations and the later performed better. The adsorption isotherm data were fitted well by both Langmuir and Freundlich models. Meanwhile in desorption study, HP-20 and XAD-7HP gave 90.52% and 88.28% recoveries, respectively. Considering the desorption results of the macroporous resins, HP-20 and XAD-7HP were packed in chromatography column to further purify the phenolics. For dynamic adsorption, breakthrough capacity of HP-20 (0.522) was found to be higher than XAD-7HP (0.131). Different ethanol concentrations (30% to 50% (v/v)) were investigated at fixed flowrate (1 ml/min) on phenolics recovery from both types of resins. The highest recovery of bioactive phenolics was 94.3% using XAD-7HP resins at 50% (v/v) of ethanol. Only 77.1% of bioactive phenolics were recovered using HP-20 resin at the same experimental conditions. The purified extract subsequently was analyzed using HPLC. The results showed that three phenolics (gallic acid 3,4-dihydroxybenzoic acid and 4-hydroxybenzoic acid) were identified with higher concentrations as compared to non-purified extract. Finally, the purified extract was tested for scavenging activity against DPPH, and it showed that the activity increased significantly to 90.80% from 59.94% in non-purified extract.

The comparison of antioxidative and hepatoprotective activities of Codonopsis pilosula polysaccharide (CP) and sulfated CP.

PubMed

Liu, Cui; Chen, Jin; Li, Entao; Fan, Qiang; Wang, Deyun; Li, Peng; Li, Xiuping; Chen, Xingying; Qiu, Shulei; Gao, Zhenzhen; Li, Hongquan; Hu, Yuanliang

2015-02-01

Codonopsis pilosula polysaccharide (CP) was extracted, purified and modified by chlorosulfonic acid-pyridine method to obtain a sulfated CP (sCP). Their antioxidative activities in vitro were compared through the free radical-scavenging test. The results demonstrated that the scavenging capabilities of sCP were significantly stronger than those of CP. In vivo test, the mice hepatic injury model was prepared by BCG/LPS method, then administrated respectively with sCP and CP at three dosages, the biochemical indexes in serum, antioxidative indexes in liver homogenate and histopathological change in liver of the mice were compared. The results showed that in high (200mg/kg) and middle (150mg/kg) dosages of sCP groups, the contents of ALT, AST and TNF-α in serum and MDA in liver homogenate were significantly lower than those in the model group and numerically lower than those in the CP groups, the activities of SOD and GSH-Px in liver homogenate were significantly higher than those in the model group and numerically higher than those in the CP groups. In the model group there were obvious pathological changes in the liver, while in the sCP groups were near normal. These results indicate that sCP and CP possess antioxidative activity in vitro and in vivo, the activity of sCP is stronger than that of CP and sulfation modification can enhance the antioxidative and hepatoprotective activities of Codonopsis pilosula polysaccharide. Copyright © 2014 Elsevier B.V. All rights reserved.

Processing of configural and componential information in face-selective cortical areas.

PubMed

Zhao, Mintao; Cheung, Sing-Hang; Wong, Alan C-N; Rhodes, Gillian; Chan, Erich K S; Chan, Winnie W L; Hayward, William G

2014-01-01

We investigated how face-selective cortical areas process configural and componential face information and how race of faces may influence these processes. Participants saw blurred (preserving configural information), scrambled (preserving componential information), and whole faces during fMRI scan, and performed a post-scan face recognition task using blurred or scrambled faces. The fusiform face area (FFA) showed stronger activation to blurred than to scrambled faces, and equivalent responses to blurred and whole faces. The occipital face area (OFA) showed stronger activation to whole than to blurred faces, which elicited similar responses to scrambled faces. Therefore, the FFA may be more tuned to process configural than componential information, whereas the OFA similarly participates in perception of both. Differences in recognizing own- and other-race blurred faces were correlated with differences in FFA activation to those faces, suggesting that configural processing within the FFA may underlie the other-race effect in face recognition.

Method for removal of phosgene from boron trichloride

DOEpatents

Freund, S.M.

1983-09-20

Selective ultraviolet photolysis using an unfiltered mercury arc lamp has been used to substantially reduce the phosgene impurity in a mixture of boron trichloride and phosgene. Infrared spectrophotometric analysis of the sample before and after irradiation shows that it is possible to highly purify commercially available boron trichloride with this method. 5 figs.

Evolution of the human immunodeficiency virus envelope gene is dominated by purifying selection.

PubMed

Edwards, C T T; Holmes, E C; Pybus, O G; Wilson, D J; Viscidi, R P; Abrams, E J; Phillips, R E; Drummond, A J

2006-11-01

The evolution of the human immunodeficiency virus (HIV-1) during chronic infection involves the rapid, continuous turnover of genetic diversity. However, the role of natural selection, relative to random genetic drift, in governing this process is unclear. We tested a stochastic model of genetic drift using partial envelope sequences sampled longitudinally in 28 infected children. In each case the Bayesian posterior (empirical) distribution of coalescent genealogies was estimated using Markov chain Monte Carlo methods. Posterior predictive simulation was then used to generate a null distribution of genealogies assuming neutrality, with the null and empirical distributions compared using four genealogy-based summary statistics sensitive to nonneutral evolution. Because both null and empirical distributions were generated within a coalescent framework, we were able to explicitly account for the confounding influence of demography. From the distribution of corrected P-values across patients, we conclude that empirical genealogies are more asymmetric than expected if evolution is driven by mutation and genetic drift only, with an excess of low-frequency polymorphisms in the population. This indicates that although drift may still play an important role, natural selection has a strong influence on the evolution of HIV-1 envelope. A negative relationship between effective population size and substitution rate indicates that as the efficacy of selection increases, a smaller proportion of mutations approach fixation in the population. This suggests the presence of deleterious mutations. We therefore conclude that intrahost HIV-1 evolution in envelope is dominated by purifying selection against low-frequency deleterious mutations that do not reach fixation.

Genomic analysis reveals major determinants of cis-regulatory variation in Capsella grandiflora

PubMed Central

Steige, Kim A.; Laenen, Benjamin; Reimegård, Johan; Slotte, Tanja

2017-01-01

Understanding the causes of cis-regulatory variation is a long-standing aim in evolutionary biology. Although cis-regulatory variation has long been considered important for adaptation, we still have a limited understanding of the selective importance and genomic determinants of standing cis-regulatory variation. To address these questions, we studied the prevalence, genomic determinants, and selective forces shaping cis-regulatory variation in the outcrossing plant Capsella grandiflora. We first identified a set of 1,010 genes with common cis-regulatory variation using analyses of allele-specific expression (ASE). Population genomic analyses of whole-genome sequences from 32 individuals showed that genes with common cis-regulatory variation (i) are under weaker purifying selection and (ii) undergo less frequent positive selection than other genes. We further identified genomic determinants of cis-regulatory variation. Gene body methylation (gbM) was a major factor constraining cis-regulatory variation, whereas presence of nearby transposable elements (TEs) and tissue specificity of expression increased the odds of ASE. Our results suggest that most common cis-regulatory variation in C. grandiflora is under weak purifying selection, and that gene-specific functional constraints are more important for the maintenance of cis-regulatory variation than genome-scale variation in the intensity of selection. Our results agree with previous findings that suggest TE silencing affects nearby gene expression, and provide evidence for a link between gbM and cis-regulatory constraint, possibly reflecting greater dosage sensitivity of body-methylated genes. Given the extensive conservation of gbM in flowering plants, this suggests that gbM could be an important predictor of cis-regulatory variation in a wide range of plant species. PMID:28096395

Selection for low dormancy in annual ryegrass (Lolium rigidum) seeds results in high constitutive expression of a glucose-responsive α-amylase isoform

PubMed Central

Goggin, Danica E.; Powles, Stephen B.

2012-01-01

Background and Aims α-Amylase in grass caryopses (seeds) is usually expressed upon commencement of germination and is rarely seen in dry, mature seeds. A heat-stable α-amylase activity was unexpectedly selected for expression in dry annual ryegrass (Lolium rigidum) seeds during targeted selection for low primary dormancy. The aim of this study was to characterize this constitutive activity biochemically and determine if its presence conferred insensitivity to the germination inhibitors abscisic acid and benzoxazolinone. Methods α-Amylase activity in developing, mature and germinating seeds from the selected (low-dormancy) and a field-collected (dormant) population was characterized by native activity PAGE. The response of seed germination and α-amylase activity to abscisic acid and benzoxazolinone was assessed. Using an alginate affinity matrix, α-amylase was purified from dry and germinating seeds for analysis of its enzymatic properties. Key Results The constitutive α-amylase activity appeared late during seed development and was mainly localized in the aleurone; in germinating seeds, this activity was responsive to both glucose and gibberellin. It migrated differently on native PAGE compared with the major activities in germinating seeds of the dormant population, but the enzymatic properties of α-amylase purified from the low-dormancy and dormant seeds were largely indistinguishable. Seed imbibition on benzoxazolinone had little effect on the low-dormancy seeds but greatly inhibited germination and α-amylase activity in the dormant population. Conclusions The constitutive α-amylase activity in annual ryegrass seeds selected for low dormancy is electrophoretically different from that in germinating seeds and its presence confers insensitivity to benzoxazolinone. The concurrent selection of low dormancy and constitutive α-amylase activity may help to enhance seedling establishment under competitive conditions. PMID:23002268

Serum Albumin Domain Structures in Human Blood Serum by Mass Spectrometry and Computational Biology.

PubMed

Belsom, Adam; Schneider, Michael; Fischer, Lutz; Brock, Oliver; Rappsilber, Juri

2016-03-01

Chemical cross-linking combined with mass spectrometry has proven useful for studying protein-protein interactions and protein structure, however the low density of cross-link data has so far precluded its use in determining structures de novo. Cross-linking density has been typically limited by the chemical selectivity of the standard cross-linking reagents that are commonly used for protein cross-linking. We have implemented the use of a heterobifunctional cross-linking reagent, sulfosuccinimidyl 4,4'-azipentanoate (sulfo-SDA), combining a traditional sulfo-N-hydroxysuccinimide (sulfo-NHS) ester and a UV photoactivatable diazirine group. This diazirine yields a highly reactive and promiscuous carbene species, the net result being a greatly increased number of cross-links compared with homobifunctional, NHS-based cross-linkers. We present a novel methodology that combines the use of this high density photo-cross-linking data with conformational space search to investigate the structure of human serum albumin domains, from purified samples, and in its native environment, human blood serum. Our approach is able to determine human serum albumin domain structures with good accuracy: root-mean-square deviation to crystal structure are 2.8/5.6/2.9 Å (purified samples) and 4.5/5.9/4.8Å (serum samples) for domains A/B/C for the first selected structure; 2.5/4.9/2.9 Å (purified samples) and 3.5/5.2/3.8 Å (serum samples) for the best out of top five selected structures. Our proof-of-concept study on human serum albumin demonstrates initial potential of our approach for determining the structures of more proteins in the complex biological contexts in which they function and which they may require for correct folding. Data are available via ProteomeXchange with identifier PXD001692. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Stubborn contaminants: influence of detergents on the purity of the multidrug ABC transporter BmrA.

PubMed

Wiseman, Benjamin; Kilburg, Arnaud; Chaptal, Vincent; Reyes-Mejia, Gina Catalina; Sarwan, Jonathan; Falson, Pierre; Jault, Jean-Michel

2014-01-01

Despite the growing interest in membrane proteins, their crystallization remains a major challenge. In the course of a crystallographic study on the multidrug ATP-binding cassette transporter BmrA, mass spectral analyses on samples purified with six selected detergents revealed unexpected protein contamination visible for the most part on overloaded SDS-PAGE. A major contamination from the outer membrane protein OmpF was detected in purifications with Foscholine 12 (FC12) but not with Lauryldimethylamine-N-oxide (LDAO) or any of the maltose-based detergents. Consequently, in the FC12 purified BmrA, OmpF easily crystallized over BmrA in a new space group, and whose structure is reported here. We therefore devised an optimized protocol to eliminate OmpF during the FC12 purification of BmrA. On the other hand, an additional band visible at ∼110 kDa was detected in all samples purified with the maltose-based detergents. It contained AcrB that crystallized over BmrA despite its trace amounts. Highly pure BmrA preparations could be obtained using either a ΔacrAB E. coli strain and n-dodecyl-β-D-maltopyranoside, or a classical E. coli strain and lauryl maltose neopentyl glycol for the overexpression and purification, respectively. Overall our results urge to incorporate a proteomics-based purity analysis into quality control checks prior to commencing crystallization assays of membrane proteins that are notoriously arduous to crystallize. Moreover, the strategies developed here to selectively eliminate obstinate contaminants should be applicable to the purification of other membrane proteins overexpressed in E. coli.

Stubborn Contaminants: Influence of Detergents on the Purity of the Multidrug ABC Transporter BmrA

PubMed Central

Chaptal, Vincent; Reyes-Mejia, Gina Catalina; Sarwan, Jonathan; Falson, Pierre; Jault, Jean-Michel

2014-01-01

Despite the growing interest in membrane proteins, their crystallization remains a major challenge. In the course of a crystallographic study on the multidrug ATP-binding cassette transporter BmrA, mass spectral analyses on samples purified with six selected detergents revealed unexpected protein contamination visible for the most part on overloaded SDS-PAGE. A major contamination from the outer membrane protein OmpF was detected in purifications with Foscholine 12 (FC12) but not with Lauryldimethylamine-N-oxide (LDAO) or any of the maltose-based detergents. Consequently, in the FC12 purified BmrA, OmpF easily crystallized over BmrA in a new space group, and whose structure is reported here. We therefore devised an optimized protocol to eliminate OmpF during the FC12 purification of BmrA. On the other hand, an additional band visible at ∼110 kDa was detected in all samples purified with the maltose-based detergents. It contained AcrB that crystallized over BmrA despite its trace amounts. Highly pure BmrA preparations could be obtained using either a ΔacrAB E. coli strain and n-dodecyl-β-D-maltopyranoside, or a classical E. coli strain and lauryl maltose neopentyl glycol for the overexpression and purification, respectively. Overall our results urge to incorporate a proteomics-based purity analysis into quality control checks prior to commencing crystallization assays of membrane proteins that are notoriously arduous to crystallize. Moreover, the strategies developed here to selectively eliminate obstinate contaminants should be applicable to the purification of other membrane proteins overexpressed in E. coli. PMID:25517996

Evaluation of ionic air purifiers for reducing aerosol exposure in confined indoor spaces.

PubMed

Grinshpun, S A; Mainelis, G; Trunov, M; Adhikari, A; Reponen, T; Willeke, K

2005-08-01

Numerous techniques have been developed over the years for reducing aerosol exposure in indoor air environments. Among indoor air purifiers of different types, ionic emitters have gained increasing attention and are presently used for removing dust particles, aeroallergens and airborne microorganisms from indoor air. In this study, five ionic air purifiers (two wearable and three stationary) that produce unipolar air ions were evaluated with respect to their ability to reduce aerosol exposure in confined indoor spaces. The concentration decay of respirable particles of different properties was monitored in real time inside the breathing zone of a human manikin, which was placed in a relatively small (2.6 m3) walk-in chamber during the operation of an ionic air purifier in calm air and under mixing air condition. The particle removal efficiency as a function of particle size was determined using the data collected with a size-selective optical particle counter. The removal efficiency of the more powerful of the two wearable ionic purifiers reached about 50% after 15 min and almost 100% after 1.5 h of continuous operation in the chamber under calm air conditions. In the absence of external ventilation, air mixing, especially vigorous one (900 CFM), enhanced the air cleaning effect. Similar results were obtained when the manikin was placed inside a partial enclosure that simulated an aircraft seating configuration. All three stationary ionic air purifiers tested in this study were found capable of reducing the aerosol concentration in a confined indoor space. The most powerful stationary unit demonstrated an extremely high particle removal efficiency that increased sharply to almost 90% within 5-6 min, reaching about 100% within 10-12 min for all particle sizes (0.3-3 microm) tested in the chamber. For the units of the same emission rate, the data suggest that the ion polarity per se (negative vs. positive) does not affect the performance but the ion emission rate does. The effects of particle size (within the tested range) and properties (NaCl, PSL, Pseudomonas fluorescens bacteria) as well as the effects of the manikin's body temperature and its breathing on the ionic purifier performance were either small or insignificant. The data suggest that the unipolar ionic air purifiers are particularly efficient in reducing aerosol exposure in the breathing zone when used inside confined spaces with a relatively high surface-to-volume ratio. Ionic air purifiers have become increasingly popular for removing dust particles, aeroallergens and airborne microorganisms from indoor air in various settings. While the indoor air cleaning effect, resulting from unipolar and bipolar ion emission, has been tested by several investigators, there are still controversial claims (favorable and unfavorable) about the performance of commercially available ionic air purifiers. Among the five tested ionic air purifiers (two wearable and three stationary) producing unipolar air ions, the units with a higher ion emission rate provided higher particle removal efficiency. The ion polarity (negative vs. positive), the particle size (0.3-3 microm) and properties (NaCl, PSL, Pseudomonas fluorescens bacteria), as well as the body temperature and breathing did not considerable affected the ionization-driven particle removal. The data suggest that the unipolar ionic air purifiers are particularly efficient in reducing aerosol exposure in the breathing zone when they are used inside confined spaces with a relatively high surface-to-volume ratio (such as automobile cabins, aircraft seating areas, bathrooms, cellular offices, small residential rooms, and animal confinements). Based on our experiments, we proposed that purifiers with a very high ion emission rate be operated in an intermittent mode if used indoors for extended time periods. As the particles migrate to and deposit on indoor surfaces during the operation of ionic air purifiers, some excessive surface contamination may occur, which introduces the need of periodic cleaning these surfaces.

Inferring Selective Constraint from Population Genomic Data Suggests Recent Regulatory Turnover in the Human Brain

PubMed Central

Schrider, Daniel R.; Kern, Andrew D.

2015-01-01

The comparative genomics revolution of the past decade has enabled the discovery of functional elements in the human genome via sequence comparison. While that is so, an important class of elements, those specific to humans, is entirely missed by searching for sequence conservation across species. Here we present an analysis based on variation data among human genomes that utilizes a supervised machine learning approach for the identification of human-specific purifying selection in the genome. Using only allele frequency information from the complete low-coverage 1000 Genomes Project data set in conjunction with a support vector machine trained from known functional and nonfunctional portions of the genome, we are able to accurately identify portions of the genome constrained by purifying selection. Our method identifies previously known human-specific gains or losses of function and uncovers many novel candidates. Candidate targets for gain and loss of function along the human lineage include numerous putative regulatory regions of genes essential for normal development of the central nervous system, including a significant enrichment of gain of function events near neurotransmitter receptor genes. These results are consistent with regulatory turnover being a key mechanism in the evolution of human-specific characteristics of brain development. Finally, we show that the majority of the genome is unconstrained by natural selection currently, in agreement with what has been estimated from phylogenetic methods but in sharp contrast to estimates based on transcriptomics or other high-throughput functional methods. PMID:26590212

Electrochemical Glucose Biosensor Based on Glucose Oxidase Displayed on Yeast Surface.

PubMed

Wang, Hongwei; Lang, Qiaolin; Liang, Bo; Liu, Aihua

2015-01-01

The conventional enzyme-based biosensor requires chemical or physical immobilization of purified enzymes on electrode surface, which often results in loss of enzyme activity and/or fractions immobilized over time. It is also costly. A major advantage of yeast surface display is that it enables the direct utilization of whole cell catalysts with eukaryote-produced proteins being displayed on the cell surface, providing an economic alternative to traditional production of purified enzymes. Herein, we describe the details of the display of glucose oxidase (GOx) on yeast cell surface and its application in the development of electrochemical glucose sensor. In order to achieve a direct electrochemistry of GOx, the entire cell catalyst (yeast-GOx) was immobilized together with multiwalled carbon nanotubes on the electrode, which allowed sensitive and selective glucose detection.

Purification and properties of aryl acylamidase from Pseudomonas fluorescens ATCC 39004.

PubMed

Hammond, P M; Price, C P; Scawen, M D

1983-05-16

Aryl acylamidase has been purified from a strain of Pseudomonas fluorescens ATCC 39004, selected from soil on the basis of its ability to utilise acylanilide compounds as a sole source of carbon. The enzyme was purified to homogeneity by a combination of ion-exchange, hydrophobic and gel-permeation chromatography. A relative molecular mass of about 52 500 was estimated by gel filtration. The native enzyme was shown to be a monomeric protein by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The enzyme was maximally active at a pH of 8.6 and at a temperature of 45 degrees C. The enzyme shows Michaelis-Menten kinetics; Km values for nitroacetanilide (69 microM) and hydroxyacetanilide (6.1 microM) were low, indicating that the enzyme has a very high affinity for both substrates.

Selection and Reduced Population Size Cannot Explain Higher Amounts of Neandertal Ancestry in East Asian than in European Human Populations

PubMed Central

Kim, Bernard Y.; Lohmueller, Kirk E.

2015-01-01

It has been hypothesized that the greater proportion of Neandertal ancestry in East Asians than in Europeans is due to the fact that purifying selection is less effective at removing weakly deleterious Neandertal alleles from East Asian populations. Using simulations of a broad range of models of selection and demography, we have shown that this hypothesis cannot account for the higher proportion of Neandertal ancestry in East Asians than in Europeans. Instead, more complex demographic scenarios, most likely involving multiple pulses of Neandertal admixture, are required to explain the data. PMID:25683122

Aptamer facilitated purification of functional proteins.

PubMed

Beloborodov, Stanislav S; Bao, Jiayin; Krylova, Svetlana M; Shala-Lawrence, Agnesa; Johnson, Philip E; Krylov, Sergey N

2018-01-15

DNA aptamers are attractive capture probes for affinity chromatography since, in contrast to antibodies, they can be chemically synthesized and, in contrast to tag-specific capture probes (such as Nickel-NTA or Glutathione), they can be used for purification of proteins free of genetic modifications (such as His or GST tags). Despite these attractive features of aptamers as capture probes, there are only a few reports on aptamer-based protein purification and none of them includes a test of the purified protein's activity, thus, leaving discouraging doubts about method's ability to purify proteins in their active state. The goal of this work was to prove that aptamers could facilitate isolation of active proteins. We refined a complete aptamer-based affinity purification procedure, which takes 4 h to complete. We further applied this procedure to purify two recombinant proteins, MutS and AlkB, from bacterial cell culture: 0.21 mg of 85%-pure AlkB from 4 mL of culture and 0.24 mg of 82%-pure MutS from 0.5 mL of culture. Finally, we proved protein activity by two capillary electrophoresis based assays: an enzymatic assay for AlkB and a DNA-binding assay for MutS. We suggest that in combination with aptamer selection for non-purified protein targets in crude cell lysate, aptamer-based purification provides a means of fast isolation of tag-free recombinant proteins in their native state without the use of antibodies. Copyright © 2017 Elsevier B.V. All rights reserved.

[Identification and production of monoclonal antibody of Siberian tiger's immunoglobulin].

PubMed

Zhang, Yaonglong; Zhang, Duanling; Zhou, Ming; Xue, Yuan; Hua, Yuping; Ma, Jianzhang

2010-03-01

To purify immunoglobulin (Ig) of Siberian Tiger and prepare monoclonal antibody (mAb) against the Ig,which can be used to develop immunological diagnostic kits for diagnosing infectious disease in Siberian Tiger. The Ig of Siberian tigers was purified with saturated ammonium sulfate combined with recombinant Protein G. The C57BL/6 mice were immunized with the purified Ig. Spleno-cytes of the mice immunized were collected and fused with the mouse myeloma cell line (Sp2/0-Ag14). The positive hybridoma clones were selected by ELISA and were identified by western blot. The sandwich ELISA was used to detect immunocompetence of the purified Ig and the mAb. We obtained three mouse hybridoma clones that produced mAbs against Ig of Siberian Tiger. The derived McAbs could recognize Ig heavy chain of Siberian Tiger specifically. The biological activity of the Ig and obtained McAbs also could be identified by detecting the antibody induced by panleukopenia virus (FPV-HLJ) vaccine in Siberian Tiger. The antibody also would be useful for assess the vaccine efficacy against the infectious disease on the Siberian Tiger. Protein G can be used in Ig purification of Siberian Tiger. The obtained McAbs from the hybridoma ADT11 in this study owned strong ability to bind Ig of Siberian Tiger and have a stable immunocompetence. They can be used to develop diagnostic methods for detecting infectious disease in Siberian Tiger and vaccine research.

Expression, Identification and Purification of Dictyostelium Acetoacetyl-CoA Thiolase Expressed in Escherichia coli

PubMed Central

Tanaka, Takeshi; Shima, Yasuyuki; Ogawa, Naoki; Nagayama, Koki; Yoshida, Takashi; Ohmachi, Tetsuo

2011-01-01

Acetoacetyl-CoA thiolase (AT) is an enzyme that catalyses the CoA-dependent thiolytic cleavage of acetoacetyl-CoA to yield 2 molecules of acetyl-CoA, or the reverse condensation reaction. A full-length cDNA clone pBSGT-3, which has homology to known thiolases, was isolated from Dictyostelium cDNA library. Expression of the protein encoded in pBSGT-3 in Escherichia coli, its thiolase enzyme activity, and the amino acid sequence homology search revealed that pBSGT-3 encodes an AT. The recombinant AT (r-thiolase) was expressed in an active form in an E. coli expression system, and purified to homogeneity by selective ammonium sulfate fractionation and two steps of column chromatography. The purified enzyme exhibited a specific activity of 4.70 mU/mg protein. Its N-terminal sequence was (NH2)-Arg-Met-Tyr-Thr-Thr-Ala-Lys-Asn-Leu-Glu-, which corresponds to the sequence from positions 15 to 24 of the amino acid sequence deduced from pBSGT-3 clone. The r-thiolase in the inclusion body expressed highly in E. coli was the precursor form, which is slightly larger than the purified r-thiolase. When incubated with the cell-free extract of Dictyostelium cells, the precursor was converted to the same size to the purified r-thiolase, suggesting that the presequence at the N-terminus is removed by a Dictyostelium processing peptidase. PMID:21209787

Application of aqueous biphasic systems as strategy to purify tannase from Aspergillus tamarii URM 7115.

PubMed

de Sena, Amanda Reges; Barros Oliveira, Flávio Manoel; Campos Leite, Tonny Cley; Evaristo da Silva Nascimento, Talita Camila; Moreira, Keila Aparecida; de Assis, Sandra Aparecida

2017-10-21

The aims of the current study are to assess the influence of polyethylene glycol (PEG) concentration, molar mass, pH, and citrate concentrations on aqueous biphasic systems based on 2 4 factorial designs, as well as to check their capacity to purify tannase secreted by Aspergillus tamarii URM 7115. Tannase was produced through submerged fermentation at 26°C for 67 h in Czapeck-Dox modified broth and added with yeast extract and tannic acid. The factorial design was followed to assess the influence of PEG molar mass (M PEG 600; 4,000 and 8,000 g/ mol), and PEG (C PEG 20.0; 22.0 and 24.0% w/w) and citrate concentrations (C CIT 15.0, 17.5, and 20.0%, w/w), as well as of pH (6.0, 7.0, and 8.0) on the response variables; moreover, partition coefficient (K), yield (Y), and purification factor (PF) were analyzed. The most suitable parameters to purify tannase secreted by A. tamarii URM 7115 through a biphasic system were 600 (g/mol) M PEG , 24% (w/w) C PEG , 15% (w/w) C CIT at pH 6.0 and they resulted in 6.33 enzyme partition, 131.25% yield, 19.80 purification factor and 195.08 selectivity. Tannase secreted by A. tamarii URM 7115 purified through aqueous biphasic systems composed of PEG/citrate can be used for industrial purposes, since it presents suitable purification factor and yield.

A Ten-Week Biochemistry Lab Project Studying Wild-Type and Mutant Bacterial Alkaline Phosphatase

ERIC Educational Resources Information Center

Witherow, D. Scott

2016-01-01

This work describes a 10-week laboratory project studying wild-type and mutant bacterial alkaline phosphatase, in which students purify, quantitate, and perform kinetic assays on wild-type and selected mutants of the enzyme. Students also perform plasmid DNA purification, digestion, and gel analysis. In addition to simply learning important…

Method for removal of phosgene from boron trichloride. [DOE patent application; mercury arc lamp

DOEpatents

Freund, S.M.

1981-09-03

Selective ultraviolet photolysis using an unfiltered mercury arc lamp has been used to substantially reduce the phosgene impurity in a mixture of boron trichloride and phosgene. Infrared spectrophotometric analysis of the sample before and after irradiation shows that it is possible to highly purify commercially available boron trichloride with this method.

Molecular and biological characterization of 'Rs551, a filamentous bacteriophage isolated from a race 3 biovar 2 strain of Ralstonia solanacearum

USDA-ARS?s Scientific Manuscript database

A filamentous bacteriophage, designated 'Rs551, was isolated and purified from the quarantine and select agent phytopathogen Ralstonia solanacearum race 3 biovar 2 strain UW551 (phylotype IIB sequevar 1) grown under normal culture conditions. Electron microscopy suggested that 'Rs551 is a member of ...

Antisocial Peer Affiliation and Externalizing Disorders in the Transition from Adolescence to Young Adulthood: Selection versus Socialization Effects

PubMed Central

Samek, Diana R.; Goodman, Rebecca J.; Erath, Stephen A.; McGue, Matt; Iacono, William G.

2016-01-01

Prior research has demonstrated both socialization and selection effects for the relationship between antisocial peer affiliation and externalizing problems in adolescence. Less research has evaluated such effects post-adolescence. In this study, a cross-lagged panel analysis was used to evaluate the extent of socialization (i.e., the effect of antisocial peer affiliation on subsequent externalizing disorders) and selection (i.e., the effect of externalizing disorders on subsequent antisocial peer affiliation) in the prospective relationships between antisocial peer affiliation and externalizing disorders from adolescence through young adulthood. Data from a community sample of 2,769 individuals (52% female) with assessments at ages 17, 20, 24, and 29 were used. Analyses with a latent externalizing measure (estimated using clinical symptom counts of nicotine dependence, alcohol use disorder, illicit drug use disorder, and adult antisocial behavior) and self-reported antisocial peer affiliation revealed significantly stronger socialization effects from age 17 to 20, followed by significantly stronger selection effects from age 20 to 24 and 24 to 29. To better understand the impact of college experience, moderation by college status was evaluated at each developmental transition. Results were generally consistent for those who were in or were not in college. Results suggest selection effects are more important in later developmental periods than earlier periods, particularly in relation to an overall liability towards externalizing disorders, likely due to more freedom in peer selection post-adolescence. PMID:26914216

Assessing the effects of hybridization and precipitation on invasive weed demography using strength of selection on vital rates.

PubMed

Teitel, Zachary; Klimowski, Agnieszka; Campbell, Lesley G

2016-12-07

As global climate change transforms average temperature and rainfall, species distributions may meet, increasing the potential for hybridization and altering individual fitness and population growth. Altered rainfall specifically may shift the strength and direction of selection, also manipulating population trajectories. Here, we investigated the role of interspecific hybridization and selection imposed by rainfall on the evolution of weedy life-history in non-hybrid (Raphanus raphanistrum) and hybrid (R. raphanistrum x R. sativus) populations using a life table response experiment. In documenting long-term population dynamics, we determined intrinsic (r) and asymptotic (λ) population growth rates and sensitivities, a measure of selection imposed on demographic rates. Hybrid populations experienced 8.7-10.3 times stronger selection than wild populations for increased seedling survival. Whereas crop populations generally exhibit little dormancy and wild populations often exhibit dormancy, non-hybrid populations experienced 10% stronger selection than hybrid populations for exhibiting seed dormancy. Selection on survival-to-flowering in wild, not hybrid, populations declined marginally with increasing soil moisture. Hybrid populations exhibited greater r, but not λ, than wild populations regardless of moisture environment. In general, fecundity contributed most to differences in λ but fecundity only contributed positively to hybrid λ relative to wild λ when precipitation was altered (either higher or lower than control) and not under control watering conditions. Selection on key demographic traits may not change dramatically in response to rainfall, and hybridization may more strongly influence the demography of these weedy species than rainfall. If hybrid populations can respond to selection for increased dormancy, this may make it more difficult to deplete weed seed banks and increase the persistence of crop genes in weed populations.

α-l-rhamnosidase selective for rutin to isoquercitrin transformation from Penicillium griseoroseum MTCC-9224.

PubMed

Yadav, Sarita; Yadava, Sudha; Yadav, Kapil D S

2017-02-01

An α-l-rhamnosidase secreting fungal strain has been isolated from the decaying goose berry (Emblica officinalis) fruit peel. The fungal strain has been identified as Penicillium greoroseum MTCC-9224. The α-l-rhamnosidase of this fungal strain has been purified to homogeneity using a simple procedure involving concentration by ultra filtration and an anion exchange chromatography on DEAE-cellulose. The purified enzyme gave a single protein band corresponding to molecular mass of 97kDa in SDS-PAGE analysis. The native-PAGE analysis also gave a single protein band confirming the purity of the enzyme. Using p-nitrophenyl-α-l-rhamnopyranoside as the substrate, K m and k cat values of the enzyme were 0.65mM and 43.65s -1 , respectively. The pH and temperature optima of the enzyme were 6.5 and 57°C, respectively. The activation energy for the thermal denaturation of the enzyme was 27.9kJ/mol. The purified α-l-rhamnosidase hydrolyzed rutin to isoquercitrin and l-rhamnose but has no effect on naringin and hesperidin. Copyright © 2017 Elsevier Inc. All rights reserved.

Derivation of highly purified cardiomyocytes from human induced pluripotent stem cells using small molecule-modulated differentiation and subsequent glucose starvation.

PubMed

Sharma, Arun; Li, Guang; Rajarajan, Kuppusamy; Hamaguchi, Ryoko; Burridge, Paul W; Wu, Sean M

2015-03-18

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/β-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Metabolism of aspartame by human and pig intestinal microvillar peptidases.

PubMed Central

Hooper, N M; Hesp, R J; Tieku, S

1994-01-01

The artificial sweetener aspartame (N-L-alpha-aspartyl-L-phenyl-alanine-1-methyl ester; Nutrasweet), its decomposition product alpha Asp-Phe and the related peptide alpha Asp-PheNH2 were rapidly hydrolysed by microvillar membranes prepared from human duodenum, jejunum and ileum, and from pig duodenum and kidney. The metabolism of aspartame by the human and pig intestinal microvillar membrane preparations was inhibited significantly (> 78%) by amastatin or 1,10-phenanthroline, and partially (> 38%) by actinonin or bestatin, and was activated 2.9-4.5-fold by CaCl2. The inhibition by amastatin and 1,10-phenanthroline, and the activation by CaCl2 are characteristic of the cell-surface ectoenzyme aminopeptidase A (EC 3.4.11.7) and a purified preparation of this enzyme hydrolysed aspartame with a Km of 0.25 mM and a Vmax of 126 mumol/min per mg. A purified preparation of aminopeptidase W (EC 3.4.11.16) also hydrolysed aspartame but with a Km of 4.96 mM and a Vmax of 110 mumol/min per mg. However, rentiapril, an inhibitor of aminopeptidase W, caused only slight inhibition (maximally 19%) of the hydrolysis of aspartame by the microvillar membrane preparations. Similar patterns of inhibition and kinetic parameters were observed for alpha Asp-Phe and alpha Asp-PheNH2. Two other decomposition products of aspartame, beta Asp-PheMe and cyclo-Asp-Phe, were essentially resistant to hydrolysis by both the human and pig intestinal microvillar membrane preparations and the purified preparations of aminopeptidases A and W. Although the relatively selective inhibitor of aminopeptidase N (EC 3.4.11.2), actinonin, partially inhibited the metabolism of aspartame, alpha Asp-Phe and alpha Asp-PheNH2 by the human and pig intestinal microvillar membrane preparations, these peptides were not hydrolysed by a purified preparation of aminopeptidase N. Membrane dipeptidase (EC 3.4.13.19) only hydrolysed the unblocked dipeptide, alpha Asp-Phe, but the selective inhibitor of this enzyme, cilastatin, did not block the metabolism of alpha Asp-Phe by the microvillar membrane preparations. PMID:8141778

Gene copy number variation and its significance in cyanobacterial phylogeny

PubMed Central

2012-01-01

Background In eukaryotes, variation in gene copy numbers is often associated with deleterious effects, but may also have positive effects. For prokaryotes, studies on gene copy number variation are rare. Previous studies have suggested that high numbers of rRNA gene copies can be advantageous in environments with changing resource availability, but further association of gene copies and phenotypic traits are not documented. We used one of the morphologically most diverse prokaryotic phyla to test whether numbers of gene copies are associated with levels of cell differentiation. Results We implemented a search algorithm that identified 44 genes with highly conserved copies across 22 fully sequenced cyanobacterial taxa. For two very basal cyanobacterial species, Gloeobacter violaceus and a thermophilic Synechococcus species, distinct phylogenetic positions previously found were supported by identical protein coding gene copy numbers. Furthermore, we found that increased ribosomal gene copy numbers showed a strong correlation to cyanobacteria capable of terminal cell differentiation. Additionally, we detected extremely low variation of 16S rRNA sequence copies within the cyanobacteria. We compared our results for 16S rRNA to three other eubacterial phyla (Chroroflexi, Spirochaetes and Bacteroidetes). Based on Bayesian phylogenetic inference and the comparisons of genetic distances, we could confirm that cyanobacterial 16S rRNA paralogs and orthologs show significantly stronger conservation than found in other eubacterial phyla. Conclusions A higher number of ribosomal operons could potentially provide an advantage to terminally differentiated cyanobacteria. Furthermore, we suggest that 16S rRNA gene copies in cyanobacteria are homogenized by both concerted evolution and purifying selection. In addition, the small ribosomal subunit in cyanobacteria appears to evolve at extraordinary slow evolutionary rates, an observation that has been made previously for morphological characteristics of cyanobacteria. PMID:22894826

Lower prevalence but similar fitness in a parasitic fungus at higher radiation levels near Chernobyl.

PubMed

Aguileta, Gabriela; Badouin, Helene; Hood, Michael E; Møller, Anders P; Le Prieur, Stephanie; Snirc, Alodie; Siguenza, Sophie; Mousseau, Timothy A; Shykoff, Jacqui A; Cuomo, Christina A; Giraud, Tatiana

2016-07-01

Nuclear disasters at Chernobyl and Fukushima provide examples of effects of acute ionizing radiation on mutations that can affect the fitness and distribution of species. Here, we investigated the prevalence of Microbotryum lychnidis-dioicae, a pollinator-transmitted fungal pathogen of plants causing anther-smut disease in Chernobyl, its viability, fertility and karyotype variation, and the accumulation of nonsynonymous mutations in its genome. We collected diseased flowers of Silene latifolia from locations ranging by more than two orders of magnitude in background radiation, from 0.05 to 21.03 μGy/h. Disease prevalence decreased significantly with increasing radiation level, possibly due to lower pollinator abundance and altered pollinator behaviour. Viability and fertility, measured as the budding rate of haploid sporidia following meiosis from the diploid teliospores, did not vary with increasing radiation levels and neither did karyotype overall structure and level of chromosomal size heterozygosity. We sequenced the genomes of twelve samples from Chernobyl and of four samples collected from uncontaminated areas and analysed alignments of 6068 predicted genes, corresponding to 1.04 × 10(7)  base pairs. We found no dose-dependent differences in substitution rates (neither dN, dS, nor dN/dS). Thus, we found no significant evidence of increased deleterious mutation rates at higher levels of background radiation in this plant pathogen. We even found lower levels of nonsynonymous substitution rates in contaminated areas compared to control regions, suggesting that purifying selection was stronger in contaminated than uncontaminated areas. We briefly discuss the possibilities for a mechanistic basis of radio resistance in this nonmelanized fungus. © 2016 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.

Yeast-expressed recombinant protein of the receptor-binding domain in SARS-CoV spike protein with deglycosylated forms as a SARS vaccine candidate

PubMed Central

Chen, Wen-Hsiang; Du, Lanying; Chag, Shivali M; Ma, Cuiqing; Tricoche, Nancy; Tao, Xinrong; Seid, Christopher A; Hudspeth, Elissa M; Lustigman, Sara; Tseng, Chien-Te K; Bottazzi, Maria Elena; Hotez, Peter J; Zhan, Bin; Jiang, Shibo

2014-01-01

Development of vaccines for preventing a future pandemic of severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV) and for biodefense preparedness is urgently needed. Our previous studies have shown that a candidate SARS vaccine antigen consisting of the receptor-binding domain (RBD) of SARS-CoV spike protein can induce potent neutralizing antibody responses and protection against SARS-CoV challenge in vaccinated animals. To optimize expression conditions for scale-up production of the RBD vaccine candidate, we hypothesized that this could be potentially achieved by removing glycosylation sites in the RBD protein. In this study, we constructed two RBD protein variants: 1) RBD193-WT (193-aa, residues 318–510) and its deglycosylated forms (RBD193-N1, RBD193-N2, RBD193-N3); 2) RBD219-WT (219-aa, residues 318–536) and its deglycosylated forms (RBD219-N1, RBD219-N2, and RBD219-N3). All constructs were expressed as recombinant proteins in yeast. The purified recombinant proteins of these constructs were compared for their antigenicity, functionality and immunogenicity in mice using alum as the adjuvant. We found that RBD219-N1 exhibited high expression yield, and maintained its antigenicity and functionality. More importantly, RBD219-N1 induced significantly stronger RBD-specific antibody responses and a higher level of neutralizing antibodies in immunized mice than RBD193-WT, RBD193-N1, RBD193-N3, or RBD219-WT. These results suggest that RBD219-N1 could be selected as an optimal SARS vaccine candidate for further development. PMID:24355931

Yeast-expressed recombinant protein of the receptor-binding domain in SARS-CoV spike protein with deglycosylated forms as a SARS vaccine candidate.

PubMed

Chen, Wen-Hsiang; Du, Lanying; Chag, Shivali M; Ma, Cuiqing; Tricoche, Nancy; Tao, Xinrong; Seid, Christopher A; Hudspeth, Elissa M; Lustigman, Sara; Tseng, Chien-Te K; Bottazzi, Maria Elena; Hotez, Peter J; Zhan, Bin; Jiang, Shibo

2014-01-01

Development of vaccines for preventing a future pandemic of severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV) and for biodefense preparedness is urgently needed. Our previous studies have shown that a candidate SARS vaccine antigen consisting of the receptor-binding domain (RBD) of SARS-CoV spike protein can induce potent neutralizing antibody responses and protection against SARS-CoV challenge in vaccinated animals. To optimize expression conditions for scale-up production of the RBD vaccine candidate, we hypothesized that this could be potentially achieved by removing glycosylation sites in the RBD protein. In this study, we constructed two RBD protein variants: 1) RBD193-WT (193-aa, residues 318-510) and its deglycosylated forms (RBD193-N1, RBD193-N2, RBD193-N3); 2) RBD219-WT (219-aa, residues 318-536) and its deglycosylated forms (RBD219-N1, RBD219-N2, and RBD219-N3). All constructs were expressed as recombinant proteins in yeast. The purified recombinant proteins of these constructs were compared for their antigenicity, functionality and immunogenicity in mice using alum as the adjuvant. We found that RBD219-N1 exhibited high expression yield, and maintained its antigenicity and functionality. More importantly, RBD219-N1 induced significantly stronger RBD-specific antibody responses and a higher level of neutralizing antibodies in immunized mice than RBD193-WT, RBD193-N1, RBD193-N3, or RBD219-WT. These results suggest that RBD219-N1 could be selected as an optimal SARS vaccine candidate for further development.

[Mineral elements analysis of Momordica charantiap seeds by ICP-AES and fatty acid profile identification of seed oil by GC-MS].

PubMed

Liu, Xiao-ru; Deng, Ze-yuan; Fan, Ya-wei; Li, Jing; Liu, Zhi-han

2010-08-01

In the present study, a special kind of Momordica charantia seeds produced in Hai Nan was selected and analyzed. Firstly, inductively coupled plasma-atomic emission spectrophotometry (ICP-AES) was used to determine the mineral elements. It was clear that the contents of K, Mg and P are the highest in the seeds; Cr and Zn takes up to 5.65% and 45.45% high, especially, which are rare in plant foods. These minerals, especially Cr and Zn might have a complex effect on those proteins or polysaccharides and form a stronger anticipation of hyperlipidemia, hyperglycemia and cholesterol. Secondly, seed oil was extracted by supercritical CO2 extraction with a yield ratio of 36.89, and the fatty acids were treated by methylation in alkaline process and purified by thin-layer chromatography, then analyzed by gas chromatography-mass spectrometer (GC-MS) identification. The saturated fatty acids (SFA) take up 36.712, and mainly are stearic acid; monounsaturated fatty acid (MUFA) is only 3.33% which is dominantly linoleic acid (LA); Polyunsaturated fatty acid (PUFA) accounted for 59.96%, and the alpha-eleostearic acid takes up 54.26% as the main fatty acids in all. The plentiful alpha-eleostearic acid leads to strong effects of inhibiting tumor cell proliferation, lowering blood fat, anti-cancer, anti-inflammatory and preventing cardiovascular diseases, and so on. Knowing clearly the mineral elements distribution and identifying the composition of fatty acid, especially the main fatty acids in the oil, are both of great guiding importance to further exploit the clinical and edible value in Momordica charantiap seeds.

Increased fire frequency promotes stronger spatial genetic structure and natural selection at regional and local scales in Pinus halepensis Mill

PubMed Central

González-Martínez, Santiago C.; Navascués, Miguel; Burgarella, Concetta; Mosca, Elena; Lorenzo, Zaida; Zabal-Aguirre, Mario; Vendramin, Giovanni G.; Verdú, Miguel; Pausas, Juli G.

2017-01-01

Background and Aims The recurrence of wildfires is predicted to increase due to global climate change, resulting in severe impacts on biodiversity and ecosystem functioning. Recurrent fires can drive plant adaptation and reduce genetic diversity; however, the underlying population genetic processes have not been studied in detail. In this study, the neutral and adaptive evolutionary effects of contrasting fire regimes were examined in the keystone tree species Pinus halepensis Mill. (Aleppo pine), a fire-adapted conifer. The genetic diversity, demographic history and spatial genetic structure were assessed at local (within-population) and regional scales for populations exposed to different crown fire frequencies. Methods Eight natural P. halepensis stands were sampled in the east of the Iberian Peninsula, five of them in a region exposed to frequent crown fires (HiFi) and three of them in an adjacent region with a low frequency of crown fires (LoFi). Samples were genotyped at nine neutral simple sequence repeats (SSRs) and at 251 single nucleotide polymorphisms (SNPs) from coding regions, some of them potentially important for fire adaptation. Key Results Fire regime had no effects on genetic diversity or demographic history. Three high-differentiation outlier SNPs were identified between HiFi and LoFi stands, suggesting fire-related selection at the regional scale. At the local scale, fine-scale spatial genetic structure (SGS) was overall weak as expected for a wind-pollinated and wind-dispersed tree species. HiFi stands displayed a stronger SGS than LoFi stands at SNPs, which probably reflected the simultaneous post-fire recruitment of co-dispersed related seeds. SNPs with exceptionally strong SGS, a proxy for microenvironmental selection, were only reliably identified under the HiFi regime. Conclusions An increasing fire frequency as predicted due to global change can promote increased SGS with stronger family structures and alter natural selection in P. halepensis and in plants with similar life history traits. PMID:28159988

Group-size-mediated habitat selection and group fusion-fission dynamics of bison under predation risk.

PubMed

Fortin, Daniel; Fortin, Marie-Eve; Beyer, Hawthorne L; Duchesne, Thierry; Courant, Sabrina; Dancose, Karine

2009-09-01

For gregarious animals the cost-benefit trade-offs that drive habitat selection may vary dynamically with group size, which plays an important role in foraging and predator avoidance strategies. We examined how habitat selection by bison (Bison bison) varied as a function of group size and interpreted these patterns by testing whether habitat selection was more strongly driven by the competing demands of forage intake vs. predator avoidance behavior. We developed an analytical framework that integrated group size into resource selection functions (RSFs). These group-size-dependent RSFs were based on a matched case-control design and were estimated using conditional logistic regression (mixed and population-averaged models). Fitting RSF models to bison revealed that bison groups responded to multiple aspects of landscape heterogeneity and that selection varied seasonally and as a function of group size. For example, roads were selected in summer, but not in winter. Bison groups avoided areas of high snow water equivalent in winter. They selected areas composed of a large proportion of meadow area within a 700-m radius, and within those areas, bison selected meadows. Importantly, the strength of selection for meadows varied as a function of group size, with stronger selection being observed in larger groups. Hence the bison-habitat relationship depended in part on the dynamics of group formation and division. Group formation was most likely in meadows. In contrast, risk of group fission increased when bison moved into the forest and was higher during the time of day when movements are generally longer and more variable among individuals. We also found that stronger selection for meadows by large rather than small bison groups was caused by longer residence time in individual meadows by larger groups and that departure from meadows appears unlikely to result from a depression in food intake rate. These group-size-dependent patterns were consistent with the hypothesis that avoidance of predation risk is the strongest driver of habitat selection.

Myosin light chain 2-based selection of human iPSC-derived early ventricular cardiac myocytes

PubMed Central

Bizy, Alexandra; Guerrero-Serna, Guadalupe; Hu, Bin; Ponce-Balbuena, Daniela; Willis, B. Cicero; Zarzoso, Manuel; Ramirez, Rafael J.; Sener, Michelle F.; Mundada, Lakshmi V.; Klos, Matthew; Devaney, Eric J.; Vikstrom, Karen L.; Herron, Todd J.; Jalife, José

2014-01-01

Applications of human induced pluripotent stemcell derived-cardiac myocytes (hiPSC-CMs) would be strengthened by the ability to generate specific cardiac myocyte (CM) lineages. However, purification of lineage-specific hiPSC-CMs is limited by the lack of cell marking techniques. Here, we have developed an iPSC-CM marking system using recombinant adenoviral reporter constructs with atrial- or ventricular-specific myosin light chain-2 (MLC-2) promoters. MLC-2a and MLC-2v selected hiPSC-CMs were purified by fluorescence-activated cell sorting and their biochemical and electrophysiological phenotypes analyzed. We demonstrate that the phenotype of both populations remained stable in culture and they expressed the expected sarcomeric proteins, gap junction proteins and chamber-specific transcription factors. Compared to MLC-2a cells, MLC-2v selected CMs had larger action potential amplitudes and durations. In addition, by immunofluorescence, we showed that MLC-2 isoform expression can be used to enrich hiPSC-CM consistent with early atrial and ventricularmyocyte lineages. However, only the ventricular myosin light chain-2 promoter was able to purify a highly homogeneous population of iPSC-CMs. Using this approach, it is now possible to develop ventricular-specific disease models using iPSC-CMs while atrial-specific iPSC-CM cultures may require additional chamber-specific markers. PMID:24095945

Myosin light chain 2-based selection of human iPSC-derived early ventricular cardiac myocytes.

PubMed

Bizy, Alexandra; Guerrero-Serna, Guadalupe; Hu, Bin; Ponce-Balbuena, Daniela; Willis, B Cicero; Zarzoso, Manuel; Ramirez, Rafael J; Sener, Michelle F; Mundada, Lakshmi V; Klos, Matthew; Devaney, Eric J; Vikstrom, Karen L; Herron, Todd J; Jalife, José

2013-11-01

Applications of human induced pluripotent stem cell derived-cardiac myocytes (hiPSC-CMs) would be strengthened by the ability to generate specific cardiac myocyte (CM) lineages. However, purification of lineage-specific hiPSC-CMs is limited by the lack of cell marking techniques. Here, we have developed an iPSC-CM marking system using recombinant adenoviral reporter constructs with atrial- or ventricular-specific myosin light chain-2 (MLC-2) promoters. MLC-2a and MLC-2v selected hiPSC-CMs were purified by fluorescence-activated cell sorting and their biochemical and electrophysiological phenotypes analyzed. We demonstrate that the phenotype of both populations remained stable in culture and they expressed the expected sarcomeric proteins, gap junction proteins and chamber-specific transcription factors. Compared to MLC-2a cells, MLC-2v selected CMs had larger action potential amplitudes and durations. In addition, by immunofluorescence, we showed that MLC-2 isoform expression can be used to enrich hiPSC-CM consistent with early atrial and ventricular myocyte lineages. However, only the ventricular myosin light chain-2 promoter was able to purify a highly homogeneous population of iPSC-CMs. Using this approach, it is now possible to develop ventricular-specific disease models using iPSC-CMs while atrial-specific iPSC-CM cultures may require additional chamber-specific markers. © 2013.

Dual Functions of Lip6 and Its Regulation of Lipid Metabolism in the Oleaginous Fungus Mucor circinelloides.

PubMed

Zan, Xinyi; Tang, Xin; Chu, Linfang; Song, Yuanda

2018-03-21

Although multiple roles of lipases have been reported in yeasts and microalgae, the functions of lipases have not been studied in oleaginous filamentous fungi. Lipase Lip6 has been reported in the oleaginous filamentous fungus Mucor circinelloides with the consensus lipase motif GXSXG and the typical acyltransferase motif of H-(X) 4 -D. To demonstrate that Lip6 might play dual roles as a lipase and an acyltransferase, we performed site-directed mutagenesis in the lipase motif and the acyltransferase motif of Lip6. Mutation in the lipase motif increased cell biomass by 12%-18% and promoted lipid accumulation by 9%-24%, while mutation in the acyltransferase motif induced lipid degradation. In vitro, purified Lip6 had a slight lipase activity but had a stronger phospholipid:DAG acyltransferase activity. Enzyme activity assays in vivo and phospholipid synthesis pathway analysis suggested that phosphatidyl serine and phosphatidyl ethanolamine can be the supplier of a fatty acyl moiety to form TAG in M. circinelloides.

Enzymatic synthesis of chlorogenic acid glucoside using dextransucrase and its physical and functional properties.

PubMed

Nam, Seung-Hee; Ko, Jin-A; Jun, Woojin; Wee, Young-Jung; Walsh, Marie K; Yang, Kwang-Yeol; Choi, Jin-Ho; Eun, Jon-Bang; Choi, Jeong; Kim, Young-Min; Han, Songhee; Nguyen, Thi Thanh Hanh; Kim, Doman

2017-12-01

Chlorogenic acid, a major polyphenol in edible plants, possesses strong antioxidant activity, anti-lipid peroxidation and anticancer effects. It used for industrial applications; however, this is limited by its instability to heat or light. In this study, we for the first time synthesized chlorogenic acid glucoside (CHG) via transglycosylation using dextransucrase from Leuconostoc mesenteroides and sucrose. CHG was purified and its structure determined by nuclear magnetic resonance and matrix-associated laser desorption ionization-time-of-flight mass spectroscopy. The production yield of CHG was 44.0% or 141mM, as determined by response surface methodology. CHG possessed a 65% increased water solubility and 2-fold browning resistance while it displayed stronger inhibition of lipid peroxidation and of colon cancer cell growth by MTT assay, compared to chlorogenic acid. Therefore, this study may expand the industrial applications of chlorogenic acid as water-soluble or browning resistant compound (CHG) through enzymatic glycosylation. Copyright © 2017 Elsevier Inc. All rights reserved.

Purification, characterisation and salt-tolerance molecular mechanisms of aspartyl aminopeptidase from Aspergillus oryzae 3.042.

PubMed

Gao, Xianli; Yin, Yiyun; Zhou, Cunshan

2018-02-01

A salt-tolerant aspartyl aminopeptidase (approximately 57kDa) from Aspergillus oryzae 3.042 was purified and identified. Specific inhibitor experiments indicated that it was an aminopeptidase containing Zn 2+ . Its optimal and stable pH values and temperatures were 7 and 50°C, respectively. Its relative activity remained beyond 30% in 3M NaCl solution for 15d, and its K m and V max were slightly affected in 3M NaCl solution, indicating its excellent salt-tolerance. A comprehensive analysis including protein homology modelling, molecular dynamics simulation, secondary structure, acidic residues and hydrophobicity of interior residues demonstrated that aspartyl aminopeptidase had a greater stability than non-salt-tolerant protease in high salinity. Higher contents of ordered secondary structures, more salt bridges between hydrated surface acidic residues and specific basic residues and stronger hydrophobicity of interior residues were the salt-tolerance mechanisms of aspartyl aminopeptidase. Copyright © 2017. Published by Elsevier Ltd.

Structural diversity of pectins isolated from the Styrian oil-pumpkin (Cucurbita pepo var. styriaca) fruit.

PubMed

Košťálová, Zuzana; Hromádková, Zdenka; Ebringerová, Anna

2013-03-01

To evaluate the seeded fruit biomass of the Styrian oil-pumpkin in view of its pectin component, a series of acidic polysaccharides were isolated by a six-step sequential extraction using hot water, EDTA, dilute HCl (twice) and dilute and stronger NaOH solutions. Chemical, physicochemical and spectroscopy analyses revealed that the first four fractions comprised partially methyl-esterified and acetylated pectins with varying proportions of rhamnogalacturonan regions ramified with galactose- and arabinose-containing side chains and showed considerable polymolecularity. The alkali-extracted polysaccharides contained lower amounts of pectins with homogalacturonan and arabinose-rich rhamnogalacturonan regions next to hemicelluloses prevailing in the last polysaccharide. Using (1)H-(13)C HSQC and HMBC spectroscopy, the resonances of free and methylesterified galacturonic acid residues in the purified acid-extracted pectin were unambiguously established and various diads formed by both residues identified. The results might serve as a basis for searching technological conditions to produce pectin from the oil-pumpkin fruit biomass. Copyright © 2012 Elsevier Ltd. All rights reserved.

Studies on an inactivated vaccine against rabies virus in domestic animals.

PubMed

Monaco, F; Franchi, P M; Lelli, R

2006-01-01

An inactivated vaccine against rabies virus was prepared from the attenuated ATCC PV-12 viral rabbit Pasteur strain. The virus was grown on Baby Hamster Kidney (BHK21) cells, and the supernatant was purified by filtration and inactivated with beta-propriolactone. The inactivated product was checked according to the NHI and European Pharmacopoeia methods. Part of the product was then lyophilised and the other part was adjuvanted with Al(OH)3. Both parts were used to vaccinate and boost groups of horses, cattle and sheep at different intervals. Their immunogenicity was compared with a similar commercial product. Blood samples were collected on a regular basis and the antibody titre was determined by the Fluorescence Antibody Virus Neutralisation (FAVN) test. No significant differences were found between species after both inoculations even though the immune response increased in intensity and duration after the booster dose in all the animals tested and was stronger and lasted longer with the adjuvanted aliquot.

Force feedback controls motor activity and mechanical properties of self-assembling branched actin networks

PubMed Central

Bieling, Peter; Li, Tai-De; Weichsel, Julian; McGorty, Ryan; Jreij, Pamela; Huang, Bo; Fletcher, Daniel A.; Mullins, R. Dyche

2016-01-01

Branched actin networks–created by the Arp2/3 complex, capping protein, and a nucleation promoting factor– generate and transmit forces required for many cellular processes, but their response to force is poorly understood. To address this, we assembled branched actin networks in vitro from purified components and used simultaneous fluorescence and atomic force microscopy to quantify their molecular composition and material properties under various forces. Remarkably, mechanical loading of these self-assembling materials increases their density, power, and efficiency. Microscopically, increased density reflects increased filament number and altered geometry, but no change in average length. Macroscopically, increased density enhances network stiffness and resistance to mechanical failure beyond those of isotropic actin networks. These effects endow branched actin networks with memory of their mechanical history that shapes their material properties and motor activity. This work reveals intrinsic force feedback mechanisms by which mechanical resistance makes self-assembling actin networks stiffer, stronger, and more powerful. PMID:26771487

Foundational errors in the Neutral and Nearly-Neutral theories of evolution in relation to the Synthetic Theory: is a new evolutionary paradigm necessary?

PubMed

Valenzuela, Carlos Y

2013-01-01

The Neutral Theory of Evolution (NTE) proposes mutation and random genetic drift as the most important evolutionary factors. The most conspicuous feature of evolution is the genomic stability during paleontological eras and lack of variation among taxa; 98% or more of nucleotide sites are monomorphic within a species. NTE explains this homology by random fixation of neutral bases and negative selection (purifying selection) that does not contribute either to evolution or polymorphisms. Purifying selection is insufficient to account for this evolutionary feature and the Nearly-Neutral Theory of Evolution (N-NTE) included negative selection with coefficients as low as mutation rate. These NTE and N-NTE propositions are thermodynamically (tendency to random distributions, second law), biotically (recurrent mutation), logically and mathematically (resilient equilibria instead of fixation by drift) untenable. Recurrent forward and backward mutation and random fluctuations of base frequencies alone in a site make life organization and fixations impossible. Drift is not a directional evolutionary factor, but a directional tendency of matter-energy processes (second law) which threatens the biotic organization. Drift cannot drive evolution. In a site, the mutation rates among bases and selection coefficients determine the resilient equilibrium frequency of bases that genetic drift cannot change. The expected neutral random interaction among nucleotides is zero; however, huge interactions and periodicities were found between bases of dinucleotides separated by 1, 2... and more than 1,000 sites. Every base is co-adapted with the whole genome. Neutralists found that neutral evolution is independent of population size (N); thus neutral evolution should be independent of drift, because drift effect is dependent upon N. Also, chromosome size and shape as well as protein size are far from random.

A nanobody directed to a functional epitope on VEGF, as a novel strategy for cancer treatment

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

Farajpour, Zahra; Rahbarizadeh, Fatemeh, E-mail: rahbarif@modares.ac.ir; Kazemi, Bahram

Highlights: • A novel nanobody directed to antigenic regions on VEGF was identified. • Our nanobody was successfully purified. • Our nanobody significantly inhibited VEGF-induced proliferation of HUVECs in a dose dependent manner. - Abstract: Compelling evidence suggests that vascular endothelial growth factor (VEGF), due to its essential role in angiogenesis, is a critical target for cancer treatment. Neutralizing monoclonal antibodies against VEGF are important class of drugs used in cancer therapy. However, the cost of production, large size, and immunogenicity are main drawbacks of conventional monoclonal therapy. Nanobodies are the smallest antigen-binding antibody fragments, which occur naturally in camelidae.more » Because of their remarkable features, we decided to use an immune library of nanobody to direct phage display to recognition of novel functional epitopes on VEGF. Four rounds of selection were performed and six phage-displayed nanobodies were obtained from an immune phage library. The most reactive clone in whole-cell ELISA experiments, was purified and assessed in proliferation inhibition assay. Purified ZFR-5 not only blocked interaction of VEGF with its receptor in cell ELISA experiments, but also was able to significantly inhibit proliferation response of human umbilical vein endothelial cells to VEGF in a dose-dependent manner. Taken together, our study demonstrates that by using whole-cell ELISA experiments, nanobodies against antigenic regions included in interaction of VEGF with its receptors can be directed. Because of unique and intrinsic properties of a nanobody and the ability of selected nanobody for blocking the epitope that is important for biological function of VEGF, it represents novel potential drug candidate.« less

A robust robotic high-throughput antibody purification platform.

PubMed

Schmidt, Peter M; Abdo, Michael; Butcher, Rebecca E; Yap, Min-Yin; Scotney, Pierre D; Ramunno, Melanie L; Martin-Roussety, Genevieve; Owczarek, Catherine; Hardy, Matthew P; Chen, Chao-Guang; Fabri, Louis J

2016-07-15

Monoclonal antibodies (mAbs) have become the fastest growing segment in the drug market with annual sales of more than 40 billion US$ in 2013. The selection of lead candidate molecules involves the generation of large repertoires of antibodies from which to choose a final therapeutic candidate. Improvements in the ability to rapidly produce and purify many antibodies in sufficient quantities reduces the lead time for selection which ultimately impacts on the speed with which an antibody may transition through the research stage and into product development. Miniaturization and automation of chromatography using micro columns (RoboColumns(®) from Atoll GmbH) coupled to an automated liquid handling instrument (ALH; Freedom EVO(®) from Tecan) has been a successful approach to establish high throughput process development platforms. Recent advances in transient gene expression (TGE) using the high-titre Expi293F™ system have enabled recombinant mAb titres of greater than 500mg/L. These relatively high protein titres reduce the volume required to generate several milligrams of individual antibodies for initial biochemical and biological downstream assays, making TGE in the Expi293F™ system ideally suited to high throughput chromatography on an ALH. The present publication describes a novel platform for purifying Expi293F™-expressed recombinant mAbs directly from cell-free culture supernatant on a Perkin Elmer JANUS-VariSpan ALH equipped with a plate shuttle device. The purification platform allows automated 2-step purification (Protein A-desalting/size exclusion chromatography) of several hundred mAbs per week. The new robotic method can purify mAbs with high recovery (>90%) at sub-milligram level with yields of up to 2mg from 4mL of cell-free culture supernatant. Copyright © 2016 Elsevier B.V. All rights reserved.

Selective Expansion of Memory CD4+ T cells By Mitogenic Human CD28 Generates Inflammatory Cytokines and Regulatory T cells

PubMed Central

Singh, Manisha; Basu, Sreemanti; Camell, Christina; Couturier, Jacob; Nudelman, Rodolfo J.; Medina, Miguel A.; Rodgers, John R.; Lewis, Dorothy E.

2009-01-01

Co-stimulatory signals are important for development of effector and regulatory T cells. In this case, CD28 signaling is usually considered inert in the absence of signaling through the TCR. By contrast, mitogenic rat CD28 mAbs reportedly expand regulatory T cells without TCR stimulation. We found that a commercially available human CD28 mAb (ANC28) stimulated PBMCs without TCR co-ligation or cross-linking; ANC28 selectively expanded CD4+CD25+FoxP3−(T effector) and CD4+CD25+FoxP3+ (Treg) cells. ANC28 stimulated the CD45RO+ CD4+ (memory) population whereas CD45RA+CD4+ (naïve) cells did not respond. ANC28 also induced inflammatory cytokines. Treg induced by ANC28 retain the Treg phenotype longer than did co-stimulated Treg. Treg induced by ANC28 suppressed CD25− T cells through a contact-dependent mechanism. Purity influenced the response of CD4+CD25+ cells because bead-purified CD4+CD25+ cells (85–90% pure) responded strongly to ANC28, whereas 98% pure FACS-sorted CD4+CD25 bright (T-reg) did not respond. Purified CD4+CD25int cells responded similarly to the bead-purified CD4+CD25+ cells. Thus, pre-activated CD4+ T cells (CD25int) respond to ANC28 rather than Treg (CD25bright). The ability of ANC28 to expand both effectors producing inflammatory cytokines as well as suppressive regulatory T cells might be useful for ex vivo expansion of therapeutic T cells. PMID:18446791

A lateralized functional auditory network is involved in anuran sexual selection.

PubMed

Xue, Fei; Fang, Guangzhan; Yue, Xizi; Zhao, Ermi; Brauth, Steven E; Tang, Yezhong

2016-12-01

Right ear advantage (REA) exists in many land vertebrates in which the right ear and left hemisphere preferentially process conspecific acoustic stimuli such as those related to sexual selection. Although ecological and neural mechanisms for sexual selection have been widely studied, the brain networks involved are still poorly understood. In this study we used multi-channel electroencephalographic data in combination with Granger causal connectivity analysis to demonstrate, for the first time, that auditory neural network interconnecting the left and right midbrain and forebrain function asymmetrically in the Emei music frog (Babina daunchina), an anuran species which exhibits REA. The results showed the network was lateralized. Ascending connections between the mesencephalon and telencephalon were stronger in the left side while descending ones were stronger in the right, which matched with the REA in this species and implied that inhibition from the forebrainmay induce REA partly. Connections from the telencephalon to ipsilateral mesencephalon in response to white noise were the highest in the non-reproductive stage while those to advertisement calls were the highest in reproductive stage, implying the attention resources and living strategy shift when entered the reproductive season. Finally, these connection changes were sexually dimorphic, revealing sex differences in reproductive roles.

Developmental changes in the inferior frontal cortex for selecting semantic representations

PubMed Central

Lee, Shu-Hui; Booth, James R.; Chen, Shiou-Yuan; Chou, Tai-Li

2012-01-01

Functional magnetic resonance imaging (fMRI) was used to examine the neural correlates of semantic judgments to Chinese words in a group of 10–15 year old Chinese children. Two semantic tasks were used: visual–visual versus visual–auditory presentation. The first word was visually presented (i.e. character) and the second word was either visually or auditorily presented, and the participant had to determine if these two words were related in meaning. Different from English, Chinese has many homophones in which each spoken word corresponds to many characters. The visual–auditory task, therefore, required greater engagement of cognitive control for the participants to select a semantically appropriate answer for the second homophonic word. Weaker association pairs produced greater activation in the mid-ventral region of left inferior frontal gyrus (BA 45) for both tasks. However, this effect was stronger for the visual–auditory task than for the visual–visual task and this difference was stronger for older compared to younger children. The findings suggest greater involvement of semantic selection mechanisms in the cross-modal task requiring the access of the appropriate meaning of homophonic spoken words, especially for older children. PMID:22337757

Male preference for female pubic hair: an evolutionary view.

PubMed

Prokop, Pavol

The ectoparasite avoidance hypothesis proposes that human hairlessness was favoured by sexual selection, because hairless individuals suffered from lower parasite loads. Females have seemingly less hairy bodies than men suggesting that the selection toward hairlessness is more intense in females than in males. This study examined male preference for hairy and shaved female genitalia. Pubic hair, although still functional in terms of dissipation of phermononal secretions, was perceived by heterosexual males recruited in the university (age range: 19-38 years, N = 96) as much less attractive as shaved female genitalia. Males who were more disgust sensitive and those who were sexually unrestricted showed a stronger preference for shaved genitalia than others. Self-reported frequency of pornography consumption was associated, contrary to expectations, with a stronger preference for hairy genitalia which suggests that this may be a result of negative frequency dependent selection. Older males also preferred hairy genitalia more than younger males. Overall, these results suggest that a preference for shaved genitalia may be explained by the superficial resemblance of pubic hair with chest hair, which is less developed as in our evolutionary past, perhaps due to the benefits associated with ectoparasite avoidance.

Antibody Characterization Process | Office of Cancer Clinical Proteomics Research

Cancer.gov

The goal of the NCI's Antibody Characterization Program (ACP) is to have three monoclonal antibodies produced for each successfully expressed/purified recombinant antigen and one antibody per peptide (1 to 3 peptides per protein). To date, over 4000 clones have been screened before selecting the current 393 antibodies. They are winnowed down based on the projected end use of the antibody.

Antimalarial drug discovery: screening of Brazilian medicinal plants and purified compounds.

PubMed

Krettli, Antoniana Ursine

2009-02-01

Malaria is the most important parasitic disease and its control depends on specific chemotherapy, now complicated by Plasmodium falciparum that has become resistant to most commonly available antimalarials. Treatment of the disease requires quinine or drug combinations of artemisinin derivatives and other antimalarials. Further drug resistance is expected. New active compounds need to be discovered. To find new antimalarials from medicinal and randomly collected plants, crude extracts are screened against P. falciparum in cultures and in malaria animal models, following bioassays of purified fractions, and cytotoxicity tests. For antimalarial research, screening medicinal plants is more efficient than screening randomly chosen plants. Biomonitored fractionation allows selection of new active molecules identified as potential antimalarials in multidisciplinary projects in Brazil; no new molecule is available for human testing. The advantages of projects based on ethnopharmacology are discussed.

Anti-tumour potential of a gallic acid-containing phenolic fraction from Oenothera biennis.

PubMed

Pellegrina, Chiara Dalla; Padovani, Giorgia; Mainente, Federica; Zoccatelli, Gianni; Bissoli, Gaetano; Mosconi, Silvia; Veneri, Gianluca; Peruffo, Angelo; Andrighetto, Giancarlo; Rizzi, Corrado; Chignola, Roberto

2005-08-08

A phenolic fraction purified form defatted seeds of Oenothera biennis promoted selective apoptosis of human and mouse bone marrow-derived cell lines following first-order kinetics through a caspase-dependent pathway. In non-leukemia tumour cell lines, such as human colon carcinoma CaCo(2) cells and mouse fibrosarcoma WEHI164 cells, this fraction inhibited (3)H-thymidine incorporation but not cell death or cell cycle arrest. Human peripheral blood mononuclear cells showed low sensitivity to treatment. Single bolus injection of the phenolic fraction could delay the growth of established myeloma tumours in syngeneic animals. HPLC and mass spectrometry analysis revealed that the fraction contains gallic acid. However, the biological activity of the fraction differs from the activity of this phenol and hence it should be attributed to other co-purified molecules which remain still unidentified.

Process for forming a nickel foil with controlled and predetermined permeability to hydrogen

DOEpatents

Engelhaupt, Darell E.

1981-09-22

The present invention provides a novel process for forming a nickel foil having a controlled and predetermined hydrogen permeability. This process includes the steps of passing a nickel plating bath through a suitable cation exchange resin to provide a purified nickel plating bath free of copper and gold cations, immersing a nickel anode and a suitable cathode in the purified nickel plating bath containing a selected concentration of an organic sulfonic acid such as a napthalene-trisulfonic acid, electrodepositing a nickel layer having the thickness of a foil onto the cathode, and separating the nickel layer from the cathode to provide a nickel foil. The anode is a readily-corrodible nickel anode. The present invention also provides a novel nickel foil having a greater hydrogen permeability than palladium at room temperature.

Anti-Legionella activity of staphylococcal hemolytic peptides.

PubMed

Marchand, A; Verdon, J; Lacombe, C; Crapart, S; Héchard, Y; Berjeaud, J M

2011-05-01

A collection of various Staphylococci was screened for their anti-Legionella activity. Nine of the tested strains were found to secrete anti-Legionella compounds. The culture supernatants of the strains, described in the literature to produce hemolytic peptides, were successfully submitted to a two step purification process. All the purified compounds, except one, corresponded to previously described hemolytic peptides and were not known for their anti-Legionella activity. By comparison of the minimal inhibitory concentrations, minimal permeabilization concentrations, decrease in the number of cultivable bacteria, hemolytic activity and selectivity, the purified peptides could be separated in two groups. First group, with warnericin RK as a leader, corresponds to the more hemolytic and bactericidal peptides. The peptides of the second group, represented by the PSMα from Staphylococcus epidermidis, appeared bacteriostatic and poorly hemolytic. Copyright © 2011 Elsevier Inc. All rights reserved.

Induction of specific immune unresponsiveness with purified mixed leukocyte culture-activated T lymphoblasts as autoimmunogen. II. An analysis of the effects measured at the cellular and serological levels

PubMed Central

1978-01-01

T lymphoblasts specific for foreign histocompatibility antigens and purified via mixed leukocyte culture (MLC) and 1 g velocity sedimentation procedures can be used as autoimmunogen to produce specific immunological unresponsiveness in adult animals. This unresponsiveness is positively correlated to the production of autoanti- idiotypic antibodies in the blast immunized animals and no evidence of coexisting alloimmunity was found. We consider this autoanti-idiotypic immunity to be the specific inducing agent of the immune tolerance. The blast immunization procedure will lead to selective reduction in T-cell reactivity against the relevant alloantigens as measured by MLC, cell- mediated lympholysis, or graft-versus-host assays. However, in individual animals, dichtomy in suppression between two T-cell assays could sometimes be observed indicating elimination of only a select group of idiotypic functionally distinct population of T cells in these blast-immunized animals. Attempts to abrogate already immune animals by the autoblast procedure were successful, in part suggesting the use of the present procedure when trying to induce in accelerated reversion of such immunity. PMID:75235

Accumulation of slightly deleterious mutations in the mitochondrial genome: a hallmark of animal domestication.

PubMed

Hughes, Austin L

2013-02-15

The hypothesis that domestication leads to a relaxation of purifying selection on mitochondrial (mt) genomes was tested by comparative analysis of mt genes from dog, pig, chicken, and silkworm. The three vertebrate species showed mt genome phylogenies in which domestic and wild isolates were intermingled, whereas the domestic silkworm (Bombyx mori) formed a distinct cluster nested within its closest wild relative (Bombyx mandarina). In spite of these differences in phylogenetic pattern, significantly greater proportions of nonsynonymous SNPs than of synonymous SNPs were unique to the domestic populations of all four species. Likewise, in all four species, significantly greater proportions of RNA-encoding SNPs than of synonymous SNPs were unique to the domestic populations. Thus, domestic populations were characterized by an excess of unique polymorphisms in two categories generally subject to purifying selection: nonsynonymous sites and RNA-encoding sites. Many of these unique polymorphisms thus seem likely to be slightly deleterious; the latter hypothesis was supported by the generally lower gene diversities of polymorphisms unique to domestic populations in comparison to those of polymorphisms shared by domestic and wild populations. Copyright © 2012 Elsevier B.V. All rights reserved.

An integrated map of genetic variation from 1,092 human genomes

PubMed Central

2012-01-01

Summary Through characterising the geographic and functional spectrum of human genetic variation, the 1000 Genomes Project aims to build a resource to help understand the genetic contribution to disease. We describe the genomes of 1,092 individuals from 14 populations, constructed using a combination of low-coverage whole-genome and exome sequencing. By developing methodologies to integrate information across multiple algorithms and diverse data sources we provide a validated haplotype map of 38 million SNPs, 1.4 million indels and over 14 thousand larger deletions. We show that individuals from different populations carry different profiles of rare and common variants and that low-frequency variants show substantial geographic differentiation, which is further increased by the action of purifying selection. We show that evolutionary conservation and coding consequence are key determinants of the strength of purifying selection, that rare-variant load varies substantially across biological pathways and that each individual harbours hundreds of rare non-coding variants at conserved sites, such as transcription-factor-motif disrupting changes. This resource, which captures up to 98% of accessible SNPs at a frequency of 1% in populations of medical genetics focus, enables analysis of common and low-frequency variants in individuals from diverse, including admixed, populations. PMID:23128226

Multi-branched gold nanostars with fractal structure for SERS detection of the pesticide thiram

NASA Astrophysics Data System (ADS)

Zhu, Jian; Liu, Mei-Jin; Li, Jian-Jun; Li, Xin; Zhao, Jun-Wu

2018-01-01

The surface-enhanced Raman scattering (SERS) activity of multi-branched gold nanostars with fractal structure has been investigated for trace detection of pesticide thiram. Raman spectrum results show that the gold nanostars substrate can produce about 102 fold stronger signal than the thiram alone with the thiram concentration increase of 103 times and 1.4 fold stronger signal than the gold nanostars without fractal feature. In the detection procedure, the most prominent SERS peak at 1376 cm- 1 has been chosen to characterize and quantify the concentration of thiram. Experimental results indicate this Raman substrate based on fractal gold nanostars exhibits excellent selective probing performance for thiram with a detection limit as low as 10- 10 M in solution and 0.24 ng/cm2 in apple peels. Interference experiment results show that the effects from the interfering pesticides could be neglected in the detection procedure. Therefore, the gold nanostars as a SERS substrate have excellent sensitivity and selectivity.

Synthesis of α-MoC1-x Nanoparticles with a Surface-Modified SBA-15 Hard Template: Determination of Structure-Function Relationships in Acetic Acid Deoxygenation.

PubMed

Baddour, Frederick G; Nash, Connor P; Schaidle, Joshua A; Ruddy, Daniel A

2016-07-25

Surface modification of mesoporous SBA-15 silica generated a hydrophobic environment for a molybdenum diamine (Mo-diamine) precursor solution, enabling direct growth of isolated 1.9±0.4 nm α-MoC1-x nanoparticles (NPs) inside the pores of the support. The resulting NP catalysts are bifunctional, and compared to bulk α-MoC1-x and β-Mo2 C, the NPs exhibit a greater acid-site:H-site ratio and a fraction of stronger acid sites. The greater acid-site:H-site ratio results in higher decarbonylation (DCO) selectivity during acetic acid hydrodeoxygenation (HDO) reactions, and the stronger acid sites lead to higher activity and ketonization (KET) selectivity at high temperatures. The hard-templating synthetic method could be a versatile route toward carbide NPs of varying size, composition, and phase, on a range of mesoporous oxide supports. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Co-Pt core-shell nanostructured catalyst prepared by selective chemical vapor pulse deposition of Pt on Co as a cathode in polymer electrolyte fuel cells

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

Seo, Sang-Joon; Chung, Ho-Kyoon; Yoo, Ji-Beom

2014-01-15

A new type of PtCo/C catalyst for use as a cathode in polymer electrolyte fuel cells was prepared by selective chemical vapor pulse deposition (CVPD) of Pt on the surface of Co. The activity of the prepared catalyst for oxygen reduction was higher than that of a catalyst prepared by sequential impregnation (IMP) with the two metallic components. This catalytic activity difference occurs because the former catalyst has smaller Pt crystallites that produce stronger Pt-Co interactions and have a larger Pt surface area. Consequently, the CVPD catalyst has a great number of Co particles that are in close contact withmore » the added Pt. The Pt surface was also electronically modified by interactions with Co, which were stronger in the CVPD catalyst than in the IMP catalyst, as indicated by X-ray diffraction, X-ray photoemission spectroscopy, and cyclic voltammetry measurements of the catalysts.« less

Synthesis of α-MoC 1-x Nanoparticles with a Surface-Modified SBA-15 Hard Template: Determination of Structure-Function Relationships in Acetic Acid Deoxygenation

DOE PAGES

Baddour, Frederick G.; Nash, Connor P.; Schaidle, Joshua A.; ...

2016-06-07

Surface modification of mesoporous SBA-15 silica generated a hydrophobic environment for a molybdenum diamine (Mo-diamine) precursor solution, enabling direct growth of isolated 1.9 ± 0.4 nm α-MoC 1-x nanoparticles (NPs) inside the pores of the support. The resulting NP catalysts are bifunctional, and compared to bulk α-MoC 1-x and β-Mo 2C, the NPs exhibit a greater acid-site:H-site ratio and a fraction of stronger acid sites. The greater acid-site:H-site ratio results in higher decarbonylation (DCO) selectivity during acetic acid hydrodeoxygenation (HDO) reactions, and the stronger acid sites lead to higher activity and ketonization (KET) selectivity at high temperatures. Lastly, the hard-templatingmore » synthetic method could be a versatile route toward carbide NPs of varying size, composition, and phase, on a range of mesoporous oxide supports.« less

Topology of evolving, mutagenized viral populations: quasispecies expansion, compression, and operation of negative selection.

PubMed

Ojosnegros, Samuel; Agudo, Rubén; Sierra, Macarena; Briones, Carlos; Sierra, Saleta; González-López, Claudia; Domingo, Esteban; Cristina, Juan

2008-07-17

The molecular events and evolutionary forces underlying lethal mutagenesis of virus (or virus extinction through an excess of mutations) are not well understood. Here we apply for the first time phylogenetic methods and Partition Analysis of Quasispecies (PAQ) to monitor genetic distances and intra-population structures of mutant spectra of foot-and-mouth disease virus (FMDV) quasispecies subjected to mutagenesis by base and nucleoside analogues. Phylogenetic and PAQ analyses have revealed a highly dynamic variation of intrapopulation diversity of FMDV quasispecies. The population diversity first suffers striking expansions in the presence of mutagens and then compressions either when the presence of the mutagenic analogue was discontinued or when a mutation that decreased sensitivity to a mutagen was selected. The pattern of mutations found in the populations was in agreement with the behavior of the corresponding nucleotide analogues with FMDV in vitro. Mutations accumulated at preferred genomic sites, and dn/ds ratios indicate the operation of negative (or purifying) selection in populations subjected to mutagenesis. No evidence of unusually elevated genetic distances has been obtained for FMDV populations approaching extinction. Phylogenetic and PAQ analysis provide adequate procedures to describe the evolution of viral sequences subjected to lethal mutagenesis. These methods define the changes of intra-population structure more precisely than mutation frequencies and Shannon entropies. PAQ is very sensitive to variations of intrapopulation genetic distances. Strong negative (or purifying) selection operates in FMDV populations subjected to enhanced mutagenesis. The quantifications provide evidence that extinction does not imply unusual increases of intrapopulation complexity, in support of the lethal defection model of virus extinction.

Selection and reduced population size cannot explain higher amounts of Neandertal ancestry in East Asian than in European human populations.

PubMed

Kim, Bernard Y; Lohmueller, Kirk E

2015-03-05

It has been hypothesized that the greater proportion of Neandertal ancestry in East Asians than in Europeans is due to the fact that purifying selection is less effective at removing weakly deleterious Neandertal alleles from East Asian populations. Using simulations of a broad range of models of selection and demography, we have shown that this hypothesis cannot account for the higher proportion of Neandertal ancestry in East Asians than in Europeans. Instead, more complex demographic scenarios, most likely involving multiple pulses of Neandertal admixture, are required to explain the data. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Single cell gene expression profiling of cortical osteoblast lineage cells.

PubMed

Flynn, James M; Spusta, Steven C; Rosen, Clifford J; Melov, Simon

2013-03-01

In tissues with complex architectures such as bone, it is often difficult to purify and characterize specific cell types via molecular profiling. Single cell gene expression profiling is an emerging technology useful for characterizing transcriptional profiles of individual cells isolated from heterogeneous populations. In this study we describe a novel procedure for the isolation and characterization of gene expression profiles of single osteoblast lineage cells derived from cortical bone. Mixed populations of different cell types were isolated from adult long bones of C57BL/6J mice by enzymatic digestion, and subsequently subjected to FACS to purify and characterize osteoblast lineage cells via a selection strategy using antibodies against CD31, CD45, and alkaline phosphatase (AP), specific for mature osteoblasts. The purified individual osteoblast lineage cells were then profiled at the single cell level via nanofluidic PCR. This method permits robust gene expression profiling on single osteoblast lineage cells derived from mature bone, potentially from anatomically distinct sites. In conjunction with this technique, we have also shown that it is possible to carry out single cell profiling on cells purified from fixed and frozen bone samples without compromising the gene expression signal. The latter finding means the technique can be extended to biopsies of bone from diseased individuals. Our approach for single cell expression profiling provides a new dimension to the transcriptional profile of the primary osteoblast lineage population in vivo, and has the capacity to greatly expand our understanding of how these cells may function in vivo under normal and diseased states. Copyright © 2012 Elsevier Inc. All rights reserved.

Application of CRISPR technology in genome editing in agriculture -swine

USDA-ARS?s Scientific Manuscript database

Decades of selective breeding in agricultural species has led to the derivation of stronger and fitter animals with improved production traits. However, often co-segregating with beneficial traits are less desirable traits. With the plethora of genome data and annotation, has come the technology t...

Insights into recent and ancient trends in the co-evolution of Earth and life as revealed by microbial genomics

NASA Astrophysics Data System (ADS)

Anderson, R. E.; Huber, J. A.; Parsons, C.; Stüeken, E.

2017-12-01

Since the origin of life over 4 billion years ago, life has fundamentally altered the habitability of Earth. Similarly, the environment molds the evolutionary trajectory of life itself through natural selection. Microbial genomes retain a "memory" of the co-evolution of life and Earth and can be analyzed to better understand trends and events in both the recent and distant past. To examine evolutionary trends in the more recent past, we have used metagenomics analyses to investigate which environmental factors play the strongest role in driving the evolution of microbes in deep-sea hydrothermal vents, which are thought to have been important habitats in the earliest stages of life's evolution. We have shown that microbial populations in a deep, basalt-hosted system appear to be under stronger purifying selection than populations inhabiting a cooler serpentinizing system less than 20 km away, suggesting that environmental context and geochemistry have an important impact on evolutionary rates and trends. We also found evidence that viruses play an important role in driving evolution in these habitats. Changing environmental conditions may also effect long-term evolutionary trends in Earth's distant past, as revealed by comparative genomics. By reconciling phylogenetic trees for microbial species with trees of metabolic genes, we can determine approximately when crucial metabolic genes began to spread across the tree of life through horizontal gene transfer. Using these methods, we conducted an analysis of the relative timing of the spread of genes related to the nitrogen cycle. Our results indicate that the rate of horizontal gene transfer for important genes related to denitrification increased after the Great Oxidation Event, concurrent with geochemical evidence for increasing availability of nitrate, suggesting that the oxygenation of the atmosphere and surface ocean may have been an important determining factor for the spread of denitrification genes across the tree of life. In contrast, genes related to nitrogen fixation display much more consistent rates of horizontal gene transfer throughout Earth's history. Studies that couple genomics approaches with geochemistry have the potential to reveal insights into the co-evolution of life and Earth both in the recent and distant past.

Comparative study on the selective chalcopyrite bioleaching of a molybdenite concentrate with mesophilic and thermophilic bacteria.

PubMed

Romano, P; Blázquez, M L; Alguacil, F J; Muñoz, J A; Ballester, A; González, F

2001-03-01

This study evaluates different bioleaching treatments of a molybdenite concentrate using mesophilic and thermophilic bacterial cultures. Further studies on the chemical leaching and the electrochemical behavior of the MoS(2) concentrate were carried out. Bioleaching tests showed a progressive removal of chalcopyrite from the molybdenite concentrate with an increase in temperature. Chemical leaching tests support the idea of an indirect attack of the concentrate. Electrochemical tests indicate that chalcopyrite dissolution is favored when molybdenite is present. Therefore, this type of bioleaching treatment could be applied to purify molybdenite flotation concentrates by selectively dissolving chalcopyrite.

Whole-Gene Positive Selection, Elevated Synonymous Substitution Rates, Duplication, and Indel Evolution of the Chloroplast clpP1 Gene

PubMed Central

Erixon, Per; Oxelman, Bengt

2008-01-01

Background Synonymous DNA substitution rates in the plant chloroplast genome are generally relatively slow and lineage dependent. Non-synonymous rates are usually even slower due to purifying selection acting on the genes. Positive selection is expected to speed up non-synonymous substitution rates, whereas synonymous rates are expected to be unaffected. Until recently, positive selection has seldom been observed in chloroplast genes, and large-scale structural rearrangements leading to gene duplications are hitherto supposed to be rare. Methodology/Principle Findings We found high substitution rates in the exons of the plastid clpP1 gene in Oenothera (the Evening Primrose family) and three separate lineages in the tribe Sileneae (Caryophyllaceae, the Carnation family). Introns have been lost in some of the lineages, but where present, the intron sequences have substitution rates similar to those found in other introns of their genomes. The elevated substitution rates of clpP1 are associated with statistically significant whole-gene positive selection in three branches of the phylogeny. In two of the lineages we found multiple copies of the gene. Neighboring genes present in the duplicated fragments do not show signs of elevated substitution rates or positive selection. Although non-synonymous substitutions account for most of the increase in substitution rates, synonymous rates are also markedly elevated in some lineages. Whereas plant clpP1 genes experiencing negative (purifying) selection are characterized by having very conserved lengths, genes under positive selection often have large insertions of more or less repetitive amino acid sequence motifs. Conclusions/Significance We found positive selection of the clpP1 gene in various plant lineages to correlated with repeated duplication of the clpP1 gene and surrounding regions, repetitive amino acid sequences, and increase in synonymous substitution rates. The present study sheds light on the controversial issue of whether negative or positive selection is to be expected after gene duplications by providing evidence for the latter alternative. The observed increase in synonymous substitution rates in some of the lineages indicates that the detection of positive selection may be obscured under such circumstances. Future studies are required to explore the functional significance of the large inserted repeated amino acid motifs, as well as the possibility that synonymous substitution rates may be affected by positive selection. PMID:18167545

Molecular evolution of the odorant and gustatory receptor genes in lepidopteran insects: implications for their adaptation and speciation.

PubMed

Engsontia, Patamarerk; Sangket, Unitsa; Chotigeat, Wilaiwan; Satasook, Chutamas

2014-08-01

Lepidoptera (comprised of butterflies and moths) is one of the largest groups of insects, including more than 160,000 described species. Chemoreception plays important roles in the adaptation of these species to a wide range of niches, e.g., plant hosts, egg-laying sites, and mates. This study investigated the molecular evolution of the lepidopteran odorant (Or) and gustatory receptor (Gr) genes using recently identified genes from Bombyx mori, Danaus plexippus, Heliconius melpomene, Plutella xylostella, Heliothis virescens, Manduca sexta, Cydia pomonella, and Spodoptera littoralis. A limited number of cases of large lineage-specific gene expansion are observed (except in the P. xylostella lineage), possibly due to selection against tandem gene duplication. There has been strong purifying selection during the evolution of both lepidopteran odorant and gustatory genes, as shown by the low ω values estimated through CodeML analysis, ranging from 0.0093 to 0.3926. However, purifying selection has been relaxed on some amino acid sites in these receptors, leading to sequence divergence, which is a precursor of positive selection on these sequences. Signatures of positive selection were detected only in a few loci from the lineage-specific analysis. Estimation of gene gains and losses suggests that the common ancestor of the Lepidoptera had fewer Or genes compared to extant species and an even more reduced number of Gr genes, particularly within the bitter receptor clade. Multiple gene gains and a few gene losses occurred during the evolution of Lepidoptera. Gene family expansion may be associated with the adaptation of lepidopteran species to plant hosts, especially after angiosperm radiation. Phylogenetic analysis of the moth sex pheromone receptor genes suggested that chromosomal translocations have occurred several times. New sex pheromone receptors have arisen through tandem gene duplication. Positive selection was detected at some amino acid sites predicted to be in the extracellular and transmembrane regions of the newly duplicated genes, which might be associated with the evolution of the new pheromone receptors.

Production of capsular polysaccharide of Streptococcus pneumoniae type 14 and its purification by affinity chromatography.

PubMed

Suárez, N; Fraguas, L F; Texeira, E; Massaldi, H; Batista-Viera, F; Ferreira, F

2001-02-01

We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents.

Production of Capsular Polysaccharide of Streptococcus pneumoniae Type 14 and Its Purification by Affinity Chromatography

PubMed Central

Suárez, Norma; Fraguas, Laura Franco; Texeira, Esther; Massaldi, Hugo; Batista-Viera, Francisco; Ferreira, Fernando

2001-01-01

We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents. PMID:11157270

Antagonistic Effect of Nonpathogenic Fusarium oxysporum Fo47 and Pseudobactin 358 upon Pathogenic Fusarium oxysporum f. sp. dianthi

PubMed Central

Lemanceau, Philippe; Bakker, Peter A. H. M.; De Kogel, Willem Jan; Alabouvette, Claude; Schippers, Bob

1993-01-01

Pseudobactin production by Pseudomonas putida WCS358 significantly improves biological control of fusarium wilt caused by nonpathogenic Fusarium oxysporum Fo47b10 (P. Lemanceau, P. A. H. M. Bakker, W. J. de Kogel, C. Alabouvette, and B. Schippers, Appl. Environ. Microbiol. 58:2978-2982, 1992). The antagonistic effect of Fo47b10 and purified pseudobactin 358 was studied by using an in vitro bioassay. This bioassay allows studies on interactions among nonpathogenic F. oxysporum Fo47b10, pathogenic F. oxysporum f. sp. dianthi WCS816, and purified pseudobactin 358, the fluorescent siderophore produced by P. putida WCS358. Both nonpathogenic and pathogenic F. oxysporum reduced each other's growth when grown together. However, in these coinoculation experiments, pathogenic F. oxysporum WCS816 was relatively more inhibited in its growth than nonpathogenic F. oxysporum Fo47b10. The antagonism of nonpathogenic F. oxysporum against pathogenic F. oxysporum strongly depends on the ratio of nonpathogenic to pathogenic F. oxysporum densities: the higher this ratio, the stronger the antagonism. This fungal antagonism appears to be mainly associated with the competition for glucose. Pseudobactin 358 reduced the growth of both F. oxysporum strains, whereas ferric pseudobactin 358 did not; antagonism by pseudobactin 358 was then related to competition for iron. However, the pathogenic F. oxysporum strain was more sensitive to this antagonism than the nonpathogenic strain. Pseudobactin 358 reduced the efficiency of glucose metabolism by the fungi. These results suggest that pseudobactin 358 increases the intensity of the antagonism of nonpathogenic F. oxysporum Fo47b10 against pathogenic F. oxysporum WCS816 by making WCS816 more sensitive to the glucose competition by Fo47b10. PMID:16348860

Reconstituted High-Density Lipoprotein Containing Human Growth Hormone-1 Shows Potent Tissue Regeneration Activity with Enhancement of Anti-Oxidant and Anti-Atherosclerotic Activities.

PubMed

Lee, Eun-Young; Kim, So-Hee; Cho, Kyung-Hyun

2015-06-01

Human growth hormone-1 (GH-1), somatotropin, is a peptide hormone that stimulates cell division in tissues such as bone and cartilage. To compare physiological activities in lipid-free and lipid-bound states, we expressed and incorporated GH-1 in reconstituted high-density lipoprotein (rHDL). GH-1 was expressed and purified using the pET30(a) vector and an Escherichia coli expression system. Purified GH-1 (at least 98% purity, 23 kD) was characterized and synthesized with apolipoproteinA-I (apoA-I), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and cholesterol. Secondary structure analysis of GH-1 revealed 54% α-helical content in a lipid-free state and 65% α-helical content in a lipid-bound state along with blue-shifted tryptophan movement (around 2 nm). GH-1 caused less uptake of oxidized low-density lipoprotein (oxLDL) into macrophages and inhibited senescence of dermal cells in a dose-dependent manner. GH-1 in a lipid-bound state exerted stronger inhibitory activity than in a lipid-free state, indicating improved anti-atherosclerotic activity due to the lipid formulation. In a fin regeneration experiment using zebrafish (17 weeks old, n=9), GH-1 showed its highest regeneration speed without any side effects. GH-1-rHDL containing apoA-I showed 2.3-fold and 1.6-fold higher regeneration speeds than lipid-free GH-1 (in native state) and lipid-bound GH-1, respectively. Incorporation of GH-1 and apoA-I in HDL enhanced tissue regeneration activity of amputated tail fin, indicating a synergetic effect between GH-1 and apoA-I in a lipid-bound state.

Identification of conformational epitopes for human IgG on Chemotaxis inhibitory protein of Staphylococcus aureus

PubMed Central

Gustafsson, Erika; Haas, Pieter-Jan; Walse, Björn; Hijnen, Marcel; Furebring, Christina; Ohlin, Mats; van Strijp, Jos AG; van Kessel, Kok PM

2009-01-01

Background The Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) blocks the Complement fragment C5a receptor (C5aR) and formylated peptide receptor (FPR) and is thereby a potent inhibitor of neutrophil chemotaxis and activation of inflammatory responses. The majority of the healthy human population has antibodies against CHIPS that have been shown to interfere with its function in vitro. The aim of this study was to define potential epitopes for human antibodies on the CHIPS surface. We also initiate the process to identify a mutated CHIPS molecule that is not efficiently recognized by preformed anti-CHIPS antibodies and retains anti-inflammatory activity. Results In this paper, we panned peptide displaying phage libraries against a pool of CHIPS specific affinity-purified polyclonal human IgG. The selected peptides could be divided into two groups of sequences. The first group was the most dominant with 36 of the 48 sequenced clones represented. Binding to human affinity-purified IgG was verified by ELISA for a selection of peptide sequences in phage format. For further analysis, one peptide was chemically synthesized and antibodies affinity-purified on this peptide were found to bind the CHIPS molecule as studied by ELISA and Surface Plasmon Resonance. Furthermore, seven potential conformational epitopes responsible for antibody recognition were identified by mapping phage selected peptide sequences on the CHIPS surface as defined in the NMR structure of the recombinant CHIPS31–121 protein. Mapped epitopes were verified by in vitro mutational analysis of the CHIPS molecule. Single mutations introduced in the proposed antibody epitopes were shown to decrease antibody binding to CHIPS. The biological function in terms of C5aR signaling was studied by flow cytometry. A few mutations were shown to affect this biological function as well as the antibody binding. Conclusion Conformational epitopes recognized by human antibodies have been mapped on the CHIPS surface and amino acid residues involved in both antibody and C5aR interaction could be defined. This information has implications for the development of an effective anti-inflammatory agent based on a functional CHIPS molecule with low interaction with human IgG. PMID:19284584

Purifying Selection and Birth-and-Death Evolution in the Class II Hydrophobin Gene Families of the Ascomycete Trichoderma/Hypocrea

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

kubicek, Christian P.; Baker, Scott E.; Gamauf, Christian

2008-01-10

Hydrophobins are proteins containing eight conserved cysteine residues that occur uniquely in mycelial fungi, where their main function is to confer hydrophobicity to fungal surfaces in contact with air and during attachment of hyphae to hydrophobic surfaces of hosts, symbiotic partners or of themselves resulting in morphogenetic signals. Based on their hydropathy patterns and their solubility characteristics, they are classified in class I and class II hydrophobins, the latter being found only in ascomycetes. Here we have investigated the mechanisms driving the evolution of the class II hydrophobins in nine species of the mycoparasitic ascomycetous genus Trichoderma/Hypocrea, using three fullymore » sequenced genomes (H. jecorina=T. reesei, H. atroviridis=T. atroviride; H. virens=T. virens) and a total of 14.000 ESTs of six others (T. asperellum, H. lixii=T. harzianum, T. aggressivum var. europeae, T. longibrachiatum, T. cf. viride). The former three contained six, ten and nine members, which is the highest number found in any other ascomycete so far. They all showed the conserved four beta-strands/one helix structure, which is stabilized by four disulfide bonds. In addition, a small number of these HFBs contained an extended N-terminus rich in either praline and aspartate, or glycine-asparagine. Phylogenetic analysis reveals a mosaic of terminal clades contain duplicated genes and shows only three reasonably supported clades. Calculation of the ratio of differences in synonymous vs. non-synonymous nucleotide substitutions provides evidence for strong purifying selection (KS/Ka >> 1). A genome database search for class II HFBs from other ascomycetes retrieved a much smaller number of hydrophobins (2-4) from each species, and most of them were from Pyrenomycetes. A combined phylogeny of these sequences with those of Trichoderma showed that the Trichoderma HFBs mostly formed their own clades, whereas those of other pyrenomycetes occured in shared clades. Our study shows that the genus Trichoderma/Hypocrea has a proliferated arsenal of class II hydrophobins which arose by purifying selection and birth-and-death evolution.« less

Weaving a Stronger Fabric for Improved Outcomes

ERIC Educational Resources Information Center

Lobry de Bruyn, Lisa; Prior, Julian; Lenehan, Jo

2014-01-01

Purpose: To explain how training and education events (TEEs) can be designed to increase the likelihood of achieving behavioural objectives. Approach: The approach combined both a quantitative review of evaluation surveys undertaken at the time of the TEE, and qualitative telephone interviews with selected attendees (2025% of the total population…

Dropout Prevention Fieldbook: Best Practices from the Field

ERIC Educational Resources Information Center

Schargel, Franklin P.

2012-01-01

Reduce your school's dropout rate, help improve teaching and learning, and develop stronger relationships with parents and the community. This book showcases the collected efforts of dedicated educators from across the country, selected and presented by one of today's leading experts in dropout prevention, Franklin Schargel. Easily indexed…

KIR2DL2/2DL3-E35 alleles are functionally stronger than -Q35 alleles

NASA Astrophysics Data System (ADS)

Bari, Rafijul; Thapa, Rajoo; Bao, Ju; Li, Ying; Zheng, Jie; Leung, Wing

2016-03-01

KIR2DL2 and KIR2DL3 segregate as alleles of a single locus in the centromeric motif of the killer cell immunoglobulin-like receptor (KIR) gene family. Although KIR2DL2/L3 polymorphism is known to be associated with many human diseases and is an important factor for donor selection in allogeneic hematopoietic stem cell transplantation, the molecular determinant of functional diversity among various alleles is unclear. In this study we found that KIR2DL2/L3 with glutamic acid at position 35 (E35) are functionally stronger than those with glutamine at the same position (Q35). Cytotoxicity assay showed that NK cells from HLA-C1 positive donors with KIR2DL2/L3-E35 could kill more target cells lacking their ligands than NK cells with the weaker -Q35 alleles, indicating better licensing of KIR2DL2/L3+ NK cells with the stronger alleles. Molecular modeling analysis reveals that the glutamic acid, which is negatively charged, interacts with positively charged histidine located at position 55, thereby stabilizing KIR2DL2/L3 dimer and reducing entropy loss when KIR2DL2/3 binds to HLA-C ligand. The results of this study will be important for future studies of KIR2DL2/L3-associated diseases as well as for donor selection in allogeneic stem cell transplantation.

Electrodrift purification of materials for room temperature radiation detectors

DOEpatents

James, R.B.; Van Scyoc, J.M. III; Schlesinger, T.E.

1997-06-24

A method of purifying nonmetallic, crystalline semiconducting materials useful for room temperature radiation detecting devices by applying an electric field across the material is disclosed. The present invention discloses a simple technology for producing purified ionic semiconducting materials, in particular PbI{sub 2} and preferably HgI{sub 2}, which produces high yields of purified product, requires minimal handling of the material thereby reducing the possibility of introducing or reintroducing impurities into the material, is easy to control, is highly selective for impurities, retains the stoichiometry of the material and employs neither high temperatures nor hazardous materials such as solvents or liquid metals. An electric field is applied to a bulk sample of the material causing impurities present in the sample to drift in a preferred direction. After all of the impurities have been transported to the ends of the sample the current flowing through the sample, a measure of the rate of transport of mobile impurities, falls to a low, steady state value, at which time the end sections of the sample where the impurities have concentrated are removed leaving a bulk sample of higher purity material. Because the method disclosed here only acts on the electrically active impurities, the stoichiometry of the host material remains substantially unaffected. 4 figs.

Electrodrift purification of materials for room temperature radiation detectors

DOEpatents

James, Ralph B.; Van Scyoc, III, John M.; Schlesinger, Tuviah E.

1997-06-24

A method of purifying nonmetallic, crystalline semiconducting materials useful for room temperature radiation detecting devices by applying an electric field across the material. The present invention discloses a simple technology for producing purified ionic semiconducting materials, in particular PbI.sub.2 and preferably HgI.sub.2, which produces high yields of purified product, requires minimal handling of the material thereby reducing the possibility of introducing or reintroducing impurities into the material, is easy to control, is highly selective for impurities, retains the stoichiometry of the material and employs neither high temperatures nor hazardous materials such as solvents or liquid metals. An electric field is applied to a bulk sample of the material causing impurities present in the sample to drift in a preferred direction. After all of the impurities have been transported to the ends of the sample the current flowing through the sample, a measure of the rate of transport of mobile impurities, falls to a low, steady state value, at which time the end sections of the sample where the impurities have concentrated are removed leaving a bulk sample of higher purity material. Because the method disclosed here only acts on the electrically active impurities, the stoichiometry of the host material remains substantially unaffected.

Expression and purification of human and Saccharomyces cerevisiae equilibrative nucleoside transporters.

PubMed

Boswell-Casteel, Rebba C; Johnson, Jennifer M; Roe-Žurž, Zygy; Duggan, Kelli D; Schmitz, Hannah; Hays, Franklin A

2018-02-01

Nucleosides play an essential role in the physiology of eukaryotes by acting as metabolic precursors in de novo nucleic acid synthesis and energy metabolism. Nucleosides also act as ligands for purinergic receptors. Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that aid in regulating plasmalemmal flux of purine and pyrimidine nucleosides and nucleobases. ENTs exhibit broad substrate selectivity across different isoforms and utilize diverse mechanisms to drive substrate flux across membranes. However, the molecular mechanisms and chemical determinants of ENT-mediated substrate recognition, binding, inhibition, and transport are poorly understood. To determine how ENT-mediated transport occurs at the molecular level, greater chemical insight and assays employing purified protein are essential. This article focuses on the expression and purification of human ENT1, human ENT2, and Saccharomyces cerevisiae ScENT1 using novel expression and purification strategies to isolate recombinant ENTs. ScENT1, hENT1, and hENT2 were expressed in W303 Saccharomyces cerevisiae cells and detergent solubilized from the membrane. After detergent extraction, these ENTs were further purified using immobilized metal affinity chromatography and size exclusion chromatography. This effort resulted in obtaining quantities of purified protein sufficient for future biophysical analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

Purification and characterization of an alginate lyase from marine Bacterium Vibrio sp. mutant strain 510-64.

PubMed

Hu, Xiaoke; Jiang, Xiaolu; Hwang, Huey-Min

2006-08-01

Marine Vibrio sp. 510 was chosen as a parent strain for screening high producers of alginate lyase using the complex mutagenesis of Ethyl Methanesulphonate and UV radiation treatments. The mutant strain Vibrio sp. 510-64 was selected and its alginate lyase activity was increased by 3.87-fold (reaching 46.12 EU/mg) over that of the parent strain. An extracellular alginate lyase was purified from Vibrio sp. 510-64 cultural supernatant by successive fractionation on DEAE Sepharose FF and two steps of Superdex 75. The purified enzyme yielded a single band on SDS-PAGE with the molecular weight of 34.6 kDa. Data of the N-terminal amino acid sequence indicated that this protein might be a novel alginate lyase. The substrate specificity results demonstrated that the alginate lyase had the specificity for poly G block.

Bacterial Reaction Centers Purified with Styrene Maleic Acid Copolymer Retain Native Membrane Functional Properties and Display Enhanced Stability**

PubMed Central

Swainsbury, David J K; Scheidelaar, Stefan; van Grondelle, Rienk; Killian, J Antoinette; Jones, Michael R

2014-01-01

Integral membrane proteins often present daunting challenges for biophysical characterization, a fundamental issue being how to select a surfactant that will optimally preserve the individual structure and functional properties of a given membrane protein. Bacterial reaction centers offer a rare opportunity to compare the properties of an integral membrane protein in different artificial lipid/surfactant environments with those in the native bilayer. Here, we demonstrate that reaction centers purified using a styrene maleic acid copolymer remain associated with a complement of native lipids and do not display the modified functional properties that typically result from detergent solubilization. Direct comparisons show that reaction centers are more stable in this copolymer/lipid environment than in a detergent micelle or even in the native membrane, suggesting a promising new route to exploitation of such photovoltaic integral membrane proteins in device applications. PMID:25212490

The metabolism of neuropeptides. Neurokinin A (substance K) is a substrate for endopeptidase-24.11 but not for peptidyl dipeptidase A (angiotensin-converting enzyme).

PubMed Central

Hooper, N M; Kenny, A J; Turner, A J

1985-01-01

Both endopeptidase-24.11 and peptidyl dipeptidase A have previously been shown to hydrolyse the neuropeptide substance P. The structurally related peptide neurokinin A is also shown to be hydrolysed by pig kidney endopeptidase-24.11. The identified products indicated hydrolysis at two sites, Ser5-Phe6 and Gly8-Leu9, consistent with the known specificity of the enzyme. The pattern of hydrolysis of neurokinin A by synaptic membranes prepared from pig striatum was similar to that observed with purified endopeptidase-24.11, and hydrolysis was substantially abolished by the selective inhibitor phosphoramidon. Peptidyl dipeptidase A purified from pig kidney was shown to hydrolyse substance P but not neurokinin A. It is concluded that endopeptidase-24.11 has the general capacity to hydrolyse and inactivate the family of tachykinin peptides, including substance P and neurokinin A. PMID:2998348

The metabolism of neuropeptides. Neurokinin A (substance K) is a substrate for endopeptidase-24.11 but not for peptidyl dipeptidase A (angiotensin-converting enzyme).

PubMed

Hooper, N M; Kenny, A J; Turner, A J

1985-10-15

Both endopeptidase-24.11 and peptidyl dipeptidase A have previously been shown to hydrolyse the neuropeptide substance P. The structurally related peptide neurokinin A is also shown to be hydrolysed by pig kidney endopeptidase-24.11. The identified products indicated hydrolysis at two sites, Ser5-Phe6 and Gly8-Leu9, consistent with the known specificity of the enzyme. The pattern of hydrolysis of neurokinin A by synaptic membranes prepared from pig striatum was similar to that observed with purified endopeptidase-24.11, and hydrolysis was substantially abolished by the selective inhibitor phosphoramidon. Peptidyl dipeptidase A purified from pig kidney was shown to hydrolyse substance P but not neurokinin A. It is concluded that endopeptidase-24.11 has the general capacity to hydrolyse and inactivate the family of tachykinin peptides, including substance P and neurokinin A.

Molecular Evolution of the Neural Crest Regulatory Network in Ray-Finned Fish

PubMed Central

Kratochwil, Claudius F.; Geissler, Laura; Irisarri, Iker; Meyer, Axel

2015-01-01

Abstract Gene regulatory networks (GRN) are central to developmental processes. They are composed of transcription factors and signaling molecules orchestrating gene expression modules that tightly regulate the development of organisms. The neural crest (NC) is a multipotent cell population that is considered a key innovation of vertebrates. Its derivatives contribute to shaping the astounding morphological diversity of jaws, teeth, head skeleton, or pigmentation. Here, we study the molecular evolution of the NC GRN by analyzing patterns of molecular divergence for a total of 36 genes in 16 species of bony fishes. Analyses of nonsynonymous to synonymous substitution rate ratios (dN/dS) support patterns of variable selective pressures among genes deployed at different stages of NC development, consistent with the developmental hourglass model. Model-based clustering techniques of sequence features support the notion of extreme conservation of NC-genes across the entire network. Our data show that most genes are under strong purifying selection that is maintained throughout ray-finned fish evolution. Late NC development genes reveal a pattern of increased constraints in more recent lineages. Additionally, seven of the NC-genes showed signs of relaxation of purifying selection in the famously species-rich lineage of cichlid fishes. This suggests that NC genes might have played a role in the adaptive radiation of cichlids by granting flexibility in the development of NC-derived traits—suggesting an important role for NC network architecture during the diversification in vertebrates. PMID:26475317

Stearoyl-acyl carrier protein desaturases are associated with floral isolation in sexually deceptive orchids

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

Schluter, P.M.; Shanklin, J.; Xu, S.

The orchids Ophrys sphegodes and O. exaltata are reproductively isolated from each other by the attraction of two different, highly specific pollinator species. For pollinator attraction, flowers chemically mimic the pollinators sex pheromones, the key components of which are alkenes with different double-bond positions. This study identifies genes likely involved in alkene biosynthesis, encoding stearoyl-acyl carrier protein (ACP) desaturase (SAD) homologs. The expression of two isoforms, SAD1 and SAD2, is flower-specific and broadly parallels alkene production during flower development. SAD2 shows a significant association with alkene production, and in vitro assays show that O. sphegodes SAD2 has activity both asmore » an 18:0-ACP {Delta}{sup 9} and a 16:0-ACP {Delta}{sup 4} desaturase. Downstream metabolism of the SAD2 reaction products would give rise to alkenes with double-bonds at position 9 or position 12, matching double-bond positions observed in alkenes in the odor bouquet of O. sphegodes. SAD1 and SAD2 show evidence of purifying selection before, and positive or relaxed purifying selection after gene duplication. By contributing to the production of species-specific alkene bouquets, SAD2 is suggested to contribute to differential pollinator attraction and reproductive isolation among these species. Taken together, these data are consistent with the hypothesis that SAD2 is a florally expressed barrier gene of large phenotypic effect and, possibly, a genic target of pollinator-mediated selection.« less

Stearoyl-acyl carrier protein desaturases are associated with floral isolation in sexually deceptive orchids

PubMed Central

Schlüter, Philipp M.; Xu, Shuqing; Gagliardini, Valeria; Whittle, Edward; Shanklin, John; Grossniklaus, Ueli; Schiestl, Florian P.

2011-01-01

The orchids Ophrys sphegodes and O. exaltata are reproductively isolated from each other by the attraction of two different, highly specific pollinator species. For pollinator attraction, flowers chemically mimic the pollinators’ sex pheromones, the key components of which are alkenes with different double-bond positions. This study identifies genes likely involved in alkene biosynthesis, encoding stearoyl-acyl carrier protein (ACP) desaturase (SAD) homologs. The expression of two isoforms, SAD1 and SAD2, is flower-specific and broadly parallels alkene production during flower development. SAD2 shows a significant association with alkene production, and in vitro assays show that O. sphegodes SAD2 has activity both as an 18:0-ACP Δ9 and a 16:0-ACP Δ4 desaturase. Downstream metabolism of the SAD2 reaction products would give rise to alkenes with double-bonds at position 9 or position 12, matching double-bond positions observed in alkenes in the odor bouquet of O. sphegodes. SAD1 and SAD2 show evidence of purifying selection before, and positive or relaxed purifying selection after gene duplication. By contributing to the production of species-specific alkene bouquets, SAD2 is suggested to contribute to differential pollinator attraction and reproductive isolation among these species. Taken together, these data are consistent with the hypothesis that SAD2 is a florally expressed barrier gene of large phenotypic effect and, possibly, a genic target of pollinator-mediated selection. PMID:21436056

Emergence and Evolution of Hominidae-Specific Coding and Noncoding Genomic Sequences

PubMed Central

Saber, Morteza Mahmoudi; Adeyemi Babarinde, Isaac; Hettiarachchi, Nilmini; Saitou, Naruya

2016-01-01

Family Hominidae, which includes humans and great apes, is recognized for unique complex social behavior and intellectual abilities. Despite the increasing genome data, however, the genomic origin of its phenotypic uniqueness has remained elusive. Clade-specific genes and highly conserved noncoding sequences (HCNSs) are among the high-potential evolutionary candidates involved in driving clade-specific characters and phenotypes. On this premise, we analyzed whole genome sequences along with gene orthology data retrieved from major DNA databases to find Hominidae-specific (HS) genes and HCNSs. We discovered that Down syndrome critical region 4 (DSCR4) is the only experimentally verified gene uniquely present in Hominidae. DSCR4 has no structural homology to any known protein and was inferred to have emerged in several steps through LTR/ERV1, LTR/ERVL retrotransposition, and transversion. Using the genomic distance as neutral evolution threshold, we identified 1,658 HS HCNSs. Polymorphism coverage and derived allele frequency analysis of HS HCNSs showed that these HCNSs are under purifying selection, indicating that they may harbor important functions. They are overrepresented in promoters/untranslated regions, in close proximity of genes involved in sensory perception of sound and developmental process, and also showed a significantly lower nucleosome occupancy probability. Interestingly, many ancestral sequences of the HS HCNSs showed very high evolutionary rates. This suggests that new functions emerged through some kind of positive selection, and then purifying selection started to operate to keep these functions. PMID:27289096

Purification of flavonoids from licorice using an off-line preparative two-dimensional normal-phase liquid chromatography/reversed-phase liquid chromatography method.

PubMed

Fan, Yunpeng; Fu, Yanhui; Fu, Qing; Cai, Jianfeng; Xin, Huaxia; Dai, Mei; Jin, Yu

2016-07-01

An orthogonal (71.9%) off-line preparative two-dimensional normal-phase liquid chromatography/reversed-phase liquid chromatography method coupled with effective sample pretreatment was developed for separation and purification of flavonoids from licorice. Most of the nonflavonoids were firstly removed using a self-made Click TE-Cys (60 μm) solid-phase extraction. In the first dimension, an industrial grade preparative chromatography was employed to purify the crude flavonoids. Click TE-Cys (10 μm) was selected as the stationary phase that provided an excellent separation with high reproducibility. Ethyl acetate/ethanol was selected as the mobile phase owing to their excellent solubility for flavonoids. Flavonoids co-eluted in the first dimension were selected for further purification using reversed-phase liquid chromatography. Multiple compounds could be isolated from one normal-phase fraction and some compounds with bad resolution in one-dimensional liquid chromatography could be prepared in this two-dimensional system owing to the orthogonal separation. Moreover, this two-dimensional liquid chromatography method was beneficial for the preparation of relatively trace flavonoid compounds, which were enriched in the first dimension and further purified in the second dimension. Totally, 24 flavonoid compounds with high purity were obtained. The results demonstrated that the off-line two-dimensional liquid chromatography method was effective for the preparative separation and purification of flavonoids from licorice. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Darwinian natural selection: its enduring explanatory power

PubMed Central

2012-01-01

Evolutionary theory has never had a stronger scientific foundation than it does today. In a short review I hope to portray the deep commitment of today's biologists to Darwinian natural selection and to discoveries made since Darwin's time. In spite of the scientific advances in the century and a half since the publication of On the Origin of Species, Darwin still remains the principal author of modern evolutionary theory. He is one of the greatest contributors of all time to our understanding of nature. PMID:22481845

Air Vehicle Integration and Technology Research (AVIATR). Delivery Order 0023: Predictive Capability for Hypersonic Structural Response and Life Prediction: Phase 2 - Detailed Design of Hypersonic Cruise Vehicle Hot-Structure

DTIC Science & Technology

2012-05-01

30 Figure 5.0.1 Phase II Analysis Process ...panel study the panel selection process followed a review of the outer skin environment investigated during the HTV-3X program which was suitable as...Subsequently, Panel 1B was down-selected from the screening process as it was observed to be subjected to stronger thermal field contributions due to fuel

Hand-held dynamometer testing of the internal and external rotator musculature based on selected positions to establish normative data and unilateral ratios.

PubMed

Riemann, Bryan L; Davies, George J; Ludwig, Lauren; Gardenhour, Helen

2010-12-01

Objective documentation is needed of shoulder internal and external rotator strength using hand-held dynamometry in selected positions commonly used in a clinic. We compared strength measures and unilateral ratios between gender, limbs (dominant, nondominant), and 3 testing positions. We hypothesized that men would be stronger than women, the dominant shoulder would be stronger than the nondominant shoulder, and the seated neutral (0° adduction) and seated 30° abduction, 30° scaption, 30° diagonal (30°-30°-30°) positions would be stronger than the prone at 90°-90° position. Three positions (prone at 90°, seated at neutral, and seated at 30°-30°-30°) were evaluated in 181 individuals using hand-held dynamometry. Three separate 3-factor (limb by position by gender) analyses of variance were conducted on internal rotation, external rotation, and unilateral ratios. Although the dominant limb was significantly stronger (P < .001) than the nondominant for internal rotation, there was no difference for external rotation. The external rotators demonstrated significantly greater strength in the prone at 90° position compared with the seated at neutral (P = .001) and seated at 30°-30°-30° (P = .002) positions. The internal rotators demonstrated significantly greater (P = .036) strength in the neutral position than in the prone at 90° position for the women. The unilateral ratio of external rotators/internal rotators ranged from 86% to 99%. For the women, the prone at 90° ratio was significantly greater than seated at neutral (P = .001) and seated at 30°-30°-30° (P = .001) positions. Moderate strength relationships (r = 0.506 to 0.572) were revealed between body mass and all strength measures. The results of this study provide evidence to interpret normative data, bilateral comparisons and unilateral ratios of the internal/external rotators in the 3 selected positions. Because there are no differences between the seated at neutral and 30°-30°-30° positions and the advantages it offers, we recommend the 30°-30°-30° position for testing and the initiation of rehabilitation. Copyright © 2010 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Mosby, Inc. All rights reserved.

Sexual selection predicts species richness across the animal kingdom.

PubMed

Janicke, Tim; Ritchie, Michael G; Morrow, Edward H; Marie-Orleach, Lucas

2018-05-16

Our improving knowledge of the animal tree of life consistently demonstrates that some taxa diversify more rapidly than others, but what contributes to this variation remains poorly understood. An influential hypothesis proposes that selection arising from competition for mating partners plays a key role in promoting speciation. However, empirical evidence showing a link between proxies of this sexual selection and species richness is equivocal. Here, we collected standardized metrics of sexual selection for a broad range of animal taxa, and found that taxonomic families characterized by stronger sexual selection on males show relatively higher species richness. Thus, our data support the hypothesis that sexual selection elevates species richness. This could occur either by promoting speciation and/or by protecting species against extinction. © 2018 The Author(s).

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

PubMed Central

Murphy, Patrick J. M.; Stone, Orrin J.; Anderson, Michelle E.

2011-01-01

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification. HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4. The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach. PMID:21968976

Importance of NADPH oxidase-mediated redox signaling in the detrimental effect of CRP on pancreatic insulin secretion.

PubMed

Chan, Pei-Chi; Wang, Ya-Chin; Chen, Yi-Ling; Hsu, Wan-Ning; Tian, Yu-Feng; Hsieh, Po-Shiuan

2017-11-01

Elevations in C-reactive protein (CRP) levels are positively correlated with the progress of type 2 diabetes mellitus. However, the effect of CRP on pancreatic insulin secretion is unknown. Here, we showed that purified human CRP impaired insulin secretion in isolated mouse islets and NIT-1 insulin-secreting cells in dose- and time-dependent manners. CRP increased NADPH oxidase-mediated ROS (reactive oxygen species) production, which simultaneously promoted the production of nitrotyrosine (an indicator of RNS, reactive nitrogen species) and TNFα, to diminish cell viability, insulin secretion in islets and insulin-secreting cells. These CRP-mediated detrimental effects on cell viability and insulin secretion were significantly reversed by adding NAC (a potent antioxidant), apocynin (a selective NADPH oxidase inhibitor), L-NAME (a non-selective nitric oxide synthase (NOS) inhibitor), aminoguanidine (a selective iNOS inhibitor), PDTC (a selective NFκB inhibitor) or Enbrel (an anti-TNFα fusion protein). However, CRP-induced ROS production failed to change after adding L-NAME, aminoguanidine or PDTC. In isolated islets and NIT-1 cells, the elevated nitrotyrosine contents by CRP pretreatment were significantly suppressed by adding L-NAME but not PDTC. Conversely, CRP-induced increases in TNF-α production were significantly reversed by administration of PDTC but not L-NAME. In addition, wild-type mice treated with purified human CRP showed significant decreases in the insulin secretion index (HOMA-β cells) and the insulin stimulation index in isolated islets that were reversed by the addition of L-NAME, aminoguanidine or NAC. It is suggested that CRP-activated NADPH-oxidase redox signaling triggers iNOS-mediated RNS and NFκB-mediated proinflammatory cytokine production to cause β cell damage in state of inflammation. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparative transcriptome resources of two Dysosma species (Berberidaceae) and molecular evolution of the CYP719A gene in Podophylloideae.

PubMed

Mao, Yunrui; Zhang, Yonghua; Xu, Chuan; Qiu, Yingxiong

2016-01-01

Dysosma species (Berberidaceae, Podophylloideae) are of great medicinal pharmacogenetic importance and used as model systems to study the drivers and mechanisms of species diversification of temperate plants in East Asia. Recently, we have sequenced the transcriptome of the low-elevation D. versipellis. In this study, we sequenced the transcriptome of the high-elevation D. aurantiocaulis and used comparative genomic approaches to investigate the transcriptome evolution of the two species. We retrieved 53,929 unigenes from D. aurantiocaulis by de novo transcriptome assemblies using the Illumina HiSeq 2000 platform. Comparing the transcriptomes of both species, we identified 4593 orthologs. Estimation of Ka/Ks ratios for 3126 orthologs revealed that none had a Ka/Ks significantly greater than 1, whereas 1273 (Ka/Ks < 0.5, P < 0.05) were inferred to be under purifying selection. A total of 51 primer pairs were successfully designed from 461 EST-SSRs contained in 4593 orthologs. Marker validation assay revealed that 26 (51%) and 41 (80.4%) produced clear fragments with the expected sizes in all Podophylloideae species. Specifically, 19 different sequences of CYP719A were identified from PCR-amplified genomic DNA of all 12 species of Podophylloideae using primers designed from the assembled transcripts. The data further indicated that CYP719A was likely subject to strong selective constraints maintaining only one copy per genome. In Dysosma, there was relaxed purifying selection or more positive selection for high-elevation species. Overall, this study has generated a wealth of molecular resources potentially useful for pharmacogenetic and evolutionary studies in Dysosma and allied taxa. © 2015 John Wiley & Sons Ltd.

Importance of the residue Asp 290 on chain length selectivity and catalytic efficiency of recombinant Staphylococcus simulans lipase expressed in E. coli.

PubMed

Sayari, Adel; Mosbah, Habib; Gargouri, Youssef

2007-05-01

In addition to their physiological importance, microbial lipases, like staphylococcal ones, are of considerable commercial interest for biotechnological applications such as detergents, food production, and pharmaceuticals and industrial synthesis of fine chemicals. The gene encoding the extracellular lipase of Staphylococcus simulans (SSL) was subcloned in the pET-14b expression vector and expressed in Esherichia coli BL21 (DE3). The wild-type SSL was expressed as amino terminal His6-tagged recombinant protein. One-step purification of the recombinant lipase was achieved with nickel metal affinity column. The purified His-tagged SSL (His6-SSL) is able to hydrolyse triacylglycerols without chain length selectivity. The major differences among lipases are reflected in their chemical specificity in the hydrolysis of peculiar ester bonds, and their respective capacity to hydrolyse substrates having different physico-chemical properties. It has been proposed, using homology alignment, that the region around the residue 290 of Staphylococcus hyicus lipase could be involved in the selection of the substrate. To evaluate the importance of this environment, the residue Asp290 of Staphylococcus simulans lipase was mutated to Ala using site-directed mutagenesis. The mutant expression plasmid was also overexpressed in Esherichia coli and purified with a nickel metal affinity column. The substitution of Asp290 by Ala was accompanied by a significant shift of the acyl-chain length specificity of the mutant towards short chain fatty acid esters. Kinetic studies of wild-type SSL and its mutant D290A were carried out, and show essentially that the catalytic efficiency (k cat /K M ) of the mutant was affected. Our results confirmed that Asp290 is important for the chain length selectivity and catalytic efficiency of Staphylococcus simulans lipase.

Analysis of Serine Codon Conservation Reveals Diverse Phenotypic Constraints on Hepatitis C Virus Glycoprotein Evolution

PubMed Central

Koutsoudakis, George; Urbanowicz, Richard A.; Mirza, Deeman; Ginkel, Corinne; Riebesehl, Nina; Calland, Noémie; Albecka, Anna; Price, Louisa; Hudson, Natalia; Descamps, Véronique; Backx, Matthijs; McClure, C. Patrick; Duverlie, Gilles; Pecheur, Eve-Isabelle; Dubuisson, Jean; Perez-del-Pulgar, Sofia; Forns, Xavier; Steinmann, Eike; Tarr, Alexander W.; Pietschmann, Thomas

2014-01-01

Serine is encoded by two divergent codon types, UCN and AGY, which are not interchangeable by a single nucleotide substitution. Switching between codon types therefore occurs via intermediates (threonine or cysteine) or via simultaneous tandem substitutions. Hepatitis C virus (HCV) chronically infects 2 to 3% of the global population. The highly variable glycoproteins E1 and E2 decorate the surface of the viral envelope, facilitate cellular entry, and are targets for host immunity. Comparative sequence analysis of globally sampled E1E2 genes, coupled with phylogenetic analysis, reveals the signatures of multiple archaic codon-switching events at seven highly conserved serine residues. Limited detection of intermediate phenotypes indicates that associated fitness costs restrict their fixation in divergent HCV lineages. Mutational pathways underlying codon switching were probed via reverse genetics, assessing glycoprotein functionality using multiple in vitro systems. These data demonstrate selection against intermediate phenotypes can act at the structural/functional level, with some intermediates displaying impaired virion assembly and/or decreased capacity for target cell entry. These effects act in residue/isolate-specific manner. Selection against intermediates is also provided by humoral targeting, with some intermediates exhibiting increased epitope exposure and enhanced neutralization sensitivity, despite maintaining a capacity for target cell entry. Thus, purifying selection against intermediates limits their frequencies in globally sampled strains, with divergent functional constraints at the protein level restricting the fixation of deleterious mutations. Overall our study provides an experimental framework for identification of barriers limiting viral substitutional evolution and indicates that serine codon-switching represents a genomic “fossil record” of historical purifying selection against E1E2 intermediate phenotypes. PMID:24173227

Topology of evolving, mutagenized viral populations: quasispecies expansion, compression, and operation of negative selection

PubMed Central

2008-01-01

Background The molecular events and evolutionary forces underlying lethal mutagenesis of virus (or virus extinction through an excess of mutations) are not well understood. Here we apply for the first time phylogenetic methods and Partition Analysis of Quasispecies (PAQ) to monitor genetic distances and intra-population structures of mutant spectra of foot-and-mouth disease virus (FMDV) quasispecies subjected to mutagenesis by base and nucleoside analogues. Results Phylogenetic and PAQ analyses have revealed a highly dynamic variation of intrapopulation diversity of FMDV quasispecies. The population diversity first suffers striking expansions in the presence of mutagens and then compressions either when the presence of the mutagenic analogue was discontinued or when a mutation that decreased sensitivity to a mutagen was selected. The pattern of mutations found in the populations was in agreement with the behavior of the corresponding nucleotide analogues with FMDV in vitro. Mutations accumulated at preferred genomic sites, and dn/ds ratios indicate the operation of negative (or purifying) selection in populations subjected to mutagenesis. No evidence of unusually elevated genetic distances has been obtained for FMDV populations approaching extinction. Conclusion Phylogenetic and PAQ analysis provide adequate procedures to describe the evolution of viral sequences subjected to lethal mutagenesis. These methods define the changes of intra-population structure more precisely than mutation frequencies and Shannon entropies. PAQ is very sensitive to variations of intrapopulation genetic distances. Strong negative (or purifying) selection operates in FMDV populations subjected to enhanced mutagenesis. The quantifications provide evidence that extinction does not imply unusual increases of intrapopulation complexity, in support of the lethal defection model of virus extinction. PMID:18637173

Modular Organization of Residue-Level Contacts Shapes the Selection Pressure on Individual Amino Acid Sites of Ribosomal Proteins.

PubMed

Mallik, Saurav; Kundu, Sudip

2017-04-01

Understanding the molecular evolution of macromolecular complexes in the light of their structure, assembly, and stability is of central importance. Here, we address how the modular organization of native molecular contacts shapes the selection pressure on individual residue sites of ribosomal complexes. The bacterial ribosomal complex is represented as a residue contact network where nodes represent amino acid/nucleotide residues and edges represent their van der Waals interactions. We find statistically overrepresented native amino acid-nucleotide contacts (OaantC, one amino acid contacts one or multiple nucleotides, internucleotide contacts are disregarded). Contact number is defined as the number of nucleotides contacted. Involvement of individual amino acids in OaantCs with smaller contact numbers is more random, whereas only a few amino acids significantly contribute to OaantCs with higher contact numbers. An investigation of structure, stability, and assembly of bacterial ribosome depicts the involvement of these OaantCs in diverse biophysical interactions stabilizing the complex, including high-affinity protein-RNA contacts, interprotein cooperativity, intersubunit bridge, packing of multiple ribosomal RNA domains, etc. Amino acid-nucleotide constituents of OaantCs with higher contact numbers are generally associated with significantly slower substitution rates compared with that of OaantCs with smaller contact numbers. This evolutionary rate heterogeneity emerges from the strong purifying selection pressure that conserves the respective amino acid physicochemical properties relevant to the stabilizing interaction with OaantC nucleotides. An analysis of relative molecular orientations of OaantC residues and their interaction energetics provides the biophysical ground of purifying selection conserving OaantC amino acid physicochemical properties. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Pollinator-mediated natural selection in Penstemon digitalis.

PubMed

Parachnowitsch, Amy; Kessler, André

2010-12-01

Measuring the agents of natural selection is important because it allows us to understand not only which traits are expected to evolve but also why they will evolve. Natural selection by pollinators on floral traits is often assumed because in outcrossing animal-pollinated plants flowers are generally thought to function as advertisements of rewards directed at pollinators. We tested the role of bee pollinators in selection on Penstemon digitalis and found that pollinators were driving selection for larger and more flowers. However, what makes our publication unique is the additional information we gained from reviewing the few other studies that also directly tested whether pollinators were agents of selection on floral traits. As we would expect if pollinators are important agents of selection, selection on floral traits was significantly stronger when pollinators were present than when they were experimentally removed. Taken together, these results suggest that pollinators can be important drivers of selection in contemporary populations.

Experimental mutation-accumulation on the X chromosome of Drosophila melanogaster reveals stronger selection on males than females.

PubMed

Mallet, Martin A; Bouchard, Jessica M; Kimber, Christopher M; Chippindale, Adam K

2011-06-06

Sex differences in the magnitude or direction of mutational effect may be important to a variety of population processes, shaping the mutation load and affecting the cost of sex itself. These differences are expected to be greatest after sexual maturity. Mutation-accumulation (MA) experiments provide the most direct way to examine the consequences of new mutations, but most studies have focused on juvenile viability without regard to sex, and on autosomes rather than sex chromosomes; both adult fitness and X-linkage have been little studied. We therefore investigated the effects of 50 generations of X-chromosome mutation accumulation on the fitness of males and females derived from an outbred population of Drosophila melanogaster. Fitness declined rapidly in both sexes as a result of MA, but adult males showed markedly greater fitness loss relative to their controls compared to females expressing identical genotypes, even when females were made homozygous for the X. We estimate that these mutations are partially additive (h ~ 0.3) in females. In addition, the majority of new mutations appear to harm both males and females. Our data helps fill a gap in our understanding of the consequences of sexual selection for genetic load, and suggests that stronger selection on males may indeed purge deleterious mutations affecting female fitness.

Retention properties of novel beta-CD bonded stationary phases in reversed-phase HPLC mode.

PubMed

Zhao, Yanyan; Guo, Zhimou; Zhang, Yongping; Xue, Xingya; Xu, Qing; Li, Xiuling; Liang, Xinmiao; Zhang, Yukui

2009-05-15

With the given special structures, the CD bonded stationary phases are expected to have complementary retention properties with conventional C18 stationary phase, which will be helpful to enhance the polar selectivity in RP mode separation. In this work, two beta-cyclodextrin (beta-CD) bonded stationary phases for reversed-phase HPLC, including 1, 12-dodecyldiol linked beta-CD stationary phase (CD1) and olio (ethylene glycol) (OEG) linked beta-CD stationary phase (CD2), have been synthesized via click chemistry. The resulting materials were characterized with FT-IR and elemental analysis, which proved the successful immobilization of ligands. The similarities and differences in retention characteristics between the CD and C18 stationary phases have been elucidated by using comparative linear solvation energy relationships (LSERs). The force related to solute McGowan volume has no significant difference, while the hydrogen bonding and dipolar interactions between solutes and CD stationary phases are stronger than between solutes and C18, which is attributed to the special structures (CD and triazole groups) of CD stationary phases. Chemical origins are interpreted by comparison between CD1 and CD2. Similar dispersive interactions of CD1 and CD2 are attributed to their similar length of spacer arms. CD2 which contains OEG spacer arm has relative weaker HBD acidity but stronger HBA basicity. CD stationary phases display no serious different methylene selectivity and higher polar selectivity than in the case of C18. Higher acid selectivity and lower basic selectivity are observed on CD2 than on CD1. Distinctive retention properties and good complementary separation selectivity to C18 make the novel CD bonded stationary phases available for more application in RPLC.

Recombinant production of enzymatically active male contraceptive drug target hTSSK2 - Localization of the TSKS domain phosphorylated by TSSK2.

PubMed

Shetty, Jagathpala; Sinville, Rondedrick; Shumilin, Igor A; Minor, Wladek; Zhang, Jianhai; Hawkinson, Jon E; Georg, Gunda I; Flickinger, Charles J; Herr, John C

2016-05-01

The testis-specific serine/threonine kinase 2 (TSSK2) has been proposed as a candidate male contraceptive target. Development of a selective inhibitor for this kinase first necessitates the production of highly purified, soluble human TSSK2 and its substrate, TSKS, with high yields and retention of biological activity for crystallography and compound screening. Strategies to produce full-length, soluble, biologically active hTSSK2 in baculovirus expression systems were tested and refined. Soluble preparations of TSSK2 were purified by immobilized-metal affinity chromatography (IMAC) followed by gel filtration chromatography. The biological activities of rec.hTSSK2 were verified by in vitro kinase and mobility shift assays using bacterially produced hTSKS (isoform 2), casein, glycogen synthase peptide (GS peptide) and various TSKS peptides as target substrates. Purified recombinant hTSSK2 showed robust kinase activity in the in vitro kinase assay by phosphorylating hTSKS isoform 2 and casein. The ATP Km values were similar for highly and partially purified fractions of hTSSK2 (2.2 and 2.7 μM, respectively). The broad spectrum kinase inhibitor staurosporine was a potent inhibitor of rec.hTSSK2 (IC50 = 20 nM). In vitro phosphorylation experiments carried out with TSKS (isoform 1) fragments revealed particularly strong phosphorylation of a recombinant N-terminal region representing aa 1-150 of TSKS, indicating that the N-terminus of human TSKS is phosphorylated by human TSSK2. Production of full-length enzymatically active recombinant TSSK2 kinase represents the achievement of a key benchmark for future discovery of TSSK inhibitors as male contraceptive agents. Copyright © 2016 Elsevier Inc. All rights reserved.

Safety and immunogenicity of mammalian cell derived and Modified Vaccinia Ankara vectored African swine fever subunit antigens in swine.

PubMed

Lopera-Madrid, Jaime; Osorio, Jorge E; He, Yongqun; Xiang, Zuoshuang; Adams, L Garry; Laughlin, Richard C; Mwangi, Waithaka; Subramanya, Sandesh; Neilan, John; Brake, David; Burrage, Thomas G; Brown, William Clay; Clavijo, Alfonso; Bounpheng, Mangkey A

2017-03-01

A reverse vaccinology system, Vaxign, was used to identify and select a subset of five African Swine Fever (ASF) antigens that were successfully purified from human embryonic kidney 293 (HEK) cells and produced in Modified vaccinia virus Ankara (MVA) viral vectors. Three HEK-purified antigens [B646L (p72), E183L (p54), and O61R (p12)], and three MVA-vectored antigens [B646L, EP153R, and EP402R (CD2v)] were evaluated using a prime-boost immunization regimen swine safety and immunogenicity study. Antibody responses were detected in pigs following prime-boost immunization four weeks apart with the HEK-293-purified p72, p54, and p12 antigens. Notably, sera from the vaccinees were positive by immunofluorescence on ASFV (Georgia 2007/1)-infected primary macrophages. Although MVA-vectored p72, CD2v, and EP153R failed to induce antibody responses, interferon-gamma (IFN-γ + ) spot forming cell responses against all three antigens were detected one week post-boost. The highest IFN-γ + spot forming cell responses were detected against p72 in pigs primed with MVA-p72 and boosted with the recombinant p72. Antigen-specific (p12, p72, CD2v, and EP153R) T-cell proliferative responses were also detected post-boost. Collectively, these results are the first demonstration that ASFV subunit antigens purified from mammalian cells or expressed in MVA vectors are safe and can induce ASFV-specific antibody and T-cell responses following a prime-boost immunization regimen in swine. Copyright © 2017 Elsevier B.V. All rights reserved.

The production and characterization of novel heavy-chain antibodies against the tandem repeat region of MUC1 mucin.

PubMed

Rahbarizadeh, Fatemeh; Rasaee, Mohammad J; Forouzandeh, Mehdi; Allameh, Abdolamir; Sarrami, Ramin; Nasiry, Habib; Sadeghizadeh, Majid

2005-01-01

Camelidae are known to produce immunoglobulins (Igs) devoid of light chains and constant heavy-chain domains (CH1). Antigen-specific fragments of these heavy-chain IgGs (VHH) are of great interest in biotechnology applications. This paper describes the first example of successfully raised heavy-chain antibodies in Camelus dromedarius (single-humped camel) and Camelus bactrianus (two-humped camel) against a MUC1 related peptide that is found to be an important epitope expressed in cancerous tissue. Camels were immunized against a synthetic peptide corresponding to the tandem repeat region of MUC1 mucin and cancerous tissue preparation obtained from patients suffering from breast carcinoma. Three IgG subclasses with different binding properties to protein A and G were purified by affinity chromatography. Both conventional and heavy-chain IgG antibodies were produced in response to MUC1-related peptide. The elicited antibodies could react specifically with the tandem repeat region of MUC1 mucin in an enzyme linked immunosorbant assay (ELISA). Anti-peptide antibodies were purified after passing antiserum over two affinity chromatography columns. Using ELISA, immunocytochemistry and Western blotting, the interaction of purified antibodies with different antigens was evaluated. The antibodies were observed to be selectively bound to antigens namely: MUC1 peptide (tandem repeat region), human milk fat globule membrane (HMFG), deglycosylated human milk fat globule membrane (D-HMFG), homogenized cancerous breast tissue and a native MUC1 purified from ascitic fluid. Ka values of specific polyclonal antipeptide antibodies were estimated in C. dromedarius and C. bactrianus, as 7 x 10(10) M(-1) and 1.4 x 10(10) M(-1) respectively.

Phosphatidylinositol anchor of HeLa cell alkaline phosphatase

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

Jemmerson, R.; Low, M.G.

1987-09-08

Alkaline phosphatase from cancer cells, HeLa TCRC-1, was biosynthetically labeled with either /sup 3/H-fatty acids or (/sup 3/H)ethanolamine as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material. Phosphatidylinositol-specific phospholipase C (PI-PLC) released a substantial proportion of the /sup 3/H-fatty acid label from immunoaffinity-purified alkaline phosphatase but had no effect on the radioactivity of (/sup 3/H)ethanolamine-labeled material. PI-PLC also liberated catalytically active alkaline phosphatase from viable cells, and this could be selectively blocked by monoclonal antibodies to alkaline phosphatase. However, the alkaline phosphatase released from /sup 3/H-fatty acid labeled cells by PI-PLC was not radioactive. By contrast,more » treatment with bromelain removed both the /sup 3/H-fatty acid and the (/sup 3/H)ethanolamine label from purified alkaline phosphatase. Subtilisin was also able to remove the (/sup 3/H)ethanolamine label from the purified alkaline phosphatase. The /sup 3/H radioactivity in alkaline phosphatase purified from (/sup 3/H)ethanolamine-labeled cells comigrated with authentic (/sup 3/H)ethanolamine by anion-exchange chromatography after acid hydrolysis. The data suggest that the /sup 3/H-fatty acid and (/sup 3/H)ethanolamine are covalently attached to the carboxyl-terminal segment since bromelain and subtilisin both release alkaline phosphatase from the membrane by cleavage at that end of the polypeptide chain. The data are consistent with findings for other proteins recently shown to be anchored in the membrane through a glycosylphosphatidylinositol structure and indicate that a similar structure contributes to the membrane anchoring of alkaline phosphatase.« less

Purification of Cardiomyocytes from Differentiating Pluripotent Stem Cells using Molecular Beacons Targeting Cardiomyocyte-Specific mRNA

PubMed Central

Kim, Sangsung; Park, Hun-Jun; Byun, Jaemin; Cho, Kyu-Won; Saafir, Talib; Song, Ming-Ke; Yu, Shan Ping; Wagner, Mary; Bao, Gang; Yoon, Young-Sup

2013-01-01

Background While methods for generating cardiomyocytes (CMs) from pluripotent stem cells (PSCs) have been reported, current methods produce heterogeneous mixtures of CMs and non-CM cells. Here, we report an entirely novel system in which PSC-derived CMs are purified by CM-specific molecular beacons (MBs). MBs are nano-scale probes that emit a fluorescence signal when hybridized to target mRNAs. Method and Results Five MBs targeting mRNAs of either cardiac troponin T or myosin heavy chain 6/7 were generated. Among five MBs, a MB targeting myosin heavy chain 6/7 mRNA (MHC1-MB) identified up to 99% of HL-1 CMs, a mouse CM cell line, but < 3% of four non-CM cell types in flow cytometry analysis, indicating that MHC1-MB is specific for identifying CMs. We delivered MHC1-MB into cardiomyogenically differentiated PSCs through nucleofection. The detection rate of CMs was similar to the percentages of cardiac troponin T (TNNT2) or cardiac troponin I (TNNI3)-positive CMs, supporting the specificity of MBs. Finally, MHC1-MB-positive cells were FACS-sorted from mouse and human PSC differentiating cultures and ~97% cells expressed TNNT2- or TNNI3 determined by flow cytometry. These MB-based sorted cells maintained their CM characteristics verified by spontaneous beating, electrophysiologic studies, and expression of cardiac proteins. When transplanted in a myocardial infarction model, MB-based purified CMs improved cardiac function and demonstrated significant engraftment for 4 weeks without forming tumors. Conclusions We developed a novel CM selection system that allows production of highly purified CMs. These purified CMs and this system can be valuable for cell therapy and drug discovery. PMID:23995537

Purification of cardiomyocytes from differentiating pluripotent stem cells using molecular beacons that target cardiomyocyte-specific mRNA.

PubMed

Ban, Kiwon; Wile, Brian; Kim, Sangsung; Park, Hun-Jun; Byun, Jaemin; Cho, Kyu-Won; Saafir, Talib; Song, Ming-Ke; Yu, Shan Ping; Wagner, Mary; Bao, Gang; Yoon, Young-Sup

2013-10-22

Although methods for generating cardiomyocytes from pluripotent stem cells have been reported, current methods produce heterogeneous mixtures of cardiomyocytes and noncardiomyocyte cells. Here, we report an entirely novel system in which pluripotent stem cell-derived cardiomyocytes are purified by cardiomyocyte-specific molecular beacons (MBs). MBs are nanoscale probes that emit a fluorescence signal when hybridized to target mRNAs. Five MBs targeting mRNAs of either cardiac troponin T or myosin heavy chain 6/7 were generated. Among 5 MBs, an MB that targeted myosin heavy chain 6/7 mRNA (MHC1-MB) identified up to 99% of HL-1 cardiomyocytes, a mouse cardiomyocyte cell line, but <3% of 4 noncardiomyocyte cell types in flow cytometry analysis, which indicates that MHC1-MB is specific for identifying cardiomyocytes. We delivered MHC1-MB into cardiomyogenically differentiated pluripotent stem cells through nucleofection. The detection rate of cardiomyocytes was similar to the percentages of cardiac troponin T- or cardiac troponin I-positive cardiomyocytes, which supports the specificity of MBs. Finally, MHC1-MB-positive cells were sorted by fluorescence-activated cell sorter from mouse and human pluripotent stem cell differentiating cultures, and ≈97% cells expressed cardiac troponin T or cardiac troponin I as determined by flow cytometry. These MB-based sorted cells maintained their cardiomyocyte characteristics, which was verified by spontaneous beating, electrophysiological studies, and expression of cardiac proteins. When transplanted in a myocardial infarction model, MB-based purified cardiomyocytes improved cardiac function and demonstrated significant engraftment for 4 weeks without forming tumors. We developed a novel cardiomyocyte selection system that allows production of highly purified cardiomyocytes. These purified cardiomyocytes and this system can be valuable for cell therapy and drug discovery.

Body Size, Fecundity, and Sexual Size Dimorphism in the Neotropical Cricket Macroanaxipha macilenta (Saussure) (Orthoptera: Gryllidae).

PubMed

Cueva Del Castillo, R

2015-04-01

Body size is directly or indirectly correlated with fitness. Body size, which conveys maximal fitness, often differs between sexes. Sexual size dimorphism (SSD) evolves because body size tends to be related to reproductive success through different pathways in males and females. In general, female insects are larger than males, suggesting that natural selection for high female fecundity could be stronger than sexual selection in males. I assessed the role of body size and fecundity in SSD in the Neotropical cricket Macroanaxipha macilenta (Saussure). This species shows a SSD bias toward males. Females did not present a correlation between number of eggs and body size. Nonetheless, there were fluctuations in the number of eggs carried by females during the sampling period, and the size of females that were collected carrying eggs was larger than that of females collected with no eggs. Since mating induces vitellogenesis in some cricket species, differences in female body size might suggest male mate choice. Sexual selection in the body size of males of M. macilenta may possibly be stronger than the selection of female fecundity. Even so, no mating behavior was observed during the field observations, including audible male calling or courtship songs, yet males may produce ultrasonic calls due to their size. If female body size in M. macilenta is not directly related to fecundity, the lack of a correlated response to selection on female body size could represent an alternate evolutionary pathway in the evolution of body size and SSD in insects.

Masting behaviour in a Mediterranean pine tree alters seed predator selection on reproductive output.

PubMed

Moreira, X; Abdala-Roberts, L; Zas, R; Merlo, E; Lombardero, M J; Sampedro, L; Mooney, K A

2016-11-01

Context-dependency in species interactions is widespread and can produce concomitant patterns of context-dependent selection. Masting (synchronous production of large seed crops at irregular intervals by a plant population) has been shown to reduce seed predation through satiation (reduction in rates of seed predation with increasing seed cone output) and thus represents an important source of context-dependency in plant-animal interactions. However, the evolutionary consequences of such dynamics are not well understood. Here we describe masting behaviour in a Mediterranean model pine species (Pinus pinaster) and present a test of the effects of masting on selection by seed predators on reproductive output. We predicted that masting, by enhancing seed predator satiation, could in turn strengthen positive selection by seed predators for larger cone output. For this we collected six-year data (spanning one mast year and five non-mast years) on seed cone production and seed cone predation rates in a forest genetic trial composed by 116 P. pinaster genotypes. Following our prediction, we found stronger seed predator satiation during the masting year, which in turn led to stronger seed predator selection for increased cone production relative to non-masting years. These findings provide evidence that masting can alter the evolutionary outcome of plant-seed predator interactions. More broadly, our findings highlight that changes in consumer responses to resource abundance represent a widespread mechanism for predicting and understanding context dependency in plant-consumer evolutionary dynamics. © 2016 German Botanical Society and The Royal Botanical Society of the Netherlands.

Molecular evolution and expression profile of the chemerine encoding gene RARRES2 in baboon and chimpanzee.

PubMed

González-Alvarez, Rafael; Garza-Rodríguez, María de Lourdes; Delgado-Enciso, Iván; Treviño-Alvarado, Víctor Manuel; Canales-Del-Castillo, Ricardo; Martínez-De-Villarreal, Laura Elia; Lugo-Trampe, Ángel; Tejero, María Elizabeth; Schlabritz-Loutsevitch, Natalia E; Rocha-Pizaña, María Del Refugio; Cole, Shelley A; Reséndez-Pérez, Diana; Moises-Alvarez, Mario; Comuzzie, Anthony G; Barrera-Saldaña, Hugo Alberto; Garza-Guajardo, Raquel; Barboza-Quintana, Oralia; Rodríguez-Sánchez, Irám Pablo

2015-06-12

Chemerin, encoded by the retinoic acid receptor responder 2 (RARRES2) gene is an adipocytesecreted protein with autocrine/paracrine functions in adipose tissue, metabolism and inflammation with a recently described function in vascular tone regulation, liver, steatosis, etc. This molecule is believed to represent a critical endocrine signal linking obesity to diabetes. There are no data available regarding evolution of RARRES2 in non-human primates and great apes. Expression profile and orthology in RARRES2 genes are unknown aspects in the biology of this multigene family in primates. Thus; we attempt to describe expression profile and phylogenetic relationship as complementary knowledge in the function of this gene in primates. To do that, we performed A RT-PCR from different tissues obtained during necropsies. Also we tested the hypotheses of positive evolution, purifying selection, and neutrality. And finally a phylogenetic analysis was made between primates RARRES2 protein. RARRES2 transcripts were present in liver, lung, adipose tissue, ovary, pancreas, heart, hypothalamus and pituitary tissues. Expression in kidney and leukocytes were not detectable in either species. It was determined that the studied genes are orthologous. RARRES2 evolution fits the hypothesis of purifying selection. Expression profiles of the RARRES2 gene are similar in baboons and chimpanzees and are also phylogenetically related.

Common spectrum of polypeptides occurs in secretion granule membranes of different exocrine glands

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

Cameron, R.S.; Cameron, P.L.; Castle, J.D.

1986-10-01

A highly purified membrane preparation from rat parotid secretion granules has been used as a comparative probe to examine the extent of compositional overlap in granule membranes of three other exocrine secretory tissues - pancreatic, lacrimal, and submandibular - from several standpoints. First, indirect immunofluorescent studies using a polyclonal polyspecific anti-parotid granule membrane antiserum has indicated a selective staining of granule membrane profiles in all acinar cells of all tissues. Second, highly purified granule membrane subfractions have been isolated from each exocrine tissue; comparative two-dimensional (isoelectric focusing; SDS) PAGE of radioiodinated granule membranes has identified 10-15 polypeptides of identical pImore » and apparent molecular mass. These species are likely to be integral membrane components since they are not extracted by either saponin-sodium sulfate or sodium carbonate (pH 11.5) treatments, and they do not have counterparts in the granule content. Finally, the identity among selected parotid and pancreatic radioiodinated granule membrane polypeptides has been documented using two-dimensional peptide mapping of chymotryptic and tryptic digests. These findings clearly indicate that exocrine secretory granules, irrespective of the nature of stored secretion, comprise a type of vesicular carrier with a common (and probably refined) membrane composition. Conceivably, the polypeptides identified carry out general functions related to exocrine secretion.« less

Recombinant expression of extracellular domain of mutant Epidermal Growth Factor Receptor in prokaryotic and baculovirus expression systems.

PubMed

Vettath, Sunitha Kodengil; Shivashankar, Gaganashree; Menon, Krishnakumar N; Vijayachandran, Lakshmi S

2018-04-15

Epidermal Growth Factor Receptor variant III (EGFRvIII) is a tumor specific antigen detected in various tumors including gliomas, breast cancer, lung cancer, head and neck squamous cell carcinoma (HNSCC). Screening of EGFRvIII targeting drug molecules can be accelerated by developing drug screening platforms using recombinantly expressed protein. Choice of expression system is one of the major factors deciding the success of recombinant expression of a protein. In our study, we have tried to express and purify the extracellular domain (ECD) of this highly unstable protein using bacterial and baculovirus expression systems to select the expression system suited for our purpose. Even though the protein was successfully expressed in prokaryotic system, purification could be done only under denaturing conditions. But in the baculovirus expression system, the protein was expressed in soluble form and could be purified under native conditions, with single step of purification. Based on our results, we conclude that insect cells are better choice over E. coli cells for expressing EGFRvIII ECD in soluble form. This study provides insights for other researchers involved in expression of similar unstable membrane proteins, on selecting the best expression system and challenges involved. Copyright © 2018 Elsevier B.V. All rights reserved.

Affinity-based precipitation via a bivalent peptidic hapten for the purification of monoclonal antibodies.

PubMed

Handlogten, Michael W; Stefanick, Jared F; Deak, Peter E; Bilgicer, Basar

2014-09-07

In a previous study, we demonstrated a non-chromatographic affinity-based precipitation method, using trivalent haptens, for the purification of mAbs. In this study, we significantly improved this process by using a simplified bivalent peptidic hapten (BPH) design, which enables facile and rapid purification of mAbs while overcoming the limitations of the previous trivalent design. The improved affinity-based precipitation method (ABP(BPH)) combines the simplicity of salt-induced precipitation with the selectivity of affinity chromatography for the purification of mAbs. The ABP(BPH) method involves 3 steps: (i) precipitation and separation of protein contaminants larger than immunoglobulins with ammonium sulfate; (ii) selective precipitation of the target-antibody via BPH by inducing antibody-complex formation; (iii) solubilization of the antibody pellet and removal of BPH with membrane filtration resulting in the pure antibody. The ABP(BPH) method was evaluated by purifying the pharmaceutical antibody trastuzumab from common contaminants including CHO cell conditioned media, DNA, ascites fluid, other antibodies, and denatured antibody with >85% yield and >97% purity. Importantly, the purified antibody demonstrated native binding activity to cell lines expressing the target protein, HER2. Combined, the ABP(BPH) method is a rapid and scalable process for the purification of antibodies with the potential to improve product quality while decreasing purification costs.

Differential Selectivity of the Escherichia coli Cell Membrane Shifts the Equilibrium for the Enzyme-Catalyzed Isomerization of Galactose to Tagatose▿

PubMed Central

Kim, Jin-Ha; Lim, Byung-Chul; Yeom, Soo-Jin; Kim, Yeong-Su; Kim, Hye-Jung; Lee, Jung-Kul; Lee, Sook-Hee; Kim, Seon-Won; Oh, Deok-Kun

2008-01-01

An Escherichia coli galactose kinase gene knockout (ΔgalK) strain, which contains the l-arabinose isomerase gene (araA) to isomerize d-galactose to d-tagatose, showed a high conversion yield of tagatose compared with the original galK strain because galactose was not metabolized by endogenous galactose kinase. In whole cells of the ΔgalK strain, the isomerase-catalyzed reaction exhibited an equilibrium shift toward tagatose, producing a tagatose fraction of 68% at 37°C, whereas the purified l-arabinose isomerase gave a tagatose equilibrium fraction of 36%. These equilibrium fractions are close to those predicted from the measured equilibrium constants of the isomerization reaction catalyzed in whole cells and by the purified enzyme. The equilibrium shift in these cells resulted from the higher uptake and lower release rates for galactose, which is a common sugar substrate, than for tagatose, which is a rare sugar product. A ΔmglB mutant had decreased uptake rates for galactose and tagatose, indicating that a methylgalactoside transport system, MglABC, is the primary contributing transporter for the sugars. In the present study, whole-cell conversion using differential selectivity of the cell membrane was proposed as a method for shifting the equilibrium in sugar isomerization reactions. PMID:18263746

Differential selectivity of the Escherichia coli cell membrane shifts the equilibrium for the enzyme-catalyzed isomerization of galactose to tagatose.

PubMed

Kim, Jin-Ha; Lim, Byung-Chul; Yeom, Soo-Jin; Kim, Yeong-Su; Kim, Hye-Jung; Lee, Jung-Kul; Lee, Sook-Hee; Kim, Seon-Won; Oh, Deok-Kun

2008-04-01

An Escherichia coli galactose kinase gene knockout (DeltagalK) strain, which contains the l-arabinose isomerase gene (araA) to isomerize d-galactose to d-tagatose, showed a high conversion yield of tagatose compared with the original galK strain because galactose was not metabolized by endogenous galactose kinase. In whole cells of the DeltagalK strain, the isomerase-catalyzed reaction exhibited an equilibrium shift toward tagatose, producing a tagatose fraction of 68% at 37 degrees C, whereas the purified l-arabinose isomerase gave a tagatose equilibrium fraction of 36%. These equilibrium fractions are close to those predicted from the measured equilibrium constants of the isomerization reaction catalyzed in whole cells and by the purified enzyme. The equilibrium shift in these cells resulted from the higher uptake and lower release rates for galactose, which is a common sugar substrate, than for tagatose, which is a rare sugar product. A DeltamglB mutant had decreased uptake rates for galactose and tagatose, indicating that a methylgalactoside transport system, MglABC, is the primary contributing transporter for the sugars. In the present study, whole-cell conversion using differential selectivity of the cell membrane was proposed as a method for shifting the equilibrium in sugar isomerization reactions.

Antifungal Activity of Eupolauridine and Its Action on DNA Topoisomerases

PubMed Central

Khan, Shabana I.; Nimrod, Alison C.; Mehrpooya, Mohammed; Nitiss, John L.; Walker, Larry A.; Clark, Alice M.

2002-01-01

The azafluoranthene alkaloid eupolauridine has previously been shown to have in vitro antifungal activity and selective inhibition of fungal topoisomerase I. The present study was undertaken to examine further its selectivity and mode of action. Eupolauridine completely inhibits the DNA relaxation activity of purified fungal topoisomerase I at 50 μg/ml, but it does not stabilize the cleavage complex of either human or fungal topoisomerase I. Cleavage complex stabilization is the mode of action of topoisomerase I targeting drugs of the camptothecin family. Also, unlike camptothecin, eupolauridine does not cause significant cytotoxicity in mammalian cells. To determine if the inhibition of topoisomerase I is the principal mode of antifungal action of eupolauridine, Saccharomyces cerevisiae strains with alterations in topoisomerase genes were used in clonogenic assays. The antifungal activity of eupolauridine was not diminished in the absence of topoisomerase I; rather, the cells lacking the enzyme were more sensitive to the drug. Cell-killing activity of eupolauridine was also more pronounced in cells that overexpressed topoisomerase II. In vitro assays with the purified yeast enzyme confirmed that eupolauridine stabilized topoisomerase II covalent complexes. These results indicate that a major target for fungal cell killing by eupolauridine is DNA topoisomerase II rather than topoisomerase I, but does not exclude the possibility that the drug also acts against other targets. PMID:12019091

Antifungal activity of eupolauridine and its action on DNA topoisomerases.

PubMed

Khan, Shabana I; Nimrod, Alison C; Mehrpooya, Mohammed; Nitiss, John L; Walker, Larry A; Clark, Alice M

2002-06-01

The azafluoranthene alkaloid eupolauridine has previously been shown to have in vitro antifungal activity and selective inhibition of fungal topoisomerase I. The present study was undertaken to examine further its selectivity and mode of action. Eupolauridine completely inhibits the DNA relaxation activity of purified fungal topoisomerase I at 50 microg/ml, but it does not stabilize the cleavage complex of either human or fungal topoisomerase I. Cleavage complex stabilization is the mode of action of topoisomerase I targeting drugs of the camptothecin family. Also, unlike camptothecin, eupolauridine does not cause significant cytotoxicity in mammalian cells. To determine if the inhibition of topoisomerase I is the principal mode of antifungal action of eupolauridine, Saccharomyces cerevisiae strains with alterations in topoisomerase genes were used in clonogenic assays. The antifungal activity of eupolauridine was not diminished in the absence of topoisomerase I; rather, the cells lacking the enzyme were more sensitive to the drug. Cell-killing activity of eupolauridine was also more pronounced in cells that overexpressed topoisomerase II. In vitro assays with the purified yeast enzyme confirmed that eupolauridine stabilized topoisomerase II covalent complexes. These results indicate that a major target for fungal cell killing by eupolauridine is DNA topoisomerase II rather than topoisomerase I, but does not exclude the possibility that the drug also acts against other targets.

The alpha glycerophosphate cycle in Drosophila melanogaster VI. structure and evolution of enzyme paralogs in the genus Drosophila.

PubMed

Carmon, Amber; MacIntyre, Ross

2010-01-01

The genome sequences of 12 Drosophila species contain 3 paralogs for alpha glycerophosphate dehydrogenase (GPDH) and for the mitochondrial alpha glycerophosphate oxidase (GPO). These 2 enzymes participate in the alpha glycerophosphate cycle in the adult thoracic flight muscles. The flight muscle enzymes are encoded by gpdh-1 at 26A2 and gpo-1 at 52C8. In this paper, we show that the GPDH paralogs share the same evolutionarily conserved functional domains and most intron positions, whereas the GPO paralogs share only some of the functional domains of mitochondrial oxidoreductases. The GPO paralogs not expressed in the flight muscles essentially lack introns. GPDH paralogs encoded by gpdh-2 and gpdh-3 and the GPO paralogs encoded by gpo-2 and gpo-3 are expressed only in the testes. Gene trees for the GPDH and GPO paralogs indicate that the genes expressed in the flight muscles are evolving very slowly presumably under strong purifying selection whereas the paralogs expressed in the testes are evolving more rapidly. The concordance between species and gene trees, d(N)/d(S) ratios, phylogenetic analysis by maximum likelihood-based tests, and analyses of radical and conservative substitutions all indicate that the additional GPDH and GPO paralogs are also evolving under purifying selection.

Increased fire frequency promotes stronger spatial genetic structure and natural selection at regional and local scales in Pinus halepensis Mill.

PubMed

Budde, Katharina B; González-Martínez, Santiago C; Navascués, Miguel; Burgarella, Concetta; Mosca, Elena; Lorenzo, Zaida; Zabal-Aguirre, Mario; Vendramin, Giovanni G; Verdú, Miguel; Pausas, Juli G; Heuertz, Myriam

2017-04-01

The recurrence of wildfires is predicted to increase due to global climate change, resulting in severe impacts on biodiversity and ecosystem functioning. Recurrent fires can drive plant adaptation and reduce genetic diversity; however, the underlying population genetic processes have not been studied in detail. In this study, the neutral and adaptive evolutionary effects of contrasting fire regimes were examined in the keystone tree species Pinus halepensis Mill. (Aleppo pine), a fire-adapted conifer. The genetic diversity, demographic history and spatial genetic structure were assessed at local (within-population) and regional scales for populations exposed to different crown fire frequencies. Eight natural P. halepensis stands were sampled in the east of the Iberian Peninsula, five of them in a region exposed to frequent crown fires (HiFi) and three of them in an adjacent region with a low frequency of crown fires (LoFi). Samples were genotyped at nine neutral simple sequence repeats (SSRs) and at 251 single nucleotide polymorphisms (SNPs) from coding regions, some of them potentially important for fire adaptation. Fire regime had no effects on genetic diversity or demographic history. Three high-differentiation outlier SNPs were identified between HiFi and LoFi stands, suggesting fire-related selection at the regional scale. At the local scale, fine-scale spatial genetic structure (SGS) was overall weak as expected for a wind-pollinated and wind-dispersed tree species. HiFi stands displayed a stronger SGS than LoFi stands at SNPs, which probably reflected the simultaneous post-fire recruitment of co-dispersed related seeds. SNPs with exceptionally strong SGS, a proxy for microenvironmental selection, were only reliably identified under the HiFi regime. An increasing fire frequency as predicted due to global change can promote increased SGS with stronger family structures and alter natural selection in P. halepensis and in plants with similar life history traits. © The Author 2017. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Landscape genomics: natural selection drives the evolution of mitogenome in penguins.

PubMed

Ramos, Barbara; González-Acuña, Daniel; Loyola, David E; Johnson, Warren E; Parker, Patricia G; Massaro, Melanie; Dantas, Gisele P M; Miranda, Marcelo D; Vianna, Juliana A

2018-01-16

Mitochondria play a key role in the balance of energy and heat production, and therefore the mitochondrial genome is under natural selection by environmental temperature and food availability, since starvation can generate more efficient coupling of energy production. However, selection over mitochondrial DNA (mtDNA) genes has usually been evaluated at the population level. We sequenced by NGS 12 mitogenomes and with four published genomes, assessed genetic variation in ten penguin species distributed from the equator to Antarctica. Signatures of selection of 13 mitochondrial protein-coding genes were evaluated by comparing among species within and among genera (Spheniscus, Pygoscelis, Eudyptula, Eudyptes and Aptenodytes). The genetic data were correlated with environmental data obtained through remote sensing (sea surface temperature [SST], chlorophyll levels [Chl] and a combination of SST and Chl [COM]) through the distribution of these species. We identified the complete mtDNA genomes of several penguin species, including ND6 and 8 tRNAs on the light strand and 12 protein coding genes, 14 tRNAs and two rRNAs positioned on the heavy strand. The highest diversity was found in NADH dehydrogenase genes and the lowest in COX genes. The lowest evolutionary divergence among species was between Humboldt (Spheniscus humboldti) and Galapagos (S. mendiculus) penguins (0.004), while the highest was observed between little penguin (Eudyptula minor) and Adélie penguin (Pygoscelis adeliae) (0.097). We identified a signature of purifying selection (Ka/Ks < 1) across the mitochondrial genome, which is consistent with the hypothesis that purifying selection is constraining mitogenome evolution to maintain Oxidative phosphorylation (OXPHOS) proteins and functionality. Pairwise species maximum-likelihood analyses of selection at codon sites suggest positive selection has occurred on ATP8 (Fixed-Effects Likelihood, FEL) and ND4 (Single Likelihood Ancestral Counting, SLAC) in all penguins. In contrast, COX1 had a signature of strong negative selection. ND4 Ka/Ks ratios were highly correlated with SST (Mantel, p-value: 0.0001; GLM, p-value: 0.00001) and thus may be related to climate adaptation throughout penguin speciation. These results identify mtDNA candidate genes under selection which could be involved in broad-scale adaptations of penguins to their environment. Such knowledge may be particularly useful for developing predictive models of how these species may respond to severe climatic changes in the future.

The Early Childhood Community Gives Back: Exchange Center Makeover Project

ERIC Educational Resources Information Center

Exchange: The Early Childhood Leaders' Magazine Since 1978, 2010

2010-01-01

The early childhood community gives back. With the extreme generosity of the "Exchange" Makeover Project partners, Jewel's Learning Center, selected as the winner of the Center Makeover, will be awarded with new and innovative tools to help build an even stronger educational foundation for the children attending the center. Included will be new…

Family Roles and Work Values: Processes of Selection and Change

ERIC Educational Resources Information Center

Kirkpatrick Johnson, Monica

2005-01-01

This study focuses on whether marriage and parenthood influence work values after taking into account the influence of work values on family formation. In a recent panel of young adults (N=709), stronger extrinsic and weaker intrinsic work values during adolescence predicted marriage and parenthood 9 years out of high school. Controlling these…

English Proficiency and Behavioral Change in Spanish-Speaking Children.

ERIC Educational Resources Information Center

Cordova, Joe E.

The purpose of this study was to improve the English competence of Spanish-speaking students, to increase their success in school, and to help them develop stronger self-concepts. Control and experimental groups of students were selected from kindergarten, second, and fourth grades of elementary schools with a predominantly Chicano population.…

Stronger Bonds: Six Building Blocks for a Better Alumni Giving Program

ERIC Educational Resources Information Center

Skari, Lisa; Ullman, Ellen

2012-01-01

Community colleges for a long time have operated under the misguided assumption that they should not waste time or resources prospecting and cultivating their alumni in hopes of generating philanthropic support. How could two-year colleges possibly compete with the success of alumni giving at four-year universities--with their selective admissions…

Immunogenicity and protective efficacy of recombinant iron superoxide dismutase protein from Bordetella pertussis in mice models.

PubMed

Yılmaz, Çiğdem; Apak, Aycan; Özcengiz, Erkan; Özcengiz, Gülay

2016-11-01

Whooping cough (pertussis) is a highly contagious respiratory infection caused by Bordetella pertussis. Although availability of effective pertussis vaccines reportedly decreases the incidence of the disease, B. pertussis circulation in populations has not been eliminated. Thus, it is necessary to find new protein candidates with greater immune protective capacities than the currently available acellular pertussis vaccines. In this study, iron superoxide dismutase (FeSOD) gene (sodB) was cloned, expressed in Escherichia coli and recombinant FeSOD protein thence purified. The recombinant protein (rFeSOD) was formulated with aluminum hydroxide (Alum) or monophosphoryl lipid A (MPLA) and injected intraperitoneally to immunize mice, after which IgG1, IgG2a and IFN-γ titers were measured to assess humoral and cellular responses, respectively, to these immunizations. The extent of bacterial colonization in lungs of intranasally challenged mice was determined 5, 8 and 14 days post-challenge. IgG1 and IgG2a responses were significantly stronger in mice that had been immunized with rFeSOD-MPLA than in those that had received rFeSOD-Alum (P < 0.05). Additionally, IgG2a titers were higher in mice vaccinated with recombinant protein FeSOD (rFeSOD) formulated with MPLA, especially after the second immunization. Immunization with rFeSOD-MPLA also provided a modest, but significant decrease in bacterial counts in lungs of mice (P < 0.05). Antigen specific-IFN-γ responses were significantly stronger in the group vaccinated with rFeSOD-MPLA, which could account for the lower bacterial counts. These findings suggest that rFeSOD protein formulated with MPLA has potential as an acellular pertussis vaccine candidate component. © 2016 The Societies and John Wiley & Sons Australia, Ltd.

Induction of a specific strong polyantigenic cellular immune response after short-term chemotherapy controls bacillary reactivation in murine and guinea pig experimental models of tuberculosis.

PubMed

Guirado, Evelyn; Gil, Olga; Cáceres, Neus; Singh, Mahavir; Vilaplana, Cristina; Cardona, Pere-Joan

2008-08-01

RUTI is a therapeutic vaccine that is generated from detoxified and liposomed Mycobacterium tuberculosis cell fragments that has demonstrated its efficacy in the control of bacillus reactivation after short-term chemotherapy. The aim of this study was to characterize the cellular immune response generated after the therapeutic administration of RUTI and to corroborate the lack of toxicity of the vaccine. Mouse and guinea pig experimental models were infected with a low-dose M. tuberculosis aerosol. RUTI-treated animals showed the lowest bacillary load in both experimental models. RUTI also decreased the percentage of pulmonary granulomatous infiltration in the mouse and guinea pig models. This was not the case after Mycobacterium bovis BCG treatment. Cellular immunity was studied through the characterization of the intracellular gamma interferon (IFN-gamma)-producing cells after the splenocytes' stimulation with M. tuberculosis-specific structural and growth-related antigens. Our data show that the difference between the therapeutic administration of BCG and RUTI resides mainly in the stronger activation of IFN-gamma(+) CD4(+) cells and CD8(+) cells against tuberculin purified protein derivative, ESAT-6, and Ag85B that RUTI generates. Both vaccines also triggered a specific immune response against the M. tuberculosis structural antigens Ag16kDa and Ag38kDa and a marked mRNA expression of IFN-gamma, tumor necrosis factor, interleukin-12, inducible nitric oxide synthase, and RANTES in the lung. The results show that RUTI's therapeutic effect is linked not only to the induction of a Th1 response but also to the stimulation of a quicker and stronger specific immunity against structural and growth-related antigens that reduces both the bacillary load and the pulmonary pathology.

Induction of a Specific Strong Polyantigenic Cellular Immune Response after Short-Term Chemotherapy Controls Bacillary Reactivation in Murine and Guinea Pig Experimental Models of Tuberculosis▿

PubMed Central

Guirado, Evelyn; Gil, Olga; Cáceres, Neus; Singh, Mahavir; Vilaplana, Cristina; Cardona, Pere-Joan

2008-01-01

RUTI is a therapeutic vaccine that is generated from detoxified and liposomed Mycobacterium tuberculosis cell fragments that has demonstrated its efficacy in the control of bacillus reactivation after short-term chemotherapy. The aim of this study was to characterize the cellular immune response generated after the therapeutic administration of RUTI and to corroborate the lack of toxicity of the vaccine. Mouse and guinea pig experimental models were infected with a low-dose M. tuberculosis aerosol. RUTI-treated animals showed the lowest bacillary load in both experimental models. RUTI also decreased the percentage of pulmonary granulomatous infiltration in the mouse and guinea pig models. This was not the case after Mycobacterium bovis BCG treatment. Cellular immunity was studied through the characterization of the intracellular gamma interferon (IFN-γ)-producing cells after the splenocytes' stimulation with M. tuberculosis-specific structural and growth-related antigens. Our data show that the difference between the therapeutic administration of BCG and RUTI resides mainly in the stronger activation of IFN-γ+ CD4+ cells and CD8+ cells against tuberculin purified protein derivative, ESAT-6, and Ag85B that RUTI generates. Both vaccines also triggered a specific immune response against the M. tuberculosis structural antigens Ag16kDa and Ag38kDa and a marked mRNA expression of IFN-γ, tumor necrosis factor, interleukin-12, inducible nitric oxide synthase, and RANTES in the lung. The results show that RUTI's therapeutic effect is linked not only to the induction of a Th1 response but also to the stimulation of a quicker and stronger specific immunity against structural and growth-related antigens that reduces both the bacillary load and the pulmonary pathology. PMID:18524883

The merit of meritocracy.

PubMed

Son Hing, Leanne S; Bobocel, D Ramona; Zanna, Mark P; Garcia, Donna M; Gee, Stephanie S; Orazietti, Katie

2011-09-01

We argue that the preference for the merit principle is a separate construct from hierarchy-legitimizing ideologies (i.e., system justification beliefs, prejudice, social dominance orientation), including descriptive beliefs that meritocracy currently exists in society. Moreover, we hypothesized that prescriptive beliefs about merit should have a stronger influence on reactions to the status quo when hierarchy-legitimizing ideologies are weak (vs. strong). In 4 studies, participants' preference for the merit principle and hierarchy-legitimizing ideologies were assessed; later, the participants evaluated organizational selection practices that support or challenge the status quo. Participants' prescriptive and descriptive beliefs about merit were separate constructs; only the latter predicted other hierarchy-legitimizing ideologies. In addition, as hypothesized, among participants who weakly endorsed hierarchy-legitimizing ideologies, the stronger their preference for the merit principle, the more they opposed selection practices that were perceived to be merit violating but the more they supported practices that were perceived to be merit restoring. In contrast, those who strongly endorsed hierarchy-legitimizing ideologies were always motivated to support the status quo, regardless of their preference for the merit principle. PsycINFO Database Record (c) 2011 APA, all rights reserved.

Genotypic diversity effects on the performance of Taraxacum officinale populations increase with time and environmental favorability.

PubMed

Drummond, Emily B M; Vellend, Mark

2012-01-01

Within-population genetic diversity influences many ecological processes, but few studies have examined how environmental conditions may impact these short-term diversity effects. Over four growing seasons, we followed experimental populations of a clonal, ubiquitous weed, Taraxacum officinale, with different numbers of genotypes in relatively favorable fallow field and unfavorable mowed lawn environmental treatments. Population performance (measured as total leaf area, seed production or biomass) clearly and consistently increased with diversity, and this effect became stronger over the course of the experiment. Diversity effects were stronger, and with different underlying mechanisms, in the fallow field versus the mowed lawn. Large genotypes dominated in the fallow field driving overyielding (via positive selection effects), whereas in the mowed lawn, where performance was limited by regular disturbance, there was evidence for complementarity among genotypes (with one compact genotype in particular performing better in mixture than monoculture). Hence, we predict stronger genotypic diversity effects in environments where intense intraspecific competition enhances genotypic differences. Our four-year field experiment plus seedling establishment trials indicate that genotypic diversity effects have far-reaching and context-dependent consequences across generations.

Sexually selected females in the monogamous Western Australian seahorse.

PubMed

Kvarnemo, Charlotta; Moore, Glenn I; Jones, Adam G

2007-02-22

Studies of sexual selection in monogamous species have hitherto focused on sexual selection among males. Here, we provide empirical documentation that sexual selection can also act strongly on females in a natural population with a monogamous mating system. In our field-based genetic study of the monogamous Western Australian seahorse, Hippocampus subelongatus, sexual selection differentials and gradients show that females are under stronger sexual selection than males: mated females are larger than unmated ones, whereas mated and unmated males do not differ in size. In addition, the opportunity for sexual selection (variance in mating success divided by its mean squared) for females is almost three times that for males. These results, which seem to be generated by a combination of a male preference for larger females and a female-biased adult sex ratio, indicate that substantial sexual selection on females is a potentially important but under-appreciated evolutionary phenomenon in monogamous species.

Sexually selected females in the monogamous Western Australian seahorse

PubMed Central

Kvarnemo, Charlotta; Moore, Glenn I; Jones, Adam G

2006-01-01

Studies of sexual selection in monogamous species have hitherto focused on sexual selection among males. Here, we provide empirical documentation that sexual selection can also act strongly on females in a natural population with a monogamous mating system. In our field-based genetic study of the monogamous Western Australian seahorse, Hippocampus subelongatus, sexual selection differentials and gradients show that females are under stronger sexual selection than males: mated females are larger than unmated ones, whereas mated and unmated males do not differ in size. In addition, the opportunity for sexual selection (variance in mating success divided by its mean squared) for females is almost three times that for males. These results, which seem to be generated by a combination of a male preference for larger females and a female-biased adult sex ratio, indicate that substantial sexual selection on females is a potentially important but under-appreciated evolutionary phenomenon in monogamous species. PMID:17476772

Sex-linked genomic variation and its relationship to avian plumage dichromatism and sexual selection.

PubMed

Huang, Huateng; Rabosky, Daniel L

2015-09-16

Sexual dichromatism is the tendency for sexes to differ in color pattern and represents a striking form of within-species morphological variation. Conspicuous intersexual differences in avian plumage are generally thought to result from Darwinian sexual selection, to the extent that dichromatism is often treated as a surrogate for the intensity of sexual selection in phylogenetic comparative studies. Intense sexual selection is predicted to leave a footprint on genetic evolution by reducing the relative genetic diversity on sex chromosome to that on the autosomes. In this study, we test the association between plumage dichromatism and sex-linked genetic diversity using eight species pairs with contrasting levels of dichromatism. We estimated Z-linked and autosomal genetic diversity for these non-model avian species using restriction-site associated (RAD) loci that covered ~3 % of the genome. We find that monochromatic birds consistently have reduced sex-linked genomic variation relative to phylogenetically-paired dichromatic species and this pattern is robust to mutational biases. Our results are consistent with several interpretations. If present-day sexual selection is stronger in dichromatic birds, our results suggest that its impact on sex-linked genomic variation is offset by other processes that lead to proportionately lower Z-linked variation in monochromatic species. We discuss possible factors that may contribute to this discrepancy between phenotypes and genomic variation. Conversely, it is possible that present-day sexual selection -- as measured by the variance in male reproductive success -- is stronger in the set of monochromatic taxa we have examined, potentially reflecting the importance of song, behavior and other non-plumage associated traits as targets of sexual selection. This counterintuitive finding suggests that the relationship between genomic variation and sexual selection is complex and highlights the need for a more comprehensive survey of genomic variation in avian taxa that vary markedly in social and genetic mating systems.

Hydrogen bonds in the vicinity of the special pair of the bacterial reaction center probed by hydrostatic high-pressure absorption spectroscopy.

PubMed

Kangur, Liina; Jones, Michael R; Freiberg, Arvi

2017-12-01

Using the native bacteriochlorophyll a pigment cofactors as local probes, we investigated the response to external hydrostatic high pressure of reaction center membrane protein complexes from the photosynthetic bacterium Rhodobacter sphaeroides. Wild-type and engineered complexes were used with a varied number (0, 1 or 2) of hydrogen bonds that bind the reaction center primary donor bacteriochlorophyll cofactors to the surrounding protein scaffold. A pressure-induced breakage of hydrogen bonds was established for both detergent-purified and membrane-embedded reaction centers, but at rather different pressures: between 0.2 and 0.3GPa and at about 0.55GPa, respectively. The free energy change associated with the rupture of the single hydrogen bond present in wild-type reaction centers was estimated to be equal to 13-14kJ/mol. In the mutant with two symmetrical hydrogen bonds (FM197H) a single cooperative rupture of the two bonds was observed corresponding to an about twice stronger bond, rather than a sequential rupture of two individual bonds. Copyright © 2017 Elsevier B.V. All rights reserved.

The Selenylation Modification of Epimedium Polysaccharide and Isatis Root Polysaccharide and the Immune-enhancing Activity Comparison of Their Modifiers.

PubMed

Li, Xiuping; Hou, Ranran; Yue, Chanjuan; Liu, Jie; Gao, Zhenzhen; Chen, Jin; Lu, Yu; Wang, Deyun; Liu, Cui; Hu, Yuanliang

2016-05-01

Epimedium polysaccharide (EPS) and isatis root polysaccharide (IRPS) were extracted, purified, and selenizingly modified by nitric acid-sodium selenite method to obtain nine selenizing EPSs (sEPSs), sEPS1-sEPS9 and nine selenizing IRPSs (sIRPSs), sIRPS1-sIRPS9, respectively. Their effects on chicken peripheral lymphocyte proliferation in vitro were compared by MTT assay. The results showed that selenium polysaccharides at appropriate concentration could promote lymphocyte proliferation more significantly than unmodified polysaccharides, sEPS5 and sIRPS5 with stronger actions were picked out and injected into the chickens vaccinated with Newcastle disease vaccine in vivo tests. The peripheral lymphocyte proliferation and serum antibody titer were determined. The results showed that sEPS5 and sIRPS5 could elevate serum antibody titer and promote lymphocyte proliferation more significantly than unmodified polysaccharides, sEPS5 possessed the strongest efficacy. These results indicate that selenylation modification can significantly enhance the immune-enhancing activity of EPS and IRPS, and sEPS5 can be as a new-type immunopotentiator of chickens.

When transcription goes on Holliday: Double Holliday junctions block RNA polymerase II transcription in vitro.

PubMed

Pipathsouk, Anne; Belotserkovskii, Boris P; Hanawalt, Philip C

2017-02-01

Non-canonical DNA structures can obstruct transcription. This transcription blockage could have various biological consequences, including genomic instability and gratuitous transcription-coupled repair. Among potential structures causing transcription blockage are Holliday junctions (HJs), which can be generated as intermediates in homologous recombination or during processing of stalled replication forks. Of particular interest is the double Holliday junction (DHJ), which contains two HJs. Topological considerations impose the constraint that the total number of helical turns in the DNA duplexes between the junctions cannot be altered as long as the flanking DNA duplexes are intact. Thus, the DHJ structure should strongly resist transient unwinding during transcription; consequently, it is predicted to cause significantly stronger blockage than single HJ structures. The patterns of transcription blockage obtained for RNA polymerase II transcription in HeLa cell nuclear extracts were in accordance with this prediction. However, we did not detect transcription blockage with purified T7 phage RNA polymerase; we discuss a possible explanation for this difference. In general, our findings implicate naturally occurring Holliday junctions in transcription arrest. Copyright © 2016 Elsevier B.V. All rights reserved.

Efficient killing of CD22{sup +} tumor cells by a humanized diabody-RNase fusion protein

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

Krauss, Juergen; Arndt, Michaela A.E.; Vu, Bang K.

2005-06-03

We report on the generation of a dimeric immunoenzyme capable of simultaneously delivering two ribonuclease (RNase) effector domains on one molecule to CD22{sup +} tumor cells. As targeting moiety a diabody derived from the previously humanized scFv SGIII with grafted specificity of the murine anti-CD22 mAb RFB4 was constructed. Further engineering the interface of this construct (V{sub L}36{sub Leu{yields}}{sub Tyr}) resulted in a highly robust bivalent molecule that retained the same high affinity as the murine mAb RFB4 (K{sub D} 0.2 nM). A dimeric immunoenzyme comprising this diabody and Rana pipiens liver ribonuclease I (rapLRI) was generated, expressed as solublemore » protein in bacteria, and purified to homogeneity. The dimeric fusion protein killed several CD22{sup +} tumor cell lines with high efficacy (IC{sub 50} = 3-20 nM) and exhibited 9- to 48-fold stronger cytotoxicity than a monovalent rapLRI-scFv counterpart. Our results demonstrate that engineering of dimeric antibody-ribonuclease fusion proteins can markedly enhance their biological efficacy.« less

Sweeter and stronger: enhancing sweetness and stability of the single chain monellin MNEI through molecular design

NASA Astrophysics Data System (ADS)

Leone, Serena; Pica, Andrea; Merlino, Antonello; Sannino, Filomena; Temussi, Piero Andrea; Picone, Delia

2016-09-01

Sweet proteins are a family of proteins with no structure or sequence homology, able to elicit a sweet sensation in humans through their interaction with the dimeric T1R2-T1R3 sweet receptor. In particular, monellin and its single chain derivative (MNEI) are among the sweetest proteins known to men. Starting from a careful analysis of the surface electrostatic potentials, we have designed new mutants of MNEI with enhanced sweetness. Then, we have included in the most promising variant the stabilising mutation E23Q, obtaining a construct with enhanced performances, which combines extreme sweetness to high, pH-independent, thermal stability. The resulting mutant, with a sweetness threshold of only 0.28 mg/L (25 nM) is the strongest sweetener known to date. All the new proteins have been produced and purified and the structures of the most powerful mutants have been solved by X-ray crystallography. Docking studies have then confirmed the rationale of their interaction with the human sweet receptor, hinting at a previously unpredicted role of plasticity in said interaction.

Coexpression of interleukin-6 and -2 from giant panda in Escherichia coli and the biological activity of the fusion protein.

PubMed

Yi, Y; Nian, Y-Y; Ji, H-W; Zhang, H; Zhu, L; Xu, Z-W

2013-06-14

To construct a fusion cytokine protein with more and stronger bioactivities to enhance the immunity of the cytokine alone, we expressed interleukin (IL)-6/(IL)-2 from giant panda (Ailuropoda melanoleuca) in Escherichia coli as a 59.4-kDa fusion protein. Subsequently, the inclusion bodies were solubilized with 8 M urea and applied onto a Ni-nitrilotriacetic acid column. The final production of IL-6/IL-2 reached 6 mg/L in soluble form, and the purified final product was >96% pure. In Western blot assays, the recombinant IL-6/IL-2 was recognized by polyclonal antibodies against IL-6 and IL-2 of giant panda. The results demonstrated that the protein mixture contained correctly folded IL-2 and IL-6 proteins. A 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide assay demonstrated that IL-6/IL-2 can promote lymphocyte proliferation and differentiation. These data suggest that the fusion protein could be used to develop a novel immunoadjuvant to enhance the immunity of animals against infectious diseases.

(1→3)-β-d-Glucan oligosaccharides monomers purification and its H2O2 induction effect study.

PubMed

Fu, Yunbin; Wang, Mengyu; Wang, Wenxia; Tuo, Yaqin; Guo, Zhimou; Du, Yuguang; Yin, Heng

2015-11-01

In order to produce highly purified (1→3)-β-d-glucan oligosaccharides ((1→3)-β-d-GOS) monomers, a hydrophilic interaction liquid chromatography (HILIC) system with X-Amide stationary phase was performed. Nine (1→3)-β-d-GOS monomers with degree of polymerization (DP) from 2 to 10 were successfully separated. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) demonstrated that these monomers were with high purity. Furthermore, a hydrogen peroxide (H2O2) online detection method was established to monitor H2O2 releases in tobacco cells. This is the first report on nine consecutive (1→3)-β-d-GOS monomers purification and its effect upon H2O2-releasing in plants. It was found that (1→3)-β-d-GOS monomers with higher DP induced stronger defense responses in plants, which will pave the way for elucidating the relationship between (1→3)-β-d-GOS and biological activities. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparison of single-step and two-step purified coagulants from Moringa oleifera seed for turbidity and DOC removal.

PubMed

Sánchez-Martín, J; Ghebremichael, K; Beltrán-Heredia, J

2010-08-01

The coagulant proteins from Moringa oleifera purified with single-step and two-step ion-exchange processes were used for the coagulation of surface water from Meuse river in The Netherlands. The performances of the two purified coagulants and the crude extract were assessed in terms of turbidity and DOC removal. The results indicated that the optimum dosage of the single-step purified coagulant was more than two times higher compared to the two-step purified coagulant in terms of turbidity removal. And the residual DOC in the two-step purified coagulant was lower than in single-step purified coagulant or crude extract. (c) 2010 Elsevier Ltd. All rights reserved.

Contrasted evolutionary histories of two Toll-like receptors (Tlr4 and Tlr7) in wild rodents (MURINAE)

PubMed Central

2013-01-01

Background In vertebrates, it has been repeatedly demonstrated that genes encoding proteins involved in pathogen-recognition by adaptive immunity (e.g. MHC) are subject to intensive diversifying selection. On the other hand, the role and the type of selection processes shaping the evolution of innate-immunity genes are currently far less clear. In this study we analysed the natural variation and the evolutionary processes acting on two genes involved in the innate-immunity recognition of Microbe-Associated Molecular Patterns (MAMPs). Results We sequenced genes encoding Toll-like receptor 4 (Tlr4) and 7 (Tlr7), two of the key bacterial- and viral-sensing receptors of innate immunity, across 23 species within the subfamily Murinae. Although we have shown that the phylogeny of both Tlr genes is largely congruent with the phylogeny of rodents based on a comparably sized non-immune sequence dataset, we also identified several potentially important discrepancies. The sequence analyses revealed that major parts of both Tlrs are evolving under strong purifying selection, likely due to functional constraints. Yet, also several signatures of positive selection have been found in both genes, with more intense signal in the bacterial-sensing Tlr4 than in the viral-sensing Tlr7. 92% and 100% of sites evolving under positive selection in Tlr4 and Tlr7, respectively, were located in the extracellular domain. Directly in the Ligand-Binding Region (LBR) of TLR4 we identified two rapidly evolving amino acid residues and one site under positive selection, all three likely involved in species-specific recognition of lipopolysaccharide of gram-negative bacteria. In contrast, all putative sites of LBRTLR7 involved in the detection of viral nucleic acids were highly conserved across rodents. Interspecific differences in the predicted 3D-structure of the LBR of both Tlrs were not related to phylogenetic history, while analyses of protein charges clearly discriminated Rattini and Murini clades. Conclusions In consequence of the constraints given by the receptor protein function purifying selection has been a dominant force in evolution of Tlrs. Nevertheless, our results show that episodic diversifying parasite-mediated selection has shaped the present species-specific variability in rodent Tlrs. The intensity of diversifying selection was higher in Tlr4 than in Tlr7, presumably due to structural properties of their ligands. PMID:24028551

40 CFR 1065.845 - Response factor determination.

Code of Federal Regulations, 2014 CFR

2014-07-01

... that FID zero and span balance gases may be any combination of purified air or purified nitrogen that... and span balance gases may be any combination of purified air or purified nitrogen that meets the...

Adsorption of lead onto smectite from aqueous solution.

PubMed

Mhamdi, M; Galai, H; Mnasri, N; Elaloui, E; Trabelsi-Ayadi, M

2013-03-01

The purpose of this research is to study the effect of a new method of adsorption using membrane filtration to determine the maximum amount of lead adsorbed by clay and investigate the behavior of the clay after adsorption of the said metal. Treatment of wastewater contaminated with heavy metals depends on the characteristics of the effluent, the amount of final discharge, the cost of treatment, and the compatibility of the treatment process. The process of adsorption of heavy metals by clays may be a simple, selective, and economically viable alternative to the conventional physical-chemical treatment. This is justified by the importance of the surface developed by this material, the presence of negative charges on the said surface, the possibility of ion exchange taking place, and its wide availability in nature. The removal of lead from wastewater was studied by using the adsorption technique and using clay as the adsorbent. A method was optimized for adsorption through a membrane approaching natural adsorption. This new method is simple, selective, and the lead adsorption time is about 3 days. The various properties of clay were determined. It was observed that the cation exchange capacity of the clay was 56 meq/100 g of hydrated clay for the raw sample and 82 meq/100 g for the purified sample. The total surface area determined by the methylene blue method was equal to 556 and 783 m(2)/g for the raw and purified samples, respectively. The adsorption kinetics depends on several parameters. The Pb(II) clay, obeys the Langmuir, Freundlich, and the Elovich adsorption isotherms with high regression coefficients. The use of this adsorbent notably decreases the cost of treatment. It was concluded that clay shows a strong adsorption capacity on Pb(II), the maximum interaction occurring with purified clay treated at high concentration of lead. It is proposed that this adsorption through a membrane be extended for the treatment of effluents containing other metals.

Facially Selective Cu-catalyzed Carbozincation of Cyclopropenes Using Arylzinc Reagents Formed by Sequential I/Mg/Zn exchange

PubMed Central

Tarwade, Vinod; Selvaraj, Ramajeyam; Fox, Joseph M.

2012-01-01

Described is a Cu-catalyzed directed carbozincation of cyclopropenes with organozinc reagents prepared by I/Mg/Zn exchange. This protocol broadens the scope with respect to functional group tolerance and enables use of aryl iodide precursors, rather than purified diorganozinc precursors. Critical to diastereoselectivity of the carbozincation step is the removal of magnesium halide salts after transmetallation with ZnCl2. PMID:23035947

The Microanatomic Segregation of Selection by Apoptosis in the Germinal Center

PubMed Central

Mayer, Christian T.; Gazumyan, Anna; Kara, Ervin E.; Gitlin, Alexander D.; Golijanin, Jovana; Viant, Charlotte; Pai, Joy; Oliveira, Thiago Y.; Wang, Qiao; Escolano, Amelia; Medina-Ramirez, Max; Sanders, Rogier W.; Nussenzweig, Michel C.

2018-01-01

B cells undergo rapid cell division and affinity maturation in anatomically distinct sites in lymphoid organs called germinal centers (GCs). Homeostasis is maintained in part by B-cell apoptosis. However, the precise contribution of apoptosis to GC biology and selection is not well defined. We developed apoptosis-indicator mice and used them to visualize, purify, and characterize dying GC B cells. Apoptosis is prevalent in the GC with up to half of all GC B cells dying every 6h. Moreover, programmed cell death is differentially regulated in the light zone (LZ) and the dark zone (DZ): LZ B cells die by default if they are not positively selected, whereas DZ cells die when their antigen receptors are damaged by activation-induced cytidine deaminase (AID). PMID:28935768

Silencing Effect of Hominoid Highly Conserved Noncoding Sequences on Embryonic Brain Development

PubMed Central

Mahmoudi Saber, Morteza

2017-01-01

Abstract Superfamily Hominoidea, which consists of Hominidae (humans and great apes) and Hylobatidae (gibbons), is well-known for sharing human-like characteristics, however, the genomic origins of these shared unique phenotypes have mainly remained elusive. To decipher the underlying genomic basis of Hominoidea-restricted phenotypes, we identified and characterized Hominoidea-restricted highly conserved noncoding sequences (HCNSs) that are a class of potential regulatory elements which may be involved in evolution of lineage-specific phenotypes. We discovered 679 such HCNSs from human, chimpanzee, gorilla, orangutan and gibbon genomes. These HCNSs were demonstrated to be under purifying selection but with lineage-restricted characteristics different from old CNSs. A significant proportion of their ancestral sequences had accelerated rates of nucleotide substitutions, insertions and deletions during the evolution of common ancestor of Hominoidea, suggesting the intervention of positive Darwinian selection for creating those HCNSs. In contrary to enhancer elements and similar to silencer sequences, these Hominoidea-restricted HCNSs are located in close proximity of transcription start sites. Their target genes are enriched in the nervous system, development and transcription, and they tend to be remotely located from the nearest coding gene. Chip-seq signals and gene expression patterns suggest that Hominoidea-restricted HCNSs are likely to be functional regulatory elements by imposing silencing effects on their target genes in a tissue-restricted manner during fetal brain development. These HCNSs, emerged through adaptive evolution and conserved through purifying selection, represent a set of promising targets for future functional studies of the evolution of Hominoidea-restricted phenotypes. PMID:28633494

Genome Evolution in the Obligate but Environmentally Active Luminous Symbionts of Flashlight Fish

PubMed Central

Hendry, Tory A.; de Wet, Jeffrey R.; Dougan, Katherine E.; Dunlap, Paul V.

2016-01-01

The luminous bacterial symbionts of anomalopid flashlight fish are thought to be obligately dependent on their hosts for growth and share several aspects of genome evolution with unrelated obligate symbionts, including genome reduction. However, in contrast to most obligate bacteria, anomalopid symbionts have an active environmental phase that may be important for symbiont transmission. Here we investigated patterns of evolution between anomalopid symbionts compared with patterns in free-living relatives and unrelated obligate symbionts to determine if trends common to obligate symbionts are also found in anomalopid symbionts. Two symbionts, “Candidatus Photodesmus katoptron” and “Candidatus Photodesmus blepharus,” have genomes that are highly similar in gene content and order, suggesting genome stasis similar to ancient obligate symbionts present in insect lineages. This genome stasis exists in spite of the symbiont’s inferred ability to recombine, which is frequently lacking in obligate symbionts with stable genomes. Additionally, we used genome comparisons and tests of selection to infer which genes may be particularly important for the symbiont’s ecology compared with relatives. In keeping with obligate dependence, substitution patterns suggest that most symbiont genes are experiencing relaxed purifying selection compared with relatives. However, genes involved in motility and carbon storage, which are likely to be used outside the host, appear to be under increased purifying selection. Two chemoreceptor chemotaxis genes are retained by both species and show high conservation with amino acid sensing genes, suggesting that the bacteria may actively seek out hosts using chemotaxis toward amino acids, which the symbionts are not able to synthesize. PMID:27389687

Emergence and Evolution of Hominidae-Specific Coding and Noncoding Genomic Sequences.

PubMed

Saber, Morteza Mahmoudi; Adeyemi Babarinde, Isaac; Hettiarachchi, Nilmini; Saitou, Naruya

2016-07-12

Family Hominidae, which includes humans and great apes, is recognized for unique complex social behavior and intellectual abilities. Despite the increasing genome data, however, the genomic origin of its phenotypic uniqueness has remained elusive. Clade-specific genes and highly conserved noncoding sequences (HCNSs) are among the high-potential evolutionary candidates involved in driving clade-specific characters and phenotypes. On this premise, we analyzed whole genome sequences along with gene orthology data retrieved from major DNA databases to find Hominidae-specific (HS) genes and HCNSs. We discovered that Down syndrome critical region 4 (DSCR4) is the only experimentally verified gene uniquely present in Hominidae. DSCR4 has no structural homology to any known protein and was inferred to have emerged in several steps through LTR/ERV1, LTR/ERVL retrotransposition, and transversion. Using the genomic distance as neutral evolution threshold, we identified 1,658 HS HCNSs. Polymorphism coverage and derived allele frequency analysis of HS HCNSs showed that these HCNSs are under purifying selection, indicating that they may harbor important functions. They are overrepresented in promoters/untranslated regions, in close proximity of genes involved in sensory perception of sound and developmental process, and also showed a significantly lower nucleosome occupancy probability. Interestingly, many ancestral sequences of the HS HCNSs showed very high evolutionary rates. This suggests that new functions emerged through some kind of positive selection, and then purifying selection started to operate to keep these functions. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

ACP5 (Uteroferrin): Phylogeny of an Ancient and Conserved Gene Expressed in the Endometrium of Mammals1

PubMed Central

Padua, Maria B.; Lynch, Vincent J.; Alvarez, Natalia V.; Garthwaite, Mark A.; Golos, Thaddeus G.; Bazer, Fuller W.; Kalkunte, Satyan; Sharma, Surendra; Wagner, Gunter P.; Hansen, Peter J.

2012-01-01

ABSTRACT Type 5 acid phosphatase (ACP5; also known as tartrate-resistant acid phosphatase or uteroferrin) is a metalloprotein secreted by the endometrial glandular epithelium of pigs, mares, sheep, and water buffalo. In this paper, we describe the phylogenetic distribution of endometrial expression of ACP5 and demonstrate that endometrial expression arose early in evolution (i.e., before divergence of prototherian and therian mammals ∼166 million years ago). To determine expression of ACP5 in the pregnant endometrium, RNA was isolated from rhesus, mouse, rat, dog, sheep, cow, horse, armadillo, opossum, and duck-billed platypus. Results from RT-PCR and RNA-Seq experiments confirmed that ACP5 is expressed in all species examined. ACP5 was also demonstrated immunochemically in endometrium of rhesus, marmoset, sheep, cow, goat, and opossum. Alignment of inferred amino acid sequences shows a high conservation of ACP5 throughout speciation, with species-specific differences most extensive in the N-terminal and C-terminal regions of the protein. Analysis by Selecton indicated that most of the sites in ACP5 are undergoing purifying selection, and no sites undergoing positive selection were found. In conclusion, endometrial expression of ACP5 is a common feature in all orders of mammals and has been subjected to purifying selection. Expression of ACP5 in the uterus predates the divergence of therians and prototherians. ACP5 is an evolutionary conserved gene that likely exerts a common function important for pregnancy in mammals using a wide range of reproductive strategies. PMID:22278982

The environment shapes microbial enzymes: five cold-active and salt-resistant carboxylesterases from marine metagenomes.

PubMed

Tchigvintsev, Anatoli; Tran, Hai; Popovic, Ana; Kovacic, Filip; Brown, Greg; Flick, Robert; Hajighasemi, Mahbod; Egorova, Olga; Somody, Joseph C; Tchigvintsev, Dmitri; Khusnutdinova, Anna; Chernikova, Tatyana N; Golyshina, Olga V; Yakimov, Michail M; Savchenko, Alexei; Golyshin, Peter N; Jaeger, Karl-Erich; Yakunin, Alexander F

2015-03-01

Most of the Earth's biosphere is cold and is populated by cold-adapted microorganisms. To explore the natural enzyme diversity of these environments and identify new carboxylesterases, we have screened three marine metagenome gene libraries for esterase activity. The screens identified 23 unique active clones, from which five highly active esterases were selected for biochemical characterization. The purified metagenomic esterases exhibited high activity against α-naphthyl and p-nitrophenyl esters with different chain lengths. All five esterases retained high activity at 5 °C indicating that they are cold-adapted enzymes. The activity of MGS0010 increased more than two times in the presence of up to 3.5 M NaCl or KCl, whereas the other four metagenomic esterases were inhibited to various degrees by these salts. The purified enzymes showed different sensitivities to inhibition by solvents and detergents, and the activities of MGS0010, MGS0105 and MGS0109 were stimulated three to five times by the addition of glycerol. Screening of purified esterases against 89 monoester substrates revealed broad substrate profiles with a preference for different esters. The metagenomic esterases also hydrolyzed several polyester substrates including polylactic acid suggesting that they can be used for polyester depolymerization. Thus, esterases from marine metagenomes are cold-adapted enzymes exhibiting broad biochemical diversity reflecting the environmental conditions where they evolved.

Purification of influenza deoxyribonucleic acid-based vaccine using agmatine monolith.

PubMed

Bicho, D; Caramelo-Nunes, C; Sousa, A; Sousa, F; Queiroz, J A; Tomaz, C T

2016-02-15

Lately, researchers have made several efforts to improve vaccine production to fight highly contagious respiratory diseases like influenza. One of the most promising options for reducing the impact of this virus is DNA vaccination. However, a large quantity of highly pure plasmid DNA (pDNA) is necessary to attain this goal. The present work describes the production and purification of the plasmid NTC7482-41H-VA2HA expressing influenza virus hemagglutinin using an agmatine monolith. This ligand was chosen to purify supercoiled (sc) pDNA from complex lysates because of its versatile multimodal character. Its natural intervention in several biological systems together with its similarity with the highly studied arginine ligand allowed the development of a simpler and more specific purification process. Agmatine works under two strategies: descending ammonium sulfate gradient and ascending sodium chloride gradient. Furthermore, pH manipulation revealed an important role in pDNA isoforms selectivity. Dynamic binding capacity (DBC) experiments were performed varying different parameters and showed an increase with pDNA concentration, while high flow rate and high pH had the opposite effect. Sc pDNA was purified with high yield and was efficient with respect to cell transfection and cell viability. This monolith showed to be appropriate to purify the plasmid NTC7482-41H-VA2HA, providing a valuable tool for pDNA influenza vaccines preparation. Copyright © 2016. Published by Elsevier B.V.

[Partial purification of peptides present in the Tityus macrochirus (Buthidae) scorpion venom and preliminary assessment of their cytotoxicity].

PubMed

Rincón-Cortés, Clara Andrea; Reyes-Montaño, Edgar Antonio; Vega-Castro, Nohora Angélica

2017-06-01

Scorpion venom contains peptides with neurotoxic action primarily active on ion channels in the nervous system of insects and mammals. They are also characterized as cytolytic and anticancer, biological characteristics that have not yet been reported for the Tityus macrochirus venom. To assess if the total T. macrochirus venom and the fraction of partially purified peptides decrease the viability of various tumor-derived cell lines. The scorpion venom was collected by electrical stimulation and, subsequently, subjected to chromatography, electrophoresis, and ultrafiltration with Amicon Ultra 0.5® membranes for the partial identification and purification of its peptides. The cytotoxic activity of the venom and the peptides fraction trials on tumor-derived cell lines were carried out by the MTT method. The T. macrochirus scorpion venom has peptides with molecular weights ranging between 3 and 10 kDa. They were partially purified using the ultrafiltration technique, and assessed by the RP-HPLC method. Cytotoxicity trials with the whole T. macrochirus venom showed a higher viability decrease on the PC3 cell line compared to the other cell lines assessed, while the partially purified peptides decreased the HeLa cell line viability. Peptides in the T. macrochirus scorpion venom showed cytotoxic activity on some tumorderived cell lines. We observed some degree of selectivity against other cell lines assessed.

Molecular approaches towards development of purified natural products and their structurally known derivatives as efficient anti-cancer drugs: current trends.

PubMed

Saha, Santu Kumar; Khuda-Bukhsh, Anisur Rahman

2013-08-15

Several natural products and their derivatives, either in purified or structurally identified form, exhibit immense pharmacological and biological properties, some of them showing considerable anticancer potential. Although the molecular mechanisms of action of some of these products are yet to be elucidated, extensive research in this area continues to generate new data that are clinically exploitable. Recent advancement in molecular biology, high throughput screening, biomarker identifications, target selection and genomic approaches have enabled us to understand salient interactions of natural products and their derivatives with cancer cells vis-à-vis normal cells. In this review we highlight the recent approaches and application of innovative technologies made to improve quality as well as efficiency of structurally identified natural products and their derivatives, particularly in small molecular forms capable of being used in "targeted therapies" in oncology. These products preferentially involve multiple mechanistic pathways and overcome chemo-resistance in tumor types with cumulative action. We also mention briefly a few physico-chemical features that compare natural products with drugs in recent natural product discovery approaches. We further report here a few purified natural products as examples that provide molecular interventions in cancer therapeutics to give the reader a glimpse of the current trends of approach for discovering useful anticancer drugs. © 2013 Elsevier B.V. All rights reserved.

Exopolysaccharide produced by Pediococcus acidilactici M76 isolated from the Korean traditional rice wine, makgeolli.

PubMed

Song, Young-Ran; Jeong, Do-Youn; Cha, Youn-Soo; Baik, Sang-Ho

2013-05-01

This work is aimed to increase knowledge of the functional exopolysaccharide (EPS) from lactic acid bacteria (LAB) in makgeolli, a Korean fermented rice wine. Among LAB strains isolated from makgeolli, strain M76 was selected as a functional strain producing a bioactive EPS, based on its antioxidative activity on the DPPH radical. The 16S rRNA gene sequencing analysis showed a high sequence similarity (99.0%) with P. acidilactici, but had different biochemical properties with the already known P. acidilactici type strains in the aspect of carbohydrates utilization. The obtained P. acidilactici M76 produced a soluble EPS above 2 g/l. One-step chromatography using gel filtration after ethanol precipitation from the supernatant of P. acidilactici M76 was enough to obtain purified EPS with a single peak, showing a molecular mass of approximately 67 kDa. Componential and structural analyses of EPS by TLC, HPLC, and FT-IR indicated that the EPS is a glucan, consisting of glucose units. The purified EPS had antioxidant activity on the DPPH radical of 45.8% at a concentration of 1 mg/ml. The purified EPS also showed proliferative effect on the pancreatic RIN-m5F cell line and remarkable protection activity on alloxan-induced cytotoxicity. This potent antioxidant and antidiabetic EPS by LAB in makgeolli may contribute to understanding the functionality of makgeolli.

Novel Coprinopsis cinerea Polyesterase That Hydrolyzes Cutin and Suberin▿ †

PubMed Central

Kontkanen, Hanna; Westerholm-Parvinen, Ann; Saloheimo, Markku; Bailey, Michael; Rättö, Marjaana; Mattila, Ismo; Mohsina, Marzia; Kalkkinen, Nisse; Nakari-Setälä, Tiina; Buchert, Johanna

2009-01-01

Three cutinase gene-like genes from the basidiomycete Coprinopsis cinerea (Coprinus cinereus) found with a similarity search were cloned and expressed in Trichoderma reesei under the control of an inducible cbh1 promoter. The selected transformants of all three polyesterase constructs showed activity with p-nitrophenylbutyrate, used as a model substrate. The most promising transformant of the cutinase CC1G_09668.1 gene construct was cultivated in a laboratory fermentor, with a production yield of 1.4 g liter−l purified protein. The expressed cutinase (CcCUT1) was purified to homogeneity by immobilized metal affinity chromatography exploiting a C-terminal His tag. The N terminus of the enzyme was found to be blocked. The molecular mass of the purified enzyme was determined to be around 18.8 kDa by mass spectrometry. CcCUT1 had higher activity on shorter (C2 to C10) fatty acid esters of p-nitrophenol than on longer ones, and it also exhibited lipase activity. CcCUT1 had optimal activity between pH 7 and 8 but retained activity over a wide pH range. The enzyme retained 80% of its activity after 20 h of incubation at 50°C, but residual activity decreased sharply at 60°C. Microscopic analyses and determination of released hydrolysis products showed that the enzyme was able to depolymerize apple cutin and birch outer bark suberin. PMID:19201950

Novel Coprinopsis cinerea polyesterase that hydrolyzes cutin and suberin.

PubMed

Kontkanen, Hanna; Westerholm-Parvinen, Ann; Saloheimo, Markku; Bailey, Michael; Rättö, Marjaana; Mattila, Ismo; Mohsina, Marzia; Kalkkinen, Nisse; Nakari-Setälä, Tiina; Buchert, Johanna

2009-04-01

Three cutinase gene-like genes from the basidiomycete Coprinopsis cinerea (Coprinus cinereus) found with a similarity search were cloned and expressed in Trichoderma reesei under the control of an inducible cbh1 promoter. The selected transformants of all three polyesterase constructs showed activity with p-nitrophenylbutyrate, used as a model substrate. The most promising transformant of the cutinase CC1G_09668.1 gene construct was cultivated in a laboratory fermentor, with a production yield of 1.4 g liter(-l) purified protein. The expressed cutinase (CcCUT1) was purified to homogeneity by immobilized metal affinity chromatography exploiting a C-terminal His tag. The N terminus of the enzyme was found to be blocked. The molecular mass of the purified enzyme was determined to be around 18.8 kDa by mass spectrometry. CcCUT1 had higher activity on shorter (C(2) to C(10)) fatty acid esters of p-nitrophenol than on longer ones, and it also exhibited lipase activity. CcCUT1 had optimal activity between pH 7 and 8 but retained activity over a wide pH range. The enzyme retained 80% of its activity after 20 h of incubation at 50 degrees C, but residual activity decreased sharply at 60 degrees C. Microscopic analyses and determination of released hydrolysis products showed that the enzyme was able to depolymerize apple cutin and birch outer bark suberin.

Older and Stronger Object Memories are Selectively Destabilized by Reactivation in the Presence of New Information

ERIC Educational Resources Information Center

Winters, Boyer D.; Tucci, Mark C.; DaCosta-Furtado, Melynda

2009-01-01

Reactivation can destabilize previously consolidated memories, rendering them vulnerable to disruption and necessitating a process of reconsolidation in order for them to be maintained. This process of destabilization and reconsolidation has commonly been cited as a means by which established memories can be updated or modified. However, little…

No Large Convergence during Marriage for Health, Lifestyle, and Personality in a Large Sample of Norwegian Spouses.

ERIC Educational Resources Information Center

Tambs, Kristian; Moum, Torbjorn

1992-01-01

Tested notion that resemblance between spouses increases with marital duration because of common environmental or reciprocal influences. Data from 15,925 to 23,033 Norwegian couples revealed that correlations between spouses did not vary much with marital duration, implying stronger effect of mate selection than convergence. Found moderate…

A Mathematical Model for Allocation of School Resources to Optimize a Selected Output.

ERIC Educational Resources Information Center

McAfee, Jackson K.

The methodology of costing an education program by identifying the resources it utilizes places all costs within the framework of staff, equipment, materials, facilities, and services. This paper suggests that this methodology is much stronger than the more traditional budgetary and cost per pupil approach. The techniques of data collection are…

Pollinators exert natural selection on flower size and floral display in Penstemon digitalis.

PubMed

Parachnowitsch, Amy L; Kessler, André

2010-10-01

• A major gap in our understanding of floral evolution, especially micro-evolutionary processes, is the role of pollinators in generating patterns of natural selection on floral traits. Here we explicitly tested the role of pollinators in selecting floral traits in a herbaceous perennial, Penstemon digitalis. • We manipulated the effect of pollinators on fitness through hand pollinations and compared phenotypic selection in open- and hand-pollinated plants. • Despite the lack of pollen limitation in our population, pollinators mediated selection on floral size and floral display. Hand pollinations removed directional selection for larger flowers and stabilizing selection on flower number, suggesting that pollinators were the agents of selection on both of these traits. • We reviewed studies that measured natural selection on floral traits by biotic agents and generally found stronger signatures of selection imposed by pollinators than by herbivores and co-flowering plant species. © The Authors (2010). Journal compilation © New Phytologist Trust (2010).

Characterization of a monoclonal antibody against P57, the C3/C3b-cleaving proteinase expressed in human erythrocyte membranes.

PubMed

Fiandino-Tirel, A; Barel, M; Lyamani, F; Gauffre, A; Hermann, J; Frade, R

1991-08-01

A monoclonal antibody was raised against p57, a serine proteinase, characterized by an apparent molecular weight of 57 kDa, and purified from human erythrocyte membranes. P57 proteinase cleaves the human third component of complement, C3. The antibody selected, MP1, of IgG2a isotype, precipitated specifically the p57 antigen which carried the C3/C3b-cleaving activity present in membrane crude extract of human erythrocytes. P57 proteinase eluted from MP1-sepharose was inhibited by 5 x 10(-4) M PMSF, enhanced by 0.5% SDS and generated C3 fragments identical to those generated by membrane crude extract of human erythrocytes. All these properties were identical to those of the p57 previously purified by biochemical procedures. In addition, 5000 binding sites were detected on cell surface. This MP1 monoclonal antibody will be helpful to analyse the role of p57 in human erythrocytes.

A recombinant dromedary antibody fragment (VHH or nanobody) directed against human Duffy antigen receptor for chemokines.

PubMed

Smolarek, Dorota; Hattab, Claude; Hassanzadeh-Ghassabeh, Gholamreza; Cochet, Sylvie; Gutiérrez, Carlos; de Brevern, Alexandre G; Udomsangpetch, Rachanee; Picot, Julien; Grodecka, Magdalena; Wasniowska, Kazimiera; Muyldermans, Serge; Colin, Yves; Le Van Kim, Caroline; Czerwinski, Marcin; Bertrand, Olivier

2010-10-01

Fy blood group antigens are carried by the Duffy antigen receptor for chemokines (DARC), a red cells receptor for Plasmodium vivax broadly implicated in human health and diseases. Recombinant VHHs, or nanobodies, the smallest intact antigen binding fragment derivative from the heavy chain-only antibodies present in camelids, were prepared from a dromedary immunized against DARC N-terminal extracellular domain and selected for DARC binding. A described VHH, CA52, does recognize native DARC on cells. It inhibits P. vivax invasion of erythrocytes and displaces interleukin-8 bound to DARC. The targeted epitope overlaps the well-defined DARC Fy6 epitope. K (D) of CA52-DARC equilibrium is sub-nanomolar, hence ideal to develop diagnostic or therapeutic compounds. Immunocapture by immobilized CA52 yielded highly purified DARC from engineered K562 cells. This first report on a VHH with specificity for a red blood cell protein exemplifies VHHs' potentialities to target, to purify, and to modulate the function of cellular markers.

Purification and characterization of enantioselective N-acetyl-β-Phe acylases from Burkholderia sp. AJ110349.

PubMed

Imabayashi, Yuki; Suzuki, Shun'ichi; Kawasaki, Hisashi; Nakamatsu, Tsuyoshi

2016-01-01

For the production of enantiopure β-amino acids, enantioselective resolution of N-acyl β-amino acids using acylases, especially those recognizing N-acetyl-β-amino acids, is one of the most attractive methods. Burkholderia sp. AJ110349 had been reported to exhibit either (R)- or (S)-enantiomer selective N-acetyl-β-Phe amidohydrolyzing activity, and in this study, both (R)- and (S)-enantioselective N-acetyl-β-Phe acylases were purified to be electrophoretically pure and determined the sequences, respectively. They were quite different in terms of enantioselectivities and in their amino acids sequences and molecular weights. Although both the purified acylases were confirmed to catalyze N-acetyl hydrolyzing activities, neither of them show sequence similarities to the N-acetyl-α-amino acid acylases reported thus far. Both (R)- and (S)-enantioselective N-acetyl-β-Phe acylase were expressed in Escherichia coli. Using these recombinant strains, enantiomerically pure (R)-β-Phe (>99% ee) and (S)-β-Phe (>99% ee) were obtained from the racemic substrate.

Cloning, expression, and purification of the virulence-associated protein D from Xylella fastidiosa.

PubMed

Catani, Cleide Ferreira; Azzoni, Adriano Rodrigues; Paula, Débora Pires; Tada, Susely Ferraz Siqueira; Rosselli, Luciana Kauer; de Souza, Anete Pereira; Yano, Tomomasa

2004-10-01

In this study, an efficient expression system, based on the pET32Xa/LIC vector, for producing a Xylella fastidiosa virulence-associated protein D, found to have a strong similarity to Riemerella anatipestifer and Actinobacillus actinomycetencomitans VapD protein, is presented. The protein has a molecular mass of 17.637 Da and a calculated pI of 5.49. The selected XFa0052 gene was cloned in the pET32Xa/LIC vector and the plasmid was transformed into Escherichia coli BL21 (DE3) strain at 37 degrees C, with an induction time of 2 h and 1 mM IPTG concentration. The protein present in the soluble fraction was purified by immobilized metal affinity chromatography (IMAC), and had its identity determined by mass spectrometry (MALDI-TOF) and N-terminal sequencing. The purified protein was found as a single band on SDS-PAGE and its correct folding was verified by circular dichroism spectroscopy.

Recombinant scFv antibodies against infectious pancreatic necrosis virus isolated by flow cytometry.

PubMed

Xu, Li-Ming; Zhao, Jing-Zhuang; Liu, Miao; Cao, Yong-Sheng; Yin, Jia-Sheng; Liu, Hong-Bai; Lu, Tongyan

2016-11-01

Infectious pancreatic necrosis is a significant disease of farmed salmonids in China. In this study, a single chain variable fragment (scFv) antibody library derived from rainbow trout (Oncorhynchus mykiss) and viral protein VP2 of a Chinese infectious pancreatic necrosis virus (IPNV) isolate ChRtm213 were co-expressed by a bacterial display technology. The library was subjected to three rounds of screening by flow cytometry (FCM) to select IPNV specific antibodies. Six antibody clones with different mean fluorescence intensities (MFI) were obtained by picking colonies at random. The antibody clones were expressed and purified. The purified IPNV-specific scFv antibodies were used successfully in Western blotting, enzyme linked immunosorbent assay (ELISA) and an immunofluorescence antibody test (IFAT). This method provides a high throughput means to screen an antibody library by flow cytometry, and isolate a panel of antibody that can be used as potential reagents for the detection and study of IPNV that are prevalent in China. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of Metal-impregnated Single Walled Carbon Nanotubes for Toxic Gas Contaminant Control in Advanced Life Support Systems

NASA Technical Reports Server (NTRS)

Pisharody, Suresh A.; Fisher, John W.; Wignarajah, K.

2002-01-01

The success of physico-chemical waste processing and resource recovery technologies for life support application depends partly on the ability of gas clean-up systems to efficiently remove trace contaminants generated during the process with minimal use of expendables. Carbon nanotubes promise superior performance over conventional approaches to gas clean-up due to their ability to direct the selective uptake of gaseous species based on their controlled pore size, high surface area, ordered chemical structure that allows functionalization and their effectiveness also as catalyst support materials for toxic gas conversion. We present results and findings from a preliminary study on the effectiveness of metal impregnated single walled nanotubes as catalyst/catalyst support materials for toxic gas contaminate control. The study included the purification of single walled nanotubes, the catalyst impregnation of the purified nanotubes, the experimental characterization of the surface properties of purified single walled nanotubes and the characterization of physisorption and chemisorption of uptake molecules.

Purification, crystallization and preliminary X-ray diffraction analysis of the glyoxalase II from Leishmania infantum

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

Trincão, José; Sousa Silva, Marta; Barata, Lídia

2006-08-01

A glyoxalase II from L. infantum was cloned, purified and crystallized and its structure was solved by X-ray crystallography. In trypanosomatids, trypanothione replaces glutathione in all glutathione-dependent processes. Of the two enzymes involved in the glyoxalase pathway, glyoxalase I and glyoxalase II, the latter shows absolute specificity towards trypanothione thioester, making this enzyme an excellent model to understand the molecular basis of trypanothione binding. Cloned glyoxalase II from Leishmania infantum was overexpressed in Escherichia coli, purified and crystallized. Crystals belong to space group C222{sub 1} (unit-cell parameters a = 65.6, b = 88.3, c = 85.2 Å) and diffract beyondmore » 2.15 Å using synchrotron radiation. The structure was solved by molecular replacement using the human glyoxalase II structure as a search model. These results, together with future detailed kinetic characterization using lactoyltrypanothione, should shed light on the evolutionary selection of trypanothione instead of glutathione by trypano-somatids.« less

A high-throughput differential filtration assay to screen and select detergents for membrane proteins

PubMed Central

Vergis, James M.; Purdy, Michael D.; Wiener, Michael C.

2015-01-01

Structural studies on integral membrane proteins are routinely performed on protein–detergent complexes (PDCs) consisting of purified protein solubilized in a particular detergent. Of all the membrane protein crystal structures solved to date, a subset of only four detergents has been used in more than half of these structures. Unfortunately, many membrane proteins are not well behaved in these four detergents and/or fail to yield well-diffracting crystals. Identification of detergents that maintain the solubility and stability of a membrane protein is a critical step and can be a lengthy and “protein-expensive” process. We have developed an assay that characterizes the stability and size of membrane proteins exchanged into a panel of 94 commercially available and chemically diverse detergents. This differential filtration assay (DFA), using a set of filtered microplates, requires sub-milligram quantities of purified protein and small quantities of detergents and other reagents and is performed in its entirety in several hours. PMID:20667442

Substitution rate and natural selection in parvovirus B19

PubMed Central

Stamenković, Gorana G.; Ćirković, Valentina S.; Šiljić, Marina M.; Blagojević, Jelena V.; Knežević, Aleksandra M.; Joksić, Ivana D.; Stanojević, Maja P.

2016-01-01

The aim of this study was to estimate substitution rate and imprints of natural selection on parvovirus B19 genotype 1. Studied datasets included 137 near complete coding B19 genomes (positions 665 to 4851) for phylogenetic and substitution rate analysis and 146 and 214 partial genomes for selection analyses in open reading frames ORF1 and ORF2, respectively, collected 1973–2012 and including 9 newly sequenced isolates from Serbia. Phylogenetic clustering assigned majority of studied isolates to G1A. Nucleotide substitution rate for total coding DNA was 1.03 (0.6–1.27) x 10−4 substitutions/site/year, with higher values for analyzed genome partitions. In spite of the highest evolutionary rate, VP2 codons were found to be under purifying selection with rare episodic positive selection, whereas codons under diversifying selection were found in the unique part of VP1, known to contain B19 immune epitopes important in persistent infection. Analyses of overlapping gene regions identified nucleotide positions under opposite selective pressure in different ORFs, suggesting complex evolutionary mechanisms of nucleotide changes in B19 viral genomes. PMID:27775080

Capture-SELEX: Selection of DNA Aptamers for Aminoglycoside Antibiotics

PubMed Central

2012-01-01

Small organic molecules are challenging targets for an aptamer selection using the SELEX technology (SELEX—Systematic Evolution of Ligans by EXponential enrichment). Often they are not suitable for immobilization on solid surfaces, which is a common procedure in known aptamer selection methods. The Capture-SELEX procedure allows the selection of DNA aptamers for solute targets. A special SELEX library was constructed with the aim to immobilize this library on magnetic beads or other surfaces. For this purpose a docking sequence was incorporated into the random region of the library enabling hybridization to a complementary oligo fixed on magnetic beads. Oligonucleotides of the library which exhibit high affinity to the target and a secondary structure fitting to the target are released from the beads for binding to the target during the aptamer selection process. The oligonucleotides of these binding complexes were amplified, purified, and immobilized via the docking sequence to the magnetic beads as the starting point of the following selection round. Based on this Capture-SELEX procedure, the successful DNA aptamer selection for the aminoglycoside antibiotic kanamycin A as a small molecule target is described. PMID:23326761

In vitro Fab display: a cell-free system for IgG discovery

PubMed Central

Stafford, Ryan L.; Matsumoto, Marissa L.; Yin, Gang; Cai, Qi; Fung, Juan Jose; Stephenson, Heather; Gill, Avinash; You, Monica; Lin, Shwu-Hwa; Wang, Willie D.; Masikat, Mary Rose; Li, Xiaofan; Penta, Kalyani; Steiner, Alex R.; Baliga, Ramesh; Murray, Christopher J.; Thanos, Christopher D.; Hallam, Trevor J.; Sato, Aaron K.

2014-01-01

Selection technologies such as ribosome display enable the rapid discovery of novel antibody fragments entirely in vitro. It has been assumed that the open nature of the cell-free reactions used in these technologies limits selections to single-chain protein fragments. We present a simple approach for the selection of multi-chain proteins, such as antibody Fab fragments, using ribosome display. Specifically, we show that a two-chain trastuzumab (Herceptin) Fab domain can be displayed in a format which tethers either the heavy or light chain to the ribosome while retaining functional antigen binding. Then, we constructed synthetic Fab HC and LC libraries and performed test selections against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF). The Fab selection output was reformatted into full-length immunoglobulin Gs (IgGs) and directly expressed at high levels in an optimized cell-free system for immediate screening, purification and characterization. Several novel IgGs were identified using this cell-free platform that bind to purified CEA, CEA positive cells and VEGF. PMID:24586053

Situation Selection and Modification for Emotion Regulation in Younger and Older Adults.

PubMed

Livingstone, Kimberly M; Isaacowitz, Derek M

2015-11-01

This research investigated age differences in use and effectiveness of situation selection and situation modification for emotion regulation. Socioemotional selectivity theory suggests stronger emotional well-being goals in older age; emotion regulation may support this goal. Younger and older adults assigned to an emotion regulation or "just view" condition first freely chose to engage with negative, neutral, or positive material (situation selection), then chose to view or skip negative and positive material (situation modification), rating affect after each experience. In both tasks, older adults in both goal conditions demonstrated pro-hedonic emotion regulation, spending less time with negative material compared to younger adults. Younger adults in the regulate condition also engaged in pro-hedonic situation selection, but not modification. Whereas situation selection was related to affect, modification of negative material was not. This research supports more frequent pro-hedonic motivation in older age, as well as age differences in use of early-stage emotion regulation.

Simultaneous oxidation of Hg0 and NH3-SCR of NO by nanophase Ce x Zr y Mn z O2 at low temperature: the interaction and mechanism.

PubMed

Wu, Wanrong; Zeng, Zheng; Lu, Pei; Xing, Yi; Wei, Jianjun; Yue, Huifang; Li, Rui

2018-03-10

Simultaneous oxidation of Hg 0 and NH 3 -SCR of NO by catalyst is one of the key methods for co-purification of coal-fired flue gas. Till now, the interaction between the oxidation of Hg 0 and NH 3 -SCR of NO and its mechanism have not clarified. In this study, a series of nanophase Ce x Zr y Mn z O 2 was prepared for the simultaneous oxidation of Hg 0 and NH 3 -SCR of NO at low temperature. The catalysts were characterized using surface area analysis, X-ray diffraction, temperature-programmed techniques, and several types of microscopy and spectroscopy. The experimental results indicated that the Ce 0.47 Zr 0.22 Mn 0.31 O 2 exhibited superior Hg 0 removal efficiency (> 99%) and NO conversion efficiency (> 90%) even at 150 °C, and it also exhibited a good durability in the presence of SO 2 and H 2 O. The excellent performance of Ce 0.47 Zr 0.22 Mn 0.31 O 2 on co-purifying Hg 0 and NO was due to the stronger synergistic effects of Ce-Zr-Mn in Ce 0.47 Zr 0.22 Mn 0.31 O 2 than that of the others, which was illustrated by the characterization results of XPS, XRD, and FT-IR. Moreover, it was found that the NO conversion of Ce 0.47 Zr 0.22 Mn 0.31 O 2 could be slightly influenced by Hg 0 and was decreased about 4% to the max, while that of Hg 0 could rarely be affected by the selected catalytic reduction process of NO. It might be due to the co-purification mechanism of NO and Hg 0 . The mechanism of the simultaneous oxidation of Hg 0 and NH 3 -SCR of NO was mainly due to the synergetic effect on the mobility of surface oxygen and the activation of lattice oxygen of Ce 0.47 Zr 0.22 Mn 0.31 O 2 . The effect of the oxidation of Hg 0 on the NH 3 -SCR of NO was mainly due to the absorbed Hg 0 /Hg 2+ on the surface of Ce 0.47 Zr 0.22 Mn 0.31 O 2 , which attenuated the formation of NH 3(ad) , -NH 2(ad) , and NH 4 + on its acid sites. Similarly, the NH 3 -SCR of NO process could hardly influence the oxidation of Hg 0 when NO and Hg 0 were co-purified.

Isolation, purification and characterization of antimicrobial protein from seedlings of Bauhinia purpurea L.

PubMed

Sakthivel, Muthu; Palani, Perumal

2016-05-01

A novel antimicrobial protein was purified from the seedlings of Bauhinia purpurea by sequential procedures entailing ammonium sulfate precipitation, cation exchange chromatography, preparative native-PAGE and a yield of 2.7% was obtained from the crude extract. The purified antimicrobial protein appeared as a single protein band on SDS-PAGE with the molecular mass of 20.9 kDa. Purified antimicrobial protein exhibited a potent antimicrobial activity against both Gram-positive and Gram-negative bacteria. Analysis of the trypsin digested peptides of purified protein using the MALDI-TOF MS/MS resulted in the identification of 174 amino acids. The purified protein had an optimum of pH of 5.5 and was stable at 35 °C for exhibiting its maximal antibacterial activity. The addition of metal ions such as Mn(2+) and Ca(2+) to the purified protein enhanced the antimicrobial activity of purified protein. The MIC of purified protein against Bacillus cereus and Escherichia coli were 13 μg/ml and 15 μg/ml, respectively. The purified protein digested the peptidoglycan layer of bacteria which was visualized by TEM analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Spatial genetic structure in continuous and fragmented populations of Pinus pinaster Aiton.

PubMed

De-Lucas, A I; González-Martínez, S C; Vendramin, G G; Hidalgo, E; Heuertz, M

2009-11-01

Habitat fragmentation, i.e., the reduction of populations into small isolated remnants, is expected to increase spatial genetic structure (SGS) in plant populations through nonrandom mating, lower population densities and potential aggregation of reproductive individuals. We investigated the effects of population size reduction and genetic isolation on SGS in maritime pine (Pinus pinaster Aiton) using a combined experimental and simulation approach. Maritime pine is a wind-pollinated conifer which has a scattered distribution in the Iberian Peninsula as a result of forest fires and habitat fragmentation. Five highly polymorphic nuclear microsatellites were genotyped in a total of 394 individuals from two population pairs from the Iberian Peninsula, formed by one continuous and one fragmented population each. In agreement with predictions, SGS was significant and stronger in fragments (Sp = 0.020 and Sp = 0.026) than in continuous populations, where significant SGS was detected for one population only (Sp = 0.010). Simulations suggested that under fat-tailed dispersal, small population size is a stronger determinant of SGS than genetic isolation, while under normal dispersal, genetic isolation has a stronger effect. SGS was always stronger in real populations than in simulations, except if unrealistically narrow dispersal and/or high variance of reproductive success were modelled (even when accounting for potential overestimation of SGS in real populations as a result of short-distance sampling). This suggests that factors such as nonrandom mating or selection not considered in the simulations were additionally operating on SGS in Iberian maritime pine populations.

Distillation Brine Purification for Resource Recovery Applications

NASA Technical Reports Server (NTRS)

Wheeler, Raymond M.

2014-01-01

Wastewater processing systems for space generate residual brine that contains water and salts that could be recovered to life support consumables. The project assessed the use of ion-exchange resins to selectively remove salts from wastewater treatment brines. The resins were then regenerated for additional use. The intention would be to generate a Na/K and CI rich or purified brine that would then be processed into high value chemicals, such as acids, bases, and/or bleach.

Deep sequencing of the LRRK2 gene in 14,002 individuals reveals evidence of purifying selection and independent origin of the p.Arg1628Pro mutation in Europe

PubMed Central

Rubio, Justin P.; Topp, Simon; Warren, Liling; St Jean, Pamela L.; Wegmann, Daniel; Kessner, Darren; Novembre, John; Shen, Judong; Fraser, Dana; Aponte, Jennifer; Nangle, Keith; Cardon, Lon R.; Ehm, Margaret G.; Chissoe, Stephanie L.; Whittaker, John C.; Nelson, Matthew R.; Mooser, Vincent E.

2012-01-01

Genetic variation in LRRK2 predisposes to Parkinson disease (PD), which underpins its development as a therapeutic target. Here, we aimed to identify novel genotype-phenotype associations that might support developing LRRK2 therapies for other conditions. We sequenced the 51 exons of LRRK2 in cases comprising 12 common diseases (n = 9,582), and in 4,420 population controls. We identified 739 single nucleotide variants (SNVs), 62% of which were observed in only one person, including 316 novel exonic variants. We found evidence of purifying selection for the LRRK2 gene and a trend suggesting that this is more pronounced in the central (ROC-COR-kinase) core protein domains of LRRK2 than the flanking domains. Population genetic analyses revealed that LRRK2 is not especially polymorphic or differentiated in comparison to 201 other drug target genes. Amongst Europeans, we identified 17 carriers (0.13%) of pathogenic LRRK2 mutations that were not significantly enriched within any disease or in those reporting a family history of PD. Analysis of pathogenic mutations within Europe reveals that the p.Arg1628Pro (c4883G>C) mutation arose independently in Europe and Asia. Taken together, these findings demonstrate how targeted deep sequencing can help to reveal fundamental characteristics of clinically important loci. PMID:22415848

Deep sequencing of the LRRK2 gene in 14,002 individuals reveals evidence of purifying selection and independent origin of the p.Arg1628Pro mutation in Europe.

PubMed

Rubio, Justin P; Topp, Simon; Warren, Liling; St Jean, Pamela L; Wegmann, Daniel; Kessner, Darren; Novembre, John; Shen, Judong; Fraser, Dana; Aponte, Jennifer; Nangle, Keith; Cardon, Lon R; Ehm, Margaret G; Chissoe, Stephanie L; Whittaker, John C; Nelson, Matthew R; Mooser, Vincent E

2012-07-01

Genetic variation in LRRK2 predisposes to Parkinson disease (PD), which underpins its development as a therapeutic target. Here, we aimed to identify novel genotype-phenotype associations that might support developing LRRK2 therapies for other conditions. We sequenced the 51 exons of LRRK2 in cases comprising 12 common diseases (n = 9,582), and in 4,420 population controls. We identified 739 single-nucleotide variants, 62% of which were observed in only one person, including 316 novel exonic variants. We found evidence of purifying selection for the LRRK2 gene and a trend suggesting that this is more pronounced in the central (ROC-COR-kinase) core protein domains of LRRK2 than the flanking domains. Population genetic analyses revealed that LRRK2 is not especially polymorphic or differentiated in comparison to 201 other drug target genes. Among Europeans, we identified 17 carriers (0.13%) of pathogenic LRRK2 mutations that were not significantly enriched within any disease or in those reporting a family history of PD. Analysis of pathogenic mutations within Europe reveals that the p.Arg1628Pro (c4883G>C) mutation arose independently in Europe and Asia. Taken together, these findings demonstrate how targeted deep sequencing can help to reveal fundamental characteristics of clinically important loci. © 2012 Wiley Periodicals, Inc.

A Unique Report: Development of Super Anti-Human IgG Monoclone with Optical Density Over Than 3

PubMed Central

Aghebati Maleki, Leili; Baradaran, Behzad; Abdolalizadeh, Jalal; Ezzatifar, Fatemeh; Majidi, Jafar

2013-01-01

Purpose: Monoclonal antibodies and related conjugates are key reagents used in biomedical researches as well as, in treatment, purification and diagnosis of infectious and non- infectious diseases. Methods: Balb/c mice were immunized with purified human IgG. Spleen cells of the most immune mouse were fused with SP2/0 in the presence of Poly Ethylene Glycol (PEG). Supernatant of hybridoma cells was screened for detection of antibody by ELISA. Then, the sample was assessed for cross-reactivity with IgM & IgA by ELISA and confirmed by immunoblotting. The subclasses of the selected mAbs were determined. The best clone was injected intraperitoneally to some pristane-injected mice. Anti-IgG mAb was purified from the animals' ascitic fluid by Ion exchange chromatography and then, mAb was conjugated with HRP. Results: In the present study, over than 50 clones were obtained that 1 clone had optical density over than 3. We named this clone as supermonoclone which was selected for limiting dilution. The result of the immunoblotting, showed sharp band in IgG position and did not show any band in IgM&IgA position. Conclusion: Based on the findings of this study, the conjugated monoclonal antibody could have application in diagnosis of infectious diseases like Toxoplasmosis, Rubella and IgG class of other infectious and non- infectious diseases. PMID:24312857

Identification of the immunogenic epitopes of the whole venom component of the Hemiscorpius lepturus scorpion using the phage display peptide library.

PubMed

Jahdasani, Roghaye; Jamnani, Fatemeh Rahimi; Behdani, Mahdi; Habibi-Anbouhi, Mahdi; Yardehnavi, Najmeh; Shahbazzadeh, Delavar; Kazemi-Lomedasht, Fatemeh

2016-12-15

The venom of the Hemiscorpius lepturus scorpion contains mixtures of bioactive compounds that disturb biochemical and physiological functions of the victims. Hemiscorpius lepturus envenomation is recognized as a serious health concern in tropical regions. So far, there is no preventive procedure, and the main focus is on treatment of victims with an antiserum purified from hyper-immunized horses. Although antisera can neutralize the venom, they, in some cases, lead to anaphylactic shock and even death. Selection of peptides mimicking antigenic and immunogenic epitopes of toxins from random peptide libraries is a novel approach for the development of recombinant toxins and poly-epitopic vaccine. To achieve this aim, a phage display peptide library and three rounds of biopanning were performed on immobilized antibodies (IgGs) purified from the sera of hyper-immunized horses. The results show that the highest binding of the phage to immobilized horse antibodies occurred in the third round of biopanning. Over 125 individual clones carrying mimotopes of Hemiscorpius lepturus toxins were selected and subjected for sequencing. The sequencing results identified unique peptides mimicking the antigenic and immunogenic epitopes of Hemiscorpius lepturus toxins. The results of this study provide a basis for further studies and the development of a putative epitopic vaccine and a recombinant toxin. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Interplay of Temperature and Genotype on Patterns of Alternative Splicing in Drosophila melanogaster.

PubMed

Jakšić, Ana Marija; Schlötterer, Christian

2016-09-01

Alternative splicing is the highly regulated process of variation in the removal of introns from premessenger-RNA transcripts. The consequences of alternative splicing on the phenotype are well documented, but the impact of the environment on alternative splicing is not yet clear. We studied variation in alternative splicing among four different temperatures, 13, 18, 23, and 29°, in two Drosophila melanogaster genotypes. We show plasticity of alternative splicing with up to 10% of the expressed genes being differentially spliced between the most extreme temperatures for a given genotype. Comparing the two genotypes at different temperatures, we found <1% of the genes being differentially spliced at 18°. At extreme temperatures, however, we detected substantial differences in alternative splicing-with almost 10% of the genes having differential splicing between the genotypes: a magnitude similar to between species differences. Genes with differential alternative splicing between genotypes frequently exhibit dominant inheritance. Remarkably, the pattern of surplus of differences in alternative splicing at extreme temperatures resembled the pattern seen for gene expression intensity. Since different sets of genes were involved for the two phenotypes, we propose that purifying selection results in the reduction of differences at benign temperatures. Relaxed purifying selection at temperature extremes, on the other hand, may cause the divergence in gene expression and alternative splicing between the two strains in rarely encountered environments. Copyright © 2016 by the Genetics Society of America.

Highly selective water channel activity measured by voltage clamp: analysis of planar lipid bilayers reconstituted with purified AqpZ.

PubMed

Pohl, P; Saparov, S M; Borgnia, M J; Agre, P

2001-08-14

Aquaporins are membrane channels selectively permeated by water or water plus glycerol. Conflicting reports have described ion conductance associated with some water channels, raising the question of whether ion conductance is a general property of the aquaporin family. To clarify this question, a defined system was developed to simultaneously measure water permeability and ion conductance. The Escherichia coli water channel aquaporin-Z (AqpZ) was studied, because it is a highly stable tetramer. Planar lipid bilayers were formed from unilamellar vesicles containing purified AqpZ. The hydraulic conductivity of bilayers made from the total extract of E. coli lipids increased 3-fold if reconstituted with AqpZ, but electric conductance was unchanged. No channel activity was detected under voltage-clamp conditions, indicating that less than one in 10(9) transport events is electrogenic. Microelectrode measurements were simultaneously undertaken adjacent to the membrane. Changes in sodium concentration profiles accompanying transmembrane water flow permitted calculation of the activation energies: 14 kcal/mol for protein-free lipid bilayers and 4 kcal/mol for lipid bilayers containing AqpZ. Neither the water permeability nor the electric conductivity exhibited voltage dependence. This sensitive system demonstrated that AqpZ is permeated by water but not charged ions and should permit direct analyses of putative electrogenic properties of other aquaporins.

Evolution and functional divergence of the anoctamin family of membrane proteins

PubMed Central

2010-01-01

Background The anoctamin family of transmembrane proteins are found in all eukaryotes and consists of 10 members in vertebrates. Ano1 and ano2 were observed to have Ca2+ activated Cl- channel activity. Recent findings however have revealed that ano6, and ano7 can also produce chloride currents, although with different properties. In contrast, ano9 and ano10 suppress baseline Cl- conductance when co-expressed with ano1 thus suggesting that different anoctamins can interfere with each other. In order to elucidate intrinsic functional diversity, and underlying evolutionary mechanism among anoctamins, we performed comprehensive bioinformatics analysis of anoctamin gene family. Results Our results show that anoctamin protein paralogs evolved from several gene duplication events followed by functional divergence of vertebrate anoctamins. Most of the amino acid replacements responsible for the functional divergence were fixed by adaptive evolution and this seem to be a common pattern in anoctamin gene family evolution. Strong purifying selection and the loss of many gene duplication products indicate rigid structure-function relationships among anoctamins. Conclusions Our study suggests that anoctamins have evolved by series of duplication events, and that they are constrained by purifying selection. In addition we identified a number of protein domains, and amino acid residues which contribute to predicted functional divergence. Hopefully, this work will facilitate future functional characterization of the anoctamin membrane protein family. PMID:20964844

Production of a novel camel single-domain antibody specific for the type III mutant EGFR.

PubMed

Omidfar, K; Rasaee, M J; Modjtahedi, H; Forouzandeh, M; Taghikhani, M; Golmakani, N

2004-01-01

Camelids have a unique immune system capable of producing single-domain heavy-chain antibodies. The antigen-specific domain of these heavy-chain IgGs (VHH) are the smallest binding units produced by the immune system. In this study, we report the isolation and characterization of several binders against the epidermal growth factor receptor (EGFR) vIII retrieved from immune library of camels (Camelus bactrianus and Camelus dromedarius). The EGFRvIII is a ligand-independent, constitutively active, mutated form of the wild-type EGFR. The expression of EGFRvIII has been demonstrated in a wide range of human malignancies, including gliomas, and breast, prostate, ovarian and lung cancer. Camels were immunized with a synthetic peptide corresponding to a mutated sequence and tissue homogenates. Single-domain antibodies (VHH) were directly selected by panning a phage display library on successively decreasing amounts of synthetic peptide immobilized on magnetic beads. The anti-EGFRvIII camel single-domain antibodies selectively bound to the EGFRvIII peptide and reacted specifically with the immunoaffinity-purified antigen from a non-small cell lung cancer patient. These antibodies with affinities in the nanomolar range recognized the EGFRvIII peptide and affinity-purified mutated receptor. We concluded that using the phage display technique, antigen-specific VHH antibody fragments are readily accessible from the camelids. These antibodies may be good candidates for tumor-diagnostic and therapeutic applications. Copyright 2004 S. Karger AG, Basel.

Aspartate aminotransferase is potently inhibited by copper complexes: Exploring copper complex-binding proteome.

PubMed

Jia, Yuqi; Lu, Liping; Yuan, Caixia; Feng, Sisi; Zhu, Miaoli

2017-05-01

Recent researches indicated that a copper complex-binding proteome that potently interacted with copper complexes and then influenced cellular metabolism might exist in organism. In order to explore the copper complex-binding proteome, a copper chelating ion-immobilized affinity chromatography (Cu-IMAC) column and mass spectrometry were used to separate and identify putative Cu-binding proteins in primary rat hepatocytes. A total of 97 putative Cu-binding proteins were isolated and identified. Five higher abundance proteins, aspartate aminotransferase (AST), malate dehydrogenase (MDH), catalase (CAT), calreticulin (CRT) and albumin (Alb) were further purified using a SP-, and (or) Q-Sepharose Fast Flow column. The interaction between the purified proteins and selected 11 copper complexes and CuCl 2 was investigated. The enzymes inhibition tests demonstrated that AST was potently inhibited by copper complexes while MDH and CAT were weakly inhibited. Schiff-based copper complexes 6 and 7 potently inhibited AST with the IC 50 value of 3.6 and 7.2μM, respectively and exhibited better selectivity over MDH and CAT. Fluorescence titration results showed the two complexes tightly bound to AST with binding constant of 3.89×10 6 and 3.73×10 6 M -1 , respectively and a stoichiometry ratio of 1:1. Copper complex 6 was able to enter into HepG2 cells and further inhibit intracellular AST activity. Copyright © 2017 Elsevier Inc. All rights reserved.

Different level of population differentiation among human genes.

PubMed

Wu, Dong-Dong; Zhang, Ya-Ping

2011-01-14

During the colonization of the world, after dispersal out of African, modern humans encountered changeable environments and substantial phenotypic variations that involve diverse behaviors, lifestyles and cultures, were generated among the different modern human populations. Here, we study the level of population differentiation among different populations of human genes. Intriguingly, genes involved in osteoblast development were identified as being enriched with higher FST SNPs, a result consistent with the proposed role of the skeletal system in accounting for variation among human populations. Genes involved in the development of hair follicles, where hair is produced, were also found to have higher levels of population differentiation, consistent with hair morphology being a distinctive trait among human populations. Other genes that showed higher levels of population differentiation include those involved in pigmentation, spermatid, nervous system and organ development, and some metabolic pathways, but few involved with the immune system. Disease-related genes demonstrate excessive SNPs with lower levels of population differentiation, probably due to purifying selection. Surprisingly, we find that Mendelian-disease genes appear to have a significant excessive of SNPs with high levels of population differentiation, possibly because the incidence and susceptibility of these diseases show differences among populations. As expected, microRNA regulated genes show lower levels of population differentiation due to purifying selection. Our analysis demonstrates different level of population differentiation among human populations for different gene groups.

Detergent solubilization of the EGF receptor from A431 cells

NASA Technical Reports Server (NTRS)

Dayanidhi, R.; Rintoul, D. A.; Spooner, B. S. (Principal Investigator)

1993-01-01

Functional reconstitution of purified preparations of human epidermal growth factor receptor (EGFR) requires dissociation of the protein from its plasma membrane lipid environment. Solubilization of membrane proteins in this manner requires the use of detergents, which are known to disrupt plasma membrane lipid/protein interactions. We have investigated the ability of three nonionic detergents to solubilize the human EGFR selectively, and have also analyzed the effect of these various treatments on the intrinsic tyrosyl kinase activity of the receptor. The nonionic detergent known as n-octyl glucoside (n-octyl beta-D-glucopyranoside) was found to give the best combination of selectivity, yield, and maintenance of enzymatic activity of the human EGFR.

Cloning, expression and purification of d-tagatose 3-epimerase gene from Escherichia coli JM109.

PubMed

He, Xiaoliang; Zhou, Xiaohui; Yang, Zi; Xu, Le; Yu, Yuxiu; Jia, Lingling; Li, Guoqing

2015-10-01

An unknown d-tagatose 3-epimerase (DTE) containing a IoIE domain was identified and cloned from Escherichia coli. This gene was subcloned into the prokaryotic expression vector pET-15b, and induced by IPTG in E. coli BL21 expression system. Through His-select gel column purification and fast-protein liquid chromatography, highly purified and stable DTE protein was produced. The molecular weight of the DTE protein was estimated to be 29.8kDa. The latest 83 DTE sequences from public database were selected and analyzed by molecular clustering, multi-sequence alignment. DTEs were roughly divided into five categories. Copyright © 2015 Elsevier Inc. All rights reserved.

21 CFR 880.6710 - Medical ultraviolet water purifier.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Medical ultraviolet water purifier. 880.6710... Miscellaneous Devices § 880.6710 Medical ultraviolet water purifier. (a) Identification. A medical ultraviolet water purifier is a device intended for medical purposes that is used to destroy bacteria in water by...

21 CFR 880.6710 - Medical ultraviolet water purifier.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Medical ultraviolet water purifier. 880.6710... Miscellaneous Devices § 880.6710 Medical ultraviolet water purifier. (a) Identification. A medical ultraviolet water purifier is a device intended for medical purposes that is used to destroy bacteria in water by...

42 CFR 84.181 - Non-powered air-purifying particulate filter efficiency level determination.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 1 2010-10-01 2010-10-01 false Non-powered air-purifying particulate filter...-purifying particulate filter efficiency level determination. (a) Twenty filters of each non-powered air-purifying particulate respirator model shall be tested for filter efficiency against: (1) A solid sodium...

42 CFR 84.181 - Non-powered air-purifying particulate filter efficiency level determination.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 1 2011-10-01 2011-10-01 false Non-powered air-purifying particulate filter...-purifying particulate filter efficiency level determination. (a) Twenty filters of each non-powered air-purifying particulate respirator model shall be tested for filter efficiency against: (1) A solid sodium...

42 CFR 84.181 - Non-powered air-purifying particulate filter efficiency level determination.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 1 2012-10-01 2012-10-01 false Non-powered air-purifying particulate filter...-purifying particulate filter efficiency level determination. (a) Twenty filters of each non-powered air-purifying particulate respirator model shall be tested for filter efficiency against: (1) A solid sodium...

42 CFR 84.181 - Non-powered air-purifying particulate filter efficiency level determination.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 1 2014-10-01 2014-10-01 false Non-powered air-purifying particulate filter...-purifying particulate filter efficiency level determination. (a) Twenty filters of each non-powered air-purifying particulate respirator model shall be tested for filter efficiency against: (1) A solid sodium...

42 CFR 84.181 - Non-powered air-purifying particulate filter efficiency level determination.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 42 Public Health 1 2013-10-01 2013-10-01 false Non-powered air-purifying particulate filter...-purifying particulate filter efficiency level determination. (a) Twenty filters of each non-powered air-purifying particulate respirator model shall be tested for filter efficiency against: (1) A solid sodium...

21 CFR 880.6710 - Medical ultraviolet water purifier.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Medical ultraviolet water purifier. 880.6710... Miscellaneous Devices § 880.6710 Medical ultraviolet water purifier. (a) Identification. A medical ultraviolet water purifier is a device intended for medical purposes that is used to destroy bacteria in water by...

76 FR 66687 - Purified Carboxymethylcellulose From the Netherlands: Final Results of Antidumping Duty...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-10-27

... antidumping duty administrative review of purified carboxymethylcellulose (CMC) from the Netherlands, covering...) (Preliminary Results). The merchandise covered by the order is purified CMC, as described in the ``Scope of the... duty order on purified CMC from the Netherlands. See Preliminary Results. The respondent under review...

21 CFR 880.6710 - Medical ultraviolet water purifier.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Medical ultraviolet water purifier. 880.6710... Miscellaneous Devices § 880.6710 Medical ultraviolet water purifier. (a) Identification. A medical ultraviolet water purifier is a device intended for medical purposes that is used to destroy bacteria in water by...

Interactions of Multimodal Ligands with Proteins: Insights into Selectivity Using Molecular Dynamics Simulations.

PubMed

Parimal, Siddharth; Garde, Shekhar; Cramer, Steven M

2015-07-14

Fundamental understanding of protein-ligand interactions is important to the development of efficient bioseparations in multimodal chromatography. Here we employ molecular dynamics (MD) simulations to investigate the interactions of three different proteins--ubiquitin, cytochrome C, and α-chymotrypsinogen A, sampling a range of charge from +1e to +9e--with two multimodal chromatographic ligands containing similar chemical moieties--aromatic, carboxyl, and amide--in different structural arrangements. We use a spherical harmonic expansion to analyze ligand and individual moiety density profiles around the proteins. We find that the Capto MMC ligand, which contains an additional aliphatic group, displays stronger interactions than Nuvia CPrime ligand with all three proteins. Studying the ligand densities at the moiety level suggests that hydrophobic interactions play a major role in determining the locations of high ligand densities. Finally, the greater structural flexibility of the Capto MMC ligand compared to that of the Nuvia cPrime ligand allows for stronger structural complementarity and enables stronger hydrophobic interactions. These subtle and not-so-subtle differences in binding affinities and modalities for multimodal ligands can result in significantly different binding behavior towards proteins with important implications for bioprocessing.

In situ vibrational spectroscopy of adsorbed nitrogen in porous carbon materials.

PubMed

Ray, Paramita; Xu, Enshi; Crespi, Vincent H; Badding, John V; Lueking, Angela D

2018-05-25

This study uses in situ vibrational spectroscopy to probe nitrogen adsorption to porous carbon materials, including single-wall carbon nanotubes and Maxsorb super-activated carbon, demonstrating how the nitrogen Raman stretch mode is perturbed by adsorption. In all porous carbon samples upon N2 physisorption in the mesopore filling regime, the N2 Raman mode downshifts by ∼2 cm-1, a downshift comparable to liquid N2. The relative intensity of this mode increases as pressure is increased to saturation, and trends in the relative intensity parallel the volumetric gas adsorption isotherm. This mode with ∼2 cm-1 downshift is thus attributed to perturbations arising due to N2-N2 interactions in a condensed film. The mode is also observed for the activated carbon at 298 K, and the relative intensity once again parallels the gas adsorption isotherm. For select samples, a mode with a stronger downshift (>4 cm-1) is observed, and the stronger downshift is attributed to stronger N2-carbon surface interactions. Simulations for a N2 surface film support peak assignments. These results suggest that N2 vibrational spectroscopy could provide an indication of the presence or absence of porosity for very small quantities of samples.

Genotypic Diversity Effects on the Performance of Taraxacum officinale Populations Increase with Time and Environmental Favorability

PubMed Central

Drummond, Emily B. M.; Vellend, Mark

2012-01-01

Within-population genetic diversity influences many ecological processes, but few studies have examined how environmental conditions may impact these short-term diversity effects. Over four growing seasons, we followed experimental populations of a clonal, ubiquitous weed, Taraxacum officinale, with different numbers of genotypes in relatively favorable fallow field and unfavorable mowed lawn environmental treatments. Population performance (measured as total leaf area, seed production or biomass) clearly and consistently increased with diversity, and this effect became stronger over the course of the experiment. Diversity effects were stronger, and with different underlying mechanisms, in the fallow field versus the mowed lawn. Large genotypes dominated in the fallow field driving overyielding (via positive selection effects), whereas in the mowed lawn, where performance was limited by regular disturbance, there was evidence for complementarity among genotypes (with one compact genotype in particular performing better in mixture than monoculture). Hence, we predict stronger genotypic diversity effects in environments where intense intraspecific competition enhances genotypic differences. Our four-year field experiment plus seedling establishment trials indicate that genotypic diversity effects have far-reaching and context-dependent consequences across generations. PMID:22348004

Psychological Type and Undergraduate Student Achievement in Pharmacy Course in Military Medical University

ERIC Educational Resources Information Center

Shi, Ru; Shan, Shou-qin; Tian, Jian-quan

2007-01-01

The Myers-Briggs Type Indicator (MBTI) was given to 264 students in an undergraduate Pharmacy course at a military medical university. Selected MBTI personality types were compared for achievement in the course using a t-test to compare total points earned. High grades were earned by students stronger in the traits of introversion (I) and judgment…

Electrophysiological evidence for cognitive control during conflict processing in visual spatial attention.

PubMed

Kehrer, Stefanie; Kraft, Antje; Irlbacher, Kerstin; Koch, Stefan P; Hagendorf, Herbert; Kathmann, Norbert; Brandt, Stephan A

2009-11-01

Event-related potentials were measured to investigate the role of visual spatial attention mechanisms in conflict processing. We suggested that a more difficult target selection leads to stronger attentional top-down control, thereby reducing the effects of arising conflicts. This hypothesis was tested by varying the selection difficulty in a location negative priming (NP) paradigm. The difficult task resulted in prolonged responses as compared to the easy task. A behavioral NP effect was only evident in the easy task. Psychophysiologically the easy task was associated with reduced parietal N1, enhanced frontocentral N2 and N2pc components and a prolonged P3 latency for the conflict as compared to the control condition. The N2pc effect was also obvious in the difficult task. Additionally frontocentral N2 amplitudes increased and latencies of N2pc and P3 were delayed compared to the easy task. The differences at frontocentral and parietal electrodes are consistent with previous studies ascribing activity in the prefrontal and parietal cortex as the source of top-down attentional control. Thus, we propose that stronger cognitive control is involved in the difficult task, resulting in a reduced behavioral NP conflict.

Early growth, dominance acquisition and lifetime reproductive success in male and female cooperative meerkats

PubMed Central

English, Sinead; Huchard, Elise; Nielsen, Johanna F; Clutton-Brock, Tim H

2013-01-01

In polygynous species, variance in reproductive success is higher in males than females. There is consequently stronger selection for competitive traits in males and early growth can have a greater influence on later fitness in males than in females. As yet, little is known about sex differences in the effect of early growth on subsequent breeding success in species where variance in reproductive success is higher in females than males, and competitive traits are under stronger selection in females. Greater variance in reproductive success has been documented in several singular cooperative breeders. Here, we investigated consequences of early growth for later reproductive success in wild meerkats. We found that, despite the absence of dimorphism, females who exhibited faster growth until nutritional independence were more likely to become dominant, whereas early growth did not affect dominance acquisition in males. Among those individuals who attained dominance, there was no further influence of early growth on dominance tenure or lifetime reproductive success in males or females. These findings suggest that early growth effects on competitive abilities and fitness may reflect the intensity of intrasexual competition even in sexually monomorphic species. PMID:24340181

Amino acid usage is asymmetrically biased in AT- and GC-rich microbial genomes.

PubMed

Bohlin, Jon; Brynildsrud, Ola; Vesth, Tammi; Skjerve, Eystein; Ussery, David W

2013-01-01

Genomic base composition ranges from less than 25% AT to more than 85% AT in prokaryotes. Since only a small fraction of prokaryotic genomes is not protein coding even a minor change in genomic base composition will induce profound protein changes. We examined how amino acid and codon frequencies were distributed in over 2000 microbial genomes and how these distributions were affected by base compositional changes. In addition, we wanted to know how genome-wide amino acid usage was biased in the different genomes and how changes to base composition and mutations affected this bias. To carry this out, we used a Generalized Additive Mixed-effects Model (GAMM) to explore non-linear associations and strong data dependences in closely related microbes; principal component analysis (PCA) was used to examine genomic amino acid- and codon frequencies, while the concept of relative entropy was used to analyze genomic mutation rates. We found that genomic amino acid frequencies carried a stronger phylogenetic signal than codon frequencies, but that this signal was weak compared to that of genomic %AT. Further, in contrast to codon usage bias (CUB), amino acid usage bias (AAUB) was differently distributed in AT- and GC-rich genomes in the sense that AT-rich genomes did not prefer specific amino acids over others to the same extent as GC-rich genomes. AAUB was also associated with relative entropy; genomes with low AAUB contained more random mutations as a consequence of relaxed purifying selection than genomes with higher AAUB. Genomic base composition has a substantial effect on both amino acid- and codon frequencies in bacterial genomes. While phylogeny influenced amino acid usage more in GC-rich genomes, AT-content was driving amino acid usage in AT-rich genomes. We found the GAMM model to be an excellent tool to analyze the genomic data used in this study.

Amino Acid Usage Is Asymmetrically Biased in AT- and GC-Rich Microbial Genomes

PubMed Central

Bohlin, Jon; Brynildsrud, Ola; Vesth, Tammi; Skjerve, Eystein; Ussery, David W.

2013-01-01

Introduction Genomic base composition ranges from less than 25% AT to more than 85% AT in prokaryotes. Since only a small fraction of prokaryotic genomes is not protein coding even a minor change in genomic base composition will induce profound protein changes. We examined how amino acid and codon frequencies were distributed in over 2000 microbial genomes and how these distributions were affected by base compositional changes. In addition, we wanted to know how genome-wide amino acid usage was biased in the different genomes and how changes to base composition and mutations affected this bias. To carry this out, we used a Generalized Additive Mixed-effects Model (GAMM) to explore non-linear associations and strong data dependences in closely related microbes; principal component analysis (PCA) was used to examine genomic amino acid- and codon frequencies, while the concept of relative entropy was used to analyze genomic mutation rates. Results We found that genomic amino acid frequencies carried a stronger phylogenetic signal than codon frequencies, but that this signal was weak compared to that of genomic %AT. Further, in contrast to codon usage bias (CUB), amino acid usage bias (AAUB) was differently distributed in AT- and GC-rich genomes in the sense that AT-rich genomes did not prefer specific amino acids over others to the same extent as GC-rich genomes. AAUB was also associated with relative entropy; genomes with low AAUB contained more random mutations as a consequence of relaxed purifying selection than genomes with higher AAUB. Conclusion Genomic base composition has a substantial effect on both amino acid- and codon frequencies in bacterial genomes. While phylogeny influenced amino acid usage more in GC-rich genomes, AT-content was driving amino acid usage in AT-rich genomes. We found the GAMM model to be an excellent tool to analyze the genomic data used in this study. PMID:23922837

Immunogenicity of T7 bacteriophage nanoparticles displaying G-H loop of foot-and-mouth disease virus (FMDV).

PubMed

Xu, Hai; Bao, Xi; Lu, Yu; Liu, Yamei; Deng, Bihua; Wang, Yiwei; Xu, Yue; Hou, Jibo

2017-06-01

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals that causes severe economic losses worldwide. The G-H loop of the FMDV VP1 structural protein is the major neutralizing antigenic site. However, a fully protective G-H loop peptide vaccine requires the addition of promiscuous Th sites from a source outside VP1. Thus, we demonstrated the potential of T7 bacteriophage based nanoparticles displaying a genetically fused G-H loop peptide (T7-GH) as a FMDV vaccine candidate. Recombinant T7-GH phage was constructed by inserting the G-H loop coding region into the T7 Select 415-1b vector. Purified T7-GH phage nanoparticles were analyzed by SDS-PAGE, Western blot and Dot-ELISA. Pigs seronegative for FMDV exposure were immunized with T7-GH nanoparticles along with the adjuvant Montanide ISA206, and two commercially available FMDV vaccines (InactVac and PepVac). Humoral and cellular immune responses, as well as protection against virulent homologous virus challenge were assessed following single dose immunization. Pigs immunized T7-GH developed comparable anti-VP1 antibody titers to PepVac, although lower LPBE titers than was induced by InactVac. Antigen specific lymphocyte proliferation was detected in T7-GH group similar to that of PepVac group, however, weaker than InactVac group. Pigs immunized with T7-GH developed a neutralizing antibody response stronger than PepVac, but weaker than InactVac. Furthermore, 80% (4/5) of T7-GH immunized pigs were protected from challenge with virulent homologous virus. These findings demonstrate that the T7-GH phage nanoparticles were effective in eliciting antigen specific immune responses in pigs, highlighting the value of such an approach in the research and development of FMDV vaccines. Copyright © 2017 Elsevier B.V. All rights reserved.

40 CFR 1065.845 - Response factor determination.

Code of Federal Regulations, 2010 CFR

2010-07-01

... zero and span balance gases may be any combination of purified air or purified nitrogen that meets the... to respond with a value of 600 µmol/mol. (5) Zero the FID. Note that FID zero and span balance gases may be any combination of purified air or purified nitrogen that meets the specifications of § 1065...

40 CFR 1065.845 - Response factor determination.

Code of Federal Regulations, 2013 CFR

2013-07-01

... zero and span balance gases may be any combination of purified air or purified nitrogen that meets the... to respond with a value of 600 µmol/mol. (5) Zero the FID. Note that FID zero and span balance gases may be any combination of purified air or purified nitrogen that meets the specifications of § 1065...

40 CFR 1065.845 - Response factor determination.

Code of Federal Regulations, 2011 CFR

2011-07-01

... zero and span balance gases may be any combination of purified air or purified nitrogen that meets the... to respond with a value of 600 µmol/mol. (5) Zero the FID. Note that FID zero and span balance gases may be any combination of purified air or purified nitrogen that meets the specifications of § 1065...

40 CFR 1065.845 - Response factor determination.

Code of Federal Regulations, 2012 CFR

2012-07-01

... zero and span balance gases may be any combination of purified air or purified nitrogen that meets the... to respond with a value of 600 µmol/mol. (5) Zero the FID. Note that FID zero and span balance gases may be any combination of purified air or purified nitrogen that meets the specifications of § 1065...

21 CFR 880.6500 - Medical ultraviolet air purifier.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Medical ultraviolet air purifier. 880.6500 Section... Miscellaneous Devices § 880.6500 Medical ultraviolet air purifier. (a) Identification. A medical ultraviolet air purifier is a device intended for medical purposes that is used to destroy bacteria in the air by exposure...

21 CFR 880.6500 - Medical ultraviolet air purifier.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Medical ultraviolet air purifier. 880.6500 Section... Miscellaneous Devices § 880.6500 Medical ultraviolet air purifier. (a) Identification. A medical ultraviolet air purifier is a device intended for medical purposes that is used to destroy bacteria in the air by exposure...

21 CFR 880.6500 - Medical ultraviolet air purifier.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Medical ultraviolet air purifier. 880.6500 Section... Miscellaneous Devices § 880.6500 Medical ultraviolet air purifier. (a) Identification. A medical ultraviolet air purifier is a device intended for medical purposes that is used to destroy bacteria in the air by exposure...

Comparative study of disinfectants for use in low-cost gravity driven household water purifiers.

PubMed

Patil, Rajshree A; Kausley, Shankar B; Balkunde, Pradeep L; Malhotra, Chetan P

2013-09-01

Point-of-use (POU) gravity-driven household water purifiers have been proven to be a simple, low-cost and effective intervention for reducing the impact of waterborne diseases in developing countries. The goal of this study was to compare commonly used water disinfectants for their feasibility of adoption in low-cost POU water purifiers. The potency of each candidate disinfectant was evaluated by conducting a batch disinfection study for estimating the concentration of disinfectant needed to inactivate a given concentration of the bacterial strain Escherichia coli ATCC 11229. Based on the concentration of disinfectant required, the size, weight and cost of a model purifier employing that disinfectant were estimated. Model purifiers based on different disinfectants were compared and disinfectants which resulted in the most safe, compact and inexpensive purifiers were identified. Purifiers based on bromine, tincture iodine, calcium hypochlorite and sodium dichloroisocyanurate were found to be most efficient, cost effective and compact with replacement parts costing US$3.60-6.00 for every 3,000 L of water purified and are thus expected to present the most attractive value proposition to end users.

Effects of high pressure processing on activity and structure of soluble acid invertase in mango pulp, crude extract, purified form and model systems.

PubMed

Li, Renjie; Wang, Yongtao; Ling, Jiangang; Liao, Xiaojun

2017-09-15

The effects of high pressure processing (HPP) on the activity of soluble acid invertase (SAI) in mango pulp, crude extract, purified SAI and purified SAI in model systems (pectin, bovine serum albumin (BSA), sugars and pH 3-7) were investigated. The activity of SAI in mango pulp was increased after HPP, and that in crude extract stayed unchanged. The activity of purified SAI was decreased after HPP at 45 and 50°C. Pectin exhibited a concentration-dependent protection for purified SAI against HPP at 50°C/600MPa for 30min. Pectin that had an esterification degree (DE) of 85% exhibited a greater protection than pectin that had a DE of 20-34%. BSA, acidic pH (3-6) and sucrose also exhibited protection for purified SAI against HPP. HPP at 50°C/600MPa for 30min disrupted the secondary structure and tertiary structure of purified SAI, but no aggregation of purified SAI was observed after HPP. Copyright © 2017 Elsevier Ltd. All rights reserved.

Natural Selection in the Great Apes

PubMed Central

Cagan, Alexander; Theunert, Christoph; Laayouni, Hafid; Santpere, Gabriel; Pybus, Marc; Casals, Ferran; Prüfer, Kay; Navarro, Arcadi; Marques-Bonet, Tomas; Bertranpetit, Jaume; Andrés, Aida M.

2016-01-01

Natural selection is crucial for the adaptation of populations to their environments. Here, we present the first global study of natural selection in the Hominidae (humans and great apes) based on genome-wide information from population samples representing all extant species (including most subspecies). Combining several neutrality tests we create a multi-species map of signatures of natural selection covering all major types of natural selection. We find that the estimated efficiency of both purifying and positive selection varies between species and is significantly correlated with their long-term effective population size. Thus, even the modest differences in population size among the closely related Hominidae lineages have resulted in differences in their ability to remove deleterious alleles and to adapt to changing environments. Most signatures of balancing and positive selection are species-specific, with signatures of balancing selection more often being shared among species. We also identify loci with evidence of positive selection across several lineages. Notably, we detect signatures of positive selection in several genes related to brain function, anatomy, diet and immune processes. Our results contribute to a better understanding of human evolution by putting the evidence of natural selection in humans within its larger evolutionary context. The global map of natural selection in our closest living relatives is available as an interactive browser at http://tinyurl.com/nf8qmzh. PMID:27795229

The Susceptibilities of Respiratory Syncytial Virus to Nucleolin Receptor Blocking and Antibody Neutralization Are Dependent upon the Method of Virus Purification

PubMed Central

Bilawchuk, Leanne M.; Jensen, Lionel D.; Marchant, David J.

2017-01-01

Respiratory Syncytial Virus (RSV) that is propagated in cell culture is purified from cellular contaminants that can confound experimental results. A number of different purification methods have been described, including methods that utilize fast protein liquid chromatography (FPLC) and gradient ultracentrifugation. Thus, the constituents and experimental responses of RSV stocks purified by ultracentrifugation in sucrose and by FPLC were analyzed and compared by infectivity assay, Coomassie stain, Western blot, mass spectrometry, immuno-transmission electron microscopy (TEM), and ImageStream flow cytometry. The FPLC-purified RSV had more albumin contamination, but there was less evidence of host-derived exosomes when compared to ultracentrifugation-purified RSV as detected by Western blot and mass spectrometry for the exosome markers superoxide dismutase [Cu-Zn] (SOD1) and the tetraspanin CD63. Although the purified virus stocks were equally susceptible to nucleolin-receptor blocking by the DNA aptamer AS1411, the FPLC-purified RSV was significantly less susceptible to anti-RSV polyclonal antibody neutralization; there was 69% inhibition (p = 0.02) of the sucrose ultracentrifugation-purified RSV, 38% inhibition (p = 0.03) of the unpurified RSV, but statistically ineffective neutralization in the FPLC-purified RSV (22% inhibition; p = 0.30). The amount of RSV neutralization of the purified RSV stocks was correlated with anti-RSV antibody occupancy on RSV particles observed by immuno-TEM. RSV purified by different methods alters the stock composition and morphological characteristics of virions that can lead to different experimental responses. PMID:28771197

The Relationship Between Athletic Identity and Academic Major Chosen by Student-Athletes.

PubMed

Foster, Sayvon J L; Huml, Matt R

2017-01-01

This study examines the correlation between athletic identity and academic major selection among intercollegiate student-athletes. A thorough review of literature focusing on academic clustering, athletic identity, and academic development leads to the development of two hypotheses - 1) student-athletes with stronger athletic identity will have a declared major of decreased academic rigor; and 2) student-athletes with stronger athletic identity will be more likely to be undecided on their major. Data were collected through a survey administered to Division I, II, and III student-athletes recording academic major and their Athletic Identity Measurement Scale (AIMS). After analyzing the student responses, Hypothesis I is supported, while Hypothesis II is met with some limitation that leads to a lack of statistical significance. Overall, this study sheds light on a connection between academic choice and athletic identity.

The Relationship Between Athletic Identity and Academic Major Chosen by Student-Athletes

PubMed Central

FOSTER, SAYVON J.L.; HUML, MATT R.

2017-01-01

This study examines the correlation between athletic identity and academic major selection among intercollegiate student-athletes. A thorough review of literature focusing on academic clustering, athletic identity, and academic development leads to the development of two hypotheses – 1) student-athletes with stronger athletic identity will have a declared major of decreased academic rigor; and 2) student-athletes with stronger athletic identity will be more likely to be undecided on their major. Data were collected through a survey administered to Division I, II, and III student-athletes recording academic major and their Athletic Identity Measurement Scale (AIMS). After analyzing the student responses, Hypothesis I is supported, while Hypothesis II is met with some limitation that leads to a lack of statistical significance. Overall, this study sheds light on a connection between academic choice and athletic identity. PMID:29170694

Transformational leadership, initiating structure, and substitutes for leadership: a longitudinal study of research and development project team performance.

PubMed

Keller, Robert T

2006-01-01

Transformational leadership, initiating structure, and selected substitutes for leadership were studied as longitudinal predictors of performance in 118 research and development (R&D) project teams from 5 firms. As hypothesized, transformational leadership predicted 1-year-later technical quality, schedule performance, and cost performance and 5-year-later profitability and speed to market. Initiating structure predicted all the performance measures. The substitutes of subordinate ability and an intrinsically satisfying task each predicted technical quality and profitability, and ability predicted speed to market. Moderator effects for type of R&D work were hypothesized and found whereby transformational leadership was a stronger predictor of technical quality in research projects, whereas initiating structure was a stronger predictor of technical quality in development projects. Implications for leadership theory and research are discussed. (c) 2006 APA, all rights reserved.

Effects of diet quality on vulnerability to mild subchronic social defeat stress in mice.

PubMed

Goto, Tatsuhiko; Kubota, Yoshifumi; Toyoda, Atsushi

2016-09-01

The chronic social defeat stress (CSDS) mouse model is a potentially useful system for understanding stress responses to social environments. We previously developed a mouse model of subchronic and mild social defeat stress (sCSDS) that exhibits increased body weight gain and food intake following polydipsia-like features. sCSDS mice also show avoidance behavior in a social interaction test. In this study, we examined the effects of diet quality on susceptibility to sCSDS by feeding these mice semi- and non-purified diets. Male C57BL/6J (B6; n = 82) mice were exposed to sCSDS using male ICR mice. The B6 mice were divided into four test groups: semi-purified pellet diet + sCSDS, non-purified pellet diet + sCSDS, semi-purified diet + control (no sCSDS), and non-purified diet + control. Although increased body weight, and food and water intake following sCSDS exposure were consistently observed in the groups that were fed semi- and non-purified diets, social avoidance behavior was influenced by food type (i.e., sCSDS mice fed semi-purified diet showed the greatest social avoidance behavior). In addition, the rates of stress susceptibility were estimated at 73.9 and 34.8% in sCSDS mice fed semi-purified and non-purified diets, respectively (P < 0.05). For comparison, the susceptible-like phenotype rates were estimated at 12.5 and 8.3% in healthy control mice fed semi-purified and non-purified diets, respectively. These results suggest that diet quality affects the vulnerability of mice to social defeat stress.

Peptide biomarkers used for the selective breeding of a complex polygenic trait in honey bees.

PubMed

Guarna, M Marta; Hoover, Shelley E; Huxter, Elizabeth; Higo, Heather; Moon, Kyung-Mee; Domanski, Dominik; Bixby, Miriam E F; Melathopoulos, Andony P; Ibrahim, Abdullah; Peirson, Michael; Desai, Suresh; Micholson, Derek; White, Rick; Borchers, Christoph H; Currie, Robert W; Pernal, Stephen F; Foster, Leonard J

2017-08-21

We present a novel way to select for highly polygenic traits. For millennia, humans have used observable phenotypes to selectively breed stronger or more productive livestock and crops. Selection on genotype, using single-nucleotide polymorphisms (SNPs) and genome profiling, is also now applied broadly in livestock breeding programs; however, selection on protein/peptide or mRNA expression markers has not yet been proven useful. Here we demonstrate the utility of protein markers to select for disease-resistant hygienic behavior in the European honey bee (Apis mellifera L.). Robust, mechanistically-linked protein expression markers, by integrating cis- and trans- effects from many genomic loci, may overcome limitations of genomic markers to allow for selection. After three generations of selection, the resulting marker-selected stock outperformed an unselected benchmark stock in terms of hygienic behavior, and had improved survival when challenged with a bacterial disease or a parasitic mite, similar to bees selected using a phenotype-based assessment for this trait. This is the first demonstration of the efficacy of protein markers for industrial selective breeding in any agricultural species, plant or animal.

Why Do Floral Perfumes Become Different? Region-Specific Selection on Floral Scent in a Terrestrial Orchid

PubMed Central

Gross, Karin; Sun, Mimi; Schiestl, Florian P.

2016-01-01

Geographically structured phenotypic selection can lead to adaptive divergence. However, in flowering plants, such divergent selection has rarely been shown, and selection on floral signals is generally little understood. In this study, we measured phenotypic selection on display size, floral color, and floral scent in four lowland and four mountain populations of the nectar-rewarding terrestrial orchid Gymnadenia odoratissima in two years. We also quantified population differences in these traits and pollinator community composition. Our results show positive selection on display size and positive, negative, or absence of selection on different scent compounds and floral color. Selection on the main scent compounds was consistently stronger in the lowlands than in the mountains, and lowland plants emitted higher amounts of most of these compounds. Pollinator community composition also differed between regions, suggesting different pollinators select for differences in floral volatiles. Overall, our study is the first to document consistent regional differences in selection on floral scent, suggesting this pattern of selection is one of the evolutionary forces contributing to regional divergence in floral chemical signaling. PMID:26886766

Purification and properties of a novel ferricyanide-linked xanthine dehydrogenase from Pseudomonas putida 40.

PubMed Central

Woolfolk, C A

1985-01-01

The isolation of a xanthine dehydrogenase from Pseudomonas putida 40 which utilizes ferricyanide as an electron acceptor at high efficiency is presented. The new activity is separate from the NAD+ and oxygen-utilizing activities of the same organism but displays a broad pattern for reducing substrates typical of those of previously studied xanthine-oxidizing enzymes. Unlike the previously studied enzymes, the new enzyme appears to lack flavin but possess heme and is resistant to cyanide treatment. However, sensitivity of the purified enzyme to methanol and the selective elimination of the activity when tungstate is added to certain growth media suggest a role for molybdenum. The enzyme is subject to a selective proteolytic action during processing which is not accompanied by denaturation or loss of activity and which is minimized by the continuous exposure of the activity to EDTA and phenylmethylsulfonyl fluoride. Electrophoresis of the denatured enzyme in the presence of sodium dodecyl sulfate suggests that the enzyme is constructed of subunits with a molecular weight of approximately 72,000. Electrophoresis under native conditions of a purified enzyme previously exposed to magnesium ion reveals a series of major and minor activity bands which display some selectivity toward both electron donors and acceptors. An analysis of the effect of gel concentration on this pattern suggests that the enzyme forms a series of charge and size isomers with a pair of trimeric forms predominating. Comparison of the rate of sedimentation of the enzyme in sucrose gradients with its elution profile from standardized Sepharose 6B columns suggests a molecular weight of 255,000 for the major form of the native enzyme. Images PMID:3860496

Genome-Wide Identification and Evolution Analysis of Trehalose-6-Phosphate Synthase Gene Family in Nelumbo nucifera

PubMed Central

Jin, Qijiang; Hu, Xin; Li, Xin; Wang, Bei; Wang, Yanjie; Jiang, Hongwei; Mattson, Neil; Xu, Yingchun

2016-01-01

Trehalose-6-phosphate synthase (TPS) plays a key role in plant carbohydrate metabolism and the perception of carbohydrate availability. In the present work, the publicly available Nelumbo nucifera (lotus) genome sequence database was analyzed which led to identification of nine lotus TPS genes (NnTPS). It was found that at least two introns are included in the coding sequences of NnTPS genes. When the motif compositions were analyzed we found that NnTPS generally shared the similar motifs, implying that they have similar functions. The dN/dS ratios were always less than 1 for different domains and regions outside domains, suggesting purifying selection on the lotus TPS gene family. The regions outside TPS domain evolved relatively faster than NnTPS domains. A phylogenetic tree was constructed using all predicted coding sequences of lotus TPS genes, together with those from Arabidopsis, poplar, soybean, and rice. The result indicated that those TPS genes could be clearly divided into two main subfamilies (I-II), where each subfamily could be further divided into 2 (I) and 5 (II) subgroups. Analyses of divergence and adaptive evolution show that purifying selection may have been the main force driving evolution of plant TPS genes. Some of the critical sites that contributed to divergence may have been under positive selection. Transcriptome data analysis revealed that most NnTPS genes were predominantly expressed in sink tissues. Expression pattern of NnTPS genes under copper and submergence stress indicated that NNU_014679 and NNU_022788 might play important roles in lotus energy metabolism and participate in stress response. Our results can facilitate further functional studies of TPS genes in lotus. PMID:27746792

Antiviral activities of extracts and selected pure constituents of Ocimum basilicum.

PubMed

Chiang, Lien-Chai; Ng, Lean-Teik; Cheng, Pei-Win; Chiang, Win; Lin, Chun-Ching

2005-10-01

1. Ocimum basilicum (OB), also known as sweet basil, is a well known medicinal herb in traditional Chinese medicine preparations. In the present study, extracts and purified components of OB were used to identify possible antiviral activities against DNA viruses (herpes viruses (HSV), adenoviruses (ADV) and hepatitis B virus) and RNA viruses (coxsackievirus B1 (CVB1) and enterovirus 71 (EV71)). 2. The results show that crude aqueous and ethanolic extracts of OB and selected purified components, namely apigenin, linalool and ursolic acid, exhibit a broad spectrum of antiviral activity. Of these compounds, ursolic acid showed the strongest activity against HSV-1 (EC50 = 6.6 mg/L; selectivity index (SI) = 15.2), ADV-8 (EC50 = 4.2 mg/L; SI = 23.8), CVB1 (EC50 = 0.4 mg/L; SI = 251.3) and EV71 (EC50 = 0.5 mg/L; SI = 201), whereas apigenin showed the highest activity against HSV-2 (EC50 = 9.7 mg/L; SI = 6.2), ADV-3 (EC50 = 11.1 mg/L; SI = 5.4), hepatitis B surface antigen (EC50 = 7.1 mg/L; SI = 2.3) and hepatitis B e antigen (EC50 = 12.8 mg/L; SI = 1.3) and linalool showed strongest activity against AVD-II (EC50 = 16.9 mg/L; SI = 10.5). 3. No activity was noted for carvone, cineole, beta-caryophyllene, farnesol, fenchone, geraniol, beta-myrcene and alpha-thujone. 4. The action of ursolic acid against CVB1 and EV71 was found to occur during the infection process and the replication phase. 5. With SI values greater than 200, the potential use of ursolic acid for treating infection with CVB1 and EV71 merits further investigation.

Translating natural genetic variation to gene expression in a computational model of the Drosophila gap gene regulatory network

PubMed Central

Kozlov, Konstantin N.; Kulakovskiy, Ivan V.; Zubair, Asif; Marjoram, Paul; Lawrie, David S.; Nuzhdin, Sergey V.; Samsonova, Maria G.

2017-01-01

Annotating the genotype-phenotype relationship, and developing a proper quantitative description of the relationship, requires understanding the impact of natural genomic variation on gene expression. We apply a sequence-level model of gap gene expression in the early development of Drosophila to analyze single nucleotide polymorphisms (SNPs) in a panel of natural sequenced D. melanogaster lines. Using a thermodynamic modeling framework, we provide both analytical and computational descriptions of how single-nucleotide variants affect gene expression. The analysis reveals that the sequence variants increase (decrease) gene expression if located within binding sites of repressors (activators). We show that the sign of SNP influence (activation or repression) may change in time and space and elucidate the origin of this change in specific examples. The thermodynamic modeling approach predicts non-local and non-linear effects arising from SNPs, and combinations of SNPs, in individual fly genotypes. Simulation of individual fly genotypes using our model reveals that this non-linearity reduces to almost additive inputs from multiple SNPs. Further, we see signatures of the action of purifying selection in the gap gene regulatory regions. To infer the specific targets of purifying selection, we analyze the patterns of polymorphism in the data at two phenotypic levels: the strengths of binding and expression. We find that combinations of SNPs show evidence of being under selective pressure, while individual SNPs do not. The model predicts that SNPs appear to accumulate in the genotypes of the natural population in a way biased towards small increases in activating action on the expression pattern. Taken together, these results provide a systems-level view of how genetic variation translates to the level of gene regulatory networks via combinatorial SNP effects. PMID:28898266

Molecular organization and phylogenetic analysis of 5S rDNA in crustaceans of the genus Pollicipes reveal birth-and-death evolution and strong purifying selection.

PubMed

Perina, Alejandra; Seoane, David; González-Tizón, Ana M; Rodríguez-Fariña, Fernanda; Martínez-Lage, Andrés

2011-10-17

The 5S ribosomal DNA (5S rDNA) is organized in tandem arrays with repeat units that consist of a transcribing region (5S) and a variable nontranscribed spacer (NTS), in higher eukaryotes. Until recently the 5S rDNA was thought to be subject to concerted evolution, however, in several taxa, sequence divergence levels between the 5S and the NTS were found higher than expected under this model. So, many studies have shown that birth-and-death processes and selection can drive the evolution of 5S rDNA. In analyses of 5S rDNA evolution is found several 5S rDNA types in the genome, with low levels of nucleotide variation in the 5S and a spacer region highly divergent. Molecular organization and nucleotide sequence of the 5S ribosomal DNA multigene family (5S rDNA) were investigated in three Pollicipes species in an evolutionary context. The nucleotide sequence variation revealed that several 5S rDNA variants occur in Pollicipes genomes. They are clustered in up to seven different types based on differences in their nontranscribed spacers (NTS). Five different units of 5S rDNA were characterized in P. pollicipes and two different units in P. elegans and P. polymerus. Analysis of these sequences showed that identical types were shared among species and that two pseudogenes were present. We predicted the secondary structure and characterized the upstream and downstream conserved elements. Phylogenetic analysis showed an among-species clustering pattern of 5S rDNA types. These results suggest that the evolution of Pollicipes 5S rDNA is driven by birth-and-death processes with strong purifying selection.

Molecular organization and phylogenetic analysis of 5S rDNA in crustaceans of the genus Pollicipes reveal birth-and-death evolution and strong purifying selection

PubMed Central

2011-01-01

Background The 5S ribosomal DNA (5S rDNA) is organized in tandem arrays with repeat units that consist of a transcribing region (5S) and a variable nontranscribed spacer (NTS), in higher eukaryotes. Until recently the 5S rDNA was thought to be subject to concerted evolution, however, in several taxa, sequence divergence levels between the 5S and the NTS were found higher than expected under this model. So, many studies have shown that birth-and-death processes and selection can drive the evolution of 5S rDNA. In analyses of 5S rDNA evolution is found several 5S rDNA types in the genome, with low levels of nucleotide variation in the 5S and a spacer region highly divergent. Molecular organization and nucleotide sequence of the 5S ribosomal DNA multigene family (5S rDNA) were investigated in three Pollicipes species in an evolutionary context. Results The nucleotide sequence variation revealed that several 5S rDNA variants occur in Pollicipes genomes. They are clustered in up to seven different types based on differences in their nontranscribed spacers (NTS). Five different units of 5S rDNA were characterized in P. pollicipes and two different units in P. elegans and P. polymerus. Analysis of these sequences showed that identical types were shared among species and that two pseudogenes were present. We predicted the secondary structure and characterized the upstream and downstream conserved elements. Phylogenetic analysis showed an among-species clustering pattern of 5S rDNA types. Conclusions These results suggest that the evolution of Pollicipes 5S rDNA is driven by birth-and-death processes with strong purifying selection. PMID:22004418

Functional reconstitution of the voltage-regulated sodium channel purified from electroplax of Electrophorus electricus

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

Rosenberg, R.L.

1985-01-01

The voltage-regulated NA channel is responsible for the depolarization of the excitable cell membrane during the normal action potential. This research has focused on the functional properties of the Na channel, purified from detergent extracts of electroplax membranes of the electric eel, and reconstituted into vesicles of defined phospholipid. These properties were assessed by measuring neurotoxin-modulated ion flux into the reconstituted membrane vesicles and by recording the single-channel currents of the purified channel by the patch-clamp method. The binding of tritiated tetrodotoxin (TTX) was employed as a marker for the purification of the channel. Two high-resolution fractionation steps, based onmore » molecular charge and protein size, were used to obtain a preparation that is 80% homogeneous for a large peptide of 270,000 daltons. Radiotracer /sup 22/Na/sup +/ influx into the vesicles was stimulated by veratridine and by batrachotoxin (BTX) at concentrations of 100 ..mu..M and 5 ..mu..M, respectively. The stimulation by BTX was greater than that by veratridine, and can be as much as 16-fold over control influx levels. The stimulated influx is blocked by TTX with a K/sub i/ of 35 nM, and by local anesthetics in the normal pharmacological range. Large multilamellar vesicles prepared with a freeze-thaw step are suitable for single-channel recording techniques. When excised patches of the reconstituted membranes were voltage-clamped in the absence of activating neurotoxins, voltage-dependent single-channel currents were recorded. These displayed properties similar to those from native membranes of nerve and muscle. These results indicate that the protein purified on the basis of TTX binding is a functional Na channel possessing these functional domains: the ion-selective channel, the voltage sensors controlling activation and inactivation, and the sites of action of TTX, alkaloid neurotoxins, and local anesthetics.« less

Genetic Determinism of Sensitivity to Corynespora cassiicola Exudates in Rubber Tree (Hevea brasiliensis).

PubMed

Tran, Dinh Minh; Clément-Demange, André; Déon, Marine; Garcia, Dominique; Le Guen, Vincent; Clément-Vidal, Anne; Soumahoro, Mouman; Masson, Aurélien; Label, Philippe; Le, Mau Tuy; Pujade-Renaud, Valérie

2016-01-01

An indirect phenotyping method was developed in order to estimate the susceptibility of rubber tree clonal varieties to Corynespora Leaf Fall (CLF) disease caused by the ascomycete Corynespora cassiicola. This method consists in quantifying the impact of fungal exudates on detached leaves by measuring the induced electrolyte leakage (EL%). The tested exudates were either crude culture filtrates from diverse C. cassiicola isolates or the purified cassiicolin (Cas1), a small secreted effector protein produced by the aggressive isolate CCP. The test was found to be quantitative, with the EL% response proportional to toxin concentration. For eight clones tested with two aggressive isolates, the EL% response to the filtrates positively correlated to the response induced by conidial inoculation. The toxicity test applied to 18 clones using 13 toxinic treatments evidenced an important variability among clones and treatments, with a significant additional clone x treatment interaction effect. A genetic linkage map was built using 306 microsatellite markers, from the F1 population of the PB260 x RRIM600 family. Phenotyping of the population for sensitivity to the purified Cas1 effector and to culture filtrates from seven C. cassiicola isolates revealed a polygenic determinism, with six QTL detected on five chromosomes and percentages of explained phenotypic variance varying from 11 to 17%. Two common QTL were identified for the CCP filtrate and the purified cassiicolin, suggesting that Cas1 may be the main effector of CCP filtrate toxicity. The CCP filtrate clearly contrasted with all other filtrates. The toxicity test based on Electrolyte Leakage Measurement offers the opportunity to assess the sensitivity of rubber genotypes to C. cassiicola exudates or purified effectors for genetic investigations and early selection, without risk of spreading the fungus in plantations. However, the power of this test for predicting field susceptibility of rubber clones to CLF will have to be further investigated.

Genetic Determinism of Sensitivity to Corynespora cassiicola Exudates in Rubber Tree (Hevea brasiliensis)

PubMed Central

Tran, Dinh Minh; Clément-Demange, André; Déon, Marine; Garcia, Dominique; Le Guen, Vincent; Clément-Vidal, Anne; Soumahoro, Mouman; Masson, Aurélien; Label, Philippe; Le, Mau Tuy; Pujade-Renaud, Valérie

2016-01-01

An indirect phenotyping method was developed in order to estimate the susceptibility of rubber tree clonal varieties to Corynespora Leaf Fall (CLF) disease caused by the ascomycete Corynespora cassiicola. This method consists in quantifying the impact of fungal exudates on detached leaves by measuring the induced electrolyte leakage (EL%). The tested exudates were either crude culture filtrates from diverse C. cassiicola isolates or the purified cassiicolin (Cas1), a small secreted effector protein produced by the aggressive isolate CCP. The test was found to be quantitative, with the EL% response proportional to toxin concentration. For eight clones tested with two aggressive isolates, the EL% response to the filtrates positively correlated to the response induced by conidial inoculation. The toxicity test applied to 18 clones using 13 toxinic treatments evidenced an important variability among clones and treatments, with a significant additional clone x treatment interaction effect. A genetic linkage map was built using 306 microsatellite markers, from the F1 population of the PB260 x RRIM600 family. Phenotyping of the population for sensitivity to the purified Cas1 effector and to culture filtrates from seven C. cassiicola isolates revealed a polygenic determinism, with six QTL detected on five chromosomes and percentages of explained phenotypic variance varying from 11 to 17%. Two common QTL were identified for the CCP filtrate and the purified cassiicolin, suggesting that Cas1 may be the main effector of CCP filtrate toxicity. The CCP filtrate clearly contrasted with all other filtrates. The toxicity test based on Electrolyte Leakage Measurement offers the opportunity to assess the sensitivity of rubber genotypes to C. cassiicola exudates or purified effectors for genetic investigations and early selection, without risk of spreading the fungus in plantations. However, the power of this test for predicting field susceptibility of rubber clones to CLF will have to be further investigated. PMID:27736862

Who chooses a consumer-directed health plan?

PubMed

Barry, Colleen L; Cullen, Mark R; Galusha, Deron; Slade, Martin D; Busch, Susan H

2008-01-01

Consumer-directed health plans (CDHPs) hold the promise of reining in health spending by giving consumers a greater stake in health care purchasing, yet little is known about employers' experience with these products. In examining the characteristics of those selecting a CDHP offered by one large employer, we found stronger evidence of selection than has been identified in prior research. Our findings suggest that in the context of plan choice, CDHPs may offer little opportunity to greatly lower employers' cost burden, and they highlight concerns about the potential for risk segmentation and the value of conferring preferential tax treatment to CDHPs.

Who Chooses A Consumer-Directed Health Plan?

PubMed Central

Barry, Colleen L.; Cullen, Mark R.; Galusha, Deron; Slade, Martin D.; Busch, Susan H.

2012-01-01

Consumer-directed health plans (CDHPs) hold the promise of reining in health spending by giving consumers a greater stake in health care purchasing, yet little is known about employers’ experience with these products. In examining the characteristics of those selecting a CDHP offered by one large employer, we found stronger evidence of selection than has been identified in prior research. Our findings suggest that in the context of plan choice, CDHPs may offer little opportunity to greatly lower employers’ cost burden, and they highlight concerns about the potential for risk segmentation and the value of conferring preferential tax treatment to CDHPs. PMID:18997225

The Landscape of A-to-I RNA Editome Is Shaped by Both Positive and Purifying Selection

PubMed Central

Kong, Yimeng; Pan, Bohu; Chen, Longxian; Wang, Hongbing; Hao, Pei; Li, Xuan

2016-01-01

The hydrolytic deamination of adenosine to inosine (A-to-I editing) in precursor mRNA induces variable gene products at the post-transcription level. How and to what extent A-to-I RNA editing diversifies transcriptome is not fully characterized in the evolution, and very little is known about the selective constraints that drive the evolution of RNA editing events. Here we present a study on A-to-I RNA editing, by generating a global profile of A-to-I editing for a phylogeny of seven Drosophila species, a model system spanning an evolutionary timeframe of approximately 45 million years. Of totally 9281 editing events identified, 5150 (55.5%) are located in the coding sequences (CDS) of 2734 genes. Phylogenetic analysis places these genes into 1,526 homologous families, about 5% of total gene families in the fly lineages. Based on conservation of the editing sites, the editing events in CDS are categorized into three distinct types, representing events on singleton genes (type I), and events not conserved (type II) or conserved (type III) within multi-gene families. While both type I and II events are subject to purifying selection, notably type III events are positively selected, and highly enriched in the components and functions of the nervous system. The tissue profiles are documented for three editing types, and their critical roles are further implicated by their shifting patterns during holometabolous development and in post-mating response. In conclusion, three A-to-I RNA editing types are found to have distinct evolutionary dynamics. It appears that nervous system functions are mainly tested to determine if an A-to-I editing is beneficial for an organism. The coding plasticity enabled by A-to-I editing creates a new class of binary variations, which is a superior alternative to maintain heterozygosity of expressed genes in a diploid mating system. PMID:27467689

Genome-wide sequence variations between wild and cultivated tomato species revisited by whole genome sequence mapping.

PubMed

Sahu, Kamlesh Kumar; Chattopadhyay, Debasis

2017-06-02

Cultivated tomato (Solanum lycopersicum L.) is the second most important vegetable crop after potato and a member of thirteen interfertile species of Solanum genus. Domestication and continuous selection for desirable traits made cultivated tomato species susceptible to many stresses as compared to the wild species. In this study, we analyzed and compared the genomes of wild and cultivated tomato accessions to identify the genomic regions that encountered changes during domestication. Analysis was based on SNP and InDel mining of twentynine accessions of twelve wild tomato species and forty accessions of cultivated tomato. Percentage of common SNPs among the accessions within a species corresponded with the reproductive behavior of the species. SNP profiles of the wild tomato species within a phylogenetic subsection varied with their geographical distribution. Interestingly, the ratio of genic SNP to total SNPs increased with phylogenetic distance of the wild tomato species from the domesticated species, suggesting that variations in gene-coding region play a major role in speciation. We retrieved 2439 physical positions in 1594 genes including 32 resistance related genes where all the wild accessions possessed a common wild variant allele different from all the cultivated accessions studied. Tajima's D analysis predicted a very strong purifying selection associated with domestication in nearly 1% of its genome, half of which is contributed by chromosome 11. This genomic region with a low Tajima's D value hosts a variety of genes associated with important agronomic trait such as, fruit size, tiller number and wax deposition. Our analysis revealed a broad-spectrum genetic base in wild tomato species and erosion of that in cultivated tomato due to recurrent selection for agronomically important traits. Identification of the common wild variant alleles and the genomic regions undergoing purifying selection during cultivation would facilitate future breeding program by introgression from wild species.

The population genetics of human disease: The case of recessive, lethal mutations

PubMed Central

Gao, Ziyue; Baker, Zachary; Diesel, José Francisco; Simons, Yuval B.; Haque, Imran S.; Pickrell, Joseph; Przeworski, Molly

2017-01-01

Do the frequencies of disease mutations in human populations reflect a simple balance between mutation and purifying selection? What other factors shape the prevalence of disease mutations? To begin to answer these questions, we focused on one of the simplest cases: recessive mutations that alone cause lethal diseases or complete sterility. To this end, we generated a hand-curated set of 417 Mendelian mutations in 32 genes reported to cause a recessive, lethal Mendelian disease. We then considered analytic models of mutation-selection balance in infinite and finite populations of constant sizes and simulations of purifying selection in a more realistic demographic setting, and tested how well these models fit allele frequencies estimated from 33,370 individuals of European ancestry. In doing so, we distinguished between CpG transitions, which occur at a substantially elevated rate, and three other mutation types. Intriguingly, the observed frequency for CpG transitions is slightly higher than expectation but close, whereas the frequencies observed for the three other mutation types are an order of magnitude higher than expected, with a bigger deviation from expectation seen for less mutable types. This discrepancy is even larger when subtle fitness effects in heterozygotes or lethal compound heterozygotes are taken into account. In principle, higher than expected frequencies of disease mutations could be due to widespread errors in reporting causal variants, compensation by other mutations, or balancing selection. It is unclear why these factors would have a greater impact on disease mutations that occur at lower rates, however. We argue instead that the unexpectedly high frequency of disease mutations and the relationship to the mutation rate likely reflect an ascertainment bias: of all the mutations that cause recessive lethal diseases, those that by chance have reached higher frequencies are more likely to have been identified and thus to have been included in this study. Beyond the specific application, this study highlights the parameters likely to be important in shaping the frequencies of Mendelian disease alleles. PMID:28957316

Impact of speciation on the electron charge transfer properties of nanodiamond drug carriers

NASA Astrophysics Data System (ADS)

Sun, Baichuan; Barnard, Amanda S.

2016-07-01

Unpassivated diamond nanoparticles (bucky-diamonds) exhibit a unique surface reconstruction involving graphitization of certain crystal facets, giving rise to hybrid core-shell particles containing both aromatic and aliphatic carbon. Considerable effort is directed toward eliminating the aromatic shell, but persistent graphitization of subsequent subsurface-layers makes perdurable purification a challenge. In this study we use some simple statistical methods, in combination with electronic structure simulations, to predict the impact of different fractions of aromatic and aliphatic carbon on the charge transfer properties of the ensembles of bucky-diamonds. By predicting quality factors for a variety of cases, we find that perfect purification is not necessary to preserve selectivity, and there is a clear motivation for purifying samples to improve the sensitivity of charge transfer reactions. This may prove useful in designing drug delivery systems where the release of (selected) drugs needs to be sensitive to specific conditions at the point of delivery.Unpassivated diamond nanoparticles (bucky-diamonds) exhibit a unique surface reconstruction involving graphitization of certain crystal facets, giving rise to hybrid core-shell particles containing both aromatic and aliphatic carbon. Considerable effort is directed toward eliminating the aromatic shell, but persistent graphitization of subsequent subsurface-layers makes perdurable purification a challenge. In this study we use some simple statistical methods, in combination with electronic structure simulations, to predict the impact of different fractions of aromatic and aliphatic carbon on the charge transfer properties of the ensembles of bucky-diamonds. By predicting quality factors for a variety of cases, we find that perfect purification is not necessary to preserve selectivity, and there is a clear motivation for purifying samples to improve the sensitivity of charge transfer reactions. This may prove useful in designing drug delivery systems where the release of (selected) drugs needs to be sensitive to specific conditions at the point of delivery. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr03068h

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

Howard, A.D.

The aim of this research was to purify and characterize active opioid receptors and elucidate molecular aspects of opioid receptor heterogeneity. Purification to apparent homogeneity of an opioid binding protein from bovine caudate was achieved by solubilization in the non-ionic detergent, digitonin, followed by sequential chromatography on the opiate affinity matrix, ..beta..-naltrexylethylenediamine-CH-Sepharose 4B, and on the lectine affinity matrix, wheat germ agglutinin-agarose. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) followed by autoradiography revealed that radioiodinated purified receptor gave a single band. Purified receptor preparations showed a specific activity of 12,000-15,000 fmol of opiate bound per mgmore » of protein. Radioiodinated human beta-endorphin (/sup 125/I-beta-end/sub H/) was used as a probe to investigate the ligand binding subunits of mu and delta opioid receptors. /sup 125/I-beta-end/sub H/ was shown to bind to a variety of opioid receptor-containing tissues with high affinity and specificity with preference for mu and delta sites, and with little, if any, binding to kappa sites. Affinity crosslinking techniques were employed to covalently link /sup 125/I-beta-end/sub H/ to opioid receptors, utilizing derivatives of bis-succinimidyl esters that are bifunctional crosslinkers with specificities for amino and sulfhydryl groups. This, and competition experiments with high type-selective ligands, permitted the assignment of two labeled peptides to their receptor types, namely a peptide of M/sub r/ = 65,000 for mu receptors and one of M/sub r/ = 53,000 for delta receptors.« less

Simultaneous separation and purification of flavonoids and oleuropein from Olea europaea L. (olive) leaves using macroporous resin.

PubMed

Li, Chen; Zheng, Yuanyuan; Wang, Xiaofei; Feng, Shilan; Di, Duolong

2011-12-01

This study developed a feasible process to simultaneously separate and purify polyphenols, including flavonoids and oleuropein, from the leaves of Olea europaea L. Macroporous resins were used as the separation and purification materials. The performance and separation capabilities of eight resins (D101, DM130, HPD450, LSA-21, LSA-40, 07C, LSD001 and HPD600) were systematically evaluated. The contents of target polyphenols in different extracts were determined using ultraviolet (for flavonoids) and high-performance liquid chromatographic (for oleuropein) methods. The static adsorption and desorption results showed that resin LSA-21 had better adsorption properties among the eight resins. Influential factors such as extraction method, pH value of feeding solution, desorption solution, adsorption kinetics and adsorption isotherm, etc. to the extraction and purification of these polyphenols were successively investigated on resin LSA-21. The target flavonoids and oleuropein were selectively purified using resin LSA-21. Compared with the contents in raw leaves, the contents of total flavonoids and oleuropein in the final purified products were increased 13.2-fold (from 16 to 211 g kg(-1) ) and 7.5-fold (from 120 to 902 g kg(-1) ) with recovery yields of 87.9% and 85.6%, respectively. This extraction and purification method could be used in the large-scale enrichment or purification of flavonoids, oleuropein and other polyphenols from O. europaea L. leaves or other herbal materials in industrial, food processing and medical manufacture. Copyright © 2011 Society of Chemical Industry.

Recombinant Mip-PilE-FlaA dominant epitopes vaccine candidate against Legionella pneumophila.

PubMed

He, Jinlei; Huang, Fan; Chen, Han; Chen, Qiwei; Zhang, Junrong; Li, Jiao; Chen, Dali; Chen, Jianping

2017-06-01

Legionella pneumophila is the main causative agent of Legionnaires' disease, which is a severe multi-system disease with pneumonia as the primary manifestation. We designed a recombinant Mip-PilE-FlaA dominant epitopes vaccine against Legionella pneumophila to prevent the disease and evaluated its immunogenicity and protective immunity. The protein structures of Mip, PilE and FlaA were analyzed using a computer, and the gene sequences of the dominant epitopes of the three proteins were selected to construct and optimize the vaccine. The optimized mip, pilE, flaA and recombinant mip-pilE-flaA gene sequences were cloned, expressed and purified. The purified proteins were used as dominant epitopes vaccines to immunize BALB/c mice and determine the protective immunity and immunogenicity of these purified proteins. The identification confirmed that the recombinant mip-pilE-flaA was successfully cloned and expressed. ELISA revealed that the Mip-PilE-FlaA group produced the highest IgG response, and this protein may considerably improve the production of some cytokines in BALB/c mice. Histopathology analyses of lungs from mice immunized with Mip-PilE-FlaA revealed a certain protective effect. Our work demonstrated that the recombinant dominant epitopes of Mip-PilE-FlaA exhibited strong immunogenicity and immune protection, and this protein may be an efficient epitopes vaccine candidate against Legionella pneumophila. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Composition and Potency Characterization of Mycobacterium avium subsp. paratuberculosis Purified Protein Derivatives

PubMed Central

Capsel, Randal T.; Thoen, Charles O.; Reinhardt, Timothy A.; Lippolis, John D.; Olsen, Renee; Stabel, Judith R.; Bannantine, John P.

2016-01-01

Mycobacterium avium subsp. paratuberculosis (MAP) purified protein derivatives (PPDs) are immunologic reagents prepared from cultured filtrates of the type strain. Traditional production consists of floating culture incubation at 37°C, organism inactivation by autoclaving, coarse filtration, and protein precipitation. Three traditional production PPDs were used in this study including lot 9801, which served as a reference and has been used in the field for decades. Alternative production PPDs (0902A and 0902B), in which the autoclaving step was removed, were also analyzed in this study. SDS-PAGE analysis revealed protein smearing in traditional PPDs, but distinct bands were observed in the alternative PPD preparations. Antibody bound distinct protein bands in the alternative PPDs by immunoblot analysis, whereas an immunoreactive smear was observed with the traditional PPDs. Mass spectrometry identified 194 proteins among three PPD lots representing the two different production methods, ten of which were present in all PPDs examined. Selected proteins identified by mass spectrometry were recombinantly expressed and purified from E. coli and evaluated by the guinea pig potency test. Seven recombinant proteins showed greater erythema as compared to the reference PPD lot 9801 in paired guinea pigs and were able to stimulate interferon-gamma production in blood from Johne’s positive animals. These results suggest that autoclaving culture suspensions is not a necessary step in PPD production and specific proteins could supplant the PPD antigen for intradermal skin testing procedures and for use as in-vitro assay reagents. PMID:27136199

Irradiated and activated autologous PBMCs induce expansion of highly cytotoxic human NK cells in vitro.

PubMed

Ahn, Yong-Oon; Kim, Saerom; Kim, Tae Min; Song, Eun Young; Park, Myoung Hee; Heo, Dae Seog

2013-09-01

Adoptive cell transfer of ex vivo-activated natural killer (NK) cells is a promising therapy for cancer treatment. Because of inhibitory signaling through killer immunoglobulin-like receptor (KIR)-KIR ligands, KIR-mismatched allogeneic NK cell transfer is considered to be a more effective strategy than is autologous transfer. However, purified NK cells do not expand well enough in vitro with good manufacturing practice-compliant components for clinical use. Some investigators have developed selective expansion of NK cells from peripheral blood mononuclear cells, but these cells have the risk of graft-versus-host disease in allogeneic settings because of T cells contamination. In this study, we developed a novel method for NK cell activation and expansion. Using only good manufacturing practice-compliant components and autologous feeder cells, once purified NK cells were effectively expanded (2500-fold at day 17). The expanded cells were highly purified NK cells, and the use of these cells is suitable for allogeneic transfer without the risk of graft-versus-host disease induction. Importantly, the expanded NK cells also showed enhanced cytotoxicity compared with NK cells conventionally expanded by recombinant human interleukin 2. Finally, induction of NKG2D ligand expression on feeder cells implies that the NKG2D-NKG2DL interaction may play a role in NK cell expansion. In conclusion, this method can be used to obtain NK cells for more successful allogeneic NK cell adoptive transfer for use in antitumor immune therapy.

Expression level, cellular compartment and metabolic network position all influence the average selective constraint on mammalian enzymes

PubMed Central

2011-01-01

Background A gene's position in regulatory, protein interaction or metabolic networks can be predictive of the strength of purifying selection acting on it, but these relationships are neither universal nor invariably strong. Following work in bacteria, fungi and invertebrate animals, we explore the relationship between selective constraint and metabolic function in mammals. Results We measure the association between selective constraint, estimated by the ratio of nonsynonymous (Ka) to synonymous (Ks) substitutions, and several, primarily metabolic, measures of gene function. We find significant differences between the selective constraints acting on enzyme-coding genes from different cellular compartments, with the nucleus showing higher constraint than genes from either the cytoplasm or the mitochondria. Among metabolic genes, the centrality of an enzyme in the metabolic network is significantly correlated with Ka/Ks. In contrast to yeasts, gene expression magnitude does not appear to be the primary predictor of selective constraint in these organisms. Conclusions Our results imply that the relationship between selective constraint and enzyme centrality is complex: the strength of selective constraint acting on mammalian genes is quite variable and does not appear to exclusively follow patterns seen in other organisms. PMID:21470417

40 CFR 91.312 - Analytical gases.

Code of Federal Regulations, 2014 CFR

2014-07-01

...) Purified nitrogen, also referred to as “zero-grade nitrogen” (Contamination≤1 ppm C, ≤1 ppm CO, ≤400 ppm... percent hydrogen, balance helium) (Contamination≤1 ppm C, ≤400 ppm CO) (4) Purified synthetic air, also... must be available: C3 H8 and purified synthetic air (dilute measurements); C3 H8 and purified nitrogen...

Measurement of Ozone Emission and Particle Removal Rates from Portable Air Purifiers

ERIC Educational Resources Information Center

Mang, Stephen A.; Walser, Maggie L.; Nizkorodov, Sergey A.; Laux, John M.

2009-01-01

Portable air purifiers are popular consumer items, especially in areas with poor air quality. Unfortunately, most users of these air purifiers have minimal understanding of the factors affecting their efficiency in typical indoor settings. Emission of the air pollutant ozone (O[subscript 3]) by certain air purifiers is of particular concern. In an…

Gas stream purifier

NASA Technical Reports Server (NTRS)

Adam, Steven J.

1994-01-01

A gas stream purifier has been developed that is capable of removing corrosive acid, base, solvent, organic, inorganic, and water vapors as well as particulates from an inert mixed gas stream using only solid scrubbing agents. This small, lightweight purifier has demonstrated the ability to remove contaminants from an inert gas stream with a greater than 99 percent removal efficiency. The Gas Stream Purifier has outstanding market and sales potential in manufacturing, laboratory and science industries, medical, automotive, or any commercial industry where pollution, contamination, or gas stream purification is a concern. The purifier was developed under NASA contract NAS9-18200 Schedule A for use in the international Space Station. A patent application for the Gas Stream Purifier is currently on file with the United States Patent and Trademark Office.

The selective recognition of antibody IgY for digestive system cancers.

PubMed

Yang, J; Jin, Z; Yu, Q; Yang, T; Wang, H; Liu, L

1997-01-01

Biological methods for cancer therapies are very important. A small and efficient target carrier is the key component for anti-cancer drugs. In our laboratory, the antibody IgY was extracted from egg yolk of a SPF hen. The SPF hen was immunized with an antigene of P110 protein which was purified from human stomach cancer MGC-803 cells. Results indicated that the antibody IgY can specifically recognize gastrointestinal system cancers. It may become an important carrier for antitumorigenic drugs.

Feed gas contaminant removal in ion transport membrane systems

DOEpatents

Carolan, Michael Francis [Allentown, PA; Miller, Christopher Francis [Macungie, PA

2008-09-16

Method for gas purification comprising (a) obtaining a feed gas stream containing one or more contaminants selected from the group consisting of volatile metal oxy-hydroxides, volatile metal oxides, and volatile silicon hydroxide; (b) contacting the feed gas stream with a reactive solid material in a guard bed and reacting at least a portion of the contaminants with the reactive solid material to form a solid reaction product in the guard bed; and (c) withdrawing from the guard bed a purified gas stream.

Applications Research of Microbial Ecological Preparation in Sea Cucumber Culture

NASA Astrophysics Data System (ADS)

Jiang, Jiahui; Wang, Guangyu

2017-12-01

At present, micro ecological preparation is widely applied in aquaculture with good effect. The application of micro ecological preparation in sea cucumber culture can effectively improve the economic benefits. The micro ecological preparation can play the role of inhibiting harmful bacteria, purifying water quality and saving culture cost in the process of sea cucumber culture. We should select appropriate bacteria, guarantee stable environment and use with long-term in the applications of microbial ecological preparation in sea cucumber culture to obtain good effects.

A New Type of Heterogeneous Catalyst with Isolated FE-RH Diatomic Sites

DTIC Science & Technology

1988-06-01

the logical step was to take advantage of the hoped-for changes in reactivity and selectivity of the new sites. They showed that characterization can...These gases were further purified by passing through Oxy-traps ( Alltech Associates) and molecular sieve traps (Linde 4A) to remove any remainin oxygen or...on a support surface. One of the advantages of the N Mossbauer Effect is that only certain atoms are Mossbauer active and the source used will dictate

Evolution of dosage compensation under sexual selection differs between X and Z chromosomes

PubMed Central

Mullon, Charles; Wright, Alison E.; Reuter, Max; Pomiankowski, Andrew; Mank, Judith E.

2015-01-01

Complete sex chromosome dosage compensation has more often been observed in XY than ZW species. In this study, using a population genetic model and the chicken transcriptome, we assess whether sexual conflict can account for this difference. Sexual conflict over expression is inevitable when mutation effects are correlated across the sexes, as compensatory mutations in the heterogametic sex lead to hyperexpression in the homogametic sex. Coupled with stronger selection and greater reproductive variance in males, this results in slower and less complete evolution of Z compared with X dosage compensation. Using expression variance as a measure of selection strength, we find that, as predicted by the model, dosage compensation in the chicken is most pronounced in genes that are under strong selection biased towards females. Our study explains the pattern of weak dosage compensation in ZW systems, and suggests that sexual selection plays a major role in shaping sex chromosome dosage compensation. PMID:26212613

From Evolutionary Allometry to Sexual Display: (A Reply to Holman and Bro-Jørgensen).

PubMed

Raia, Pasquale; Passaro, Federico; Carotenuto, Francesco; Meiri, Shai; Piras, Paolo

2016-08-01

Conventional wisdom holds that the complex shapes of deer antlers are produced under the sole influence of sexual selection. We questioned this view by demonstrating that trends for increased body size evolution passively yield more-complex ornaments, even in organisms where no effect of sexual selection is possible, with similar allometric slopes. Recent investigations suggest that sexual selection on antlers of larger deer species is stronger than that in smaller species; hence, the use of conspicuous antlers for display in large male deer is a secondary function driven by especially intense sexual selection on these large-bodied species. Since ancestral deer were small and had very simple antlers, such an intense selection on antlers shape was probably absent in early deer. Therefore, the evolution of complex ornaments is coupled with body size evolution, even in deer.

Positive selection and ancient duplications in the evolution of class B floral homeotic genes of orchids and grasses

PubMed Central

Mondragón-Palomino, Mariana; Hiese, Luisa; Härter, Andrea; Koch, Marcus A; Theißen, Günter

2009-01-01

Background Positive selection is recognized as the prevalence of nonsynonymous over synonymous substitutions in a gene. Models of the functional evolution of duplicated genes consider neofunctionalization as key to the retention of paralogues. For instance, duplicate transcription factors are specifically retained in plant and animal genomes and both positive selection and transcriptional divergence appear to have played a role in their diversification. However, the relative impact of these two factors has not been systematically evaluated. Class B MADS-box genes, comprising DEF-like and GLO-like genes, encode developmental transcription factors essential for establishment of perianth and male organ identity in the flowers of angiosperms. Here, we contrast the role of positive selection and the known divergence in expression patterns of genes encoding class B-like MADS-box transcription factors from monocots, with emphasis on the family Orchidaceae and the order Poales. Although in the monocots these two groups are highly diverse and have a strongly canalized floral morphology, there is no information on the role of positive selection in the evolution of their distinctive flower morphologies. Published research shows that in Poales, class B-like genes are expressed in stamens and in lodicules, the perianth organs whose identity might also be specified by class B-like genes, like the identity of the inner tepals of their lily-like relatives. In orchids, however, the number and pattern of expression of class B-like genes have greatly diverged. Results The DEF-like genes from Orchidaceae form four well-supported, ancient clades of orthologues. In contrast, orchid GLO-like genes form a single clade of ancient orthologues and recent paralogues. DEF-like genes from orchid clade 2 (OMADS3-like genes) are under less stringent purifying selection than the other orchid DEF-like and GLO-like genes. In comparison with orchids, purifying selection was less stringent in DEF-like and GLO-like genes from Poales. Most importantly, positive selection took place before the major organ reduction and losses in the floral axis that eventually yielded the zygomorphic grass floret. Conclusion In DEF-like genes of Poales, positive selection on the region mediating interactions with other proteins or DNA could have triggered the evolution of the regulatory mechanisms behind the development of grass-specific reproductive structures. Orchidaceae show a different trend, where gene duplication and transcriptional divergence appear to have played a major role in the canalization and modularization of perianth development. PMID:19383167

Primer-Free Aptamer Selection Using A Random DNA Library

PubMed Central

Pan, Weihua; Xin, Ping; Patrick, Susan; Dean, Stacey; Keating, Christine; Clawson, Gary

2010-01-01

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified through an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) to recognize a wide variety of targets, from small molecules to proteins and other macromolecules 2-4. Aptamers have properties that are well suited for in vivo diagnostic and/or therapeutic applications: Besides good specificity and affinity, they are easily synthesized, survive more rigorous processing conditions, they are poorly immunogenic, and their relatively small size can result in facile penetration of tissues. Aptamers that are identified through the standard SELEX process usually comprise ~80 nucleotides (nt), since they are typically selected from nucleic acid libraries with ~40 nt long randomized regions plus fixed primer sites of ~20 nt on each side. The fixed primer sequences thus can comprise nearly ~50% of the library sequences, and therefore may positively or negatively compromise identification of aptamers in the selection process 3, although bioinformatics approaches suggest that the fixed sequences do not contribute significantly to aptamer structure after selection 5. To address these potential problems, primer sequences have been blocked by complementary oligonucleotides or switched to different sequences midway during the rounds of SELEX 6, or they have been trimmed to 6-9 nt 7, 8. Wen and Gray 9 designed a primer-free genomic SELEX method, in which the primer sequences were completely removed from the library before selection and were then regenerated to allow amplification of the selected genomic fragments. However, to employ the technique, a unique genomic library has to be constructed, which possesses limited diversity, and regeneration after rounds of selection relies on a linear reamplification step. Alternatively, efforts to circumvent problems caused by fixed primer sequences using high efficiency partitioning are met with problems regarding PCR amplification 10. We have developed a primer-free (PF) selection method that significantly simplifies SELEX procedures and effectively eliminates primer-interference problems 11, 12. The protocols work in a straightforward manner. The central random region of the library is purified without extraneous flanking sequences and is bound to a suitable target (for example to a purified protein or complex mixtures such as cell lines). Then the bound sequences are obtained, reunited with flanking sequences, and re-amplified to generate selected sub-libraries. As an example, here we selected aptamers to S100B, a protein marker for melanoma. Binding assays showed Kd s in the 10-7 - 10-8 M range after a few rounds of selection, and we demonstrate that the aptamers function effectively in a sandwich binding format. PMID:20689511

Comparative transcriptome analysis and identification of candidate effectors in two related rust species (Gymnosporangium yamadae and Gymnosporangium asiaticum).

PubMed

Tao, Si-Qi; Cao, Bin; Tian, Cheng-Ming; Liang, Ying-Mei

2017-08-23

Rust fungi constitute the largest group of plant fungal pathogens. However, a paucity of data, including genomic sequences, transcriptome sequences, and associated molecular markers, hinders the development of inhibitory compounds and prevents their analysis from an evolutionary perspective. Gymnosporangium yamadae and G. asiaticum are two closely related rust fungal species, which are ecologically and economically important pathogens that cause apple rust and pear rust, respectively, proved to be devastating to orchards. In this study, we investigated the transcriptomes of these two Gymnosporangium species during the telial stage of their lifecycles. The aim of this study was to understand the evolutionary patterns of these two related fungi and to identify genes that developed by selection. The transcriptomes of G. yamadae and G. asiaticum were generated from a mixture of RNA from three biological replicates of each species. We obtained 49,318 and 54,742 transcripts, with N50 values of 1957 and 1664, for G. yamadae and G. asiaticum, respectively. We also identified a repertoire of candidate effectors and other gene families associated with pathogenicity. A total of 4947 pairs of putative orthologues between the two species were identified. Estimation of the non-synonymous/synonymous substitution rate ratios for these orthologues identified 116 pairs with Ka/Ks values greater than1 that are under positive selection and 170 pairs with Ka/Ks values of 1 that are under neutral selection, whereas the remaining 4661 genes are subjected to purifying selection. We estimate that the divergence time between the two species is approximately 5.2 Mya. This study constitutes a de novo assembly and comparative analysis between the transcriptomes of the two rust species G. yamadae and G. asiaticum. The results identified several orthologous genes, and many expressed genes were identified by annotation. Our analysis of Ka/Ks ratios identified orthologous genes subjected to positive or purifying selection. An evolutionary analysis of these two species provided a relatively precise divergence time. Overall, the information obtained in this study increases the genetic resources available for research on the genetic diversity of the Gymnosporangium genus.

Purification and immunochemical characterization of type e polysaccharide antigen of Streptococcus mutans.

PubMed

Hamada, S; Slade, H D

1976-07-01

The type-specific antigen of Streptococcus mutans strain MT703, serotype e, has been chromatographically purified and characterized. Two chromatographic fractions were obtained from saline extracts which reacted with both anti-MT703 whole-cell serum and Lancefield group E serum. The major fraction (eI) was identified as a polysaccharide composed of 37% glucose, 56% rhamnose, 5% protein, and 0.3% phosphorus, whereas the minor fraction (eII) contained 66% protein in addition to 10% glucose and 17% rhamnose. The immunological specificity of these antigens was found to be the same by immunodiffusion in agar gel. Another fraction with a negative charge (eIII) reacted with polyglycerophosphate antisera from Streptococcus mutans and Streptococcus pyogenes. For comparison, the MT703 antigen in a hot trichloroacetic acid extract (eA) and the group E antigen from a saline extract of cells of strain K129 (EI) were similarly purified by anionic ion-exchange chromatography. Although the ratio of glucose and rhamnose in eA was 1:0.9 and in eI and eII approximately 1:1.5, reactions of identity were obtained in gel diffusion against specific anti-e serum. This difference in ratio is probably a result of the extraction procedures. Both the type e and group E antisera were reactive with both eI and EI antigens. The adsorption of group E antiserum with MT703 cells removed all E antibody, whereas type e-specific antibody remained after adsorption with K129 cells. These results suggest that eI antigen possesses both e and E specificities, whereas EI possesses E only. These findings were supported by the quantitative precipitin test and immunodiffusion and/or immunoelectrophoretic patterns in agar gel. Methyl-beta-D-glucopyranoside markedly inhibited the precipitin reaction in both type e and group E sera. However, a significantly stronger inhibition by cellobiose of type e serum than of group E serum indicates that a beta-linked glucose-glucose dimer is the predominant antigenic determinant of the e specificity. The presence of both e and E specificities on a single polysaccharide molecule was demonstrated by the use of purified e antigen released from a specific e-anti-e complex. This antigen reacted with a group E-specific serum as well as a type e-specific serum. An examination of five S. mutans type e strains showed the presence of group E specificity also, whereas the I, II, and IV serotypes of group E streptococci only possessed the group E specificity.

Predictors of longitudinal substance use and mental health outcomes for patients in two integrated service delivery systems.

PubMed

Grella, Christine E; Stein, Judith A; Weisner, Constance; Chi, Felicia; Moos, Rudolf

2010-07-01

Individuals who have both substance use disorders and mental health problems have poorer treatment outcomes. This study examines the relationship of service utilization and 12-step participation to outcomes at 1 and 5 years for patients treated in one of two integrated service delivery systems: the Department of Veterans Affairs (VA) system and a health maintenance organization (HMO). Sub-samples from each system were selected using multiple criteria indicating severity of mental health problems at admission to substance use disorder treatment (VA=401; HMO=331). Separate and multiple group structural equation model analyses used baseline characteristics, service use, and 12-step participation as predictors of substance use and mental health outcomes at 1 and 5 years following admission. Substance use and related problems showed stability across time, however, these relationships were stronger among VA patients. More continuing care substance use outpatient visits were associated with reductions in mental health symptoms in both groups, whereas receipt of outpatient mental health services was associated with more severe psychological symptoms. Participation in 12-step groups had a stronger effect on reducing cocaine use among VA patients, whereas it had a stronger effect on reducing alcohol use among HMO patients. More outpatient psychological services had a stronger effect on reducing alcohol use among HMO patients. Common findings across these two systems demonstrate the persistence of substance use and related psychological problems, but also show that continuing care services and participation in 12-step groups are associated with better outcomes in both systems. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Alkali metal nitrate purification

DOEpatents

Fiorucci, Louis C.; Morgan, Michael J.

1986-02-04

A process is disclosed for removing contaminants from impure alkali metal nitrates containing them. The process comprises heating the impure alkali metal nitrates in solution form or molten form at a temperature and for a time sufficient to effect precipitation of solid impurities and separating the solid impurities from the resulting purified alkali metal nitrates. The resulting purified alkali metal nitrates in solution form may be heated to evaporate water therefrom to produce purified molten alkali metal nitrates suitable for use as a heat transfer medium. If desired, the purified molten form may be granulated and cooled to form discrete solid particles of purified alkali metal nitrates.

PROCESS FOR SEPARATING AMERICIUM AND CURIUM FROM RARE EARTH ELEMENTS

DOEpatents

Baybarz, R.D.; Lloyd, M.H.

1963-02-26

This invention relates to methods of separating americium and curium values from rare earth values. In accordance with the invention americium, curium, and rare earth values are sorbed on an anion exchange resin. A major portion of the rare earth values are selectively stripped from the resin with a concentrated aqueous solution of lithium chloride, and americium, curium, and a minor portion of rare earth values are then stripped from the resin with a dilute aqueous solution of lithium chloride. The americium and curium values are further purified by increasing the concentration of lithium chloride in the solution to at least 8 molar and selectively extracting rare earth values from the resulting solution with a monoalkylphosphoric acid. (AEC)

Selective counting and sizing of single virus particles using fluorescent aptamer-based nanoparticle tracking analysis.

PubMed

Szakács, Zoltán; Mészáros, Tamás; de Jonge, Marien I; Gyurcsányi, Róbert E

2018-05-30

Detection and counting of single virus particles in liquid samples are largely limited to narrow size distribution of viruses and purified formulations. To address these limitations, here we propose a calibration-free method that enables concurrently the selective recognition, counting and sizing of virus particles as demonstrated through the detection of human respiratory syncytial virus (RSV), an enveloped virus with a broad size distribution, in throat swab samples. RSV viruses were selectively labeled through their attachment glycoproteins (G) with fluorescent aptamers, which further enabled their identification, sizing and counting at the single particle level by fluorescent nanoparticle tracking analysis. The proposed approach seems to be generally applicable to virus detection and quantification. Moreover, it could be successfully applied to detect single RSV particles in swab samples of diagnostic relevance. Since the selective recognition is associated with the sizing of each detected particle, this method enables to discriminate viral elements linked to the virus as well as various virus forms and associations.

Purification-Free, Target-Selective Immobilization of a Protein from Cell Lysates.

PubMed

Cha, Jaehyun; Kwon, Inchan

2018-02-27

Protein immobilization has been widely used for laboratory experiments and industrial processes. Preparation of a recombinant protein for immobilization usually requires laborious and expensive purification steps. Here, a novel purification-free, target-selective immobilization technique of a protein from cell lysates is reported. Purification steps are skipped by immobilizing a target protein containing a clickable non-natural amino acid (p-azidophenylalanine) in cell lysates onto alkyne-functionalized solid supports via bioorthogonal azide-alkyne cycloaddition. In order to achieve a target protein-selective immobilization, p-azidophenylalanine was introduced into an exogenous target protein, but not into endogenous non-target proteins using host cells with amber codon-free genomic DNAs. Immobilization of superfolder fluorescent protein (sfGFP) from cell lysates is as efficient as that of the purified sfGFP. Using two fluorescent proteins (sfGFP and mCherry), the authors also demonstrated that the target proteins are immobilized with a minimal immobilization of non-target proteins (target-selective immobilization). © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Immunodiagnosis of Canine Visceral Leishmaniasis Using Mimotope Peptides Selected from Phage Displayed Combinatorial Libraries

PubMed Central

Toledo-Machado, Christina Monerat; Machado de Avila, Ricardo Andrez; NGuyen, Christophe; Granier, Claude; Bueno, Lilian Lacerda; Carneiro, Claudia Martins; Menezes-Souza, Daniel; Carneiro, Rubens Antonio; Chávez-Olórtegui, Carlos; Fujiwara, Ricardo Toshio

2015-01-01

ELISA and RIFI are currently used for serodiagnosis of canine visceral leishmaniasis (CVL). The accuracy of these tests is controversial in endemic areas where canine infections by Trypanosoma cruzi may occur. We evaluated the usefulness of synthetic peptides that were selected through phage display technique in the serodiagnosis of CVL. Peptides were chosen based on their ability to bind to IgGs purified from infected dogs pooled sera. We selected three phage clones that reacted only with those IgGs. Peptides were synthesized, polymerized with glutaraldehyde, and used as antigens in ELISA assays. Each individual peptide or a mix of them was reactive with infected dogs serum. The assay was highly sensitive and specific when compared to soluble Leishmania antigen that showed cross-reactivity with anti-T. cruzi IgGs. Our results demonstrate that phage display technique is useful for selection of peptides that may represent valuable synthetic antigens for an improved serodiagnosis of CVL. PMID:25710003

Signatures of negative selection in the genetic architecture of human complex traits.

PubMed

Zeng, Jian; de Vlaming, Ronald; Wu, Yang; Robinson, Matthew R; Lloyd-Jones, Luke R; Yengo, Loic; Yap, Chloe X; Xue, Angli; Sidorenko, Julia; McRae, Allan F; Powell, Joseph E; Montgomery, Grant W; Metspalu, Andres; Esko, Tonu; Gibson, Greg; Wray, Naomi R; Visscher, Peter M; Yang, Jian

2018-05-01

We develop a Bayesian mixed linear model that simultaneously estimates single-nucleotide polymorphism (SNP)-based heritability, polygenicity (proportion of SNPs with nonzero effects), and the relationship between SNP effect size and minor allele frequency for complex traits in conventionally unrelated individuals using genome-wide SNP data. We apply the method to 28 complex traits in the UK Biobank data (N = 126,752) and show that on average, 6% of SNPs have nonzero effects, which in total explain 22% of phenotypic variance. We detect significant (P < 0.05/28) signatures of natural selection in the genetic architecture of 23 traits, including reproductive, cardiovascular, and anthropometric traits, as well as educational attainment. The significant estimates of the relationship between effect size and minor allele frequency in complex traits are consistent with a model of negative (or purifying) selection, as confirmed by forward simulation. We conclude that negative selection acts pervasively on the genetic variants associated with human complex traits.

[Relation between frequency modulation direction selectivity and forward masking of inferior collicular neurons: a study on in vivo intracellular recording in mice].

PubMed

Fu, Zi-Ying; Zeng, Hong; Tang, Jia; Li, Jie; Li, Juan; Chen, Qi-Cai

2013-06-25

It has been reported that the frequency modulation (FM) or FM direction sensitivity and forward masking of central auditory neurons are related with the neural inhibition, but there are some arguments, because no direct evidence of inhibitory synaptic input was obtained in previous studies using extracellular recording. In the present study, we studied the relation between FM direction sensitivity and forward masking of the inferior collicular (IC) neurons using in vivo intracellular recordings in 20 Mus musculus Km mice. Thirty seven with complete data among 93 neurons were analyzed and discussed. There was an inhibitory area which consisted of inhibitory postsynaptic potentials (IPSP) at high frequency side of frequency tuning of up-sweep FM (FMU) sensitive neurons (n = 12) and at low frequency side of frequency tuning of down-sweep FM (FMD) selective neurons (n = 8), while there was no any inhibitory area at both sides of frequency tuning of non-FM sweep direction (FMN) sensitive neurons (n = 17). Therefore, these results show that the inhibitory area at low or high frequency side of frequency tuning is one of the mechanisms for forming FM sweep direction sensitivity of IC neurons. By comparison of forward masking produced by FMU and FMD sound stimuli in FMU, FMD and FMN neurons, the selective FM sounds could produce stronger forward masking than the non-selective in FMU and FMD neurons, while there was no forward masking difference between FMU and FMD stimuli in the FMN neurons. We suggest that the post-action potential IPSP is a potential mechanism for producing stronger forward masking in FMU and FMD neurons.

M3 cholinoreceptors alter electrical activity of rat left atrium via suppression of L-type Ca2+ current without affecting K+ conductance.

PubMed

Filatova, Tatiana S; Naumenko, Nikolay; Galenko-Yaroshevsky, Pavel A; Abramochkin, Denis V

2017-05-01

Electrophysiological effects produced by selective activation of M3 cholinoreceptors were studied in isolated left atrium preparations from rat using the standard sharp glass microelectrode technique. The stimulation of M3 receptors was obtained by application of muscarinic agonist pilocarpine (10 -5  M) in the presence of selective M2 antagonist methoctramine (10 -7  M). Stimulation of M3 receptors induced marked reduction of action potential duration by 14.4 ± 2.4% and 16.1 ± 2.5% of control duration measured at 50 and 90% of repolarization, respectively. This effect was completely abolished by selective M3 blocker 4-DAMP (10 -8  M). In isolated myocytes obtained from the rat left atrium, similar pharmacological stimulation of M3 receptors led to suppression of peak L-type calcium current by 13.9 ± 2.6% of control amplitude (measured at +10 mV), but failed to affect K + currents I to , I Kur , and I Kir . In the absence of M2 blocker methoctramine, pilocarpine (10 -5  M) produced stronger attenuation of I CaL and induced an increase in I Kir . This additive inward rectifier current could be abolished by highly selective blocker of K ir 3.1/3.4 channels tertiapin-Q (10 -6  M) and therefore was identified as I KACh . Thus, in the rat atrial myocardium activation of M3 receptors leads to shortening of action potentials via suppression of I CaL , but does not enhance the major potassium currents involved in repolarization. Joint stimulation of M2 and M3 receptors produces stronger action potential shortening due to M2-mediated activation of I KACh.

The effect of parasites on sex differences in selection.

PubMed

Sharp, N P; Vincent, C M

2015-04-01

The life history strategies of males and females are often divergent, creating the potential for sex differences in selection. Deleterious mutations may be subject to stronger selection in males, owing to sexual selection, which can improve the mean fitness of females and reduce mutation load in sexual populations. However, sex differences in selection might also maintain sexually antagonistic genetic variation, creating a sexual conflict load. The overall impact of separate sexes on fitness is unclear, but the net effect is likely to be positive when there is a large sex difference in selection against deleterious mutations. Parasites can also have sex-specific effects on fitness, and there is evidence that parasites can intensify the fitness consequences of deleterious mutations. Using lines that accumulated mutations for over 60 generations, we studied the effect of the pathogenic bacterium Pseudomonas aeruginosa on sex differences in selection in the fruit fly Drosophila melanogaster. Pseudomonas infection increased the sex difference in selection, but may also have weakened the intersexual correlation for fitness. Our results suggest that parasites may increase the benefits of sexual selection.

A biotin-conjugated pyridine-based isatoic anhydride, a selective room temperature RNA-acylating agent for the nucleic acid separation.

PubMed

Ursuegui, S; Yougnia, R; Moutin, S; Burr, A; Fossey, C; Cailly, T; Laayoun, A; Laurent, A; Fabis, F

2015-03-28

Isatoic anhydride derivatives, including a biotin and a disulfide linker were specifically designed for nucleic acid separation. 2'-OH selective RNA acylation, capture of biotinylated RNA adducts by streptavidin-coated magnetic beads and disulfide chemical cleavage led to isolation of highly enriched RNA samples from an initial 9/1 DNA-RNA mixture. Starting from the parent compound N-methylisatoic anhydride A which was used at 65 °C, we improved the extraction process by designing a new generation of isatoic anhydrides that are able to react under smoother conditions. Among them, a pyridine-based isatoic anhydride derivative 15f was found to be reactive at room temperature, leading to enhance the efficiency and selectivity of the extraction process by significantly reducing DNA side extraction. The extracted and purified RNAs can then be detected by RT-PCR.

Mutation-profile-based methods for understanding selection forces in cancer somatic mutations: a comparative analysis.

PubMed

Zhou, Zhan; Zou, Yangyun; Liu, Gangbiao; Zhou, Jingqi; Wu, Jingcheng; Zhao, Shimin; Su, Zhixi; Gu, Xun

2017-08-29

Human genes exhibit different effects on fitness in cancer and normal cells. Here, we present an evolutionary approach to measure the selection pressure on human genes, using the well-known ratio of the nonsynonymous to synonymous substitution rate in both cancer genomes ( C N / C S ) and normal populations ( p N / p S ). A new mutation-profile-based method that adopts sample-specific mutation rate profiles instead of conventional substitution models was developed. We found that cancer-specific selection pressure is quite different from the selection pressure at the species and population levels. Both the relaxation of purifying selection on passenger mutations and the positive selection of driver mutations may contribute to the increased C N / C S values of human genes in cancer genomes compared with the p N / p S values in human populations. The C N / C S values also contribute to the improved classification of cancer genes and a better understanding of the onco-functionalization of cancer genes during oncogenesis. The use of our computational pipeline to identify cancer-specific positively and negatively selected genes may provide useful information for understanding the evolution of cancers and identifying possible targets for therapeutic intervention.

Signatures of selection acting on the innate immunity gene Toll-like receptor 2 (TLR2) during the evolutionary history of rodents.

PubMed

Tschirren, B; Råberg, L; Westerdahl, H

2011-06-01

Patterns of selection acting on immune defence genes have recently been the focus of considerable interest. Yet, when it comes to vertebrates, studies have mainly focused on the acquired branch of the immune system. Consequently, the direction and strength of selection acting on genes of the vertebrate innate immune defence remain poorly understood. Here, we present a molecular analysis of selection on an important receptor of the innate immune system of vertebrates, the Toll-like receptor 2 (TLR2), across 17 rodent species. Although purifying selection was the prevalent evolutionary force acting on most parts of the rodent TLR2, we found that codons in close proximity to pathogen-binding and TLR2-TLR1 heterodimerization sites have been subject to positive selection. This indicates that parasite-mediated selection is not restricted to acquired immune system genes like the major histocompatibility complex, but also affects innate defence genes. To obtain a comprehensive understanding of evolutionary processes in host-parasite systems, both innate and acquired immunity thus need to be considered. © 2011 The Authors. Journal of Evolutionary Biology © 2011 European Society For Evolutionary Biology.

Anti-platelet activity of a three-finger toxin (3FTx) from Indian monocled cobra (Naja kaouthia) venom.

PubMed

Chanda, Chandrasekhar; Sarkar, Angshuman; Sistla, Srinivas; Chakrabarty, Dibakar

2013-11-22

A low molecular weight anti-platelet peptide (6.9 kDa) has been purified from Naja kaouthia venom and was named KT-6.9. MALDI-TOF/TOF mass spectrometry analysis revealed the homology of KT-6.9 peptide sequence with many three finger toxin family members. KT-6.9 inhibited human platelet aggregation process in a dose dependent manner. It has inhibited ADP, thrombin and arachidonic acid induced platelet aggregation process in dose dependent manner, but did not inhibit collagen and ristocetin induced platelet aggregation. Strong inhibition (70%) of the ADP induced platelet aggregation by KT-6.9 suggests competition with ADP for its receptors on platelet surface. Anti-platelet activity of KT-6.9 was found to be 25 times stronger than that of anti-platelet drug clopidogrel. Binding of KT-6.9 to platelet surface was confirmed by surface plasma resonance analysis using BIAcore X100. Binding was also observed by a modified sandwich ELISA method using anti-KT-6.9 antibodies. KT-6.9 is probably the first 3 FTx from Indian monocled cobra venom reported as a platelet aggregation inhibitor. Copyright © 2013 Elsevier Inc. All rights reserved.

Dual targeting and enhanced cytotoxicity to HER2-overexpressing tumors by immunoapoptotin-armored mesenchymal stem cells.

PubMed

Cai, Yanhui; Xi, Yujing; Cao, Zhongyuan; Xiang, Geng; Ni, Qingrong; Zhang, Rui; Chang, Jing; Du, Xiao; Yang, Angang; Yan, Bo; Zhao, Jing

2016-10-10

Mesenchymal stem cells (MSCs) are promising vehicles for the delivery of anticancer agents in cancer therapy. However, the tumor targeting of loaded therapeutics is essential. Here, we explored a dual-targeting strategy to incorporate tumor-tropic MSC delivery with HER2-specific killing by the immunoapoptotin e23sFv-Fdt-tBid generated in our previous studies. The MSC engineering allowed simultaneous immunoapoptotin secretion and bioluminescence detection of the modified MSCs. Systemic administration of the immunoapoptotin-engineered MSCs was investigated in human HER2-reconstituted syngeneic mouse models of orthotopic and metastatic breast cancer, as well as in a xenograft nude mouse model of orthotopic gastric cancer. In vivo dual tumor targeting was confirmed by local accumulation of the bioluminescence-imaged MSCs and persistence of His-immunostained immunoapoptotins in tumor sites. The added tumor preference of MSC-secreted immunoapoptotins resulted in a significantly stronger antitumor effect compared with purified immunoapoptotins and Jurkat-delivered immunoapoptotins. This immunoapoptotin-armored MSC strategy provides a rationale for its use in extended malignancies by combining MSC mobility with redirected immunoapoptotins against a given tumor antigen. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Purification, characterization, antioxidant and anticancer activities of novel polysaccharides extracted from Bachu mushroom.

PubMed

Zeng, Di; Zhu, Siming

2018-02-01

Two novel polysaccharide fractions (HLP1-1 and HLP2-1) were purified from crude polysaccharides of Helvella leucopus by using DEAE-52 column (2.6cm×20cm) and Sephadex G-150 column (1.6×60cm). The characterization, antioxidant and anticancer activities of HLP1-1 and HLP2-1 were investigated. The GPC results showed that HLP1-1 and HLP2-1 had similar molecular weight (21,382Da and 23,063Da, respectively). Tertiary structure analyses indicated that HLP2-1 had triple-helical conformation, but HLP1-1 not. The monosaccharide compositions of HLP1-1 included rhamnose, glucosamine and mannose at a molar ratio of 11.8:1:78.6, and HLP2-1 included of rhamnose, glucosamine, glucose and mannose at a molar ratio of 4.2:1:18.1:27.3. Both HLP1-1 and HLP2-1 showed a certain antioxidant activity, and HLP2-1 showed stronger antioxidant activities than HLP1-1. Both HLP1-1 and HLP2-1 exhibited a relatively inhibition on HepG2. Copyright © 2017 Elsevier B.V. All rights reserved.

Metal-Chelate Affinity Precipitation with Thermo-Responsive Polymer for Purification of ε-Poly-L-Lysine.

PubMed

Li, Sipeng; Ding, Zhaoyang; Liu, Jifu; Cao, Xuejun

2017-12-01

ε-Poly-L-lysine (ε-PL) is a natural preservative for food processing industry. A thermo-responsive polymer, attached with Cu 2+ or Ni 2+ , was prepared for metal-chelate affinity precipitation for purification of ε-PL. The low critical solution temperatures (LCSTs) of these polymers were close to the room temperature (31.0-35.0 °C). The optimal adsorption conditions were as follows: pH 4.0, 0 mol/L NaCl, ligand density 75.00 μmol/g, and 120 min. The ligand Cu 2+ showed a stronger affinity interaction with ε-PL and the highest adsorption amount reached 251.93 mg/g polymer. The elution recovery of ε-PL could be 98.42% with 0.50 mol/L imidazole (pH = 8.0) as the eluent. The method could purify ε-PL from fermentation broth and the final product was proved as electrophoretic pure by SDS-PAGE. Moreover, these affinity polymers could be recycled after the purification of ε-PL and the recoveries were above 95.00%. Graphical Abstract Scheme for affinity precipitation of ε-PL.

Extracts and Constituents of Rubus chingii with 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Free Radical Scavenging Activity

PubMed Central

Ding, Hsiou-Yu

2011-01-01

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity of the fruits of Rubus chingii was studied in vitro. Ethanolic extract, ethyl acetate and n-butanol fractions from dried R. chingii fruits revealed strong DPPH free radical scavenging activity with IC50 values of 17.9, 3.4 and 4.0 μg/mL, respectively. The ethyl acetate and n-butanol fractions were further purified by a combination of silica gel chromatography, Lobar RP-8 chromatography, and high-pressure liquid chromatography (HPLC). Nine compounds were isolated, where methyl (3-hydroxy-2-oxo-2,3-dihydroindol-3-yl)-acetate (2), vanillic acid (5), kaempferol (7), and tiliroside (9) showed stronger DPPH free radical scavenging activity than that of ascorbic acid (131.8 μM) with IC50 values of 45.2, 34.9, 78.5, and 13.7 μM, respectively. In addition, rubusine (1) is a new compound discovered in the present study and methyl (3-hydroxy-2-oxo-2,3-dihydroindol-3-yl)-acetate (2), methyl dioxindole-3-acetate (3), and 2-oxo-1,2-dihydroquinoline-4-carboxylic acid (4) were isolated from the fruits for the first time. PMID:21747716

Purification, characterization and anti-proliferation activities of polysaccharides extracted from Viscum coloratum (Kom.) Nakai.

PubMed

Chai, Yangyang; Zhao, Min

2016-09-20

Three polysaccharides, VCP1, VCP2 and VCP3 were isolated from Viscum coloratum (Kom.) Nakai using DEAE-cellulose chromatography. VCP1 (32KDa) was composed of glucose, galactose, arabinose, rhamnose and mannose, while VCP2 (280KDa) and VCP3 (21KDa) were consisted of glucose, galactose, arabinose, rhamnose, mannose, glucuronic acid and galacturonic acid. The optical rotation was measured at 20+1°C. The characteristic absorptive bands of purified fraction were determined by FT-IR. (13)C NMR spectroscopy analysis showed that VCP1 was a neutral polysaccharide, and VCP2 and VCP3 were RG-I type pectin. The linkage patterns of VCP2 were evaluated by methylation analysis: 1,5-linked Araf, 1,4-linked Galp, 1,2-linked Rhap, and 1,2,4-linked Rhap. The degree of esterification was 50%. The anti-proliferation ability against HepG2 cells and HepG2.2.15 cells of VCP2 was stronger than VCP1 and VCP3. So the polysaccharides from Viscum coloratum (Kom.) Nakai could be used as potential natural sources with inhibiting tumor cells proliferation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Antioxidant activity and oxidative stress protection of duck proteins hydrolysates in SK-N-SH cells.

PubMed

Guo, Yuxing; Pan, Daodong; Wu, Zhen; Zhao, Chuanchuan; Cao, Jinxuan

2013-02-26

Studies have found that natural antioxidants, which are free-radical scavengers, can reduce the risk of diseases caused by free radicals. This work investigated the antioxidant properties of duck proteins hydrolysates. The free-radical scavenging function of CP-1 (M(r) > 10 kDa), CP-2 (5 kDa < M(r) < 10 kDa) and CP-3 (M(r) < 5 kDa), obtained through ultrafiltration and gel filtration were evaluated. The results showed that the lower molecular weight fraction exhibited a stronger free-radical scavenging ability. The highest free-radical scavenging activity was detected in the fraction of p4 purified from CP-3 using Sephadex G-15 column chromatography. The 50% inhibitory value (IC(50)) of p4 for scavenging radicals of superoxide, hydroxyl and 1,1-diphenyl-2-pycrylhydrazyl (DPPH) were, respectively, 0.97 mg mL(-1), 0.84 mg mL(-1) and 1.84 mg mL(-1). Furthermore, the p4 fraction at a concentration of 10 μg mL(-1) increased cell viability from 84.8% to 94% under antioxidative stress in neuroblastoma SK-N-SH cells.

Heat-shock protein-25/27 phosphorylation by the delta isoform of protein kinase C.

PubMed Central

Maizels, E T; Peters, C A; Kline, M; Cutler, R E; Shanmugam, M; Hunzicker-Dunn, M

1998-01-01

Small heat-shock proteins (sHSPs) are widely expressed 25-28 kDa proteins whose functions are dynamically regulated by phosphorylation. While recent efforts have clearly delineated a stress-responsive p38 mitogen-activated protein-kinase (MAPK)-dependent kinase pathway culminating in activation of the heat-shock (HSP)-kinases, mitogen-activated protein-kinase-activated protein kinase-2 and -3, not all sHSP phosphorylation events can be explained by the p38 MAPK-dependent pathway. The contribution of protein kinase C (PKC) to sHSP phosphorylation was suggested by early studies but later questioned on the basis of the reported poor ability of purified PKC to phosphorylate sHSP in vitro. The current study re-evaluates the role of PKC in sHSP phosphorylation in the light of the isoform complexity of the PKC family. We evaluated the sHSP phosphorylation status in rat corpora lutea obtained from two stages of pregnancy, mid-pregnancy and late-pregnancy, which express different levels of the novel PKC isoform, PKC-delta. Two-dimensional Western blot analysis showed that HSP-27 was more highly phosphorylated in vivo in corpora lutea of late pregnancy, corresponding to the developmental stage in which PKC-delta is abundant and active. Late-pregnant luteal extracts contained a lipid-sensitive HSP-kinase activity which exactly co-purified with PKC-delta using hydroxyapatite and S-Sepharose column chromatography. To determine whether there might be preferential phosphorylation of sHSP by a particular PKC isoform, purified recombinant PKC isoforms corresponding to those PKC isoforms detected in rat corpora lutea were evaluated for HSP-kinase activity in vitro. Recombinant PKC-delta effectively catalysed the phosphorylation of sHSP in vitro, and PKC-alpha was 30-50% as effective as an HSP-kinase; other PKCs tested (beta1, beta2, epsilon and zeta) were poor HSP-kinases. These results show that select PKC family members can function as direct HSP-kinases in vitro. Moreover, the observation of enhanced luteal HSP-27 phosphorylation in vivo, in late pregnancy, when PKC-delta is abundant and active, suggests that select PKC family members contribute to sHSP phosphorylation events in vivo. PMID:9620873

Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi

DOE PAGES

Xu, Qi; Knoshaug, Eric P.; Wang, Wei; ...

2017-07-24

Lipomyces starkeyi is one of the leading lipid-producing microorganisms reported to date; its genetic transformation was only recently reported. Our aim is to engineer L. starkeyi to serve in consolidated bioprocessing (CBP) to produce lipid or fatty acid-related biofuels directly from abundant and low-cost lignocellulosic substrates. To evaluate L. starkeyi in this role, we first conducted a genome analysis, which revealed the absence of key endo- and exocellulases in this yeast, prompting us to select and screen four signal peptides for their suitability for the overexpression and secretion of cellulase genes. To compensate for the cellulase deficiency, we chose twomore » prominent cellulases, Trichoderma reesei endoglucanase II (EG II) and a chimeric cellobiohydrolase I (TeTrCBH I) formed by fusion of the catalytic domain from Talaromyces emersonii CBH I with the linker peptide and cellulose-binding domain from T. reesei CBH I. The systematically tested signal peptides included three peptides from native L. starkeyi and one from Yarrowia lipolytica. We found that all four signal peptides permitted secretion of active EG II. We also determined that three of these signal peptides worked for expression of the chimeric CBH I; suggesting that our design criteria for selecting these signal peptides was effective. Encouragingly, the Y. lipolytica signal peptide was able to efficiently guide secretion of the chimeric TeTrCBH I protein from L. starkeyi. The purified chimeric TeTrCBH I showed high activity against the cellulose in pretreated corn stover and the purified EG II showed high endocellulase activity measured by the CELLG3 (Megazyme) method. Our results suggest that L. starkeyi is capable of expressing and secreting core fungal cellulases. Moreover, the purified EG II and chimeric TeTrCBH I displayed significant and potentially useful enzymatic activities, demonstrating that engineered L. starkeyi has the potential to function as an oleaginous CBP strain for biofuel production. Lastly, the effectiveness of the tested secretion signals will also benefit future secretion of other heterologous proteins in L. starkeyi and, given the effectiveness of the cross-genus secretion signal, possibly other oleaginous yeasts as well.« less

Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi

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

Xu, Qi; Knoshaug, Eric P.; Wang, Wei

Lipomyces starkeyi is one of the leading lipid-producing microorganisms reported to date; its genetic transformation was only recently reported. Our aim is to engineer L. starkeyi to serve in consolidated bioprocessing (CBP) to produce lipid or fatty acid-related biofuels directly from abundant and low-cost lignocellulosic substrates. To evaluate L. starkeyi in this role, we first conducted a genome analysis, which revealed the absence of key endo- and exocellulases in this yeast, prompting us to select and screen four signal peptides for their suitability for the overexpression and secretion of cellulase genes. To compensate for the cellulase deficiency, we chose twomore » prominent cellulases, Trichoderma reesei endoglucanase II (EG II) and a chimeric cellobiohydrolase I (TeTrCBH I) formed by fusion of the catalytic domain from Talaromyces emersonii CBH I with the linker peptide and cellulose-binding domain from T. reesei CBH I. The systematically tested signal peptides included three peptides from native L. starkeyi and one from Yarrowia lipolytica. We found that all four signal peptides permitted secretion of active EG II. We also determined that three of these signal peptides worked for expression of the chimeric CBH I; suggesting that our design criteria for selecting these signal peptides was effective. Encouragingly, the Y. lipolytica signal peptide was able to efficiently guide secretion of the chimeric TeTrCBH I protein from L. starkeyi. The purified chimeric TeTrCBH I showed high activity against the cellulose in pretreated corn stover and the purified EG II showed high endocellulase activity measured by the CELLG3 (Megazyme) method. Our results suggest that L. starkeyi is capable of expressing and secreting core fungal cellulases. Moreover, the purified EG II and chimeric TeTrCBH I displayed significant and potentially useful enzymatic activities, demonstrating that engineered L. starkeyi has the potential to function as an oleaginous CBP strain for biofuel production. Lastly, the effectiveness of the tested secretion signals will also benefit future secretion of other heterologous proteins in L. starkeyi and, given the effectiveness of the cross-genus secretion signal, possibly other oleaginous yeasts as well.« less

Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi.

PubMed

Xu, Qi; Knoshaug, Eric P; Wang, Wei; Alahuhta, Markus; Baker, John O; Yang, Shihui; Vander Wall, Todd; Decker, Stephen R; Himmel, Michael E; Zhang, Min; Wei, Hui

2017-07-24

Lipomyces starkeyi is one of the leading lipid-producing microorganisms reported to date; its genetic transformation was only recently reported. Our aim is to engineer L. starkeyi to serve in consolidated bioprocessing (CBP) to produce lipid or fatty acid-related biofuels directly from abundant and low-cost lignocellulosic substrates. To evaluate L. starkeyi in this role, we first conducted a genome analysis, which revealed the absence of key endo- and exocellulases in this yeast, prompting us to select and screen four signal peptides for their suitability for the overexpression and secretion of cellulase genes. To compensate for the cellulase deficiency, we chose two prominent cellulases, Trichoderma reesei endoglucanase II (EG II) and a chimeric cellobiohydrolase I (TeTrCBH I) formed by fusion of the catalytic domain from Talaromyces emersonii CBH I with the linker peptide and cellulose-binding domain from T. reesei CBH I. The systematically tested signal peptides included three peptides from native L. starkeyi and one from Yarrowia lipolytica. We found that all four signal peptides permitted secretion of active EG II. We also determined that three of these signal peptides worked for expression of the chimeric CBH I; suggesting that our design criteria for selecting these signal peptides was effective. Encouragingly, the Y. lipolytica signal peptide was able to efficiently guide secretion of the chimeric TeTrCBH I protein from L. starkeyi. The purified chimeric TeTrCBH I showed high activity against the cellulose in pretreated corn stover and the purified EG II showed high endocellulase activity measured by the CELLG3 (Megazyme) method. Our results suggest that L. starkeyi is capable of expressing and secreting core fungal cellulases. Moreover, the purified EG II and chimeric TeTrCBH I displayed significant and potentially useful enzymatic activities, demonstrating that engineered L. starkeyi has the potential to function as an oleaginous CBP strain for biofuel production. The effectiveness of the tested secretion signals will also benefit future secretion of other heterologous proteins in L. starkeyi and, given the effectiveness of the cross-genus secretion signal, possibly other oleaginous yeasts as well.