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Sample records for identify eggshell proteins

  1. Gene expression profiling to identify eggshell proteins involved in physical defense of the chicken egg

    PubMed Central

    2010-01-01

    Background As uricoletic animals, chickens produce cleidoic eggs, which are self-contained bacteria-resistant biological packages for extra-uterine development of the chick embryo. The eggshell constitutes a natural physical barrier against bacterial penetration if it forms correctly and remains intact. The eggshell's remarkable mechanical properties are due to interactions among mineral components and the organic matrix proteins. The purpose of our study was to identify novel eggshell proteins by examining the transcriptome of the uterus during calcification of the eggshell. An extensive bioinformatic analysis on genes over-expressed in the uterus allowed us to identify novel eggshell proteins that contribute to the egg's natural defenses. Results Our 14 K Del-Mar Chicken Integrated Systems microarray was used for transcriptional profiling in the hen's uterus during eggshell deposition. A total of 605 transcripts were over-expressed in the uterus compared with the magnum or white isthmus across a wide range of abundance (1.1- to 79.4-fold difference). The 605 highly-expressed uterine transcripts correspond to 469 unique genes, which encode 437 different proteins. Gene Ontology (GO) analysis was used for interpretation of protein function. The most over-represented GO terms are related to genes encoding ion transport proteins, which provide eggshell mineral precursors. Signal peptide sequence was found for 54 putative proteins secreted by the uterus during eggshell formation. Many functional proteins are involved in calcium binding or biomineralization--prerequisites for interacting with the mineral phase during eggshell fabrication. While another large group of proteins could be involved in proper folding of the eggshell matrix. Many secreted uterine proteins possess antibacterial properties, which would protect the egg against microbial invasion. A final group includes proteases and protease inhibitors that regulate protein activity in the acellular uterine fluid

  2. Differential label-free quantitative proteomic analysis of avian eggshell matrix and uterine fluid proteins associated with eggshell mechanical property.

    PubMed

    Sun, Congjiao; Xu, Guiyun; Yang, Ning

    2013-12-01

    Eggshell strength is a crucial economic trait for table egg production. During the process of eggshell formation, uncalcified eggs are bathed in uterine fluid that plays regulatory roles in eggshell calcification. In this study, a label-free MS-based protein quantification technology was used to detect differences in protein abundance between eggshell matrix from strong and weak eggs (shell matrix protein from strong eggshells and shell matrix protein from weak eggshells) and between the corresponding uterine fluids bathing strong and weak eggs (uterine fluid bathing strong eggs and uterine fluid bathing weak eggs) in a chicken population. Here, we reported the first global proteomic analysis of uterine fluid. A total of 577 and 466 proteins were identified in uterine fluid and eggshell matrix, respectively. Of 447 identified proteins in uterine fluid bathing strong eggs, up to 357 (80%) proteins were in common with proteins in uterine fluid bathing weak eggs. Similarly, up to 83% (328/396) of the proteins in shell matrix protein from strong eggshells were in common with the proteins in shell matrix protein from weak eggshells. The large amount of common proteins indicated that the difference in protein abundance should play essential roles in influencing eggshell strength. Ultimately, 15 proteins mainly relating to eggshell matrix specific proteins, calcium binding and transportation, protein folding and sorting, bone development or diseases, and thyroid hormone activity were considered to have closer association with the formation of strong eggshell. PMID:24151251

  3. Minor proteins and enzymes of the Drosophila eggshell matrix.

    PubMed

    Fakhouri, Mazen; Elalayli, Maggie; Sherling, Daniel; Hall, Jacklyn D; Miller, Eric; Sun, Xutong; Wells, Lance; LeMosy, Ellen K

    2006-05-01

    The Drosophila eggshell provides an in vivo model system for extracellular matrix assembly, in which programmed gene expression, cell migrations, extracellular protein trafficking, proteolytic processing, and cross-linking are all required to generate a multi-layered and regionally complex architecture. While abundant structural components of the eggshell are known and are being characterized, less is known about non-abundant structural, regulatory, and enzymatic components that are likely to play critical roles in eggshell assembly. We have used sensitive mass spectrometry-based analyses of fractionated eggshell matrices to validate six previously predicted eggshell proteins and to identify eleven novel components, and have characterized the expression patterns of many of their mRNAs. Among these are several putative structural or regulatory (non-enzymatic) proteins, most larger in mass than the major eggshell proteins and often showing preferential expression in follicle cells overlying specific structural features of the eggshell. Of particular note are the putative enzymes, some likely to be involved in matrix cross-linking (two yellow family members previously implicated in eggshell integrity, a heme peroxidase, and a small-molecule oxidoreductase) and others possibly involved in matrix proteolysis or adhesion (proteins related to cathepsins B and D). This work provides a framework for future molecular studies of eggshell assembly. PMID:16515779

  4. Possible eggshell protein gene from Schistosoma mansoni.

    PubMed

    Johnson, K S; Taylor, D W; Cordingley, J S

    1987-01-01

    We have identified and sequenced a cDNA clone of a mRNA found only in mature female schistosomes. This mRNA is not detectably synthesized by female worms from single sex infections (unisexual females), by males or by the developing miracidia in the eggs. The clone hybridises to a highly abundant polyadenylated mRNA of approximately 1500 nucleotides. The nucleotide sequence of the clone predicts a polypeptide comprising two repetitive regions. A pentapeptide repeat with the consensus sequence Gly-Tyr-Asp-Lys-Tyr, and a region rich in histidine residues. Hybrid selected mRNA translated in vitro with [3H]tyrosine as labelled amino acid yields a polypeptide of 48 kDa (p48) that corresponds to the major [3H]tyrosine labelled translation product of female worm total mRNA. p48 does not label with [35S]methionine and is absent from the translation products of male and unisexual female mRNAs. The amino acid sequence of p48 has significant homologies to silk moth chorion proteins and we suggest that it is one of the major components of the schistosome eggshell probably accounting for the high level of [3H]tyrosine incorporation into the vitellaria of Schistosoma mansoni. The tyrosine content of the polypeptide suggests that it may play a role in phenol oxidase mediated cross-linking of the schistosome eggshell and in support of this we find that mushroom phenol oxidase will cause the specific cross-linking of p48 in in vitro translation products. PMID:3100949

  5. Proteins of insoluble matrix of avian (gallus gallus) eggshell.

    PubMed

    Miksík, Ivan; Eckhardt, Adam; Sedláková, Pavla; Mikulikova, Katerina

    2007-01-01

    The protein composition of the insoluble avian eggshell matrix was studied. The determination of these proteins insoluble in water (EDTA-insoluble) was carried out using enzymatic cleavage followed by a high-performance liquid chromatography-mass spectrometry method. The influence of various enzymes on the protein splitting also was studied. The distribution of proteins depends on the type of layer (localization within the eggshell): ovocalyxin-32 was found mainly in the outer layer (the cuticle); ovocleidin-116 and 17 and ovocalyxin-36 were found throughout the whole eggshell, whereas ovalbumin was only found in the inner layer, the mammillary. The pigment (protoporphyrin IX) was mainly found in the cuticle and is incorporated into the protein network. PMID:17364661

  6. Integrating transcriptome and genome re-sequencing data to identify key genes and mutations affecting chicken eggshell qualities.

    PubMed

    Zhang, Quan; Zhu, Feng; Liu, Long; Zheng, Chuan Wei; Wang, De He; Hou, Zhuo Cheng; Ning, Zhong Hua

    2015-01-01

    Eggshell damages lead to economic losses in the egg production industry and are a threat to human health. We examined 49-wk-old Rhode Island White hens (Gallus gallus) that laid eggs having shells with significantly different strengths and thicknesses. We used HiSeq 2000 (Illumina) sequencing to characterize the chicken transcriptome and whole genome to identify the key genes and genetic mutations associated with eggshell calcification. We identified a total of 14,234 genes expressed in the chicken uterus, representing 89% of all annotated chicken genes. A total of 889 differentially expressed genes were identified by comparing low eggshell strength (LES) and normal eggshell strength (NES) genomes. The DEGs are enriched in calcification-related processes, including calcium ion transport and calcium signaling pathways as revealed by gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. Some important matrix proteins, such as OC-116, LTF and SPP1, were also expressed differentially between two groups. A total of 3,671,919 single-nucleotide polymorphisms (SNPs) and 508,035 Indels were detected in protein coding genes by whole-genome re-sequencing, including 1775 non-synonymous variations and 19 frame-shift Indels in DEGs. SNPs and Indels found in this study could be further investigated for eggshell traits. This is the first report to integrate the transcriptome and genome re-sequencing to target the genetic variations which decreased the eggshell qualities. These findings further advance our understanding of eggshell calcification in the chicken uterus. PMID:25974068

  7. Integrating Transcriptome and Genome Re-Sequencing Data to Identify Key Genes and Mutations Affecting Chicken Eggshell Qualities

    PubMed Central

    Liu, Long; Zheng, Chuan Wei; Wang, De He; Hou, Zhuo Cheng; Ning, Zhong Hua

    2015-01-01

    Eggshell damages lead to economic losses in the egg production industry and are a threat to human health. We examined 49-wk-old Rhode Island White hens (Gallus gallus) that laid eggs having shells with significantly different strengths and thicknesses. We used HiSeq 2000 (Illumina) sequencing to characterize the chicken transcriptome and whole genome to identify the key genes and genetic mutations associated with eggshell calcification. We identified a total of 14,234 genes expressed in the chicken uterus, representing 89% of all annotated chicken genes. A total of 889 differentially expressed genes were identified by comparing low eggshell strength (LES) and normal eggshell strength (NES) genomes. The DEGs are enriched in calcification-related processes, including calcium ion transport and calcium signaling pathways as reveled by gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. Some important matrix proteins, such as OC-116, LTF and SPP1, were also expressed differentially between two groups. A total of 3,671,919 single-nucleotide polymorphisms (SNPs) and 508,035 Indels were detected in protein coding genes by whole-genome re-sequencing, including 1775 non-synonymous variations and 19 frame-shift Indels in DEGs. SNPs and Indels found in this study could be further investigated for eggshell traits. This is the first report to integrate the transcriptome and genome re-sequencing to target the genetic variations which decreased the eggshell qualities. These findings further advance our understanding of eggshell calcification in the chicken uterus. PMID:25974068

  8. Fabrication of a biocomposite reinforced with hydrophilic eggshell proteins

    NASA Astrophysics Data System (ADS)

    Kim, Geun Hyung; Min, Taijin; Park, Su A.; Doo Kim, Wan; Koh, Young Ho

    2007-12-01

    Soluble eggshell proteins were used as a reinforcing material of electrospun micro/nanofibers for tissue engineering. A biocomposite composed of poly(ɛ-caprolactone) (PCL) micro/nanofibers and soluble eggshell protein was fabricated with a two-step fabrication method, which is an electrospinning process followed by an air-spraying process. To achieve a stable electrospinning process, we used an auxiliary cylindrical electrode connected with a spinning nozzle. PCL biocomposite was characterized in water contact angle and mechanical properties as well as cell proliferation for its application as a tissue engineering material. It showed an improved hydrophilic characteristic compared with that of a micro/nanofiber web generated from a pure PCL solution using a typical electrospinning process. Moreover, the fabricated biocomposite had good mechanical properties compared to a typical electrospun micro/nanofiber mat. The fabricated biocomposite made human dermal fibroblasts grow better than pure PCL. From the results, the reinforced polymeric micro/nanofiber scaffold can be easily achieved with these modified processes.

  9. Organic solvent-tolerant elastase efficiently hydrolyzes insoluble, cross-linked, protein fiber of eggshell membranes.

    PubMed

    Takenaka, Shinji; Hano, Shinpei; Cheng, Minyi; Yoshida, Ken-ichi; Aoki, Kenji

    2012-05-01

    Eggshell membrane is a mechanically stable and insoluble cross-linked fibrous protein. Pseudomonas aeruginosa strain ME-4 synthesizes a metalloprotease that degrades the eggshell membrane. We cloned the encoding gene in Escherichia coli. The recombinant protease, over-expressed in E. coli, was inactive but addition of acetone to crude cell extracts restored the activity and removed many E. coli proteins. We purified the active, acetone-treated protease to homogeneity in a single chromatography step with 57% recovery. The recombinant protease partially hydrolyzed eggshell membrane and produced more soluble peptides and proteins than commercial elastase, α-chymotrypsin, and collagenase. The soluble peptides produced from hydrolyzed eggshell membrane inhibited angiotensin-I-converting enzyme activity. The degradation of eggshell membrane by the recombinant elastase could be applied to the production of soluble bioactive peptides. PMID:22286207

  10. Structure of struthiocalcin-1, an intramineral protein from Struthio camelus eggshell, in two crystal forms.

    PubMed

    Ruiz-Arellano, Rayana R; Medrano, Francisco J; Moreno, Abel; Romero, Antonio

    2015-04-01

    Biomineralization is the process by which living organisms produce minerals. One remarkable example is the formation of eggshells in birds. Struthiocalcins present in the ostrich (Struthio camellus) eggshell matrix act as biosensors of calcite growth during eggshell formation. Here, the crystal structure of struthiocalcin-1 (SCA-1) is reported in two different crystal forms. The structure is a compact single domain with an α/β fold characteristic of the C-type lectin family. In contrast to the related avian ovocleidin OC17, the electrostatic potential on the molecular surface is dominated by an acidic patch. Scanning electron microscopy combined with Raman spectroscopy indicates that these intramineral proteins (SCA-1 and SCA-2) induce calcium carbonate precipitation, leading to the formation of a stable form of calcite in the mature eggshell. Finally, the implications of these two intramineral proteins SCA-1 and SCA-2 in the nucleation of calcite during the formation of eggshells in ratite birds are discussed. PMID:25849392

  11. Drosophila dec-1 eggshell proteins are differentially distributed via a multistep extracellular processing and localization pathway.

    PubMed

    Noguerón, M I; Mauzy-Melitz, D; Waring, G L

    2000-09-15

    In Drosophila the multilayered eggshell forms during late oogenesis between the oocyte and the overlaying follicle cells. Proper eggshell assembly requires wild-type dec-1 gene function. Alternatively spliced dec-1 transcripts encode three proproteins that are cleaved extracellularly in a stage-specific manner to at least five distinct derivatives. Using polyclonal antibodies raised against fusion proteins containing different regions of the dec-1 proteins, we have localized several dec-1 derivatives in the assembling and completed eggshell. Although all of the dec-1 derivatives are generated in the oocyte proximal vitelline membrane layer, they are differentially distributed in the mature egg. Some derivatives are gradually released from the vitelline membrane and become localized within distinct regions of the chorion, while others are taken up by the oocyte or become concentrated in the endochorionic spaces or cavities. The diverse distributions of the dec-1 derivatives suggest that each derivative plays a distinct role in eggshell assembly. These results also suggest that the vitelline membrane layer, by acting as a transient storage site, may control the availability of molecules active in eggshell assembly and by extension perhaps other follicle cell products important in early embryonic pattern formation. PMID:10985863

  12. Quantitative expression of candidate genes affecting eggshell color.

    PubMed

    Zheng, Chuanwei; Li, Zesheng; Yang, Ning; Ning, Zhonghua

    2014-05-01

    There are three pigments that affect the color of an eggshell: protoporphyrin, biliverdin and biliverdin-zinc chelate. Protoporphyrin is the main pigment in brown and light-brown eggshells, whereas very little protoporphyrin is found in white eggshells. Eggshell protoporphyrin is derived from the heme formation in birds. Coproporphyrinogen III oxidase (CPOX) and ferrochelatase (FECH) represent rate-limiting enzymes for the heme-biosynthetic pathway. Breast cancer resistance protein (BCRP), feline leukemia virus receptor (FLVCR), and heme-responsive gene-1 (HRG1) serve as primary transporters for both protoporphyrinogen and heme. Finally, four organic anion transporting polypeptide family members (including solute carrier organic anion transporter family, SLCO1C1, SLCO1A2, SLCO1B3 and LOC418189) may affect pigment transport within eggshells. Here we measured gene expression levels in key tissues of egg-producing hens. We analyzed three different types of hens that generated distinct eggshell colors: white, pink or brown. Our data revealed three ways in which eggshell color was genetically influenced. First, high-level expression of CPOX generated more protoporphyrinogen and a brown eggshell color. In contrast, high expression of FECH likely converted more protoporphyrinogen into heme, reduced protoporphyrinogen levels within the eggshell and generated a light color. Second, heme transporters also affected eggshell color. High-level expression of BCRP, HRG1 and FLVCR were associated with brown, white and generally lighter eggshell colors, respectively. Finally, protoporphyrin precipitation also affected eggshell color, as high expression of both SLCO1A2 and SLCO1C1 were associated with brown eggshell color. As such, we have identified seven genes in which expression levels in different tissues were associated with eggshell color. PMID:24612318

  13. Genetic variation in eggshell crystal size and orientation is large and these traits are correlated with shell thickness and are associated with eggshell matrix protein markers.

    PubMed

    Dunn, I C; Rodríguez-Navarro, A B; Mcdade, K; Schmutz, M; Preisinger, R; Waddington, D; Wilson, P W; Bain, M M

    2012-08-01

    The size and orientation of calcium carbonate crystals influence the structure and strength of the eggshells of chickens. In this study, estimates of heritability were found to be high (0.6) for crystal size and moderate (0.3) for crystal orientation. There was a strong positive correlation (0.65) for crystal size and orientation with the thickness of the shell and, in particular, with the thickness of the mammillary layer. Correlations with shell breaking strength were positive but with a high standard error. This was contrary to expectations, as in man-made materials smaller crystals would be stronger. We believe the results of this study support the hypothesis that the structural organization of shell, and in particular the mammillary layer, is influenced by crystal size and orientation, especially during the initial phase of calcification. Genetic associations for crystal measurements were observed between haplotype blocks or individual markers for a number of eggshell matrix proteins. Ovalbumin and ovotransferrin (LTF) markers for example were associated with crystal size, while ovocleidin-116 and ovocalyxin-32 (RARRES1) markers were associated with crystal orientation. The location of these proteins in the eggshell is consistent with different phases of the shell-formation process. In conclusion, the variability of crystal size, and to a lesser extent orientation, appears to have a large genetic component, and the formation of calcite crystals are intimately related to the ultrastructure of the eggshell. Moreover, this study also provides evidence that proteins in the shell influence the variability of crystal traits and, in turn, the shell's thickness profile. The crystal measurements and/or the associated genetic markers may therefore prove to be useful in selection programs to improve eggshell quality. PMID:22497523

  14. Antimicrobial properties of avian eggshell-specific C-type lectin-like proteins.

    PubMed

    Wellman-Labadie, Olivier; Lakshminarayanan, Rajamani; Hincke, Maxwell T

    2008-03-01

    C-type lectin-like proteins are major components of the calcified eggshell of multiple avian species. In this study, two representative avian C-type lectin-like proteins, ovocleidin-17 and ansocalcin, were purified from decalcified chicken and goose eggshell protein extracts and investigated for carbohydrate binding activity as well as antimicrobial activity. Purified ovocleidin-17 and ansocalcin were found to bind bacterial polysaccharides, and were bactericidal against Bacillus subtilis, Staphylococcus aureus and Pseudomona aeruginosa. Bactericidal activity was found to be enhanced in the presence of calcium but was not dependent on its presence. The results suggest that avian C-type lectin-like proteins may play an important antimicrobial role in defence of the avian embryo. PMID:18258195

  15. Ovocalyxin-36 is a pattern recognition protein in chicken eggshell membranes.

    PubMed

    Cordeiro, Cristianne M M; Esmaili, Hamed; Ansah, George; Hincke, Maxwell T

    2013-01-01

    The avian eggshell membranes are essential elements in the fabrication of the calcified shell as a defense against bacterial penetration. Ovocalyxin-36 (OCX-36) is an abundant avian eggshell membrane protein, which shares protein sequence homology to bactericidal permeability-increasing protein (BPI), lipopolysaccharide-binding protein (LBP) and palate, lung and nasal epithelium clone (PLUNC) proteins. We have developed an efficient method to extract OCX-36 from chicken eggshell membranes for purification with cation and anion exchange chromatographies. Purified OCX-36 protein exhibited lipopolysaccharide (LPS) binding activity and bound lipopolysaccharide (LPS) from Escherichia coli O111:B4 in a dose-dependent manner. OCX-36 showed inhibitory activity against growth of Staphylococcus aureus ATCC 6538. OCX-36 single nucleotide polymorphisms (SNPs) were verified at cDNA 211 position and the corresponding proteins proline-71 (Pro-71) or serine-71 (Ser-71) were purified from eggs collected from genotyped hens. A significant difference between Pro-71 and Ser-71 OCX-36 for S. aureus lipoteichoic acid (LTA) binding activity was detected. The current study is a starting point to understand the innate immune role that OCX-36 may play in protection against bacterial invasion of both embryonated eggs (relevant to avian reproductive success) and unfertilized table eggs (relevant to food safety). PMID:24391897

  16. Solubilization and identification of hen eggshell membrane proteins during different times of chicken embryo development using the proteomic approach.

    PubMed

    Kaweewong, Kritsda; Garnjanagoonchorn, Wunwiboon; Jirapakkul, Wannee; Roytrakul, Sittiruk

    2013-04-01

    A fertilized chicken egg is a unit of life. During hatching, transport of nutrients, including calcium, have been reported from the egg components to the developing embryo. Calcium is mobilized from the eggshell with the involvement of Ca(2+)-binding proteins. In addition, other unknown proteins may also play some important roles during embryo developing process. Therefore identification and prediction of biological functions of eggshell membrane (ESM) proteins during chick embryo development was conducted by proteome analysis. Comparison of different lysis solutions indicated that the highest ability to extract ESM proteins could be obtained with 1 % sodium dodecyl sulfate in 5 mM Tris-HCl buffer pH 8.8 containing 0.1 % 2-mercaptoethanol. In this study fertilized Cornish chicken eggs were incubated at 37 °C in humidified incubators for up to 21 days. At selected times (days 1, 9, 15 and 21), samples were taken and the ESMs were carefully separated by hand, washed with distilled water, and air-dried at room temperature. The ESM proteins were then solubilized and analyzed by proteome analysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis combined with high performance liquid chromatography and mass spectrometry revealed 62 proteins in the ESM; only keratin is known ESM protein, 8 of which are egg white proteins and related while 53 others have not previously been reported. Some differences in the types of proteins and their molecular functions were noted in ESM at different incubation times. One protein which was present only at days 15 and 21 of egg incubation was identified as a calcium binding protein i.e. EGF like repeats and discoidin I like domain 3 (EDIL3 homologous protein). PMID:23636516

  17. Enhanced protein delivery by multi-ion containing eggshell derived apatitic-alginate composite nanocarriers.

    PubMed

    Sampath Kumar, T S; Madhumathi, K; Rajkamal, B; Zaheatha, S; Rajathi Malar, A; Alamelu Bai, S

    2014-11-01

    Eggshell is an attractive natural source of calcium for the synthesis of hydroxyapatite (HA) as it contains minor amounts of biologically relevant elements such as Mg, Sr, and Si. The mineral phase of the human bone is essentially a calcium deficient hydroxyapatite (CDHA) which shows more bioactivities and absorbance than stoichiometric HA does. Hence, we have attempted to develop a protein delivery system based on eggshell derived CDHA (ECDHA) nanoparticles for bone tissue engineering. Nanoparticles with Ca/P molar ratio of 1.67, 1.61 and 1.51 to form CDHAs with compositions covering the properties of stable HA phase (Ca/P=1.67) to degradable tricalcium phosphate (TCP) phase (Ca/P=1.5) were synthesized by microwave-accelerated wet chemical synthesis using eggshell as well as synthetic calcium hydroxide as calcium precursors. The delivery profiles of bovine serum albumin (BSA), a model protein by the nanocarriers, were studied. Both eggshells derived and synthetic CDHA samples showed maximum amount of loading of 57% and 37%, respectively at a Ca/P ratio of 1.51, comparing to stoichiometric HA. ECDHA also showed a much more BSA release (25%) than synthetically derived CDHA (6.5%) did. To further improve the release profile, alginate coating was carried out on CDHA nanoparticles and the BSA release profiles were evaluated. A maximum release of 65% was observed for alginate coated ECDHA at a Ca/P ratio of 1.51 for a period of 2 days. The ECDHA nanoparticle with a Ca/P ratio similar to degradable TCP and with alginate coating seems to be an ideal protein delivery agent. PMID:25444657

  18. Small gene family encoding an eggshell (chorion) protein of the human parasite Schistosoma mansoni

    SciTech Connect

    Bobek, L.A.; Rekosh, D.M.; Lo Verde, P.T.

    1988-08-01

    The authors isolated six independent genomic clones encoding schistosome chorion or eggshell proteins from a Schistosoma mansoni genomic library. A linkage map of five of the clones spanning 35 kilobase pairs (kbp) of the S. mansoni genome was constructed. The region contained two eggshell protein genes closely linked, separated by 7.5 kbp of intergenic DNA. The two genes of the cluster were arranged in the same orientation, that is, they were transcribed from the same strand. The sixth clone probably represents a third copy of the eggshell gene that is not contained within the 35-kbp region. The 5- end of the mRNA transcribed from these genes was defined by primer extension directly off the RNA. The ATCAT cap site sequence was homologous to a silkmoth chorion PuTCATT cap site sequence, where Pu indicates any purine. DNA sequence analysis showed that there were no introns in these genes. The DNA sequences of the three genes were very homologous to each other and to a cDNA clone, pSMf61-46, differing only in three or four nucleotices. A multiple TATA box was located at positions -23 to -31, and a CAAAT sequence was located at -52 upstream of the eggshell transcription unit. Comparison of sequences in regions further upstream with silkmoth and Drosophila sequences revealed very short elements that were shared. One such element, TCACGT, recently shown to be an essential cis-regulatory element for silkmoth chorion gene promoter function, was found at a similar position in all three organisms.

  19. Accumulation of the Drosophila Torso-like protein at the blastoderm plasma membrane suggests that it translocates from the eggshell.

    PubMed

    Mineo, Alessandro; Furriols, Marc; Casanova, Jordi

    2015-04-01

    The eggshell serves as a depository for proteins that play an important role in early embryonic development. In particular, the Drosophila eggshell is responsible for transferring asymmetries from the egg chamber to specify the regions at both ends of the embryo through the uneven activation of the Torso (Tor) receptor in its membrane. This process relies on the restricted expression of the gene torso-like (tsl) in subpopulations of follicle cells during oogenesis and its protein accumulation at both poles of the eggshell, but it is not known how this signal is transmitted to the embryo. Here, we show that Tsl accumulates at the embryonic plasma membrane, even in the absence of the Tor receptor. However, during oogenesis, we detected Tsl accumulation only at the eggshell. These results suggest that there is a two-step mechanism to transfer the asymmetric positional cues from the egg chamber into the early embryo: initial anchoring of Tsl at the eggshell as it is secreted, followed by its later translocation to the egg plasma membrane, where it enables Tor receptor activation. Translocation of anchored determinants from the eggshell might then regulate the spatial and temporal control of early embryonic developmental processes. PMID:25758463

  20. Hen uterine gene expression profiling during eggshell formation reveals putative proteins involved in the supply of minerals or in the shell mineralization process

    PubMed Central

    2014-01-01

    Background The chicken eggshell is a natural mechanical barrier to protect egg components from physical damage and microbial penetration. Its integrity and strength is critical for the development of the embryo or to ensure for consumers a table egg free of pathogens. This study compared global gene expression in laying hen uterus in the presence or absence of shell calcification in order to characterize gene products involved in the supply of minerals and / or the shell biomineralization process. Results Microarrays were used to identify a repertoire of 302 over-expressed genes during shell calcification. GO terms enrichment was performed to provide a global interpretation of the functions of the over-expressed genes, and revealed that the most over-represented proteins are related to reproductive functions. Our analysis identified 16 gene products encoding proteins involved in mineral supply, and allowed updating of the general model describing uterine ion transporters during eggshell calcification. A list of 57 proteins potentially secreted into the uterine fluid to be active in the mineralization process was also established. They were classified according to their potential functions (biomineralization, proteoglycans, molecular chaperone, antimicrobials and proteases/antiproteases). Conclusions Our study provides detailed descriptions of genes and corresponding proteins over-expressed when the shell is mineralizing. Some of these proteins involved in the supply of minerals and influencing the shell fabric to protect the egg contents are potentially useful biological markers for the genetic improvement of eggshell quality. PMID:24649854

  1. Data set for the proteomic inventory and quantitative analysis of chicken eggshell matrix proteins during the primary events of eggshell mineralization and the active growth phase of calcification.

    PubMed

    Marie, Pauline; Labas, Valérie; Brionne, Aurélien; Harichaux, Grégoire; Hennequet-Antier, Christelle; Rodriguez-Navarro, Alejandro B; Nys, Yves; Gautron, Joël

    2015-09-01

    Chicken eggshell is a biomineral composed of 95% calcite calcium carbonate mineral and of 3.5% organic matrix proteins. The assembly of mineral and its structural organization is controlled by its organic matrix. In a recent study [1], we have used quantitative proteomic, bioinformatic and functional analyses to explore the distribution of 216 eggshell matrix proteins at four key stages of shell mineralization defined as: (1) widespread deposition of amorphous calcium carbonate (ACC), (2) ACC transformation into crystalline calcite aggregates, (3) formation of larger calcite crystal units and (4) rapid growth of calcite as columnar structure with preferential crystal orientation. The current article detailed the quantitative analysis performed at the four stages of shell mineralization to determine the proteins which are the most abundant. Additionally, we reported the enriched GO terms and described the presence of 35 antimicrobial proteins equally distributed at all stages to keep the egg free of bacteria and of 81 proteins, the function of which could not be ascribed. PMID:26306314

  2. Data set for the proteomic inventory and quantitative analysis of chicken eggshell matrix proteins during the primary events of eggshell mineralization and the active growth phase of calcification

    PubMed Central

    Marie, Pauline; Labas, Valérie; Brionne, Aurélien; Harichaux, Grégoire; Hennequet-Antier, Christelle; Rodriguez-Navarro, Alejandro B.; Nys, Yves; Gautron, Joël

    2015-01-01

    Chicken eggshell is a biomineral composed of 95% calcite calcium carbonate mineral and of 3.5% organic matrix proteins. The assembly of mineral and its structural organization is controlled by its organic matrix. In a recent study [1], we have used quantitative proteomic, bioinformatic and functional analyses to explore the distribution of 216 eggshell matrix proteins at four key stages of shell mineralization defined as: (1) widespread deposition of amorphous calcium carbonate (ACC), (2) ACC transformation into crystalline calcite aggregates, (3) formation of larger calcite crystal units and (4) rapid growth of calcite as columnar structure with preferential crystal orientation. The current article detailed the quantitative analysis performed at the four stages of shell mineralization to determine the proteins which are the most abundant. Additionally, we reported the enriched GO terms and described the presence of 35 antimicrobial proteins equally distributed at all stages to keep the egg free of bacteria and of 81 proteins, the function of which could not be ascribed. PMID:26306314

  3. Outer eggshell membrane as delivery vehicle for polysaccharide/protein microcapsules incorporated with vitamin E.

    PubMed

    Chai, Zhi; Li, Yuanyuan; Liu, Fei; Du, Bingjian; Jiao, Tong; Zhang, Chunyue; Leng, Xiaojing

    2013-01-23

    This study investigates the features of a new type of delivery system prepared by combining a natural outer eggshell membrane (OESM) with emulsified microcapsules. The loading efficiency, controlled release properties, and forming mechanisms of the prepared system were studied. The polysaccharide/protein microcapsules incorporated with vitamin E can be attached to highly cross-linked protein fiber networks of OESM. This attachment could be reinforced more than 2-fold using glutaraldehyde as a cross-linking agent. The combined OESM/microcapsule delivery system significantly exhibited better controlled release properties than the microcapsules alone because of the steric blocking effect. Moreover, the OESM delivery system incorporated with microcapsules formed by pectin/protein as wall material showed more resistance against enzymatic attacks because of the formation of compact aggregates promoted by electrostatic effects. PMID:23244530

  4. Protective effect of soluble eggshell membrane protein hydrolysate on cardiac ischemia/reperfusion injury

    PubMed Central

    Yang, Tao; Li, Yan; Ma, Meihu; Lin, Qinlu; Sun, Shuguo; Zhang, Bin; Feng, Xi; Liu, Junwen

    2015-01-01

    Background Soluble eggshell membrane protein (SEP) has been proved to hold the antioxidant activity. The functional role of SEP on cardioprotection was investigated in vivo and in vitro. Methods Rats and cardiomyocytes were pretreated with SP2, a hydrolysate attained from SEP, and then subjected to ischemia/reperfusion (I/R) or hypoxia/reoxygenation (H/R) and hydrogen peroxide, respectively. The measurement of myocardial infarct size, cell apoptosis assay, cell viability assay, and caspase activity assay were performed on rats and cardiomyocytes. Results The results showed that the treatment of SP2 induced the resistance to I/R or H/R injury on rats and cardiomyocytes as indicated by decreased infarct size and decreased cellular apoptosis. The cardioprotective roles of SP2 were partly resulted from the downregulated expression and activity of caspase-3 in which the effect was similar to the caspase inhibitor, z-VAD-fmk, and could be rescued by caspase activator, PAC-1. Conclusions This investigation has demonstrated that SP2 attenuated the damage of I/R and H/R on rats and cardiomyocytes by the caspase-dependent pathway. This cardioprotective effect of SP2 suggested a novel therapeutic agent of SEP for ischemic-related heart diseases. PMID:26699793

  5. Correlation between hammerhead ribozyme-mediated eggshell protein gene cleavage and reproduction inhibition of Schistosoma japonicum

    PubMed Central

    LIANG, YU; ZHOU, YUELAN; YIN, WEIGUO; LI, YINGJU; YANG, QIULIN; GAO, YUAN; ZHANG, YUKUAI; YANG, YAOFEI; PENG, LI; XIAO, JIANHUA

    2012-01-01

    Schistosoma japonicum (S. japonicum) is an extremely harmful pathogen, which infects humans and causes severe public health problems. To date, no effective therapeutic drugs for this pathogen are available. In this study, we designed and constructed three hammerhead ribozymes targeting the eggshell protein gene of S. japonicum (SjESG). The cleavage activities of these three ribozymes were determined using cleavage experiments. The in vitro cleavage results showed that among the three synthesized ribozymes (Rz1, Rz2 and Rz3), Rz1 and Rz3 cleaved their target RNAs effectively. However, Rz2 did not cleave its target RNA detectably. The putative therapeutic roles of these three ribozymes to inhibit the reproduction of S. japonicum in mice were studied in vivo. Compared with the negative controls, Rz1 and Rz3 treatments resulted in increased levels of IFN-γ but decreased levels of IL-4 in mice. Rz2 affected levels of IFN-γ and IL-4 to degrees similar with those caused by the vector controls. In addition, Rz1 and Rz3 reduced the amounts of adult worms and eggs in the livers of mice more extensively than Rz2 and the vector controls. Altogether, these results suggest a correlation between the in vitro cleavage abilities of Rz1 and Rz3 and their roles in reproduction inhibition of S. japonicum. PMID:22246067

  6. Influence of eggshell matrix proteins on the precipitation of calcium carbonate (CaCO 3)

    NASA Astrophysics Data System (ADS)

    Hernández-Hernández, A.; Vidal, M. L.; Gómez-Morales, J.; Rodríguez-Navarro, A. B.; Labas, V.; Gautron, J.; Nys, Y.; García Ruiz, J. M.

    2008-04-01

    To understand the role of eggshell organic matrix on the biomineralization process, we have tested the influence of different purified fractions of the eggshell organic matrix on calcium carbonate (CaCO 3) precipitation. Purification was carried out after successive anion-exchange chromatography, hydrophobic interaction chromatography and gel filtration chromatography of two different prepurified eggshell extracts (A) and (B); the purified fractions (named g, h, n and r) and ( c', g', i', k') respectively were diluted to 50 μg/ml before being tested in vitro and analysed by the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) procedure and mass spectrometry. The precipitation experiments were carried out by the method of vapour diffusion on crystallization mushrooms. Each purified fraction showed a different effect on CaCO 3 precipitation. Some of them exhibited a strong inhibitory effect on nucleation, thus suppressing the precipitation of CaCO 3 almost totally while the others did not produce any notable effect. However, all fractions favoured the precipitation of calcite over the other CaCO 3 polymorphs. Additionally, all fractions modified in a different manner the size and morphology of the precipitated calcite crystals.

  7. Soluble eggshell membrane: A natural protein to improve the properties of biomaterials used for tissue engineering applications.

    PubMed

    Sah, Mahesh Kumar; Rath, Subha Narayan

    2016-10-01

    Extracellular matrix (ECM) acts as an instructing template for the cells contained in tissues. It plays a vital role in regulating cellular behavior by holding and interacting with various growth factors and signaling molecules. The ECM materials are either directly derived from a natural origin, or synthesized mimicking the natural ECM. In this review, we have addressed the ECM derived from eggshell membrane (ESM). The development of porous structures from natural biopolymers, such as ESM holds a number of advantages for tissue engineering applications. By using ESM in tissue engineering application, the cells attach and function to make a required tissue. Thereafter, the scaffold provides mechanical support as well as a platform for cellular interaction, hence, forming a fully functional tissue. The present review summarizes the structure-function relationship of ESM and advancement in its processing methods; the contribution of its soluble form (soluble eggshell membrane protein, SEP) in the development of promising hybrid biomaterials; and the recent advancement of their applications. In addition, this comprehensive review highlights the use of ESM for guided tissue regeneration; promising future applications of SEP in tissue engineering and regenerative medicine. PMID:27287179

  8. Characterization and adsorption properties of eggshells and eggshell membrane.

    PubMed

    Tsai, W T; Yang, J M; Lai, C W; Cheng, Y H; Lin, C C; Yeh, C W

    2006-02-01

    The objective of this work was to study the chemical and physical characterization of eggshell and eggshell membrane particles prepared from the hen eggshell waste. Under the characterization measurements investigated, it was found that the pore structures of the two biomaterials belong to a typical Type II, indicating that they should be basically characteristic of nonporous materials or materials with macropores or open voids. Further, the chemical composition of the resulting eggshell particle was strongly associated with the presence of carbonate minerals from the Fourier transform infrared (FTIR) spectra. In contrast to the resulting eggshell membrane particle, the presence of functional groups of amines and amides was observable because of its chemical composition of fibrous proteins. From the isotherm data of methylene blue at 25 degrees C, the Freundlich model yielded a somewhat better fit than the Langmuir model. The adsorption isotherms revealed the eggshell biosorbents could only uptake the basic dye of less than 1.0mg/g in aqueous medium, which was attributed to their poor pore properties. PMID:15896954

  9. Eggshells as an index of aedine mosquito production. 1: Distribution, movement and sampling of Aedes taeniorhynchus eggshells.

    PubMed

    Ritchie, S A; Addison, D S; van Essen, F

    1992-03-01

    The distribution of Aedes taeniorhynchus eggshells in Florida mangrove basin forests was determined and used to design a sampling plan. Eggshells were found in 10/11 sites (91%), with a mean +/- SE density of 1.45 +/- 0.75/cc; density did not change significantly year to year. Highest densities were located on the sloping banks of hummocks, ponds and potholes. Eggshells were less clumped in distribution than eggs and larvae and thus required a smaller sample size for a given precision level. While eggshells were flushed from compact soil that was subject to runoff during heavy rain, mangrove peat, the dominant soil of eggshell-bearing sites, was less dense and had little runoff or eggshell flushing. We suggest that eggshell surveys could be used to identify Ae. taeniorhynchus oviposition sites and oviposition patterns. PMID:1583486

  10. The eggshell: structure, composition and mineralization.

    PubMed

    Hincke, Maxwell T; Nys, Yves; Gautron, Joel; Mann, Karlheinz; Rodriguez-Navarro, Alejandro B; McKee, Marc D

    2012-01-01

    The calcareous egg is produced by all birds and most reptiles. Current understanding of eggshell formation and mineralization is mainly based on intensive studies of one species - the domesticated chicken Gallus gallus. The majority of constituents of the chicken eggshell have been identified. In this article we review eggshell microstructure and ultrastructure, and the results of recent genomic, transcriptomic and proteomic analyses of the chicken eggshell matrix to draw attention to areas of current uncertainty such as the potential role of amorphous calcium carbonate and the specific nature of the molecules that initiate (nucleate) mammillary cone formation and terminate palisade layer calcification. Comparative avian genomics and proteomics have only recently become possible with the publication of the Taeniopygia guttata (zebra finch) genome. Further rapid progress is highly anticipated with the soon-to-be-released genomes of turkey (Meleagris gallopavo) and duck (Anas platyrhynchos). These resources will allow rapid advances in comparative studies of the organic constituents of avian eggshell and their functional implications. PMID:22201802

  11. Quantitative proteomics provides new insights into chicken eggshell matrix protein functions during the primary events of mineralisation and the active calcification phase.

    PubMed

    Marie, Pauline; Labas, Valérie; Brionne, Aurélien; Harichaux, Grégoire; Hennequet-Antier, Christelle; Rodriguez-Navarro, Alejandro B; Nys, Yves; Gautron, Joël

    2015-08-01

    Eggshell is a bioceramic composed of 95% calcium carbonate mineral and 3.5% organic matrix. Its structural organisation is controlled by its organic matrix. We have used quantitative proteomics to study four key stages of shell mineralisation: 1) widespread deposition of amorphous calcium carbonate (ACC), 2) ACC transformation into crystalline calcite aggregates, 3) formation of larger calcite crystal units and 4) development of a columnar structure with preferential calcite crystal orientation. This approach explored the distribution of 216 shell matrix proteins found at the four stages. Variations in abundance according to these calcification events were observed for 175 proteins. A putative function related to the mineralisation process was predicted by bioinformatics for 77 of them and was further characterised. We confirmed the important role of lysozyme, ovotransferrin, ovocleidin-17 and ovocleidin-116 for shell calcification process, characterised major calcium binding proteins (EDIL3, ALB, MFGE8, NUCB2), and described novel proteoglycans core proteins (GPC4, HAPLN3). We suggest that OVAL and OC-17 play a role in the stabilisation of ACC. Finally, we report proteins involved in the regulation of proteins driving the mineralisation. They correspond to numerous molecular chaperones including CLU, PPIB and OCX21, protease and protease inhibitors including OVM and CST3, and regulators of phosphorylation. PMID:26049031

  12. Data set for the proteomic inventory and quantitative analysis of chicken uterine fluid during eggshell biomineralization.

    PubMed

    Marie, Pauline; Labas, Valérie; Brionne, Aurélien; Harichaux, Grégoire; Hennequet-Antier, Christelle; Nys, Yves; Gautron, Joël

    2014-12-01

    Chicken eggshell is the protective barrier of the egg. It is a biomineral composed of 95% calcium carbonate on calcitic form and 3.5% organic matrix proteins. Mineralization process occurs in uterus into the uterine fluid. This acellular fluid contains ions and organic matrix proteins precursors which are interacting with the mineral phase and control crystal growth, eggshell structure and mechanical properties. We performed a proteomic approach and identified 308 uterine fluid proteins. Gene Ontology terms enrichments were determined to investigate their potential functions. Mass spectrometry analyses were also combined to label free quantitative analysis to determine the relative abundance of 96 proteins at initiation, rapid growth phase and termination of shell calcification. Sixty four showed differential abundance according to the mineralization stage. Their potential functions have been annotated. The complete proteomic, bioinformatic and functional analyses are reported in Marie et al., J. Proteomics (2015) [1]. PMID:26217689

  13. Amorphous calcium carbonate controls avian eggshell mineralization: A new paradigm for understanding rapid eggshell calcification.

    PubMed

    Rodríguez-Navarro, Alejandro B; Marie, Pauline; Nys, Yves; Hincke, Maxwell T; Gautron, Joel

    2015-06-01

    Avian eggshell mineralization is the fastest biogenic calcification process known in nature. How this is achieved while producing a highly crystalline material composed of large calcite columnar single crystals remains largely unknown. Here we report that eggshell mineral originates from the accumulation of flat disk-shaped amorphous calcium carbonate (ACC) particles on specific organic sites on the eggshell membrane, which are rich in proteins and sulfated proteoglycans. These structures known as mammillary cores promote the nucleation and stabilization of a amorphous calcium carbonate with calcitic short range order which predetermine the calcite composition of the mature eggshell. The amorphous nature of the precursor phase was confirmed by the diffuse scattering of X-rays and electrons. The nascent calcitic short-range order of this transient mineral phase was revealed by infrared spectroscopy and HRTEM. The ACC mineral deposited around the mammillary core sites progressively transforms directly into calcite crystals without the occurrence of any intermediate phase. Ionic speciation data suggest that the uterine fluid is equilibrated with amorphous calcium carbonate, throughout the duration of eggshell mineralization process, supporting that this mineral phase is constantly forming at the shell mineralization front. On the other hand, the transient amorphous calcium carbonate mineral deposits, as well as the calcite crystals into which they are converted, form by the ordered aggregation of nanoparticles that support the rapid mineralization of the eggshell. The results of this study alter our current understanding of avian eggshell calcification and provide new insights into the genesis and formation of calcium carbonate biominerals in vertebrates. PMID:25934395

  14. Manganese supplementation enhances the synthesis of glycosaminoglycan in eggshell membrane: a strategy to improve eggshell quality in laying hens.

    PubMed

    Xiao, J F; Zhang, Y N; Wu, S G; Zhang, H J; Yue, H Y; Qi, G H

    2014-02-01

    This study investigated the effect of dietary Mn supplementation on eggshell quality, ultrastructure, glycosaminoglycan (GAG), and uronic acid content, and mRNA and protein expression of Galβ1,3-glucuronosyltransferase (GlcAT-I). A total of 216 layers (Hy-Line Grey) at age of 50 wk were divided into 3 groups. In the first 8 wk of the 12-wk feeding trial, all groups were fed a basal diet that met all layer nutrient requirements except for Mn. In the last 4 wk, each group was fed 1 of 3 diets supplemented with Mn levels at 0, 25, or 100 mg Mn/kg. Dietary Mn deficiency did not affect the egg performance of layers. Dietary Mn supplementation significantly improved the breaking strength, thickness, and fracture toughness of eggshells (P < 0.05). In photographs of eggshell ultrastructure, the size of mammillary cones and cracks in the outer surface were decreased by dietary Mn supplementation. The contents of GAG and uronic acids in eggshell membrane were significantly increased by dietary Mn addition (P < 0.05). This result was further confirmed by increased mRNA expression and protein expression of GlcAT-I when Mn was added to the diet. This study suggests that dietary Mn supplementation can improve eggshell quality by enhancing the GAG and uronic acid synthesis in the eggshell glands, which can affect the ultrastructure of eggshells. PMID:24570460

  15. Investigation of the role of ansocalcin in the biomineralization in goose eggshell matrix

    PubMed Central

    Lakshminarayanan, Rajamani; Kini, R. Manjunatha; Valiyaveettil, Suresh

    2002-01-01

    The role of proteins in biomineralization and the mechanism of eggshell formation are not well understood. We have isolated and purified the major protein, ansocalcin from goose eggshell matrix. The amino acid sequence study indicates that ansocalcin is homologous to the chicken eggshell protein, ovocleidin 17, and C-type lectins. Ansocalcin nucleates polycrystalline aggregates of calcite crystals in in vitro mineralization experiments. The polycrystalline aggregates obtained at higher concentration of ansocalcin appears to be similar to the crystals observed at the mamillary layer of the eggshell. PMID:11959964

  16. High ambient temperature influences eggshell quality and calbindin-D28k localization of eggshell gland and all intestinal segments of laying hens.

    PubMed

    Ebeid, T A; Suzuki, T; Sugiyama, T

    2012-09-01

    Eggshell quality deterioration associated with heat stress is a well-known phenomenon. The involvement of the 28-kDa calcium-binding protein (calbindin, CaBP-D28k) localization in this failure is not clearly understood. To test a possible direct effect of ambient temperature on calbindin-D28k localization, 40 White Leghorn laying hens were housed in individual cages and exposed to high ambient temperature (30-33°C) and thermoneutral temperature (20-22°C) which served as a control. Eggshell quality characteristics and immunohistochemical localization of all intestinal segments and eggshell gland calbindin-D28k were performed under both environmental conditions. As expected, egg weight, eggshell thickness, eggshell percentage, and eggshell density were negatively affected by high ambient temperature (P ≤ 0.01). Immunohistochemistry showed that calbindin was localized in the intestinal enterocyte cytoplasm and glandular cell cytoplasm under thermoneutral conditions. However, the calbindin intensity was prominently decreased in ileum, cecum, colon, and eggshell gland under heat stress conditions. Therefore, it could be concluded that calbindin-D28k localization in intestinal segments and eggshell gland is negatively affected by high ambient temperature which might be related to the deterioration of eggshell quality characteristics under heat stress conditions. PMID:22912464

  17. Purification, crystallization and preliminary X-ray analysis of struthiocalcin 1 from ostrich (Struthio camelus) eggshell

    SciTech Connect

    Reyes-Grajeda, Juan Pablo; Marín-García, Liliana; Stojanoff, Vivian; Moreno, Abel

    2007-11-01

    The purification, crystallization and preliminary X-ray diffraction data of the protein struthiocalcin 1 isolated from ostrich eggshell are reported. The purification, crystallization and preliminary X-ray analysis of struthiocalcin 1 (SCA-1), a protein obtained from the intramineral part of ostrich (Struthio camelus) eggshell, is reported.

  18. Polymorphisms in Ion Transport Genes Are Associated with Eggshell Mechanical Property

    PubMed Central

    Sun, Congjiao; Shi, Fengying; Wu, Guiqin; Liu, Aiqiao; Xu, Guiyun; Yang, Ning

    2015-01-01

    Eggshell mechanical property traits such as eggshell breaking strength (ESS), eggshell thickness (EST) and eggshell weight (ESW) are most common and important indexes to evaluate eggshell quality in poultry industry. Uterine ion transporters involve in eggshell formation and might be associated with eggshell mechanical property traits. In this study, 99 SNPs in 15 ion transport genes were selected to genotype 976 pedigreed hens of Rhode Island Red. ESS, EST and ESW were measured for each bird at 55 weeks of age. The association study showed that 14 SNPs in 8 genes were significantly related (p < 0.05) with at least one trait, and their contributions to phenotypic variance ranged from 0.23% to 4.14%. Both ATP2A3 and SLC4A5 had a significant effect on all the three traits. Strong linkage disequilibrium (LD) was detected among SNPs in four genes: ATP2A3, ITPR1, SLC8A3, SCNN1a. The significant effects of those diplotypes on eggshell mechanical property traits were found, and their contributions to phenotypic variance ranged from 0.50% to 0.70%. It was concluded that the identified SNPs and diplotypes in this study were potential markers influencing the eggshell mechanical properties, which could contribute to the genetic improvement of eggshell quality. PMID:26106883

  19. Polymorphisms in Ion Transport Genes Are Associated with Eggshell Mechanical Property.

    PubMed

    Duan, Zhongyi; Chen, Sirui; Sun, Congjiao; Shi, Fengying; Wu, Guiqin; Liu, Aiqiao; Xu, Guiyun; Yang, Ning

    2015-01-01

    Eggshell mechanical property traits such as eggshell breaking strength (ESS), eggshell thickness (EST) and eggshell weight (ESW) are most common and important indexes to evaluate eggshell quality in poultry industry. Uterine ion transporters involve in eggshell formation and might be associated with eggshell mechanical property traits. In this study, 99 SNPs in 15 ion transport genes were selected to genotype 976 pedigreed hens of Rhode Island Red. ESS, EST and ESW were measured for each bird at 55 weeks of age. The association study showed that 14 SNPs in 8 genes were significantly related (p < 0.05) with at least one trait, and their contributions to phenotypic variance ranged from 0.23% to 4.14%. Both ATP2A3 and SLC4A5 had a significant effect on all the three traits. Strong linkage disequilibrium (LD) was detected among SNPs in four genes: ATP2A3, ITPR1, SLC8A3, SCNN1a. The significant effects of those diplotypes on eggshell mechanical property traits were found, and their contributions to phenotypic variance ranged from 0.50% to 0.70%. It was concluded that the identified SNPs and diplotypes in this study were potential markers influencing the eggshell mechanical properties, which could contribute to the genetic improvement of eggshell quality. PMID:26106883

  20. The eggshell in the C. elegans oocyte-to-embryo transition.

    PubMed

    Johnston, Wendy L; Dennis, James W

    2012-04-01

    In egg-laying animals, embryonic development takes place within the highly specialized environment provided by the eggshell and its underlying extracellular matrix. Far from being simply a passive physical support, the eggshell is a key player in many early developmental events. Herein, we review current understanding of eggshell structure, biosynthesis, and function in zygotic development of the nematode, C. elegans. Beginning at sperm contact or entry, eggshell layers are produced sequentially. The earlier outer layers are required for secretion or organization of inner layers, and layers differ in composition and function. Developmental events that depend on the eggshell include polyspermy barrier generation, high fidelity meiotic chromosome segregation, osmotic barrier synthesis, polar body extrusion, anterior-posterior polarization, and organization of membrane and cortical proteins. The C. elegans eggshell is proving to be an excellent, tractable system to study the molecular cues of the extracellular matrix that instruct cell polarity and early development. PMID:22083685

  1. Identifying Key Attributes for Protein Beverages.

    PubMed

    Oltman, A E; Lopetcharat, K; Bastian, E; Drake, M A

    2015-06-01

    This study identified key attributes of protein beverages and evaluated effects of priming on liking of protein beverages. An adaptive choice-based conjoint study was conducted along with Kano analysis to gain insight on protein beverage consumers (n = 432). Attributes evaluated included label claim, protein type, amount of protein, carbohydrates, sweeteners, and metabolic benefits. Utility scores for levels and importance scores for attributes were determined. Subsequently, two pairs of clear acidic whey protein beverages were manufactured that differed by age of protein source or the amount of whey protein per serving. Beverages were evaluated by 151 consumers on two occasions with or without priming statements. One priming statement declared "great flavor," the other priming statement declared 20 g protein per serving. A two way analysis of variance was applied to discern the role of each priming statement. The most important attribute for protein beverages was sweetener type, followed by amount of protein, followed by type of protein followed by label claim. Beverages with whey protein, naturally sweetened, reduced sugar and ≥15 g protein per serving were most desired. Three consumer clusters were identified, differentiated by their preferences for protein type, sweetener and amount of protein. Priming statements positively impacted concept liking (P < 0.05) but had no effect on overall liking (P > 0.05). Consistent with trained panel profiles of increased cardboard flavor with higher protein content, consumers liked beverages with 10 g protein more than beverages with 20 g protein (6.8 compared with 5.7, P < 0.05). Protein beverages must have desirable flavor for wide consumer appeal. PMID:25943857

  2. The avian eggshell as a model of biomineralization

    SciTech Connect

    Arias, J.L.; Fernandez, M.S. ); Laraia, V.J.; Janicki, J.; Heuer, A.H.; Caplan, A.I. )

    1990-11-01

    The avian eggshell is one of the most rapidly mineralizing biological systems known. By understanding the key components and steps in this process, we hope to provide relevant information for fabrication of ceramic composites. The calcification of the eggshell occurs in three main steps: (1) fabrication of an organic matrix, (2) nucleation of an inorganic phase on the organic matrix, and (3) space-filling growth of the calcite phase. The different layers of an eggshell can be separately isolated and studied. In this preliminary communication, the organization of the shell matrix and membranes and their association with the crystal phase, the immunohistochemical occurrence and distribution of types I and X collagen, and of different proteoglycans are reviewed. Also the preliminary findings of the remineralization of the intact or modified eggshell are presented. These experiments allow us to identify the essential steps in forming a natural composite ceramic. 47 refs., 6 figs.

  3. Eggshell thickness in mallards fed methylmercury

    SciTech Connect

    Heinz, G.H.

    1980-09-01

    Eggshell thinning has been linked to impaired reproduction in many wild birds. Previous work of my own and others led me to believe that methylmercury may cause some eggshell thinning in birds. The present study was designed to determine whether methylmercury in the diet of mallards would thin their eggshells and whether it would add to eggshell thinning caused by DDE.

  4. Extreme multifunctional proteins identified from a human protein interaction network

    PubMed Central

    Chapple, Charles E.; Robisson, Benoit; Spinelli, Lionel; Guien, Céline; Becker, Emmanuelle; Brun, Christine

    2015-01-01

    Moonlighting proteins are a subclass of multifunctional proteins whose functions are unrelated. Although they may play important roles in cells, there has been no large-scale method to identify them, nor any effort to characterize them as a group. Here, we propose the first method for the identification of ‘extreme multifunctional' proteins from an interactome as a first step to characterize moonlighting proteins. By combining network topological information with protein annotations, we identify 430 extreme multifunctional proteins (3% of the human interactome). We show that the candidates form a distinct sub-group of proteins, characterized by specific features, which form a signature of extreme multifunctionality. Overall, extreme multifunctional proteins are enriched in linear motifs and less intrinsically disordered than network hubs. We also provide MoonDB, a database containing information on all the candidates identified in the analysis and a set of manually curated human moonlighting proteins. PMID:26054620

  5. Identifying Protein-Calorie Malnutrition Workshop.

    ERIC Educational Resources Information Center

    Walker, Susan S.; Barker, Ellen M.

    Instructional materials are provided for a workshop to enable participants to assist in identifying patients at risk with protein-calorie malnutrition and in corrrecting this nutritional deficiency. Representative topics are nutrients; protein, mineral, and vitamin sources, functions, and deficiency symptoms; malnutrition; nutritional deficiency…

  6. Identifying the hub proteins from complicated membrane protein network systems.

    PubMed

    Shen, Yi-Zhen; Ding, Yong-Sheng; Gu, Quan; Chou, Kuo-Chen

    2010-05-01

    The so-called "hub proteins" are those proteins in a protein-protein interaction network system that have remarkably higher interaction relations (or degrees) than the others. Therefore, the information of hub proteins can provide very useful insights for selecting or prioritizing targets during drug development. In this paper, by combining the multi-agent-based method with the graphical spectrum analysis and immune-genetic algorithm, a novel simulator for identifying the hub proteins from membrane protein interaction networks is proposed. As a demonstration of using the simulator, two hub membrane proteins, YPL227C and YIL147C, were identified from a complicated network system consisting of 1500 membrane proteins. Meanwhile, along with the two identified hub proteins, their molecular functions, biological processes, and cellular components were also revealed. It is anticipated that the hub-protein-simulator may become a very useful tool for system biology and drug development, particularly in deciphering unknown protein functions, determining protein complexes, and in identifying the key targets from a complicated disease system. PMID:20507268

  7. Antimicrobial properties of a nanostructured eggshell from a compost-nesting bird.

    PubMed

    D'Alba, Liliana; Jones, Darryl N; Badawy, Hope T; Eliason, Chad M; Shawkey, Matthew D

    2014-04-01

    Infection is an important source of mortality for avian embryos but parental behaviors and eggs themselves can provide a network of antimicrobial defenses. Mound builders (Aves: Megapodiidae) are unique among birds in that they produce heat for developing embryos not by sitting on eggs but by burying them in carefully tended mounds of soil and microbially decomposing vegetation. The low infection rate of eggs of one species in particular, the Australian brush-turkey (Alectura lathami), suggests that they possess strong defensive mechanisms. To identify some of these mechanisms, we first quantified antimicrobial albumen proteins and characterized eggshell structure, finding that albumen was not unusually antimicrobial, but that eggshell cuticle was composed of nanometer-sized calcite spheres. Experimental tests revealed that these modified eggshells were significantly more hydrophobic and better at preventing bacterial attachment and penetration into the egg contents than chicken eggs. Our results suggest that these mechanisms may contribute to the antimicrobial defense system of these eggs, and may provide inspiration for new biomimetic anti-fouling surfaces. PMID:24311808

  8. Eggshell Types and Their Evolutionary Correlation with Life-History Strategies in Squamates

    PubMed Central

    Hallmann, Konstantin; Griebeler, Eva Maria

    2015-01-01

    The eggshell is an important physiological structure for the embryo. It enables gas exchange, physical protection and is a calcium reserve. Most squamates (lizards, snakes, worm lizards) lay parchment-shelled eggs, whereas only some gekkotan species, a subgroup of lizards, have strongly calcified eggshells. In viviparous (live-bearing) squamates the eggshell is reduced or completely missing (hereafter “shell-less”). Recent studies showed that life-history strategies of gekkotan species differ between species with parchment- and rigid-shelled eggshells. Here we test if the three different eggshell types found in the squamates are also associated with different life-history strategies. We first investigated the influence of the phylogeny on the trait “eggshell type” and on six life-history traits of 32 squamate species. Phylogenetic principal component analysis (pPCA) was then conducted to identify an association between life-history strategies and eggshell types. Finally, we also considered adult weight in the pPCA to examine its potential effect on this association. Eggshell types in squamates show a strong phylogenetic signal at a low taxonomical level. Four out of the six life-history traits showed also a phylogenetic signal (birth size, clutch size, clutches per year and age at female maturity), while two had none (incubation time, maximum longevity). The pPCA suggested an association of life-history strategies and eggshell types, which disappeared when adult weight was included in the analysis. We conclude that the variability seen in eggshell types of squamates is weakly influenced by phylogeny. Eggshell types correlate with different life-history strategies, and mainly reflect differences in adult weights of species. PMID:26393343

  9. Eggshell Types and Their Evolutionary Correlation with Life-History Strategies in Squamates.

    PubMed

    Hallmann, Konstantin; Griebeler, Eva Maria

    2015-01-01

    The eggshell is an important physiological structure for the embryo. It enables gas exchange, physical protection and is a calcium reserve. Most squamates (lizards, snakes, worm lizards) lay parchment-shelled eggs, whereas only some gekkotan species, a subgroup of lizards, have strongly calcified eggshells. In viviparous (live-bearing) squamates the eggshell is reduced or completely missing (hereafter "shell-less"). Recent studies showed that life-history strategies of gekkotan species differ between species with parchment- and rigid-shelled eggshells. Here we test if the three different eggshell types found in the squamates are also associated with different life-history strategies. We first investigated the influence of the phylogeny on the trait "eggshell type" and on six life-history traits of 32 squamate species. Phylogenetic principal component analysis (pPCA) was then conducted to identify an association between life-history strategies and eggshell types. Finally, we also considered adult weight in the pPCA to examine its potential effect on this association. Eggshell types in squamates show a strong phylogenetic signal at a low taxonomical level. Four out of the six life-history traits showed also a phylogenetic signal (birth size, clutch size, clutches per year and age at female maturity), while two had none (incubation time, maximum longevity). The pPCA suggested an association of life-history strategies and eggshell types, which disappeared when adult weight was included in the analysis. We conclude that the variability seen in eggshell types of squamates is weakly influenced by phylogeny. Eggshell types correlate with different life-history strategies, and mainly reflect differences in adult weights of species. PMID:26393343

  10. The Protein Identifier Cross-Referencing (PICR) service: reconciling protein identifiers across multiple source databases

    PubMed Central

    Côté, Richard G; Jones, Philip; Martens, Lennart; Kerrien, Samuel; Reisinger, Florian; Lin, Quan; Leinonen, Rasko; Apweiler, Rolf; Hermjakob, Henning

    2007-01-01

    Background Each major protein database uses its own conventions when assigning protein identifiers. Resolving the various, potentially unstable, identifiers that refer to identical proteins is a major challenge. This is a common problem when attempting to unify datasets that have been annotated with proteins from multiple data sources or querying data providers with one flavour of protein identifiers when the source database uses another. Partial solutions for protein identifier mapping exist but they are limited to specific species or techniques and to a very small number of databases. As a result, we have not found a solution that is generic enough and broad enough in mapping scope to suit our needs. Results We have created the Protein Identifier Cross-Reference (PICR) service, a web application that provides interactive and programmatic (SOAP and REST) access to a mapping algorithm that uses the UniProt Archive (UniParc) as a data warehouse to offer protein cross-references based on 100% sequence identity to proteins from over 70 distinct source databases loaded into UniParc. Mappings can be limited by source database, taxonomic ID and activity status in the source database. Users can copy/paste or upload files containing protein identifiers or sequences in FASTA format to obtain mappings using the interactive interface. Search results can be viewed in simple or detailed HTML tables or downloaded as comma-separated values (CSV) or Microsoft Excel (XLS) files suitable for use in a local database or a spreadsheet. Alternatively, a SOAP interface is available to integrate PICR functionality in other applications, as is a lightweight REST interface. Conclusion We offer a publicly available service that can interactively map protein identifiers and protein sequences to the majority of commonly used protein databases. Programmatic access is available through a standards-compliant SOAP interface or a lightweight REST interface. The PICR interface, documentation and

  11. The thin eggshell problem

    USGS Publications Warehouse

    Stickel, L.F.; Rhodes, L.I.

    1970-01-01

    It has long been known that DDT and related chemicals can impair the reproduction of birds. In early years of organochlorine pesticide use, widespread mortality occurred immediately following heavy applications of these chemicals, and survivors contained substantial amounts of toxicant in their tissues. Repopulation from untreated areas tended to conceal the extent of the effects. DDT and dieldrin have become ubiquitous and the original source of the chemicals producing bird deaths often cannot be traced. The extent of sublethal effects cannot be fully appraised, although laboratory experiments continually reveal new and potentially deleterious physiological reactions. Thin eggshells have become prevalent among certain declining species of predatory birds. Shell thinning and associated reproductive effects have been produced experimentally in mallard ducks and in sparrow hawks. Coturnix quail fed dietary dosages of p,p'-DDT produced fewer eggs than did untreated birds and the eggs had thinner shells. Hatchability was not significantly altered. Comparisons between these results and those obtained in other studies indicate significant species differences.

  12. Eggshell apex abnormalities associated with Mycoplasma synoviae infection in layers.

    PubMed

    Jeon, Eun-Ok; Kim, Jong-Nyeo; Lee, Hae-Rim; Koo, Bon-Sang; Min, Kyeong-Cheol; Han, Moo-Sung; Lee, Seung-Baek; Bae, Yeon-Ji; Mo, Jong-Suk; Cho, Sun-Hyung; Lee, Chang-Hee; Mo, In-Pil

    2014-12-01

    Eggs exhibiting eggshell apex abnormalities (EAA) were evaluated for changes in shell characteristics such as strength, thickness, and ultrastructure. Mycoplasma synoviae (MS) infection was confirmed by serological assay along with isolation of MS from the trachea and oviduct. Changes in eggshell quality were shown to be statistically significant (p < 0.01). We also identified ultrastructural changes in the mammillary knob layer by Scanning Electron Microscopy. While eggs may seem to be structurally sound, ultrastructural evaluation showed that affected eggs do not regain their former quality. In our knowledge, this is the first report describing the occurrence of EAA in Korea. PMID:24962418

  13. Laser Cleaning of Avian Eggshell

    NASA Astrophysics Data System (ADS)

    Cornish, L.; Ball, A.; Russell, D.

    A low vacuum SEM was used to evaluate the effect of using an Nd:YAG laser as a non-contact technique for cleaning avian eggshells. The technique shows potential, since there are no obvious deleterious effects from cleaning, but further study is required to understand how the laser is interacting with the sample surface.

  14. Embryonic sex steroid hormones accumulate in the eggshell of loggerhead sea turtle (Caretta caretta).

    PubMed

    Kobayashi, Shohei; Saito, Yoshimichi; Osawa, Akihisa; Katsumata, Etsuko; Karaki, Isuke; Nagaoka, Kentaro; Taya, Kazuyoshi; Watanabe, Gen

    2015-12-01

    Steroids hormones such as estradiol-17β (E2) and testosterone (T) are involved in gonadal differentiation of oviparous animals with temperature-dependent sex determination (TSD), and are greatly distributed. This hypothesizes that these embryonic steroid hormones probably accumulate in the eggshell throughout blood or/and chorioallantoic fluid in sea turtle species with TSD, producing females at higher temperature. To demonstrate this hypothesis, concentrations of E2 and T in the blood plasma from the hatchling loggerhead sea turtle (Caretta caretta) and in their eggshells were measured by radioimmunoassay. In the present study we propose that both concentrations of E2 and T in the blood plasma are correlated with amounts of these sex steroids in the eggshell. Moreover, contents of E2 in the eggshell showed a significant positive correlation with mean incubation temperatures during a thermosensitive period in the experimental nests, whereas T contents in the eggshell did not. Taken together, these findings indicated that embryonic E2 and T that accumulated in the eggshell can be extracted and measured. Furthermore, the present study suggested that contents of E2 in the eggshell may differ between male and female, and monitoring of these steroids is a useful method to identify the sex of loggerhead sea turtle hatchling. PMID:26050561

  15. Microwave Irradiation of Nanohydroxyapatite from Chicken Eggshells and Duck Eggshells

    PubMed Central

    Sajahan, Nor Adzliana; Wan Ibrahim, Wan Mohd Azhar

    2014-01-01

    Due to similarity in composition to the mineral component of bones and human hard tissues, hydroxyapatite with chemical formula Ca10(PO4)6(OH)2 has been widely used in medical field. Both chicken and duck eggshells are mainly composed of calcium carbonate. An attempt has been made to fabricate nanohydroxyapatite (nHA) by chicken (CES) and duck eggshells (DES) as calcium carbonate source (CaCO3). CES and DES were reacted with diammonium hydrogen [(NH4)2HPO4] solution and subjected to microwave heating at 15 mins. Under the effect of microwave irradiation, nHA was produced directly in the solution and involved in crystallographic transformation. Sample characterization was done using by X-ray diffraction (XRD), fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). PMID:25383364

  16. Structural neighboring property for identifying protein-protein binding sites

    PubMed Central

    2015-01-01

    Background The protein-protein interaction plays a key role in the control of many biological functions, such as drug design and functional analysis. Determination of binding sites is widely applied in molecular biology research. Therefore, many efficient methods have been developed for identifying binding sites. In this paper, we calculate structural neighboring property through Voronoi diagram. Using 6,438 complexes, we study local biases of structural neighboring property on interface. Results We propose a novel statistical method to extract interacting residues, and interacting patches can be clustered as predicted interface residues. In addition, structural neighboring property can be adopted to construct a new energy function, for evaluating docking solutions. It includes new statistical property as well as existing energy items. Comparing to existing methods, our approach improves overall Fnat value by at least 3%. On Benchmark v4.0, our method has average Irmsd value of 3.31Å and overall Fnat value of 63%, which improves upon Irmsd of 3.89 Å and Fnat of 49% for ZRANK, and Irmsd of 3.99Å and Fnat of 46% for ClusPro. On the CAPRI targets, our method has average Irmsd value of 3.46 Å and overall Fnat value of 45%, which improves upon Irmsd of 4.18 Å and Fnat of 40% for ZRANK, and Irmsd of 5.12 Å and Fnat of 32% for ClusPro. Conclusions Experiments show that our method achieves better results than some state-of-the-art methods for identifying protein-protein binding sites, with the prediction quality improved in terms of CAPRI evaluation criteria. PMID:26356630

  17. The evolution of eggshell cuticle in relation to nesting ecology.

    PubMed

    D'Alba, Liliana; Maia, Rafael; Hauber, Mark E; Shawkey, Matthew D

    2016-08-17

    Avian eggs are at risk of microbial infection prior to and during incubation. A large number of defence mechanisms have evolved in response to the severe costs imposed by these infections. The eggshell's cuticle is an important component of antimicrobial defence, and its role in preventing contamination by microorganisms in domestic chickens is well known. Nanometer-scale cuticular spheres that reduce microbial attachment and penetration have recently been identified on eggs of several wild avian species. However, whether these spheres have evolved specifically for antimicrobial defence is unknown. Here, we use comparative data on eggshell cuticular structure and nesting ecology to test the hypothesis that birds nesting in habitats with higher risk of infection (e.g. wetter and warmer) are more likely to evolve cuticular nanospheres on their eggshells than those nesting in less risky habitats. We found that nanostructuring, present in 54 of 296 analysed species, is the ancestral condition of avian eggshells and has been retained more often in taxa that nest in humid infection-prone environments, suggesting that they serve critical roles in antimicrobial egg defence. PMID:27488648

  18. Fossil avian eggshell preserves ancient DNA

    PubMed Central

    Oskam, Charlotte L.; Haile, James; McLay, Emma; Rigby, Paul; Allentoft, Morten E.; Olsen, Maia E.; Bengtsson, Camilla; Miller, Gifford H.; Schwenninger, Jean-Luc; Jacomb, Chris; Walter, Richard; Baynes, Alexander; Dortch, Joe; Parker-Pearson, Michael; Gilbert, M. Thomas P.; Holdaway, Richard N.; Willerslev, Eske; Bunce, Michael

    2010-01-01

    Owing to exceptional biomolecule preservation, fossil avian eggshell has been used extensively in geochronology and palaeodietary studies. Here, we show, to our knowledge, for the first time that fossil eggshell is a previously unrecognized source of ancient DNA (aDNA). We describe the successful isolation and amplification of DNA from fossil eggshell up to 19 ka old. aDNA was successfully characterized from eggshell obtained from New Zealand (extinct moa and ducks), Madagascar (extinct elephant birds) and Australia (emu and owl). Our data demonstrate excellent preservation of the nucleic acids, evidenced by retrieval of both mitochondrial and nuclear DNA from many of the samples. Using confocal microscopy and quantitative PCR, this study critically evaluates approaches to maximize DNA recovery from powdered eggshell. Our quantitative PCR experiments also demonstrate that moa eggshell has approximately 125 times lower bacterial load than bone, making it a highly suitable substrate for high-throughput sequencing approaches. Importantly, the preservation of DNA in Pleistocene eggshell from Australia and Holocene deposits from Madagascar indicates that eggshell is an excellent substrate for the long-term preservation of DNA in warmer climates. The successful recovery of DNA from this substrate has implications in a number of scientific disciplines; most notably archaeology and palaeontology, where genotypes and/or DNA-based species identifications can add significantly to our understanding of diets, environments, past biodiversity and evolutionary processes. PMID:20219731

  19. Evolution of avian eggshell structure.

    PubMed

    Osterström, Ola; Lilja, Clas

    2012-03-01

    Data are presented suggesting that birds have evolved eggs with shells containing different structures (numbers of mammillae per unit of inner eggshell surface area, i.e., mammillary densities) to cope up with different calcium requirements imposed by different growth rates and modes of development. Precocial bird species grow slowly, but have high mammillary density, while altricial bird species grow rapidly, but have low mammillary density. These results suggest an adaptation associated with growth rate and mode of development and show, moreover, that the mammillary layer is indicative of the breeding biology of the bird. PMID:21987469

  20. Eggshell composition of squamate reptiles: relationship between eggshell permeability and amino acid distribution.

    PubMed

    Sexton, Owen J; Bramble, Judith E; Heisler, I Lorraine; Phillips, Christopher A; Cox, David L

    2005-10-01

    Most snakes and lizards produce eggs with flexible shells that interact with the environment to maintain water balance. Geckos produce rigid eggshells that are independent of an external source of water and can be oviposited in more open, dryer locations. In this study, we analyzed and compared the amino acid composition of 24 lizard species, six snake species, and four outgroups (including avian and reptilian elastin and chicken eggshell). Rigid Gecko eggshells had significantly lower levels of seven of the 17 amino acids evaluated. Multivariate analysis showed that proline was the most important amino acid in distinguishing between these two groups of eggshells, occurring at significantly higher levels in flexible eggshells. High levels of proline have also been observed in the eggshells of other species. Proline and other amino acids are associated with the alleviation of water and salt stress in plants. PMID:16195850

  1. Variability in Avian Eggshell Colour: A Comparative Study of Museum Eggshells

    PubMed Central

    Cassey, Phillip; Portugal, Steven J.; Maurer, Golo; Ewen, John G.; Boulton, Rebecca L.; Hauber, Mark E.; Blackburn, Tim M.

    2010-01-01

    Background The exceptional diversity of coloration found in avian eggshells has long fascinated biologists and inspired a broad range of adaptive hypotheses to explain its evolution. Three main impediments to understanding the variability of eggshell appearance are: (1) the reliable quantification of the variation in eggshell colours; (2) its perception by birds themselves, and (3) its relation to avian phylogeny. Here we use an extensive museum collection to address these problems directly, and to test how diversity in eggshell coloration is distributed among different phylogenetic levels of the class Aves. Methodology and Results Spectrophotometric data on eggshell coloration were collected from a taxonomically representative sample of 251 bird species to determine the change in reflectance across different wavelengths and the taxonomic level where the variation resides. As many hypotheses for the evolution of eggshell coloration assume that egg colours provide a communication signal for an avian receiver, we also modelled reflectance spectra of shell coloration for the avian visual system. We found that a majority of species have eggs with similar background colour (long wavelengths) but that striking differences are just as likely to occur between congeners as between members of different families. The region of greatest variability in eggshell colour among closely related species coincided with the medium-wavelength sensitive region around 500 nm. Conclusions The majority of bird species share similar background eggshell colours, while the greatest variability among species aligns with differences along a red-brown to blue axis that most likely corresponds with variation in the presence and concentration of two tetrapyrrole pigments responsible for eggshell coloration. Additionally, our results confirm previous findings of temporal changes in museum collections, and this will be of particular concern for studies testing intraspecific hypotheses relating

  2. A nanostructural basis for gloss of avian eggshells.

    PubMed

    Igic, Branislav; Fecheyr-Lippens, Daphne; Xiao, Ming; Chan, Andrew; Hanley, Daniel; Brennan, Patricia R L; Grim, Tomas; Waterhouse, Geoffrey I N; Hauber, Mark E; Shawkey, Matthew D

    2015-02-01

    The role of pigments in generating the colour and maculation of birds' eggs is well characterized, whereas the effects of the eggshell's nanostructure on the visual appearance of eggs are little studied. Here, we examined the nanostructural basis of glossiness of tinamou eggs. Tinamou eggs are well known for their glossy appearance, but the underlying mechanism responsible for this optical effect is unclear. Using experimental manipulations in conjunction with angle-resolved spectrophotometry, scanning electron microscopy, atomic force microscopy and chemical analyses, we show that the glossy appearance of tinamou eggshells is produced by an extremely smooth cuticle, composed of calcium carbonate, calcium phosphate and, potentially, organic compounds such as proteins and pigments. Optical calculations corroborate surface smoothness as the main factor producing gloss. Furthermore, we reveal the presence of weak iridescence on eggs of the great tinamou (Tinamus major), an optical effect never previously documented for bird eggs. These data highlight the need for further exploration into the nanostructural mechanisms for the production of colour and other optical effects of avian eggshells. PMID:25505139

  3. Subtleties of biomineralisation revealed by manipulation of the eggshell membrane.

    PubMed

    Li, Nan; Niu, Li-na; Qi, Yi-pin; Yiu, Cynthia K Y; Ryou, Heonjune; Arola, Dwayne D; Chen, Ji-hua; Pashley, David H; Tay, Franklin R

    2011-12-01

    Biocalcification of collagen matrices with calcium phosphate and biosilicification of diatom frustules with amorphous silica are two discrete processes that have intrigued biologists and materials scientists for decades. Recent advancements in the understanding of the mechanisms involved in these two biomineralisation processes have resulted in the use of biomimetic strategies to replicate these processes separately using polyanionic, polycationic or zwitterionic analogues of extracellular matrix proteins to stabilise amorphous mineral precursor phases. To date, there is a lack of a universal model that enables the subtleties of these two apparently dissimilar biomineralisation processes to be studied together. Here, we utilise the eggshell membrane as a universal model for differential biomimetic calcification and silicification. By manipulating the eggshell membrane to render it permeable to stabilised mineral precursors, it is possible to introduce nanostructured calcium phosphate or silica into eggshell membrane fibre cores or mantles. We provide a model for infiltrating the two compartmental niches of a biopolymer membrane with different intrafibre minerals to obtain materials with potentially improved structure-property relationships. PMID:21864897

  4. A nanostructural basis for gloss of avian eggshells

    PubMed Central

    Igic, Branislav; Fecheyr-Lippens, Daphne; Xiao, Ming; Chan, Andrew; Hanley, Daniel; Brennan, Patricia R. L.; Grim, Tomas; Waterhouse, Geoffrey I. N.; Hauber, Mark E.; Shawkey, Matthew D.

    2015-01-01

    The role of pigments in generating the colour and maculation of birds' eggs is well characterized, whereas the effects of the eggshell's nanostructure on the visual appearance of eggs are little studied. Here, we examined the nanostructural basis of glossiness of tinamou eggs. Tinamou eggs are well known for their glossy appearance, but the underlying mechanism responsible for this optical effect is unclear. Using experimental manipulations in conjunction with angle-resolved spectrophotometry, scanning electron microscopy, atomic force microscopy and chemical analyses, we show that the glossy appearance of tinamou eggshells is produced by an extremely smooth cuticle, composed of calcium carbonate, calcium phosphate and, potentially, organic compounds such as proteins and pigments. Optical calculations corroborate surface smoothness as the main factor producing gloss. Furthermore, we reveal the presence of weak iridescence on eggs of the great tinamou (Tinamus major), an optical effect never previously documented for bird eggs. These data highlight the need for further exploration into the nanostructural mechanisms for the production of colour and other optical effects of avian eggshells. PMID:25505139

  5. Systematic Characterization of Human Protein Complexes Identifies Chromosome Segregation Proteins

    PubMed Central

    Hutchins, James R.A.; Toyoda, Yusuke; Hegemann, Björn; Poser, Ina; Hériché, Jean-Karim; Sykora, Martina M.; Augsburg, Martina; Hudecz, Otto; Buschhorn, Bettina A.; Bulkescher, Jutta; Conrad, Christian; Comartin, David; Schleiffer, Alexander; Sarov, Mihail; Pozniakovsky, Andrei; Slabicki, Mikolaj Michal; Schloissnig, Siegfried; Steinmacher, Ines; Leuschner, Marit; Ssykor, Andrea; Lawo, Steffen; Pelletier, Laurence; Stark, Holger; Nasmyth, Kim; Ellenberg, Jan; Durbin, Richard; Buchholz, Frank; Mechtler, Karl; Hyman, Anthony A.; Peters, Jan-Michael

    2010-01-01

    Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference (RNAi) screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization and tandem affinity purification-mass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex (APC/C) and the γ-tubulin ring complex (γ-TuRC), large complexes which are essential for spindle assembly and chromosome segregation. The approaches we describe here are generally applicable to high throughput follow-up analyses of phenotypic screens in mammalian cells. PMID:20360068

  6. Systematic analysis of human protein complexes identifies chromosome segregation proteins.

    PubMed

    Hutchins, James R A; Toyoda, Yusuke; Hegemann, Björn; Poser, Ina; Hériché, Jean-Karim; Sykora, Martina M; Augsburg, Martina; Hudecz, Otto; Buschhorn, Bettina A; Bulkescher, Jutta; Conrad, Christian; Comartin, David; Schleiffer, Alexander; Sarov, Mihail; Pozniakovsky, Andrei; Slabicki, Mikolaj Michal; Schloissnig, Siegfried; Steinmacher, Ines; Leuschner, Marit; Ssykor, Andrea; Lawo, Steffen; Pelletier, Laurence; Stark, Holger; Nasmyth, Kim; Ellenberg, Jan; Durbin, Richard; Buchholz, Frank; Mechtler, Karl; Hyman, Anthony A; Peters, Jan-Michael

    2010-04-30

    Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization, and tandem-affinity purification-mass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or had only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex and the gamma-tubulin ring complex--large complexes that are essential for spindle assembly and chromosome segregation. The approaches we describe here are generally applicable to high-throughput follow-up analyses of phenotypic screens in mammalian cells. PMID:20360068

  7. Identification of uterine ion transporters for mineralisation precursors of the avian eggshell

    PubMed Central

    2012-01-01

    Background In Gallus gallus, eggshell formation takes place daily in the hen uterus and requires large amounts of the ionic precursors for calcium carbonate (CaCO3). Both elements (Ca2+, HCO3-) are supplied by the blood via trans-epithelial transport. Our aims were to identify genes coding for ion transporters that are upregulated in the uterine portion of the oviduct during eggshell calcification, compared to other tissues and other physiological states, and incorporate these proteins into a general model for mineral transfer across the tubular gland cells during eggshell formation. Results A total of 37 candidate ion transport genes were selected from our database of overexpressed uterine genes associated with eggshell calcification, and by analogy with mammalian transporters. Their uterine expression was compared by qRTPCR in the presence and absence of eggshell formation, and with relative expression levels in magnum (low Ca2+/HCO3- movement) and duodenum (high rates of Ca2+/HCO3- trans-epithelial transfer). We identified overexpression of eleven genes related to calcium movement: the TRPV6 Ca2+ channel (basolateral uptake of Ca2+), 28 kDa calbindin (intracellular Ca2+ buffering), the endoplasmic reticulum type 2 and 3 Ca2+ pumps (ER uptake), and the inositol trisphosphate receptors type 1, 2 and 3 (ER release). Ca2+ movement across the apical membrane likely involves membrane Ca2+ pumps and Ca2+/Na+ exchangers. Our data suggests that Na+ transport involved the SCNN1 channel and the Na+/Ca2+ exchangers SLC8A1, 3 for cell uptake, the Na+/K+ ATPase for cell output. K+ uptake resulted from the Na+/K+ ATPase, and its output from the K+ channels (KCNJ2, 15, 16 and KCNMA1). We propose that the HCO3- is mainly produced from CO2 by the carbonic anhydrase 2 (CA2) and that HCO3- is secreted through the HCO3-/Cl- exchanger SLC26A9. HCO3- synthesis and precipitation with Ca2+ produce two H+. Protons are absorbed via the membrane’s Ca2+ pumps ATP2B1, 2 in the apical

  8. Quantitative proteomic profiling identifies protein correlates to EGFR kinase inhibition.

    PubMed

    Kani, Kian; Faca, Vitor M; Hughes, Lindsey D; Zhang, Wenxuan; Fang, Qiaojun; Shahbaba, Babak; Luethy, Roland; Erde, Jonathan; Schmidt, Joanna; Pitteri, Sharon J; Zhang, Qing; Katz, Jonathan E; Gross, Mitchell E; Plevritis, Sylvia K; McIntosh, Martin W; Jain, Anjali; Hanash, Samir; Agus, David B; Mallick, Parag

    2012-05-01

    Clinical oncology is hampered by lack of tools to accurately assess a patient's response to pathway-targeted therapies. Serum and tumor cell surface proteins whose abundance, or change in abundance in response to therapy, differentiates patients responding to a therapy from patients not responding to a therapy could be usefully incorporated into tools for monitoring response. Here, we posit and then verify that proteomic discovery in in vitro tissue culture models can identify proteins with concordant in vivo behavior and further, can be a valuable approach for identifying tumor-derived serum proteins. In this study, we use stable isotope labeling of amino acids in culture (SILAC) with proteomic technologies to quantitatively analyze the gefitinib-related protein changes in a model system for sensitivity to EGF receptor (EGFR)-targeted tyrosine kinase inhibitors. We identified 3,707 intracellular proteins, 1,276 cell surface proteins, and 879 shed proteins. More than 75% of the proteins identified had quantitative information, and a subset consisting of 400 proteins showed a statistically significant change in abundance following gefitinib treatment. We validated the change in expression profile in vitro and screened our panel of response markers in an in vivo isogenic resistant model and showed that these were markers of gefitinib response and not simply markers of phospho-EGFR downregulation. In doing so, we also were able to identify which proteins might be useful as markers for monitoring response and which proteins might be useful as markers for a priori prediction of response. PMID:22411897

  9. Quantitative Proteomic profiling identifies protein correlates to EGFR kinase inhibition

    PubMed Central

    Kani, Kian; Faca, Vitor M.; Hughes, Lindsey D.; Zhang, Wenxuan; Fang, Qiaojun; Shahbaba, Babak; Luethy, Roland; Erde, Jonathan; Schmidt, Joanna; Pitteri, Sharon J.; Zhang, Qing; Katz, Jonathan E.; Gross, Mitchell E.; Plevritis, Sylvia K.; McIntosh, Martin W.; Jain, Anjali; Hanash, Sam; Agus, David B.; Mallick, Parag

    2014-01-01

    Clinical oncology is hampered by a lack of tools to accurately assess a patient’s response to pathway-targeted therapies. Serum and tumor cell surface proteins whose abundance, or change in abundance in response to therapy, differentiates patients responding to a therapy from patients not-responding to a therapy could be usefully incorporated into tools for monitoring response. Here we posit and then verify that proteomic discovery in in vitro tissue culture models can identify proteins with concordant in vivo behavior and further, can be a valuable approach for identifying tumor-derived serum proteins. In this study we use Stable Isotope Labeling of Amino acids in Culture (SILAC) with proteomic technologies to quantitatively analyze the gefitinib-related protein changes in a model system for sensitivity to EGFR targeted tyrosine kinase inhibitors. We identified 3,707 intracellular proteins, 1,276 cell surface proteins, and 879 shed proteins. More than 75% of the proteins identified had quantitative information and a subset consisting of [400] proteins showed a statistically significant change in abundance following gefitinib treatment. We validated the change in expression profile in vitro and screened our panel of response markers in an in vivo isogenic resistant model and demonstrated that these were markers of gefitinib response and not simply markers of phospho-EGFR downregulation. In doing so, we also were able to identify which proteins might be useful as markers for monitoring response and which proteins might be useful as markers for a priori prediction of response. PMID:22411897

  10. Productive performance, eggshell quality, and eggshell ultrastructure of laying hens fed diets supplemented with organic trace minerals.

    PubMed

    Stefanello, C; Santos, T C; Murakami, A E; Martins, E N; Carneiro, T C

    2014-01-01

    This study was carried out with the purpose of evaluating the effect of supplementing hens' diets with trace minerals from inorganic or organic sources on the productive performance, eggshell quality, and eggshell ultrastructure of laying hens. Three hundred sixty Hy-Line W36 laying hens between 47 to 62 wk of age were used and distributed in a completely randomized experimental design with 9 treatments, 5 replicates, and 8 birds for each experimental unit. The treatments consisted of a control diet without supplementation of the trace minerals Mn, Zn, and Cu; 4 supplementation levels of these trace minerals from an inorganic source; and the same levels of supplementation from an organic source (proteinates). The supplementation levels in milligrams per kilogram for Mn, Zn, and Cu, were, respectively, 35-30-05, 65-60-10, 95-90-15, and 125-120-20. There was no effect of supplementation of trace minerals on the rate of posture, feed intake, feed conversion, specific weight, and Haugh unit of eggs. However, there was a quadratic effect (P < 0.05) of the levels of trace mineral supplementation on average egg weight and egg mass; the results did not differ regarding the source used. The increase in the levels of supplementation of Mn, Zn, and Cu provided a linear increase (P < 0.05) in the breaking strength and the percentage of eggshell. There was a linear decrease (P < 0.05) in the egg loss and the number of mammillary buttons in the shell. The best results were obtained using diets supplemented with trace minerals from an organic source because these diets provided lower egg loss, higher thickness, and increased strength of the shell. Structurally, organic Mn, Zn, and Cu provided higher thickness of the palisade layer and lower mammillary density. The trace mineral supplementation improved the structural characteristics and the quality of the eggshells. PMID:24570429

  11. Direct visualization of identified and newly synthesized proteins in situ

    PubMed Central

    Dieck, Susanne tom; Kochen, Lisa; Hanus, Cyril; Bartnik, Ina; Nassim-Assir, Belquis; Merk, Katrin; Mosler, Thorsten; Garg, Sakshi; Bunse, Stefanie; Tirrell, David A.; Schuman, Erin M.

    2015-01-01

    Protein synthesis is a dynamic process to tune the cellular proteome to internal and external demands. Metabolic labeling approaches identify the general proteomic response but missing is a tool to visualize within cells specific newly synthesized proteins. Here we describe a technique that couples non-canonical amino acid tagging or puromycylation with the proximity-ligation assay to visualize identified newly synthesized proteins and monitor their origin, redistribution and turnover in situ. PMID:25775042

  12. A comprehensive strategy to identify stoichiometric membrane protein interactomes

    PubMed Central

    Gokhale, Avanti; Perez-Cornejo, Patricia; Duran, Charity; Hartzell, H. Criss; Faundez, Victor

    2012-01-01

    There are numerous experimental approaches to identify the interaction networks of soluble proteins, but strategies for the identification of membrane protein interactomes remain limited. We discuss in detail the logic of an experimental design that led us to identify the interactome of a membrane protein of complex membrane topology, the calcium activated chloride channel Anoctamin 1/Tmem16a (Ano1). We used covalent chemical stabilizers of protein-protein interactions combined with magnetic bead immuno-affinity chromatography, quantitative SILAC mass-spectrometry and in silico network construction. This strategy led us to define a putative Ano1 interactome from which we selected key components for functional testing. We propose a combination of procedures to narrow down candidate proteins interacting with a membrane protein of interest for further functional studies. PMID:23676845

  13. Not so colourful after all: eggshell pigments constrain avian eggshell colour space.

    PubMed

    Hanley, Daniel; Grim, Tomáš; Cassey, Phillip; Hauber, Mark E

    2015-05-01

    Birds' eggshells are renowned for their striking colours and varied patterns. Although often considered exceptionally diverse, we report that avian eggshell coloration, sampled here across the full phylogenetic diversity of birds, occupies only 0.08-0.10% of the avian perceivable colour space. The concentrations of the two known tetrapyrrole eggshell pigments (protoporphyrin and biliverdin) are generally poor predictors of colour, both intra- and interspecifically. Here, we show that the constrained diversity of eggshell coloration can be accurately predicted by colour mixing models based on the relative contribution of both pigments and we demonstrate that the models' predictions can be improved by accounting for the reflectance of the eggshell's calcium carbonate matrix. The establishment of these proximate links between pigmentation and colour will enable future tests of hypotheses on the functions of perceived avian eggshell colours that depend on eggshell chemistry. More generally, colour mixing models are not limited to avian eggshell colours but apply to any natural colour. Our approach illustrates how modelling can aid the understanding of constraints on phenotypic diversity. PMID:25994009

  14. Not so colourful after all: eggshell pigments constrain avian eggshell colour space

    PubMed Central

    Hanley, Daniel; Grim, Tomáš; Cassey, Phillip; Hauber, Mark E.

    2015-01-01

    Birds' eggshells are renowned for their striking colours and varied patterns. Although often considered exceptionally diverse, we report that avian eggshell coloration, sampled here across the full phylogenetic diversity of birds, occupies only 0.08–0.10% of the avian perceivable colour space. The concentrations of the two known tetrapyrrole eggshell pigments (protoporphyrin and biliverdin) are generally poor predictors of colour, both intra- and interspecifically. Here, we show that the constrained diversity of eggshell coloration can be accurately predicted by colour mixing models based on the relative contribution of both pigments and we demonstrate that the models' predictions can be improved by accounting for the reflectance of the eggshell's calcium carbonate matrix. The establishment of these proximate links between pigmentation and colour will enable future tests of hypotheses on the functions of perceived avian eggshell colours that depend on eggshell chemistry. More generally, colour mixing models are not limited to avian eggshell colours but apply to any natural colour. Our approach illustrates how modelling can aid the understanding of constraints on phenotypic diversity. PMID:25994009

  15. Shotgun Proteomics Identifies Proteins Specific for Acute Renal Transplant Rejection

    SciTech Connect

    Sigdel, Tara K.; Kaushal, Amit; Gritsenko, Marina A.; Norbeck, Angela D.; Qian, Weijun; Xiao, Wenzhong; Camp, David G.; Smith, Richard D.; Sarwal, Minnie M.

    2010-01-04

    Acute rejection (AR) remains the primary risk factor for renal transplant outcome; development of non-invasive diagnostic biomarkers for AR is an unmet need. We used shotgun proteomics using LC-MS/MS and ELISA to analyze a set of 92 urine samples, from patients with AR, stable grafts (STA), proteinuria (NS), and healthy controls (HC). A total of 1446 urinary proteins were identified along with a number of NS specific, renal transplantation specific and AR specific proteins. Relative abundance of identified urinary proteins was measured by protein-level spectral counts adopting a weighted fold-change statistic, assigning increased weight for more frequently observed proteins. We have identified alterations in a number of specific urinary proteins in AR, primarily relating to MHC antigens, the complement cascade and extra-cellular matrix proteins. A subset of proteins (UMOD, SERPINF1 and CD44), have been further cross-validated by ELISA in an independent set of urine samples, for significant differences in the abundance of these urinary proteins in AR. This label-free, semi-quantitative approach for sampling the urinary proteome in normal and disease states provides a robust and sensitive method for detection of urinary proteins for serial, non-invasive clinical monitoring for graft rejection after

  16. How the oxygen isotope ratio of rain water influences the isotope ratio of chicken eggshell carbonate

    NASA Astrophysics Data System (ADS)

    Price, Gregory; Grimes, Stephen

    2015-04-01

    The stable oxygen isotope ratio of chicken eggshell carbonate was analysed from chicken eggs laid under free range, and organic farming regimes from across the UK. The eggshell carbonate oxygen isotope data shows a clear depletion in delta18O distribution from the southwest to the northeast. Although consistently offset by around 1 permil, the same isotopic distribution as that seen in eggshell carbonate is observed in the delta18O ratio of rainfall and groundwater from across the UK. This distribution is related to the Rayleigh distillation of rainfall driven by westerly winds across the UK landmass. The clear relationship observed between eggshell delta18O values and that of rainwater presumably reflects the nature of free range chickens which must be drinking locally derived rainwater and supplementing their diet and water intake with locally derived food. These results suggest that the oxygen isotope value of chicken eggshells can be used as a forensic tool to identify the locality that free range and organic eggs were laid within the UK. Furthermore, if suitable material is preserved in the archaeological and geological record then such a relationship can potentially be used to establish the oxygen isotope value of rainwater from which ancient and / or ancestral birds lived.

  17. The location of protoporphyrin in the eggshell of brown-shelled eggs.

    PubMed

    Samiullah, S; Roberts, J R

    2013-10-01

    Protoporphyrin has been identified as the main eggshell pigment in brown-shelled eggs. However, there has been some uncertainty concerning the distribution of the pigment within the shell (and cuticle) in brown-shelled eggs. Most previous studies have suggested that the bulk of the shell pigment is deposited in the cuticle of the shell. The present study measured the levels of protoporphyrin in intact eggshells and in shells from which the cuticle had been removed, using eggs from flocks at 3 different ages. This enabled the calculation of the relative amount of protoporphyrin in the calcareous eggshell and the cuticle layer of the eggshell. The majority of the protoporphyrin pigment was located in the calcareous part of the eggshell (80-87%) with a minority contained within the cuticle (13-20%). These findings suggest that studies focused on maintenance of shell color in brown-shelled eggs need to consider the stage of egg formation at which the reduction in pigment deposition is occurring. PMID:24046428

  18. Shifts in Bacterial Communities of Eggshells and Antimicrobial Activities in Eggs during Incubation in a Ground-Nesting Passerine

    PubMed Central

    Grizard, Stéphanie; Versteegh, Maaike A.; Ndithia, Henry K.; Salles, Joana F.; Tieleman, B. Irene

    2015-01-01

    Microbial invasion of egg contents is a cause of embryonic death. To counter infection risks, the embryo is protected physically by the eggshell and chemically by antimicrobial proteins. If microbial pressure drives embryo mortality, then females may have evolved, through natural selection, to adapt their immune investment into eggs. Although frequently hypothesized, this match between immune allocation and microorganisms has not been explored yet. To examine if correlations between microbes on eggs and immunity in eggs exist, we collected eggs from red-capped larks (Calandrella cinerea) and simultaneously examined their bacterial communities and antimicrobial components—pH, lysozyme and ovotransferrin—during natural incubation. Using molecular techniques, we find that bacterial communities are highly dynamic: bacterial abundance increases from the onset to late incubation, Shannon’s α-diversity index increases during early incubation stages, and β-diversity analysis shows that communities from 1 day-old clutches are phylogenetically more similar to each other than the older ones. Regarding the antimicrobials, we notice a decrease of pH and lysozyme concentration, while ovotransferrin concentration increases during incubation. Interestingly, we show that two eggs of the same clutch share equivalent immune protection, independent of clutch age. Lastly, our results provide limited evidence of significant correlation between antimicrobial compounds and bacterial communities. Our study examined simultaneously, for the first time in a wild bird, the dynamics of bacterial communities present on eggshells and of albumen-associated antimicrobial components during incubation and investigated their relationship. However, the link between microorganisms and immunity of eggs remains to be elucidated further. Identifying invading microbes and their roles in embryo mortality, as well as understanding the role of the eggshell microbiome, might be key to better understand

  19. Nutritional supplement of hatchery eggshell membrane improves poultry performance and provides resistance against endotoxin stress

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Eggshells are significant part of hatchery waste which consist of calcium carbonate crust, membranes, and proteins and peptides of embryonic origins along with other entrapped contaminants including microbes. We hypothesized that using this product as a nutritional additive in poultry diet may confe...

  20. Dietary supplementation with sodium bicarbonate improves calcium absorption and eggshell quality of laying hens during peak production.

    PubMed

    Jiang, M J; Zhao, J P; Jiao, H C; Wang, X J; Zhang, Q; Lin, H

    2015-01-01

    The advantage of supplemental sodium bicarbonate (NaHCO3) on eggshell quality in laying hens changes with age. Besides increasing calcium (Ca) secretion in the eggshell gland, it may improve Ca absorption in the intestine or kidney. Hy-Line Brown layers (n = 384), 25 weeks of age, were allocated to two treatment groups in two experiments, each of which included 4 replicates of 24 hens. Hens were fed a basal diet (control) or the basal diet containing 3 g NaHCO3 g/kg for 50 or 20 weeks in Experiment 1 or 2, respectively. A 24-h continuous lighting regimen was used to allow hens to consume the dietary supplements during the period of active eggshell formation. In Experiment 1, particularly from 25 to 50 weeks of age, and in Experiment 2, NaHCO3 supplementation favoured hen-d egg production at the expense of lower egg weight. The increased eggshell thickness should have nothing to do with the additional eggshell formation, because of the unchanged egg mass and daily eggshell calcification. At 35 weeks of age in both experiments, NaHCO3 supplementation increased duodenal expression of calbindin-d28k (CaBP-D28k) protein, contributing to higher Ca retention and balance. From 50 to 75 weeks of age in Experiment 1, the hens had little response to NaHCO3 supplementation and showed a negative trend on eggshell thickness and strength. It is concluded that dietary supplementation with 3 g NaHCO3 g/kg improves Ca absorption and eggshell quality of laying hens during the peak but not late production period, with the introduction of continuous lighting. PMID:26569471

  1. Novel approaches to identify protein adducts produced by lipid peroxidation.

    PubMed

    Codreanu, S G; Liebler, D C

    2015-01-01

    Lipid peroxidation is responsible for the generation of chemically reactive, diffusible lipid-derived electrophiles (LDEs) that covalently modify cellular protein targets. These protein modifications modulate protein activity and macromolecular interactions and induce adaptive and toxic cell signaling. Protein modifications induced by LDEs can be identified and quantified by affinity enrichment and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based techniques. Tagged LDE analog probes with different electrophilic groups can be covalently captured by click chemistry for LC-MS/MS analyses, thereby enabling in-depth studies of proteome damage at the protein and peptide sequence levels. Conversely, click-reactive, thiol-directed probes can be used to evaluate thiol damage caused by LDE by difference. These analytical approaches permit systematic study of the dynamics of protein damage caused by LDE and mechanisms by which oxidative stress contribute to toxicity and diseases. PMID:25819163

  2. A Least Square Method Based Model for Identifying Protein Complexes in Protein-Protein Interaction Network

    PubMed Central

    Dai, Qiguo; Guo, Maozu; Guo, Yingjie; Liu, Xiaoyan; Liu, Yang; Teng, Zhixia

    2014-01-01

    Protein complex formed by a group of physical interacting proteins plays a crucial role in cell activities. Great effort has been made to computationally identify protein complexes from protein-protein interaction (PPI) network. However, the accuracy of the prediction is still far from being satisfactory, because the topological structures of protein complexes in the PPI network are too complicated. This paper proposes a novel optimization framework to detect complexes from PPI network, named PLSMC. The method is on the basis of the fact that if two proteins are in a common complex, they are likely to be interacting. PLSMC employs this relation to determine complexes by a penalized least squares method. PLSMC is applied to several public yeast PPI networks, and compared with several state-of-the-art methods. The results indicate that PLSMC outperforms other methods. In particular, complexes predicted by PLSMC can match known complexes with a higher accuracy than other methods. Furthermore, the predicted complexes have high functional homogeneity. PMID:25405206

  3. 40 CFR 174.529 - Bacillus thuringiensis modified Cry1Ab protein as identified under OECD Unique Identifier SYN...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... protein as identified under OECD Unique Identifier SYN-IR67B-1 in cotton; exemption from the requirement... Tolerance Exemptions § 174.529 Bacillus thuringiensis modified Cry1Ab protein as identified under OECD... Bacillus thuringiensis modified Cry1Ab protein as identified under OECD Unique Identifier SYN-IR67B-1...

  4. 40 CFR 174.529 - Bacillus thuringiensis modified Cry1Ab protein as identified under OECD Unique Identifier SYN...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... protein as identified under OECD Unique Identifier SYN-IR67B-1 in cotton; exemption from the requirement... Tolerance Exemptions § 174.529 Bacillus thuringiensis modified Cry1Ab protein as identified under OECD... Bacillus thuringiensis modified Cry1Ab protein as identified under OECD Unique Identifier SYN-IR67B-1...

  5. 40 CFR 174.529 - Bacillus thuringiensis modified Cry1Ab protein as identified under OECD Unique Identifier SYN...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... protein as identified under OECD Unique Identifier SYN-IR67B-1 in cotton; exemption from the requirement... Tolerance Exemptions § 174.529 Bacillus thuringiensis modified Cry1Ab protein as identified under OECD... Bacillus thuringiensis modified Cry1Ab protein as identified under OECD Unique Identifier SYN-IR67B-1...

  6. 40 CFR 174.529 - Bacillus thuringiensis modified Cry1Ab protein as identified under OECD Unique Identifier SYN...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... protein as identified under OECD Unique Identifier SYN-IR67B-1 in cotton; exemption from the requirement... Tolerance Exemptions § 174.529 Bacillus thuringiensis modified Cry1Ab protein as identified under OECD... Bacillus thuringiensis modified Cry1Ab protein as identified under OECD Unique Identifier SYN-IR67B-1...

  7. Proteomic Analysis of the Soybean Symbiosome Identifies New Symbiotic Proteins*

    PubMed Central

    Clarke, Victoria C.; Loughlin, Patrick C.; Gavrin, Aleksandr; Chen, Chi; Brear, Ella M.; Day, David A.; Smith, Penelope M.C.

    2015-01-01

    Legumes form a symbiosis with rhizobia in which the plant provides an energy source to the rhizobia bacteria that it uses to fix atmospheric nitrogen. This nitrogen is provided to the legume plant, allowing it to grow without the addition of nitrogen fertilizer. As part of the symbiosis, the bacteria in the infected cells of a new root organ, the nodule, are surrounded by a plant-derived membrane, the symbiosome membrane, which becomes the interface between the symbionts. Fractions containing the symbiosome membrane (SM) and material from the lumen of the symbiosome (peribacteroid space or PBS) were isolated from soybean root nodules and analyzed using nongel proteomic techniques. Bicarbonate stripping and chloroform-methanol extraction of isolated SM were used to reduce complexity of the samples and enrich for hydrophobic integral membrane proteins. One hundred and ninety-seven proteins were identified as components of the SM, with an additional fifteen proteins identified from peripheral membrane and PBS protein fractions. Proteins involved in a range of cellular processes such as metabolism, protein folding and degradation, membrane trafficking, and solute transport were identified. These included a number of proteins previously localized to the SM, such as aquaglyceroporin nodulin 26, sulfate transporters, remorin, and Rab7 homologs. Among the proteome were a number of putative transporters for compounds such as sulfate, calcium, hydrogen ions, peptide/dicarboxylate, and nitrate, as well as transporters for which the substrate is not easy to predict. Analysis of the promoter activity for six genes encoding putative SM proteins showed nodule specific expression, with five showing expression only in infected cells. Localization of two proteins was confirmed using GFP-fusion experiments. The data have been deposited to the ProteomeXchange with identifier PXD001132. This proteome will provide a rich resource for the study of the legume-rhizobium symbiosis. PMID

  8. Proteomic analysis of the soybean symbiosome identifies new symbiotic proteins.

    PubMed

    Clarke, Victoria C; Loughlin, Patrick C; Gavrin, Aleksandr; Chen, Chi; Brear, Ella M; Day, David A; Smith, Penelope M C

    2015-05-01

    Legumes form a symbiosis with rhizobia in which the plant provides an energy source to the rhizobia bacteria that it uses to fix atmospheric nitrogen. This nitrogen is provided to the legume plant, allowing it to grow without the addition of nitrogen fertilizer. As part of the symbiosis, the bacteria in the infected cells of a new root organ, the nodule, are surrounded by a plant-derived membrane, the symbiosome membrane, which becomes the interface between the symbionts. Fractions containing the symbiosome membrane (SM) and material from the lumen of the symbiosome (peribacteroid space or PBS) were isolated from soybean root nodules and analyzed using nongel proteomic techniques. Bicarbonate stripping and chloroform-methanol extraction of isolated SM were used to reduce complexity of the samples and enrich for hydrophobic integral membrane proteins. One hundred and ninety-seven proteins were identified as components of the SM, with an additional fifteen proteins identified from peripheral membrane and PBS protein fractions. Proteins involved in a range of cellular processes such as metabolism, protein folding and degradation, membrane trafficking, and solute transport were identified. These included a number of proteins previously localized to the SM, such as aquaglyceroporin nodulin 26, sulfate transporters, remorin, and Rab7 homologs. Among the proteome were a number of putative transporters for compounds such as sulfate, calcium, hydrogen ions, peptide/dicarboxylate, and nitrate, as well as transporters for which the substrate is not easy to predict. Analysis of the promoter activity for six genes encoding putative SM proteins showed nodule specific expression, with five showing expression only in infected cells. Localization of two proteins was confirmed using GFP-fusion experiments. The data have been deposited to the ProteomeXchange with identifier PXD001132. This proteome will provide a rich resource for the study of the legume-rhizobium symbiosis. PMID

  9. Biodegradation of thermoplastic starch/eggshell powder composites.

    PubMed

    Bootklad, Munlika; Kaewtatip, Kaewta

    2013-09-12

    Thermoplastic starch (TPS) was prepared using compression molding and chicken eggshell was used as a filler. The effect of the eggshell powder (EP) on the properties of TPS was compared with the effect of commercial calcium carbonate (CC). The organic compound on the surface of the eggshell powder acted as a coupling agent that resulted in a strong adhesion between the eggshell powder and the TPS matrix, as confirmed by SEM micrographs. The biodegradation was determined by the soil burial test. The TPS/EP composites were more rapidly degraded than the TPS/CC composites. In addition, the eggshell powder improved the water resistance and thermal stability of the TPS. PMID:23911451

  10. Proteomic Approach to Identify Nuclear Proteins in Wheat Grain.

    PubMed

    Bancel, Emmanuelle; Bonnot, Titouan; Davanture, Marlène; Branlard, Gérard; Zivy, Michel; Martre, Pierre

    2015-10-01

    The nuclear proteome of the grain of the two cultivated wheat species Triticum aestivum (hexaploid wheat; genomes A, B, and D) and T. monococcum (diploid wheat; genome A) was analyzed in two early stages of development using shotgun-based proteomics. A procedure was optimized to purify nuclei, and an improved protein sample preparation was developed to efficiently remove nonprotein substances (starch and nucleic acids). A total of 797 proteins corresponding to 528 unique proteins were identified, 36% of which were classified in functional groups related to DNA and RNA metabolism. A large number (107 proteins) of unknown functions and hypothetical proteins were also found. Some identified proteins may be multifunctional and may present multiple localizations. On the basis of the MS/MS analysis, 368 proteins were present in the two species, and in two stages of development, some qualitative differences between species and stages of development were also found. All of these data illustrate the dynamic function of the grain nucleus in the early stages of development. PMID:26228564

  11. The cuticle modulates ultraviolet reflectance of avian eggshells

    PubMed Central

    Fecheyr-Lippens, Daphne C.; Igic, Branislav; D'Alba, Liliana; Hanley, Daniel; Verdes, Aida; Holford, Mande; Waterhouse, Geoffrey I. N.; Grim, Tomas; Hauber, Mark E.; Shawkey, Matthew D.

    2015-01-01

    ABSTRACT Avian eggshells are variedly coloured, yet only two pigments, biliverdin and protoporphyrin IX, are known to contribute to the dramatic diversity of their colours. By contrast, the contributions of structural or other chemical components of the eggshell are poorly understood. For example, unpigmented eggshells, which appear white to the human eye, vary in their ultraviolet (UV) reflectance, which may be detectable by birds. We investigated the proximate mechanisms for the variation in UV-reflectance of unpigmented bird eggshells using spectrophotometry, electron microscopy, chemical analyses, and experimental manipulations. We specifically tested how UV-reflectance is affected by the eggshell cuticle, the outermost layer of most avian eggshells. The chemical dissolution of the outer eggshell layers, including the cuticle, increased UV-reflectance for only eggshells that contained a cuticle. Our findings demonstrate that the outer eggshell layers, including the cuticle, absorb UV-light, probably because they contain higher levels of organic components and other chemicals, such as calcium phosphates, compared to the predominantly calcite-based eggshell matrix. These data highlight the need to examine factors other than the known pigments in studies of avian eggshell colour. PMID:25964661

  12. The cuticle modulates ultraviolet reflectance of avian eggshells.

    PubMed

    Fecheyr-Lippens, Daphne C; Igic, Branislav; D'Alba, Liliana; Hanley, Daniel; Verdes, Aida; Holford, Mande; Waterhouse, Geoffrey I N; Grim, Tomas; Hauber, Mark E; Shawkey, Matthew D

    2015-01-01

    Avian eggshells are variedly coloured, yet only two pigments, biliverdin and protoporphyrin IX, are known to contribute to the dramatic diversity of their colours. By contrast, the contributions of structural or other chemical components of the eggshell are poorly understood. For example, unpigmented eggshells, which appear white to the human eye, vary in their ultraviolet (UV) reflectance, which may be detectable by birds. We investigated the proximate mechanisms for the variation in UV-reflectance of unpigmented bird eggshells using spectrophotometry, electron microscopy, chemical analyses, and experimental manipulations. We specifically tested how UV-reflectance is affected by the eggshell cuticle, the outermost layer of most avian eggshells. The chemical dissolution of the outer eggshell layers, including the cuticle, increased UV-reflectance for only eggshells that contained a cuticle. Our findings demonstrate that the outer eggshell layers, including the cuticle, absorb UV-light, probably because they contain higher levels of organic components and other chemicals, such as calcium phosphates, compared to the predominantly calcite-based eggshell matrix. These data highlight the need to examine factors other than the known pigments in studies of avian eggshell colour. PMID:25964661

  13. Thermal emissivity of avian eggshells.

    PubMed

    Björn, Lars Olof; Bengtson, Sven-Axel; Li, Shaoshan; Hecker, Christoph; Ullah, Saleem; Roos, Arne; Nilsson, Annica M

    2016-04-01

    The hypothesis has been tested that evolution has resulted in lower thermal emissivity of eggs of birds breeding openly in cold climates than of eggs of birds that nest under protective covering or in warmer climates. Directional thermal emissivity has been estimated from directional-hemispherical reflectance spectra. Due to several methodological difficulties the absolute emissivity is not accurately determined, but differences between species are obvious. Most notably, small waders of the genus Calidris, breeding in cold climates on the tundra, and in most cases with uniparental nest attendance, have low directional emissivity of their eggshells, about 0.92 when integration is carried out for wavelengths up to 16μm. Species belonging to Galloanserinae have the highest directional emissivity, about 0.96, of their eggs. No differences due to climate or breeding conditions were found within this group. Eggs of most other birds tested possess intermediate emissivity, but the values for Pica pica and Corvus corone cornix are as low as for Calidris. Large species-dependent differences in spectral reflectance were found at specific wavelengths. For instance, at 4.259μm the directional-hemispherical reflectance for galliforms range from 0.05 to 0.09, while for Fratercula arctica and Fulmarus glacialis it is about 0.3. The reflection peaks at 6.5 and 11.3μm due to calcite are differentially attenuated in different species. In conclusion, the hypothesis that evolution has resulted in lower thermal emissivity of bird eggs being exposed in cold climates is not supported by our results. The emissivity is not clearly related to nesting habits or climate, and it is unlikely that the small differences observed are ecologically important. The spectral differences between eggs that nevertheless exist should be taken into account when using infrared thermometers for estimating the surface temperature of avian eggs. PMID:27033033

  14. Recovery of Salmonella from eggshell wash, eggshell crush, and egg internal contents of unwashed commercial shell eggs in Australia.

    PubMed

    Chousalkar, K K; Roberts, J R

    2012-07-01

    The experiment was conducted to study the prevalence of Salmonella spp. on the eggshell surface, eggshell membranes or pores, and in egg internal contents from unwashed eggs collected from commercial caged layer farms in Australia. Eggshell rinsate, shell crush, and egg internal contents (yolk and albumen) of eggs were processed for Salmonella spp. Salmonella Infantis and Salmonella subspecies 1, serotype 4,12:d were isolated from the eggshell surface. Salmonella spp. were not isolated from any eggshell crush or egg internal contents. It would appear that the occurrence of Salmonella in the Australian egg industry is low. PMID:22700522

  15. Identifying protein complexes in protein-protein interaction networks by using clique seeds and graph entropy.

    PubMed

    Chen, Bolin; Shi, Jinhong; Zhang, Shenggui; Wu, Fang-Xiang

    2013-01-01

    The identification of protein complexes plays a key role in understanding major cellular processes and biological functions. Various computational algorithms have been proposed to identify protein complexes from protein-protein interaction (PPI) networks. In this paper, we first introduce a new seed-selection strategy for seed-growth style algorithms. Cliques rather than individual vertices are employed as initial seeds. After that, a result-modification approach is proposed based on this seed-selection strategy. Predictions generated by higher order clique seeds are employed to modify results that are generated by lower order ones. The performance of this seed-selection strategy and the result-modification approach are tested by using the entropy-based algorithm, which is currently the best seed-growth style algorithm to detect protein complexes from PPI networks. In addition, we investigate four pairs of strategies for this algorithm in order to improve its accuracy. The numerical experiments are conducted on a Saccharomyces cerevisiae PPI network. The group of best predictions consists of 1711 clusters, with the average f-score at 0.68 after removing all similar and redundant clusters. We conclude that higher order clique seeds can generate predictions with higher accuracy and that our improved entropy-based algorithm outputs more reasonable predictions than the original one. PMID:23112006

  16. A Functional Genomic Yeast Screen to Identify Pathogenic Bacterial Proteins

    PubMed Central

    Slagowski, Naomi L; Kramer, Roger W; Morrison, Monica F; LaBaer, Joshua; Lesser, Cammie F

    2008-01-01

    Many bacterial pathogens promote infection and cause disease by directly injecting into host cells proteins that manipulate eukaryotic cellular processes. Identification of these translocated proteins is essential to understanding pathogenesis. Yet, their identification remains limited. This, in part, is due to their general sequence uniqueness, which confounds homology-based identification by comparative genomic methods. In addition, their absence often does not result in phenotypes in virulence assays limiting functional genetic screens. Translocated proteins have been observed to confer toxic phenotypes when expressed in the yeast Saccharomyces cerevisiae. This observation suggests that yeast growth inhibition can be used as an indicator of protein translocation in functional genomic screens. However, limited information is available regarding the behavior of non-translocated proteins in yeast. We developed a semi-automated quantitative assay to monitor the growth of hundreds of yeast strains in parallel. We observed that expression of half of the 19 Shigella translocated proteins tested but almost none of the 20 non-translocated Shigella proteins nor ∼1,000 Francisella tularensis proteins significantly inhibited yeast growth. Not only does this study establish that yeast growth inhibition is a sensitive and specific indicator of translocated proteins, but we also identified a new substrate of the Shigella type III secretion system (TTSS), IpaJ, previously missed by other experimental approaches. In those cases where the mechanisms of action of the translocated proteins are known, significant yeast growth inhibition correlated with the targeting of conserved cellular processes. By providing positive rather than negative indication of activity our assay complements existing approaches for identification of translocated proteins. In addition, because this assay only requires genomic DNA it is particularly valuable for studying pathogens that are difficult to

  17. Fossil struthionid eggshells from Laetoli, Tanzania: Taxonomic and biostratigraphic significance

    NASA Astrophysics Data System (ADS)

    Harrison, Terry; Msuya, Charles P.

    2005-04-01

    Recent paleontological investigations at Laetoli and neighboring localities in northern Tanzania have produced a large collection of fossil ostrich eggshells from the Pliocene-aged Laetolil Beds (˜3.5-4.5 Ma) and Ndolanya Beds (˜2.6-2.7 Ma). A detailed analysis of the morphology of the eggshells and their taxonomic affinities indicates that two different species of Struthio are represented. In the Lower Laetolil Beds and in the Upper Laetolil Beds below Tuff 3 a new species is recognized— Struthio kakesiensis. This is replaced in the Upper Laetolil Beds by Struthio camelus, the modern species of ostrich. Since radiometric age determinations are available for the stratigraphic sequence at Laetoli, it is possible to precisely date the first appearance of S. camelus at ˜3.6-3.8 Ma. Comparisons of the Laetoli material with specimens from the well-dated sequences at Lothagam and Kanapoi in northern Kenya, allow the taxonomic and biochronological analysis to be extended back in time to the late Miocene. At about 6.5 Ma, Diamantornis and elephant birds were replaced in East Africa by ostriches belonging to the genus Struthio. Three time-successive species of ostriches are identified in the fossil record of East Africa, beginning with Struthio. cf. karingarabensis (˜6.5-4.2 Ma), followed by S. kakesiensis (˜4.5-3.6 Ma) and then S. camelus (˜3.8 Ma onwards). A similar sequence of taxa has previously been recorded from localities in Namibia, but at these sites there is no possibility to precisely calibrate the ages of the different species using radiometric dating. Nevertheless, the broadly similar evolutionary sequence and the close correspondence in inferred ages for the succession of species in East Africa and Namibia suggest that ostrich eggshells are a very useful tool for biochronological correlation of paleontological sites in sub-Saharan Africa.

  18. A novel approach to tag and identify geranylgeranylated proteins

    PubMed Central

    Chan, Lai N.; Hart, Courtenay; Guo, Lea; Nyberg, Tamara; Davies, Brandon S.J.; Fong, Loren G.; Young, Stephen G.; Agnew, Brian J.; Tamanoi, Fuyuhiko

    2010-01-01

    A recently developed proteomic strategy, the “GG-azide”-labeling approach, is described for the detection and proteomic analysis of geranylgeranylated proteins. This approach involves metabolic incorporation of a synthetic azido-geranylgeranyl analog and chemoselective derivatization of azido-geranylgeranyl-modified proteins by the “click” chemistry, using a tetramethylrhodamine-alkyne. The resulting conjugated proteins can be separated by 1-D or 2-D and pH fractionation, and detected by fluorescence imaging. This method is compatible with downstream LC-MS/MS analysis. Proteomic analysis of conjugated proteins by this approach identified several known geranylgeranylated proteins as well as Rap2c, a novel member of the Ras family. Furthermore, prenylation of progerin in mouse embryonic fibroblast cells was examined using this approach, demonstrating that this strategy can be used to study prenylation of specific proteins. The “GG-azide”-labeling approach provides a new tool for the detection and proteomic analysis of geranylgeranylated proteins, and it can readily be extended to other post-translational modifications. PMID:19784953

  19. EFFECTS OF DICOFOL ON MALLARD EGGSHELL QUALITY

    EPA Science Inventory

    Dicofol is an organochlorine acaricide structurally similar to DDE and there is concern that it may effect avian reproduction. Effects of dietary dicofol on mallard (Anas platyrhynchos) egg production and eggshell quality were evaluated; the mallard is moderately sensitive to ODE...

  20. Advanced oxidation process sanitization of eggshell surfaces.

    PubMed

    Gottselig, Steven M; Dunn-Horrocks, Sadie L; Woodring, Kristy S; Coufal, Craig D; Duong, Tri

    2016-06-01

    The microbial quality of eggs entering the hatchery represents an important critical control point for biosecurity and pathogen reduction programs in integrated poultry production. The development of safe and effective interventions to reduce microbial contamination on the surface of eggs will be important to improve the overall productivity and microbial food safety of poultry and poultry products. The hydrogen peroxide (H2O2) and ultraviolet (UV) light advanced oxidation process is a potentially important alternative to traditional sanitizers and disinfectants for egg sanitation. The H2O2/UV advanced oxidation process was demonstrated previously to be effective in reducing surface microbial contamination on eggs. In this study, we evaluated treatment conditions affecting the efficacy of H2O2/UV advanced oxidation in order to identify operational parameters for the practical application of this technology in egg sanitation. The effect of the number of application cycles, UV intensity, duration of UV exposure, and egg rotation on the recovery of total aerobic bacteria from the surface of eggs was evaluated. Of the conditions evaluated, we determined that reduction of total aerobic bacteria from naturally contaminated eggs was optimized when eggs were sanitized using 2 repeated application cycles with 5 s exposure to 14 mW cm(-2) UV light, and that rotation of the eggs between application cycles was unnecessary. Additionally, using these optimized conditions, the H2O2/UV process reduced Salmonella by greater than 5 log10 cfu egg(-1) on the surface of experimentally contaminated eggs. This study demonstrates the potential for practical application of the H2O2/UV advanced oxidation process in egg sanitation and its effectiveness in reducing Salmonella on eggshell surfaces. PMID:27030693

  1. Identifying Antioxidant Proteins by Using Optimal Dipeptide Compositions.

    PubMed

    Feng, Pengmian; Chen, Wei; Lin, Hao

    2016-06-01

    Antioxidant proteins are a kind of molecules that can terminate cellular and DNA damages caused by free radical intermediates. The use of antioxidant proteins for prevention of diseases has been intensively studied in recent years. Thus, accurate identification of antioxidant proteins is essential for understanding their roles in pharmacology. In this study, a support vector machine-based predictor called AodPred was developed for identifying antioxidant proteins. In this predictor, the sequence was formulated by using the optimal 3-gap dipeptides obtained by using feature selection method. It was observed by jackknife cross-validation test that AodPred can achieve an overall accuracy of 74.79 % in identifying antioxidant proteins. As a user-friendly tool, AodPred is freely accessible at http://lin.uestc.edu.cn/server/AntioxiPred . To maximize the convenience of the vast majority of experimental scientists, a step-by-step guide is provided on how to use the web server to obtain the desired results. PMID:26345449

  2. SRide: a server for identifying stabilizing residues in proteins

    PubMed Central

    Magyar, Csaba; Gromiha, M. Michael; Pujadas, Gerard; Tusnády, Gábor E.; Simon, István

    2005-01-01

    Residues expected to play key roles in the stabilization of proteins [stabilizing residues (SRs)] are selected by combining several methods based mainly on the interactions of a given residue with its spatial, rather than its sequential neighborhood and by considering the evolutionary conservation of the residues. A residue is selected as a stabilizing residue if it has high surrounding hydrophobicity, high long-range order, high conservation score and if it belongs to a stabilization center. The definition of all these parameters and the thresholds used to identify the SRs are discussed in detail. The algorithm for identifying SRs was originally developed for TIM-barrel proteins [M. M. Gromiha, G. Pujadas, C. Magyar, S. Selvaraj, and I. Simon (2004), Proteins, 55, 316–329] and is now generalized for all proteins of known 3D structure. SRs could be applied in protein engineering and homology modeling and could also help to explain certain folds with significant stability. The SRide server is located at . PMID:15980477

  3. SRide: a server for identifying stabilizing residues in proteins.

    PubMed

    Magyar, Csaba; Gromiha, M Michael; Pujadas, Gerard; Tusnády, Gábor E; Simon, István

    2005-07-01

    Residues expected to play key roles in the stabilization of proteins [stabilizing residues (SRs)] are selected by combining several methods based mainly on the interactions of a given residue with its spatial, rather than its sequential neighborhood and by considering the evolutionary conservation of the residues. A residue is selected as a stabilizing residue if it has high surrounding hydrophobicity, high long-range order, high conservation score and if it belongs to a stabilization center. The definition of all these parameters and the thresholds used to identify the SRs are discussed in detail. The algorithm for identifying SRs was originally developed for TIM-barrel proteins [M. M. Gromiha, G. Pujadas, C. Magyar, S. Selvaraj, and I. Simon (2004), Proteins, 55, 316-329] and is now generalized for all proteins of known 3D structure. SRs could be applied in protein engineering and homology modeling and could also help to explain certain folds with significant stability. The SRide server is located at http://sride.enzim.hu. PMID:15980477

  4. Identifying Unstable Regions of Proteins Involved in Misfolding Diseases

    NASA Astrophysics Data System (ADS)

    Guest, Will; Cashman, Neil; Plotkin, Steven

    2009-05-01

    Protein misfolding is a necessary step in the pathogenesis of many diseases, including Creutzfeldt-Jakob disease (CJD) and familial amyotrophic lateral sclerosis (fALS). Identifying unstable structural elements in their causative proteins elucidates the early events of misfolding and presents targets for inhibition of the disease process. An algorithm was developed to calculate the Gibbs free energy of unfolding for all sequence-contiguous regions of a protein using three methods to parameterize energy changes: a modified G=o model, changes in solvent-accessible surface area, and all-atoms molecular dynamics. The entropic effects of disulfide bonds and post-translational modifications are treated analytically. It incorporates a novel method for finding local dielectric constants inside a protein to accurately handle charge effects. We have predicted the unstable parts of prion protein and superoxide dismutase 1, the proteins involved in CJD and fALS respectively, and have used these regions as epitopes to prepare antibodies that are specific to the misfolded conformation and show promise as therapeutic agents.

  5. Immobilization of the urease on eggshell membrane and its application in biosensor.

    PubMed

    D'Souza, S F; Kumar, Jitendra; Jha, Sandeep Kumar; Kubal, B S

    2013-03-01

    Eggshell membrane is a natural material, essentially made up of protein fibers having flexibility in the aqueous solution and possessing gas and water permeability. It is used as a biomembrane for immobilization of urease for the development of a potentiometric urea biosensor. Eggshell membrane was treated with polyethyleneimine (PEI) to impart polycation characteristics. Urease was immobilized on the PEI treated eggshell membrane through adsorption. SEM study was carried out to observe the changes in surface morphology after immobilization. FTIR study of membrane was carried out to observe the changes in IR spectra after immobilization of enzyme. Immobilized membrane was associated with ammonium ion selective electrode. Biosensor exhibited sigmoidal responses for the urea concentration range from 0.5 to 10mM. The response time of the biosensor was 120 s. A single membrane was reused for 270 reactions without loss of activity. The urease-eggshell membranes were stable for 2 months when stored in buffer even at room temperature. PMID:25427497

  6. Multivariate proteomic profiling identifies novel accessory proteins of coated vesicles

    PubMed Central

    Antrobus, Robin; Hirst, Jennifer; Bhumbra, Gary S.; Kozik, Patrycja; Jackson, Lauren P.; Sahlender, Daniela A.

    2012-01-01

    Despite recent advances in mass spectrometry, proteomic characterization of transport vesicles remains challenging. Here, we describe a multivariate proteomics approach to analyzing clathrin-coated vesicles (CCVs) from HeLa cells. siRNA knockdown of coat components and different fractionation protocols were used to obtain modified coated vesicle-enriched fractions, which were compared by stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative mass spectrometry. 10 datasets were combined through principal component analysis into a “profiling” cluster analysis. Overall, 136 CCV-associated proteins were predicted, including 36 new proteins. The method identified >93% of established CCV coat proteins and assigned >91% correctly to intracellular or endocytic CCVs. Furthermore, the profiling analysis extends to less well characterized types of coated vesicles, and we identify and characterize the first AP-4 accessory protein, which we have named tepsin. Finally, our data explain how sequestration of TACC3 in cytosolic clathrin cages causes the severe mitotic defects observed in auxilin-depleted cells. The profiling approach can be adapted to address related cell and systems biological questions. PMID:22472443

  7. Eukaryotic protein synthesis inhibitors identified by comparison of cytotoxicity profiles

    PubMed Central

    CHAN, JENNY; KHAN, SHAKILA N.; HARVEY, ISABELLE; MERRICK, WILLIAM; PELLETIER, JERRY

    2004-01-01

    The National Cancer Institute (NCI) Human Tumor Cell Line Anti-Cancer Drug Screen has evaluated the cytotoxicity profiles of a large number of synthetic compounds, natural products, and plant extracts on 60 different cell lines. The data for each compound/extract can be assessed for similarity of cytotoxicity pattern, relative to a given test compound, using an algorithm called COMPARE. In applying a chemical biology approach to better understand the mechanism of eukaryotic protein synthesis, we used these resources to search for novel inhibitors of translation. The cytotoxicity profiles of 31 known protein synthesis inhibitors were used to identify compounds from the NCI database with similar activity profiles. Using this approach, two natural products, phyllanthoside and nagilactone C, were identified and characterized as novel protein synthesis inhibitors. Both compounds are specific for the eukaryotic translation apparatus, function in vivo and in vitro, and interfere with translation elongation. Our results demonstrate the feasibility of utilizing cytotoxicity profiles to identify new inhibitors of translation. PMID:14970397

  8. Irregularly calcified eggs and eggshells of Caiman latirostris (Alligatoridae: Crocodylia).

    PubMed

    Fernández, Mariela Soledad; Simoncini, Melina Soledad; Dyke, Gareth

    2013-05-01

    We describe irregularly calcified egg and eggshell morphologies for the first time in nests of the broad-snouted caiman, Caiman latirostris. Research is based on detailed descriptions of 270 eggs from a total sample of 46,800 collected between 2005 and 2011 in Santa Fe Province, Argentina, and encompasses animals from both natural habitats and held in captivity. We discuss possible reasons for the occurrence of eggs with different mineralisation patterns in our extensive C. latirostris field sample and its conservation significance; the chemistry of egg laying in amniotes is sensitive to environmental contamination which, in turn, has biological implications. Based on our egg sample, we identify two caiman eggshell abnormalities: (1) regularly calcified eggs with either calcitic nodules or superficial wrinkles at one egg end and (2) irregularly calcified eggs with structural gaps that weaken the shell. Some recently laid clutches we examined included eggs with most of the shell broken and detached from the flexible membrane. Most type 1 regularly calcified eggs lost their initial calcified nodules during incubation, suggesting that these deposits do not affect embryo survival rates. In contrast, irregularly calcified caiman eggs have a mean hatching success rate of 8.9% (range 0-38%) across our sample compared to a mean normal success of 75%. Most irregularly calcified caiman eggs probably die because of infections caused by fungi and bacteria in the organic nest material, although another possible explanation that merits further investigation could be an increase in permeability, leading to embryo dehydration. PMID:23604383

  9. Irregularly calcified eggs and eggshells of Caiman latirostris (Alligatoridae: Crocodylia)

    NASA Astrophysics Data System (ADS)

    Fernández, Mariela Soledad; Simoncini, Melina Soledad; Dyke, Gareth

    2013-05-01

    We describe irregularly calcified egg and eggshell morphologies for the first time in nests of the broad-snouted caiman, Caiman latirostris. Research is based on detailed descriptions of 270 eggs from a total sample of 46,800 collected between 2005 and 2011 in Santa Fe Province, Argentina, and encompasses animals from both natural habitats and held in captivity. We discuss possible reasons for the occurrence of eggs with different mineralisation patterns in our extensive C. latirostris field sample and its conservation significance; the chemistry of egg laying in amniotes is sensitive to environmental contamination which, in turn, has biological implications. Based on our egg sample, we identify two caiman eggshell abnormalities: (1) regularly calcified eggs with either calcitic nodules or superficial wrinkles at one egg end and (2) irregularly calcified eggs with structural gaps that weaken the shell. Some recently laid clutches we examined included eggs with most of the shell broken and detached from the flexible membrane. Most type 1 regularly calcified eggs lost their initial calcified nodules during incubation, suggesting that these deposits do not affect embryo survival rates. In contrast, irregularly calcified caiman eggs have a mean hatching success rate of 8.9 % (range 0-38 %) across our sample compared to a mean normal success of 75 %. Most irregularly calcified caiman eggs probably die because of infections caused by fungi and bacteria in the organic nest material, although another possible explanation that merits further investigation could be an increase in permeability, leading to embryo dehydration.

  10. Engineering ecotin for identifying proteins with a trypsin fold.

    PubMed

    Sathler, Plínio C; Craik, Charles S; Takeuchi, Toshihiko; Zingali, Russolina B; Castro, Helena C

    2010-04-01

    Ecotin is a bidentate, fold-specific inhibitor of mammalian serine-proteases produced by Escherichia coli. This molecule may be engineered to increase and/or change its affinity and specificity providing significant biotechnological potential. Since ecotin binds tightly to serine proteases of the trypsin fold, it may help to identify the role of these enzymes in different biological processes. In this work, we tested ecotin variants as an affinity purification reagent for identifying enzymes in samples of tumor progression and mammary gland involution. Initially, we used a commercial source of urokinase-type plasminogen activator (u-PA) that remained fully active after elution from an affinity column of the ecotin variant (M84R, M85R). We then successfully identified u-PA from more complex mixtures including lysates from a prostate cancer cell line and involuting mouse mammary glands. Interestingly, a membrane-type serine protease 1 was isolated from the Triton X-100-solubilized PC-3 cell lysates, and surprisingly, haptoglobin, a serine-protease homolog protein, was also identified in mammary gland lysates and in blood. Haptoglobin does not prevent ecotin inhibition of u-PA, but it may act as a carrier within blood when ecotin is used in vivo. Finally, this affinity purification matrix was also able to identify a thrombin-like enzyme from snake venom using an ecotin variant directed against thrombin. Overall, the ecotin variants acted as robust tools for the isolation and characterization of proteins with a trypsin fold. Thus, they may assist in the understanding of the role of these serine proteases and homologous proteins in different biological processes. PMID:19728173

  11. Mitochondrial Protein Interaction Mapping Identifies Regulators of Respiratory Chain Function.

    PubMed

    Floyd, Brendan J; Wilkerson, Emily M; Veling, Mike T; Minogue, Catie E; Xia, Chuanwu; Beebe, Emily T; Wrobel, Russell L; Cho, Holly; Kremer, Laura S; Alston, Charlotte L; Gromek, Katarzyna A; Dolan, Brendan K; Ulbrich, Arne; Stefely, Jonathan A; Bohl, Sarah L; Werner, Kelly M; Jochem, Adam; Westphall, Michael S; Rensvold, Jarred W; Taylor, Robert W; Prokisch, Holger; Kim, Jung-Ja P; Coon, Joshua J; Pagliarini, David J

    2016-08-18

    Mitochondria are essential for numerous cellular processes, yet hundreds of their proteins lack robust functional annotation. To reveal functions for these proteins (termed MXPs), we assessed condition-specific protein-protein interactions for 50 select MXPs using affinity enrichment mass spectrometry. Our data connect MXPs to diverse mitochondrial processes, including multiple aspects of respiratory chain function. Building upon these observations, we validated C17orf89 as a complex I (CI) assembly factor. Disruption of C17orf89 markedly reduced CI activity, and its depletion is found in an unresolved case of CI deficiency. We likewise discovered that LYRM5 interacts with and deflavinates the electron-transferring flavoprotein that shuttles electrons to coenzyme Q (CoQ). Finally, we identified a dynamic human CoQ biosynthetic complex involving multiple MXPs whose topology we map using purified components. Collectively, our data lend mechanistic insight into respiratory chain-related activities and prioritize hundreds of additional interactions for further exploration of mitochondrial protein function. PMID:27499296

  12. An in vivo platform for identifying inhibitors of protein aggregation

    PubMed Central

    Mahood, Rachel A.; Jackson, Matthew P.; Revill, Charlotte H.; Foster, Richard J.; Smith, D. Alastair; Ashcroft, Alison E.; Brockwell, David J.; Radford, Sheena E.

    2015-01-01

    Protein aggregation underlies an array of human diseases, yet only one small molecule therapeutic has been successfully developed to date. Here, we introduce an in vivo system, based on a β-lactamase tripartite fusion construct, capable of identifying aggregation-prone sequences in the periplasm of Escherichia coli and inhibitors that prevent their aberrant self-assembly. We demonstrate the power of the system using a range of proteins, from small unstructured peptides (islet amyloid polypeptide and amyloid β) to larger, folded immunoglobulin domains. Configured in a 48-well format, the split β-lactamase sensor readily differentiates between aggregation-prone and soluble sequences. Performing the assay in the presence of 109 compounds enabled a rank ordering of inhibition and revealed a new inhibitor of IAPP aggregation. This platform can be applied to both amyloidogenic and other aggregation-prone systems, independent of sequence or size, and can identify small molecules or other factors able to ameliorate or inhibit protein aggregation. PMID:26656088

  13. An in vivo platform for identifying inhibitors of protein aggregation.

    PubMed

    Saunders, Janet C; Young, Lydia M; Mahood, Rachel A; Jackson, Matthew P; Revill, Charlotte H; Foster, Richard J; Smith, D Alastair; Ashcroft, Alison E; Brockwell, David J; Radford, Sheena E

    2016-02-01

    Protein aggregation underlies an array of human diseases, yet only one small-molecule therapeutic targeting this process has been successfully developed to date. Here, we introduce an in vivo system, based on a β-lactamase tripartite fusion construct, that is capable of identifying aggregation-prone sequences in the periplasm of Escherichia coli and inhibitors that prevent their aberrant self-assembly. We demonstrate the power of the system using a range of proteins, from small unstructured peptides (islet amyloid polypeptide and amyloid β) to larger, folded immunoglobulin domains. Configured in a 48-well format, the split β-lactamase sensor readily differentiates between aggregation-prone and soluble sequences. Performing the assay in the presence of 109 compounds enabled a rank ordering of inhibition and revealed a new inhibitor of islet amyloid polypeptide aggregation. This platform can be applied to both amyloidogenic and other aggregation-prone systems, independent of sequence or size, and can identify small molecules or other factors able to ameliorate or inhibit protein aggregation. PMID:26656088

  14. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells.

    PubMed

    Roux, Kyle J; Kim, Dae In; Raida, Manfred; Burke, Brian

    2012-03-19

    We have developed a new technique for proximity-dependent labeling of proteins in eukaryotic cells. Named BioID for proximity-dependent biotin identification, this approach is based on fusion of a promiscuous Escherichia coli biotin protein ligase to a targeting protein. BioID features proximity-dependent biotinylation of proteins that are near-neighbors of the fusion protein. Biotinylated proteins may be isolated by affinity capture and identified by mass spectrometry. We apply BioID to lamin-A (LaA), a well-characterized intermediate filament protein that is a constituent of the nuclear lamina, an important structural element of the nuclear envelope (NE). We identify multiple proteins that associate with and/or are proximate to LaA in vivo. The most abundant of these include known interactors of LaA that are localized to the NE, as well as a new NE-associated protein named SLAP75. Our results suggest BioID is a useful and generally applicable method to screen for both interacting and neighboring proteins in their native cellular environment. PMID:22412018

  15. Eggshell Spottiness Reflects Maternally Transferred Antibodies in Blue Tits

    PubMed Central

    Holveck, Marie-Jeanne; Grégoire, Arnaud; Staszewski, Vincent; Guerreiro, Romain; Perret, Philippe; Boulinier, Thierry; Doutrelant, Claire

    2012-01-01

    Blue-green and brown-spotted eggshells in birds have been proposed as sexual signals of female physiological condition and egg quality, reflecting maternal investment in the egg. Testing this hypothesis requires linking eggshell coloration to egg content, which is lacking for brown protoporphyrin-based pigmentation. As protoporphyrins can induce oxidative stress, and a large amount in eggshells should indicate either high female and egg quality if it reflects the female's high oxidative tolerance, or conversely poor quality if it reflects female physiological stress. Different studies supported either predictions but are difficult to compare given the methodological differences in eggshell-spottiness measurements. Using the blue tit Cyanistes caeruleus as a model species, we aimed at disentangling both predictions in testing if brown-spotted eggshell could reflect the quality of maternal investment in antibodies and carotenoids in the egg, and at improving between-study comparisons in correlating several common measurements of eggshell coloration (spectral and digital measures, spotted surface, pigmentation indices). We found that these color variables were weakly correlated highlighting the need for comparable quantitative measurements between studies and for multivariate regressions incorporating several eggshell-color characteristics. When evaluating the potential signaling function of brown-spotted eggshells, we thus searched for the brown eggshell-color variables that best predicted the maternal transfer of antibodies and carotenoids to egg yolks. We also tested the effects of several parental traits and breeding parameters potentially affecting this transfer. While eggshell coloration did not relate to yolk carotenoids, the eggs with larger and less evenly-distributed spots had higher antibody concentrations, suggesting that both the quantity and distribution of brown pigments reflected the transfer of maternal immune compounds in egg yolks. As yolk antibody

  16. Eggshell spottiness reflects maternally transferred antibodies in blue tits.

    PubMed

    Holveck, Marie-Jeanne; Grégoire, Arnaud; Staszewski, Vincent; Guerreiro, Romain; Perret, Philippe; Boulinier, Thierry; Doutrelant, Claire

    2012-01-01

    Blue-green and brown-spotted eggshells in birds have been proposed as sexual signals of female physiological condition and egg quality, reflecting maternal investment in the egg. Testing this hypothesis requires linking eggshell coloration to egg content, which is lacking for brown protoporphyrin-based pigmentation. As protoporphyrins can induce oxidative stress, and a large amount in eggshells should indicate either high female and egg quality if it reflects the female's high oxidative tolerance, or conversely poor quality if it reflects female physiological stress. Different studies supported either predictions but are difficult to compare given the methodological differences in eggshell-spottiness measurements. Using the blue tit Cyanistes caeruleus as a model species, we aimed at disentangling both predictions in testing if brown-spotted eggshell could reflect the quality of maternal investment in antibodies and carotenoids in the egg, and at improving between-study comparisons in correlating several common measurements of eggshell coloration (spectral and digital measures, spotted surface, pigmentation indices). We found that these color variables were weakly correlated highlighting the need for comparable quantitative measurements between studies and for multivariate regressions incorporating several eggshell-color characteristics. When evaluating the potential signaling function of brown-spotted eggshells, we thus searched for the brown eggshell-color variables that best predicted the maternal transfer of antibodies and carotenoids to egg yolks. We also tested the effects of several parental traits and breeding parameters potentially affecting this transfer. While eggshell coloration did not relate to yolk carotenoids, the eggs with larger and less evenly-distributed spots had higher antibody concentrations, suggesting that both the quantity and distribution of brown pigments reflected the transfer of maternal immune compounds in egg yolks. As yolk antibody

  17. Blot-MS of Carbonylated Proteins: A Tool to Identify Oxidized Proteins.

    PubMed

    Ferreira, Rita; Domingues, Pedro; Amado, Francisco; Vitorino, Rui

    2016-01-01

    The efficiency of proteostasis regulation declines during aging and the failure of protein homeostasis is common in age-related diseases. Protein oxidation is a major contributor to the loss of proteome homeostasis, also called "proteostasis," precluding protein misfolding and aggregation. So, the identification of the molecular pathways impaired by protein oxidation will increase the understanding of proteostasis and the pathophysiological conditions related to the loss of proteostasis. Sample derivatization with dinitrophenyl hydrazine and western blot immunoassay detection of carbonylated proteins (commonly known as Oxyblot™) coupled to mass spectrometry (blot-MS) is an attractive methodological approach to identify proteins that are more prone to carbonylation, a typical oxidative modification of amino acid residues. The integration of blot-MS data of carbonylated proteins with bioinformatics tools allows the identification of the biological processes more affected by protein oxidation and that, eventually, result in the loss of proteostasis.In this chapter, we describe a blot-MS methodology to identify the proteins more prone to oxidation in biological samples, as cell and tissue extracts, and biofluids. Analysis of mitochondria isolated from cardiac tissue is provided as an example. Bioinformatic strategy to deal with data retrieved from blot-MS experiments are proposed for the identification of relevant biological processes modulated by oxidative stress stimuli. PMID:27613049

  18. Dataset of integrin-linked kinase protein: Protein interactions in cardiomyocytes identified by mass spectrometry.

    PubMed

    Traister, Alexandra; Lu, Mingliang; Coles, John G; Maynes, Jason T

    2016-06-01

    Using hearts from mice overexpressing integrin linked kinase (ILK) behind the cardiac specific promoter αMHC, we have performed immunoprecipitation and mass spectrometry to identify novel ILK protein:protein interactions that regulate cardiomyocyte activity and calcium flux. Integrin linked kinase complexes were captured from mouse heart lysates using a commercial antibody, with subsequent liquid chromatography tandem mass spectral analysis. Interacting partners were identified using the MASCOT server, and important interactions verified using reverse immunoprecipitation and mass spectrometry. All ILK interacting proteins were identified in a non-biased manner, and are stored in the ProteomeXchange Consortium via the PRIDE partner repository (reference ID PRIDE: PXD001053). The functional role of identified ILK interactions in cardiomyocyte function and arrhythmia were subsequently confirmed in human iPSC-cardiomyocytes. PMID:27408918

  19. Quantitative Tagless Copurification: A Method to Validate and Identify Protein-Protein Interactions.

    PubMed

    Shatsky, Maxim; Dong, Ming; Liu, Haichuan; Yang, Lee Lisheng; Choi, Megan; Singer, Mary E; Geller, Jil T; Fisher, Susan J; Hall, Steven C; Hazen, Terry C; Brenner, Steven E; Butland, Gareth; Jin, Jian; Witkowska, H Ewa; Chandonia, John-Marc; Biggin, Mark D

    2016-06-01

    Identifying protein-protein interactions (PPIs) at an acceptable false discovery rate (FDR) is challenging. Previously we identified several hundred PPIs from affinity purification - mass spectrometry (AP-MS) data for the bacteria Escherichia coli and Desulfovibrio vulgaris These two interactomes have lower FDRs than any of the nine interactomes proposed previously for bacteria and are more enriched in PPIs validated by other data than the nine earlier interactomes. To more thoroughly determine the accuracy of ours or other interactomes and to discover further PPIs de novo, here we present a quantitative tagless method that employs iTRAQ MS to measure the copurification of endogenous proteins through orthogonal chromatography steps. 5273 fractions from a four-step fractionation of a D. vulgaris protein extract were assayed, resulting in the detection of 1242 proteins. Protein partners from our D. vulgaris and E. coli AP-MS interactomes copurify as frequently as pairs belonging to three benchmark data sets of well-characterized PPIs. In contrast, the protein pairs from the nine other bacterial interactomes copurify two- to 20-fold less often. We also identify 200 high confidence D. vulgaris PPIs based on tagless copurification and colocalization in the genome. These PPIs are as strongly validated by other data as our AP-MS interactomes and overlap with our AP-MS interactome for D.vulgaris within 3% of expectation, once FDRs and false negative rates are taken into account. Finally, we reanalyzed data from two quantitative tagless screens of human cell extracts. We estimate that the novel PPIs reported in these studies have an FDR of at least 85% and find that less than 7% of the novel PPIs identified in each screen overlap. Our results establish that a quantitative tagless method can be used to validate and identify PPIs, but that such data must be analyzed carefully to minimize the FDR. PMID:27099342

  20. Structure-based protocol for identifying mutations that enhance protein-protein binding affinities.

    PubMed

    Sammond, Deanne W; Eletr, Ziad M; Purbeck, Carrie; Kimple, Randall J; Siderovski, David P; Kuhlman, Brian

    2007-08-31

    The ability to manipulate protein binding affinities is important for the development of proteins as biosensors, industrial reagents, and therapeutics. We have developed a structure-based method to rationally predict single mutations at protein-protein interfaces that enhance binding affinities. The protocol is based on the premise that increasing buried hydrophobic surface area and/or reducing buried hydrophilic surface area will generally lead to enhanced affinity if large steric clashes are not introduced and buried polar groups are not left without a hydrogen bond partner. The procedure selects affinity enhancing point mutations at the protein-protein interface using three criteria: (1) the mutation must be from a polar amino acid to a non-polar amino acid or from a non-polar amino acid to a larger non-polar amino acid, (2) the free energy of binding as calculated with the Rosetta protein modeling program should be more favorable than the free energy of binding calculated for the wild-type complex and (3) the mutation should not be predicted to significantly destabilize the monomers. The performance of the computational protocol was experimentally tested on two separate protein complexes; Galpha(i1) from the heterotrimeric G-protein system bound to the RGS14 GoLoco motif, and the E2, UbcH7, bound to the E3, E6AP from the ubiquitin pathway. Twelve single-site mutations that were predicted to be stabilizing were synthesized and characterized in the laboratory. Nine of the 12 mutations successfully increased binding affinity with five of these increasing binding by over 1.0 kcal/mol. To further assess our approach we searched the literature for point mutations that pass our criteria and have experimentally determined binding affinities. Of the eight mutations identified, five were accurately predicted to increase binding affinity, further validating the method as a useful tool to increase protein-protein binding affinities. PMID:17603074

  1. Identifying relevant positions in proteins by Critical Variable Selection.

    PubMed

    Grigolon, Silvia; Franz, Silvio; Marsili, Matteo

    2016-06-21

    Evolution in its course has found a variety of solutions to the same optimisation problem. The advent of high-throughput genomic sequencing has made available extensive data from which, in principle, one can infer the underlying structure on which biological functions rely. In this paper, we present a new method aimed at the extraction of sites encoding structural and functional properties from a set of protein primary sequences, namely a multiple sequence alignment. The method, called critical variable selection, is based on the idea that subsets of relevant sites correspond to subsequences that occur with a particularly broad frequency distribution in the dataset. By applying this algorithm to in silico sequences, to the response regulator receiver and to the voltage sensor domain of ion channels, we show that this procedure recovers not only the information encoded in single site statistics and pairwise correlations but also captures dependencies going beyond pairwise correlations. The method proposed here is complementary to statistical coupling analysis, in that the most relevant sites predicted by the two methods differ markedly. We find robust and consistent results for datasets as small as few hundred sequences that reveal a hidden hierarchy of sites that are consistent with the present knowledge on biologically relevant sites and evolutionary dynamics. This suggests that critical variable selection is capable of identifying a core of sites encoding functional and structural information in a multiple sequence alignment. PMID:26974515

  2. Automated Analysis of Fluorescence Microscopy Images to Identify Protein-Protein Interactions

    DOE PAGESBeta

    Venkatraman, S.; Doktycz, M. J.; Qi, H.; Morrell-Falvey, J. L.

    2006-01-01

    The identification of protein interactions is important for elucidating biological networks. One obstacle in comprehensive interaction studies is the analyses of large datasets, particularly those containing images. Development of an automated system to analyze an image-based protein interaction dataset is needed. Such an analysis system is described here, to automatically extract features from fluorescence microscopy images obtained from a bacterial protein interaction assay. These features are used to relay quantitative values that aid in the automated scoring of positive interactions. Experimental observations indicate that identifying at least 50% positive cells in an image is sufficient to detect a protein interaction.more » Based on this criterion, the automated system presents 100% accuracy in detecting positive interactions for a dataset of 16 images. Algorithms were implemented using MATLAB and the software developed is available on request from the authors.« less

  3. Antimicrobial Characteristics of Heated Eggshell Powder.

    PubMed

    Ohshima, Yuki; Takada, Daisuke; Namai, Satoe; Sawai, Jun; Kikuchi, Mikio; Hotta, Mikinori

    2015-01-01

    Eggshells have high bioavailability and can be used as a source of calcium. The main component is CaCO3, which, when heated, is converted to CaO. Seashells are also mainly composed of CaCO3 and were previously found to exhibit antimicrobial activity after being heated. In this study, heated eggshell powder (HESP) was found to have antimicrobial activity against bacterial vegetative cells, fungi and bacterial spores. Parameters, such as the minimum inhibitory concentration, were determined with kinetic analysis using an indirect conductimetric assay. Moreover, HESP was able to kill the Bacillus subtilis spores. There were no significant differences in the activity between HESP, heated scallop-shell powder and pure CaO. The MIC values for HESP against bacteria and fungi were 0.29-0.43 and 1.3-1.5 mg/mL, respectively. Against B. subtilis spores, a reduction of two orders of magnitude of viability was confirmed following 20 min of treatment at 10 mg/mL at 60 ℃. The active oxygen generated from the HESP slurry was examined with chemiluminescence. The intensity of this increased with increasing concentrations of the HESP slurry. This suggests that HESP could be used as a natural antimicrobial agent. Although a high pH is the main contributor to this antimicrobial activity, active oxygen species generated from HESP are likely to be the main antimicrobial agents.. PMID:26699855

  4. Identifying hierarchical and overlapping protein complexes based on essential protein-protein interactions and "seed-expanding" method.

    PubMed

    Ren, Jun; Zhou, Wei; Wang, Jianxin

    2014-01-01

    Many evidences have demonstrated that protein complexes are overlapping and hierarchically organized in PPI networks. Meanwhile, the large size of PPI network wants complex detection methods have low time complexity. Up to now, few methods can identify overlapping and hierarchical protein complexes in a PPI network quickly. In this paper, a novel method, called MCSE, is proposed based on λ-module and "seed-expanding." First, it chooses seeds as essential PPIs or edges with high edge clustering values. Then, it identifies protein complexes by expanding each seed to a λ-module. MCSE is suitable for large PPI networks because of its low time complexity. MCSE can identify overlapping protein complexes naturally because a protein can be visited by different seeds. MCSE uses the parameter λ_th to control the range of seed expanding and can detect a hierarchical organization of protein complexes by tuning the value of λ_th. Experimental results of S. cerevisiae show that this hierarchical organization is similar to that of known complexes in MIPS database. The experimental results also show that MCSE outperforms other previous competing algorithms, such as CPM, CMC, Core-Attachment, Dpclus, HC-PIN, MCL, and NFC, in terms of the functional enrichment and matching with known protein complexes. PMID:25143945

  5. Identifying Hierarchical and Overlapping Protein Complexes Based on Essential Protein-Protein Interactions and “Seed-Expanding” Method

    PubMed Central

    Ren, Jun; Zhou, Wei; Wang, Jianxin

    2014-01-01

    Many evidences have demonstrated that protein complexes are overlapping and hierarchically organized in PPI networks. Meanwhile, the large size of PPI network wants complex detection methods have low time complexity. Up to now, few methods can identify overlapping and hierarchical protein complexes in a PPI network quickly. In this paper, a novel method, called MCSE, is proposed based on λ-module and “seed-expanding.” First, it chooses seeds as essential PPIs or edges with high edge clustering values. Then, it identifies protein complexes by expanding each seed to a λ-module. MCSE is suitable for large PPI networks because of its low time complexity. MCSE can identify overlapping protein complexes naturally because a protein can be visited by different seeds. MCSE uses the parameter λ_th to control the range of seed expanding and can detect a hierarchical organization of protein complexes by tuning the value of λ_th. Experimental results of S. cerevisiae show that this hierarchical organization is similar to that of known complexes in MIPS database. The experimental results also show that MCSE outperforms other previous competing algorithms, such as CPM, CMC, Core-Attachment, Dpclus, HC-PIN, MCL, and NFC, in terms of the functional enrichment and matching with known protein complexes. PMID:25143945

  6. Dynamics of bacterial and fungal communities associated with eggshells during incubation

    PubMed Central

    Grizard, Stéphanie; Dini-Andreote, Francisco; Tieleman, B Irene; Salles, Joana F

    2014-01-01

    Microorganisms are closely associated with eggs and may play a determinant role in embryo survival. Yet, the majority of studies focusing on this association relied on culture-based methodology, eventually leading to a skewed assessment of microbial communities. By targeting the 16S rRNA gene and internal transcribed spacer (ITS) region, we, respectively, described bacterial and fungal communities on eggshells of the homing pigeon Columba livia. We explored their structure, abundance, and composition. Firstly, we showed that sampling technique affected the outcome of the results. While broadly used, the egg swabbing procedure led to a lower DNA extraction efficiency and provided different profiles of bacterial communities than those based on crushed eggshell pieces. Secondly, we observed shifts in bacterial and fungal communities during incubation. At late incubation, bacterial communities showed a reduction in diversity, while their abundance increased, possibly due to the competitive advantage of some species. When compared to their bacterial counterparts, fungal communities also decreased in diversity at late incubation. In that case, however, the decline was associated with a diminution of their overall abundance. Conclusively, our results showed that although incubation might inhibit microbial growth when compared to unincubated eggs, we observed the selective growth of specific bacterial species during incubation. Moreover, we showed that fungi are a substantial component of the microbial communities associated with eggshells and require further investigations in avian ecology. Identifying the functional roles of these microorganisms is likely to provide news insights into the evolutionary strategies that control embryo survival. We aimed to describe the dynamics of bacterial and fungal communities on homing pigeon eggshell surfaces. We investigated these communities at early and late incubation stages. PMID:24772289

  7. A simple and efficient method for extraction of PCR-amplifiable DNA from chicken eggshells.

    PubMed

    Rikimaru, Kazuhiro; Takahashi, Hideaki

    2009-04-01

    Recently, we reported a method for discriminating a Japanese brand of chicken, the Hinai-jidori. As an application of this method for discriminating Hinai-jidori eggs, we here report an efficient method for extracting maternal DNA from eggshells. Eggshell powder was completely decalcified with EDTA solution, and then DNA was isolated by conventional phenol-chloroform extraction and ethanol precipitation. The efficiency of DNA recovery from eggshells was 50-fold higher than that of a previously reported method. The recovered DNA could be used for PCR, and 10 markers for identifying the Hinai-jidori chicken were detected. The genotypes of the Hinai-jidori exactly matched those of the Hinai-dori breed. Using this method, Hinai-jidori and Hinai-dori eggs could be distinguished from the eggs of Rhode Island Reds. This is the first report of a technique that can be used to discriminate the eggs of Hinai-jidori from those of other chickens, and it can also be utilized to validate the labeling of Hinai-jidori eggs in the market. PMID:20163594

  8. Nanosized hydroxyapatite powder synthesized from eggshell and phosphoric acid.

    PubMed

    Lee, Sang-Jin; Yoon, Young-Soo; Lee, Myung-Hyun; Oh, Nam-Sik

    2007-11-01

    The present research describes synthesis of highly sinterable, nano-sized hydroxyapatite (HAp) powders using a wet chemical route with recycled eggshell and phosphoric acid as calcium and phosphorous sources. The raw eggshell was easily turned to CaO by the calcining process, and phosphoric acid was mixed with the calcined eggshell by the wet, ball-milling method. The crystalline development and microstructures of the synthesized powders and sintered samples were examined by X-ray diffractometry and scanning electron microscopy, respectively. The observed phases on the powder synthesis process were dependent on the mixing ratio (wt%) of the calcined eggshell to phosphoric acid and the heating temperature. The ball-milled, nano-sized HAp powder, which has an average particle size of 70 nm, was fully densified at 1300 degrees C for 1h. The Ca/P ratio for stoichiometric composition of HAp was controlled by adjustment of the mixing ratio. PMID:18047119

  9. Respiratory Pores on Ostrich Struthio camelus (Aves: Struthionidae) Eggshells.

    PubMed

    Koyama, T; Tennyson, A J D

    2016-01-01

    Respiratory pores are essential for the survival of the embryo within the eggshell. Distribution patterns of such pores on ostrich (Struthio camelus) eggshells show remarkable variations in bird group. Eggshells preserved in the museum of New Zealand have long, superficial, winding grooves and ridges, with pores distributed densely in the bottom of grooves. Both the grooves and ridges that separate them are twisted. By contrast, the surfaces of eggs from farmed ostriches are mostly smooth, with only occasional, short grooves, and respiratory pores distributed more evenly. The cause of ridging and grooving of the surface of eggs from wild birds is unclear but may be due to the need for stronger shells and effects of environmental stresses. It appears that the arrangement of respiratory pores on ostrich eggshells seems to be changeable by surrounding stresses. PMID:27526124

  10. 15. April 1963 SPIRAL STAIRS AND EGGSHELL DORMER Shaker ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    15. April 1963 SPIRAL STAIRS AND EGG-SHELL DORMER - Shaker Centre Family Trustees' Office, South side of Village Road, North of U.S. Route 68 & State Route 33 intersection, Shakertown, Mercer County, KY

  11. 40 CFR 174.529 - Bacillus thuringiensis modified Cry1Ab protein as identified under OECD Unique Identifier SYN...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus thuringiensis modified Cry1Ab protein as identified under OECD Unique Identifier SYN-IR67B-1 in cotton; exemption from the requirement of a tolerance. 174.529 Section 174.529 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS PROCEDURES...

  12. Avian eggshell thickness: Variability and sampling

    USGS Publications Warehouse

    Klaas, E.E.; Ohlendorf, H.M.; Heath, R.G.

    1974-01-01

    Measurements of shell thickness of the eggs of five species were subjected to nested analyses of variance. The analyses separated variation into two or three levels for which variances and percentages of the total variation were derived. The results show that differences among measurements of the same egg contribute little to the sample variance whereas differences among eggs within clutches contribute nearly as much as differences among clutches. It is more efficient and less costly to collect entire clutches of eggs in most studies of shell thickness. Using entire clutches, sample sizes needed to detect differences of 10 percent in shell thickness (at given significance levels and power) were estimated to be eight to 11 clutches for the species studied. For differences of five percent, 26 to 38 clutches are required. Guidelines are presented which may assist other workers in evaluating the efficiency of their sampling designs, and in estimating sample sizes for detecting differences in eggshell thickness in wild birds.

  13. Eggshell defects detection based on color processing

    NASA Astrophysics Data System (ADS)

    Garcia-Alegre, Maria C.; Ribeiro, Angela; Guinea, Domingo; Cristobal, Gabriel

    2000-03-01

    The automatic classification of defective eggs constitutes a fundamental issue at the poultry industry for both economical and sanitary reasons. The early separation of eggs with spots and cracks is a relevant task as the stains can leak while progressing on the conveyor-belts, degrading all the mechanical parts. Present work is focused on the implementation of an artificial vision system for detecting in real time defective eggs at the poultry farm. First step of the algorithmic process is devoted to the detection of the egg shape to fix the region of interest. A color processing is then performed only on the eggshell to obtain an image segmentation that allows the discrimination of defective eggs from clean ones in critic time. The results are presented to demonstrate the validity of the proposed visual process on a wide sample of both defective and non-defective eggs.

  14. Application of waste eggshell as low-cost solid catalyst for biodiesel production.

    PubMed

    Wei, Ziku; Xu, Chunli; Li, Baoxin

    2009-06-01

    Waste eggshell was investigated in triglyceride transesterification with a view to determine its viability as a solid catalyst for use in biodiesel synthesis. Effect of calcination temperature on structure and activity of eggshell catalysts was investigated. Reusability of eggshell catalysts was also examined. It was found that high active, reusable solid catalyst was obtained by just calcining eggshell. Utilization of eggshell as a catalyst for biodiesel production not only provides a cost-effective and environmental friendly way of recycling this solid eggshell waste, significantly reducing its environmental effects, but also reduces the price of biodiesel to make biodiesel competitive with petroleum diesel. PMID:19201602

  15. Eggshell calcium in the prevention and treatment of osteoporosis.

    PubMed

    Rovenský, J; Stancíková, M; Masaryk, P; Svík, K; Istok, R

    2003-01-01

    In this paper the most significant biological and clinical aspects of a biopreparation made of chicken eggshells are reviewed. Eggshell powder is a natural source of calcium and other elements (e.g. strontium and fluorine) which may have a positive effect on bone metabolism. Experimental and clinical studies performed to date have shown a number of positive properties of eggshell powder, such as antirachitic effects in rats and humans. A positive effect was observed on bone density in animal models of postmenopausal osteoporosis in ovariectomized female rats. In vitro eggshell powder stimulates chondrocyte differentiation and cartilage growth. Clinical studies in postmenopausal women and women with senile osteoporosis showed that eggshell powder reduces pain and osteoresorption and increases mobility and bone density or arrests its loss. The bioavailability of calcium from this source, as tested in piglets, was similar or better than that of food grade purified calcium carbonate. Clinical and experimental studies showed that eggshell powder has positive effects on bone and cartilage and that it is suitable in the prevention and treatment of osteoporosis. PMID:15018022

  16. Eggshell thickness and DDE residue levels in vlulture eggs

    USGS Publications Warehouse

    Kiff, L.F.; Peakall, D.B.; Morrison, M.L.; Wilbur, S.R.

    1983-01-01

    Post-DDT (post-1947) eggshell thickness was examined in samples of Turkey Vulture, Black Vulture, and Crested Caracara eggs from several parts of the United States. Highly significant post-DDT decreases in eggshell thickness indices of at least 10 percent were found in Turkey Vulture eggs from California, Florida, and Texas and in Black Vulture eggs from Texas and Florida. Over one-third of the Black VUlture eggs and about 30 percent of the Turkey Vulture eggs from Texas showed thinning exceeding 20 percent, a level associated with reproductive failure and population decline in other species. A strong negative correlation was found between eggshell thickness indices and DDE residues extracted from eggshell membranes in California and Texas samples of Turkey Vulture eggs and in Texas Black Vulture eggs. Crested Caracara eggs from Texas and Florida showed mean changes in eggshell thickness indices of only -5.6 and -8.2 percent, respectively, although thinning in a few eggs from both states exceeded 20 percent. Most of the post-DDT Old World vulture eggs examined appeared to be of normal thickness, with low DDE residue levels in eggshell membranes; but single eggs of Egyptian Vulture from India, Cinereous Vulture from Spain, and White-headed Vulture from Zambia showed apparent thinning. Further monitoring of vulture populations in tropical regions, where DDT use is still increasing, is recommended.

  17. Playing biology's name game: identifying protein names in scientific text.

    PubMed

    Hanisch, Daniel; Fluck, Juliane; Mevissen, Heinz-Theodor; Zimmer, Ralf

    2003-01-01

    A growing body of work is devoted to the extraction of protein or gene interaction information from the scientific literature. Yet, the basis for most extraction algorithms, i.e. the specific and sensitive recognition of protein and gene names and their numerous synonyms, has not been adequately addressed. Here we describe the construction of a comprehensive general purpose name dictionary and an accompanying automatic curation procedure based on a simple token model of protein names. We designed an efficient search algorithm to analyze all abstracts in MEDLINE in a reasonable amount of time on standard computers. The parameters of our method are optimized using machine learning techniques. Used in conjunction, these ingredients lead to good search performance. A supplementary web page is available at http://cartan.gmd.de/ProMiner/. PMID:12603045

  18. Identifying Protein Stabilizing Ligands Using GroEL

    PubMed Central

    Naik, Subhashchandra; Haque, Inamul; Degner, Nick; Kornilayev, Boris; Bomhoff, Gregory; Hodges, Jacob; Khorassani, Ara-Azad; Katayama, Hiroo; Morris, Jill; Kelly, Jeffery; Seed, John; Fisher, Mark T.

    2010-01-01

    Over the past five years, it has become increasingly apparent to researchers that the initial promise and excitement of using gene replacement therapies to ameliorate folding diseases are still far from being broadly or easily applicable. Because a large number of human diseases are protein folding diseases (~30 to 50%), many researchers now realize that more directed approaches to target and reverse the fundamental misfolding reactions preceding disease are highly feasible and offer the potential of developing more targeted drug therapies. This is also true with a large number of so called “orphan protein folding diseases”. The development of a broad-based general screening array method using the chaperonin as a detection platform will enable us to screen large chemical combinatorial libraries for specific ligands against the elusive transient, primary reactions that often lead to protein misfolding. This development will provide a highly desirable tool for the pharmaceutical, academic and medical professions. PMID:19802819

  19. Transcriptome analyses of inhibitor-treated schistosome females provide evidence for cooperating Src-kinase and TGFβ receptor pathways controlling mitosis and eggshell formation.

    PubMed

    Buro, Christin; Oliveira, Katia C; Lu, Zhigang; Leutner, Silke; Beckmann, Svenja; Dissous, Colette; Cailliau, Katia; Verjovski-Almeida, Sergio; Grevelding, Christoph G

    2013-01-01

    Schistosome parasites cause schistosomiasis, one of the most prevalent parasitemias worldwide affecting humans and animals. Constant pairing of schistosomes is essential for female sexual maturation and egg production, which causes pathogenesis. Female maturation involves signaling pathways controlling mitosis and differentiation within the gonads. In vitro studies had shown before that a Src-specific inhibitor, Herbimycin A (Herb A), and a TGFβ receptor (TβR) inhibitor (TRIKI) have physiological effects such as suppressed mitoses and egg production in paired females. As one Herb A target, the gonad-specifically expressed Src kinase SmTK3 was identified. Here, we comparatively analyzed the transcriptome profiles of Herb A- and TRIKI-treated females identifying transcriptional targets of Src-kinase and TβRI pathways. After demonstrating that TRIKI inhibits the schistosome TGFβreceptor SmTβRI by kinase assays in Xenopus oocytes, couples were treated with Herb A, TRIKI, or both inhibitors simultaneously in vitro. RNA was isolated from females for microarray hybridizations and transcription analyses. The obtained data were evaluated by Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA), but also by manual classification and intersection analyses. Finally, extensive qPCR experiments were done to verify differential transcription of candidate genes under inhibitor influence but also to functionally reinforce specific physiological effects. A number of genes found to be differentially regulated are associated with mitosis and differentiation. Among these were calcium-associated genes and eggshell-forming genes. In situ hybridization confirmed transcription of genes coding for the calcium sensor hippocalcin, the calcium transporter ORAI-1, and the calcium-binding protein calmodulin-4 in the reproductive system pointing to a role of calcium in parasite reproduction. Functional qPCR results confirmed an inhibitor-influenced, varying dependence of the

  20. Identifying protein interactions with metal-modified DNA using microarray technology.

    PubMed

    Stansfield, Hope E; Kulczewski, Bethany P; Lybrand, Kyle E; Jamieson, Elizabeth R

    2009-02-01

    Protein microarrays have been used extensively to identify protein-protein interactions; however, this technology has not been widely applied to protein-DNA interactions. In particular, this work demonstrates the utility of this technique for rapidly identifying interactions of proteins with metal-modified DNA. Protein macroarray experiments were carried out with high mobility group protein 1 (HMG-1) and cisplatin- and chromium-modified 50-mer oligonucleotides to demonstrate "proof of principle." Commercially available protein microarrays containing many different classes of human proteins were then employed to search for additional interactions with cisplatin-modified DNA. The results of the microarray experiments confirmed some known interactions and, more importantly, identified many novel protein interactions, demonstrating the utility of this method as a rapid, high-throughput technique to discover proteins that interact with metal-modified DNA. PMID:18936984

  1. Identifying Novel Candidate Genes Related to Apoptosis from a Protein-Protein Interaction Network

    PubMed Central

    Wang, Baoman; Yuan, Fei; Kong, Xiangyin; Hu, Lan-Dian; Cai, Yu-Dong

    2015-01-01

    Apoptosis is the process of programmed cell death (PCD) that occurs in multicellular organisms. This process of normal cell death is required to maintain the balance of homeostasis. In addition, some diseases, such as obesity, cancer, and neurodegenerative diseases, can be cured through apoptosis, which produces few side effects. An effective comprehension of the mechanisms underlying apoptosis will be helpful to prevent and treat some diseases. The identification of genes related to apoptosis is essential to uncover its underlying mechanisms. In this study, a computational method was proposed to identify novel candidate genes related to apoptosis. First, protein-protein interaction information was used to construct a weighted graph. Second, a shortest path algorithm was applied to the graph to search for new candidate genes. Finally, the obtained genes were filtered by a permutation test. As a result, 26 genes were obtained, and we discuss their likelihood of being novel apoptosis-related genes by collecting evidence from published literature. PMID:26543496

  2. Paramagnetic Ligand Tagging To Identify Protein Binding Sites

    PubMed Central

    2015-01-01

    Transient biomolecular interactions are the cornerstones of the cellular machinery. The identification of the binding sites for low affinity molecular encounters is essential for the development of high affinity pharmaceuticals from weakly binding leads but is hindered by the lack of robust methodologies for characterization of weakly binding complexes. We introduce a paramagnetic ligand tagging approach that enables localization of low affinity protein–ligand binding clefts by detection and analysis of intermolecular protein NMR pseudocontact shifts, which are invoked by the covalent attachment of a paramagnetic lanthanoid chelating tag to the ligand of interest. The methodology is corroborated by identification of the low millimolar volatile anesthetic interaction site of the calcium sensor protein calmodulin. It presents an efficient route to binding site localization for low affinity complexes and is applicable to rapid screening of protein–ligand systems with varying binding affinity. PMID:26289584

  3. Protein networks identify novel symbiogenetic genes resulting from plastid endosymbiosis.

    PubMed

    Méheust, Raphaël; Zelzion, Ehud; Bhattacharya, Debashish; Lopez, Philippe; Bapteste, Eric

    2016-03-29

    The integration of foreign genetic information is central to the evolution of eukaryotes, as has been demonstrated for the origin of the Calvin cycle and of the heme and carotenoid biosynthesis pathways in algae and plants. For photosynthetic lineages, this coordination involved three genomes of divergent phylogenetic origins (the nucleus, plastid, and mitochondrion). Major hurdles overcome by the ancestor of these lineages were harnessing the oxygen-evolving organelle, optimizing the use of light, and stabilizing the partnership between the plastid endosymbiont and host through retargeting of proteins to the nascent organelle. Here we used protein similarity networks that can disentangle reticulate gene histories to explore how these significant challenges were met. We discovered a previously hidden component of algal and plant nuclear genomes that originated from the plastid endosymbiont: symbiogenetic genes (S genes). These composite proteins, exclusive to photosynthetic eukaryotes, encode a cyanobacterium-derived domain fused to one of cyanobacterial or another prokaryotic origin and have emerged multiple, independent times during evolution. Transcriptome data demonstrate the existence and expression of S genes across a wide swath of algae and plants, and functional data indicate their involvement in tolerance to oxidative stress, phototropism, and adaptation to nitrogen limitation. Our research demonstrates the "recycling" of genetic information by photosynthetic eukaryotes to generate novel composite genes, many of which function in plastid maintenance. PMID:26976593

  4. Protein networks identify novel symbiogenetic genes resulting from plastid endosymbiosis

    PubMed Central

    Méheust, Raphaël; Zelzion, Ehud; Bhattacharya, Debashish; Lopez, Philippe; Bapteste, Eric

    2016-01-01

    The integration of foreign genetic information is central to the evolution of eukaryotes, as has been demonstrated for the origin of the Calvin cycle and of the heme and carotenoid biosynthesis pathways in algae and plants. For photosynthetic lineages, this coordination involved three genomes of divergent phylogenetic origins (the nucleus, plastid, and mitochondrion). Major hurdles overcome by the ancestor of these lineages were harnessing the oxygen-evolving organelle, optimizing the use of light, and stabilizing the partnership between the plastid endosymbiont and host through retargeting of proteins to the nascent organelle. Here we used protein similarity networks that can disentangle reticulate gene histories to explore how these significant challenges were met. We discovered a previously hidden component of algal and plant nuclear genomes that originated from the plastid endosymbiont: symbiogenetic genes (S genes). These composite proteins, exclusive to photosynthetic eukaryotes, encode a cyanobacterium-derived domain fused to one of cyanobacterial or another prokaryotic origin and have emerged multiple, independent times during evolution. Transcriptome data demonstrate the existence and expression of S genes across a wide swath of algae and plants, and functional data indicate their involvement in tolerance to oxidative stress, phototropism, and adaptation to nitrogen limitation. Our research demonstrates the “recycling” of genetic information by photosynthetic eukaryotes to generate novel composite genes, many of which function in plastid maintenance. PMID:26976593

  5. Safety evaluation of a natural eggshell membrane-derived product.

    PubMed

    Ruff, Kevin J; Endres, John R; Clewell, Amy E; Szabo, James R; Schauss, Alexander G

    2012-03-01

    Natural Eggshell Membrane (NEM®) is a novel dietary ingredient that contains naturally occurring glycosaminoglycans and proteins essential for maintaining healthy joint and connective tissues. NEM® was evaluated for safety via in vitro and in vivo toxicological studies. This included testing for cytotoxicity, genotoxicity, acute oral toxicity, and 90-day repeated-dose oral toxicity. NEM® did not exhibit any cytotoxic effects at a dose of 100 μg in an in vitro human cell viability assay after incubation for up to 20 h. NEM® did not exhibit any genotoxic effects in an in vitro assay of four strains of histidine-dependent Salmonella typhimurium and one strain of tryptophan-dependent Escherichia coli at a dose of up to 5000 μg/plate. NEM® did not exhibit any signs of acute toxicity in rats at a single oral dose of up to 2000 mg/kg body weight, nor signs of toxicity (via urinalysis, hematology, clinical chemistry, or histopathological evaluation) in rats at a repeated oral dose of up to 2000 mg/kg body weight per day for 90 days. The results of these studies suggest that NEM® may be safe for human consumption. PMID:22245377

  6. Identifying and tracking proteins through the marine water column: insights into the inputs and preservation mechanisms of protein in sediments

    PubMed Central

    Moore, Eli K.; Nunn, Brook L.; Goodlett, David R.; Harvey, H. Rodger

    2012-01-01

    Proteins generated during primary production represent an important fraction of marine organic nitrogen and carbon, and have the potential to provide organism-specific information in the environment. The Bering Sea is a highly productive system dominated by seasonal blooms and was used as a model system for algal proteins to be tracked through the water column and incorporated into detrital sedimentary material. Samples of suspended and sinking particles were collected at multiple depths along with surface sediments on the continental shelf and deeper basin of the Bering Sea. Modified standard proteomic preparations were used in conjunction with high pressure liquid chromatography-tandem mass spectrometry to identify the suite of proteins present and monitor changes in their distribution. In surface waters 207 proteins were identified, decreasing through the water column to 52 proteins identified in post-bloom shelf surface sediments and 24 proteins in deeper (3490 m) basin sediments. The vast majority of identified proteins in all samples were diatom in origin, reflecting their dominant contribution of biomass during the spring bloom. Identified proteins were predominantly from metabolic, binding/structural, and transport-related protein groups. Significant linear correlations were observed between the number of proteins identified and the concentration of total hydrolysable amino acids normalized to carbon and nitrogen. Organelle-bound, transmembrane, photosynthetic, and other proteins involved in light harvesting were preferentially retained during recycling. These findings suggest that organelle and membrane protection represent important mechanisms that enhance the preservation of protein during transport and incorporation into sediments. PMID:22711915

  7. Identifying and tracking proteins through the marine water column: Insights into the inputs and preservation mechanisms of protein in sediments

    NASA Astrophysics Data System (ADS)

    Moore, Eli K.; Nunn, Brook L.; Goodlett, David R.; Harvey, H. Rodger

    2012-04-01

    Proteins generated during primary production represent an important fraction of marine organic nitrogen and carbon, and have the potential to provide organism-specific information in the environment. The Bering Sea is a highly productive system dominated by seasonal blooms and was used as a model system for algal proteins to be tracked through the water column and incorporated into detrital sedimentary material. Samples of suspended and sinking particles were collected at multiple depths along with surface sediments on the continental shelf and deeper basin of the Bering Sea. Modified standard proteomic preparations were used in conjunction with high pressure liquid chromatography-tandem mass spectrometry to identify the suite of proteins present and monitor changes in their distribution. In surface waters 207 proteins were identified, decreasing through the water column to 52 proteins identified in post-bloom shelf surface sediments and 24 proteins in deeper (3490 m) basin sediments. The vast majority of identified proteins in all samples were diatom in origin, reflecting their dominant contribution of biomass during the spring bloom. Identified proteins were predominantly from metabolic, binding/structural, and transport-related protein groups. Significant linear correlations were observed between the number of proteins identified and the concentration of total hydrolysable amino acids normalized to carbon and nitrogen. Organelle-bound, transmembrane, photosynthetic, and other proteins involved in light harvesting were preferentially retained during recycling. These findings suggest that organelle and membrane protection represent important mechanisms that enhance the preservation of protein during transport and incorporation into sediments.

  8. New method for effectively and quantitatively labeling cysteine residues on chicken eggshell membrane.

    PubMed

    Wang, Xiaojing; Li, Qian; Yuan, Yue; Mei, Bin; Huang, Rui; Tian, Ying; Sun, Jing; Cao, Chunyan; Lu, Guangming; Liang, Gaolin

    2012-10-28

    Using maleimidoethylmonoamide cysteine (Fmoc)(StBu) (1) as a medium, cysteine residues on proteins of chicken eggshell membrane (ESM) were successfully converted into N-terminal cysteines. After a biocompatible condensation reaction between the N-terminal cysteine and fluorescent probe 2-cyanobenzothiazole-Gly-Gly-Gly-fluorescein isothiocyanate (2), a new fluorogenic structure luciferin-Gly-Gly-Gly-FITC (3) was obtained, which exhibits a 2-fold fluorescence emission increase compared to that of 2. Thus, a new method for effectively labeling cysteine residues on ESMs was developed. Enhanced fluorescence images of ESMs were directly observed under a microscope and a small animal imaging machine. PMID:22961406

  9. Eggshell color in brown-egg laying hens - a review.

    PubMed

    Samiullah, S; Roberts, J R; Chousalkar, K

    2015-10-01

    The major pigment in eggshells of brown-egg laying hens is protoporphyrin IX, but traces of biliverdin and its zinc chelates are also present. The pigment appears to be synthesized in the shell gland. The protoporphyrin IX synthetic pathway is well defined, but precisely where and how it is synthesized in the shell gland of the brown-egg laying hen is still ambiguous. The pigment is deposited onto all shell layers including the shell membranes, but most of it is concentrated in the outermost layer of the calcareous shell and in the cuticle. Recently, the genes that are involved in pigment synthesis have been identified, but the genetic control of synthesis and deposition of brown pigment in the commercial laying hen is not fully understood. The brown coloration of the shell is an important shell quality parameter and has a positive influence on consumer preference. The extent of pigment deposition is influenced by the housing system, hen age, hen strain, diet, stressors, and certain diseases such as infectious bronchitis. In this article, the physiological and biochemical characteristics of the brown pigment in commercial brown-egg layers are reviewed in relation to its various functions in the poultry industry. PMID:26240390

  10. U-Th Burial Dates on Ostrich Eggshell

    NASA Astrophysics Data System (ADS)

    Sharp, W. D.; Fylstra, N. D.; Tryon, C. A.; Faith, J. T.; Peppe, D. J.

    2015-12-01

    Obtaining precise and accurate dates at archaeological sites beyond the range of radiocarbon dating is challenging but essential for understanding human origins. Eggshells of ratites (large flightless birds including ostrich, emu and others) are common in many archaeological sequences in Africa, Australia and elsewhere. Ancient eggshells are geochemically suitable for the U-Th technique (1), which has about ten times the range of radiocarbon dating (>500 rather than 50 ka), making eggshells attractive dating targets. Moreover, C and N isotopic studies of eggshell provide insights into paleovegetation and paleoprecipitation central to assessing past human-environment interactions (2,3). But until now, U-Th dates on ratite eggshell have not accounted for the secondary origin of essentially all of their U. We report a novel approach to U-Th dating of eggshell that explicitly accounts for secondary U uptake that begins with burial. Using ostrich eggshell (OES) from Pleistocene-Holocene east African sites, we have measured U and 232Th concentration profiles across OES by laser ablation ICP-MS. U commonly peaks at 10s to 100s of ppb and varies 10-fold or more across the ~2 mm thickness of OES, with gradients modulated by the layered structure of the eggshell. Common Th is high near the shell surfaces, but low in the middle "pallisade" layer of OES, making it optimal for U-Th dating. We determine U-Th ages along the U concentration gradient by solution ICP-MS analyses of two or more fractions of the pallisade layer. We then estimate OES burial dates using a simple model for diffusive uptake of uranium. Comparing such "U-Th burial dates" with radiocarbon dates for OES calcite from the same shells, we find good agreement in 7 out of 9 cases, consistent with rapid burial and confirming the accuracy of the approach. The remaining 2 eggshells have anomalous patterns of apparent ages that reveal they are unsuitable for U-Th dating, thereby providing reliability criteria innate

  11. Experimental strategies for cloning or identifying genes encoding DNA-binding proteins.

    PubMed

    Carey, Michael F; Peterson, Craig L; Smale, Stephen T

    2012-02-01

    This article describes experimental strategies for cloning or identifying genes encoding DNA-binding proteins. DNA-binding proteins are most commonly identified by electrophoretic mobility-shift assay (EMSA) or DNase I footprinting. To identify the gene encoding a protein detected by EMSA or DNase footprinting, the protein often needs to be purified and its sequence analyzed, as described here. Other methods are also available which do not resort to protein purification, including the one-hybrid screen, in vitro expression library screen, and mammalian expression cloning. These methods are outlined, and their advantages and disadvantages are discussed. PMID:22301659

  12. Identifying Interactions that Determine Fragment Binding at Protein Hotspots.

    PubMed

    Radoux, Chris J; Olsson, Tjelvar S G; Pitt, Will R; Groom, Colin R; Blundell, Tom L

    2016-05-12

    Locating a ligand-binding site is an important first step in structure-guided drug discovery, but current methods do little to suggest which interactions within a pocket are the most important for binding. Here we illustrate a method that samples atomic hotspots with simple molecular probes to produce fragment hotspot maps. These maps specifically highlight fragment-binding sites and their corresponding pharmacophores. For ligand-bound structures, they provide an intuitive visual guide within the binding site, directing medicinal chemists where to grow the molecule and alerting them to suboptimal interactions within the original hit. The fragment hotspot map calculation is validated using experimental binding positions of 21 fragments and subsequent lead molecules. The ligands are found in high scoring areas of the fragment hotspot maps, with fragment atoms having a median percentage rank of 97%. Protein kinase B and pantothenate synthetase are examined in detail. In each case, the fragment hotspot maps are able to rationalize a Free-Wilson analysis of SAR data from a fragment-based drug design project. PMID:27043011

  13. Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual

    DOEpatents

    Thompson, Vicki S; Lacey, Jeffrey A; Gentillon, Cynthia A; Apel, William A

    2015-03-03

    A method for determining a plurality of proteins for discriminating and positively identifying an individual based from a biological sample. The method may include profiling a biological sample from a plurality of individuals against a protein array including a plurality of proteins. The protein array may include proteins attached to a support in a preselected pattern such that locations of the proteins are known. The biological sample may be contacted with the protein array such that a portion of antibodies in the biological sample reacts with and binds to the proteins forming immune complexes. A statistical analysis method, such as discriminant analysis, may be performed to determine discriminating proteins for distinguishing individuals. Proteins of interest may be used to form a protein array. Such a protein array may be used, for example, to compare a forensic sample from an unknown source with a sample from a known source.

  14. Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual

    DOEpatents

    Apel, William A.; Thompson, Vicki S; Lacey, Jeffrey A.; Gentillon, Cynthia A.

    2016-08-09

    A method for determining a plurality of proteins for discriminating and positively identifying an individual based from a biological sample. The method may include profiling a biological sample from a plurality of individuals against a protein array including a plurality of proteins. The protein array may include proteins attached to a support in a preselected pattern such that locations of the proteins are known. The biological sample may be contacted with the protein array such that a portion of antibodies in the biological sample reacts with and binds to the proteins forming immune complexes. A statistical analysis method, such as discriminant analysis, may be performed to determine discriminating proteins for distinguishing individuals. Proteins of interest may be used to form a protein array. Such a protein array may be used, for example, to compare a forensic sample from an unknown source with a sample from a known source.

  15. Identifying protein complexes from interaction networks based on clique percolation and distance restriction

    PubMed Central

    2010-01-01

    Background Identification of protein complexes in large interaction networks is crucial to understand principles of cellular organization and predict protein functions, which is one of the most important issues in the post-genomic era. Each protein might be subordinate multiple protein complexes in the real protein-protein interaction networks. Identifying overlapping protein complexes from protein-protein interaction networks is a considerable research topic. Result As an effective algorithm in identifying overlapping module structures, clique percolation method (CPM) has a wide range of application in social networks and biological networks. However, the recognition accuracy of algorithm CPM is lowly. Furthermore, algorithm CPM is unfit to identifying protein complexes with meso-scale when it applied in protein-protein interaction networks. In this paper, we propose a new topological model by extending the definition of k-clique community of algorithm CPM and introduced distance restriction, and develop a novel algorithm called CP-DR based on the new topological model for identifying protein complexes. In this new algorithm, the protein complex size is restricted by distance constraint to conquer the shortcomings of algorithm CPM. The algorithm CP-DR is applied to the protein interaction network of Sacchromyces cerevisiae and identifies many well known complexes. Conclusion The proposed algorithm CP-DR based on clique percolation and distance restriction makes it possible to identify dense subgraphs in protein interaction networks, a large number of which correspond to known protein complexes. Compared to algorithm CPM, algorithm CP-DR has more outstanding performance. PMID:21047377

  16. Proteome Analysis. Novel Proteins Identified at the Peribacteroid Membrane from Lotus japonicus Root Nodules1

    PubMed Central

    Wienkoop, Stefanie; Saalbach, Gerhard

    2003-01-01

    The peribacteroid membrane (PBM) forms the structural and functional interface between the legume plant and the rhizobia. The model legume Lotus japonicus was chosen to study the proteins present at the PBM by proteome analysis. PBM was purified from root nodules by an aqueous polymer two-phase system. Extracted proteins were subjected to a global trypsin digest. The peptides were separated by nanoscale liquid chromatography and analyzed by tandem mass spectrometry. Searching the nonredundant protein database and the green plant expressed sequence tag database using the tandem mass spectrometry data identified approximately 94 proteins, a number far exceeding the number of proteins reported for the PBM hitherto. In particular, a number of membrane proteins like transporters for sugars and sulfate; endomembrane-associated proteins such as GTP-binding proteins and vesicle receptors; and proteins involved in signaling, for example, receptor kinases, calmodulin, 14-3-3 proteins, and pathogen response-related proteins, including a so-called HIR protein, were detected. Several ATPases and aquaporins were present, indicating a more complex situation than previously thought. In addition, the unexpected presence of a number of proteins known to be located in other compartments was observed. Two characteristic protein complexes obtained from native gel electrophoresis of total PBM proteins were also analyzed. Together, the results identified specific proteins at the PBM involved in important physiological processes and localized proteins known from nodule-specific expressed sequence tag databases to the PBM. PMID:12644660

  17. Improving reproducibility and sensitivity in identifying human proteins by shotgun proteomics.

    PubMed

    Resing, Katheryn A; Meyer-Arendt, Karen; Mendoza, Alex M; Aveline-Wolf, Lauren D; Jonscher, Karen R; Pierce, Kevin G; Old, William M; Cheung, Hiu T; Russell, Steven; Wattawa, Joy L; Goehle, Geoff R; Knight, Robin D; Ahn, Natalie G

    2004-07-01

    Identifying proteins in cell extracts by shotgun proteomics involves digesting the proteins, sequencing the resulting peptides by data-dependent mass spectrometry (MS/MS), and searching protein databases to identify the proteins from which the peptides are derived. Manual analysis and direct spectral comparison reveal that scores from two commonly used search programs (Sequest and Mascot) validate less than half of potentially identifiable MS/MS spectra (class positive) from shotgun analyses of the human erythroleukemia K562 cell line. Here we demonstrate increased sensitivity and accuracy using a focused search strategy along with a peptide sequence validation script that does not rely exclusively on XCorr or Mowse scores generated by Sequest or Mascot, but uses consensus between the search programs, along with chemical properties and scores describing the nature of the fragmentation spectrum (ion score and RSP). The approach yielded 4.2% false positive and 8% false negative frequencies in peptide assignments. The protein profile is then assembled from peptide assignments using a novel peptide-centric protein nomenclature that more accurately reports protein variants that contain identical peptide sequences. An Isoform Resolver algorithm ensures that the protein count is not inflated by variants in the protein database, eliminating approximately 25% of redundant proteins. Analysis of soluble proteins from a human K562 cells identified 5130 unique proteins, with approximately 100 false positive protein assignments. PMID:15228325

  18. Dating lacustrine episodes in the eastern Sahara by the epimerization of isoleucine in ostrich eggshells

    USGS Publications Warehouse

    Miller, G.H.; Wendorf, F.; Ernst, R.; Schild, R.; Close, A.E.; Friedman, I.; Schwarcz, H.P.

    1991-01-01

    The eggshell of the African ostrich, Struthio camelus, closely approximates a closed system for the retention of indigenous proteinaceous residues. Epimerization of the protein amino acid isoleucine follows linear first-order kinetics in laboratory simulations nearly to racemic equilibrium, and the variation in D/L ratio within a single fragment, or between fragments of the same age, is significantly less than in other carbonate systems. These observations suggest that the extent of isoleucine epimerization (aIle/Ile ratio) in ostrich eggshell offers the potential for high-resolution geochronology of Quaternary deposits. From the simulation experiments, and dated early Holocene samples for which we have in situ mean annual sediment temperature measurements, Arrhenius parameters have been calculated; the activation energy is 30.33 kcal mol-1, similar to that of other carbonate systems. We have measured the aIle/Ile ratio in ostrich eggshell associated with lacustrine episodes at Bir Tarfawi and Bir Sahara East, two depressions in what is currently the hyperarid eastern Sahara. The ratios can be used directly to indicate qualitatively the time represented by each series of lake sediment, and to correlate disjunct lacustrine deposits within and between the basins. Uranium-series disequilibrium dating of algal mats contained within some of the lake beds indicate that a major wet interval occurred about 130 ka ago. Using the U-series date for calibration, the amino acid ratios are used to date the most recent lacustrine interval to about 100 ka B.P., and two older intervals, one about 200 ?? 25 ka B.P., and an older interval that occurred prior to 250 ka ago. ?? 1991.

  19. Pathway Analysis Incorporating Protein-Protein Interaction Networks Identified Candidate Pathways for the Seven Common Diseases.

    PubMed

    Lin, Peng-Lin; Yu, Ya-Wen; Chung, Ren-Hua

    2016-01-01

    Pathway analysis has become popular as a secondary analysis strategy for genome-wide association studies (GWAS). Most of the current pathway analysis methods aggregate signals from the main effects of single nucleotide polymorphisms (SNPs) in genes within a pathway without considering the effects of gene-gene interactions. However, gene-gene interactions can also have critical effects on complex diseases. Protein-protein interaction (PPI) networks have been used to define gene pairs for the gene-gene interaction tests. Incorporating the PPI information to define gene pairs for interaction tests within pathways can increase the power for pathway-based association tests. We propose a pathway association test, which aggregates the interaction signals in PPI networks within a pathway, for GWAS with case-control samples. Gene size is properly considered in the test so that genes do not contribute more to the test statistic simply due to their size. Simulation studies were performed to verify that the method is a valid test and can have more power than other pathway association tests in the presence of gene-gene interactions within a pathway under different scenarios. We applied the test to the Wellcome Trust Case Control Consortium GWAS datasets for seven common diseases. The most significant pathway is the chaperones modulate interferon signaling pathway for Crohn's disease (p-value = 0.0003). The pathway modulates interferon gamma, which induces the JAK/STAT pathway that is involved in Crohn's disease. Several other pathways that have functional implications for the seven diseases were also identified. The proposed test based on gene-gene interaction signals in PPI networks can be used as a complementary tool to the current existing pathway analysis methods focusing on main effects of genes. An efficient software implementing the method is freely available at http://puppi.sourceforge.net. PMID:27622767

  20. Polymorphisms in eggshell organic matrix genes are associated with eggshell quality measurements in pedigree Rhode Island Red hens.

    PubMed

    Dunn, I C; Joseph, N T; Bain, M; Edmond, A; Wilson, P W; Milona, P; Nys, Y; Gautron, J; Schmutz, M; Preisinger, R; Waddington, D

    2009-02-01

    Novel and traditional eggshell quality measurements were made from up to 2000 commercial pedigree hens for a candidate gene association analysis with organic eggshell matrix genes: ovocleidin-116, osteopontin (SPP1), ovocalyxin-32 (RARRES1), ovotransferrin (LTF), ovalbumin and ovocalyxin-36, as well as key genes in the maintenance and function of the shell gland [estrogen receptor (ESR1) and carbonic anhydrase II (CAII)]. Associations were found for (i) ovalbumin with breaking strength and shell thickness; (ii) ovocleidin-116 with elastic modulus, shell thickness and egg shape; (iii) RARRES1 with mammillary layer thickness; (iv) ESR1 with dynamic stiffness; (v) SPP1 with fracture toughness and (vi) CAII with egg shape. The marker effects are as large as 17% of trait standard deviations and could be used to improve eggshell quality. PMID:18828860

  1. Embryonic eggshell thickness erosion: A literature survey re-assessing embryo-induced eggshell thinning in birds.

    PubMed

    Orłowski, Grzegorz; Hałupka, Lucyna

    2015-10-01

    Although eggshell thinning has been described mainly in the context of environmental pollution, it can also be the effect of reproductive changes induced by a developing embryo. On the basis of a literature survey of 25 bird species (26 published papers) we reviewed data on embryo-induced eggshell thinning (EET) in three groups of birds: precocials, semi-precocials and altricials. The average EET at the equator of the eggs was 6.4% (median = 4.7%). Our review did not confirm a general prediction of elevated EET at the egg equator in precocial species: altricial birds exhibited the highest EET (average = 12.0%), followed by precocials (7.6%) and semi-precocials (4.2%). We make certain critical recommendations based on the results of this study. Studies aiming to assess variation in eggshell thickness should examine intrinsic factors affecting shell properties of avian eggs, like thickness, which are the result of anatomical or reproductive changes. PMID:26074163

  2. Benchmark data for identifying multi-functional types of membrane proteins.

    PubMed

    Wan, Shibiao; Mak, Man-Wai; Kung, Sun-Yuan

    2016-09-01

    Identifying membrane proteins and their multi-functional types is an indispensable yet challenging topic in proteomics and bioinformatics. In this article, we provide data that are used for training and testing Mem-ADSVM (Wan et al., 2016. "Mem-ADSVM: a two-layer multi-label predictor for identifying multi-functional types of membrane proteins" [1]), a two-layer multi-label predictor for predicting multi-functional types of membrane proteins. PMID:27294176

  3. Which Came First, the Eggshell or the Egg? Answering Biomineralization Riddles (442nd Brookhaven Lecture)

    SciTech Connect

    DiMasi, Elaine

    2008-11-12

    Some of the hardest and sturdiest materials are not made in the factory; they are made inside the bodies of animals through a process called biomineralization. Look no further than your refrigerator for one of the simplest products of this natural construction company: a chicken's eggshell. Made out of just about a half-millimeter of layered calcium carbonate and protein, eggshells might be thought of as fragile, but they also provide vital protection for the chick forming inside. Biomineralization, the process by which organisms form materials such as bones, mollusk shells, and other structures, has captured the attention of scientists for years. The cells in an animal's body have special ways of controlling the sizes and shapes of these mineral compounds and incorporating organic materials into the mix, making many materials that are stronger, harder, and more wear-resistant than rocks. Finding a way to mimic the properties of these sturdy and naturally made materials could lead to the medical engineering of replacement bone, teeth, and cartilage, as well as the development of new electronic and industrial materials. With collaborators at Stony Brook University, physicist Elaine DiMasi develops different biomineralization models, including a protein network that resembles real tissue. Then, the researchers use x-rays at the NSLS and a technique called shear modulation force microscopy to determine what biominerals look like and how they grow. In particular, DiMasi is interested in studying some of the earliest stages of biomineralization to find out what sets the process in motion.

  4. A local average connectivity-based method for identifying essential proteins from the network level.

    PubMed

    Li, Min; Wang, Jianxin; Chen, Xiang; Wang, Huan; Pan, Yi

    2011-06-01

    Identifying essential proteins is very important for understanding the minimal requirements of cellular survival and development. Fast growth in the amount of available protein-protein interactions has produced unprecedented opportunities for detecting protein essentiality from the network level. Essential proteins have been found to be more abundant among those highly connected proteins. However, there exist a number of highly connected proteins which are not essential. By analyzing these proteins, we find that few of their neighbors interact with each other. Thus, we propose a new local method, named LAC, to determine a protein's essentiality by evaluating the relationship between a protein and its neighbors. The performance of LAC is validated based on the yeast protein interaction networks obtained from two different databases: DIP and BioGRID. The experimental results of the two networks show that the number of essential proteins predicted by LAC clearly exceeds that explored by Degree Centrality (DC). More over, LAC is also compared with other seven measures of protein centrality (Neighborhood Component (DMNC), Betweenness Centrality (BC), Closeness Centrality (CC), Bottle Neck (BN), Information Centrality (IC), Eigenvector Centrality (EC), and Subgraph Centrality (SC)) in identifying essential proteins. The comparison results based on the validations of sensitivity, specificity, F-measure, positive predictive value, negative predictive value, and accuracy consistently show that LAC outweighs these seven previous methods. PMID:21704260

  5. Engineering and Identifying Supercharged Proteins for Macromolecule Delivery into Mammalian Cells

    PubMed Central

    Thompson, David B.; Cronican, James J.; Liu, David R.

    2012-01-01

    Supercharged proteins are a class of engineered or naturally occurring proteins with unusually high net positive or negative theoretical charge. Both supernegatively and superpositively charged proteins exhibit a remarkable ability to withstand thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. Associating cargo with these proteins, such as plasmid DNA, siRNA, or other proteins, can enable the functional delivery of these macromolecules into mammalian cells both in vitro and in vivo. The potency of functional delivery in some cases can exceed that of other current methods for macromolecule delivery, including the use of cell-penetrating peptides such as Tat, and adenoviral delivery vectors. This chapter summarizes methods for engineering supercharged proteins, optimizing cell penetration, identifying naturally occurring supercharged proteins, and using these proteins for macromolecule delivery into mammalian cells. PMID:22230574

  6. Development of ‘Redox Arrays’ for identifying novel glutathionylated proteins in the secretome

    PubMed Central

    Mullen, Lisa; Seavill, Miles; Hammouz, Raneem; Bottazzi, Barbara; Chan, Philippe; Vaudry, David; Ghezzi, Pietro

    2015-01-01

    Proteomics techniques for analysing the redox status of individual proteins in complex mixtures tend to identify the same proteins due to their high abundance. We describe here an array-based technique to identify proteins undergoing glutathionylation and apply it to the secretome and the proteome of human monocytic cells. The method is based on incorporation of biotinylated glutathione (GSH) into proteins, which can then be identified following binding to a 1000-protein antibody array. We thus identify 38 secreted and 55 intracellular glutathionylated proteins, most of which are novel candidates for glutathionylation. Two of the proteins identified in these experiments, IL-1 sRII and Lyn, were then confirmed to be susceptible to glutathionylation. Comparison of the redox array with conventional proteomic methods confirmed that the redox array is much more sensitive, and can be performed using more than 100-fold less protein than is required for methods based on mass spectrometry. The identification of novel targets of glutathionylation, particularly in the secretome where the protein concentration is much lower, shows that redox arrays can overcome some of the limitations of established redox proteomics techniques. PMID:26416726

  7. A novel method to identify nucleic acid binding sites in proteins by scanning mutagenesis: application to iron regulatory protein.

    PubMed Central

    Neupert, B; Menotti, E; Kühn, L C

    1995-01-01

    We describe a new procedure to identify RNA or DNA binding sites in proteins, based on a combination of UV cross-linking and single-hit chemical peptide cleavage. Site-directed mutagenesis is used to create a series of mutants with single Asn-Gly sequences in the protein to be analysed. Recombinant mutant proteins are incubated with their radiolabelled target sequence and UV irradiated. Covalently linked RNA- or DNA-protein complexes are digested with hydroxylamine and labelled peptides identified by SDS-PAGE and autoradiography. The analysis requires only small amounts of protein and is achieved within a relatively short time. Using this method we mapped the site at which human iron regulatory protein (IRP) is UV cross-linked to iron responsive element RNA to amino acid residues 116-151. Images PMID:7544459

  8. A computational tool for identifying minimotifs in protein-protein interactions and improving the accuracy of minimotif predictions.

    PubMed

    Rajasekaran, Sanguthevar; Merlin, Jerlin Camilus; Kundeti, Vamsi; Mi, Tian; Oommen, Aaron; Vyas, Jay; Alaniz, Izua; Chung, Keith; Chowdhury, Farah; Deverasatty, Sandeep; Irvey, Tenisha M; Lacambacal, David; Lara, Darlene; Panchangam, Subhasree; Rathnayake, Viraj; Watts, Paula; Schiller, Martin R

    2011-01-01

    Protein-protein interactions are important to understanding cell functions; however, our theoretical understanding is limited. There is a general discontinuity between the well-accepted physical and chemical forces that drive protein-protein interactions and the large collections of identified protein-protein interactions in various databases. Minimotifs are short functional peptide sequences that provide a basis to bridge this gap in knowledge. However, there is no systematic way to study minimotifs in the context of protein-protein interactions or vice versa. Here we have engineered a set of algorithms that can be used to identify minimotifs in known protein-protein interactions and implemented this for use by scientists in Minimotif Miner. By globally testing these algorithms on verified data and on 100 individual proteins as test cases, we demonstrate the utility of these new computation tools. This tool also can be used to reduce false-positive predictions in the discovery of novel minimotifs. The statistical significance of these algorithms is demonstrated by an ROC analysis (P = 0.001). PMID:20938975

  9. Protein-protein interaction networks identify targets which rescue the MPP+ cellular model of Parkinson’s disease

    PubMed Central

    Keane, Harriet; Ryan, Brent J.; Jackson, Brendan; Whitmore, Alan; Wade-Martins, Richard

    2015-01-01

    Neurodegenerative diseases are complex multifactorial disorders characterised by the interplay of many dysregulated physiological processes. As an exemplar, Parkinson’s disease (PD) involves multiple perturbed cellular functions, including mitochondrial dysfunction and autophagic dysregulation in preferentially-sensitive dopamine neurons, a selective pathophysiology recapitulated in vitro using the neurotoxin MPP+. Here we explore a network science approach for the selection of therapeutic protein targets in the cellular MPP+ model. We hypothesised that analysis of protein-protein interaction networks modelling MPP+ toxicity could identify proteins critical for mediating MPP+ toxicity. Analysis of protein-protein interaction networks constructed to model the interplay of mitochondrial dysfunction and autophagic dysregulation (key aspects of MPP+ toxicity) enabled us to identify four proteins predicted to be key for MPP+ toxicity (P62, GABARAP, GBRL1 and GBRL2). Combined, but not individual, knockdown of these proteins increased cellular susceptibility to MPP+ toxicity. Conversely, combined, but not individual, over-expression of the network targets provided rescue of MPP+ toxicity associated with the formation of autophagosome-like structures. We also found that modulation of two distinct proteins in the protein-protein interaction network was necessary and sufficient to mitigate neurotoxicity. Together, these findings validate our network science approach to multi-target identification in complex neurological diseases. PMID:26608097

  10. Protein-protein interaction networks identify targets which rescue the MPP+ cellular model of Parkinson’s disease

    NASA Astrophysics Data System (ADS)

    Keane, Harriet; Ryan, Brent J.; Jackson, Brendan; Whitmore, Alan; Wade-Martins, Richard

    2015-11-01

    Neurodegenerative diseases are complex multifactorial disorders characterised by the interplay of many dysregulated physiological processes. As an exemplar, Parkinson’s disease (PD) involves multiple perturbed cellular functions, including mitochondrial dysfunction and autophagic dysregulation in preferentially-sensitive dopamine neurons, a selective pathophysiology recapitulated in vitro using the neurotoxin MPP+. Here we explore a network science approach for the selection of therapeutic protein targets in the cellular MPP+ model. We hypothesised that analysis of protein-protein interaction networks modelling MPP+ toxicity could identify proteins critical for mediating MPP+ toxicity. Analysis of protein-protein interaction networks constructed to model the interplay of mitochondrial dysfunction and autophagic dysregulation (key aspects of MPP+ toxicity) enabled us to identify four proteins predicted to be key for MPP+ toxicity (P62, GABARAP, GBRL1 and GBRL2). Combined, but not individual, knockdown of these proteins increased cellular susceptibility to MPP+ toxicity. Conversely, combined, but not individual, over-expression of the network targets provided rescue of MPP+ toxicity associated with the formation of autophagosome-like structures. We also found that modulation of two distinct proteins in the protein-protein interaction network was necessary and sufficient to mitigate neurotoxicity. Together, these findings validate our network science approach to multi-target identification in complex neurological diseases.

  11. New proteins identified in epididymal fluid from the platypus (Ornithorhynchus anatinus).

    PubMed

    Dacheux, Jean-Louis; Dacheux, Francoise; Labas, Valerie; Ecroyd, Heath; Nixon, Brett; Jones, Russell C

    2009-01-01

    The platypus epididymal proteome is being studied because epididymal proteins are essential for male fertility in mammals and it is considered that knowledge of the epididymal proteome in an early mammal would be informative in assessing the convergence and divergence of proteins that are important in the function of the mammalian epididymis. Few of the epididymal proteins that have been identified in eutherian mammals were found in platypus caudal epididymal fluid, and the major epididymal proteins in the platypus (PXN-FBPL, SPARC and E-OR20) have never been identified in the epididymis of any other mammal. PMID:19874724

  12. KESTREL: a powerful method for identifying the physiological substrates of protein kinases

    PubMed Central

    Cohen, Philip; Knebel, Axel

    2005-01-01

    The identification of all the substrates of every protein kinase is one of the major challenges of post-genomic research. Here we review a powerful method for tackling this problem that we have developed over the last 5 years. The method has so far been used to identify novel substrates for eight different protein kinases, demonstrating that it is of general utility. Importantly, the method can be used to identify distinct physiological substrates of protein kinases, such as PKB (protein kinase B) and SGK (serum- and glucocorticoid-induced kinase), that are closely related in structure and have similar specificity determinants. PMID:16336195

  13. Organochlorine residues and eggshell thinning in wood storks and anhingas

    USGS Publications Warehouse

    Ohlendorf, H.M.; Klaas, E.E.; Kaiser, T.E.

    1978-01-01

    All 10 Wood Stork eggs collected at Merritt Island National Wildlife Refuge in 1973 contained residues of DDE (geometric mean 4.0 ppm wet weight) and PCBs (1.2 ppm). Nine other organochlorines were found at lower frequencies in the eggs. Eggshells from the recent period were 8.9% thinner (P < 0.001) than pre-1947 samples; decrease in eggshell thickness was more closely correlated with DDE than other organochlorines and correlation of DDE and eggshell thickness approached significance (P = 0.115).....Anhinga eggs were collected at 7 localities; 45 of the 46 eggs analyzed contained DDE residues and 24 contained PCBs. Residues of other organochlorines were found less frequently. Shell thickness of recent eggs from Louisiana and Mississippi was significantly less (-7.5%; P < 0.05) than the mean for pre-1947 eggs, but there was no significant change in shell thickness of eggs from Florida. The change in clutch mean eggshell thickness was significantly negatively correlated (P < 0.05) with the concentration of DDE in the eggs.

  14. Dieldrin and DDT: effects on sparrow hawk eggshells and reproduction

    USGS Publications Warehouse

    Porter, Ron; Wiemeyer, Stanley N.

    1969-01-01

    Patterns of reproductive failure in declining populations of several European and North American raptorial species were duplicated experimentally with captive American sparrow hawks Falco sparverius that were given a diet containing two commonly used organochlorine insecticides. Major effects on reproduction were increased egg disappearance, increased egg destruction by parent birds, and reduced eggshell thickness.

  15. MAS C-Terminal Tail Interacting Proteins Identified by Mass Spectrometry- Based Proteomic Approach

    PubMed Central

    Tirupula, Kalyan C.; Zhang, Dongmei; Osbourne, Appledene; Chatterjee, Arunachal; Desnoyer, Russ; Willard, Belinda; Karnik, Sadashiva S.

    2015-01-01

    Propagation of signals from G protein-coupled receptors (GPCRs) in cells is primarily mediated by protein-protein interactions. MAS is a GPCR that was initially discovered as an oncogene and is now known to play an important role in cardiovascular physiology. Current literature suggests that MAS interacts with common heterotrimeric G-proteins, but MAS interaction with proteins which might mediate G protein-independent or atypical signaling is unknown. In this study we hypothesized that MAS C-terminal tail (Ct) is a major determinant of receptor-scaffold protein interactions mediating MAS signaling. Mass-spectrometry based proteomic analysis was used to comprehensively identify the proteins that interact with MAS Ct comprising the PDZ-binding motif (PDZ-BM). We identified both PDZ and non-PDZ proteins from human embryonic kidney cell line, mouse atrial cardiomyocyte cell line and human heart tissue to interact specifically with MAS Ct. For the first time our study provides a panel of PDZ and other proteins that potentially interact with MAS with high significance. A ‘cardiac-specific finger print’ of MAS interacting PDZ proteins was identified which includes DLG1, MAGI1 and SNTA. Cell based experiments with wild-type and mutant MAS lacking the PDZ-BM validated MAS interaction with PDZ proteins DLG1 and TJP2. Bioinformatics analysis suggested well-known multi-protein scaffold complexes involved in nitric oxide signaling (NOS), cell-cell signaling of neuromuscular junctions, synapses and epithelial cells. Majority of these protein hits were predicted to be part of disease categories comprising cancers and malignant tumors. We propose a ‘MAS-signalosome’ model to stimulate further research in understanding the molecular mechanism of MAS function. Identifying hierarchy of interactions of ‘signalosome’ components with MAS will be a necessary step in future to fully understand the physiological and pathological functions of this enigmatic receptor. PMID

  16. A Simple Approach for an Eggshell-Based 3D-Printed Osteoinductive Multiphasic Calcium Phosphate Scaffold.

    PubMed

    Dadhich, Prabhash; Das, Bodhisatwa; Pal, Pallabi; Srivas, Pavan K; Dutta, Joy; Ray, Sabyasachi; Dhara, Santanu

    2016-05-18

    Natural origin bioceramics are widely used for bone grafts. In the present study, an eggshell-derived bioceramic scaffold is fabricated by 3D printing as a potential bone-graft analogue. The eggshell, a biological waste material, was mixed with a specific ratio of phosphoric acid and chitosan to form a precursor toward the fabrication of an osteoinductive multiphasic calcium phosphate scaffold via a coagulation-assisted extrusion and sintering for a multiscalar hierarchical porous structure with improved mechanical properties. Physicochemical characterization of the formed scaffolds was carried out for phase analysis, surface morphology, and mechanical properties. A similar scaffold was prepared using a chemically synthesized calcium phosphate powder that was compared with the natural origin one. The higher surface area associated with the interconnected porosity along with multiple phases of the natural origin scaffold facilitated higher cell adhesion and proliferation compared to the chemically synthesized one. Further, the natural origin scaffold displayed relatively higher cell differentiation activity, as is evident by protein and gene expression studies. On subcutaneous implantation for 30 days, promising vascular tissue in-growth was observed, circumventing a major foreign body response. Collagen-rich vascular extracellular matrix deposition and osteocalcin secretion indicated bonelike tissue formation. Finally, the eggshell-derived multiphasic calcium phosphate scaffold displayed improvement in the mechanical properties with higher porosity and osteoinductivity compared to the chemically derived apatite and unveiled a new paradigm for utilization of biological wastes in bone-graft application. PMID:26853051

  17. Utilizing Yeast Surface Human Proteome Display Libraries to Identify Small Molecule-Protein Interactions

    PubMed Central

    Bidlingmaier, Scott; Liu, Bin

    2016-01-01

    The identification of proteins that interact with small bioactive molecules is a critical but often difficult and time-consuming step in understanding cellular signaling pathways or molecular mechanisms of drug action. Numerous methods for identifying small molecule-interacting proteins have been developed and utilized, including affinity-based purification followed by mass spectrometry analysis, protein microarrays, phage display, and three-hybrid approaches. Although all these methods have been used successfully, there remains a need for additional techniques for analyzing small molecule-protein interactions. A promising method for identifying small molecule-protein interactions is affinity-based selection of yeast surface-displayed human proteome libraries. Large and diverse libraries displaying human protein fragments on the surface of yeast cells have been constructed and subjected to FACS-based enrichment followed by comprehensive exon microarray-based output analysis to identify protein fragments with affinity for small molecule ligands. In a recent example, a proteome-wide search has been successfully carried out to identify cellular proteins binding to the signaling lipids PtdIns(4,5)P2 and PtdIns(3,4,5)P3. Known phosphatidylinositide-binding proteins such as pleckstrin homology domains were identified, as well as many novel interactions. Intriguingly, many novel nuclear phosphatidylinositide-binding proteins were discovered. Although the existence of an independent pool of nuclear phosphatidylinositides has been known about for some time, their functions and mechanism of action remain obscure. Thus, the identification and subsequent study of nuclear phosphatidylinositide-binding proteins is expected to bring new insights to this important biological question. Based on the success with phosphatidylinositides, it is expected that the screening of yeast surface-displayed human proteome libraries will be of general use for the discovery of novel small

  18. Utilizing Yeast Surface Human Proteome Display Libraries to Identify Small Molecule-Protein Interactions.

    PubMed

    Bidlingmaier, Scott; Liu, Bin

    2015-01-01

    The identification of proteins that interact with small bioactive molecules is a critical but often difficult and time-consuming step in understanding cellular signaling pathways or molecular mechanisms of drug action. Numerous methods for identifying small molecule-interacting proteins have been developed and utilized, including affinity-based purification followed by mass spectrometry analysis, protein microarrays, phage display, and three-hybrid approaches. Although all these methods have been used successfully, there remains a need for additional techniques for analyzing small molecule-protein interactions. A promising method for identifying small molecule-protein interactions is affinity-based selection of yeast surface-displayed human proteome libraries. Large and diverse libraries displaying human protein fragments on the surface of yeast cells have been constructed and subjected to FACS-based enrichment followed by comprehensive exon microarray-based output analysis to identify protein fragments with affinity for small molecule ligands. In a recent example, a proteome-wide search has been successfully carried out to identify cellular proteins binding to the signaling lipids PtdIns(4,5)P2 and PtdIns(3,4,5)P3. Known phosphatidylinositide-binding proteins such as pleckstrin homology domains were identified, as well as many novel interactions. Intriguingly, many novel nuclear phosphatidylinositide-binding proteins were discovered. Although the existence of an independent pool of nuclear phosphatidylinositides has been known about for some time, their functions and mechanism of action remain obscure. Thus, the identification and subsequent study of nuclear phosphatidylinositide-binding proteins is expected to bring new insights to this important biological question. Based on the success with phosphatidylinositides, it is expected that the screening of yeast surface-displayed human proteome libraries will be of general use for the discovery of novel small

  19. A novel approach to identify genes that determine grain protein deviation in cereals.

    PubMed

    Mosleth, Ellen F; Wan, Yongfang; Lysenko, Artem; Chope, Gemma A; Penson, Simon P; Shewry, Peter R; Hawkesford, Malcolm J

    2015-06-01

    Grain yield and protein content were determined for six wheat cultivars grown over 3 years at multiple sites and at multiple nitrogen (N) fertilizer inputs. Although grain protein content was negatively correlated with yield, some grain samples had higher protein contents than expected based on their yields, a trait referred to as grain protein deviation (GPD). We used novel statistical approaches to identify gene transcripts significantly related to GPD across environments. The yield and protein content were initially adjusted for nitrogen fertilizer inputs and then adjusted for yield (to remove the negative correlation with protein content), resulting in a parameter termed corrected GPD. Significant genetic variation in corrected GPD was observed for six cultivars grown over a range of environmental conditions (a total of 584 samples). Gene transcript profiles were determined in a subset of 161 samples of developing grain to identify transcripts contributing to GPD. Principal component analysis (PCA), analysis of variance (ANOVA) and means of scores regression (MSR) were used to identify individual principal components (PCs) correlating with GPD alone. Scores of the selected PCs, which were significantly related to GPD and protein content but not to the yield and significantly affected by cultivar, were identified as reflecting a multivariate pattern of gene expression related to genetic variation in GPD. Transcripts with consistent variation along the selected PCs were identified by an approach hereby called one-block means of scores regression (one-block MSR). PMID:25400203

  20. Tandem Affinity Purification Combined with Mass Spectrometry to Identify Components of Protein Complexes

    PubMed Central

    Kaiser, Peter; Meierhofer, David; Wang, Xiaorong; Huang, Lan

    2011-01-01

    Most biological processes are governed by multiprotein complexes rather than individual proteins. Identification of protein complexes therefore is becoming increasingly important to gain a molecular understanding of cells and organisms. Mass spectrometry–based proteomics combined with affinity-tag-based protein purification is one of the most effective strategies to isolate and identify protein complexes. The development of tandem-affinity purification approaches has revolutionized proteomics experiments. These two-step affinity purification strategies allow rapid, effective purification of protein complexes and, at the same time, minimize background. Identification of even very low-abundant protein complexes with modern sensitive mass spectrometers has become routine. Here, we describe two general strategies for tandem-affinity purification followed by mass spectrometric identification of protein complexes. PMID:18370112

  1. Proteomic Analysis to Identify Tightly-Bound Cell Wall Protein in Rice Calli.

    PubMed

    Cho, Won Kyong; Hyun, Tae Kyung; Kumar, Dhinesh; Rim, Yeonggil; Chen, Xiong Yan; Jo, Yeonhwa; Kim, Suwha; Lee, Keun Woo; Park, Zee-Yong; Lucas, William J; Kim, Jae-Yean

    2015-08-01

    Rice is a model plant widely used for basic and applied research programs. Plant cell wall proteins play key roles in a broad range of biological processes. However, presently, knowledge on the rice cell wall proteome is rudimentary in nature. In the present study, the tightly-bound cell wall proteome of rice callus cultured cells using sequential extraction protocols was developed using mass spectrometry and bioinformatics methods, leading to the identification of 1568 candidate proteins. Based on bioinformatics analyses, 389 classical rice cell wall proteins, possessing a signal peptide, and 334 putative non-classical cell wall proteins, lacking a signal peptide, were identified. By combining previously established rice cell wall protein databases with current data for the classical rice cell wall proteins, a comprehensive rice cell wall proteome, comprised of 496 proteins, was constructed. A comparative analysis of the rice and Arabidopsis cell wall proteomes revealed a high level of homology, suggesting a predominant conservation between monocot and eudicot cell wall proteins. This study importantly increased information on cell wall proteins, which serves for future functional analyses of these identified rice cell wall proteins. PMID:26194822

  2. Proteomic Analysis to Identify Tightly-Bound Cell Wall Protein in Rice Calli

    PubMed Central

    Cho, Won Kyong; Hyun, Tae Kyung; Kumar, Dhinesh; Rim, Yeonggil; Chen, Xiong Yan; Jo, Yeonhwa; Kim, Suwha; Lee, Keun Woo; Park, Zee-Yong; Lucas, William J.; Kim, Jae-Yean

    2015-01-01

    Rice is a model plant widely used for basic and applied research programs. Plant cell wall proteins play key roles in a broad range of biological processes. However, presently, knowledge on the rice cell wall proteome is rudimentary in nature. In the present study, the tightly-bound cell wall proteome of rice callus cultured cells using sequential extraction protocols was developed using mass spectrometry and bioinformatics methods, leading to the identification of 1568 candidate proteins. Based on bioinformatics analyses, 389 classical rice cell wall proteins, possessing a signal peptide, and 334 putative non-classical cell wall proteins, lacking a signal peptide, were identified. By combining previously established rice cell wall protein databases with current data for the classical rice cell wall proteins, a comprehensive rice cell wall proteome, comprised of 496 proteins, was constructed. A comparative analysis of the rice and Arabidopsis cell wall proteomes revealed a high level of homology, suggesting a predominant conservation between monocot and eudicot cell wall proteins. This study importantly increased information on cell wall proteins, which serves for future functional analyses of these identified rice cell wall proteins. PMID:26194822

  3. Identifying transient protein-protein interactions in EphB2 signaling by Blue Native PAGE and Mass Spectrometry

    PubMed Central

    Darie, Costel C.; Deinhardt, Katrin; Zhang, Guoan; Cardasis, Helene S.; Chao, Moses V.; Neubert, Thomas A.

    2012-01-01

    Receptor tyrosine kinases (RTKs) are proteins that upon ligand stimulation undergo dimerization and autophosphorylation. Eph receptors (EphRs) are RTKs that are found in different cell types, from both tissues that are developing and from mature tissues and play important roles in the development of the central nervous system and peripheral nervous system. EphRs also play roles in synapse formation, neural crest formation, angiogenesis and in remodeling the vascular system. Interaction of EphRs with their ephrin ligands leads to activation of signal transduction pathways and to formation of many transient protein-protein interactions that ultimately leads to cytoskeletal remodeling. However, the sequence of events at the molecular level is not well-understood. We used Blue Native PAGE (BN-PAGE) and mass spectrometry (MS) to analyze the transient protein-protein interactions that resulted from stimulation of EphB2 receptors by their ephrinB1-Fc ligands. We analyzed the phosphotyrosine-containing protein complexes immunoprecipitated (pY-IPs) from the cell lysates of both unstimulated (−) and ephrinB1-Fc-stimulated (+) NG108 cells. Our experiments allowed us to identify many signaling proteins, either known to be part of EphB2 signaling or new for this pathway, which are involved in transient protein-protein interactions upon ephrinB1-Fc stimulation. These data led us to investigate the roles in EphB2 signaling of proteins such as FAK, WAVEs, and Nischarin. PMID:21932443

  4. A New Method for Identifying Essential Proteins Based on Network Topology Properties and Protein Complexes.

    PubMed

    Qin, Chao; Sun, Yongqi; Dong, Yadong

    2016-01-01

    Essential proteins are indispensable to the viability and reproduction of an organism. The identification of essential proteins is necessary not only for understanding the molecular mechanisms of cellular life but also for disease diagnosis, medical treatments and drug design. Many computational methods have been proposed for discovering essential proteins, but the precision of the prediction of essential proteins remains to be improved. In this paper, we propose a new method, LBCC, which is based on the combination of local density, betweenness centrality (BC) and in-degree centrality of complex (IDC). First, we introduce the common centrality measures; second, we propose the densities Den1(v) and Den2(v) of a node v to describe its local properties in the network; and finally, the combined strategy of Den1, Den2, BC and IDC is developed to improve the prediction precision. The experimental results demonstrate that LBCC outperforms traditional topological measures for predicting essential proteins, including degree centrality (DC), BC, subgraph centrality (SC), eigenvector centrality (EC), network centrality (NC), and the local average connectivity-based method (LAC). LBCC also improves the prediction precision by approximately 10 percent on the YMIPS and YMBD datasets compared to the most recently developed method, LIDC. PMID:27529423

  5. A New Method for Identifying Essential Proteins Based on Network Topology Properties and Protein Complexes

    PubMed Central

    Qin, Chao; Sun, Yongqi; Dong, Yadong

    2016-01-01

    Essential proteins are indispensable to the viability and reproduction of an organism. The identification of essential proteins is necessary not only for understanding the molecular mechanisms of cellular life but also for disease diagnosis, medical treatments and drug design. Many computational methods have been proposed for discovering essential proteins, but the precision of the prediction of essential proteins remains to be improved. In this paper, we propose a new method, LBCC, which is based on the combination of local density, betweenness centrality (BC) and in-degree centrality of complex (IDC). First, we introduce the common centrality measures; second, we propose the densities Den1(v) and Den2(v) of a node v to describe its local properties in the network; and finally, the combined strategy of Den1, Den2, BC and IDC is developed to improve the prediction precision. The experimental results demonstrate that LBCC outperforms traditional topological measures for predicting essential proteins, including degree centrality (DC), BC, subgraph centrality (SC), eigenvector centrality (EC), network centrality (NC), and the local average connectivity-based method (LAC). LBCC also improves the prediction precision by approximately 10 percent on the YMIPS and YMBD datasets compared to the most recently developed method, LIDC. PMID:27529423

  6. Unique secreted–surface protein complex of Lactobacillus rhamnosus, identified by phage display

    PubMed Central

    Gagic, Dragana; Wen, Wesley; Collett, Michael A; Rakonjac, Jasna

    2013-01-01

    Proteins are the most diverse structures on bacterial surfaces; hence, they are candidates for species- and strain-specific interactions of bacteria with the host, environment, and other microorganisms. Genomics has decoded thousands of bacterial surface and secreted proteins, yet the function of most cannot be predicted because of the enormous variability and a lack of experimental data that would allow deduction of function through homology. Here, we used phage display to identify a pair of interacting extracellular proteins in the probiotic bacterium Lactobacillus rhamnosus HN001. A secreted protein, SpcA, containing two bacterial immunoglobulin-like domains type 3 (Big-3) and a domain distantly related to plant pathogen response domain 1 (PR-1-like) was identified by screening of an L. rhamnosus HN001 library using HN001 cells as bait. The SpcA-“docking” protein, SpcB, was in turn detected by another phage display library screening, using purified SpcA as bait. SpcB is a 3275-residue cell-surface protein that contains general features of large glycosylated Serine-rich adhesins/fibrils from gram-positive bacteria, including the hallmark signal sequence motif KxYKxGKxW. Both proteins are encoded by genes within a L. rhamnosus-unique gene cluster that distinguishes this species from other lactobacilli. To our knowledge, this is the first example of a secreted-docking protein pair identified in lactobacilli. PMID:23233310

  7. Method for early detection of infectious mononucleosis by identifying Inmono proteins

    DOEpatents

    Willard, Karen E.

    1984-01-01

    Early detection of infectious mononucleosis is carried out using a sample of human blood by isolating and identifying the presence of Inmono proteins in the sample from a two-dimensional protein map with the proteins being characterized by having isoelectric banding as measured in urea of about -16 to -17 with respect to certain isoelectric point standards and molecular mass of about 70 to 75 K daltons as measured in the presence of sodium dodecylsulfate containing polyacrylamide gels, the presence of the Inmono proteins being correlated with the existence of infectious mononucleosis.

  8. Dissecting Protein Function: An Efficient Protocol for Identifying Separation-of-Function Mutations That Encode Structurally Stable Proteins

    PubMed Central

    Lubin, Johnathan W.; Rao, Timsi; Mandell, Edward K.; Wuttke, Deborah S.; Lundblad, Victoria

    2013-01-01

    Mutations that confer the loss of a single biochemical property (separation-of-function mutations) can often uncover a previously unknown role for a protein in a particular biological process. However, most mutations are identified based on loss-of-function phenotypes, which cannot differentiate between separation-of-function alleles vs. mutations that encode unstable/unfolded proteins. An alternative approach is to use overexpression dominant-negative (ODN) phenotypes to identify mutant proteins that disrupt function in an otherwise wild-type strain when overexpressed. This is based on the assumption that such mutant proteins retain an overall structure that is comparable to that of the wild-type protein and are able to compete with the endogenous protein (Herskowitz 1987). To test this, the in vivo phenotypes of mutations in the Est3 telomerase subunit from Saccharomyces cerevisiae were compared with the in vitro secondary structure of these mutant proteins as analyzed by circular-dichroism spectroscopy, which demonstrates that ODN is a more sensitive assessment of protein stability than the commonly used method of monitoring protein levels from extracts. Reverse mutagenesis of EST3, which targeted different categories of amino acids, also showed that mutating highly conserved charged residues to the oppositely charged amino acid had an increased likelihood of generating a severely defective est3− mutation, which nevertheless encoded a structurally stable protein. These results suggest that charge-swap mutagenesis directed at a limited subset of highly conserved charged residues, combined with ODN screening to eliminate partially unfolded proteins, may provide a widely applicable and efficient strategy for generating separation-of-function mutations. PMID:23307900

  9. Juvenile hormone-binding proteins of Melanoplus bivittatus identified by EFDA photoaffinity labeling

    SciTech Connect

    Winder, B.S.

    1988-01-01

    Proteins that bind juvenile hormone in the hemolymph and fat body of the grasshopper, Melanoplus bivittatus were identified by photoaffinity labeling with radiolabeled epoxyfarnesyl diazoacetate ({sup 3}H-EFDA), and were characterized by electrophoretic analysis. A protocol was developed which allowed detection of {sup 3}H-EFDA that was covalently linked to proteins upon exposure to ultraviolet light at 254 nm. Quantification of protein-linked {sup 3}H-EFDA by liquid scintillation spectrometry took advantage of the differential solubility of unlinked {sup 3}H-EFDA in toluene alone, and of the protein-linked {sup 3}H-EFDA in toluene plus the detergent, Triton X-100. Competition between EFDA and juvenile hormone (JH) for binding to JH-specific binding sites was measured by hydroxyapatite protein binding assays in the presence of radiolabeled JH or EFDA and competing non-radiolabeled hormone. The protein-linked EFDA was detected on fluorograms of SDS or nondenaturing polyacrylamide gels (PAGE), and by liquid scintillation spectrometry of membranes to which the proteins had been electrophoretically transferred. Proteins which specifically bound JH were identified by photolabeling proteins in the presence and absence of nonlabeled JH-III.

  10. Protein Abundance Changes and Ubiquitylation Targets Identified after Inhibition of the Proteasome with Syringolin A*

    PubMed Central

    Svozil, Julia; Hirsch-Hoffmann, Matthias; Dudler, Robert; Gruissem, Wilhelm; Baerenfaller, Katja

    2014-01-01

    As proteins are the main effectors inside cells, their levels need to be tightly regulated. This is partly achieved by specific protein degradation via the Ubiquitin-26S proteasome system (UPS). In plants, an exceptionally high number of proteins are involved in Ubiquitin-26S proteasome system-mediated protein degradation and it is known to regulate most, if not all, important cellular processes. Here, we investigated the response to the inhibition of the proteasome at the protein level treating leaves with the specific inhibitor Syringolin A (SylA) in a daytime specific manner and found 109 accumulated and 140 decreased proteins. The patterns of protein level changes indicate that the accumulating proteins cause proteotoxic stress that triggers various responses. Comparing protein level changes in SylA treated with those in a transgenic line over-expressing a mutated ubiquitin unable to form polyubiquitylated proteins produced little overlap pointing to different response pathways. To distinguish between direct and indirect targets of the UPS we also enriched and identified ubiquitylated proteins after inhibition of the proteasome, revealing a total of 1791 ubiquitylated proteins in leaves and roots and 1209 that were uniquely identified in our study. The comparison of the ubiquitylated proteins with those changing in abundance after SylA-mediated inhibition of the proteasome confirmed the complexity of the response and revealed that some proteins are regulated both at transcriptional and post-transcriptional level. For the ubiquitylated proteins that accumulate in the cytoplasm but are targeted to the plastid or the mitochondrion, we often found peptides in their target sequences, demonstrating that the UPS is involved in controlling organellar protein levels. Attempts to identify the sites of ubiquitylation revealed that the specific properties of this post-translational modification can lead to incorrect peptide spectrum assignments in complex peptide mixtures

  11. Identifying mutated proteins secreted by colon cancer cell lines using mass spectrometry.

    PubMed

    Mathivanan, Suresh; Ji, Hong; Tauro, Bow J; Chen, Yuan-Shou; Simpson, Richard J

    2012-12-01

    Secreted proteins encoded by mutated genes (mutant proteins) are a particularly rich source of biomarkers being not only components of the cancer secretome but also actually implicated in tumorigenesis. One of the challenges of proteomics-driven biomarker discovery research is that the bulk of secreted mutant proteins cannot be identified directly and quantified by mass spectrometry due to the lack of mutated peptide information in extant proteomics databases. Here we identify, using an integrated genomics and proteomics strategy (referred to iMASp - identification of Mutated And Secreted proteins), 112 putative mutated tryptic peptides (corresponding to 57 proteins) in the collective secretomes derived from a panel of 18 human colorectal cancer (CRC) cell lines. Central to this iMASp was the creation of Human Protein Mutant Database (HPMD), against which experimentally-derived secretome peptide spectra were searched. Eight of the identified mutated tryptic peptides were confirmed by RT-PCR and cDNA sequencing of RNA extracted from those CRC cells from which the mutation was identified by mass spectrometry. The iMASp technology promises to improve the link between proteomics and genomic mutation data thereby providing an effective tool for targeting tryptic peptides with mutated amino acids as potential cancer biomarker candidates. This article is part of a Special Issue entitled: Integrated omics. PMID:22796352

  12. Proteomics informed by transcriptomics identifies novel secreted proteins in Dermacentor andersoni saliva

    SciTech Connect

    Mudenda, Lwiindi; Aguilar Pierle, Sebastian; Turse, Joshua E.; Scoles, Glen A.; Purvine, Samuel O.; Nicora, Carrie D.; Clauss, Therese RW; Ueti, Massaro W.; Brown, Wendy C.; Brayton, Kelly A.

    2014-08-07

    Dermacentor andersoni, known as the Rocky Mountain wood tick, is found in the western United States and transmits pathogens that cause diseases of veterinary and public health importance including Rocky Mountain spotted fever, tularemia, Colorado tick fever and bovine anaplasmosis. Tick saliva is known to modulate both innate and acquired immune responses, enabling ticks to feed for several days without detection. During feeding ticks subvert host defences such as hemostasis and inflammation, which would otherwise result in coagulation, wound repair and rejection of the tick. Molecular characterization of the proteins and pharmacological molecules secreted in tick saliva offers an opportunity to develop tick vaccines as an alternative to the use of acaricides, as well as new anti-inflammatory drugs. We performed proteomics informed by transcriptomics to identify D. andersoni saliva proteins that are secreted during feeding. The transcript data generated a database of 21,797 consensus sequences, which we used to identify 677 proteins secreted in the saliva of D. andersoni ticks fed for 2 and 5 days, following proteomic investigations of whole saliva using mass spectrometry. Salivary gland transcript levels of unfed ticks were compared with 2 and 5 day fed ticks to identify genes upregulated early during tick feeding. We cross-referenced the proteomic data with the transcriptomic data to identify 157 proteins of interest for immunomodulation and blood feeding. Proteins of unknown function as well as known immunomodulators were identified.

  13. Proteomics informed by transcriptomics identifies novel secreted proteins in Dermacentor andersoni saliva.

    PubMed

    Mudenda, Lwiindi; Pierlé, Sebastián Aguilar; Turse, Joshua E; Scoles, Glen A; Purvine, Samuel O; Nicora, Carrie D; Clauss, Therese R W; Ueti, Massaro W; Brown, Wendy C; Brayton, Kelly A

    2014-11-01

    Dermacentor andersoni, known as the Rocky Mountain wood tick, is found in the western United States and transmits pathogens that cause diseases of veterinary and public health importance including Rocky Mountain spotted fever, tularemia, Colorado tick fever and bovine anaplasmosis. Tick saliva is known to modulate both innate and acquired immune responses, enabling ticks to feed for several days without detection. During feeding ticks subvert host defences such as hemostasis and inflammation, which would otherwise result in coagulation, wound repair and rejection of the tick. Molecular characterization of the proteins and pharmacological molecules secreted in tick saliva offers an opportunity to develop tick vaccines as an alternative to the use of acaricides, as well as new anti-inflammatory drugs. We performed proteomics informed by transcriptomics to identify D. andersoni saliva proteins that are secreted during feeding. The transcript data generated a database of 21,797 consensus sequences, which we used to identify 677 proteins secreted in the saliva of D. andersoni ticks fed for 2 and 5days, following proteomic investigations of whole saliva using mass spectrometry. Salivary gland transcript levels of unfed ticks were compared with 2 and 5day fed ticks to identify genes upregulated early during tick feeding. We cross-referenced the proteomic data with the transcriptomic data to identify 157 proteins of interest for immunomodulation and blood feeding. Proteins of unknown function as well as known immunomodulators were identified. PMID:25110293

  14. Hydrogen peroxide treatment of eggshell membrane to control porosity.

    PubMed

    Hsieh, Shuchen; Chou, Hsuan-Hung; Hsieh, Chiung-Wen; Wu, Deng-Chyang; Kuo, Chao-Hung; Lin, Feng-Huei

    2013-12-01

    The eggshell membrane (ESM) is a naturally occurring biological polymer, which can be extracted from eggshells, and has been used for adsorption of dyes or heavy metals, as a semipermeable membrane to control particle transport, and as a natural biocompatible material for tissue replacement. In this study, we used hydrogen peroxide to control the pore size and fibre crossing density of the ESM. Structural and chemical properties were investigated using AFM, optical microscopy, contact angle, and FTIR. We show that the structure and permeability of the ESM can be controlled by timed exposure to H2O2 and we demonstrate this effect using red blood cells. This process provides a simple method for preparing biocompatible membranes, with controlled selectivity for biofiltration applications. PMID:23870936

  15. Influence of milling on the adsorption ability of eggshell waste.

    PubMed

    Baláž, Matej; Ficeriová, Jana; Briančin, Jaroslav

    2016-03-01

    Eggshell waste was successfully used for the removal of heavy metal ions from model solutions. The effect of ball milling on the structure and adsorption ability of eggshell (ES) and its membrane (ESM) was investigated, with the conclusion that milling is benefitial only for the ES. The adsorption experiments showed that the ESM is a selective adsorbent, as the adsorption ability toward different ions decreased in the following order: Ag(I) > Cd(II) > Zn(II). The obtained Qm values for Ag(I) adsorption on the ESM and ES were 52.9 and 55.7 mg g(-1), respectively. The potential industrial application of ES was also demonstrated by successful removal of Ag(I) from the technological waste. PMID:26741552

  16. HydroCalc Proteome: a tool to identify distinct characteristics of effector proteins.

    PubMed

    da Silva, G J; da Silva, R G T M; Silva, V A; C Caritá, E; Fachin, A L; Marins, M

    2016-01-01

    Bacterial pathogenicity is associated with secretion of effector proteins into intra- and extracellular spaces. These proteins interfere with cellular processes such as inhibition of phagosome-lysosome fusion, induction of apoptosis and autophagy, activation and suppression of kinases, regulation of receptor activity, and modulation of transcription factors. Knowledge regarding the characteristics of these proteins would assist in pathogenicity studies, and help to identify possible and novel targets for antibacterial drugs. Amino acid hydropathy is a property that can affect behavior patterns in effector proteins. The HydroCalc Proteome tool analyzes total hydropathy, average hydropathy, C-terminal hydropathy, C-terminal load, and basic polar amino acids at the C-terminus. These five properties could contribute to the identification of proteins with an effector potential. HydroCalc Proteome is a web tool that provides a simple interface for the analysis of hydropathy properties in proteins. This tool permits the analysis of a single protein or even the complete proteome, which cannot be achieved by using other hydropathy tools. The tool displays the result of five properties related to effector proteins in a single table. The HydroCalc Proteome (www.gmb.bio.br/hydrocalc) is a powerful tool for protein analysis, and can contribute to the study of effector proteins. PMID:27525880

  17. Network analysis identifies protein clusters of functional importance in juvenile idiopathic arthritis

    PubMed Central

    2014-01-01

    Introduction Our objective was to utilise network analysis to identify protein clusters of greatest potential functional relevance in the pathogenesis of oligoarticular and rheumatoid factor negative (RF-ve) polyarticular juvenile idiopathic arthritis (JIA). Methods JIA genetic association data were used to build an interactome network model in BioGRID 3.2.99. The top 10% of this protein:protein JIA Interactome was used to generate a minimal essential network (MEN). Reactome FI Cytoscape 2.83 Plugin and the Disease Association Protein-Protein Link Evaluator (Dapple) algorithm were used to assess the functionality of the biological pathways within the MEN and to statistically rank the proteins. JIA gene expression data were integrated with the MEN and clusters of functionally important proteins derived using MCODE. Results A JIA interactome of 2,479 proteins was built from 348 JIA associated genes. The MEN, representing the most functionally related components of the network, comprised of seven clusters, with distinct functional characteristics. Four gene expression datasets from peripheral blood mononuclear cells (PBMC), neutrophils and synovial fluid monocytes, were mapped onto the MEN and a list of genes enriched for functional significance identified. This analysis revealed the genes of greatest potential functional importance to be PTPN2 and STAT1 for oligoarticular JIA and KSR1 for RF-ve polyarticular JIA. Clusters of 23 and 14 related proteins were derived for oligoarticular and RF-ve polyarticular JIA respectively. Conclusions This first report of the application of network biology to JIA, integrating genetic association findings and gene expression data, has prioritised protein clusters for functional validation and identified new pathways for targeted pharmacological intervention. PMID:24886659

  18. Newly identified RNAs of raspberry leaf blotch virus encoding a related group of proteins.

    PubMed

    Lu, Yuwen; McGavin, Wendy; Cock, Peter J A; Schnettler, Esther; Yan, Fei; Chen, Jianping; MacFarlane, Stuart

    2015-11-01

    Members of the genus Emaravirus, including Raspberry leaf blotch virus (RLBV), are enveloped plant viruses with segmented genomes of negative-strand RNA, although the complete genome complement for any of these viruses is not yet clear. Currently, wheat mosaic virus has the largest emaravirus genome comprising eight RNAs. Previously, we identified five genomic RNAs for RLBV; here, we identify a further three RNAs (RNA6-8). RNA6-8 encode proteins that have clear homologies to one another, but not to any other emaravirus proteins. The proteins self-interacted in yeast two-hybrid and bimolecular fluorescence complementation (BiFC) experiments, and the P8 protein interacted with the virus nucleocapsid protein (P3) using BiFC. Expression of two of the proteins (P6 and P7) using potato virus X led to an increase in virus titre and symptom severity, suggesting that these proteins may play a role in RLBV pathogenicity; however, using two different tests, RNA silencing suppression activity was not detected for any of the RLBV proteins encoded by RNA2-8. PMID:26358478

  19. In Silico Designing and Analysis of Inhibitors against Target Protein Identified through Host-Pathogen Protein Interactions in Malaria

    PubMed Central

    Samant, Monika; Chadha, Nidhi; Tiwari, Anjani K.; Hasija, Yasha

    2016-01-01

    Malaria, a life-threatening blood disease, has been a major concern in the field of healthcare. One of the severe forms of malaria is caused by the parasite Plasmodium falciparum which is initiated through protein interactions of pathogen with the host proteins. It is essential to analyse the protein-protein interactions among the host and pathogen for better understanding of the process and characterizing specific molecular mechanisms involved in pathogen persistence and survival. In this study, a complete protein-protein interaction network of human host and Plasmodium falciparum has been generated by integration of the experimental data and computationally predicting interactions using the interolog method. The interacting proteins were filtered according to their biological significance and functional roles. α-tubulin was identified as a potential protein target and inhibitors were designed against it by modification of amiprophos methyl. Docking and binding affinity analysis showed two modified inhibitors exhibiting better docking scores of −10.5 kcal/mol and −10.43 kcal/mol and an improved binding affinity of −83.80 kJ/mol and −98.16 kJ/mol with the target. These inhibitors can further be tested and validated in vivo for their properties as an antimalarial drug. PMID:27057354

  20. Genetically encoded protein photocrosslinker with a transferable mass spectrometry-identifiable label

    PubMed Central

    Yang, Yi; Song, Haiping; He, Dan; Zhang, Shuai; Dai, Shizhong; Lin, Shixian; Meng, Rong; Wang, Chu; Chen, Peng R.

    2016-01-01

    Coupling photocrosslinking reagents with mass spectrometry has become a powerful tool for studying protein–protein interactions in living systems, but it still suffers from high rates of false-positive identifications as well as the lack of information on interaction interface due to the challenges in deciphering crosslinking peptides. Here we develop a genetically encoded photo-affinity unnatural amino acid that introduces a mass spectrometry-identifiable label (MS-label) to the captured prey proteins after photocrosslinking and prey–bait separation. This strategy, termed IMAPP (In-situ cleavage and MS-label transfer After Protein Photocrosslinking), enables direct identification of photo-captured substrate peptides that are difficult to uncover by conventional genetically encoded photocrosslinkers. Taking advantage of the MS-label, the IMAPP strategy significantly enhances the confidence for identifying protein–protein interactions and enables simultaneous mapping of the binding interface under living conditions. PMID:27460181

  1. Preparation and properties of calcium oxide from eggshells via calcination

    NASA Astrophysics Data System (ADS)

    Tangboriboon, N.; Kunanuruksapong, R.; Sirivat, A.

    2012-12-01

    Duck eggs are one of the most versatile cooking ingredients in which residue eggshells are discarded. Raw duck eggshells were calcined at temperatures between 300 to 900 °C, for 1, 3, and 5 h. Both the raw and calcined duck eggshells were characterized by FTIR, STA, XRD, XRF, TEM, BET, a particle size analyzer, and an impedance analyzer. The proper calcination conditions are: 900 °C and 1 h, yielding calcium oxide with a purity of 99.06 % w/w. The calcium carbonate of the rhombohedral form (CaCO3) transforms completely into the calcium oxide or lime of the face centered cubic form (CaO) at 900 °C, as shown by XRD diffraction patterns. The transmission electron microscopy (TEM) images of the calcium oxide reveal a moderately good dispersion of nearly uniform particles. The calcium oxide has a white color, a spherical shape, high porosity, and narrow particles size distribution. The percentage of ceramic yield of the calcium oxide is 53.53, as measured by STA (TG-DTA-DTG). The calcium oxide has a N2 adsorption-desorption isotherm indicating the meso-porosity range. The dielectric constant and the electrical conductivity of the calcined calcium oxide are 35 and 1:0×10-6(Ω·m)-1, respectively, at the frequency of 500 Hz.

  2. SCMMTP: identifying and characterizing membrane transport proteins using propensity scores of dipeptides

    PubMed Central

    2015-01-01

    Background Identifying putative membrane transport proteins (MTPs) and understanding the transport mechanisms involved remain important challenges for the advancement of structural and functional genomics. However, the transporter characters are mainly acquired from MTP crystal structures which are hard to crystalize. Therefore, it is desirable to develop bioinformatics tools for the effective large-scale analysis of available sequences to identify novel transporters and characterize such transporters. Results This work proposes a novel method (SCMMTP) based on the scoring card method (SCM) using dipeptide composition to identify and characterize MTPs from an existing dataset containing 900 MTPs and 660 non-MTPs which are separated into a training dataset consisting 1,380 proteins and an independent dataset consisting 180 proteins. The SCMMTP produced estimating propensity scores for amino acids and dipeptides as MTPs. The SCMMTP training and test accuracy levels respectively reached 83.81% and 76.11%. The test accuracy of support vector machine (SVM) using a complicated classification method with a low possibility for biological interpretation and position-specific substitution matrix (PSSM) as a protein feature is 80.56%, thus SCMMTP is comparable to SVM-PSSM. To identify MTPs, SCMMTP is applied to three datasets including: 1) human transmembrane proteins, 2) a photosynthetic protein dataset, and 3) a human protein database. MTPs showing α-helix rich structure is agreed with previous studies. The MTPs used residues with low hydration energy. It is hypothesized that, after filtering substrates, the hydrated water molecules need to be released from the pore regions. Conclusions SCMMTP yields estimating propensity scores for amino acids and dipeptides as MTPs, which can be used to identify novel MTPs and characterize transport mechanisms for use in further experiments. Availability http://iclab.life.nctu.edu.tw/iclab_webtools/SCMMTP/ PMID:26677931

  3. A Novel Proteomics Approach to Identify SUMOylated Proteins and Their Modification Sites in Human Cells*

    PubMed Central

    Galisson, Frederic; Mahrouche, Louiza; Courcelles, Mathieu; Bonneil, Eric; Meloche, Sylvain; Chelbi-Alix, Mounira K.; Thibault, Pierre

    2011-01-01

    The small ubiquitin-related modifier (SUMO) is a small group of proteins that are reversibly attached to protein substrates to modify their functions. The large scale identification of protein SUMOylation and their modification sites in mammalian cells represents a significant challenge because of the relatively small number of in vivo substrates and the dynamic nature of this modification. We report here a novel proteomics approach to selectively enrich and identify SUMO conjugates from human cells. We stably expressed different SUMO paralogs in HEK293 cells, each containing a His6 tag and a strategically located tryptic cleavage site at the C terminus to facilitate the recovery and identification of SUMOylated peptides by affinity enrichment and mass spectrometry. Tryptic peptides with short SUMO remnants offer significant advantages in large scale SUMOylome experiments including the generation of paralog-specific fragment ions following CID and ETD activation, and the identification of modified peptides using conventional database search engines such as Mascot. We identified 205 unique protein substrates together with 17 precise SUMOylation sites present in 12 SUMO protein conjugates including three new sites (Lys-380, Lys-400, and Lys-497) on the protein promyelocytic leukemia. Label-free quantitative proteomics analyses on purified nuclear extracts from untreated and arsenic trioxide-treated cells revealed that all identified SUMOylated sites of promyelocytic leukemia were differentially SUMOylated upon stimulation. PMID:21098080

  4. Use of a Probabilistic Motif Search to Identify Histidine Phosphotransfer Domain-Containing Proteins.

    PubMed

    Surujon, Defne; Ratner, David I

    2016-01-01

    The wealth of newly obtained proteomic information affords researchers the possibility of searching for proteins of a given structure or function. Here we describe a general method for the detection of a protein domain of interest in any species for which a complete proteome exists. In particular, we apply this approach to identify histidine phosphotransfer (HPt) domain-containing proteins across a range of eukaryotic species. From the sequences of known HPt domains, we created an amino acid occurrence matrix which we then used to define a conserved, probabilistic motif. Examination of various organisms either known to contain (plant and fungal species) or believed to lack (mammals) HPt domains established criteria by which new HPt candidates were identified and ranked. Search results using a probabilistic motif matrix compare favorably with data to be found in several commonly used protein structure/function databases: our method identified all known HPt proteins in the Arabidopsis thaliana proteome, confirmed the absence of such motifs in mice and humans, and suggests new candidate HPts in several organisms. Moreover, probabilistic motif searching can be applied more generally, in a manner both readily customized and computationally compact, to other protein domains; this utility is demonstrated by our identification of histones in a range of eukaryotic organisms. PMID:26751210

  5. Linking patient outcome to high throughput protein expression data identifies novel regulators of colorectal adenocarcinoma aggressiveness

    PubMed Central

    French, Christi L.; Ye, Fei; Revetta, Frank; Zhang, Bing; Coffey, Robert J.; Washington, M. Kay; Deane, Natasha G.; Beauchamp, R. Daniel; Weaver, Alissa M.

    2015-01-01

    A key question in cancer systems biology is how to use molecular data to predict the biological behavior of tumors from individual patients. While genomics data have been heavily used, protein signaling data are more directly connected to biological phenotype and might predict cancer phenotypes such as invasion, metastasis, and patient survival. In this study, we mined publicly available data for colorectal adenocarcinoma from the Cancer Genome Atlas and identified protein expression and signaling changes that are statistically associated with patient outcome. Our analysis identified a number of known and potentially new regulators of colorectal cancer. High levels of insulin growth factor binding protein 2 (IGFBP2) were associated with both recurrence and death, and this was validated by immunohistochemical staining of a tissue microarray for a secondary patient dataset. Interestingly, GATA binding protein 3 (GATA3) was the protein most frequently associated with death in our analysis, and GATA3 expression was significantly decreased in tumor samples from stage I-II deceased patients. Experimental studies using engineered colon cancer cell lines show that exogenous expression of GATA3 decreases three-dimensional colony growth and invasiveness of colon cancer cells but does not affect two-dimensional proliferation. These findings suggest that protein data are useful for biomarker discovery and identify GATA3 as a regulator of colorectal cancer  aggressiveness. PMID:26097693

  6. Use of a Probabilistic Motif Search to Identify Histidine Phosphotransfer Domain-Containing Proteins

    PubMed Central

    Surujon, Defne; Ratner, David I.

    2016-01-01

    The wealth of newly obtained proteomic information affords researchers the possibility of searching for proteins of a given structure or function. Here we describe a general method for the detection of a protein domain of interest in any species for which a complete proteome exists. In particular, we apply this approach to identify histidine phosphotransfer (HPt) domain-containing proteins across a range of eukaryotic species. From the sequences of known HPt domains, we created an amino acid occurrence matrix which we then used to define a conserved, probabilistic motif. Examination of various organisms either known to contain (plant and fungal species) or believed to lack (mammals) HPt domains established criteria by which new HPt candidates were identified and ranked. Search results using a probabilistic motif matrix compare favorably with data to be found in several commonly used protein structure/function databases: our method identified all known HPt proteins in the Arabidopsis thaliana proteome, confirmed the absence of such motifs in mice and humans, and suggests new candidate HPts in several organisms. Moreover, probabilistic motif searching can be applied more generally, in a manner both readily customized and computationally compact, to other protein domains; this utility is demonstrated by our identification of histones in a range of eukaryotic organisms. PMID:26751210

  7. Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast

    PubMed Central

    Huang, Mingtao; Bai, Yunpeng; Sjostrom, Staffan L.; Hallström, Björn M.; Liu, Zihe; Petranovic, Dina; Uhlén, Mathias; Joensson, Haakan N.; Andersson-Svahn, Helene; Nielsen, Jens

    2015-01-01

    There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant α-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories. PMID:26261321

  8. Preparation of gold nanoparticles on eggshell membrane and their biosensing application.

    PubMed

    Zheng, Baozhan; Qian, Lei; Yuan, Hongyan; Xiao, Dan; Yang, Xiupei; Paau, Man Chin; Choi, Martin M F

    2010-06-30

    A facile green biosynthesis method has been successfully developed to prepare gold nanoparticles (AuNPs) of various core sizes (25+/-7 nm) using a natural biomaterial, eggshell membrane (ESM) at ambient conditions. In situ synthesis of AuNPs-immobilized ESM is conducted in a simple manner by immersing ESM in a pH 6.0 aqueous solution of HAuCl(4) without adding any reductant. The formation of AuNPs on ESM protein fibers is attributed to the reduction of Au(III) ions to Au(0) by the aldehyde moieties of the natural ESM fibers. Energy dispersive X-ray spectroscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, and X-ray powder diffraction unambiguously identify the presence of AuNPs on ESM. The effect of pH on the in situ synthesis of AuNPs on ESM has been investigated in detail. The pH of the gold precursor (HAuCl(4)) solution can influence the formation rate, dispersion and size of AuNPs on ESM. At pH < or =3.0 and > or =7.0, no AuNPs are observed on ESM while small AuNPs are homogeneously dispersed on ESM at pH 4.0-6.0. The optimal pH for AuNPs formation on ESM is 6.0. AuNPs/ESMs are used to immobilize glucose oxidase (GO(x)) for glucose biosensing. AuNPs on ESM can increase the enzyme activity of GO(x). The linear response range of the glucose biosensor is 20 microM to 0.80 mM glucose with a detection limit of 17 microM (S/N=3). The biosensor has been successfully applied to determine the glucose content in commercial glucose injections. Our work provides a very simple, non-toxic, convenient, and green route to synthesize AuNPs on ESM which is potentially useful in the biosensing field. PMID:20685454

  9. Laser capture microdissection to identify septum-associated proteins in Aspergillus nidulans.

    PubMed

    Zhang, Ying; Fischer, Reinhard; Teichert, Ines; Kück, Ulrich

    2016-01-01

    To spatially resolve genetic differences at the cellular level, the laser-capture microdissection technique was developed. With this method cells can be cut from tissues with a laser beam and analyzed for DNA, RNA or protein composition. Here we adapted the technique to isolate septal microtubule-organizing center (MTOC)-associated proteins in Aspergillus nidulans About 3000 septa were collected and subjected to peptide fingerprinting by mass-spectrometric analysis. We identified the microtubule polymerase AlpA and found it interacts with ApsB specifically at sMTOCs, suggesting that AlpA might be involved in the assembly or the functioning of this protein complex. PMID:26951366

  10. Proteomics Approaches to Identify Mono(ADP-ribosyl)ated and Poly(ADP-ribosyl)ated proteins

    PubMed Central

    Vivelo, Christina A.; Leung, Anthony K. L.

    2015-01-01

    ADP-ribosylation refers to the addition of one or more ADP-ribose units onto protein substrates and this protein modification has been implicated in various cellular processes including DNA damage repair, RNA metabolism, transcription and cell cycle regulation. This review focuses on a compilation of large-scale proteomics studies that identify ADP-ribosylated proteins and their associated proteins by mass spectrometry using a variety of enrichment strategies. Some methods, such as the use of a poly(ADP-ribose)-specific antibody and boronate affinity chromatography and NAD+ analogues, have been employed for decades while others, such as the use of protein microarrays and recombinant proteins that bind ADP-ribose moieties (such as macrodomains), have only recently been developed. The advantages and disadvantages of each method and whether these methods are specific for identifying mono(ADP-ribosyl)ated and poly(ADP-ribosyl)ated proteins will be discussed. Lastly, since poly(ADP-ribose) is heterogeneous in length, it has been difficult to attain a mass signature associated with the modification sites. Several strategies on how to reduce polymer chain length heterogeneity for site identification will be reviewed. PMID:25263235

  11. Several novel nuclear envelope transmembrane proteins identified in skeletal muscle have cytoskeletal associations.

    PubMed

    Wilkie, Gavin S; Korfali, Nadia; Swanson, Selene K; Malik, Poonam; Srsen, Vlastimil; Batrakou, Dzmitry G; de las Heras, Jose; Zuleger, Nikolaj; Kerr, Alastair R W; Florens, Laurence; Schirmer, Eric C

    2011-01-01

    Nuclear envelopes from liver and a neuroblastoma cell line have previously been analyzed by proteomics; however, most diseases associated with the nuclear envelope affect muscle. To determine whether muscle has unique nuclear envelope proteins, rat skeletal muscle nuclear envelopes were prepared and analyzed by multidimensional protein identification technology. Many novel muscle-specific proteins were identified that did not appear in previous nuclear envelope data sets. Nuclear envelope residence was confirmed for 11 of these by expression of fusion proteins and by antibody staining of muscle tissue cryosections. Moreover, transcript levels for several of the newly identified nuclear envelope transmembrane proteins increased during muscle differentiation using mouse and human in vitro model systems. Some of these proteins tracked with microtubules at the nuclear surface in interphase cells and accumulated at the base of the microtubule spindle in mitotic cells, suggesting they may associate with complexes that connect the nucleus to the cytoskeleton. The finding of tissue-specific proteins in the skeletal muscle nuclear envelope proteome argues the importance of analyzing nuclear envelopes from all tissues linked to disease and suggests that general investigation of tissue differences in organellar proteomes might yield critical insights. PMID:20876400

  12. Several Novel Nuclear Envelope Transmembrane Proteins Identified in Skeletal Muscle Have Cytoskeletal Associations*

    PubMed Central

    Wilkie, Gavin S.; Korfali, Nadia; Swanson, Selene K.; Malik, Poonam; Srsen, Vlastimil; Batrakou, Dzmitry G.; de las Heras, Jose; Zuleger, Nikolaj; Kerr, Alastair R. W.; Florens, Laurence; Schirmer, Eric C.

    2011-01-01

    Nuclear envelopes from liver and a neuroblastoma cell line have previously been analyzed by proteomics; however, most diseases associated with the nuclear envelope affect muscle. To determine whether muscle has unique nuclear envelope proteins, rat skeletal muscle nuclear envelopes were prepared and analyzed by multidimensional protein identification technology. Many novel muscle-specific proteins were identified that did not appear in previous nuclear envelope data sets. Nuclear envelope residence was confirmed for 11 of these by expression of fusion proteins and by antibody staining of muscle tissue cryosections. Moreover, transcript levels for several of the newly identified nuclear envelope transmembrane proteins increased during muscle differentiation using mouse and human in vitro model systems. Some of these proteins tracked with microtubules at the nuclear surface in interphase cells and accumulated at the base of the microtubule spindle in mitotic cells, suggesting they may associate with complexes that connect the nucleus to the cytoskeleton. The finding of tissue-specific proteins in the skeletal muscle nuclear envelope proteome argues the importance of analyzing nuclear envelopes from all tissues linked to disease and suggests that general investigation of tissue differences in organellar proteomes might yield critical insights. PMID:20876400

  13. Proteomics approaches to identify mono-(ADP-ribosyl)ated and poly(ADP-ribosyl)ated proteins.

    PubMed

    Vivelo, Christina A; Leung, Anthony K L

    2015-01-01

    ADP-ribosylation refers to the addition of one or more ADP-ribose units onto protein substrates and this protein modification has been implicated in various cellular processes including DNA damage repair, RNA metabolism, transcription, and cell cycle regulation. This review focuses on a compilation of large-scale proteomics studies that identify ADP-ribosylated proteins and their associated proteins by MS using a variety of enrichment strategies. Some methods, such as the use of a poly(ADP-ribose)-specific antibody and boronate affinity chromatography and NAD(+) analogues, have been employed for decades while others, such as the use of protein microarrays and recombinant proteins that bind ADP-ribose moieties (such as macrodomains), have only recently been developed. The advantages and disadvantages of each method and whether these methods are specific for identifying mono(ADP-ribosyl)ated and poly(ADP-ribosyl)ated proteins will be discussed. Lastly, since poly(ADP-ribose) is heterogeneous in length, it has been difficult to attain a mass signature associated with the modification sites. Several strategies on how to reduce polymer chain length heterogeneity for site identification will be reviewed. PMID:25263235

  14. Egg-in-cube: design and fabrication of a novel artificial eggshell with functionalized surface.

    PubMed

    Huang, Wenjing; Arai, Fumihito; Kawahara, Tomohiro

    2015-01-01

    An eggshell is a porous microstructure that regulates the passage of gases to allow respiration. The chick embryo and its circulatory system enclosed by the eggshell has become an important model for biomedical research such as the control of angiogenesis, cancer therapy, and drug delivery test, because the use of embryo is ethically acceptable and it is inexpensive and small. However, chick embryo and extra-embryonic blood vessels cannot be accessed freely and has poor observability because the eggshell is tough and cannot be seen through, which limits its application. In this study, a novel artificial eggshell with functionalized surface is proposed, which allows the total amount of oxygen to pass into the egg for the chick embryo culturing and has high observability and accessibility for embryo manipulation. First, a 40-mm enclosed cubic-shaped eggshell consisting of a membrane structure and a rigid frame structure is designed, and then the threshold of the membrane thickness suitable for the embryo survival is figured out according to the oxygen-permeability of the membrane structure. The designed artificial eggshell was actually fabricated by using polydimethylsiloxane (PDMS) and polycarbonate (PC) in the current study. Using the fabricated eggshell, chick embryo and extra-embryonic blood vessels can be observed from multiple directions. To test the effectiveness of the design, the cubic eggshells were used to culture chick embryos and survivability was confirmed when PDMS membranes with adequate oxygen permeability were used. Since the surface of the eggshell is transparent, chick embryo tissue development could be observed during the culture period. Additionally, the chick embryo tissues could be accessed and manipulated from outside the cubic eggshell, by using mechanical tools without breakage of the eggshell. The proposed "Egg-in-Cube" with functionalized surface has great potential to serve as a promising platform for biomedical research. PMID:25768929

  15. Egg-in-Cube: Design and Fabrication of a Novel Artificial Eggshell with Functionalized Surface

    PubMed Central

    Huang, Wenjing; Arai, Fumihito; Kawahara, Tomohiro

    2015-01-01

    An eggshell is a porous microstructure that regulates the passage of gases to allow respiration. The chick embryo and its circulatory system enclosed by the eggshell has become an important model for biomedical research such as the control of angiogenesis, cancer therapy, and drug delivery test, because the use of embryo is ethically acceptable and it is inexpensive and small. However, chick embryo and extra-embryonic blood vessels cannot be accessed freely and has poor observability because the eggshell is tough and cannot be seen through, which limits its application. In this study, a novel artificial eggshell with functionalized surface is proposed, which allows the total amount of oxygen to pass into the egg for the chick embryo culturing and has high observability and accessibility for embryo manipulation. First, a 40-mm enclosed cubic-shaped eggshell consisting of a membrane structure and a rigid frame structure is designed, and then the threshold of the membrane thickness suitable for the embryo survival is figured out according to the oxygen-permeability of the membrane structure. The designed artificial eggshell was actually fabricated by using polydimethylsiloxane (PDMS) and polycarbonate (PC) in the current study. Using the fabricated eggshell, chick embryo and extra-embryonic blood vessels can be observed from multiple directions. To test the effectiveness of the design, the cubic eggshells were used to culture chick embryos and survivability was confirmed when PDMS membranes with adequate oxygen permeability were used. Since the surface of the eggshell is transparent, chick embryo tissue development could be observed during the culture period. Additionally, the chick embryo tissues could be accessed and manipulated from outside the cubic eggshell, by using mechanical tools without breakage of the eggshell. The proposed “Egg-in-Cube” with functionalized surface has great potential to serve as a promising platform for biomedical research. PMID

  16. Nutritional Supplement of Hatchery Eggshell Membrane Improves Poultry Performance and Provides Resistance against Endotoxin Stress

    PubMed Central

    Makkar, S. K.; Rath, N. C.; Packialakshmi, B.; Zhou, Z. Y.; Huff, G. R.; Donoghue, A. M.

    2016-01-01

    Eggshells are significant part of hatchery waste which consist of calcium carbonate crust, membranes, and proteins and peptides of embryonic origins along with other entrapped contaminants including microbes. We hypothesized that using this product as a nutritional additive in poultry diet may confer better immunity to the chickens in the paradigm of mammalian milk that enhances immunity. Therefore, we investigated the effect of hatchery eggshell membranes (HESM) as a short term feed supplement on growth performance and immunity of chickens under bacterial lipopolysaccharide (LPS) challenged condition. Three studies were conducted to find the effect of HESM supplement on post hatch chickens. In the first study, the chickens were fed either a control diet or diets containing 0.5% whey protein or HESM as supplement and evaluated at 5 weeks of age using growth, hematology, clinical chemistry, plasma immunoglobulins, and corticosterone as variables. The second and third studies were done to compare the effects of LPS on control and HESM fed birds at 5 weeks of age following at 4 and 24 h of treatment where the HESM was also sterilized with ethanol to deplete bacterial factors. HESM supplement caused weight gain in 2 experiments and decreased blood corticosterone concentrations. While LPS caused a significant loss in body weight at 24 h following its administration, the HESM supplemented birds showed significantly less body weight loss compared with the control fed birds. The WBC, heterophil/lymphocyte ratio, and the levels of IgG were low in chickens fed diets with HESM supplement compared with control diet group. LPS challenge increased the expression of pro-inflammatory cytokine gene IL-6 but the HESM fed birds showed its effect curtailed, also, which also, favored the up-regulation of anti-inflammatory genes compared with control diet fed chickens. Post hatch supplementation of HESM appears to improve performance, modulate immunity, and increase resistance of

  17. Nutritional Supplement of Hatchery Eggshell Membrane Improves Poultry Performance and Provides Resistance against Endotoxin Stress.

    PubMed

    Makkar, S K; Rath, N C; Packialakshmi, B; Zhou, Z Y; Huff, G R; Donoghue, A M

    2016-01-01

    Eggshells are significant part of hatchery waste which consist of calcium carbonate crust, membranes, and proteins and peptides of embryonic origins along with other entrapped contaminants including microbes. We hypothesized that using this product as a nutritional additive in poultry diet may confer better immunity to the chickens in the paradigm of mammalian milk that enhances immunity. Therefore, we investigated the effect of hatchery eggshell membranes (HESM) as a short term feed supplement on growth performance and immunity of chickens under bacterial lipopolysaccharide (LPS) challenged condition. Three studies were conducted to find the effect of HESM supplement on post hatch chickens. In the first study, the chickens were fed either a control diet or diets containing 0.5% whey protein or HESM as supplement and evaluated at 5 weeks of age using growth, hematology, clinical chemistry, plasma immunoglobulins, and corticosterone as variables. The second and third studies were done to compare the effects of LPS on control and HESM fed birds at 5 weeks of age following at 4 and 24 h of treatment where the HESM was also sterilized with ethanol to deplete bacterial factors. HESM supplement caused weight gain in 2 experiments and decreased blood corticosterone concentrations. While LPS caused a significant loss in body weight at 24 h following its administration, the HESM supplemented birds showed significantly less body weight loss compared with the control fed birds. The WBC, heterophil/lymphocyte ratio, and the levels of IgG were low in chickens fed diets with HESM supplement compared with control diet group. LPS challenge increased the expression of pro-inflammatory cytokine gene IL-6 but the HESM fed birds showed its effect curtailed, also, which also, favored the up-regulation of anti-inflammatory genes compared with control diet fed chickens. Post hatch supplementation of HESM appears to improve performance, modulate immunity, and increase resistance of

  18. Comparative interactomics analysis of different ALS-associated proteins identifies converging molecular pathways.

    PubMed

    Blokhuis, Anna M; Koppers, Max; Groen, Ewout J N; van den Heuvel, Dianne M A; Dini Modigliani, Stefano; Anink, Jasper J; Fumoto, Katsumi; van Diggelen, Femke; Snelting, Anne; Sodaar, Peter; Verheijen, Bert M; Demmers, Jeroen A A; Veldink, Jan H; Aronica, Eleonora; Bozzoni, Irene; den Hertog, Jeroen; van den Berg, Leonard H; Pasterkamp, R Jeroen

    2016-08-01

    Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment available. An increasing number of genetic causes of ALS are being identified, but how these genetic defects lead to motor neuron degeneration and to which extent they affect common cellular pathways remains incompletely understood. To address these questions, we performed an interactomic analysis to identify binding partners of wild-type (WT) and ALS-associated mutant versions of ATXN2, C9orf72, FUS, OPTN, TDP-43 and UBQLN2 in neuronal cells. This analysis identified several known but also many novel binding partners of these proteins. Interactomes of WT and mutant ALS proteins were very similar except for OPTN and UBQLN2, in which mutations caused loss or gain of protein interactions. Several of the identified interactomes showed a high degree of overlap: shared binding partners of ATXN2, FUS and TDP-43 had roles in RNA metabolism; OPTN- and UBQLN2-interacting proteins were related to protein degradation and protein transport, and C9orf72 interactors function in mitochondria. To confirm that this overlap is important for ALS pathogenesis, we studied fragile X mental retardation protein (FMRP), one of the common interactors of ATXN2, FUS and TDP-43, in more detail in in vitro and in vivo model systems for FUS ALS. FMRP localized to mutant FUS-containing aggregates in spinal motor neurons and bound endogenous FUS in a direct and RNA-sensitive manner. Furthermore, defects in synaptic FMRP mRNA target expression, neuromuscular junction integrity, and motor behavior caused by mutant FUS in zebrafish embryos, could be rescued by exogenous FMRP expression. Together, these results show that interactomics analysis can provide crucial insight into ALS disease mechanisms and they link FMRP to motor neuron dysfunction caused by FUS mutations. PMID:27164932

  19. vProtein: Identifying Optimal Amino Acid Complements from Plant-Based Foods

    PubMed Central

    Woolf, Peter J.; Fu, Leeann L.; Basu, Avik

    2011-01-01

    Background Indispensible amino acids (IAAs) are used by the body in different proportions. Most animal-based foods provide these IAAs in roughly the needed proportions, but many plant-based foods provide different proportions of IAAs. To explore how these plant-based foods can be better used in human nutrition, we have created the computational tool vProtein to identify optimal food complements to satisfy human protein needs. Methods vProtein uses 1251 plant-based foods listed in the United States Department of Agriculture standard release 22 database to determine the quantity of each food or pair of foods required to satisfy human IAA needs as determined by the 2005 daily recommended intake. The quantity of food in a pair is found using a linear programming approach that minimizes total calories, total excess IAAs, or the total weight of the combination. Results For single foods, vProtein identifies foods with particularly balanced IAA patterns such as wheat germ, quinoa, and cauliflower. vProtein also identifies foods with particularly unbalanced IAA patterns such as macadamia nuts, degermed corn products, and wakame seaweed. Although less useful alone, some unbalanced foods provide unusually good complements, such as Brazil nuts to legumes. Interestingly, vProtein finds no statistically significant bias toward grain/legume pairings for protein complementation. These analyses suggest that pairings of plant-based foods should be based on the individual foods themselves instead of based on broader food group-food group pairings. Overall, the most efficient pairings include sweet corn/tomatoes, apple/coconut, and sweet corn/cherry. The top pairings also highlight the utility of less common protein sources such as the seaweeds laver and spirulina, pumpkin leaves, and lambsquarters. From a public health perspective, many of the food pairings represent novel, low cost food sources to combat malnutrition. Full analysis results are available online at http

  20. Mass Spectrometry-Based Methods for Identifying Oxidized Proteins in Disease: Advances and Challenges

    PubMed Central

    Verrastro, Ivan; Pasha, Sabah; Tveen Jensen, Karina; Pitt, Andrew R.; Spickett, Corinne M.

    2015-01-01

    Many inflammatory diseases have an oxidative aetiology, which leads to oxidative damage to biomolecules, including proteins. It is now increasingly recognized that oxidative post-translational modifications (oxPTMs) of proteins affect cell signalling and behaviour, and can contribute to pathology. Moreover, oxidized proteins have potential as biomarkers for inflammatory diseases. Although many assays for generic protein oxidation and breakdown products of protein oxidation are available, only advanced tandem mass spectrometry approaches have the power to localize specific oxPTMs in identified proteins. While much work has been carried out using untargeted or discovery mass spectrometry approaches, identification of oxPTMs in disease has benefitted from the development of sophisticated targeted or semi-targeted scanning routines, combined with chemical labeling and enrichment approaches. Nevertheless, many potential pitfalls exist which can result in incorrect identifications. This review explains the limitations, advantages and challenges of all of these approaches to detecting oxidatively modified proteins, and provides an update on recent literature in which they have been used to detect and quantify protein oxidation in disease. PMID:25874603

  1. Using distant supervised learning to identify protein subcellular localizations from full-text scientific articles.

    PubMed

    Zheng, Wu; Blake, Catherine

    2015-10-01

    Databases of curated biomedical knowledge, such as the protein-locations reflected in the UniProtKB database, provide an accurate and useful resource to researchers and decision makers. Our goal is to augment the manual efforts currently used to curate knowledge bases with automated approaches that leverage the increased availability of full-text scientific articles. This paper describes experiments that use distant supervised learning to identify protein subcellular localizations, which are important to understand protein function and to identify candidate drug targets. Experiments consider Swiss-Prot, the manually annotated subset of the UniProtKB protein knowledge base, and 43,000 full-text articles from the Journal of Biological Chemistry that contain just under 11.5 million sentences. The system achieves 0.81 precision and 0.49 recall at sentence level and an accuracy of 57% on held-out instances in a test set. Moreover, the approach identifies 8210 instances that are not in the UniProtKB knowledge base. Manual inspection of the 50 most likely relations showed that 41 (82%) were valid. These results have immediate benefit to researchers interested in protein function, and suggest that distant supervision should be explored to complement other manual data curation efforts. PMID:26220461

  2. Crystallization Optimum Solubility Screening: using crystallization results to identify the optimal buffer for protein crystal formation

    SciTech Connect

    Collins, Bernard; Stevens, Raymond C.; Page, Rebecca

    2005-12-01

    It is shown how protein crystallization results can be used to identify buffers that improve protein solubility and, in turn, crystallization success. An optimal solubility screen is described that uses the results of crystallization trials to identify buffers that improve protein solubility and, in turn, crystallization success. This screen is useful not only for standard crystallization experiments, but also can easily be implemented into any high-throughput structure-determination pipeline. As a proof of principle, the predicted novel-fold protein AF2059 from Archaeoglobus fulgidus, which was known to precipitate in most buffers and particularly during concentration experiments, was selected. Using the crystallization results of 192 independent crystallization trials, it was possible to identify a buffer containing 100 mM CHES pH 9.25 that significantly improves its solubility. After transferring AF2059 into this ‘optimum-solubility’ buffer, the protein was rescreened for crystal formation against these same 192 conditions. Instead of extensive precipitation, as observed initially, it was found that 24 separate conditions produced crystals and the exchange of AF2059 into CHES buffer significantly improved crystallization success. Fine-screen optimization of these conditions led to the production of a crystal suitable for high-resolution (2.2 Å) structure determination.

  3. N-Ace: using solvent accessibility and physicochemical properties to identify protein N-acetylation sites.

    PubMed

    Lee, Tzong-Yi; Hsu, Justin Bo-Kai; Lin, Feng-Mao; Chang, Wen-Chi; Hsu, Po-Chiang; Huang, Hsien-Da

    2010-11-30

    Protein acetylation, which is catalyzed by acetyltransferases, is a type of post-translational modification and crucial to numerous essential biological processes, including transcriptional regulation, apoptosis, and cytokine signaling. As the experimental identification of protein acetylation sites is time consuming and laboratory intensive, several computational approaches have been developed for identifying the candidates of experimental validation. In this work, solvent accessibility and the physicochemical properties of proteins are utilized to identify acetylated alanine, glycine, lysine, methionine, serine, and threonine. A two-stage support vector machine was applied to learn the computational models with combinations of amino acid sequences, and the accessible surface area and physicochemical properties of proteins. The predictive accuracy thus achieved is 5% to 14% higher than that of models trained using only amino acid sequences. Additionally, the substrate specificity of the acetylated site was investigated in detail with reference to the subcellular colocalization of acetyltransferases and acetylated proteins. The proposed method, N-Ace, is evaluated using independent test sets in various acetylated residues and predictive accuracies of 90% were achieved, indicating that the performance of N-Ace is comparable with that of other acetylation prediction methods. N-Ace not only provides a user-friendly input/output interface but also is a creative method for predicting protein acetylation sites. This novel analytical resource is now freely available at http://N-Ace.mbc.NCTU.edu.tw/. PMID:20839302

  4. Interactions between the Powdery Mildew Effector BEC1054 and Barley Proteins Identify Candidate Host Targets.

    PubMed

    Pennington, Helen G; Gheorghe, Dana M; Damerum, Annabelle; Pliego, Clara; Spanu, Pietro D; Cramer, Rainer; Bindschedler, Laurence V

    2016-03-01

    There are over 500 candidate secreted effector proteins (CSEPs) or Blumeria effector candidates (BECs) specific to the barley powdery mildew pathogen Blumeria graminis f.sp. hordei. The CSEP/BEC proteins are expressed and predicted to be secreted by biotrophic feeding structures called haustoria. Eight BECs are required for the formation of functional haustoria. These include the RNase-like effector BEC1054 (synonym CSEP0064). In order to identify host proteins targeted by BEC1054, recombinant BEC1054 was expressed in E. coli, solubilized, and used in pull-down assays from barley protein extracts. Many putative interactors were identified by LC-MS/MS after subtraction of unspecific binders in negative controls. Therefore, a directed yeast-2-hybrid assay, developed to measure the effectiveness of the interactions in yeast, was used to validate putative interactors. We conclude that BEC1054 may target several host proteins, including a glutathione-S-transferase, a malate dehydrogenase, and a pathogen-related-5 protein isoform, indicating a possible role for BEC1054 in compromising well-known key players of defense and response to pathogens. In addition, BEC1054 interacts with an elongation factor 1 gamma. This study already suggests that BEC1054 plays a central role in barley powdery mildew virulence by acting at several levels. PMID:26813582

  5. Identifying Similar Patterns of Structural Flexibility in Proteins by Disorder Prediction and Dynamic Programming

    PubMed Central

    Petrovich, Aidan; Borne, Adam; Uversky, Vladimir N.; Xue, Bin

    2015-01-01

    Computational methods are prevailing in identifying protein intrinsic disorder. The results from predictors are often given as per-residue disorder scores. The scores describe the disorder propensity of amino acids of a protein and can be further represented as a disorder curve. Many proteins share similar patterns in their disorder curves. The similar patterns are often associated with similar functions and evolutionary origins. Therefore, finding and characterizing specific patterns of disorder curves provides a unique and attractive perspective of studying the function of intrinsically disordered proteins. In this study, we developed a new computational tool named IDalign using dynamic programming. This tool is able to identify similar patterns among disorder curves, as well as to present the distribution of intrinsic disorder in query proteins. The disorder-based information generated by IDalign is significantly different from the information retrieved from classical sequence alignments. This tool can also be used to infer functions of disordered regions and disordered proteins. The web server of IDalign is available at (http://labs.cas.usf.edu/bioinfo/service.html). PMID:26086829

  6. Towards identifying Brassica proteins involved in mediating resistance to Leptosphaeria maculans: a proteomics-based approach.

    PubMed

    Sharma, Nidhi; Hotte, Naomi; Rahman, Muhammad H; Mohammadi, Mohsen; Deyholos, Michael K; Kav, Nat N V

    2008-09-01

    To better understand the pathogen-stress response of Brassica species against the ubiquitous hemi-biotroph fungus Leptosphaeria maculans, we conducted a comparative proteomic analysis between blackleg-susceptible Brassica napus and blackleg-resistant Brassica carinata following pathogen inoculation. We examined temporal changes (6, 12, 24, 48 and 72 h) in protein profiles of both species subjected to pathogen-challenge using two-dimensional gel electrophoresis. A total of 64 proteins were found to be significantly affected by the pathogen in the two species, out of which 51 protein spots were identified using tandem mass spectrometry. The proteins identified included antioxidant enzymes, photosynthetic and metabolic enzymes, and those involved in protein processing and signaling. Specifically, we observed that in the tolerant B. carinata, enzymes involved in the detoxification of free radicals increased in response to the pathogen whereas no such increase was observed in the susceptible B. napus. The expression of genes encoding four selected proteins was validated using quantitative real-time PCR and an additional one by Western blotting. Our findings are discussed with respect to tolerance or susceptibility of these species to the pathogen. PMID:18668695

  7. Multiple biomarker tissue arrays: A computational approach to identifying protein-protein interactions in the EGFR/ERK signalling pathway

    PubMed Central

    2012-01-01

    Background Many studies have demonstrated genetic and environmental factors that lead to renal cell carcinoma (RCC) and that occur during a protracted period of tumourigenesis. It appears suitable to identify and characterise potential molecular markers that appear during tumourigenesis and that might provide rapid and effective possibilities for the early detection of RCC. EGFR activation induces cell cycle progression, inhibition of apoptosis and angiogenesis, promotion of invasion/metastasis, and other tumour promoting activities. Over-expression of EGFR is thought to play an important role in tumour initiation and progression of RCC because up-regulation of EGFR has been associated with high grade cancers and a worse prognosis. Methods Characterisation of the protein profile interacting with EGFR was performed using the following: an immunohistochemical (IHC) study of EGFR, a comprehensive computational study of EGFR protein-protein interactions, an analysis correlating the expression levels of EGFR with other significant markers in the tumourigenicity of RCC, and finally, an analysis of the utility of EGFR for prognosis in a cohort of patients with renal cell carcinoma. Results The cases that showed a higher level of this protein fell within the clear cell histological subtype (p = 0.001). The EGFR significance statistic was found with respect to a worse prognosis. In vivo significant correlations were found with PDGFR-β, Flk-1, Hif1-α, proteins related to differentiation (such as DLL3 and DLL4 ligands), and certain metabolic proteins such as Glut5. In silico significant associations gave us a panel of 32 EGFR-interacting proteins (EIP) using the APID and STRING databases. Conclusions This work summarises the multifaceted role of EGFR in the pathology of RCC, and it identifies EIPs that could help to provide mechanistic explanations for the different behaviours observed in tumours. PMID:22937740

  8. Proteomic profiling of human plasma exosomes identifies PPAR{gamma} as an exosome-associated protein

    SciTech Connect

    Looze, Christopher; Yui, David; Leung, Lester; Ingham, Matthew; Kaler, Maryann; Yao, Xianglan; Wu, Wells W.; Shen Rongfong; Daniels, Mathew P.; Levine, Stewart J.

    2009-01-16

    Exosomes are nanovesicles that are released from cells as a mechanism of cell-free intercellular communication. Only a limited number of proteins have been identified from the plasma exosome proteome. Here, we developed a multi-step fractionation scheme incorporating gel exclusion chromatography, rate zonal centrifugation through continuous sucrose gradients, and high-speed centrifugation to purify exosomes from human plasma. Exosome-associated proteins were separated by SDS-PAGE and 66 proteins were identified by LC-MS/MS, which included both cellular and extracellular proteins. Furthermore, we identified and characterized peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}), a nuclear receptor that regulates adipocyte differentiation and proliferation, as well as immune and inflammatory cell functions, as a novel component of plasma-derived exosomes. Given the important role of exosomes as intercellular messengers, the discovery of PPAR{gamma} as a component of human plasma exosomes identifies a potential new pathway for the paracrine transfer of nuclear receptors.

  9. Peptides identified in soybean protein increase plasma cholesterol in mice on hypercholesterolemic diets

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The in vitro micellar cholesterol displacement assay has been used to identify peptides that may potentially reduce cholesterol in vivo. We tested two of these peptides, LPYPR and WGAPSI, derived from soybean protein (SP) that have been reported to displace cholesterol from micelles by feeding them...

  10. Sorption mechanism of Cd(II) from water solution onto chicken eggshell

    NASA Astrophysics Data System (ADS)

    Flores-Cano, Jose Valente; Leyva-Ramos, Roberto; Mendoza-Barron, Jovita; Guerrero-Coronado, Rosa María; Aragón-Piña, Antonio; Labrada-Delgado, Gladis Judith

    2013-07-01

    The mechanism and capacity of eggshell for sorbing Cd(II) from aqueous solution was examined in detail. The eggshell was characterized by several techniques. The eggshell was mainly composed of Calcite (CaCO3). The surface charge distribution was determined by acid-base titration and the point of zero charge (PZC) of the eggshell was found to be 11.4. The sorption equilibrium data were obtained in a batch adsorber, and the adsorption isotherm of Langmuir fitted the data quite well. The sorption capacity of eggshell increased while raising the pH from 4 to 6, this tendency was attributed to the electrostatic interaction between the Cd2+ in solution and the surface of the eggshell. Furthermore, the sorption capacity was augmented by increasing the temperature from 15 to 35 °C because the sorption was endothermic. The sorption of Cd(II) occurred mainly onto the calcareous layer of the eggshell, but slightly on the membrane layer. It was demonstrated that the sorption of Cd(II) was not reversible, and the main sorption mechanisms were precipitation and ion exchange. The precipitation of (Cd,Ca)CO3 on the surface of the eggshell was corroborated by SEM and XRD analysis.

  11. Trace-element interactions in Rook Corvus frugilegus eggshells along an urbanisation gradient.

    PubMed

    Orłowski, Grzegorz; Kasprzykowski, Zbigniew; Dobicki, Wojciech; Pokorny, Przemysław; Wuczyński, Andrzej; Polechoński, Ryszard; Mazgajski, Tomasz D

    2014-11-01

    Concentrations of seven trace elements [arsenic (As), chromium (Cr), nickel (Ni), lead (Pb), copper (Cu), zinc (Zn), and cadmium (Cd)] in the eggshells of Rooks Corvus frugilegus, a focal bird species of Eurasian agricultural environments, are increased above background levels and exceed levels of toxicological concern. The concentrations of Cr, Ni, Pb, Cu, and Zn are greater in eggshells from urban rookeries (large cities) compared with rural areas (small towns and villages) suggesting an urbanisation gradient effect among eggs laid by females. In the present study, the investigators assessed whether the pattern of relationships among the seven trace elements in eggshells change along an urbanisation/pollution gradient. Surprisingly, we found that eggshells with the greatest contaminant burden, i.e., from urban rookeries, showed far fewer significant relationships (n = 4) than eggshells from villages (n = 10), small towns (n = 6), or rural areas (n = 8). In most cases, the relationships were positive. As was an exception: Its concentration was negatively correlated with Ni and Cd levels in eggshells from small town rookeries (where As levels were the highest), whereas eggshells from villages (with a lower As level) showed positive relationships between As and Cd. Our findings suggest that at low to intermediate levels, interactions between the trace elements in Rook eggshells are of a synergistic character and appear to operate as parallel coaccumulation. A habitat-specific excess of some elements (primarily Cr, Ni, Cu, As) suggests their more competitively selective sequestration. PMID:24793193

  12. 9 CFR 147.13 - Procedure for bacteriological culturing of eggshells for colon bacilli organisms.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... culturing of eggshells for colon bacilli organisms. 147.13 Section 147.13 Animals and Animal Products ANIMAL... bacteriological culturing of eggshells for colon bacilli organisms. Proper precautions to avoid environmental... conclusion of the presence of colon bacilli organisms. (Approved by the Office of Management and Budget...

  13. 9 CFR 147.13 - Procedure for bacteriological culturing of eggshells for colon bacilli organisms.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... culturing of eggshells for colon bacilli organisms. 147.13 Section 147.13 Animals and Animal Products ANIMAL... bacteriological culturing of eggshells for colon bacilli organisms. Proper precautions to avoid environmental... conclusion of the presence of colon bacilli organisms. (Approved by the Office of Management and Budget...

  14. 9 CFR 147.13 - Procedure for bacteriological culturing of eggshells for colon bacilli organisms.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... culturing of eggshells for colon bacilli organisms. 147.13 Section 147.13 Animals and Animal Products ANIMAL... bacteriological culturing of eggshells for colon bacilli organisms. Proper precautions to avoid environmental... conclusion of the presence of colon bacilli organisms. (Approved by the Office of Management and Budget...

  15. 9 CFR 147.13 - Procedure for bacteriological culturing of eggshells for colon bacilli organisms.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... culturing of eggshells for colon bacilli organisms. 147.13 Section 147.13 Animals and Animal Products ANIMAL... bacteriological culturing of eggshells for colon bacilli organisms. Proper precautions to avoid environmental... conclusion of the presence of colon bacilli organisms. (Approved by the Office of Management and Budget...

  16. 9 CFR 147.13 - Procedure for bacteriological culturing of eggshells for colon bacilli organisms.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... culturing of eggshells for colon bacilli organisms. 147.13 Section 147.13 Animals and Animal Products ANIMAL... bacteriological culturing of eggshells for colon bacilli organisms. Proper precautions to avoid environmental... conclusion of the presence of colon bacilli organisms. (Approved by the Office of Management and Budget...

  17. Distinct Host Tropism Protein Signatures to Identify Possible Zoonotic Influenza A Viruses.

    PubMed

    Eng, Christine L P; Tong, Joo Chuan; Tan, Tin Wee

    2016-01-01

    Zoonotic influenza A viruses constantly pose a health threat to humans as novel strains occasionally emerge from the avian population to cause human infections. Many past epidemic as well as pandemic strains have originated from avian species. While most viruses are restricted to their primary hosts, zoonotic strains can sometimes arise from mutations or reassortment, leading them to acquire the capability to escape host species barrier and successfully infect a new host. Phylogenetic analyses and genetic markers are useful in tracing the origins of zoonotic infections, but there are still no effective means to identify high risk strains prior to an outbreak. Here we show that distinct host tropism protein signatures can be used to identify possible zoonotic strains in avian species which have the potential to cause human infections. We have discovered that influenza A viruses can now be classified into avian, human, or zoonotic strains based on their host tropism protein signatures. Analysis of all influenza A viruses with complete proteome using the host tropism prediction system, based on machine learning classifications of avian and human viral proteins has uncovered distinct signatures of zoonotic strains as mosaics of avian and human viral proteins. This is in contrast with typical avian or human strains where they show mostly avian or human viral proteins in their signatures respectively. Moreover, we have found that zoonotic strains from the same influenza outbreaks carry similar host tropism protein signatures characteristic of a common ancestry. Our results demonstrate that the distinct host tropism protein signature in zoonotic strains may prove useful in influenza surveillance to rapidly identify potential high risk strains circulating in avian species, which may grant us the foresight in anticipating an impending influenza outbreak. PMID:26915079

  18. An Ensemble Method with Hybrid Features to Identify Extracellular Matrix Proteins

    PubMed Central

    Yang, Runtao; Zhang, Chengjin; Gao, Rui; Zhang, Lina

    2015-01-01

    The extracellular matrix (ECM) is a dynamic composite of secreted proteins that play important roles in numerous biological processes such as tissue morphogenesis, differentiation and homeostasis. Furthermore, various diseases are caused by the dysfunction of ECM proteins. Therefore, identifying these important ECM proteins may assist in understanding related biological processes and drug development. In view of the serious imbalance in the training dataset, a Random Forest-based ensemble method with hybrid features is developed in this paper to identify ECM proteins. Hybrid features are employed by incorporating sequence composition, physicochemical properties, evolutionary and structural information. The Information Gain Ratio and Incremental Feature Selection (IGR-IFS) methods are adopted to select the optimal features. Finally, the resulting predictor termed IECMP (Identify ECM Proteins) achieves an balanced accuracy of 86.4% using the 10-fold cross-validation on the training dataset, which is much higher than results obtained by other methods (ECMPRED: 71.0%, ECMPP: 77.8%). Moreover, when tested on a common independent dataset, our method also achieves significantly improved performance over ECMPP and ECMPRED. These results indicate that IECMP is an effective method for ECM protein prediction, which has a more balanced prediction capability for positive and negative samples. It is anticipated that the proposed method will provide significant information to fully decipher the molecular mechanisms of ECM-related biological processes and discover candidate drug targets. For public access, we develop a user-friendly web server for ECM protein identification that is freely accessible at http://iecmp.weka.cc. PMID:25680094

  19. Proteomic analysis of exosomes from nasopharyngeal carcinoma cell identifies intercellular transfer of angiogenic proteins.

    PubMed

    Chan, Yuk-Kit; Zhang, Huoming; Liu, Pei; Tsao, Sai-Wah; Lung, Maria Li; Mak, Nai-Ki; Ngok-Shun Wong, Ricky; Ying-Kit Yue, Patrick

    2015-10-15

    Exosomes, a group of secreted extracellular nanovesicles containing genetic materials and signaling molecules, play a critical role in intercellular communication. During tumorigenesis, exosomes have been demonstrated to promote tumor angiogenesis and metastasis while their biological functions in nasopharyngeal carcinoma (NPC) are poorly understood. In this study, we focused on the role of NPC-derived exosomes on angiogenesis. Exosomes derived from the NPC C666-1 cells and immortalized nasopharyngeal epithelial cells (NP69 and NP460) were isolated using ultracentrifugation. The molecular profile and biophysical characteristics of exosomes were verified by Western blotting, sucrose density gradient and electron microscopy. We showed that the C666-1 exosomes (10 and 20 μg/ml) could significantly increase the tubulogenesis, migration and invasion of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. Subsequently, an iTRAQ-based quantitative proteomics was used to identify the differentially expressed proteins in C666-1 exosomes. Among the 640 identified proteins, 51 and 89 proteins were considered as up- and down-regulated (≥ 1.5-fold variations) in C666-1 exosomes compared to the normal counterparts, respectively. As expected, pro-angiogenic proteins including intercellular adhesion molecule-1 (ICAM-1) and CD44 variant isoform 5 (CD44v5) are among the up-regulated proteins, whereas angio-suppressive protein, thrombospondin-1 (TSP-1) was down-regulated in C666-1 exosomes. Further confocal microscopic study and Western blotting clearly demonstrated that the alteration of ICAM-1 and TSP-1 expressions in recipient HUVECs are due to internalization of exosomes. Taken together, these data strongly indicated the critical roles of identified angiogenic proteins in the involvement of exosomes-induced angiogenesis, which could potentially be developed as therapeutic targets in future. PMID:25857718

  20. Distinct Host Tropism Protein Signatures to Identify Possible Zoonotic Influenza A Viruses

    PubMed Central

    Eng, Christine L. P.; Tong, Joo Chuan; Tan, Tin Wee

    2016-01-01

    Zoonotic influenza A viruses constantly pose a health threat to humans as novel strains occasionally emerge from the avian population to cause human infections. Many past epidemic as well as pandemic strains have originated from avian species. While most viruses are restricted to their primary hosts, zoonotic strains can sometimes arise from mutations or reassortment, leading them to acquire the capability to escape host species barrier and successfully infect a new host. Phylogenetic analyses and genetic markers are useful in tracing the origins of zoonotic infections, but there are still no effective means to identify high risk strains prior to an outbreak. Here we show that distinct host tropism protein signatures can be used to identify possible zoonotic strains in avian species which have the potential to cause human infections. We have discovered that influenza A viruses can now be classified into avian, human, or zoonotic strains based on their host tropism protein signatures. Analysis of all influenza A viruses with complete proteome using the host tropism prediction system, based on machine learning classifications of avian and human viral proteins has uncovered distinct signatures of zoonotic strains as mosaics of avian and human viral proteins. This is in contrast with typical avian or human strains where they show mostly avian or human viral proteins in their signatures respectively. Moreover, we have found that zoonotic strains from the same influenza outbreaks carry similar host tropism protein signatures characteristic of a common ancestry. Our results demonstrate that the distinct host tropism protein signature in zoonotic strains may prove useful in influenza surveillance to rapidly identify potential high risk strains circulating in avian species, which may grant us the foresight in anticipating an impending influenza outbreak. PMID:26915079

  1. The use of Saccharomyces cerevisiae proteomic libraries to identify RNA-modifying proteins.

    PubMed

    Jackman, Jane E; Grayhack, Elizabeth J; Phizicky, Eric M

    2008-01-01

    Biochemical assay of proteomic libraries derived from the Saccharomyces cerevisiae genome provides a powerful new tool for the assignment of activities to proteins. Particular advantages of this approach include the speed with which a protein can be identified and the generality for any biological activity for which an assay can be developed. We discuss the utility of this approach for the identification of RNA-modifying enzymes using a yeast proteomic library derived from a genomic set of strains expressing GST-ORF fusion proteins. This technique is also broadly applicable to other classes of RNA-protein interactions, including RNA binding and RNA degradation, and can be used with any of the proteomic libraries that are available. PMID:18982304

  2. A Machine Learning Approach To Identify Hydrogenosomal Proteins in Trichomonas vaginalis

    PubMed Central

    Burstein, David; Gould, Sven B.; Zimorski, Verena; Kloesges, Thorsten; Kiosse, Fuat; Major, Peter; Martin, William F.; Pupko, Tal

    2012-01-01

    The protozoan parasite Trichomonas vaginalis is the causative agent of trichomoniasis, the most widespread nonviral sexually transmitted disease in humans. It possesses hydrogenosomes—anaerobic mitochondria that generate H2, CO2, and acetate from pyruvate while converting ADP to ATP via substrate-level phosphorylation. T. vaginalis hydrogenosomes lack a genome and translation machinery; hence, they import all their proteins from the cytosol. To date, however, only 30 imported proteins have been shown to localize to the organelle. A total of 226 nuclear-encoded proteins inferred from the genome sequence harbor a characteristic short N-terminal presequence, reminiscent of mitochondrial targeting peptides, which is thought to mediate hydrogenosomal targeting. Recent studies suggest, however, that the presequences might be less important than previously thought. We sought to identify new hydrogenosomal proteins within the 59,672 annotated open reading frames (ORFs) of T. vaginalis, independent of the N-terminal targeting signal, using a machine learning approach. Our training set included 57 gene and protein features determined for all 30 known hydrogenosomal proteins and 576 nonhydrogenosomal proteins. Several classifiers were trained on this set to yield an import score for all proteins encoded by T. vaginalis ORFs, predicting the likelihood of hydrogenosomal localization. The machine learning results were tested through immunofluorescence assay and immunodetection in isolated cell fractions of 14 protein predictions using hemagglutinin constructs expressed under the homologous SCSα promoter in transiently transformed T. vaginalis cells. Localization of 6 of the 10 top predicted hydrogenosome-localized proteins was confirmed, and two of these were found to lack an obvious N-terminal targeting signal. PMID:22140228

  3. A yeast-based genetic screening to identify human proteins that increase homologous recombination.

    PubMed

    Collavoli, Anita; Comelli, Laura; Rainaldi, Giuseppe; Galli, Alvaro

    2008-05-01

    To identify new human proteins implicated in homologous recombination (HR), we set up 'a papillae assay' to screen a human cDNA library using the RS112 strain of Saccharomyces cerevisiae containing an intrachromosomal recombination substrate. We isolated 23 cDNAs, 11 coding for complete proteins and 12 for partially deleted proteins that increased HR when overexpressed in yeast. We characterized the effect induced by the overexpression of the complete human proteasome subunit beta 2, the partially deleted proteasome subunits alpha 3 and beta 8, the ribosomal protein L12, the brain abundant membrane signal protein (BASP1) and the human homologue to v-Ha-RAS (HRAS), which elevated HR by 2-6.5-fold over the control. We found that deletion of the RAD52 gene, which has a key role in most HR events, abolished the increase of HR induced by the proteasome subunits and HRAS; by contrast, the RAD52 deletion did not affect the high level of HR due to BASP1 and RPL12. This suggests that the proteins stimulated yeast HR via different mechanisms. Overexpression of the complete beta 2 human proteasome subunit or the partially deleted alpha 3 and beta 8 subunits increased methyl methanesulphonate (MMS) resistance much more in the rad52 Delta mutant than in the wild-type. Overexpression of RPL12 and BASP1 did not affect MMS resistance in both the wild-type and the rad52 Delta mutant, whereas HRAS decreased MMS resistance in the rad52 Delta mutant. The results indicate that these proteins may interfere with the pathway(s) involved in the repair of MMS-induced DNA damage. Finally, we provide further evidence that yeast is a helpful tool to identify human proteins that may have a regulatory role in HR. PMID:18248415

  4. Cardiovascular-related proteins identified in human plasma by the HUPO Plasma Proteome Project pilot phase.

    PubMed

    Berhane, Beniam T; Zong, Chenggong; Liem, David A; Huang, Aaron; Le, Steven; Edmondson, Ricky D; Jones, Richard C; Qiao, Xin; Whitelegge, Julian P; Ping, Peipei; Vondriska, Thomas M

    2005-08-01

    Proteomic profiling of accessible bodily fluids, such as plasma, has the potential to accelerate biomarker/biosignature development for human diseases. The HUPO Plasma Proteome Project pilot phase examined human plasma with distinct proteomic approaches across multiple laboratories worldwide. Through this effort, we confidently identified 3020 proteins, each requiring a minimum of two high-scoring MS/MS spectra. A critical step subsequent to protein identification is functional annotation, in particular with regard to organ systems and disease. Performing exhaustive literature searches, we have manually annotated a subset of these 3020 proteins that have cardiovascular-related functions on the basis of an existing body of published information. These cardiovascular-related proteins can be organized into eight groups: markers of inflammation and/or cardiovascular disease, vascular and coagulation, signaling, growth and differentiation, cytoskeletal, transcription factors, channels/receptors and heart failure and remodeling. In addition, analysis of the peptide per protein ratio for MS/MS identification reveals group-specific trends. These findings serve as a resource to interrogate the functions of plasma proteins, and moreover, the list of cardiovascular-related proteins in plasma constitutes a baseline proteomic blueprint for the future development of biosignatures for diseases such as myocardial ischemia and atherosclerosis. PMID:16052623

  5. Evaluation of chemical labeling methods for identifying functional arginine residues of proteins by mass spectrometry.

    PubMed

    Wanigasekara, Maheshika S K; Chowdhury, Saiful M

    2016-09-01

    Arginine residues undergo several kinds of post-translational modifications (PTMs). These PTMs are associated with several inflammatory diseases, such as rheumatoid arthritis, atherosclerosis, and diabetes. Mass spectrometric studies of arginine modified proteins and peptides are very important, not only to identify the reactive arginine residues but also to understand the tandem mass spectrometry behavior of these peptides for assigning the sequences unambiguously. Herein, we utilize tandem mass spectrometry to report the performance of two widely used arginine labeling reagents, 1,2-cyclohexanedione (CHD) and phenylglyoxal (PG) with several arginine containing peptides and proteins. Time course labeling studies were performed to demonstrate the selectivity of the reagents in proteins or protein digests. Structural studies on the proteins were also explored to better understand the reaction sites and position of arginine residues. We found CHD showed better labeling efficiencies compared to phenylglyoxal. Reactive arginine profiling on a purified albumin protein clearly pointed out the cellular glycation modification site for this protein with high confidence. We believe these detailed mass-spectrometric studies will provide significant input to profile reactive arginine residues in large-scale studies; therefore, targeted proteomics can be performed to the short listed reactive sites for cellular arginine modifications. PMID:27543028

  6. Quantitative proteome analyses identify PrfA-responsive proteins and phosphoproteins in Listeria monocytogenes.

    PubMed

    Misra, Sandeep Kumar; Moussan Désirée Aké, Francine; Wu, Zongfu; Milohanic, Eliane; Cao, Thanh Nguyen; Cossart, Pascale; Deutscher, Josef; Monnet, Véronique; Archambaud, Cristel; Henry, Céline

    2014-12-01

    Protein phosphorylation is a major mechanism of signal transduction in bacteria. Here, we analyzed the proteome and phosphoproteome of a wild-type strain of the food-borne pathogen Listeria monocytogenes that was grown in either chemically defined medium or rich medium containing glucose. We then compared these results with those obtained from an isogenic prfA* mutant that produced a constitutively active form of PrfA, the main transcriptional activator of virulence genes. In the prfA* mutant grown in rich medium, we identified 256 peptides that were phosphorylated on serine (S), threonine (T), or tyrosine (Y) residues, with a S/T/Y ratio of 155:75:12. Strikingly, we detected five novel phosphosites on the virulence protein ActA. This protein was known to be phosphorylated by a cellular kinase in the infected host, but phosphorylation by a listerial kinase had not previously been reported. Unexpectedly, SILAC experiments with the prfA* mutant grown in chemically defined medium revealed that, in addition to previously described PrfA-regulated proteins, several other proteins were significantly overproduced, among them were several proteins involved in purine biosynthesis. This work provides new information for our understanding of the correlation among protein phosphorylation, virulence mechanisms, and carbon metabolism. PMID:25383790

  7. A simple contact mapping algorithm for identifying potential peptide mimetics in protein–protein interaction partners

    PubMed Central

    Krall, Alex; Brunn, Jonathan; Kankanala, Spandana; Peters, Michael H

    2014-01-01

    A simple, static contact mapping algorithm has been developed as a first step at identifying potential peptide biomimetics from protein interaction partner structure files. This rapid and simple mapping algorithm, “OpenContact” provides screened or parsed protein interaction files based on specified criteria for interatomic separation distances and interatomic potential interactions. The algorithm, which uses all-atom Amber03 force field models, was blindly tested on several unrelated cases from the literature where potential peptide mimetics have been experimentally developed to varying degrees of success. In all cases, the screening algorithm efficiently predicted proposed or potential peptide biomimetics, or close variations thereof, and provided complete atom-atom interaction data necessary for further detailed analysis and drug development. In addition, we used the static parsing/mapping method to develop a peptide mimetic to the cancer protein target, epidermal growth factor receptor. In this case, secondary, loop structure for the peptide was indicated from the intra-protein mapping, and the peptide was subsequently synthesized and shown to exhibit successful binding to the target protein. The case studies, which all involved experimental peptide drug advancement, illustrate many of the challenges associated with the development of peptide biomimetics, in general. Proteins 2014; 82:2253–2262. © 2014 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. PMID:24756879

  8. Controllability analysis of the directed human protein interaction network identifies disease genes and drug targets.

    PubMed

    Vinayagam, Arunachalam; Gibson, Travis E; Lee, Ho-Joon; Yilmazel, Bahar; Roesel, Charles; Hu, Yanhui; Kwon, Young; Sharma, Amitabh; Liu, Yang-Yu; Perrimon, Norbert; Barabási, Albert-László

    2016-05-01

    The protein-protein interaction (PPI) network is crucial for cellular information processing and decision-making. With suitable inputs, PPI networks drive the cells to diverse functional outcomes such as cell proliferation or cell death. Here, we characterize the structural controllability of a large directed human PPI network comprising 6,339 proteins and 34,813 interactions. This network allows us to classify proteins as "indispensable," "neutral," or "dispensable," which correlates to increasing, no effect, or decreasing the number of driver nodes in the network upon removal of that protein. We find that 21% of the proteins in the PPI network are indispensable. Interestingly, these indispensable proteins are the primary targets of disease-causing mutations, human viruses, and drugs, suggesting that altering a network's control property is critical for the transition between healthy and disease states. Furthermore, analyzing copy number alterations data from 1,547 cancer patients reveals that 56 genes that are frequently amplified or deleted in nine different cancers are indispensable. Among the 56 genes, 46 of them have not been previously associated with cancer. This suggests that controllability analysis is very useful in identifying novel disease genes and potential drug targets. PMID:27091990

  9. A comparative protein profile of mammalian erythrocyte membranes identified by mass spectrometry.

    PubMed

    Sharma, Savita; Punjabi, Vinny; Zingde, Surekha M; Gokhale, Sadashiv M

    2014-11-01

    A comparative analysis of erythrocyte membrane proteins of economically important animals, goat (Capra aegagrus hircus), buffalo (Bubalus bubalis), pig (Sus scrofa), cow (Bos tauras), and human (Homo sapiens) was performed. Solubilized erythrocyte membrane proteins were separated by sodium dodecyl sulfate-polyacryamide gel electrophoresis (SDS-PAGE), visualized by staining the gels with Commassie Brilliant Blue (CBB), and identified by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Emerging results show that all major erythrocyte membrane proteins present in human are also seen in all the animals except for band 4.5 which could not be identified. Band 3 is seen as more intense and compact, band 4.1 appears as a doublet in all the animal erythrocyte membranes, band 4.2 exhibits a slightly higher molecular weight (Mr) in buffalo, and cow and band 4.9 has a higher Mr in all the animals relative to the human protein. In addition, there are two new bands in the goat membrane, band G1, identified as HSP 90α, and band G2 identified as HSP 70. A new band C2 identified as HSP 70 is also seen in cow membranes. Peroxiredoxin II is of lower intensity and/or higher Mr in the animals. The difference in size of the proteins possibly indicates the variations in the composition of the amino acids. The difference in intensity of the proteins among these mammalians highlights the presence of less or more number of copies of that protein per cell. This data complement the earlier observations of differences in the sialoglycoprotein profile and effect of proteases and neuraminidase on agglutination among the mammalian erythrocytes. This study provides a platform to understand the molecular architecture of the individual erythrocytes, and in turn the dependent disorders, their phylogenetic relationship and also generates a database of erythrocyte membrane proteins of mammals. The animals selected for this study are of economic importance as

  10. Alumina supported carbon composite material with exceptionally high defluoridation property from eggshell waste.

    PubMed

    Lunge, Sneha; Thakre, Dilip; Kamble, Sanjay; Labhsetwar, Nitin; Rayalu, Sadhana

    2012-10-30

    A new alumina supported carbon composite material called "Eggshell Composite" (EC) was synthesized from eggshell waste as calcium source for selective fluoride adsorption from water. The effect of various synthesis parameters like eggshell (ES): Eggshell membrane (ESM) ratio, aluminium loading, mixing time and calcinations temperature to optimize the synthesis conditions for selective fluoride removal has been studied. It was observed that the synthesis parameters have significant influence on development of EC and in turn on fluoride removal capacity. EC synthesized was characterized for elemental composition, morphology, functionality and textural properties. Results showed that EC obtained from eggshell modified with alumina precursor is more selective and efficient for fluoride removal. Langmuir and Freundlich isotherm were used to obtain ultimate fluoride removal capacity. The calcium and alumina species in EC shows synergistic effect in fluoride adsorption process. Fluoride sorption studies were carried out in synthetic, groundwater and wastewater. EC proved to be a potential, indigenous and economic adsorbent for fluoride removal. PMID:22944591

  11. A machine learning strategy to identify candidate binding sites in human protein-coding sequence

    PubMed Central

    Down, Thomas; Leong, Bernard; Hubbard, Tim JP

    2006-01-01

    Background The splicing of RNA transcripts is thought to be partly promoted and regulated by sequences embedded within exons. Known sequences include binding sites for SR proteins, which are thought to mediate interactions between splicing factors bound to the 5' and 3' splice sites. It would be useful to identify further candidate sequences, however identifying them computationally is hard since exon sequences are also constrained by their functional role in coding for proteins. Results This strategy identified a collection of motifs including several previously reported splice enhancer elements. Although only trained on coding exons, the model discriminates both coding and non-coding exons from intragenic sequence. Conclusion We have trained a computational model able to detect signals in coding exons which seem to be orthogonal to the sequences' primary function of coding for proteins. We believe that many of the motifs detected here represent binding sites for both previously unrecognized proteins which influence RNA splicing as well as other regulatory elements. PMID:17002805

  12. The inhibitory G protein G(i) identified as pertussis toxin-catalyzed ADP-ribosylation.

    PubMed

    Katada, Toshiaki

    2012-01-01

    Pertussis toxin (PTX) produced by Bordetella pertussis was first introduced by Ui and his colleagues in research on signal transduction under the name islet-activating protein in 1979, when the mechanism of toxin-induced stimulation of insulin release from pancreatic islets was reported in the rat. The stimulatory effect of PTX in vivo results from the blockage of α(2)-adrenergic receptor-mediated inhibition of insulin release. The receptor-induced inhibition of cAMP formation was also abolished in pancreatic islets isolated from PTX-treated rats, suggesting that the toxin caused uncoupling of adenylyl cyclase inhibition from receptor stimulation. The action of PTX on isolated membranes required a cytosolic factor, nicotinamide adenine dinucleotide (NAD), and the uncoupling induced by PTX was shown to be due to the toxin-catalyzed ADP-ribosylation of a 41-kDa protein with NAD as another substrate. The 41-kDa PTX substrate was soon identified and purified as the α-subunit of the inhibitory G protein that transmits an inhibitory signal from membrane receptors to adenylyl cyclase. After demonstration of the molecular mechanism of PTX, the toxin was widely utilized as a probe for identifying and analyzing major αβγ-trimeric G proteins. Thus, PTX-sensitive G proteins appeared to carry positive and negative signals from many membrane receptors to a variety of effectors other than adenylyl cyclase. PMID:23207763

  13. Identifying paths of allosteric communication in the protein BirA through simulations

    NASA Astrophysics Data System (ADS)

    Custer, Gregory; Beckett, Dorothy; Matysiak, Silvina

    Biotin ligase/repressor (BirA) is a bifunctional enzyme which adenylates biotin and transfers the product, biotinyl-5'-AMP (bio-5'-AMP) to biotin carboxyl carrier protein (BCCP). In the absence of BCCP, bio-5'-AMP promotes the dimerization of BirA. In dimer form, the BirA.bio-5'-AMP complex is able to bind to the biotin operator and prevents further synthesis of biotin. The bio-5'-AMP binds away from the dimer interface, so it is acting as an allosteric activator. We perform all-atom molecular dynamics simulations with BirA to look at fluctuations within the protein at equilibrium. We simulate apoBirA, liganded BirA, as well as two mutants, M211A and V219A. In agreement with experimental observations, several loops of the protein become stabilized for the liganded BirA when compared to the apo protein. In addition, changes in the dimer interface are observed for the M211A and V219A mutations, which are located in the ligand binding region. Using inter-residue correlation coefficients and pair energies a communication network through the protein is constructed. With this network we have identified paths which have the potential to be important in allosteric activation of BirA. These paths and the methods we use to identify them will be presented.

  14. Using a neural network to identify potential HLA-DR1 binding sites within proteins.

    PubMed

    Bisset, L R; Fierz, W

    1993-03-01

    The presentation by antigen-presenting cells of immunodominant peptide segments in association with major histocompatibility complex (MHC) encoded proteins is fundamental to the efficacy of a specific immune response. One approach used to identify immunodominant segments within proteins has involved the development of predictive algorithms which utilize amino acid sequence data to identify structural characteristics or motifs associated with in vivo antigenicity. The parallel-computing technique termed 'neural networking' has recently been shown to be remarkably efficient at addressing the problem of pattern recognition and can be applied to predict protein secondary structure attributes directly from amino acid sequence data. In order to examine the potential of a neural network to generalize peptide structural features related to binding within class II MHC-encoded proteins, we have trained a neural network to determine whether or not any given amino acid of a protein is part of a peptide segment capable of binding to HLA-DR1. We report that a neural network trained on a data base consisting of peptide segments known to bind to HLA-DR1 is able to generalize features relating to HLA-DR1-binding capacity (r = 0.17 and p = 0.0001). PMID:8251191

  15. Plasmodesmal receptor-like kinases identified through analysis of rice cell wall extracted proteins.

    PubMed

    Jo, Yeonhwa; Cho, Won Kyong; Rim, Yeonggil; Moon, Juyeon; Chen, Xiong-Yan; Chu, Hyosub; Kim, Cha Young; Park, Zee-Yong; Lucas, William J; Kim, Jae-Yean

    2011-01-01

    In plants, plasmodesmata (PD) are intercellular channels that function in both metabolite exchange and the transport of proteins and RNAs. Currently, many of the PD structural and regulatory components remain to be elucidated. Receptor-like kinases (RLKs) belonging to a notably expanded protein family in plants compared to the animal kingdom have been shown to play important roles in plant growth, development, pathogen resistance, and cell death. In this study, cell biological approaches were used to identify potential PD-associated RLK proteins among proteins contained within cell walls isolated from rice callus cultured cells. A total of 15 rice RLKs were investigated to determine their subcellular localization, using an Agrobacterium-mediated transient expression system. Of these six PD-associated RLKs were identified based on their co-localization with a viral movement protein that served as a PD marker, plasmolysis experiments, and subcellular localization at points of wall contact between spongy mesophyll cells. These findings suggest potential PD functions in apoplasmic signaling in response to environmental stimuli and developmental inputs. PMID:21161304

  16. The effect of developmental stage on eggshell thickness variation in endangered falcons.

    PubMed

    Castilla, Aurora M; Herrel, Anthony; Robles, Hugo; Malone, Jim; Negro, Juan José

    2010-05-01

    We compared eggshell thickness of hatched eggs with that of non-developed eggs in endangered falcon taxa to explore the effect of embryo development on eggshell thinning. To our knowledge, this has never been examined before in falcons, despite the fact that eggshell thinning due to pollutants and environmental contamination is often considered the most common cause of egg failure in falcons. Because of the endangered nature of these birds, and the difficulty in gaining access to the nests and their eggs, there is a large gap in our knowledge regarding eggshell thickness variation and the factors affecting it. We used a linear mixed-effects (LME) model to explore the variation in eggshell thickness (n=335 eggs) in relation to the developmental stage of the eggs, but also in relation to the falcon taxa, the laying sequence and the study zone. Female identity (n=69) and clutch identity (n=98) were also included in the LME model. Our results are consistent with the prediction that eggshell thickness decreases during incubation because of the important effect of calcium uptake by the embryo during development. Our results also show that eggs laid later in the sequence had significantly thinner eggshells. In this study, we provide the first quantitative data on eggshell thickness variation of hatched eggs in different falcon taxa that were not subjected to contamination or food limitation (i.e., bred under captive conditions). Because eggshell thickness strongly influences survival and because the species examined in this study are endangered, our data represent a valuable control for future studies on the effects of pollution on eggshells from wild populations and thus are an important contribution to the conservation of falcons. PMID:20435452

  17. Improved mutation tagging with gene identifiers applied to membrane protein stability prediction

    PubMed Central

    Winnenburg, Rainer; Plake, Conrad; Schroeder, Michael

    2009-01-01

    Background The automated retrieval and integration of information about protein point mutations in combination with structure, domain and interaction data from literature and databases promises to be a valuable approach to study structure-function relationships in biomedical data sets. Results We developed a rule- and regular expression-based protein point mutation retrieval pipeline for PubMed abstracts, which shows an F-measure of 87% for the mutation retrieval task on a benchmark dataset. In order to link mutations to their proteins, we utilize a named entity recognition algorithm for the identification of gene names co-occurring in the abstract, and establish links based on sequence checks. Vice versa, we could show that gene recognition improved from 77% to 91% F-measure when considering mutation information given in the text. To demonstrate practical relevance, we utilize mutation information from text to evaluate a novel solvation energy based model for the prediction of stabilizing regions in membrane proteins. For five G protein-coupled receptors we identified 35 relevant single mutations and associated phenotypes, of which none had been annotated in the UniProt or PDB database. In 71% reported phenotypes were in compliance with the model predictions, supporting a relation between mutations and stability issues in membrane proteins. Conclusion We present a reliable approach for the retrieval of protein mutations from PubMed abstracts for any set of genes or proteins of interest. We further demonstrate how amino acid substitution information from text can be utilized for protein structure stability studies on the basis of a novel energy model. PMID:19758467

  18. A predicted protein interactome identifies conserved global networks and disease resistance subnetworks in maize

    PubMed Central

    Musungu, Bryan; Bhatnagar, Deepak; Brown, Robert L.; Fakhoury, Ahmad M.; Geisler, Matt

    2015-01-01

    Interactomes are genome-wide roadmaps of protein-protein interactions. They have been produced for humans, yeast, the fruit fly, and Arabidopsis thaliana and have become invaluable tools for generating and testing hypotheses. A predicted interactome for Zea mays (PiZeaM) is presented here as an aid to the research community for this valuable crop species. PiZeaM was built using a proven method of interologs (interacting orthologs) that were identified using both one-to-one and many-to-many orthology between genomes of maize and reference species. Where both maize orthologs occurred for an experimentally determined interaction in the reference species, we predicted a likely interaction in maize. A total of 49,026 unique interactions for 6004 maize proteins were predicted. These interactions are enriched for processes that are evolutionarily conserved, but include many otherwise poorly annotated proteins in maize. The predicted maize interactions were further analyzed by comparing annotation of interacting proteins, including different layers of ontology. A map of pairwise gene co-expression was also generated and compared to predicted interactions. Two global subnetworks were constructed for highly conserved interactions. These subnetworks showed clear clustering of proteins by function. Another subnetwork was created for disease response using a bait and prey strategy to capture interacting partners for proteins that respond to other organisms. Closer examination of this subnetwork revealed the connectivity between biotic and abiotic hormone stress pathways. We believe PiZeaM will provide a useful tool for the prediction of protein function and analysis of pathways for Z. mays researchers and is presented in this paper as a reference tool for the exploration of protein interactions in maize. PMID:26089837

  19. Interaction of Proteus mirabilis urease apoenzyme and accessory proteins identified with yeast two-hybrid technology.

    PubMed

    Heimer, S R; Mobley, H L

    2001-02-01

    Proteus mirabilis, a gram-negative bacterium associated with complicated urinary tract infections, produces a metalloenzyme urease which hydrolyzes urea to ammonia and carbon dioxide. The apourease is comprised of three structural subunits, UreA, UreB, and UreC, assembled as a homotrimer of individual UreABC heterotrimers (UreABC)(3). To become catalytically active, apourease acquires divalent nickel ions through a poorly understood process involving four accessory proteins, UreD, UreE, UreF, and UreG. While homologues of UreD, UreF, and UreG have been copurified with apourease, it remains unclear specifically how these polypeptides associate with the apourease or each other. To identify interactions among P. mirabilis accessory proteins, in vitro immunoprecipitation and in vivo yeast two-hybrid assays were employed. A complex containing accessory protein UreD and structural protein UreC was isolated by immunoprecipitation and characterized with immunoblots. This association occurs independently of coaccessory proteins UreE, UreF, and UreG and structural protein UreA. In a yeast two-hybrid screen, UreD was found to directly interact in vivo with coaccessory protein UreF. Unique homomultimeric interactions of UreD and UreF were also detected in vivo. To substantiate the study of urease proteins with a yeast two-hybrid assay, previously described UreE dimers and homomultimeric UreA interactions among apourease trimers were confirmed in vivo. Similarly, a known structural interaction involving UreA and UreC was also verified. This report suggests that in vivo, P. mirabilis UreD may be important for recruitment of UreF to the apourease and that crucial homomultimeric associations occur among these accessory proteins. PMID:11157956

  20. Maize MeJA-responsive proteins identified by high-resolution 2-DE PAGE.

    PubMed

    Zhang, Yuliang; Pennerman, Kayla K; Yang, Fengshan; Yin, Guohua

    2015-12-01

    Exogenous methyl jasmonate (MeJA) is well-known to induce plant defense mechanisms effective against a wide variety of insect and microbial pests. High-resolution 2-DE gel electrophoresis was used to discover changes in the leaf proteome of maize exposed to MeJA. We sequenced 62 MeJA-responsive proteins by tandem mass spectroscopy, and deposited the mass spectra and identities in the EMBL-EBI PRIDE repository under reference number PXD001793. An analysis and discussion of the identified proteins in relation to maize defense against Asian corn borer is published by Zhang et al. (2015) [1]. PMID:26509185

  1. A comparative hidden Markov model analysis pipeline identifies proteins characteristic of cereal-infecting fungi

    PubMed Central

    2013-01-01

    Background Fungal pathogens cause devastating losses in economically important cereal crops by utilising pathogen proteins to infect host plants. Secreted pathogen proteins are referred to as effectors and have thus far been identified by selecting small, cysteine-rich peptides from the secretome despite increasing evidence that not all effectors share these attributes. Results We take advantage of the availability of sequenced fungal genomes and present an unbiased method for finding putative pathogen proteins and secreted effectors in a query genome via comparative hidden Markov model analyses followed by unsupervised protein clustering. Our method returns experimentally validated fungal effectors in Stagonospora nodorum and Fusarium oxysporum as well as the N-terminal Y/F/WxC-motif from the barley powdery mildew pathogen. Application to the cereal pathogen Fusarium graminearum reveals a secreted phosphorylcholine phosphatase that is characteristic of hemibiotrophic and necrotrophic cereal pathogens and shares an ancient selection process with bacterial plant pathogens. Three F. graminearum protein clusters are found with an enriched secretion signal. One of these putative effector clusters contains proteins that share a [SG]-P-C-[KR]-P sequence motif in the N-terminal and show features not commonly associated with fungal effectors. This motif is conserved in secreted pathogenic Fusarium proteins and a prime candidate for functional testing. Conclusions Our pipeline has successfully uncovered conservation patterns, putative effectors and motifs of fungal pathogens that would have been overlooked by existing approaches that identify effectors as small, secreted, cysteine-rich peptides. It can be applied to any pathogenic proteome data, such as microbial pathogen data of plants and other organisms. PMID:24252298

  2. The effect on egg-shell thickness of the inclusion of the calcinogenic plant Solanum malacoxylon in the diet of laying hens.

    PubMed

    Morris, K M; Jenkins, S A; Simonite, J P

    1977-12-17

    The leaves of the plant Solanum malacoxylon contain 1,25 dihydroxycholecalciferol, the active form of vitamin D. The effect on egg-shell thickness of supplementing the diet of laying hens with a dry powdered preparation of the leaves (DLSM) has been studied. A significnat increase in shell thickness was evident for eggs laid on the second and subsequent days of the DLSM-regimen but nor for those laid during the first 24 hours. It is suggested that the provision of high doses of the vitamin D metabolite in the form of a DLSM supplement may restore calcium binding protein levels in birds approaching the end of the first laying year and hence improve dietary calcium absorption and consequently egg-shell quality. PMID:605486

  3. Prolonged Fasting Identifies Heat Shock Protein 10 as a Sirtuin 3 Substrate

    PubMed Central

    Lu, Zhongping; Chen, Yong; Aponte, Angel M.; Battaglia, Valentina; Gucek, Marjan; Sack, Michael N.

    2015-01-01

    Although Sirtuin 3 (SIRT3), a mitochondrially enriched deacetylase and activator of fat oxidation, is down-regulated in response to high fat feeding, the rate of fatty acid oxidation and mitochondrial protein acetylation are invariably enhanced in this dietary milieu. These paradoxical data implicate that additional acetylation modification-dependent levels of regulation may be operational under nutrient excess conditions. Because the heat shock protein (Hsp) Hsp10-Hsp60 chaperone complex mediates folding of the fatty acid oxidation enzyme medium-chain acyl-CoA dehydrogenase, we tested whether acetylation-dependent mitochondrial protein folding contributes to this regulatory discrepancy. We demonstrate that Hsp10 is a functional SIRT3 substrate and that, in response to prolonged fasting, SIRT3 levels modulate mitochondrial protein folding. Acetyl mutagenesis of Hsp10 lysine 56 alters Hsp10-Hsp60 binding, conformation, and protein folding. Consistent with Hsp10-Hsp60 regulation of fatty acid oxidation enzyme integrity, medium-chain acyl-CoA dehydrogenase activity and fat oxidation are elevated by Hsp10 acetylation. These data identify acetyl modification of Hsp10 as a nutrient-sensing regulatory node controlling mitochondrial protein folding and metabolic function. PMID:25505263

  4. HUGE: a database for human large proteins identified in the Kazusa cDNA sequencing project.

    PubMed

    Kikuno, R; Nagase, T; Suyama, M; Waki, M; Hirosawa, M; Ohara, O

    2000-01-01

    HUGE is a database for human large proteins newly identified in the Kazusa cDNA project, the aim of which is to predict the primary structure of proteins from the sequences of human large cDNAs (>4 kb). In particular, cDNA clones capable of coding for large proteins (>50 kDa) are the current targets of the project. HUGE contains >1100 cDNA sequences and detailed information obtained through analysis of the sequences of cDNAs and the predicted proteins. Besides an increase in the number of cDNA entries, the amount of experimental data for expression profiling has been largely increased and data on chromosomal locations have been newly added. All of the protein-coding regions were examined by GeneMark analysis, and the results of a motif/domain search of each predicted protein sequence against the Pfam database have been newly added. HUGE is available through the WWW at http://www.kazusa.or.jp/huge PMID:10592264

  5. Ecological importance of the thermal emissivity of avian eggshells.

    PubMed

    Björn, Lars Olof; Uvdal, Per; Li, Shaoshan

    2012-05-21

    Breeding birds have to divide their time between egg incubation and foraging. Particularly in cases when only one parent incubates the eggs, and especially in cold climates, the cooling of the eggs during absence from the nest may be problematic. In the present study we find that the thermal emissivity of eggshells may be ecologically important, and that an evolutionary pressure towards lower emissivity for exposed eggs in cold climates exists. We plan to experimentally compare emissivities among species in a future study. PMID:22586725

  6. Stable-isotope analyses of dinosaur eggshells: Paleoenvironmental implications

    NASA Astrophysics Data System (ADS)

    Sarkar, A.; Bhattacharya, S. K.; Mohabey, D. M.

    1991-11-01

    Well-preserved clutches of dinosaur (sauropod) eggshells and skeletal remains have been discovered in the Upper Cretaceous Lameta limestones of the Kheda district, Gujarat, India, indicating a dinosaur nesting site. Oxygen-isotope analyses of the eggs show that the dinosaurs drank from a variety of freshwater bodies such as rivers and small evaporative pools, whereas the carbon-isotope values indicate that the reptiles were consuming plants that utilize the C3 photosynthetic pathway, e.g., small palms, shrubs, conifers, etc. Similar analyses of the host limestones suggest that they were deposited in a freshwater environment that provided the niche for large-scale breeding and nesting of the dinosaurs.

  7. Immobilization of lead in a Korean military shooting range soil using eggshell waste: an integrated mechanistic approach.

    PubMed

    Ahmad, Mahtab; Hashimoto, Yohey; Moon, Deok Hyun; Lee, Sang Soo; Ok, Yong Sik

    2012-03-30

    This study evaluated the effectiveness of eggshell and calcined eggshell on lead (Pb) immobilization in a shooting range soil. Destructive and non-destructive analytical techniques were employed to determine the mechanism of Pb immobilization. The 5% additions of eggshell and calcined eggshell significantly decreased the TCLP-Pb concentration by 68.8% due mainly to increasing soil pH. Eggshell and calcined-eggshell amendments decreased the exchangeable Pb fraction to ≈ 1% of the total Pb in the soil, while the carbonate-associated Pb fraction was increased to 40.0-47.1% at >15% application rates. The thermodynamic modeling on Pb speciation in the soil solution predicted the precipitation of Pb-hydroxide [Pb(OH)(2)] in soils amended with eggshell and calcined eggshell. The SEM-EDS, XAFS and elemental dot mapping revealed that Pb in soil amended with calcined eggshell was associated with Si and Ca, and may be immobilized by entrapping into calcium-silicate-hydrate. Comparatively, in the soil amended with eggshell, Pb was immobilized via formation of Pb-hydroxide or lanarkite [Pb(2)O(SO(4))]. Applications of amendments increased activities of alkaline phosphatase up to 3.7 times greater than in the control soil. The use of eggshell amendments may have potential as an integrated remediation strategy that enables Pb immobilization and soil biological restoration in shooting range soils. PMID:22309654

  8. Characterization of a nitrogen-regulated protein identified by cell surface biotinylation of a marine phytoplankton

    SciTech Connect

    Palenik, B.; Koke, J.A.

    1995-09-01

    The biotinylating reagent succinimidyl 6-(biotinamido) hexanoate was used to label the cell surfaces of the cosmopolitan, marine, eukaryotic microorganism Emiliania huxleyi under different growth conditions. Proteins characteristic of different nutrient conditions could be identified. In particular, a nitrogen-regulated protein, nrp1, has an 82-kDa subunit that is present under nitrogen limitation and during growth on urea. It is absent under phosphate limitation or during exponential growth on nitrate or ammonia. nrp1 is the major membrane or wall protein in nitrogen-limited cells and is found in several strains of E. huxleyi. It may be a useful biomarker for examining the physiological state of E. huxleyi cells in their environment. 35 refs., 4 figs.

  9. A coprecipitation-based validation methodology for interactions identified using protein microarrays.

    PubMed

    Marina, Ovidiu; Duke-Cohan, Jonathan S; Wu, Catherine J

    2011-01-01

    Candidate interactions identified by high-throughput protein microarray screening require rigorous -confirmation. Such validation is time-consuming and labor-intensive using conventional techniques. We describe a medium-throughput validation protocol based on coprecipitation of biotin-labeled -proteins synthesized in vitro using a rabbit reticulocyte lysate-coupled transcription and translation system. As our experimental system is based on screening for serum antibodies, we also present methods on purifying immunoglobulin from serum and quantifying the amount of coprecipitated (immunoprecipitated) target protein on Western blot. This technique provides a sensitive confirmatory test allowing for the rapid elimination of false positives prior to more extensive validation and analysis of target interactions in their native environment. PMID:21370070

  10. Extensive dataset of boar seminal plasma proteome displaying putative reproductive functions of identified proteins.

    PubMed

    Perez-Patiño, Cristina; Barranco, Isabel; Parrilla, Inmaculada; Martinez, Emilio A; Rodriguez-Martinez, Heriberto; Roca, Jordi

    2016-09-01

    A complete proteomic profile of seminal plasma (SP) remains challenging, particularly in porcine. The data reports on the analysis of boar SP-proteins by using a combination of SEC, 1-D SDS PAGE and NanoLC-ESI-MS/MS from 33 pooled SP-samples (11 boars, 3 ejaculates/boar). A complete dataset of the 536 SP-proteins identified and validated with confidence ≥95% (Unused Score >1.3) and a false discovery rate (FDR) ≤1%, is provided. In addition, the relative abundance of 432 of them is also shown. Gene ontology annotation of the complete SP-proteome complemented by an extensive description of the putative reproductive role of SP-proteins, providing a valuable source for a better understanding of SP role in the reproductive success. This data article refers to the article entitled "Characterization of the porcine seminal plasma proteome comparing ejaculate portions" (Perez-Patiño et al., 2016) [1]. PMID:27583342

  11. Cell-Surface Protein Profiling Identifies Distinctive Markers of Progenitor Cells in Human Skeletal Muscle.

    PubMed

    Uezumi, Akiyoshi; Nakatani, Masashi; Ikemoto-Uezumi, Madoka; Yamamoto, Naoki; Morita, Mitsuhiro; Yamaguchi, Asami; Yamada, Harumoto; Kasai, Takehiro; Masuda, Satoru; Narita, Asako; Miyagoe-Suzuki, Yuko; Takeda, Shin'ichi; Fukada, So-Ichiro; Nishino, Ichizo; Tsuchida, Kunihiro

    2016-08-01

    Skeletal muscle contains two distinct stem/progenitor populations. One is the satellite cell, which acts as a muscle stem cell, and the other is the mesenchymal progenitor, which contributes to muscle pathogeneses such as fat infiltration and fibrosis. Detailed and accurate characterization of these progenitors in humans remains elusive. Here, we performed comprehensive cell-surface protein profiling of the two progenitor populations residing in human skeletal muscle and identified three previously unrecognized markers: CD82 and CD318 for satellite cells and CD201 for mesenchymal progenitors. These markers distinguish myogenic and mesenchymal progenitors, and enable efficient isolation of the two types of progenitors. Functional study revealed that CD82 ensures expansion and preservation of myogenic progenitors by suppressing excessive differentiation, and CD201 signaling favors adipogenesis of mesenchymal progenitors. Thus, cell-surface proteins identified here are not only useful markers but also functionally important molecules, and provide valuable insight into human muscle biology and diseases. PMID:27509136

  12. Microstructure, crystallography and diagenetic alteration in fossil ostrich eggshells from Upper Palaeolithic sites of Indian peninsular region.

    PubMed

    Jain, Sonal; Bajpai, Sunil; Kumar, Giriraj; Pruthi, Vikas

    2016-05-01

    Biominerals studies are of importance as they provide an understanding of natural evolutionary processes. In this study we have investigated the fossil ostrich eggshells using Scanning Electron Microscopy (SEM), X-ray Diffraction (XRD) and Electron Backscatter Diffraction (EBSD). SEM studies demonstrated the ultrastructure of fossil eggshells and formation of calcified cuticular layer. The presence of calcified cuticle layer in eggshell is the basis for ancient DNA studies as it contains preserved biomolecules. EBSD accentuates the crystallographic structure of the ostrich eggshells with sub-micrometer resolution. It is a non-destructive tool for evaluating the extent of diagenesis in a biomineral. EBSD analysis revealed the presence of dolomite in the eggshells. This research resulted in the complete recognition of the structure of ostrich eggshells as well as the nature and extent of diagenesis in these eggshells which is vital for genetic and paleoenvironmental studies. PMID:26994328

  13. Revealing divergent evolution, identifying circular permutations and detecting active-sites by protein structure comparison

    PubMed Central

    Chen, Luonan; Wu, Ling-Yun; Wang, Yong; Zhang, Shihua; Zhang, Xiang-Sun

    2006-01-01

    Background Protein structure comparison is one of the most important problems in computational biology and plays a key role in protein structure prediction, fold family classification, motif finding, phylogenetic tree reconstruction and protein docking. Results We propose a novel method to compare the protein structures in an accurate and efficient manner. Such a method can be used to not only reveal divergent evolution, but also identify circular permutations and further detect active-sites. Specifically, we define the structure alignment as a multi-objective optimization problem, i.e., maximizing the number of aligned atoms and minimizing their root mean square distance. By controlling a single distance-related parameter, theoretically we can obtain a variety of optimal alignments corresponding to different optimal matching patterns, i.e., from a large matching portion to a small matching portion. The number of variables in our algorithm increases with the number of atoms of protein pairs in almost a linear manner. In addition to solid theoretical background, numerical experiments demonstrated significant improvement of our approach over the existing methods in terms of quality and efficiency. In particular, we show that divergent evolution, circular permutations and active-sites (or structural motifs) can be identified by our method. The software SAMO is available upon request from the authors, or from and . Conclusion A novel formulation is proposed to accurately align protein structures in the framework of multi-objective optimization, based on a sequence order-independent strategy. A fast and accurate algorithm based on the bipartite matching algorithm is developed by exploiting the special features. Convergence of computation is shown in experiments and is also theoretically proven. PMID:16948858

  14. Systematic analysis of mutation distribution in three dimensional protein structures identifies cancer driver genes

    PubMed Central

    Fujimoto, Akihiro; Okada, Yukinori; Boroevich, Keith A.; Tsunoda, Tatsuhiko; Taniguchi, Hiroaki; Nakagawa, Hidewaki

    2016-01-01

    Protein tertiary structure determines molecular function, interaction, and stability of the protein, therefore distribution of mutation in the tertiary structure can facilitate the identification of new driver genes in cancer. To analyze mutation distribution in protein tertiary structures, we applied a novel three dimensional permutation test to the mutation positions. We analyzed somatic mutation datasets of 21 types of cancers obtained from exome sequencing conducted by the TCGA project. Of the 3,622 genes that had ≥3 mutations in the regions with tertiary structure data, 106 genes showed significant skew in mutation distribution. Known tumor suppressors and oncogenes were significantly enriched in these identified cancer gene sets. Physical distances between mutations in known oncogenes were significantly smaller than those of tumor suppressors. Twenty-three genes were detected in multiple cancers. Candidate genes with significant skew of the 3D mutation distribution included kinases (MAPK1, EPHA5, ERBB3, and ERBB4), an apoptosis related gene (APP), an RNA splicing factor (SF1), a miRNA processing factor (DICER1), an E3 ubiquitin ligase (CUL1) and transcription factors (KLF5 and EEF1B2). Our study suggests that systematic analysis of mutation distribution in the tertiary protein structure can help identify cancer driver genes. PMID:27225414

  15. Computational approaches to identifying and characterizing protein binding sites for ligand design.

    PubMed

    Henrich, Stefan; Salo-Ahen, Outi M H; Huang, Bingding; Rippmann, Friedrich F; Cruciani, Gabriele; Wade, Rebecca C

    2010-01-01

    Given the three-dimensional structure of a protein, how can one find the sites where other molecules might bind to it? Do these sites have the properties necessary for high affinity binding? Is this protein a suitable target for drug design? Here, we discuss recent developments in computational methods to address these and related questions. Geometric methods to identify pockets on protein surfaces have been developed over many years but, with new algorithms, their performance is still improving. Simulation methods show promise in accounting for protein conformational variability to identify transient pockets but lack the ease of use of many of the (rigid) shape-based tools. Sequence and structure comparison approaches are benefiting from the constantly increasing size of sequence and structure databases. Energetic methods can aid identification and characterization of binding pockets, and have undergone recent improvements in the treatment of solvation and hydrophobicity. The "druggability" of a binding site is still difficult to predict with an automated procedure. The methodologies available for this purpose range from simple shape and hydrophobicity scores to computationally demanding free energy simulations. PMID:19746440

  16. Systematic analysis of mutation distribution in three dimensional protein structures identifies cancer driver genes.

    PubMed

    Fujimoto, Akihiro; Okada, Yukinori; Boroevich, Keith A; Tsunoda, Tatsuhiko; Taniguchi, Hiroaki; Nakagawa, Hidewaki

    2016-01-01

    Protein tertiary structure determines molecular function, interaction, and stability of the protein, therefore distribution of mutation in the tertiary structure can facilitate the identification of new driver genes in cancer. To analyze mutation distribution in protein tertiary structures, we applied a novel three dimensional permutation test to the mutation positions. We analyzed somatic mutation datasets of 21 types of cancers obtained from exome sequencing conducted by the TCGA project. Of the 3,622 genes that had ≥3 mutations in the regions with tertiary structure data, 106 genes showed significant skew in mutation distribution. Known tumor suppressors and oncogenes were significantly enriched in these identified cancer gene sets. Physical distances between mutations in known oncogenes were significantly smaller than those of tumor suppressors. Twenty-three genes were detected in multiple cancers. Candidate genes with significant skew of the 3D mutation distribution included kinases (MAPK1, EPHA5, ERBB3, and ERBB4), an apoptosis related gene (APP), an RNA splicing factor (SF1), a miRNA processing factor (DICER1), an E3 ubiquitin ligase (CUL1) and transcription factors (KLF5 and EEF1B2). Our study suggests that systematic analysis of mutation distribution in the tertiary protein structure can help identify cancer driver genes. PMID:27225414

  17. Identifying the role of Wilms tumor 1 associated protein in cancer prediction using integrative genomic analyses.

    PubMed

    Wu, Li-Sheng; Qian, Jia-Yi; Wang, Minghai; Yang, Haiwei

    2016-09-01

    The Wilms tumor suppressor, WT1 was first identified due to its essential role in the normal development of the human genitourinary system. Wilms tumor 1 associated protein (WTAP) was subsequently revealed to interact with WT1 using yeast two-hybrid screening. The present study identified 44 complete WTAP genes in the genomes of vertebrates, including fish, amphibians, birds and mammals. The vertebrate WTAP proteins clustered into the primate, rodent and teleost lineages using phylogenetic tree analysis. From 1,347 available SNPs in the human WTAP gene, 19 were identified to cause missense mutations. WTAP was expressed in bladder, blood, brain, breast, colorectal, esophagus, eye, head and neck, lung, ovarian, prostate, skin and soft tissue cancers. A total of 17 out of 328 microarrays demonstrated an association between WTAP gene expression and cancer prognosis. However, the association between WTAP gene expression and prognosis varied in distinct types of cancer, and even in identical types of cancer from separate microarray databases. By searching the Catalogue of Somatic Mutations in Cancer database, 65 somatic mutations were identified in the human WTAP gene from the cancer tissue samples. These results suggest that the function of WTAP in tumor formation may be multidimensional. Furthermore, signal transducer and activator of transcription 1, forkhead box protein O1, interferon regulatory factor 1, glucocorticoid receptor and peroxisome proliferator-activated receptor γ transcription factor binding sites were identified in the upstream (promoter) region of the human WTAP gene, suggesting that these transcription factors may be involved in WTAP functions in tumor formation. PMID:27430156

  18. Host Protein Biomarkers Identify Active Tuberculosis in HIV Uninfected and Co-infected Individuals

    PubMed Central

    Achkar, Jacqueline M.; Cortes, Laetitia; Croteau, Pascal; Yanofsky, Corey; Mentinova, Marija; Rajotte, Isabelle; Schirm, Michael; Zhou, Yiyong; Junqueira-Kipnis, Ana Paula; Kasprowicz, Victoria O.; Larsen, Michelle; Allard, René; Hunter, Joanna; Paramithiotis, Eustache

    2015-01-01

    Biomarkers for active tuberculosis (TB) are urgently needed to improve rapid TB diagnosis. The objective of this study was to identify serum protein expression changes associated with TB but not latent Mycobacterium tuberculosis infection (LTBI), uninfected states, or respiratory diseases other than TB (ORD). Serum samples from 209 HIV uninfected (HIV−) and co-infected (HIV+) individuals were studied. In the discovery phase samples were analyzed via liquid chromatography and mass spectrometry, and in the verification phase biologically independent samples were analyzed via a multiplex multiple reaction monitoring mass spectrometry (MRM-MS) assay. Compared to LTBI and ORD, host proteins were significantly differentially expressed in TB, and involved in the immune response, tissue repair, and lipid metabolism. Biomarker panels whose composition differed according to HIV status, and consisted of 8 host proteins in HIV− individuals (CD14, SEPP1, SELL, TNXB, LUM, PEPD, QSOX1, COMP, APOC1), or 10 host proteins in HIV+ individuals (CD14, SEPP1, PGLYRP2, PFN1, VASN, CPN2, TAGLN2, IGFBP6), respectively, distinguished TB from ORD with excellent accuracy (AUC = 0.96 for HIV− TB, 0.95 for HIV+ TB). These results warrant validation in larger studies but provide promise that host protein biomarkers could be the basis for a rapid, blood-based test for TB. PMID:26501113

  19. Host Protein Biomarkers Identify Active Tuberculosis in HIV Uninfected and Co-infected Individuals.

    PubMed

    Achkar, Jacqueline M; Cortes, Laetitia; Croteau, Pascal; Yanofsky, Corey; Mentinova, Marija; Rajotte, Isabelle; Schirm, Michael; Zhou, Yiyong; Junqueira-Kipnis, Ana Paula; Kasprowicz, Victoria O; Larsen, Michelle; Allard, René; Hunter, Joanna; Paramithiotis, Eustache

    2015-09-01

    Biomarkers for active tuberculosis (TB) are urgently needed to improve rapid TB diagnosis. The objective of this study was to identify serum protein expression changes associated with TB but not latent Mycobacterium tuberculosis infection (LTBI), uninfected states, or respiratory diseases other than TB (ORD). Serum samples from 209 HIV uninfected (HIV(-)) and co-infected (HIV(+)) individuals were studied. In the discovery phase samples were analyzed via liquid chromatography and mass spectrometry, and in the verification phase biologically independent samples were analyzed via a multiplex multiple reaction monitoring mass spectrometry (MRM-MS) assay. Compared to LTBI and ORD, host proteins were significantly differentially expressed in TB, and involved in the immune response, tissue repair, and lipid metabolism. Biomarker panels whose composition differed according to HIV status, and consisted of 8 host proteins in HIV(-) individuals (CD14, SEPP1, SELL, TNXB, LUM, PEPD, QSOX1, COMP, APOC1), or 10 host proteins in HIV(+) individuals (CD14, SEPP1, PGLYRP2, PFN1, VASN, CPN2, TAGLN2, IGFBP6), respectively, distinguished TB from ORD with excellent accuracy (AUC = 0.96 for HIV(-) TB, 0.95 for HIV(+) TB). These results warrant validation in larger studies but provide promise that host protein biomarkers could be the basis for a rapid, blood-based test for TB. PMID:26501113

  20. Integration of evolutionary and desolvation energy analysis identifies functional sites in a plant immunity protein

    PubMed Central

    Casasoli, Manuela; Federici, Luca; Spinelli, Francesco; Di Matteo, Adele; Vella, Nicoletta; Scaloni, Flavio; Fernandez-Recio, Juan; Cervone, Felice; De Lorenzo, Giulia

    2009-01-01

    Plant immune responses often depend on leucine-rich repeat receptors that recognize microbe-associated molecular patterns or pathogen-specific virulence proteins, either directly or indirectly. When the recognition is direct, a molecular arms race takes place where plant receptors continually and rapidly evolve in response to virulence factor evolution. A useful model system to study ligand-receptor coevolution dynamics at the protein level is represented by the interaction between pathogen-derived polygalacturonases (PGs) and plant polygalacturonase-inhibiting proteins (PGIPs). We have applied codon substitution models to PGIP sequences of different eudicotyledonous families to identify putative positively selected sites and then compared these sites with the propensity of protein surface residues to interact with protein partners, based on desolvation energy calculations. The 2 approaches remarkably correlated in pinpointing several residues in the concave face of the leucine-rich repeat domain. These residues were mutated into alanine and their effect on the recognition of several PGs was tested, leading to the identification of unique hotspots for the PGIP-PG interaction. The combined approach used in this work can be of general utility in cases where structural information about a pattern-recognition receptor or resistance-gene product is available. PMID:19372373

  1. Novel Host Proteins and Signaling Pathways in Enteropathogenic E. coli Pathogenesis Identified by Global Phosphoproteome Analysis.

    PubMed

    Scholz, Roland; Imami, Koshi; Scott, Nichollas E; Trimble, William S; Foster, Leonard J; Finlay, B Brett

    2015-07-01

    Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (T3SS) to directly translocate effector proteins into host cells where they play a pivotal role in subverting host cell signaling needed for disease. However, our knowledge of how EPEC affects host protein phosphorylation is limited to a few individual protein studies. We employed a quantitative proteomics approach to globally map alterations in the host phosphoproteome during EPEC infection. By characterizing host phosphorylation events at various time points throughout infection, we examined how EPEC dynamically impacts the host phosphoproteome over time. This experimental setup also enabled identification of T3SS-dependent and -independent changes in host phosphorylation. Specifically, T3SS-regulated events affected various cellular processes that are known EPEC targets, including cytoskeletal organization, immune signaling, and intracellular trafficking. However, the involvement of phosphorylation in these events has thus far been poorly studied. We confirmed the MAPK family as an established key host player, showed its central role in signal transduction during EPEC infection, and extended the repertoire of known signaling hubs with previously unrecognized proteins, including TPD52, CIN85, EPHA2, and HSP27. We identified altered phosphorylation of known EPEC targets, such as cofilin, where the involvement of phosphorylation has so far been undefined, thus providing novel mechanistic insights into the roles of these proteins in EPEC infection. An overlap of regulated proteins, especially those that are cytoskeleton-associated, was observed when compared with the phosphoproteome of Shigella-infected cells. We determined the biological relevance of the phosphorylation of a novel protein in EPEC pathogenesis, septin-9 (SEPT9). Both siRNA knockdown and a phosphorylation-impaired SEPT9 mutant decreased bacterial adherence and EPEC-mediated cell death. In contrast, a phosphorylation

  2. Comparative Proteomics Identifies Host Immune System Proteins Affected by Infection with Mycobacterium bovis.

    PubMed

    López, Vladimir; Villar, Margarita; Queirós, João; Vicente, Joaquín; Mateos-Hernández, Lourdes; Díez-Delgado, Iratxe; Contreras, Marinela; Alves, Paulo C; Alberdi, Pilar; Gortázar, Christian; de la Fuente, José

    2016-03-01

    Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly impact human and animal health worldwide. The mycobacterial life cycle is complex, and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Eurasian wild boar (Sus scrofa) are natural reservoir hosts for MTBC and a model for mycobacterial infection and tuberculosis (TB). In the wild boar TB model, mycobacterial infection affects the expression of innate and adaptive immune response genes in mandibular lymph nodes and oropharyngeal tonsils, and biomarkers have been proposed as correlates with resistance to natural infection. However, the mechanisms used by mycobacteria to manipulate host immune response are not fully characterized. Our hypothesis is that the immune system proteins under-represented in infected animals, when compared to uninfected controls, are used by mycobacteria to guarantee pathogen infection and transmission. To address this hypothesis, a comparative proteomics approach was used to compare host response between uninfected (TB-) and M. bovis-infected young (TB+) and adult animals with different infection status [TB lesions localized in the head (TB+) or affecting multiple organs (TB++)]. The results identified host immune system proteins that play an important role in host response to mycobacteria. Calcium binding protein A9, Heme peroxidase, Lactotransferrin, Cathelicidin and Peptidoglycan-recognition protein were under-represented in TB+ animals when compared to uninfected TB- controls, but protein levels were higher as infection progressed in TB++ animals when compared to TB- and/or TB+ adult wild boar. MHCI was the only protein over-represented in TB+ adult wild boar when compared to uninfected TB- controls. The results reported here suggest that M. bovis manipulates host immune response by reducing the production of immune system proteins. However, as infection progresses, wild boar immune response recovers to limit pathogen

  3. Comparative Proteomics Identifies Host Immune System Proteins Affected by Infection with Mycobacterium bovis

    PubMed Central

    López, Vladimir; Villar, Margarita; Queirós, João; Vicente, Joaquín; Mateos-Hernández, Lourdes; Díez-Delgado, Iratxe; Contreras, Marinela; Alves, Paulo C.; Alberdi, Pilar; Gortázar, Christian; de la Fuente, José

    2016-01-01

    Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly impact human and animal health worldwide. The mycobacterial life cycle is complex, and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Eurasian wild boar (Sus scrofa) are natural reservoir hosts for MTBC and a model for mycobacterial infection and tuberculosis (TB). In the wild boar TB model, mycobacterial infection affects the expression of innate and adaptive immune response genes in mandibular lymph nodes and oropharyngeal tonsils, and biomarkers have been proposed as correlates with resistance to natural infection. However, the mechanisms used by mycobacteria to manipulate host immune response are not fully characterized. Our hypothesis is that the immune system proteins under-represented in infected animals, when compared to uninfected controls, are used by mycobacteria to guarantee pathogen infection and transmission. To address this hypothesis, a comparative proteomics approach was used to compare host response between uninfected (TB-) and M. bovis-infected young (TB+) and adult animals with different infection status [TB lesions localized in the head (TB+) or affecting multiple organs (TB++)]. The results identified host immune system proteins that play an important role in host response to mycobacteria. Calcium binding protein A9, Heme peroxidase, Lactotransferrin, Cathelicidin and Peptidoglycan-recognition protein were under-represented in TB+ animals when compared to uninfected TB- controls, but protein levels were higher as infection progressed in TB++ animals when compared to TB- and/or TB+ adult wild boar. MHCI was the only protein over-represented in TB+ adult wild boar when compared to uninfected TB- controls. The results reported here suggest that M. bovis manipulates host immune response by reducing the production of immune system proteins. However, as infection progresses, wild boar immune response recovers to limit pathogen

  4. Genetic architecture dissection by genome-wide association analysis reveals avian eggshell ultrastructure traits.

    PubMed

    Duan, Zhongyi; Sun, Congjiao; Shen, ManMan; Wang, Kehua; Yang, Ning; Zheng, Jiangxia; Xu, Guiyun

    2016-01-01

    The ultrastructure of an eggshell is considered the major determinant of eggshell quality, which has biological and economic significance for the avian and poultry industries. However, the interrelationships and genome-wide architecture of eggshell ultrastructure remain to be elucidated. Herein, we measured eggshell thickness (EST), effective layer thickness (ET), mammillary layer thickness (MT), and mammillary density (MD) and conducted genome-wide association studies in 927 F2 hens. The SNP-based heritabilities of eggshell ultrastructure traits were estimated to be 0.39, 0.36, 0.17 and 0.19 for EST, ET, MT and MD, respectively, and a total of 719, 784, 1 and 10 genome-wide significant SNPs were associated with EST, ET, MT and MD, respectively. ABCC9, ITPR2, KCNJ8 and WNK1, which are involved in ion transport, were suggested to be the key genes regulating EST and ET. ITM2C and KNDC1 likely affect MT and MD, respectively. Additionally, there were linear relationships between the chromosome lengths and the variance explained per chromosome for EST (R(2) = 0.57) and ET (R(2) = 0.67). In conclusion, the interrelationships and genetic architecture of eggshell ultrastructure traits revealed in this study are valuable for our understanding of the avian eggshell and contribute to research on a variety of other calcified shells. PMID:27456605

  5. Genetic architecture dissection by genome-wide association analysis reveals avian eggshell ultrastructure traits

    PubMed Central

    Duan, Zhongyi; Sun, Congjiao; Shen, ManMan; Wang, Kehua; Yang, Ning; Zheng, Jiangxia; Xu, Guiyun

    2016-01-01

    The ultrastructure of an eggshell is considered the major determinant of eggshell quality, which has biological and economic significance for the avian and poultry industries. However, the interrelationships and genome-wide architecture of eggshell ultrastructure remain to be elucidated. Herein, we measured eggshell thickness (EST), effective layer thickness (ET), mammillary layer thickness (MT), and mammillary density (MD) and conducted genome-wide association studies in 927 F2 hens. The SNP-based heritabilities of eggshell ultrastructure traits were estimated to be 0.39, 0.36, 0.17 and 0.19 for EST, ET, MT and MD, respectively, and a total of 719, 784, 1 and 10 genome-wide significant SNPs were associated with EST, ET, MT and MD, respectively. ABCC9, ITPR2, KCNJ8 and WNK1, which are involved in ion transport, were suggested to be the key genes regulating EST and ET. ITM2C and KNDC1 likely affect MT and MD, respectively. Additionally, there were linear relationships between the chromosome lengths and the variance explained per chromosome for EST (R2 = 0.57) and ET (R2 = 0.67). In conclusion, the interrelationships and genetic architecture of eggshell ultrastructure traits revealed in this study are valuable for our understanding of the avian eggshell and contribute to research on a variety of other calcified shells. PMID:27456605

  6. Proteins that bind regulatory regions identified by histone modification chromatin immunoprecipitations and mass spectrometry

    PubMed Central

    Engelen, Erik; Brandsma, Johannes H.; Moen, Maaike J.; Signorile, Luca; Dekkers, Dick H. W.; Demmers, Jeroen; Kockx, Christel E. M.; Ozgür, Zehila; van IJcken, Wilfred F. J.; van den Berg, Debbie L. C.; Poot, Raymond A.

    2015-01-01

    The locations of transcriptional enhancers and promoters were recently mapped in many mammalian cell types. Proteins that bind those regulatory regions can determine cell identity but have not been systematically identified. Here we purify native enhancers, promoters or heterochromatin from embryonic stem cells by chromatin immunoprecipitations (ChIP) for characteristic histone modifications and identify associated proteins using mass spectrometry (MS). 239 factors are identified and predicted to bind enhancers or promoters with different levels of activity, or heterochromatin. Published genome-wide data indicate a high accuracy of location prediction by ChIP-MS. A quarter of the identified factors are important for pluripotency and includes Oct4, Esrrb, Klf5, Mycn and Dppa2, factors that drive reprogramming to pluripotent stem cells. We determined the genome-wide binding sites of Dppa2 and find that Dppa2 operates outside the classical pluripotency network. Our ChIP-MS method provides a detailed read-out of the transcriptional landscape representative of the investigated cell type. PMID:25990348

  7. A Genome-Wide Association Study Identifies Protein Quantitative Trait Loci (pQTLs)

    PubMed Central

    Corsi, Anna-Maria; Stevens, Kara; Rafferty, Ian; Lauretani, Fulvio; Murray, Anna; Gibbs, J. Raphael; Paolisso, Giuseppe; Rafiq, Sajjad; Simon-Sanchez, Javier; Lango, Hana; Scholz, Sonja; Weedon, Michael N.; Arepalli, Sampath; Rice, Neil; Washecka, Nicole; Hurst, Alison; Britton, Angela; Henley, William; van de Leemput, Joyce; Li, Rongling; Newman, Anne B.; Tranah, Greg; Harris, Tamara; Panicker, Vijay; Dayan, Colin; Bennett, Amanda; McCarthy, Mark I.; Ruokonen, Aimo; Jarvelin, Marjo-Riitta; Guralnik, Jack; Bandinelli, Stefania; Frayling, Timothy M.; Singleton, Andrew; Ferrucci, Luigi

    2008-01-01

    There is considerable evidence that human genetic variation influences gene expression. Genome-wide studies have revealed that mRNA levels are associated with genetic variation in or close to the gene coding for those mRNA transcripts – cis effects, and elsewhere in the genome – trans effects. The role of genetic variation in determining protein levels has not been systematically assessed. Using a genome-wide association approach we show that common genetic variation influences levels of clinically relevant proteins in human serum and plasma. We evaluated the role of 496,032 polymorphisms on levels of 42 proteins measured in 1200 fasting individuals from the population based InCHIANTI study. Proteins included insulin, several interleukins, adipokines, chemokines, and liver function markers that are implicated in many common diseases including metabolic, inflammatory, and infectious conditions. We identified eight Cis effects, including variants in or near the IL6R (p = 1.8×10−57), CCL4L1 (p = 3.9×10−21), IL18 (p = 6.8×10−13), LPA (p = 4.4×10−10), GGT1 (p = 1.5×10−7), SHBG (p = 3.1×10−7), CRP (p = 6.4×10−6) and IL1RN (p = 7.3×10−6) genes, all associated with their respective protein products with effect sizes ranging from 0.19 to 0.69 standard deviations per allele. Mechanisms implicated include altered rates of cleavage of bound to unbound soluble receptor (IL6R), altered secretion rates of different sized proteins (LPA), variation in gene copy number (CCL4L1) and altered transcription (GGT1). We identified one novel trans effect that was an association between ABO blood group and tumour necrosis factor alpha (TNF-alpha) levels (p = 6.8×10−40), but this finding was not present when TNF-alpha was measured using a different assay , or in a second study, suggesting an assay-specific association. Our results show that protein levels share some of the features of the genetics of gene expression. These

  8. Preparation and characterization of polyurethane/soluble eggshell membrane nanofibers.

    PubMed

    Chen, Long; Kang, Jian; Sukigara, Sachiko

    2014-01-01

    Superfine Particles (SP) of soluble eggshell membrane and medical grade polyurethane (MPU) blend nanofibers were prepared by electrospinning different blend ratios of SP/MPU suspensions for regeneration of the natural fiber-like structure of the eggshell membrane. The addition of SP had no obvious effect on the electrospinning process of MPU nanofibers, and the SP were randomly dispersed in the MPU nanofibers with no agglomeration of SP when the amount of SP was less than 20 wt%. Although the average diameter of the blend nanofibers is approximately 30% larger than that of the pure MPU nanofibers, they exhibit excellent tensile strength and tensile resilience that are close to those for pure MPU nanofibers. In addition, the blend nanofibers become fully hydrophilic, and the water contact angle of the blend nanofibers decreases dramatically to 0°. Therefore, with the advantages of a collagen ingredient and good hydrophilicity, these blend nanofibers are suitable for applications such as facial masks, wound dressings, and pharmaceutical carrier materials. PMID:25226894

  9. Preliminary studies on immobilization of lipase using chicken eggshell

    NASA Astrophysics Data System (ADS)

    Salleh, S.; Serri, N. A.; Hena, S.; Tajarudin, H. A.

    2016-06-01

    A few advantages of enzyme immobilization are reusability of expensive enzyme, improvement of stability and activity compared to crude enzyme. Various organic components can be used as carrier for enzyme immobilization such as chicken eggshell. It can be used as a carrier for immobilization as its mineral component mostly contains of calcium carbonate. In the present study, Tributyrin method was used to test enzyme activity of Rhizomucour Miehei, Candida Antarctica and Candida Rugosa. Rhizomucour Miehei shows the highest enzyme activity (360.8 mol/min/mL lipase) and was used in further experiment. Experiment was continued to study incubation time for lipase immobilization on eggshell (1-4 hours) and reaction time of esterification of sugar ester (0-72 hours). Two hours incubation time for lipase immobilization was observed and gives the highest yield of sugar ester (78.13%). Fructose and stearic acid as substrate was used for the production of sugar ester. The highest percentage of sugar ester production was shown at 36 hours of reaction time.

  10. Dicofol (Kelthane?)-induced eggshell thinning in captive American kestrels

    USGS Publications Warehouse

    Clark, D.R., Jr.; Spann, J.W.; Bunck, C.M.

    1990-01-01

    Reproductive parameters of American kestrels (Falco sparverius) were measured through two breeding seasons. Exposure to Kelthane? (containing no DDT-related compounds) at dietary concentrations of 0 (contro!), 1, 3, 10 and 30 ug/g (wet weight) began in late November before, and continued through, the second season. Kelthane thinned eggshells and lowered the thickness index at dietary concentrations >3 ?g/g and it reduced shell weight at >10 ?g/g when comparisons were to concurrent controls. Kelthane reduced the thickness index at >3 ug/g and it reduced shell thickness and weight at >10 ug/g when comparisons were to the same birds during the previous season. All changes were dose-related. It was not previously known that as little as 3 ug/g dicofol could cause these effects. Kestrels resembled previously studied eastern screech-owls (Otus asio) in that 10 ug/ g reduced hatchability of eggs. Both these raptors showed eggshell changes without serious effects on production of young. Available data show dicofol only equal to or less effective than DDE as a shell-thinning agent. Also, DDE may have more impact than dicofol on such critical aspects of reproduction as egg hatchability and survivability of hatchlings. Field studies of dicofol residues in food chains and of residue concentrations in eggs vs. nesting success from areas of heavy dicofol use are needed to judge this chemical's ecological impact.

  11. Diversity of epithelial morphogenesis during eggshell formation in drosophilids

    PubMed Central

    Osterfield, Miriam; Schüpbach, Trudi; Wieschaus, Eric; Shvartsman, Stanislav Y.

    2015-01-01

    ABSTRACT The eggshells of drosophilid species provide a powerful model for studying the origins of morphological diversity. The dorsal appendages, or respiratory filaments, of these eggshells display a remarkable interspecies variation in number and shape, and the epithelial patterning underlying the formation of these structures is an area of active research. To extend the analysis of dorsal appendage formation to include morphogenesis, we developed an improved 3D image reconstruction approach. This approach revealed considerable interspecies variation in the cell shape changes and neighbor exchanges underlying appendage formation. Specifically, although the appendage floor in Drosophila melanogaster is formed through spatially ordered neighbor exchanges, the same structure in Scaptodrosophila pattersoni is formed through extreme changes in cell shape, whereas Drosophila funebris appears to display a combination of both cellular mechanisms. Furthermore, localization patterns of Par3/Bazooka suggest a self-organized, cell polarity-based origin for the variability of appendage number in S. pattersoni. Our results suggest that species deploy different combinations of apically and basally driven mechanisms to convert a two-dimensional primordium into a three-dimensional structure, and provide new directions for exploring the molecular origins of interspecies morphological variation. PMID:25953345

  12. EST mining identifies proteins putatively secreted by the anthracnose pathogen Colletotrichum truncatum

    PubMed Central

    2011-01-01

    Background Colletotrichum truncatum is a haploid, hemibiotrophic, ascomycete fungal pathogen that causes anthracnose disease on many economically important leguminous crops. This pathogen exploits sequential biotrophic- and necrotrophic- infection strategies to colonize the host. Transition from biotrophy to a destructive necrotrophic phase called the biotrophy-necrotrophy switch is critical in symptom development. C. truncatum likely secretes an arsenal of proteins that are implicated in maintaining a compatible interaction with its host. Some of them might be transition specific. Results A directional cDNA library was constructed from mRNA isolated from infected Lens culinaris leaflet tissues displaying the biotrophy-necrotrophy switch of C. truncatum and 5000 expressed sequence tags (ESTs) with an average read of > 600 bp from the 5-prime end were generated. Nearly 39% of the ESTs were predicted to encode proteins of fungal origin and among these, 162 ESTs were predicted to contain N-terminal signal peptides (SPs) in their deduced open reading frames (ORFs). The 162 sequences could be assembled into 122 tentative unigenes comprising 32 contigs and 90 singletons. Sequence analyses of unigenes revealed four potential groups: hydrolases, cell envelope associated proteins (CEAPs), candidate effectors and other proteins. Eleven candidate effector genes were identified based on features common to characterized fungal effectors, i.e. they encode small, soluble (lack of transmembrane domain), cysteine-rich proteins with a putative SP. For a selected subset of CEAPs and candidate effectors, semiquantitative RT-PCR showed that these transcripts were either expressed constitutively in both in vitro and in planta or induced during plant infection. Using potato virus X (PVX) based transient expression assays, we showed that one of the candidate effectors, i. e. contig 8 that encodes a cerato-platanin (CP) domain containing protein, unlike CP proteins from other fungal

  13. Retrograde Intraflagellar Transport Mutants Identify Complex A Proteins With Multiple Genetic Interactions in Chlamydomonas reinhardtii

    PubMed Central

    Iomini, Carlo; Li, Linya; Esparza, Jessica M.; Dutcher, Susan K.

    2009-01-01

    The intraflagellar transport machinery is required for the assembly of cilia. It has been investigated by biochemical, genetic, and computational methods that have identified at least 21 proteins that assemble into two subcomplexes. It has been hypothesized that complex A is required for retrograde transport. Temperature-sensitive mutations in FLA15 and FLA17 show defects in retrograde intraflagellar transport (IFT) in Chlamydomonas. We show that IFT144 and IFT139, two complex A proteins, are encoded by FLA15 and FLA17, respectively. The fla15 allele is a missense mutation in a conserved cysteine and the fla17 allele is an in-frame deletion of three exons. The flagellar assembly defect of each mutant is rescued by the respective transgenes. In fla15 and fla17 mutants, bulges form in the distal one-third of the flagella at the permissive temperature and this phenotype is also rescued by the transgenes. These bulges contain the complex B component IFT74/72, but not α-tubulin or p28, a component of an inner dynein arm, which suggests specificity with respect to the proteins that accumulate in these bulges. IFT144 and IFT139 are likely to interact with each other and other proteins on the basis of three distinct genetic tests: (1) Double mutants display synthetic flagellar assembly defects at the permissive temperature, (2) heterozygous diploid strains exhibit second-site noncomplemention, and (3) transgenes confer two-copy suppression. Since these tests show different levels of phenotypic sensitivity, we propose they illustrate different gradations of gene interaction between complex A proteins themselves and with a complex B protein (IFT172). PMID:19720863

  14. A novel DNA replication origin identified in the human heat shock protein 70 gene promoter.

    PubMed Central

    Taira, T; Iguchi-Ariga, S M; Ariga, H

    1994-01-01

    A general and sensitive method for the mapping of initiation sites of DNA replication in vivo, developed by Vassilev and Johnson, has revealed replication origins in the region of simian virus 40 ori, in the regions upstream from the human c-myc gene and downstream from the Chinese hamster dihydrofolate reductase gene, and in the enhancer region of the mouse immunoglobulin heavy-chain gene. Here we report that the region containing the promoter of the human heat shock protein 70 (hsp70) gene was identified as a DNA replication origin in HeLa cells by this method. Several segments of the region were cloned into pUC19 and examined for autonomously replicating sequence (ARS) activity. The plasmids carrying the segments replicated episomally and semiconservatively when transfected into HeLa cells. The segments of ARS activity contained the sequences previously identified as binding sequences for a c-myc protein complex (T. Taira, Y. Negishi, F. Kihara, S. M. M. Iguchi-Ariga, and H. Ariga, Biochem. Biophys. Acta 1130:166-174, 1992). Mutations introduced within the c-myc protein complex binding sequences abolished the ARS activity. Moreover, the ARS plasmids stably replicated at episomal state for a long time in established cell lines. The results suggest that the promoter region of the human hsp70 gene plays a role in DNA replication as well as in transcription. Images PMID:8065368

  15. A Mutant Library Approach to Identify Improved Meningococcal Factor H Binding Protein Vaccine Antigens

    PubMed Central

    Konar, Monica; Rossi, Raffaella; Walter, Helen; Pajon, Rolando; Beernink, Peter T.

    2015-01-01

    Factor H binding protein (FHbp) is a virulence factor used by meningococci to evade the host complement system. FHbp elicits bactericidal antibodies in humans and is part of two recently licensed vaccines. Using human complement Factor H (FH) transgenic mice, we previously showed that binding of FH decreased the protective antibody responses to FHbp vaccination. Therefore, in the present study we devised a library-based method to identify mutant FHbp antigens with very low binding of FH. Using an FHbp sequence variant in one of the two licensed vaccines, we displayed an error-prone PCR mutant FHbp library on the surface of Escherichia coli. We used fluorescence-activated cell sorting to isolate FHbp mutants with very low binding of human FH and preserved binding of control anti-FHbp monoclonal antibodies. We sequenced the gene encoding FHbp from selected clones and introduced the mutations into a soluble FHbp construct. Using this approach, we identified several new mutant FHbp vaccine antigens that had very low binding of FH as measured by ELISA and surface plasmon resonance. The new mutant FHbp antigens elicited protective antibody responses in human FH transgenic mice that were up to 20-fold higher than those elicited by the wild-type FHbp antigen. This approach offers the potential to discover mutant antigens that might not be predictable even with protein structural information and potentially can be applied to other microbial vaccine antigens that bind host proteins. PMID:26057742

  16. A Mutant Library Approach to Identify Improved Meningococcal Factor H Binding Protein Vaccine Antigens.

    PubMed

    Konar, Monica; Rossi, Raffaella; Walter, Helen; Pajon, Rolando; Beernink, Peter T

    2015-01-01

    Factor H binding protein (FHbp) is a virulence factor used by meningococci to evade the host complement system. FHbp elicits bactericidal antibodies in humans and is part of two recently licensed vaccines. Using human complement Factor H (FH) transgenic mice, we previously showed that binding of FH decreased the protective antibody responses to FHbp vaccination. Therefore, in the present study we devised a library-based method to identify mutant FHbp antigens with very low binding of FH. Using an FHbp sequence variant in one of the two licensed vaccines, we displayed an error-prone PCR mutant FHbp library on the surface of Escherichia coli. We used fluorescence-activated cell sorting to isolate FHbp mutants with very low binding of human FH and preserved binding of control anti-FHbp monoclonal antibodies. We sequenced the gene encoding FHbp from selected clones and introduced the mutations into a soluble FHbp construct. Using this approach, we identified several new mutant FHbp vaccine antigens that had very low binding of FH as measured by ELISA and surface plasmon resonance. The new mutant FHbp antigens elicited protective antibody responses in human FH transgenic mice that were up to 20-fold higher than those elicited by the wild-type FHbp antigen. This approach offers the potential to discover mutant antigens that might not be predictable even with protein structural information and potentially can be applied to other microbial vaccine antigens that bind host proteins. PMID:26057742

  17. Strontium-90 in Canada goose eggshells: Non-fatal monitoring for contamination in wildlife

    SciTech Connect

    Rickard, W.H.; Eberhardt, L.E. )

    1989-08-01

    Strontium-90 as measured in eggshells taken form newly hatched eggs in 42 Canada goose nests. Higher-than-background levels were present in eggshells from only a few nests. The origin of the enhanced strontium-90 levels appears to be Hanford Site facilities. However, the amounts measured are too low to expect to see any deleterious health or reproductive effects on the goose population. Eggshells provide an efficient way to obtain biological samples for environmental monitoring without inducing goose mortality. 10 refs., 2 figs.

  18. Acute and chronic eggshell temperature manipulations during hatching term influence hatchability, broiler performance, and ascites incidence.

    PubMed

    Sozcu, A; Ipek, A

    2015-02-01

    The aim of the current study was to determine how a control temperature and acute and chronic high eggshell temperatures during the last three days of incubation, can affect hatchability, chick quality, and organ development on day of hatch as well as broiler performance and ascites incidence in later life. The eggshell temperature manipulations were applied during hatching term (days 19 to 21) as follows: control EST (37.3 to 38.0°C), acute high eggshell temperature manipulations (38.4- to 39.0°C for three hours daily) and chronic high eggshell temperature manipulations (38.4 to 39.0°C). The lowest hatchability and the highest cull chick rate were in the chronic high eggshell temperature manipulations group. Lower chick quality parameters correlated with lower chick weights and heavier residual yolk sac weights that were in the chronic high eggshell temperature manipulations group depending on hatch time. The live weights on the 1(st) day of the growing period were higher in the control and acute high eggshell temperature manipulations groups than the chronic high eggshell temperature manipulations group. At 6 wk of age, live weights of broilers were the highest in the control than in the acute and chronic high eggshell temperature manipulations groups. The total mortality was 2.5, 9.2, and 13.3%, the mortality due to ascites was 2.1, 8.3, and 12.9% in the control, acute ,and chronic high eggshell temperature manipulations groups, respectively. The right ventricular/total ventricular ratios for the control, acute and chronic high eggshell temperature manipulations groups were 0.22, 0.28, and 0.30%, respectively. In conclusion, short-term and long-term higher temperatures during the hatching term affect embryo development, incubation results, broiler performance, and ascites incidence. Although the acute high eggshell temperature manipulations did not affect the chick quality parameters at hatch, it negatively affected incubation results and broiler performance

  19. Engineering nanoparticles surface for biosensing: "Chemical noses" to detect and identify proteins, bacteria and cancerous cells

    NASA Astrophysics Data System (ADS)

    Miranda-Sanchez, Oscar Ramon

    Rapid and sensitive detection of biomolecules is an important issue in nanomedicine. Many disorders are manifested by changes in protein levels of serum and other biofluids. Rapid and effective differentiation between normal and cancerous cells is an important challenge for the diagnosis and treatment of tumor. Likewise, rapid and effective identification of pathogens is a key target in both biomedical and environmental monitoring. Most biological recognition processes occur via specific interactions. Gold nanoparticles (AuNP s) feature sizes commensurate with biomacromolecules, coupled with useful physical and optical properties. A key issue in the use of nanomaterials is controlling the interfacial interactions of these complex systems. Modulation of these physicochemical properties can be readily achieved by engineering nanoparticles surface. Inspired by the idea of mimicking nature, a convenient, precise and rapid method for sensing proteins, cancerous cells and bacteria has been developed by overtaking the superb performance of biological olfactory systems in odor detection, identification, tracking, and location. On the fundamental side, an array-based/'chemical nose' sensor composed of cationic functionalized AuNPs as receptors and anionic fluorescent conjugated polymers or green fluorescent proteins or enzyme/substrates as transducers that can properly detect and identify proteins, bacteria, and cancerous cells has been successfully fabricated.

  20. Bayesian hidden Markov models to identify RNA-protein interaction sites in PAR-CLIP.

    PubMed

    Yun, Jonghyun; Wang, Tao; Xiao, Guanghua

    2014-06-01

    The photoactivatable ribonucleoside enhanced cross-linking immunoprecipitation (PAR-CLIP) has been increasingly used for the global mapping of RNA-protein interaction sites. There are two key features of the PAR-CLIP experiments: The sequence read tags are likely to form an enriched peak around each RNA-protein interaction site; and the cross-linking procedure is likely to introduce a specific mutation in each sequence read tag at the interaction site. Several ad hoc methods have been developed to identify the RNA-protein interaction sites using either sequence read counts or mutation counts alone; however, rigorous statistical methods for analyzing PAR-CLIP are still lacking. In this article, we propose an integrative model to establish a joint distribution of observed read and mutation counts. To pinpoint the interaction sites at single base-pair resolution, we developed a novel modeling approach that adopts non-homogeneous hidden Markov models to incorporate the nucleotide sequence at each genomic location. Both simulation studies and data application showed that our method outperforms the ad hoc methods, and provides reliable inferences for the RNA-protein binding sites from PAR-CLIP data. PMID:24571656

  1. Genome-Wide RNAi Screen Identifies Novel Host Proteins Required for Alphavirus Entry

    PubMed Central

    Taylor, Gwen M.; Kielian, Margaret

    2013-01-01

    The enveloped alphaviruses include important and emerging human pathogens such as Chikungunya virus and Eastern equine encephalitis virus. Alphaviruses enter cells by clathrin-mediated endocytosis, and exit by budding from the plasma membrane. While there has been considerable progress in defining the structure and function of the viral proteins, relatively little is known about the host factors involved in alphavirus infection. We used a genome-wide siRNA screen to identify host factors that promote or inhibit alphavirus infection in human cells. Fuzzy homologue (FUZ), a protein with reported roles in planar cell polarity and cilia biogenesis, was required for the clathrin-dependent internalization of both alphaviruses and the classical endocytic ligand transferrin. The tetraspanin membrane protein TSPAN9 was critical for the efficient fusion of low pH-triggered virus with the endosome membrane. FUZ and TSPAN9 were broadly required for infection by the alphaviruses Sindbis virus, Semliki Forest virus, and Chikungunya virus, but were not required by the structurally-related flavivirus Dengue virus. Our results highlight the unanticipated functions of FUZ and TSPAN9 in distinct steps of alphavirus entry and suggest novel host proteins that may serve as targets for antiviral therapy. PMID:24367265

  2. JUMPg: An Integrative Proteogenomics Pipeline Identifying Unannotated Proteins in Human Brain and Cancer Cells.

    PubMed

    Li, Yuxin; Wang, Xusheng; Cho, Ji-Hoon; Shaw, Timothy I; Wu, Zhiping; Bai, Bing; Wang, Hong; Zhou, Suiping; Beach, Thomas G; Wu, Gang; Zhang, Jinghui; Peng, Junmin

    2016-07-01

    Proteogenomics is an emerging approach to improve gene annotation and interpretation of proteomics data. Here we present JUMPg, an integrative proteogenomics pipeline including customized database construction, tag-based database search, peptide-spectrum match filtering, and data visualization. JUMPg creates multiple databases of DNA polymorphisms, mutations, splice junctions, partially trypticity, as well as protein fragments translated from the whole transcriptome in all six frames upon RNA-seq de novo assembly. We use a multistage strategy to search these databases sequentially, in which the performance is optimized by re-searching only unmatched high-quality spectra and reusing amino acid tags generated by the JUMP search engine. The identified peptides/proteins are displayed with gene loci using the UCSC genome browser. Then, the JUMPg program is applied to process a label-free mass spectrometry data set of Alzheimer's disease postmortem brain, uncovering 496 new peptides of amino acid substitutions, alternative splicing, frame shift, and "non-coding gene" translation. The novel protein PNMA6BL specifically expressed in the brain is highlighted. We also tested JUMPg to analyze a stable-isotope labeled data set of multiple myeloma cells, revealing 991 sample-specific peptides that include protein sequences in the immunoglobulin light chain variable region. Thus, the JUMPg program is an effective proteogenomics tool for multiomics data integration. PMID:27225868

  3. Cross reactivity among the swine mycoplasmas as identified by protein microarray.

    PubMed

    Petersen, Andrew C; Oneal, David C; Seibel, Janice R; Poel, Kylie; Daum, Courtney L; Djordjevic, Steven P; Minion, F Chris

    2016-08-30

    Mycoplasmas are cell wall-less bacteria that infect a variety of animals in a species-specific manner. In swine, Mycoplasma hyopneumoniae is the most virulent and presents the most disease and economic problems to the swine industry. Serological assays are commonly used to assess colonization and disease, but antigenic cross-reactivity between M. hyopneumoniae and other mycoplasma species, most notably Mycoplasma hyorhinis, Mycoplasma hyosynoviae and Mycoplasma flocculare, is a concern. The extent of cross-reactivity has not been thoroughly investigated. These studies were designed to identify M. hyopneumoniae proteins that are recognized by rabbit hyperimmune sera raised against the other swine mycoplasmas. Our results indicate extensive cross-reactivity between M. flocculare and M. hyopneumoniae, which explains previous reports seen with ELISA assays. Only three of the thirty-nine M. hyopneumoniae proteins tested showed no cross reactivity with the other three swine mycoplasmas, mhp182 (42kDa C-terminal fragment), mhp638 and mhp684 (C-terminal fragment). Two proteins, mhp384 and mhp511, were cross-reactive with hyperimmune sera generated against three of the four species. None of the anti-M. hyorhinis hyperimmune sera reacted to any of the M. hyopneumoniae proteins. These results suggest that inapparent M. flocculare infections could produce positive responses in M. hyopneumoniae serological assays due to cross-reactivity, and that M. hyosynoviae infections are less likely to do so and M. hyorhinis infections unlikely to affect assay results. PMID:27527784

  4. Ratcheted molecular-dynamics simulations identify efficiently the transition state of protein folding

    NASA Astrophysics Data System (ADS)

    Tiana, Guido; Camilloni, Carlo

    2012-12-01

    The atomistic characterization of the transition state (TS) is a fundamental step to improve the understanding of the folding mechanism and the function of proteins. From a computational point of view, the identification of the conformations that build out the transition state is particularly cumbersome, mainly because of the large computational cost of generating a statistically sound set of folding trajectories. Here we show that a biasing algorithm, based on the physics of the ratchet-and-pawl, can be used to approximate efficiently the transition state. The basic idea is that the algorithmic ratchet exerts a force on the protein when it is climbing the free-energy barrier, while it is inactive when it is descending. The transition state can be identified as the point of the trajectory where the ratchet changes regime. Besides discussing this strategy in general terms, we test it within a protein model whose transition state can be studied independently by plain molecular dynamics simulations. Finally, we show its power in explicit-solvent simulations, obtaining and characterizing a set of transition-state conformations for Acyl-Coenzyme A-Binding Protein (ACBP) and Chymotrypsin Inhibitor 2 (CI2).

  5. Aberrant splicing and truncated-protein expression due to a newly identified XPA gene mutation.

    PubMed

    Sato, M; Nishigori, C; Yagi, T; Takebe, H

    1996-02-15

    A group A xeroderma pigmentosum (XPA) patient, XP2NI, is a compound heterozygote with a newly identified G to C transversion at the last nucleotide in exon 5 in one chromosome, and with the known splicing mutation in intron 3 in another chromosome in the XPA gene. XP2NI had mild skin symptoms and the cells were slightly less sensitive to UV radiation than the cells of typical severe XPA patients who have the splicing mutation in intron 3 homozygously. Reverse transcriptase (RT)-PCR and sequencing of the PCR products revealed that the mutation in exon 5 resulted in producing three types of aberrant mRNA, lacking 7 nucleotides at the end of exon 5, lacking entire exon 5, and lacking exons 3, 4 and 5. A significant amount of a truncated type of protein was produced in XP2NI cells, and the size of the protein indicated that it should have been translated from the mRNA, lacking the 7 nucleotides and retained one of the zinc-finger domains required for the DNA repair activity. The clinical mildness of XP2NI may be due to the residual DNA repair activity of the truncated XPA protein, while no XPA protein was detected in the XPA cells with the homozygous intron 3 splicing mutation. PMID:8596539

  6. Investigation of eggshell thickness and biochemical indicators of contaminant exposure in Great Blue Herons(Ardea herodias) from Mason Neck National Wildlife Refuge

    USGS Publications Warehouse

    Johnson, K.N.; Pinkney, A.E.; Melancon, M.J.; Hoffman, D.J.

    2001-01-01

    Mason Neck National Wildlife Refuge supports the largest great blue heron (Ardea herodias) rookery in the State of Virginia. The presence of bioaccumulative compounds such as polychlorinated biphenyls and DDT in fish collected from the Potomac River and tidal tributaries along the Refuge led to this study. The objective was to determine if there were any indications of pollutant-induced eggshell thinning or evidence of biochemical exposure to contaminants. We examined eggshell thickness and biomarkers of contaminant exposure in livers of embryos collected from the refuge and Coaches Island, a reference location in Chesapeake Bay. There was no evidence of eggshell thinning. Cytochrome P450 activity, measured as ethoxyresomfin-O-dealkylase (EROD) and benzyloxy-resorufin-O-dealkylase (BROD), was not significantly different in embryos from the two colonies. Biochemical indicators of oxidative stress can be reflected as changes in levels of reduced thiols, oxidized glutathione, and thiobarbituric reactive substances (TBARS). Although there were significant differences in the levels of reduced glutathione (GSH) and total thiol (TSH) activities in the embryo livers, there were no statistically significant differences in TBARS, protein-bound sulfhydryls (PBSH), oxidized glutathione (GSSG) and the ratio of GSSG to GSH. In fact, the concentrations of GSH and TSH were higher in the Mason Neck birds relative to Coaches Island. Under conditions of increased oxidative stress at least one or more of the following would be expected: decreased concentrations of reduced thiols (GSH and TSH), increased GSSG, and increased TBARS. In conclusion, we did not detect eggshell thinning or find evidence of a biochemical response to contaminant exposure in the Mason Neck great blue herons.

  7. A screen for enhancers of clearance identifies huntingtin as a heat shock protein 90 (Hsp90) client protein.

    PubMed

    Baldo, Barbara; Weiss, Andreas; Parker, Christian N; Bibel, Miriam; Paganetti, Paolo; Kaupmann, Klemens

    2012-01-01

    Mechanisms to reduce the cellular levels of mutant huntingtin (mHtt) provide promising strategies for treating Huntington disease (HD). To identify compounds enhancing the degradation of mHtt, we performed a high throughput screen using a hippocampal HN10 cell line expressing a 573-amino acid mHtt fragment. Several hit structures were identified as heat shock protein 90 (Hsp90) inhibitors. Cell treatment with these compounds reduced levels of mHtt without overt toxic effects as measured by time-resolved Förster resonance energy transfer assays and Western blots. To characterize the mechanism of mHtt degradation, we used the potent and selective Hsp90 inhibitor NVP-AUY922. In HdhQ150 embryonic stem (ES) cells and in ES cell-derived neurons, NVP-AUY922 treatment substantially reduced soluble full-length mHtt levels. In HN10 cells, Hsp90 inhibition by NVP-AUY922 enhanced mHtt clearance in the absence of any detectable Hsp70 induction. Furthermore, inhibition of protein synthesis with cycloheximide or overexpression of dominant negative heat shock factor 1 (Hsf1) in HdhQ150 ES cells attenuated Hsp70 induction but did not affect NVP-AUY922-mediated mHtt clearance. Together, these data provided evidence that direct inhibition of Hsp90 chaperone function was crucial for mHtt degradation rather than heat shock response induction and Hsp70 up-regulation. Co-immunoprecipitation experiments revealed a physical interaction of mutant and wild-type Htt with the Hsp90 chaperone. Hsp90 inhibition disrupted the interaction and induced clearance of Htt through the ubiquitin-proteasome system. Our data suggest that Htt is an Hsp90 client protein and that Hsp90 inhibition may provide a means to reduce mHtt in HD. PMID:22123826

  8. A DOC2 Protein Identified by Mutational Profiling is Essential for Apicomplexan Parasite Exocytosis

    PubMed Central

    Farrell, Andrew; Thirugnanam, Sivasakthivel; Lorestani, Alexander; Dvorin, Jeffrey D.; Eidell, Keith P.; Ferguson, David J.P.; Anderson-White, Brooke R.; Duraisingh, Manoj T.; Marth, Gabor T.; Gubbels, Marc-Jan

    2012-01-01

    Exocytosis is essential to the lytic cycle of apicomplexan parasites and required for the pathogenesis of toxoplasmosis and malaria. DOC2 proteins recruit the membrane fusion machinery required for exocytosis in a Ca2+-dependent fashion. Here, the phenotype of a Toxoplasma gondii conditional mutant impaired in host cell invasion and egress was pinpointed to a defect in secretion of the micronemes, an apicomplexan-specific organelle that contains adhesion proteins. Whole genome sequencing identified the etiological point mutation in TgDOC2.1. A conditional allele of the orthologous gene engineered into Plasmodium falciparum was also defective in microneme secretion. However, the major effect was on invasion, suggesting microneme secretion is dispensable for Plasmodium egress. PMID:22246776

  9. The newly identified yeast GRD genes are required for retention of late-Golgi membrane proteins.

    PubMed Central

    Nothwehr, S F; Bryant, N J; Stevens, T H

    1996-01-01

    Processing of A-ALP, a late-Golgi membrane protein constructed by fusing the cytosolic domain of dipeptidyl aminopeptidase A to the transmembrane and lumenal domains of alkaline phosphatase (ALP), serves as a convenient assay for loss of retention of late-Golgi membrane proteins in Saccharomyces cerevisiae. In this study, a large group of novel grd (for Golgi retention defective) yeast mutants, representing 18 complementation groups, were identified on the basis of their mislocalization of A-ALP to the vacuole, where it was proteolytically processed and thus became enzymatically activated. All of the grd mutants exhibited significant mislocalization of A-ALP, as measured by determining the kinetics of A-ALP processing and by analyzing its PMID:8649377

  10. A chemical screen to identify inducers of the mitochondrial unfolded protein response in C. elegans

    PubMed Central

    Rauthan, Manish; Pilon, Marc

    2015-01-01

    We previously showed that inhibition of the mevalonate pathway in C. elegans causes inhibition of protein prenylation, developmental arrest and lethality. We also showed that constitutive activation of the mitochondrial unfolded protein response, UPRmt, is an effective way for C. elegans to become resistant to the negative effects of mevalonate pathway inhibition. This was an important finding since statins, a drug class prescribed to lower cholesterol levels in patients, act by inhibiting the mevalonate pathway, and it is therefore possible that some of their undesirable side effects could be alleviated by activating the UPRmt. Here, we screened a chemical library and identified 4 compounds that specifically activated the UPRmt. One of these compounds, methacycline hydrochloride (a tetracycline antibiotic) also protected C. elegans and mammalian cells from statin toxicity. Methacycline hydrochloride and ethidium bromide, a known UPRmt activator, were also tested in mice: only ethidium bromide significantly activate the UPRmt in skeletal muscles. PMID:27123370

  11. Coevolution analysis of Hepatitis C virus genome to identify the structural and functional dependency network of viral proteins

    PubMed Central

    Champeimont, Raphaël; Laine, Elodie; Hu, Shuang-Wei; Penin, Francois; Carbone, Alessandra

    2016-01-01

    A novel computational approach of coevolution analysis allowed us to reconstruct the protein-protein interaction network of the Hepatitis C Virus (HCV) at the residue resolution. For the first time, coevolution analysis of an entire viral genome was realized, based on a limited set of protein sequences with high sequence identity within genotypes. The identified coevolving residues constitute highly relevant predictions of protein-protein interactions for further experimental identification of HCV protein complexes. The method can be used to analyse other viral genomes and to predict the associated protein interaction networks. PMID:27198619

  12. Coevolution analysis of Hepatitis C virus genome to identify the structural and functional dependency network of viral proteins

    NASA Astrophysics Data System (ADS)

    Champeimont, Raphaël; Laine, Elodie; Hu, Shuang-Wei; Penin, Francois; Carbone, Alessandra

    2016-05-01

    A novel computational approach of coevolution analysis allowed us to reconstruct the protein-protein interaction network of the Hepatitis C Virus (HCV) at the residue resolution. For the first time, coevolution analysis of an entire viral genome was realized, based on a limited set of protein sequences with high sequence identity within genotypes. The identified coevolving residues constitute highly relevant predictions of protein-protein interactions for further experimental identification of HCV protein complexes. The method can be used to analyse other viral genomes and to predict the associated protein interaction networks.

  13. Coevolution analysis of Hepatitis C virus genome to identify the structural and functional dependency network of viral proteins.

    PubMed

    Champeimont, Raphaël; Laine, Elodie; Hu, Shuang-Wei; Penin, Francois; Carbone, Alessandra

    2016-01-01

    A novel computational approach of coevolution analysis allowed us to reconstruct the protein-protein interaction network of the Hepatitis C Virus (HCV) at the residue resolution. For the first time, coevolution analysis of an entire viral genome was realized, based on a limited set of protein sequences with high sequence identity within genotypes. The identified coevolving residues constitute highly relevant predictions of protein-protein interactions for further experimental identification of HCV protein complexes. The method can be used to analyse other viral genomes and to predict the associated protein interaction networks. PMID:27198619

  14. Phenotypic Screening Identifies Protein Synthesis Inhibitors as H-Ras-Nanocluster-Increasing Tumor Growth Inducers.

    PubMed

    Najumudeen, Arafath K; Posada, Itziar M D; Lectez, Benoit; Zhou, Yong; Landor, Sebastian K-J; Fallarero, Adyary; Vuorela, Pia; Hancock, John; Abankwa, Daniel

    2015-12-15

    Ras isoforms H-, N-, and K-ras are each mutated in specific cancer types at varying frequencies and have different activities in cell fate control. On the plasma membrane, Ras proteins are laterally segregated into isoform-specific nanoscale signaling hubs, termed nanoclusters. As Ras nanoclusters are required for Ras signaling, chemical modulators of nanoclusters represent ideal candidates for the specific modulation of Ras activity in cancer drug development. We therefore conducted a chemical screen with commercial and in-house natural product libraries using a cell-based H-ras-nanoclustering FRET assay. Next to established Ras inhibitors, such as a statin and farnesyl-transferase inhibitor, we surprisingly identified five protein synthesis inhibitors as positive regulators. Using commonly employed cycloheximide as a representative compound, we show that protein synthesis inhibition increased nanoclustering and effector recruitment specifically of active H-ras but not of K-ras. Consistent with these data, cycloheximide treatment activated both Erk and Akt kinases and specifically promoted H-rasG12V-induced, but not K-rasG12V-induced, PC12 cell differentiation. Intriguingly, cycloheximide increased the number of mammospheres, which are enriched for cancer stem cells. Depletion of H-ras in combination with cycloheximide significantly reduced mammosphere formation, suggesting an exquisite synthetic lethality. The potential of cycloheximide to promote tumor cell growth was also reflected in its ability to increase breast cancer cell tumors grown in ovo. These results illustrate the possibility of identifying Ras-isoform-specific modulators using nanocluster-directed screening. They also suggest an unexpected feedback from protein synthesis inhibition to Ras signaling, which might present a vulnerability in certain tumor cell types. PMID:26568031

  15. Changes in pigment, spectral transmission and element content of pink chicken eggshells with different pigment intensity during incubation.

    PubMed

    Yu, Yue; Li, Zhanming; Pan, Jinming

    2016-01-01

    Objective. The objective of this study was to investigate changes in pigment, spectral transmission and element content of chicken eggshells with different intensities of pink pigment during the incubation period. We also investigated the effects of the region (small pole, equator and large pole) and pink pigment intensity of the chicken eggshell on the percent transmission of light passing through the chicken eggshells. Method. Eggs of comparable weight from a meat-type breeder (Meihuang) were used, and divided based on three levels of pink pigment (light, medium and dark) in the eggshells. During the incubation (0-21 d), the values of the eggshell pigment (ΔE, L (∗), a (∗), b (∗)) were measured. The percent transmission of light for different regions and intensities of eggshell pigmentation was measured by using the visible wavelength range of 380-780 nm. Result. Three measured indicators of eggshell color, ΔE, L (∗) and a (∗), did not change significantly during incubation. Compared with other regions and pigment intensities, eggshell at the small pole and with light pigmentation intensity showed the highest percent transmission of light. The transmission value varied significantly (P < 0.001) with incubation time. The element analysis of eggshells with different levels of pink pigment showed that the potassium content of the eggshells for all pigment levels decreased significantly during incubation. Conclusion. In summary, pigment intensity and the region of the eggshell influenced the percent transmission of light of eggshell. Differences in the spectral characteristics of different eggshells may influence the effects of photostimulation during the incubation of eggs. All of these results will be applicable for perfecting the design of light intensity for lighted incubation to improve productivity. PMID:27019785

  16. Changes in pigment, spectral transmission and element content of pink chicken eggshells with different pigment intensity during incubation

    PubMed Central

    Yu, Yue; Li, Zhanming

    2016-01-01

    Objective. The objective of this study was to investigate changes in pigment, spectral transmission and element content of chicken eggshells with different intensities of pink pigment during the incubation period. We also investigated the effects of the region (small pole, equator and large pole) and pink pigment intensity of the chicken eggshell on the percent transmission of light passing through the chicken eggshells. Method. Eggs of comparable weight from a meat-type breeder (Meihuang) were used, and divided based on three levels of pink pigment (light, medium and dark) in the eggshells. During the incubation (0–21 d), the values of the eggshell pigment (ΔE, L∗, a∗, b∗) were measured. The percent transmission of light for different regions and intensities of eggshell pigmentation was measured by using the visible wavelength range of 380–780 nm. Result. Three measured indicators of eggshell color, ΔE, L∗ and a∗, did not change significantly during incubation. Compared with other regions and pigment intensities, eggshell at the small pole and with light pigmentation intensity showed the highest percent transmission of light. The transmission value varied significantly (P < 0.001) with incubation time. The element analysis of eggshells with different levels of pink pigment showed that the potassium content of the eggshells for all pigment levels decreased significantly during incubation. Conclusion. In summary, pigment intensity and the region of the eggshell influenced the percent transmission of light of eggshell. Differences in the spectral characteristics of different eggshells may influence the effects of photostimulation during the incubation of eggs. All of these results will be applicable for perfecting the design of light intensity for lighted incubation to improve productivity. PMID:27019785

  17. A fibrinogen-related protein identified from hepatopancreas of crayfish is a potential pattern recognition receptor.

    PubMed

    Chen, Qiming; Bai, Suhua; Dong, Chaohua

    2016-09-01

    Fibrinogen-related protein (FREP) family is a large group of proteins containing fibrinogen-like (FBG) domain and plays multiple physiological roles in animals. However, their immune functions in crayfish are not fully explored. In the present study, a novel fibrinogen-like protein (designated as PcFBN1) was identified and characterized from hepatopancreas of red swamp crayfish Procambarus clarkii. The cDNA sequence of PcFBN1 contains an open reading frame (ORF) of 1353 bp encoding a protein of 450 amino acids. Sequence and structural analysis indicated that PcFBN1 contains an FBG domain in C-terminal and a putative signal peptide of 19 amino acids in N-terminal. Semi-quantitative PCR revealed that the main expression of PcFBN1 was observed in hepatopancreas and hemocyte. Temporal expression analysis exhibited that PcFBN1 expression could be significantly induced by heat-killed Aeromonas hydrophila. Tissue distribution and temporal change of PcFBN1 suggested that PcFBN1 may be involved in immune responses of red swamp crayfish. Recombinant PcFBN1 protein binds and agglutinates both gram-negative bacteria Escherichia coli and gram-positive bacteria Micrococcus lysodeikticus. Moreover, binding and agglutination is Ca(2+) dependent. Further analysis indicated that PcFBN1 recognizes some acetyl group-containing substance LPS and PGN. RNAi experiment revealed that PcFBN1 is required for bacterial clearance and survival from A. hydrophila infection. Reduction of PcFBN1 expression significantly decreased the survival and enhanced the number of A. hydrophila in the hemolymph. These results indicated that PcFBN1 plays an important role in the innate immunity of red swamp crayfish as a potential pattern recognition receptor. PMID:27417229

  18. DomHR: Accurately Identifying Domain Boundaries in Proteins Using a Hinge Region Strategy

    PubMed Central

    Zhang, Xiao-yan; Lu, Long-jian; Song, Qi; Yang, Qian-qian; Li, Da-peng; Sun, Jiang-ming; Li, Tong-hua; Cong, Pei-sheng

    2013-01-01

    Motivation The precise prediction of protein domains, which are the structural, functional and evolutionary units of proteins, has been a research focus in recent years. Although many methods have been presented for predicting protein domains and boundaries, the accuracy of predictions could be improved. Results In this study we present a novel approach, DomHR, which is an accurate predictor of protein domain boundaries based on a creative hinge region strategy. A hinge region was defined as a segment of amino acids that covers part of a domain region and a boundary region. We developed a strategy to construct profiles of domain-hinge-boundary (DHB) features generated by sequence-domain/hinge/boundary alignment against a database of known domain structures. The DHB features had three elements: normalized domain, hinge, and boundary probabilities. The DHB features were used as input to identify domain boundaries in a sequence. DomHR used a nonredundant dataset as the training set, the DHB and predicted shape string as features, and a conditional random field as the classification algorithm. In predicted hinge regions, a residue was determined to be a domain or a boundary according to a decision threshold. After decision thresholds were optimized, DomHR was evaluated by cross-validation, large-scale prediction, independent test and CASP (Critical Assessment of Techniques for Protein Structure Prediction) tests. All results confirmed that DomHR outperformed other well-established, publicly available domain boundary predictors for prediction accuracy. Availability The DomHR is available at http://cal.tongji.edu.cn/domain/. PMID:23593247

  19. A genome-wide RNAi screen identifies proteins modulating aberrant FLT3-ITD signaling

    PubMed Central

    Caldarelli, A; Müller, J P; Paskowski-Rogacz, M; Herrmann, K; Bauer, R; Koch, S; Heninger, A K; Krastev, D; Ding, L; Kasper, S; Fischer, T; Brodhun, M; Böhmer, F-D; Buchholz, F

    2013-01-01

    Fms-like tyrosine kinase-3 is a commonly mutated gene in acute myeloid leukemia, with about one-third of patients carrying an internal-tandem duplication of the juxtamembrane domain in the receptor (FLT3-ITD). FLT3-ITD exhibits altered signaling quality, including aberrant activation of STAT5. To identify genes affecting FLT3-ITD-mediated STAT5 signaling, we performed an esiRNA-based RNAi screen utilizing a STAT5-driven reporter assay. Knockdowns that caused reduced FLT3-ITD-mediated STAT5 signaling were enriched for genes encoding proteins involved in protein secretion and intracellular protein transport, indicating that modulation of protein transport processes could potentially be used to reduce constitutive STAT5 signaling in FLT3-ITD-positive cells. The relevance of KDELR1, a component involved in the Golgi-ER retrograde transport, was further analyzed. In FLT3-ITD-expressing leukemic MV4-11 cells, downregulation of KDELR1 resulted in reduced STAT5 activation, proliferation and colony-forming capacity. Stable shRNA-mediated depletion of KDELR1 in FLT3-ITD-expressing 32D cells likewise resulted in reduced STAT5 signaling and cell proliferation. Importantly, these cells also showed a reduced capacity to generate a leukemia-like disease in syngeneic C3H/HeJ mice. Together our data suggest intracellular protein transport as a potential target for FLT3-ITD driven leukemias, with KDELR1 emerging as a positive modulator of oncogenic FLT3-ITD activity. PMID:23508117

  20. FindPept, a tool to identify unmatched masses in peptide mass fingerprinting protein identification.

    PubMed

    Gattiker, Alexandre; Bienvenut, Willy V; Bairoch, Amos; Gasteiger, Elisabeth

    2002-10-01

    FindPept (http://www.expasy.org/tools/findpept.html) is a software tool designed to identify the origin of peptide masses obtained by peptide mass fingerprinting which are not matched by existing protein identification tools. It identifies masses resulting from unspecific proteolytic cleavage, missed cleavage, protease autolysis or keratin contaminants. It also takes into account post-translational modifications derived from the annotation of the SWISS-PROT database or supplied by the user, and chemical modifications of peptides. Based on a number of experimental examples, we show that the commonly held rules for the specificity of tryptic cleavage are an oversimplification, mainly because of effects of neighboring residues, experimental conditions, and contaminants present in the enzyme sample. PMID:12422360

  1. Eggshell Porosity Provides Insight on Evolution of Nesting in Dinosaurs.

    PubMed

    Tanaka, Kohei; Zelenitsky, Darla K; Therrien, François

    2015-01-01

    Knowledge about the types of nests built by dinosaurs can provide insight into the evolution of nesting and reproductive behaviors among archosaurs. However, the low preservation potential of their nesting materials and nesting structures means that most information can only be gleaned indirectly through comparison with extant archosaurs. Two general nest types are recognized among living archosaurs: 1) covered nests, in which eggs are incubated while fully covered by nesting material (as in crocodylians and megapodes), and 2) open nests, in which eggs are exposed in the nest and brooded (as in most birds). Previously, dinosaur nest types had been inferred by estimating the water vapor conductance (i.e., diffusive capacity) of their eggs, based on the premise that high conductance corresponds to covered nests and low conductance to open nests. However, a lack of statistical rigor and inconsistencies in this method render its application problematic and its validity questionable. As an alternative we propose a statistically rigorous approach to infer nest type based on large datasets of eggshell porosity and egg mass compiled for over 120 extant archosaur species and 29 archosaur extinct taxa/ootaxa. The presence of a strong correlation between eggshell porosity and nest type among extant archosaurs indicates that eggshell porosity can be used as a proxy for nest type, and thus discriminant analyses can help predict nest type in extinct taxa. Our results suggest that: 1) covered nests are likely the primitive condition for dinosaurs (and probably archosaurs), and 2) open nests first evolved among non-avian theropods more derived than Lourinhanosaurus and were likely widespread in non-avian maniraptorans, well before the appearance of birds. Although taphonomic evidence suggests that basal open nesters (i.e., oviraptorosaurs and troodontids) were potentially the first dinosaurs to brood their clutches, they still partially buried their eggs in sediment. Open nests

  2. Eggshell Porosity Provides Insight on Evolution of Nesting in Dinosaurs

    PubMed Central

    2015-01-01

    Knowledge about the types of nests built by dinosaurs can provide insight into the evolution of nesting and reproductive behaviors among archosaurs. However, the low preservation potential of their nesting materials and nesting structures means that most information can only be gleaned indirectly through comparison with extant archosaurs. Two general nest types are recognized among living archosaurs: 1) covered nests, in which eggs are incubated while fully covered by nesting material (as in crocodylians and megapodes), and 2) open nests, in which eggs are exposed in the nest and brooded (as in most birds). Previously, dinosaur nest types had been inferred by estimating the water vapor conductance (i.e., diffusive capacity) of their eggs, based on the premise that high conductance corresponds to covered nests and low conductance to open nests. However, a lack of statistical rigor and inconsistencies in this method render its application problematic and its validity questionable. As an alternative we propose a statistically rigorous approach to infer nest type based on large datasets of eggshell porosity and egg mass compiled for over 120 extant archosaur species and 29 archosaur extinct taxa/ootaxa. The presence of a strong correlation between eggshell porosity and nest type among extant archosaurs indicates that eggshell porosity can be used as a proxy for nest type, and thus discriminant analyses can help predict nest type in extinct taxa. Our results suggest that: 1) covered nests are likely the primitive condition for dinosaurs (and probably archosaurs), and 2) open nests first evolved among non-avian theropods more derived than Lourinhanosaurus and were likely widespread in non-avian maniraptorans, well before the appearance of birds. Although taphonomic evidence suggests that basal open nesters (i.e., oviraptorosaurs and troodontids) were potentially the first dinosaurs to brood their clutches, they still partially buried their eggs in sediment. Open nests

  3. Sorption on eggshell waste--a review on ultrastructure, biomineralization and other applications.

    PubMed

    Guru, Partha Sarathi; Dash, Sukalyan

    2014-07-01

    The structure, adsorption behavior and applications of eggshell waste materials have been reviewed. The ultrastructure of eggshell particles has been discussed to understand the pore structure as well as the surface geometry of the materials leading to its multifarious applicability. Besides, the ultrastructure studies give full information regarding the chemical constituents of egghell particles as well as eggshell membranes. The process of biomineralization in living organisms, their consequent effect of controlling the formation of inorganic-organic composites propelling their application in biomimetic designing of advanced composites with optimized novel properties leading to advances in materials design have been discussed. Utilization of eggshell waste materials for the removal of organic dyes and heavy inorganic ions has been reviewed with suitable models for understanding their adsorption quality and capacity. The applications of these materials in various fields of research have been extensively discussed. PMID:24456801

  4. UV, VISIBLE AND NIR SPECTRAL ANALYSIS OF EGGSHELLS IN THE CHARADRIIDAE FAMILY OF BIRDS

    EPA Science Inventory

    We employed reflectance spectrophotometry to quantify color and mineral composition of eggshells from several species of the bird family Charadriidae to characterize species physiology and to distinguish nesting habitat preferences. We used a Shimadzu spectrophotometer to measur...

  5. Filling the gaps of dinosaur eggshell phylogeny: Late Jurassic Theropod clutch with embryos from Portugal.

    PubMed

    Araújo, Ricardo; Castanhinha, Rui; Martins, Rui M S; Mateus, Octávio; Hendrickx, Christophe; Beckmann, F; Schell, N; Alves, L C

    2013-01-01

    The non-avian saurischians that have associated eggshells and embryos are represented only by the sauropodomorph Massospondylus and Coelurosauria (derived theropods), thus missing the basal theropod representatives. We report a dinosaur clutch containing several crushed eggs and embryonic material ascribed to the megalosaurid theropod Torvosaurus. It represents the first associated eggshells and embryos of megalosauroids, thus filling an important phylogenetic gap between two distantly related groups of saurischians. These fossils represent the only unequivocal basal theropod embryos found to date. The assemblage was found in early Tithonian fluvial overbank deposits of the Lourinhã Formation in West Portugal. The morphological, microstructural and chemical characterization results of the eggshell fragments indicate very mild diagenesis. Furthermore, these fossils allow unambiguous association of basal theropod osteology with a specific and unique new eggshell morphology. PMID:23722524

  6. Filling the gaps of dinosaur eggshell phylogeny: Late Jurassic Theropod clutch with embryos from Portugal

    PubMed Central

    Araújo, Ricardo; Castanhinha, Rui; Martins, Rui M. S.; Mateus, Octávio; Hendrickx, Christophe; Beckmann, F.; Schell, N.; Alves, L. C.

    2013-01-01

    The non-avian saurischians that have associated eggshells and embryos are represented only by the sauropodomorph Massospondylus and Coelurosauria (derived theropods), thus missing the basal theropod representatives. We report a dinosaur clutch containing several crushed eggs and embryonic material ascribed to the megalosaurid theropod Torvosaurus. It represents the first associated eggshells and embryos of megalosauroids, thus filling an important phylogenetic gap between two distantly related groups of saurischians. These fossils represent the only unequivocal basal theropod embryos found to date. The assemblage was found in early Tithonian fluvial overbank deposits of the Lourinhã Formation in West Portugal. The morphological, microstructural and chemical characterization results of the eggshell fragments indicate very mild diagenesis. Furthermore, these fossils allow unambiguous association of basal theropod osteology with a specific and unique new eggshell morphology. PMID:23722524

  7. Identifying intrinsically disordered protein regions likely to undergo binding-induced helical transitions.

    PubMed

    Glover, Karen; Mei, Yang; Sinha, Sangita C

    2016-10-01

    Many proteins contain intrinsically disordered regions (IDRs) lacking stable secondary and ordered tertiary structure. IDRs are often implicated in macromolecular interactions, and may undergo structural transitions upon binding to interaction partners. However, as binding partners of many protein IDRs are unknown, these structural transitions are difficult to verify and often are poorly understood. In this study we describe a method to identify IDRs that are likely to undergo helical transitions upon binding. This method combines bioinformatics analyses followed by circular dichroism spectroscopy to monitor 2,2,2-trifluoroethanol (TFE)-induced changes in secondary structure content of these IDRs. Our results demonstrate that there is no significant change in the helicity of IDRs that are not predicted to fold upon binding. IDRs that are predicted to fold fall into two groups: one group does not become helical in the presence of TFE and includes examples of IDRs that form β-strands upon binding, while the other group becomes more helical and includes examples that are known to fold into helices upon binding. Therefore, we propose that bioinformatics analyses combined with experimental evaluation using TFE may provide a general method to identify IDRs that undergo binding-induced disorder-to-helix transitions. PMID:27179590

  8. Bioinformatics analysis identifies several intrinsically disordered human E3 ubiquitin-protein ligases

    PubMed Central

    Nielsen, Sofie V.; Lindorff-Larsen, Kresten; Hartmann-Petersen, Rasmus

    2016-01-01

    The ubiquitin-proteasome system targets misfolded proteins for degradation. Since the accumulation of such proteins is potentially harmful for the cell, their prompt removal is important. E3 ubiquitin-protein ligases mediate substrate ubiquitination by bringing together the substrate with an E2 ubiquitin-conjugating enzyme, which transfers ubiquitin to the substrate. For misfolded proteins, substrate recognition is generally delegated to molecular chaperones that subsequently interact with specific E3 ligases. An important exception is San1, a yeast E3 ligase. San1 harbors extensive regions of intrinsic disorder, which provide both conformational flexibility and sites for direct recognition of misfolded targets of vastly different conformations. So far, no mammalian ortholog of San1 is known, nor is it clear whether other E3 ligases utilize disordered regions for substrate recognition. Here, we conduct a bioinformatics analysis to examine >600 human and S. cerevisiae E3 ligases to identify enzymes that are similar to San1 in terms of function and/or mechanism of substrate recognition. An initial sequence-based database search was found to detect candidates primarily based on the homology of their ordered regions, and did not capture the unique disorder patterns that encode the functional mechanism of San1. However, by searching specifically for key features of the San1 sequence, such as long regions of intrinsic disorder embedded with short stretches predicted to be suitable for substrate interaction, we identified several E3 ligases with these characteristics. Our initial analysis revealed that another remarkable trait of San1 is shared with several candidate E3 ligases: long stretches of complete lysine suppression, which in San1 limits auto-ubiquitination. We encode these characteristic features into a San1 similarity-score, and present a set of proteins that are plausible candidates as San1 counterparts in humans. In conclusion, our work indicates that San1 is

  9. The antibacterial protein lysozyme identified as the termite egg recognition pheromone.

    PubMed

    Matsuura, Kenji; Tamura, Takashi; Kobayashi, Norimasa; Yashiro, Toshihisa; Tatsumi, Shingo

    2007-01-01

    Social insects rely heavily on pheromone communication to maintain their sociality. Egg protection is one of the most fundamental social behaviours in social insects. The recent discovery of the termite-egg mimicking fungus 'termite-ball' and subsequent studies on termite egg protection behaviour have shown that termites can be manipulated by using the termite egg recognition pheromone (TERP), which strongly evokes the egg-carrying and -grooming behaviours of workers. Despite the great scientific and economic importance, TERP has not been identified because of practical difficulties. Herein we identified the antibacterial protein lysozyme as the TERP. We isolated the target protein using ion-exchange and hydrophobic interaction chromatography, and the MALDI-TOF MS analysis showed a molecular size of 14.5 kDa. We found that the TERP provided antibacterial activity against a gram-positive bacterium. Among the currently known antimicrobial proteins, the molecular size of 14.5 kDa limits the target to lysozyme. Termite lysozymes obtained from eggs and salivary glands, and even hen egg lysozyme, showed a strong termite egg recognition activity. Besides eggs themselves, workers also supply lysozyme to eggs through frequent egg-grooming, by which egg surfaces are coated with saliva containing lysozyme. Reverse transcript PCR analysis showed that mRNA of termite lysozyme was expressed in both salivary glands and eggs. Western blot analysis confirmed that lysozyme production begins in immature eggs in queen ovaries. This is the first identification of proteinaceous pheromone in social insects. Researchers have focused almost exclusively on hydrocarbons when searching for recognition pheromones in social insects. The present finding of a proteinaceous pheromone represents a major step forward in, and result in the broadening of, the search for recognition pheromones. This novel function of lysozyme as a termite pheromone illuminates the profound influence of pathogenic

  10. Macrophage Replication Screen Identifies a Novel Francisella Hydroperoxide Resistance Protein Involved in Virulence

    PubMed Central

    Llewellyn, Anna C.; Bina, James E.; Weiss, David S.

    2011-01-01

    Francisella tularensis is a Gram-negative facultative intracellular pathogen and the causative agent of tularemia. Recently, genome-wide screens have identified Francisella genes required for virulence in mice. However, the mechanisms by which most of the corresponding proteins contribute to pathogenesis are still largely unknown. To further elucidate the roles of these virulence determinants in Francisella pathogenesis, we tested whether each gene was required for replication of the model pathogen F. novicida within macrophages, an important virulence trait. Fifty-three of the 224 genes tested were involved in intracellular replication, including many of those within the Francisella pathogenicity island (FPI), validating our results. Interestingly, over one third of the genes identified are annotated as hypothetical, indicating that F. novicida likely utilizes novel virulence factors for intracellular replication. To further characterize these virulence determinants, we selected two hypothetical genes to study in more detail. As predicted by our screen, deletion mutants of FTN_0096 and FTN_1133 were attenuated for replication in macrophages. The mutants displayed differing levels of attenuation in vivo, with the FTN_1133 mutant being the most attenuated. FTN_1133 has sequence similarity to the organic hydroperoxide resistance protein Ohr, an enzyme involved in the bacterial response to oxidative stress. We show that FTN_1133 is required for F. novicida resistance to, and degradation of, organic hydroperoxides as well as resistance to the action of the NADPH oxidase both in macrophages and mice. Furthermore, we demonstrate that F. holarctica LVS, a strain derived from a highly virulent human pathogenic species of Francisella, also requires this protein for organic hydroperoxide resistance as well as replication in macrophages and mice. This study expands our knowledge of Francisella's largely uncharacterized intracellular lifecycle and demonstrates that FTN_1133 is

  11. The Antibacterial Protein Lysozyme Identified as the Termite Egg Recognition Pheromone

    PubMed Central

    Matsuura, Kenji; Tamura, Takashi; Kobayashi, Norimasa; Yashiro, Toshihisa; Tatsumi, Shingo

    2007-01-01

    Social insects rely heavily on pheromone communication to maintain their sociality. Egg protection is one of the most fundamental social behaviours in social insects. The recent discovery of the termite-egg mimicking fungus ‘termite-ball’ and subsequent studies on termite egg protection behaviour have shown that termites can be manipulated by using the termite egg recognition pheromone (TERP), which strongly evokes the egg-carrying and -grooming behaviours of workers. Despite the great scientific and economic importance, TERP has not been identified because of practical difficulties. Herein we identified the antibacterial protein lysozyme as the TERP. We isolated the target protein using ion-exchange and hydrophobic interaction chromatography, and the MALDI-TOF MS analysis showed a molecular size of 14.5 kDa. We found that the TERP provided antibacterial activity against a gram-positive bacterium. Among the currently known antimicrobial proteins, the molecular size of 14.5 kDa limits the target to lysozyme. Termite lysozymes obtained from eggs and salivary glands, and even hen egg lysozyme, showed a strong termite egg recognition activity. Besides eggs themselves, workers also supply lysozyme to eggs through frequent egg-grooming, by which egg surfaces are coated with saliva containing lysozyme. Reverse transcript PCR analysis showed that mRNA of termite lysozyme was expressed in both salivary glands and eggs. Western blot analysis confirmed that lysozyme production begins in immature eggs in queen ovaries. This is the first identification of proteinaceous pheromone in social insects. Researchers have focused almost exclusively on hydrocarbons when searching for recognition pheromones in social insects. The present finding of a proteinaceous pheromone represents a major step forward in, and result in the broadening of, the search for recognition pheromones. This novel function of lysozyme as a termite pheromone illuminates the profound influence of pathogenic

  12. Effects of Dietary Corticosterone on Yolk Colors and Eggshell Quality in Laying Hens

    PubMed Central

    Kim, Yeon-Hwa; Kim, Jimin; Yoon, Hyung-Sook; Choi, Yang-Ho

    2015-01-01

    The objective of this study was to investigate the effects of dietary corticosterone on egg quality. For 2 weeks hens received either control or experimental diet containing corticosterone at 30 mg/kg diet. Feed intake and egg production were monitored daily, and body weight measured weekly. Egg weights and egg quality were measured daily. Corticosterone treatment resulted in a remarkable increase in feed intake and sharp decrease in egg production compared with control (p<0.05) whereas body weight remained unchanged. Decreased albumen height, but no changes in egg weight, led to decreased Haugh unit (p<0.05). Corticosterone caused elevated eggshell thickness (p<0.05) without altering weight and strength, suggesting possible changes in shell structure. Yolk color and redness were increased by corticosterone (p<0.05) but lightness and yellowness were either not changed or inconsistent over the time period of measurements. Increased concentrations in plasma were also found for corticosterone, glucose, cholesterol, creatinine, uric acid, albumin, aspartate aminotransferase, creatine kinase, lactate dehydrogenase, total protein, and amylase (p<0.05), suggesting that corticosterone increased protein breakdown, renal dysfunctions and pancreatitis. Together, the current results imply that dietary corticosterone affects egg quality such as yolk colors and shell thickness, in addition to its effects on feed intake and egg production. PMID:25925061

  13. Virtual Screening and Experimental Validation Identify Novel Inhibitors of the Plasmodium falciparum Atg8-Atg3 Protein-Protein Interaction.

    PubMed

    Hain, Adelaide U P; Miller, Alexia S; Levitskaya, Jelena; Bosch, Jürgen

    2016-04-19

    New therapies are needed against malaria, a parasitic infection caused by Plasmodium falciparum, as drug resistance emerges against the current treatment, artemisinin. We previously characterized the Atg8-Atg3 protein-protein interaction (PPI), which is essential for autophagy and parasite survival. Herein we illustrate the use of virtual library screening to selectively block the PPI in the parasite without inhibiting the homologous interaction in humans by targeting the A-loop of PfAtg8. This A-loop is important for Atg3 binding in Plasmodium, but is absent from the human Atg8 homologues. In this proof-of-concept study, we demonstrate a shift in lipidation state of PfAtg8 and inhibition of P. falciparum growth in both blood- and liver-stage cultures upon drug treatment. Our results illustrate how in silico screening and structure-aided drug design against a PPI can be used to identify new hits for drug development. Additionally, as we targeted a region of Atg8 that is conserved within apicomplexans, we predict that our small molecule will have cross-reactivity against other disease-causing apicomplexans, such as Toxoplasma, Cryptosporidium, Theileria, Neospora, Eimeria, and Babesia. PMID:26748931

  14. Combined Use of Electron and Light Microscopy Techniques Reveals False Secondary Shell Units in Megaloolithidae Eggshells

    PubMed Central

    Bauluz, Blanca; Canudo, José Ignacio; Gasca, José Manuel; Torcida Fernández-Baldor, Fidel

    2016-01-01

    Abnormalities in the histo- and ultrastructure of the amniote eggshell are often related to diverse factors, such as ambient stress during egg formation, pathologies altering the physiology of the egg-laying females, or evolutionarily selected modifications of the eggshell structure that vary the physical properties of the egg, for example increasing its strength so as to avoid fracture during incubation. When dealing with fossil materials, all the above hypotheses are plausible, but a detailed taphonomical study has to be performed to rule out the possibility that secondary processes of recrystallization have occurred during fossilization. Traditional analyses, such as optical microscopy inspection and cathodoluminescence, have proven not to be enough to understand the taphonomic story of some eggshells. Recently, electron backscatter diffraction has been used, in combination with other techniques, to better understand the alteration of fossil eggshells. Here we present a combined study using scanning electron microscopy, optical microscopy, cathodoluminescence and electron backscatter diffraction of eggshell fragments assigned to Megaloolithus cf. siruguei from the Upper Cretaceous outcrops of the Cameros Basin. We focus our study on the presence of secondary shell units that mimic most aspects of the ultrastructure of the eggshell mammillae, but grow far from the inner surface of the eggshell. We call these structures extra-spherulites, describe their crystal structure and demonstrate their secondary origin. Our study has important implications for the interpretation of secondary shell units as biological or pathological structures. Thus, electron backscatter diffraction complements other microscope techniques as a useful tool for understanding taphonomical alterations in fossil eggshells. PMID:27144767

  15. Combined Use of Electron and Light Microscopy Techniques Reveals False Secondary Shell Units in Megaloolithidae Eggshells.

    PubMed

    Moreno-Azanza, Miguel; Bauluz, Blanca; Canudo, José Ignacio; Gasca, José Manuel; Torcida Fernández-Baldor, Fidel

    2016-01-01

    Abnormalities in the histo- and ultrastructure of the amniote eggshell are often related to diverse factors, such as ambient stress during egg formation, pathologies altering the physiology of the egg-laying females, or evolutionarily selected modifications of the eggshell structure that vary the physical properties of the egg, for example increasing its strength so as to avoid fracture during incubation. When dealing with fossil materials, all the above hypotheses are plausible, but a detailed taphonomical study has to be performed to rule out the possibility that secondary processes of recrystallization have occurred during fossilization. Traditional analyses, such as optical microscopy inspection and cathodoluminescence, have proven not to be enough to understand the taphonomic story of some eggshells. Recently, electron backscatter diffraction has been used, in combination with other techniques, to better understand the alteration of fossil eggshells. Here we present a combined study using scanning electron microscopy, optical microscopy, cathodoluminescence and electron backscatter diffraction of eggshell fragments assigned to Megaloolithus cf. siruguei from the Upper Cretaceous outcrops of the Cameros Basin. We focus our study on the presence of secondary shell units that mimic most aspects of the ultrastructure of the eggshell mammillae, but grow far from the inner surface of the eggshell. We call these structures extra-spherulites, describe their crystal structure and demonstrate their secondary origin. Our study has important implications for the interpretation of secondary shell units as biological or pathological structures. Thus, electron backscatter diffraction complements other microscope techniques as a useful tool for understanding taphonomical alterations in fossil eggshells. PMID:27144767

  16. Chemical Screens Identify Drugs that Enhance or Mitigate Cellular Responses to Antibody-Toxin Fusion Proteins

    PubMed Central

    Guha, Rajarshi; Simon, Nathan; Pasetto, Matteo; Keller, Jonathan; Huang, Manjie; Angelus, Evan; Pastan, Ira; Ferrer, Marc; FitzGerald, David J.; Thomas, Craig J.

    2016-01-01

    The intersection of small molecular weight drugs and antibody-based therapeutics is rarely studied in large scale. Both types of agents are currently part of the cancer armamentarium. However, very little is known about how to combine them in optimal ways. Immunotoxins are antibody-toxin gene fusion proteins engineered to target cancer cells via antibody binding to surface antigens. For fusion proteins derived from Pseudomonas exotoxin (PE), potency relies on the enzymatic domain of the toxin which catalyzes the ADP-ribosylation of EF2 causing inhibition of protein synthesis leading to cell death. Candidate immunotoxins have demonstrated clear value in clinical trials but generally have not been curative as single agents. Therefore we undertook three screens to discover effective combinations that could act synergistically. From the MIPE-3 library of compounds we identified various enhancers of immunotoxin action and at least one major class of inhibitor. Follow-up experiments confirmed the screening data and suggested that immunotoxins when administered with everolimus or nilotinib exhibit favorable combinatory activity and would be candidates for preclinical development. Mechanistic studies revealed that everolimus-immunotoxin combinations acted synergistically on elements of the protein synthetic machinery, including S61 kinase and 4E-BP1 of the mTORC1 pathway. Conversely, PARP inhibitors antagonized immunotoxins and also blocked the toxicity due to native ADP-ribosylating toxins. Thus, our goal of investigating a chemical library was justified based on the identification of several approved compounds that could be developed preclinically as ‘enhancers’ and at least one class of mitigator to be avoided. PMID:27556570

  17. Altered Protein S-Glutathionylation Identifies a Potential Mechanism of Resistance to Acetaminophen-Induced Hepatotoxicity

    PubMed Central

    McGarry, David J.; Chakravarty, Probir; Wolf, C. Roland

    2015-01-01

    Acetaminophen (APAP) is the most commonly used over-the-counter analgesic. However, hepatotoxicity induced by APAP is a major clinical issue, and the factors that define sensitivity to APAP remain unclear. We have previously demonstrated that mice nulled for glutathione S-transferase Pi (GSTP) are resistant to APAP-induced hepatotoxicity. This study aims to exploit this difference to delineate pathways of importance in APAP toxicity. We used mice nulled for GSTP and heme oxygenase-1 oxidative stress reporter mice, together with a novel nanoflow liquid chromatography–tandem mass spectrometry methodology to investigate the role of oxidative stress, cell signaling, and protein S-glutathionylation in APAP hepatotoxicity. We provide evidence that the sensitivity difference between wild-type and Gstp1/2−/− mice is unrelated to the ability of APAP to induce oxidative stress, despite observing significant increases in c-Jun N-terminal kinase and extracellular signal-regulated kinase phosphorylation in wild-type mice. The major difference in response to APAP was in the levels of protein S-glutathionylation: Gstp1/2−/− mice exhibited a significant increase in the number of S-glutathionylated proteins compared with wild-type animals. Remarkably, these S-glutathionylated proteins are involved in oxidative phosphorylation, respiratory complexes, drug metabolism, and mitochondrial apoptosis. Furthermore, we found that S-glutathionylation of the rate-limiting glutathione-synthesizing enzyme, glutamate cysteine ligase, was markedly increased in Gstp1/2−/− mice in response to APAP. The data demonstrate that S-glutathionylation provides an adaptive response to APAP and, as a consequence, suggest that this is an important determinant in APAP hepatotoxicity. This work identifies potential novel avenues associated with cell survival for the treatment of chemical-induced hepatotoxicity. PMID:26311813

  18. Organochlorine residues and eggshell thinning in anhingas and waders

    USGS Publications Warehouse

    Ohlendorf, H.M.; Klaas, E.E.; Kaiser, T.E.

    1977-01-01

    Residues of organochlorine compounds occur commonly in environmental samples and have been associated with adverse effects in numerous avian species. The affected species are usually terminal consumers, generally those feeding on aquatic organisms (primarily fish) or birds. Brown Pelicans (Pelecanus occidentalis), Double-crested Cormorants (Phalacrocorax auritus , and certain herons are among the species that have exhibited shell thinning in some regions. In 1972, we began a study to determine: (1) geographic differences in the occurrence of environmental pollutants in Anhingas (Anhinga anhinga) and waders in the eastern United States; (2) differences in environmental pollutant levels among those species nesting at the same localities; and (3) whether eggshell thickness had changed since the widespread use of organochlorine pesticides began in the mid-1940's.

  19. Comparison of the structure, crystallography and composition of eggshells of the guinea fowl and graylag goose.

    PubMed

    Pérez-Huerta, Alberto; Dauphin, Yannicke

    2016-02-01

    The structure and composition of the eggshells of two commercial species (guinea fowl and greylag goose) have been studied. Thin sections and scanning electron microcopy show the similarity of the overall structure, but the relative thickness of the layers differs in these two taxa. Atomic force microscopy shows that the different layers are composed of rounded, heterogeneous granules, the diameter of which is between 50 and 100 nm, with a thin cortex. Infrared data and thermogravimetric analyses show that both eggshells are made of calcite, but differing on the quality and quantity when the organic component is considered. Chemical maps show that chemical element distribution is not uniform within a sample, and differs between the species, but with low magnesium content. Electron back scattered diffraction confirms the eggshells are calcite, but the microtexture strongly differs between the two species. Based on the chemical-structural differences, a species-specific biological control on the biomineralization is found, despite the rapid formation of an eggshell. Overall results indicate that to estimate the quality of eggshells, such as resistance to breakage, is not a straightforward process because of the high complexity of avian eggshell biomineralization. PMID:26711013

  20. The amino acid and stable isotope biogeochemistry of elephant bird ( Aepyornis) eggshells from southern Madagascar

    NASA Astrophysics Data System (ADS)

    Clarke, Simon J.; Miller, Gifford H.; Fogel, Marilyn L.; Chivas, Allan R.; Murray-Wallace, Colin V.

    2006-09-01

    A diverse suite of animals became extinct on Madagascar during the Late Holocene. As observed on landmasses elsewhere, the extinction process broadly coincided with the arrival of people. Our research on the amino acid racemisation and the carbon and oxygen isotope biogeochemistry of elephant bird ( Aepyornis) eggshells from southern Madagascar refines models that attempt to explain the extinction process. A correlation between the extent of isoleucine epimerisation (aIle/Ile) and radiocarbon age of eggshells allows aIle/Ile to serve as a proxy for eggshell age. The aIle/Ile values indicate the majority (87%) of eggshells in this study are Holocene, with the remainder representing Pleistocene Aepyornis populations, and that further amino acid analyses would help to constrain the timing of Aepyornis extinction. Carbon isotope ratios in the organic and calcite fractions of eggshells indicate that Aepyornis primarily browsed C 3 vegetation. Oxygen isotope values are more negative and less variable than in eggshells of ostriches living in semi-arid environments, suggesting that Aepyornis populations relied upon groundwater-fed coastal wetlands for their drinking water. The isotope results require that the changing abundances of C 3 vegetation and groundwater-fed watering points be considered in models that aim to understand the extinction of Aepyornis in southern Madagascar.

  1. Changes in mineral composition of eggshells from black ducks and mallards fed DDE in the diet

    USGS Publications Warehouse

    Longcore, J.R.; Samson, F.B.; Kreitzer, J.F.; Spann, J.W.

    1971-01-01

    Diets containing 10 and 30 ppm (dry weight) DDE were fed to black ducks, and diets containing 1, 5, and 10 ppm (dry weight) DDE were fed to mallards. Among the results were the following changes in black duck eggshell composition: (a) significant increase in the percentage of Mg, (b) significant decreases in Ba and Sr, (c) increases (which approached significance) in average percentage of eggshell Na and Cu, (d) a decrease in shell Ca which approached significance, (e) patterns of mineral correlations which in some instances were distinct to dosage groups, and (f) inverse correlations in the control group between eggshell thickness Mg and Na. Changes in mallard eggshells were: (a) significant increase in percentage of magnesium at 5 and 10 ppm DDE, (b) significant decrease in Al at 5 and 10 ppm DDE, (c) a significant decrease in Ca from eggshells from the 10 ppm DDE group, and (d) an increase in average percentage of Na in eggshells from DDE dosed ducks which approached significance.

  2. Insights from the molecular characterization of mercury stress proteins identified by proteomics in E.coli nissle 1917.

    PubMed

    Seshapani, Panthangi; Rayalu, Daddam Jayasimha; Kumar, Vadde Kiran; Sekhar, Kathera Chandra; Kumari, Jasti Pramoda

    2013-01-01

    Differently expressed proteins in probiotic Escherichia coli nissle 1917 under mercury stress identified by using a proteomic approach. We applied to separate proteins by using two-dimensional gel electrophoresis and proteins were identified using MALDI-TOF-MS using PMF, by mascot database search using the NCBI database. we identified six proteins after exposure to mercury stress with respect to different functional classes. It is useful to understand the molecular insights into mercury stress in probiotic E. coli. Next we describe a structure generated by homology modelling and functional domain identification; it is interesting to study the impact of stress on protein structures. MS characterization and computational methods together provide the opportunity to examine the impact of stress arising from mercury. The role of these proteins in metal tolerance and structure relation is discussed. To the best of our knowledge, proteomics of E. coli nissle 1917 overview of mercury stress has been reported for the first time. PMID:23847405

  3. LigSearch: a knowledge-based web server to identify likely ligands for a protein target

    SciTech Connect

    Beer, Tjaart A. P. de; Laskowski, Roman A.; Duban, Mark-Eugene; Chan, A. W. Edith; Anderson, Wayne F.; Thornton, Janet M.

    2013-12-01

    LigSearch is a web server for identifying ligands likely to bind to a given protein. Identifying which ligands might bind to a protein before crystallization trials could provide a significant saving in time and resources. LigSearch, a web server aimed at predicting ligands that might bind to and stabilize a given protein, has been developed. Using a protein sequence and/or structure, the system searches against a variety of databases, combining available knowledge, and provides a clustered and ranked output of possible ligands. LigSearch can be accessed at http://www.ebi.ac.uk/thornton-srv/databases/LigSearch.

  4. Network Cluster Analysis of Protein–Protein Interaction Network–Identified Biomarker for Type 2 Diabetes

    PubMed Central

    Li, Zhonghui; Qiao, Zijun

    2015-01-01

    Abstract Type 2 diabetes mellitus (T2DM) is a complex disease that is caused by an impairment in the secretion of β-cell insulin and by a peripheral resistance to insulin. Most patients suffering from T2DM and from obesity exhibit insulin resistance in the muscles, liver, and fat, resulting in a reduced response of these tissues to insulin. In healthy individuals, pancreatic islet β-cells secrete insulin to regulate the increase in blood glucose levels. Once these β-cells fail to function, T2DM develops. Despite the progress achieved in this field in recent years, the genetic causes for insulin resistance and for T2DM have not yet been fully discovered. The present study aims to characterize T2DM by comparing its gene expression with that of normal controls, as well as to identify biomarkers for early T2DM. Gene expression profiles were downloaded from the Gene Expression Omnibus, and differentially expressed genes (DEGs) were identified for type 2 diabetes. Furthermore, functional analyses were conducted for the gene ontology and for the pathway enrichment. In total, 781 DEGs were identified in the T2DM samples relative to healthy controls. These genes were found to be involved in several biological processes, including cell communication, cell proliferation, cell shape, and apoptosis. We constructed a protein–protein interaction (PPI) network, and the clusters in the PPI were analyzed by using ClusterONE. Six functional genes that may play important roles in the initiation of T2DM were identified within the network. PMID:25879401

  5. Past Strategies and Future Directions for Identifying AMP-Activated Protein Kinase (AMPK) Modulators

    PubMed Central

    Sinnett, Sarah E.; Brenman, Jay E.

    2014-01-01

    AMP-activated protein kinase (AMPK) is a promising therapeutic target for cancer, type II diabetes, and other illnesses characterized by abnormal energy utilization. During the last decade, numerous labs have published a range of methods for identifying novel AMPK modulators. The current understanding of AMPK structure and regulation, however, has propelled a paradigm shift in which many researchers now consider ADP to be an additional regulatory nucleotide of AMPK. How can the AMPK community apply this new understanding of AMPK signaling to translational research? Recent insights into AMPK structure, regulation, and holoenzyme-sensitive signaling may provide the hindsight needed to clearly evaluate the strengths and weaknesses of past AMPK drug discovery efforts. Improving future strategies for AMPK drug discovery will require pairing the current understanding of AMPK signaling with improved experimental designs. PMID:24583089

  6. Nucleotide sequence variation of the envelope protein gene identifies two distinct genotypes of yellow fever virus.

    PubMed Central

    Chang, G J; Cropp, B C; Kinney, R M; Trent, D W; Gubler, D J

    1995-01-01

    The evolution of yellow fever virus over 67 years was investigated by comparing the nucleotide sequences of the envelope (E) protein genes of 20 viruses isolated in Africa, the Caribbean, and South America. Uniformly weighted parsimony algorithm analysis defined two major evolutionary yellow fever virus lineages designated E genotypes I and II. E genotype I contained viruses isolated from East and Central Africa. E genotype II viruses were divided into two sublineages: IIA viruses from West Africa and IIB viruses from America, except for a 1979 virus isolated from Trinidad (TRINID79A). Unique signature patterns were identified at 111 nucleotide and 12 amino acid positions within the yellow fever virus E gene by signature pattern analysis. Yellow fever viruses from East and Central Africa contained unique signatures at 60 nucleotide and five amino acid positions, those from West Africa contained unique signatures at 25 nucleotide and two amino acid positions, and viruses from America contained such signatures at 30 nucleotide and five amino acid positions in the E gene. The dissemination of yellow fever viruses from Africa to the Americas is supported by the close genetic relatedness of genotype IIA and IIB viruses and genetic evidence of a possible second introduction of yellow fever virus from West Africa, as illustrated by the TRINID79A virus isolate. The E protein genes of American IIB yellow fever viruses had higher frequencies of amino acid substitutions than did genes of yellow fever viruses of genotypes I and IIA on the basis of comparisons with a consensus amino acid sequence for the yellow fever E gene. The great variation in the E proteins of American yellow fever virus probably results from positive selection imposed by virus interaction with different species of mosquitoes or nonhuman primates in the Americas. PMID:7637022

  7. Beyond BLASTing: tertiary and quaternary structure analysis helps identify major vault proteins.

    PubMed

    Daly, Toni K; Sutherland-Smith, Andrew J; Penny, David

    2013-01-01

    We examine the advantages of going beyond sequence similarity and use both protein three-dimensional (3D) structure prediction and then quaternary structure (docking) of inferred 3D structures to help evaluate whether comparable sequences can fold into homologous structures with sufficient lateral associations for quaternary structure formation. Our test case is the major vault protein (MVP) that oligomerizes in multiple copies to form barrel-like vault particles and is relatively widespread among eukaryotes. We used the iterative threading assembly refinement server (I-TASSER) to predict whether putative MVP sequences identified by BLASTp and PSI Basic Local Alignment Search Tool are structurally similar to the experimentally determined rodent MVP tertiary structures. Then two identical predicted quaternary structures from I-TASSER are analyzed by RosettaDock to test whether a pair-wise association occurs, and hence whether the oligomeric vault complex is likely to form for a given MVP sequence. Positive controls for the method are the experimentally determined rat (Rattus norvegicus) vault X-ray crystal structure and the purple sea urchin (Strongylocentrotus purpuratus) MVP sequence that forms experimentally observed vaults. These and two kinetoplast MVP structural homologs were predicted with high confidence value, and RosettaDock predicted that these MVP sequences would dock laterally and therefore could form oligomeric vaults. As the negative control, I-TASSER did not predict an MVP-like structure from a randomized rat MVP sequence, even when constrained to the rat MVP crystal structure (PDB:2ZUO), thus further validating the method. The protocol identified six putative homologous MVP sequences in the heterobolosean Naegleria gruberi within the excavate kingdom. Two of these sequences are predicted to be structurally similar to rat MVP, despite being in excess of 300 residues shorter. The method can be used generally to help test predictions of homology via

  8. A versatile transreplication-based system to identify cellular proteins involved in geminivirus replication.

    PubMed

    Morilla, Gabriel; Castillo, Araceli G; Preiss, Werner; Jeske, Holger; Bejarano, Eduardo R

    2006-04-01

    A versatile green fluorescent protein (GFP) expression cassette containing the replication origins of the monopartite begomovirus Tomato yellow leaf curl Sardinia virus (TYLCSV) is described. Transgenic Nicotiana benthamiana plants containing one copy of the cassette stably integrated into their genome were superinfected with TYLCSV, which mobilized and replicated the cassette as an episomal replicon. The expression of the reporter gene (the GFP gene) was thereby modified. Whereas GFP fluorescence was dimmed in the intercostal areas, an increase of green fluorescence in veins of all leaves placed above the inoculation site, as well as in transport tissues of roots and stems, was observed. The release of episomal trans replicons from the transgene and the increase in GFP expression were dependent on the cognate geminiviral replication-associated protein (Rep) and required interaction between Rep and the intergenic region of TYLCSV. This expression system is able to monitor the replication status of TYLCSV in plants, as induction of GFP expression is only produced in those tissues where Rep is present. To further confirm this notion, the expression of a host factor required for geminivirus replication, the proliferating cellular nuclear antigen (PCNA) was transiently silenced. Inhibition of PCNA prevented GFP induction in veins and reduced viral DNA. We propose that these plants could be widely used to easily identify host factors required for geminivirus replication by virus-induced gene silencing. PMID:16537630

  9. A Versatile Transreplication-Based System To Identify Cellular Proteins Involved in Geminivirus Replication

    PubMed Central

    Morilla, Gabriel; Castillo, Araceli G.; Preiss, Werner; Jeske, Holger; Bejarano, Eduardo R.

    2006-01-01

    A versatile green fluorescent protein (GFP) expression cassette containing the replication origins of the monopartite begomovirus Tomato yellow leaf curl Sardinia virus (TYLCSV) is described. Transgenic Nicotiana benthamiana plants containing one copy of the cassette stably integrated into their genome were superinfected with TYLCSV, which mobilized and replicated the cassette as an episomal replicon. The expression of the reporter gene (the GFP gene) was thereby modified. Whereas GFP fluorescence was dimmed in the intercostal areas, an increase of green fluorescence in veins of all leaves placed above the inoculation site, as well as in transport tissues of roots and stems, was observed. The release of episomal trans replicons from the transgene and the increase in GFP expression were dependent on the cognate geminiviral replication-associated protein (Rep) and required interaction between Rep and the intergenic region of TYLCSV. This expression system is able to monitor the replication status of TYLCSV in plants, as induction of GFP expression is only produced in those tissues where Rep is present. To further confirm this notion, the expression of a host factor required for geminivirus replication, the proliferating cellular nuclear antigen (PCNA) was transiently silenced. Inhibition of PCNA prevented GFP induction in veins and reduced viral DNA. We propose that these plants could be widely used to easily identify host factors required for geminivirus replication by virus-induced gene silencing. PMID:16537630

  10. Human monoclonal antibodies to West Nile virus identify epitopes on the prM protein

    SciTech Connect

    Calvert, Amanda E.; Kalantarov, Gavreel F.; Chang, Gwong-Jen J.; Trakht, Ilya; Blair, Carol D.; Roehrig, John T.

    2011-02-05

    Hybridoma cell lines (2E8, 8G8 and 5G12) producing fully human monoclonal antibodies (hMAbs) specific for the pre-membrane (prM) protein of West Nile virus (WNV) were prepared using a human fusion partner cell line, MFP-2, and human peripheral blood lymphocytes from a blood donor diagnosed with WNV fever in 2004. Using site-directed mutagenesis of a WNV-like particle (VLP) we identified 4 amino acid residues in the prM protein unique to WNV and important in the binding of these hMAbs to the VLP. Residues V19 and L33 are important epitopes for the binding of all three hMAbs. Mutations at residue, T20 and T24 affected the binding of hMAbs, 8G8 and 5G12 only. These hMAbs did not significantly protect AG129 interferon-deficient mice or Swiss Webster outbred mice from WNV infection.

  11. A chemical screen to identify inducers of the mitochondrial unfolded protein response in C. elegans.

    PubMed

    Rauthan, Manish; Pilon, Marc

    2015-01-01

    We previously showed that inhibition of the mevalonate pathway in C. elegans causes inhibition of protein prenylation, developmental arrest and lethality. We also showed that constitutive activation of the mitochondrial unfolded protein response, UPR(mt), is an effective way for C. elegans to become resistant to the negative effects of mevalonate pathway inhibition. This was an important finding since statins, a drug class prescribed to lower cholesterol levels in patients, act by inhibiting the mevalonate pathway, and it is therefore possible that some of their undesirable side effects could be alleviated by activating the UPR(mt). Here, we screened a chemical library and identified 4 compounds that specifically activated the UPR(mt). One of these compounds, methacycline hydrochloride (a tetracycline antibiotic) also protected C. elegans and mammalian cells from statin toxicity. Methacycline hydrochloride and ethidium bromide, a known UPR(mt) activator, were also tested in mice: only ethidium bromide significantly activate the UPR(mt) in skeletal muscles. PMID:27123370

  12. Interactome Analysis of the Human Respiratory Syncytial Virus RNA Polymerase Complex Identifies Protein Chaperones as Important Cofactors That Promote L-Protein Stability and RNA Synthesis

    PubMed Central

    Munday, Diane C.; Wu, Weining; Smith, Nikki; Fix, Jenna; Noton, Sarah Louise; Galloux, Marie; Touzelet, Olivier; Armstrong, Stuart D.; Dawson, Jenna M.; Aljabr, Waleed; Easton, Andrew J.; Rameix-Welti, Marie-Anne; de Oliveira, Andressa Peres; Simabuco, Fernando M.; Ventura, Armando M.; Hughes, David J.; Barr, John N.; Fearns, Rachel; Digard, Paul

    2014-01-01

    ABSTRACT The human respiratory syncytial virus (HRSV) core viral RNA polymerase comprises the large polymerase protein (L) and its cofactor, the phosphoprotein (P), which associate with the viral ribonucleoprotein complex to replicate the genome and, together with the M2-1 protein, transcribe viral mRNAs. While cellular proteins have long been proposed to be involved in the synthesis of HRSV RNA by associating with the polymerase complex, their characterization has been hindered by the difficulty of purifying the viral polymerase from mammalian cell culture. In this study, enhanced green fluorescent protein (EGFP)-tagged L- and P-protein expression was coupled with high-affinity anti-GFP antibody-based immunoprecipitation and quantitative proteomics to identify cellular proteins that interacted with either the L- or the P-proteins when expressed as part of a biologically active viral RNP. Several core groups of cellular proteins were identified that interacted with each viral protein including, in both cases, protein chaperones. Ablation of chaperone activity by using small-molecule inhibitors confirmed previously reported studies which suggested that this class of proteins acted as positive viral factors. Inhibition of HSP90 chaperone function in the current study showed that HSP90 is critical for L-protein function and stability, whether in the presence or absence of the P-protein. Inhibition studies suggested that HSP70 also disrupts virus biology and might help the polymerase remodel the nucleocapsid to allow RNA synthesis to occur efficiently. This indicated a proviral role for protein chaperones in HRSV replication and demonstrates that the function of cellular proteins can be targeted as potential therapeutics to disrupt virus replication. IMPORTANCE Human respiratory syncytial virus (HRSV) represents a major health care and economic burden, being the main cause of severe respiratory infections in infants worldwide. No vaccine or effective therapy is

  13. cDNA Library Screening Identifies Protein Interactors Potentially Involved in Non-Telomeric Roles of Arabidopsis Telomerase

    PubMed Central

    Dokládal, Ladislav; Honys, David; Rana, Rajiv; Lee, Lan-Ying; Gelvin, Stanton B.; Sýkorová, Eva

    2015-01-01

    Telomerase-reverse transcriptase (TERT) plays an essential catalytic role in maintaining telomeres. However, in animal systems telomerase plays additional non-telomeric functional roles. We previously screened an Arabidopsis cDNA library for proteins that interact with the C-terminal extension (CTE) TERT domain and identified a nuclear-localized protein that contains an RNA recognition motif (RRM). This RRM-protein forms homodimers in both plants and yeast. Mutation of the gene encoding the RRM-protein had no detectable effect on plant growth and development, nor did it affect telomerase activity or telomere length in vivo, suggesting a non-telomeric role for TERT/RRM-protein complexes. The gene encoding the RRM-protein is highly expressed in leaf and reproductive tissues. We further screened an Arabidopsis cDNA library for proteins that interact with the RRM-protein and identified five interactors. These proteins are involved in numerous non-telomere-associated cellular activities. In plants, the RRM-protein, both alone and in a complex with its interactors, localizes to nuclear speckles. Transcriptional analyses in wild-type and rrm mutant plants, as well as transcriptional co-analyses, suggest that TERT, the RRM-protein, and the RRM-protein interactors may play important roles in non-telomeric cellular functions. PMID:26617625

  14. cDNA Library Screening Identifies Protein Interactors Potentially Involved in Non-Telomeric Roles of Arabidopsis Telomerase.

    PubMed

    Dokládal, Ladislav; Honys, David; Rana, Rajiv; Lee, Lan-Ying; Gelvin, Stanton B; Sýkorová, Eva

    2015-01-01

    Telomerase-reverse transcriptase (TERT) plays an essential catalytic role in maintaining telomeres. However, in animal systems telomerase plays additional non-telomeric functional roles. We previously screened an Arabidopsis cDNA library for proteins that interact with the C-terminal extension (CTE) TERT domain and identified a nuclear-localized protein that contains an RNA recognition motif (RRM). This RRM-protein forms homodimers in both plants and yeast. Mutation of the gene encoding the RRM-protein had no detectable effect on plant growth and development, nor did it affect telomerase activity or telomere length in vivo, suggesting a non-telomeric role for TERT/RRM-protein complexes. The gene encoding the RRM-protein is highly expressed in leaf and reproductive tissues. We further screened an Arabidopsis cDNA library for proteins that interact with the RRM-protein and identified five interactors. These proteins are involved in numerous non-telomere-associated cellular activities. In plants, the RRM-protein, both alone and in a complex with its interactors, localizes to nuclear speckles. Transcriptional analyses in wild-type and rrm mutant plants, as well as transcriptional co-analyses, suggest that TERT, the RRM-protein, and the RRM-protein interactors may play important roles in non-telomeric cellular functions. PMID:26617625

  15. General method to identify and enrich vicinal thiol proteins present in intact cells in the oxidized, disulfide state.

    PubMed

    Gitler, C; Zarmi, B; Kalef, E

    1997-10-01

    Some 5% of the soluble proteins of L1210 murine leukemia lymphoblasts contain surface vicinal thiols (Kalef, E., Walfish, P. G., and Gitler, C. (1993) Anal. Biochem. 212, 325-334). Redox dithiol to intraprotein disulfide conversion could regulate the cellular function of these proteins. A general method is presented to identify and enrich vicinal thiol proteins existing in cells in their oxidized, disulfide state. The method is based on the in situ blockage by cell permeable N-ethylmaleimide (NEM) of readily accessible cellular protein sulfhydryls. Following removal of the excess NEM, disulfide-containing proteins were identified by reduction with DTT and specific labeling with N-iodoacetyl-[125I]-3-iodotyrosine. The vicinal thiol proteins formed could also be enriched, prior to labeling with [125I]IAIT, by their selective binding to Sepha-rose-aminohexanoyl-4-aminophenylarsine oxide. Exponentially growing L1210 lymphoblasts contain more than 20 proteins with thiols in the oxidized, disulfide state. The majority derive from vicinal thiol proteins. The fraction oxidized, in some proteins, represents almost the totality of the protein present in the cell. Exposure of lymphoblasts to diamide increases the number and concentration of proteins with intraprotein disulfides. This method allows sensitive direct identification of vicinal thiol proteins that participate in redox regulation and those that are targets to oxidative stress conditions. PMID:9324940

  16. Freestanding eggshell membrane-based electrodes for high-performance supercapacitors and oxygen evolution reaction

    NASA Astrophysics Data System (ADS)

    Geng, Jing; Wu, Hao; Al-Enizi, Abdullah M.; Elzatahry, Ahmed A.; Zheng, Gengfeng

    2015-08-01

    A type of freestanding, light-weight eggshell membrane-based electrode is demonstrated for supercapacitors and for oxygen evolution reaction (OER) catalysis. As a widely available daily waste, eggshell membranes have unique porous three-dimensional grid-like fibrous structures with relatively high surface area and abundant macropores, allowing for effective conjugation of carbon nanotubes and growth of NiCo2O4 nanowire arrays, an effective supercapacitor material and OER catalyst. The three-dimensional fibrous eggshell membrane frameworks with carbon nanotubes offer efficient pathways for charge transport, and the macropores between adjacent fibers are fully accessible for electrolytes and bubble evolution. As a supercapacitor, the eggshell membrane/carbon nanotube/NiCo2O4 electrode shows high specific capacitances at current densities from 1 to 20 A g-1, with excellent capacitance retention (>90%) at 10 A g-1 for over 10 000 cycles. When employed as an OER catalyst, this eggshell membrane-based electrode exhibits an OER onset potential of 1.53 V vs. the reversible hydrogen electrode (RHE), and a stable catalytic current density of 20 mA cm-2 at 1.65 V vs. the RHE.A type of freestanding, light-weight eggshell membrane-based electrode is demonstrated for supercapacitors and for oxygen evolution reaction (OER) catalysis. As a widely available daily waste, eggshell membranes have unique porous three-dimensional grid-like fibrous structures with relatively high surface area and abundant macropores, allowing for effective conjugation of carbon nanotubes and growth of NiCo2O4 nanowire arrays, an effective supercapacitor material and OER catalyst. The three-dimensional fibrous eggshell membrane frameworks with carbon nanotubes offer efficient pathways for charge transport, and the macropores between adjacent fibers are fully accessible for electrolytes and bubble evolution. As a supercapacitor, the eggshell membrane/carbon nanotube/NiCo2O4 electrode shows high specific

  17. Use of Dimedone-Based Chemical Probes for Sulfenic Acid Detection: Methods to Visualize and Identify Labeled Proteins

    PubMed Central

    Nelson, Kimberly J.; Klomsiri, Chananat; Codreanu, Simona G.; Soito, Laura; Liebler, Daniel C.; Rogers, LeAnn C.; Daniel, Larry W.; Poole, Leslie B.

    2013-01-01

    Reversible thiol modification is a major component of the modulation of cell-signaling pathways by reactive oxygen species. Hydrogen peroxide, peroxynitrite, or lipid hydroperoxides are all able to oxidize cysteines to form cysteine sulfenic acids; this reactive intermediate can be directly reduced to thiol by cellular reductants such as thioredoxin or further participate in disulfide bond formation with glutathione or cysteine residues in the same or another protein. To identify the direct protein targets of cysteine modification and the conditions under which they are oxidized, a series of dimedone-based reagents linked to affinity or fluorescent tags have been developed that specifically alkylate and trap cysteine sulfenic acids. In this chapter, we provide detailed methods using one of our biotin-tagged reagents, DCP-Bio1, to identify and monitor proteins that are oxidized in vitro and in vivo. Using streptavidin-linked agarose beads, this biotin-linked reagent can be used to affinity capture labeled proteins. Stringent washing of the beads prior to elution minimizes the contamination of the enriched material with unlabeled proteins through coimmunoprecipitation or nonspecific binding. In particular, we suggest including DTT in one of the washes to remove proteins covalently linked to biotinylated proteins through a disulfide bond, except in cases where these linked proteins are of interest. We also provide methods for targeted approaches monitoring cysteine oxidation in individual proteins, global approaches to follow total cysteine oxidation in the cell, and guidelines for proteomic analyses to identify novel proteins with redox sensitive cysteines. PMID:20513473

  18. Microwave-assisted synthesis of carbon nanodots through an eggshell membrane and their fluorescent application.

    PubMed

    Wang, Qi; Liu, Xing; Zhang, Lichun; Lv, Yi

    2012-11-21

    Carbon nanodots (C-Dots) as a new form of carbonaceous nanomaterials have aroused much interest and intensive research due to their inspiring properties. Compared to traditional semiconductor quantum dots, these newly emergent nanodots possess a number of advantageous characteristics, among which low-toxicity is particularly fascinating. More and more research into C-Dots have focused on synthesis methods and biology-related applications. Microwave-assisted approaches have attracted attention because microwave treatment can provide intensive and efficient energy, and as a consequence shorten the reaction time. In this article, we designed a "green", rapid, eco-friendly and waste-reused approach to synthesize fluorescent and water-soluble C-Dots from eggshell membrane (ESM) ashes according to a microwave-assisted process. ESM selected as the carbon source was a common protein-rich waste in daily life and can be obtained easily and cheaply. The C-Dots from our method showed the maximal fluorescence emission peak at 450 nm and the fluorescence quantum yield was about 14%. We further designed a sensitive probe for glutathione based on the fluorescence turn off and on of the C-Dots-Cu(2+) system, which showed a linear range of 0.5-80 μmol L(-1) and detection limit of 0.48 μmol L(-1). In general, the C-Dots prepared briefly and inexpensively from ESM revealed excellent fluorescent property with promising potential for applications such as sample detection and biotechnology. PMID:23037913

  19. Pleiotropy among Common Genetic Loci Identified for Cardiometabolic Disorders and C-Reactive Protein

    PubMed Central

    Ligthart, Symen; de Vries, Paul S.; Uitterlinden, André G.; Hofman, Albert; Franco, Oscar H.; Chasman, Daniel I.; Dehghan, Abbas

    2015-01-01

    Pleiotropic genetic variants have independent effects on different phenotypes. C-reactive protein (CRP) is associated with several cardiometabolic phenotypes. Shared genetic backgrounds may partially underlie these associations. We conducted a genome-wide analysis to identify the shared genetic background of inflammation and cardiometabolic phenotypes using published genome-wide association studies (GWAS). We also evaluated whether the pleiotropic effects of such loci were biological or mediated in nature. First, we examined whether 283 common variants identified for 10 cardiometabolic phenotypes in GWAS are associated with CRP level. Second, we tested whether 18 variants identified for serum CRP are associated with 10 cardiometabolic phenotypes. We used a Bonferroni corrected p-value of 1.1×10-04 (0.05/463) as a threshold of significance. We evaluated the independent pleiotropic effect on both phenotypes using individual level data from the Women Genome Health Study. Evaluating the genetic overlap between inflammation and cardiometabolic phenotypes, we found 13 pleiotropic regions. Additional analyses showed that 6 regions (APOC1, HNF1A, IL6R, PPP1R3B, HNF4A and IL1F10) appeared to have a pleiotropic effect on CRP independent of the effects on the cardiometabolic phenotypes. These included loci where individuals carrying the risk allele for CRP encounter higher lipid levels and risk of type 2 diabetes. In addition, 5 regions (GCKR, PABPC4, BCL7B, FTO and TMEM18) had an effect on CRP largely mediated through the cardiometabolic phenotypes. In conclusion, our results show genetic pleiotropy among inflammation and cardiometabolic phenotypes. In addition to reverse causation, our data suggests that pleiotropic genetic variants partially underlie the association between CRP and cardiometabolic phenotypes. PMID:25768928

  20. An electrophoretic mobility shift assay identifies a mechanistically unique inhibitor of protein sumoylation.

    PubMed

    Kim, Yeong Sang; Nagy, Katelyn; Keyser, Samantha; Schneekloth, John S

    2013-04-18

    The dynamic, posttranslational modification of proteins with a small ubiquitin-like modifier (SUMO) tag has been recognized as an important cellular regulatory mechanism relevant to a number of cancers as well as normal embryonic development. As part of a program aimed toward the identification of inhibitors of SUMO-conjugating enzymes, we developed a microfluidic electrophoretic mobility shift assay to monitor sumoylation events in real time. We disclose herein the use of this assay to identify a cell-permeable compound capable of blocking the transfer of SUMO-1 from the E2 enzyme Ubc9 to the substrate. We screened a small collection of compounds and identified an oxygenated flavonoid derivative that inhibits sumoylation in vitro. Next, we carried out an in-depth mechanistic analysis that ruled out many common false-positive mechanisms such as aggregation or alkylation. Furthermore, we report that this flavonoid inhibits a single step in the sumoylation cascade: the transfer of SUMO from the E2 enzyme (Ubc9) thioester conjugate to the substrate. In addition to having a unique mechanism of action, this inhibitor has a discrete structure-activity relationship uncharacteristic of a promiscuous inhibitor. Cell-based studies showed that the flavonoid inhibits the sumoylation of topoisomerase-I in response to camptothecin treatment in two different breast cancer cell lines, while isomeric analogs are inactive. Importantly, this compound blocks sumoylation while not affecting ubiquitylation in cells. This work identifies a point of entry for pharmacologic inhibition of the sumoylation cascade and may serve as the basis for continued study of additional pharmacophores that modulate SUMO-conjugating enzymes such as Ubc9. PMID:23601649

  1. Mechanical Properties and Vulcanization Characteristics of Styrene-Butadiene Rubber (sbr) Based Compounds Filled with Eggshell Powder as a Bio-Filler

    NASA Astrophysics Data System (ADS)

    Saeb, Mohammad Reza; Dakhel, Hadi Ramezani; Ghaffari, Akbar

    2008-08-01

    Egg shell is an ordered bioceramic composite with five different layers [1, 2]. In this study, the effect of using various types of eggshell powder including after and before hatching eggshell and boiled eggshell on the mechanical properties and vulcanization characteristics of SBR compounds has been investigated. The obtained results were compared with calcium carbonate filled compounds. Evaluation of mechanical properties exhibited that incorporation of eggshell increased almost all of the mechanical properties of the compounds. BET test demonstrated higher specific area of eggshell powders compare with calcium carbonate. Some of the observed results attributed to higher specific area of eggshell powder. However, there is no definitive reason for some other results at this time. Vulcanization characteristics of the compounds showed that incorporation of eggshell powder had no considerable effect on curing time. The results of this research showed that all kinds of eggshell powders can be used in SBR based compounds successfully.

  2. Immobilisation of lead and zinc in contaminated soil using compost derived from industrial eggshell.

    PubMed

    Soares, Micaela A R; Quina, Margarida J; Quinta-Ferreira, Rosa M

    2015-12-01

    This study aims to evaluate the capacity of a compost obtained by co-composting of industrial eggshell (CES) to immobilise lead (Pb) and zinc (Zn) in an acidic soil contaminated by mining activities. Mature compost without eggshell (CWES) and natural eggshell (ES) were also tested as soil amendments for comparison purposes. Three different application rates were used for each material, ensuring the same quantity in terms of neutralizing capacity. Incubation experiments were conducted under controlled conditions and CO2 emissions monitored for 94 days. The environmental availability of Pb and Zn in the amended soil was assessed and bioassays were performed at the end of the incubation period. When eggshells were present, the CES compost raised the soil pH to values higher than 6 and reduced the soil mobile fraction for both Pb and Zn, in more than 95%. Soil toxicity towards Vibrio fischeri was also suppressed and environmental risk decreased to "low level". However, the immobilisation in the acid insoluble soil component was significantly achieved only for Zn. In addition, regarding soil carbon dynamics the CO2-C emissions were enhanced, mainly in the case of the highest rate of amendment. Both first order-E and parallel first order models may adequately describe the kinetic data of CO2-C cumulative release. Without eggshells, the CWES compost revealed limited effect on heavy metals immobilisation, likely due to its small capacity to correct soil acidity, at lower application rates. Using solely eggshells, the ES waste had similar outcomes when compared with CES, but at the higher application rate, CO2 emissions were enhanced with the eggshell compost due to the contribution of biotic carbon present therein. Therefore, this study points out that CES is an effective liming material and may be used for in situ remediation of contaminated soil with Pb and Zn. PMID:26363261

  3. Proteomic profiling identifies the inorganic pyrophosphatase (PPA1) protein as a potential biomarker of metastasis in laryngeal squamous cell carcinoma.

    PubMed

    Bodnar, Magdalena; Luczak, Magdalena; Bednarek, Kinga; Szylberg, Lukasz; Marszalek, Andrzej; Grenman, Reidar; Szyfter, Krzysztof; Jarmuz-Szymczak, Malgorzata; Giefing, Maciej

    2016-06-01

    Relapse and metastasis are the main causes of unfavorable outcome in head and neck cancers. Whereas, understanding of the molecular background of these processes is far from being complete. Therefore, in this study we aimed to identify potential biomarker candidates of relapse and metastasis in laryngeal squamous cell carcinoma (LSCC) by combining the 2D electrophoresis based protein screen and immunohistochemical analysis of candidate proteins. We screened three groups of LSCC cell lines derived from primary tumors, recurrent tumors and metastases and identified seven proteins that differed significantly in relative abundance between the analyzed groups. Among the identified proteins were the heat shock proteins HSP60 and HSP70 that were significantly downregulated both in recurrences- and metastases-derived cell lines but not in primary tumor-derived cell lines. Moreover, we identified significant upregulation of the annexin V, calreticulin and the inorganic pyrophosphatase (PPA1) exclusively in the metastases-derived cell lines. As these upregulated proteins could potentially become novel biomarkers of metastasis, we have compared their abundance in primary tumor LSCC N(0) cases, primary tumor LSCC N(+) cases as well as in LSCC metastases N(+). Our results show an intense increase of cytoplasmic PPA1 abundance in the N(+) (p = 0.000042) compared to the N(0) group. In summary, we show a group of proteins deregulated in recurrences and metastases of LSCC. Moreover, we suggest the PPA1 protein as a potential new biomarker for metastasis in this cancer. PMID:26948660

  4. A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways.

    PubMed

    Lecat, Sandra; Matthes, Hans W D; Pepperkok, Rainer; Simpson, Jeremy C; Galzi, Jean-Luc

    2015-05-01

    Several cytoplasmic proteins that are involved in G protein-coupled receptor signaling cascades are known to translocate to the plasma membrane upon receptor activation, such as beta-arrestin2. Based on this example and in order to identify new cytoplasmic proteins implicated in the ON-and-OFF cycle of G protein-coupled receptor, a live-imaging screen of fluorescently labeled cytoplasmic proteins was performed using translocation criteria. The screening of 193 fluorescently tagged human proteins identified eight proteins that responded to activation of the tachykinin NK2 receptor by a change in their intracellular localization. Previously we have presented the functional characterization of one of these proteins, REDD1, that translocates to the plasma membrane. Here we report the results of the entire screening. The process of cell activation was recorded on videos at different time points and all the videos can be visualized on a dedicated website. The proteins BAIAP3 and BIN1, partially translocated to the plasma membrane upon activation of NK2 receptors. Proteins ARHGAP12 and PKM2 translocated toward membrane blebs. Three proteins that associate with the cytoskeleton were of particular interest : PLEKHH2 rearranged from individual dots located near the cell-substrate adhesion surface into lines of dots. The speriolin-like protein, SPATC1L, redistributed to cell-cell junctions. The Chloride intracellular Channel protein, CLIC2, translocated from actin-enriched plasma membrane bundles to cell-cell junctions upon activation of NK2 receptors. CLIC2, and one of its close paralogs, CLIC4, were further shown to respond with the same translocation pattern to muscarinic M3 and lysophosphatidic LPA receptors. This screen allowed us to identify potential actors in signaling pathways downstream of G protein-coupled receptors and could be scaled-up for high-content screening. PMID:25759509

  5. B56δ-related protein phosphatase 2A dysfunction identified in patients with intellectual disability

    PubMed Central

    Houge, Gunnar; Haesen, Dorien; Vissers, Lisenka E.L.M.; Mehta, Sarju; Parker, Michael J.; Wright, Michael; Vogt, Julie; McKee, Shane; Tolmie, John L.; Cordeiro, Nuno; Kleefstra, Tjitske; Willemsen, Marjolein H.; Reijnders, Margot R.F.; Berland, Siren; Hayman, Eli; Lahat, Eli; Brilstra, Eva H.; van Gassen, Koen L.I.; Zonneveld-Huijssoon, Evelien; de Bie, Charlotte I.; Hoischen, Alexander; Eichler, Evan E.; Holdhus, Rita; Steen, Vidar M.; Døskeland, Stein Ove; Hurles, Matthew E.; FitzPatrick, David R.; Janssens, Veerle

    2015-01-01

    Here we report inherited dysregulation of protein phosphatase activity as a cause of intellectual disability (ID). De novo missense mutations in 2 subunits of serine/threonine (Ser/Thr) protein phosphatase 2A (PP2A) were identified in 16 individuals with mild to severe ID, long-lasting hypotonia, epileptic susceptibility, frontal bossing, mild hypertelorism, and downslanting palpebral fissures. PP2A comprises catalytic (C), scaffolding (A), and regulatory (B) subunits that determine subcellular anchoring, substrate specificity, and physiological function. Ten patients had mutations within a highly conserved acidic loop of the PPP2R5D-encoded B56δ regulatory subunit, with the same E198K mutation present in 6 individuals. Five patients had mutations in the PPP2R1A-encoded scaffolding Aα subunit, with the same R182W mutation in 3 individuals. Some Aα cases presented with large ventricles, causing macrocephaly and hydrocephalus suspicion, and all cases exhibited partial or complete corpus callosum agenesis. Functional evaluation revealed that mutant A and B subunits were stable and uncoupled from phosphatase activity. Mutant B56δ was A and C binding–deficient, while mutant Aα subunits bound B56δ well but were unable to bind C or bound a catalytically impaired C, suggesting a dominant-negative effect where mutant subunits hinder dephosphorylation of B56δ-anchored substrates. Moreover, mutant subunit overexpression resulted in hyperphosphorylation of GSK3β, a B56δ-regulated substrate. This effect was in line with clinical observations, supporting a correlation between the ID degree and biochemical disturbance. PMID:26168268

  6. A multiplexed analysis approach identifies new association of inflammatory proteins in patients with overactive bladder.

    PubMed

    Ma, Emily; Vetter, Joel; Bliss, Laura; Lai, H Henry; Mysorekar, Indira U; Jain, Sanjay

    2016-07-01

    Overactive bladder (OAB) is a common debilitating bladder condition with unknown etiology and limited diagnostic modalities. Here, we explored a novel high-throughput and unbiased multiplex approach with cellular and molecular components in a well-characterized patient cohort to identify biomarkers that could be reliably used to distinguish OAB from controls or provide insights into underlying etiology. As a secondary analysis, we determined whether this method could discriminate between OAB and other chronic bladder conditions. We analyzed plasma samples from healthy volunteers (n = 19) and patients diagnosed with OAB, interstitial cystitis/bladder pain syndrome (IC/BPS), or urinary tract infections (UTI; n = 51) for proinflammatory, chemokine, cytokine, angiogenesis, and vascular injury factors using Meso Scale Discovery (MSD) analysis and urinary cytological analysis. Wilcoxon rank-sum tests were used to perform univariate and multivariate comparisons between patient groups (controls, OAB, IC/BPS, and UTI). Multivariate logistic regression models were fit for each MSD analyte on 1) OAB patients and controls, 2) OAB and IC/BPS patients, and 3) OAB and UTI patients. Age, race, and sex were included as independent variables in all multivariate analysis. Receiver operating characteristic (ROC) curves were generated to determine the diagnostic potential of a given analyte. Our findings demonstrate that five analytes, i.e., interleukin 4, TNF-α, macrophage inflammatory protein-1β, serum amyloid A, and Tie2 can reliably differentiate OAB relative to controls and can be used to distinguish OAB from the other conditions. Together, our pilot study suggests a molecular imbalance in inflammatory proteins may contribute to OAB pathogenesis. PMID:27029431

  7. Exploiting CpG hypermutability to identify phenotypically significant variation within human protein-coding genes.

    PubMed

    Ying, Hua; Huttley, Gavin

    2011-01-01

    The CpG dinucleotide is disproportionately represented in human genetic variation due to the hypermutability of 5-methyl-cytosine (5mC). We exploit this hypermutability and a novel codon substitution model to identify candidate functionally important exonic nucleotides. Population genetic theory suggests that codon positions with high cross-species CpG frequency will derive from stronger purifying selection. Using the phylogeny-based maximum likelihood inference framework, we applied codon substitution models with context-dependent parameters to measure the mutagenic and selective processes affecting CpG dinucleotides within exonic sequence. The suitability of these models was validated on >2,000 protein coding genes from a naturally occurring biological control, four yeast species that do not methylate their DNA. As expected, our analyses of yeast revealed no evidence for an elevated CpG transition rate or for substitution suppression affecting CpG-containing codons. Our analyses of >12,000 protein-coding genes from four primate lineages confirm the systemic influence of 5mC hypermutability on the divergence of these genes. After adjusting for confounding influences of mutation and the properties of the encoded amino acids, we confirmed that CpG-containing codons are under greater purifying selection in primates. Genes with significant evidence of enhanced suppression of nonsynonymous CpG changes were also shown to be significantly enriched in Online Mendelian Inheritance in Man. We developed a method for ranking candidate phenotypically influential CpG positions in human genes. Application of this method indicates that of the ∼1 million exonic CpG dinucleotides within humans, ∼20% are strong candidates for both hypermutability and disease association. PMID:21398426

  8. Ultrasonic eggshell thickness measurement for selection of layers.

    PubMed

    Kibala, Lucyna; Rozempolska-Rucinska, Iwona; Kasperek, Kornel; Zieba, Grzegorz; Lukaszewicz, Marek

    2015-10-01

    This study aimed to develop a methodology for using ultrasonic technology (USG) to record eggshell thickness for selection of layers. Genetic correlations between eggshell strength and its thickness have been reported to be around 0.8, making shell thickness a selection index candidate element. Applying ultrasonic devices to measure shell thickness leaves an egg intact for further handling. In this study, eggs from 2 purebred populations of Rhode Island White (RIW) and Rhode Island Red (RIR) hens were collected on a single day in the 33rd week of the farm laying calendar from 2,414 RIR and 4,525 RIW hens. Beginning from the large end of the egg, measurements were taken at 5 latitudes: 0º (USG0), 45º (USG45), 90º (USG90), 135º (USG135), and 180º (USG180). To estimate the repeatability of readings, measurements were repeated at each parallel on 3 meridians. Electronic micrometer measurement ( EMM: ) were taken with an electronic micrometer predominantly at the wider end of eggs from 2,397 RIR and 4,447 RIW hens. A multiple-trait statistical model fit the fixed effect of year-of-hatch × hatch-within-year, and random effects due to repeated measurements (except EMM) and an animal's additive genetic component. The shell was thinnest in the region where chicks break it upon hatching (USG0, USG45). Heritabilities of shell thickness in different regions of the shell ranged from 0.09 to 0.19 (EMM) in RIW and from 0.12 to 0.23 (EMM) in RIR and were highest for USG45 and USG0. Because the measurement repeatabilities were all above 0.90, our recommendation for balancing egg strength against hatching ease is to take a single measurement of USG45. Due to high positive genetic correlations between shell thickness in different regions of the shell its thickness in the pointed end region will be modified accordingly, in response to selection for USG45. PMID:26316340

  9. A Strategy Based on Protein-Protein Interface Motifs May Help in Identifying Drug Off-Targets

    PubMed Central

    Engin, H. Billur; Keskin, Ozlem; Nussinov, Ruth; Gursoy, Attila

    2014-01-01

    Networks are increasingly used to study the impact of drugs at the systems level. From the algorithmic standpoint, a drug can ‘attack’ nodes or edges of a protein-protein interaction network. In this work, we propose a new network strategy, “The Interface Attack”, based on protein-protein interfaces. Similar interface architectures can occur between unrelated proteins. Consequently, in principle, a drug that binds to one has a certain probability of binding others. The interface attack strategy simultaneously removes from the network all interactions that consist of similar interface motifs. This strategy is inspired by network pharmacology and allows inferring potential off-targets. We introduce a network model which we call “Protein Interface and Interaction Network (P2IN)”, which is the integration of protein-protein interface structures and protein interaction networks. This interface-based network organization clarifies which protein pairs have structurally similar interfaces, and which proteins may compete to bind the same surface region. We built the P2IN of p53 signaling network and performed network robustness analysis. We show that (1) ‘hitting’ frequent interfaces (a set of edges distributed around the network) might be as destructive as eleminating high degree proteins (hub nodes); (2) frequent interfaces are not always topologically critical elements in the network; and (3) interface attack may reveal functional changes in the system better than attack of single proteins. In the off-target detection case study, we found that drugs blocking the interface between CDK6 and CDKN2D may also affect the interaction between CDK4 and CDKN2D. PMID:22817115

  10. Proteins recruited by SH3 domains of Ruk/CIN85 adaptor identified by LC-MS/MS

    PubMed Central

    Havrylov, Serhiy; Rzhepetskyy, Yuriy; Malinowska, Agata; Drobot, Lyudmyla; Redowicz, Maria Jolanta

    2009-01-01

    Background Ruk/CIN85 is a mammalian adaptor molecule with three SH3 domains. Using its SH3 domains Ruk/CIN85 can cluster multiple proteins and protein complexes, and, consequently, facilitates organisation of elaborate protein interaction networks with diverse regulatory roles. Previous research linked Ruk/CIN85 with the regulation of vesicle-mediated transport and cancer cell invasiveness. Despite the recent findings, precise molecular functions of Ruk/CIN85 in these processes remain largely elusive and further research is hampered by a lack of complete lists of its partner proteins. Results In the present study we employed a LC-MS/MS-based experimental pipeline to identify a considerable number (over 100) of proteins recruited by the SH3 domains of Ruk/CIN85 in vitro. Most of these identifications are novel Ruk/CIN85 interaction candidates. The identified proteins have diverse molecular architectures and can interact with other proteins, as well as with lipids and nucleic acids. Some of the identified proteins possess enzymatic activities. Functional profiling analyses and literature mining demonstrate that many of the proteins recruited by the SH3 domains of Ruk/CIN85 identified in this work were involved in the regulation of membranes and cytoskeletal structures necessary for vesicle-mediated transport and cancer cell invasiveness. Several groups of the proteins were also associated with few other cellular processes not previously related to Ruk/CIN85, most prominently with cell division. Conclusion Obtained data support the notion that Ruk/CIN85 regulates vesicle-mediated transport and cancer cell invasiveness through the assembly of multimeric protein complexes governing coordinated remodelling of membranes and underlying cytoskeletal structures, and imply its important roles in formation of coated vesicles and biogenesis of invadopodia. In addition, this study points to potential involvement of Ruk/CIN85 in other cellular processes, chiefly in cell division

  11. Novel mitochondria-targeted heat-soluble proteins identified in the anhydrobiotic Tardigrade improve osmotic tolerance of human cells.

    PubMed

    Tanaka, Sae; Tanaka, Junko; Miwa, Yoshihiro; Horikawa, Daiki D; Katayama, Toshiaki; Arakawa, Kazuharu; Toyoda, Atsushi; Kubo, Takeo; Kunieda, Takekazu

    2015-01-01

    Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called "anhydrobiosis". Late Embryogenesis Abundant (LEA) proteins are heat-soluble proteins involved in the desiccation tolerance of many anhydrobiotic organisms. Tardigrades, Ramazzottius varieornatus, however, express predominantly tardigrade-unique heat-soluble proteins: CAHS (Cytoplasmic Abundant Heat Soluble) and SAHS (Secretory Abundant Heat Soluble) proteins, which are secreted or localized in most intracellular compartments, except the mitochondria. Although mitochondrial integrity is crucial to ensure cellular survival, protective molecules for mitochondria have remained elusive. Here, we identified two novel mitochondrial heat-soluble proteins, RvLEAM and MAHS (Mitochondrial Abundant Heat Soluble), as potent mitochondrial protectants from Ramazzottius varieornatus. RvLEAM is a group3 LEA protein and immunohistochemistry confirmed its mitochondrial localization in tardigrade cells. MAHS-green fluorescent protein fusion protein localized in human mitochondria and was heat-soluble in vitro, though no sequence similarity with other known proteins was found, and one region was conserved among tardigrades. Furthermore, we demonstrated that RvLEAM protein as well as MAHS protein improved the hyperosmotic tolerance of human cells. The findings of the present study revealed that tardigrade mitochondria contain at least two types of heat-soluble proteins that might have protective roles in water-deficient environments. PMID:25675104

  12. Novel Mitochondria-Targeted Heat-Soluble Proteins Identified in the Anhydrobiotic Tardigrade Improve Osmotic Tolerance of Human Cells

    PubMed Central

    Tanaka, Sae; Tanaka, Junko; Miwa, Yoshihiro; Horikawa, Daiki D.; Katayama, Toshiaki; Arakawa, Kazuharu; Toyoda, Atsushi; Kubo, Takeo; Kunieda, Takekazu

    2015-01-01

    Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called “anhydrobiosis”. Late Embryogenesis Abundant (LEA) proteins are heat-soluble proteins involved in the desiccation tolerance of many anhydrobiotic organisms. Tardigrades, Ramazzottius varieornatus, however, express predominantly tardigrade-unique heat-soluble proteins: CAHS (Cytoplasmic Abundant Heat Soluble) and SAHS (Secretory Abundant Heat Soluble) proteins, which are secreted or localized in most intracellular compartments, except the mitochondria. Although mitochondrial integrity is crucial to ensure cellular survival, protective molecules for mitochondria have remained elusive. Here, we identified two novel mitochondrial heat-soluble proteins, RvLEAM and MAHS (Mitochondrial Abundant Heat Soluble), as potent mitochondrial protectants from Ramazzottius varieornatus. RvLEAM is a group3 LEA protein and immunohistochemistry confirmed its mitochondrial localization in tardigrade cells. MAHS-green fluorescent protein fusion protein localized in human mitochondria and was heat-soluble in vitro, though no sequence similarity with other known proteins was found, and one region was conserved among tardigrades. Furthermore, we demonstrated that RvLEAM protein as well as MAHS protein improved the hyperosmotic tolerance of human cells. The findings of the present study revealed that tardigrade mitochondria contain at least two types of heat-soluble proteins that might have protective roles in water-deficient environments. PMID:25675104

  13. Dietary calcium deficiency in laying ducks impairs eggshell quality by suppressing shell biomineralization.

    PubMed

    Chen, Wei; Zhao, Fei; Tian, Zhi Mei; Zhang, Han Xing; Ruan, Dong; Li, Yan; Wang, Shuang; Zheng, Chun Tian; Lin, Ying Cai

    2015-10-01

    The objective of this study was to determine the effects of dietary calcium deficiency on the process of shell formation. Four hundred and fifty female ducks (Anas platyrhynchos) at 22 weeks were randomly assigned to three groups. Ducks were fed one of two calcium-deficient diets (containing 1.8% or 0.38% calcium, respectively) or a calcium-adequate control diet (containing 3.6% calcium) for 67 days (depletion period) and then all ducks were fed a calcium-adequate diet for an additional 67 days (repletion period). Compared with the calcium-adequate control, the average shell thickness, egg shell weight, breaking strength, mammillae density and mammillary knob thickness of shell from ducks that consumed the diet with 0.38% calcium were significantly decreased (P<0.05) during the depletion period, accompanied by reduced tibia quality. The mRNA expression of both secreted phosphoprotein 1 (SPP1) and carbonic anhydrase 2 (CA2) in the uterus was decreased after feeding calcium-deficient diets (1.8% or 0.38% calcium). mRNA transcripts of calbindin 1 (CALB1), an important protein responsible for calcium transport, and the matrix protein genes ovocalyxin-32 (OCX-32) and ovocleidin-116 (OC-116) were reduced in ducks fed 0.38% calcium but not 1.8% calcium. Plasma estradiol concentration was decreased by both of the calcium-deficient diets (P<0.05). The impaired shell quality and suppressed functional proteins involved in shell formation could be reversed by repletion of dietary calcium. The results of the present study suggest that dietary calcium deficiency negatively affects eggshell quality and microarchitecture, probably by suppressing shell biomineralization. PMID:26385336

  14. APPLICATION OF A NOVEL RADIOIMMUNOASSAY TO IDENTIFY BACULOVIRUS STRUCTURAL PROTEINS THAT SHARE INTERSPECIES ANTIGENIC DETERMINANTS

    EPA Science Inventory

    Viral proteins were electrophoresed in polyacrylamide gels, transferred to nitrocellulose, and incubated with viral antisera, and the antibodies were detected with 125 I-labeled Staphylococcus aureus protein A. Antisera were prepared to purified and intact virions from five bacul...

  15. Specific inhibitors of the protein tyrosine phosphatase Shp2 identified by high-throughput docking

    PubMed Central

    Hellmuth, Klaus; Grosskopf, Stefanie; Lum, Ching Tung; Würtele, Martin; Röder, Nadine; von Kries, Jens Peter; Rosario, Marta; Rademann, Jörg; Birchmeier, Walter

    2008-01-01

    The protein tyrosine phosphatase Shp2 is a positive regulator of growth factor signaling. Gain-of-function mutations in several types of leukemia define Shp2 as a bona fide oncogene. We performed a high-throughput in silico screen for small-molecular-weight compounds that bind the catalytic site of Shp2. We have identified the phenylhydrazonopyrazolone sulfonate PHPS1 as a potent and cell-permeable inhibitor, which is specific for Shp2 over the closely related tyrosine phosphatases Shp1 and PTP1B. PHPS1 inhibits Shp2-dependent cellular events such as hepatocyte growth factor/scatter factor (HGF/SF)-induced epithelial cell scattering and branching morphogenesis. PHPS1 also blocks Shp2-dependent downstream signaling, namely HGF/SF-induced sustained phosphorylation of the Erk1/2 MAP kinases and dephosphorylation of paxillin. Furthermore, PHPS1 efficiently inhibits activation of Erk1/2 by the leukemia-associated Shp2 mutant, Shp2-E76K, and blocks the anchorage-independent growth of a variety of human tumor cell lines. The PHPS compound class is therefore suitable for further development of therapeutics for the treatment of Shp2-dependent diseases. PMID:18480264

  16. Two putative protein-tyrosine kinases identified by application of the polymerase chain reaction.

    PubMed Central

    Wilks, A F

    1989-01-01

    The pivotal role that protein-tyrosine kinases (PTKs) play in the growth regulation of eukaryotic cells is manifest in the frequent appearance of members of the PTK family as growth factor receptors or as the transforming agents of acutely transforming retroviruses. A feature common to all members of the PTK family is a highly conserved catalytic domain which is characteristic of the group as a whole and whose activity appears to be tightly regulated within the cell by other domains of the PTK. Degenerate oligonucleotide probes corresponding to two invariant amino acid sequence motifs within the catalytic domains of all PTK family members were synthesized and employed in the polymerase chain reaction (PCR) to amplify cDNA sequences between them. An M13 PCR library was produced in this way from cDNA prepared against mRNA from the murine hemopoietic cell line FDC-P1. The PCR library was then screened by DNA sequencing for PTK-related sequences. Two sequences were identified that, on the basis of sequence comparison with known PTKs, may encode representatives of a new class of PTK. Images PMID:2466296

  17. Robust Density-Based Clustering To Identify Metastable Conformational States of Proteins.

    PubMed

    Sittel, Florian; Stock, Gerhard

    2016-05-10

    A density-based clustering method is proposed that is deterministic, computationally efficient, and self-consistent in its parameter choice. By calculating a geometric coordinate space density for every point of a given data set, a local free energy is defined. On the basis of these free energy estimates, the frames are lumped into local free energy minima, ultimately forming microstates separated by local free energy barriers. The algorithm is embedded into a complete workflow to robustly generate Markov state models from molecular dynamics trajectories. It consists of (i) preprocessing of the data via principal component analysis in order to reduce the dimensionality of the problem, (ii) proposed density-based clustering to generate microstates, and (iii) dynamical clustering via the most probable path algorithm to construct metastable states. To characterize the resulting state-resolved conformational distribution, dihedral angle content color plots are introduced which identify structural differences of protein states in a concise way. To illustrate the performance of the method, three well-established model problems are adopted: conformational transitions of hepta-alanine, folding of villin headpiece, and functional dynamics of bovine pancreatic trypsin inhibitor. PMID:27058020

  18. Structure-Function Analysis of the Mcl-1 Protein Identifies a Novel Senescence-regulating Domain.

    PubMed

    Demelash, Abeba; Pfannenstiel, Lukas W; Tannenbaum, Charles S; Li, Xiaoxia; Kalady, Matthew F; DeVecchio, Jennifer; Gastman, Brian R

    2015-09-01

    Unlike other antiapoptotic Bcl-2 family members, Mcl-1 also mediates resistance to cancer therapy by uniquely inhibiting chemotherapy-induced senescence (CIS). In general, Bcl-2 family members regulate apoptosis at the level of the mitochondria through a common prosurvival binding groove. Through mutagenesis, we determined that Mcl-1 can inhibit CIS even in the absence of its apoptotically important mitochondrion-localizing domains. This finding prompted us to generate a series of Mcl-1 deletion mutants from both the N and C termini of the protein, including one that contained a deletion of all of the Bcl-2 homology domains, none of which impacted anti-CIS capabilities. Through subsequent structure-function analyses of Mcl-1, we identified a previously uncharacterized loop domain responsible for the anti-CIS activity of Mcl-1. The importance of the loop domain was confirmed in multiple tumor types, two in vivo models of senescence, and by demonstrating that a peptide mimetic of the loop domain can effectively inhibit the anti-CIS function of Mcl-1. The results from our studies appear to be highly translatable because we discerned an inverse relationship between the expression of Mcl-1 and of various senescence markers in cancerous human tissues. In summary, our findings regarding the unique structural properties of Mcl-1 provide new approaches for targeted cancer therapy. PMID:26205817

  19. Identifying protein interaction subnetworks by a bagging Markov random field-based method

    PubMed Central

    Chen, Li; Xuan, Jianhua; Riggins, Rebecca B.; Wang, Yue; Clarke, Robert

    2013-01-01

    Identification of differentially expressed subnetworks from protein–protein interaction (PPI) networks has become increasingly important to our global understanding of the molecular mechanisms that drive cancer. Several methods have been proposed for PPI subnetwork identification, but the dependency among network member genes is not explicitly considered, leaving many important hub genes largely unidentified. We present a new method, based on a bagging Markov random field (BMRF) framework, to improve subnetwork identification for mechanistic studies of breast cancer. The method follows a maximum a posteriori principle to form a novel network score that explicitly considers pairwise gene interactions in PPI networks, and it searches for subnetworks with maximal network scores. To improve their robustness across data sets, a bagging scheme based on bootstrapping samples is implemented to statistically select high confidence subnetworks. We first compared the BMRF-based method with existing methods on simulation data to demonstrate its improved performance. We then applied our method to breast cancer data to identify PPI subnetworks associated with breast cancer progression and/or tamoxifen resistance. The experimental results show that not only an improved prediction performance can be achieved by the BMRF approach when tested on independent data sets, but biologically meaningful subnetworks can also be revealed that are relevant to breast cancer and tamoxifen resistance. PMID:23161673

  20. A Serial shRNA Screen for Roadblocks to Reprogramming Identifies the Protein Modifier SUMO2.

    PubMed

    Borkent, Marti; Bennett, Brian D; Lackford, Brad; Bar-Nur, Ori; Brumbaugh, Justin; Wang, Li; Du, Ying; Fargo, David C; Apostolou, Effie; Cheloufi, Sihem; Maherali, Nimet; Elledge, Stephen J; Hu, Guang; Hochedlinger, Konrad

    2016-05-10

    The generation of induced pluripotent stem cells (iPSCs) from differentiated cells following forced expression of OCT4, KLF4, SOX2, and C-MYC (OKSM) is slow and inefficient, suggesting that transcription factors have to overcome somatic barriers that resist cell fate change. Here, we performed an unbiased serial shRNA enrichment screen to identify potent repressors of somatic cell reprogramming into iPSCs. This effort uncovered the protein modifier SUMO2 as one of the strongest roadblocks to iPSC formation. Depletion of SUMO2 both enhances and accelerates reprogramming, yielding transgene-independent, chimera-competent iPSCs after as little as 38 hr of OKSM expression. We further show that the SUMO2 pathway acts independently of exogenous C-MYC expression and in parallel with small-molecule enhancers of reprogramming. Importantly, suppression of SUMO2 also promotes the generation of human iPSCs. Together, our results reveal sumoylation as a crucial post-transcriptional mechanism that resists the acquisition of pluripotency from fibroblasts using defined factors. PMID:26947976

  1. A systems biology strategy to identify molecular mechanisms of action and protein indicators of traumatic brain injury.

    PubMed

    Yu, Chenggang; Boutté, Angela; Yu, Xueping; Dutta, Bhaskar; Feala, Jacob D; Schmid, Kara; Dave, Jitendra; Tawa, Gregory J; Wallqvist, Anders; Reifman, Jaques

    2015-02-01

    The multifactorial nature of traumatic brain injury (TBI), especially the complex secondary tissue injury involving intertwined networks of molecular pathways that mediate cellular behavior, has confounded attempts to elucidate the pathology underlying the progression of TBI. Here, systems biology strategies are exploited to identify novel molecular mechanisms and protein indicators of brain injury. To this end, we performed a meta-analysis of four distinct high-throughput gene expression studies involving different animal models of TBI. By using canonical pathways and a large human protein-interaction network as a scaffold, we separately overlaid the gene expression data from each study to identify molecular signatures that were conserved across the different studies. At 24 hr after injury, the significantly activated molecular signatures were nonspecific to TBI, whereas the significantly suppressed molecular signatures were specific to the nervous system. In particular, we identified a suppressed subnetwork consisting of 58 highly interacting, coregulated proteins associated with synaptic function. We selected three proteins from this subnetwork, postsynaptic density protein 95, nitric oxide synthase 1, and disrupted in schizophrenia 1, and hypothesized that their abundance would be significantly reduced after TBI. In a penetrating ballistic-like brain injury rat model of severe TBI, Western blot analysis confirmed our hypothesis. In addition, our analysis recovered 12 previously identified protein biomarkers of TBI. The results suggest that systems biology may provide an efficient, high-yield approach to generate testable hypotheses that can be experimentally validated to identify novel mechanisms of action and molecular indicators of TBI. PMID:25399920

  2. A systems biology strategy to identify molecular mechanisms of action and protein indicators of traumatic brain injury

    PubMed Central

    Yu, Chenggang; Boutté, Angela; Yu, Xueping; Dutta, Bhaskar; Feala, Jacob D; Schmid, Kara; Dave, Jitendra; Tawa, Gregory J; Wallqvist, Anders; Reifman, Jaques

    2015-01-01

    The multifactorial nature of traumatic brain injury (TBI), especially the complex secondary tissue injury involving intertwined networks of molecular pathways that mediate cellular behavior, has confounded attempts to elucidate the pathology underlying the progression of TBI. Here, systems biology strategies are exploited to identify novel molecular mechanisms and protein indicators of brain injury. To this end, we performed a meta-analysis of four distinct high-throughput gene expression studies involving different animal models of TBI. By using canonical pathways and a large human protein-interaction network as a scaffold, we separately overlaid the gene expression data from each study to identify molecular signatures that were conserved across the different studies. At 24 hr after injury, the significantly activated molecular signatures were nonspecific to TBI, whereas the significantly suppressed molecular signatures were specific to the nervous system. In particular, we identified a suppressed subnetwork consisting of 58 highly interacting, coregulated proteins associated with synaptic function. We selected three proteins from this subnetwork, postsynaptic density protein 95, nitric oxide synthase 1, and disrupted in schizophrenia 1, and hypothesized that their abundance would be significantly reduced after TBI. In a penetrating ballistic-like brain injury rat model of severe TBI, Western blot analysis confirmed our hypothesis. In addition, our analysis recovered 12 previously identified protein biomarkers of TBI. The results suggest that systems biology may provide an efficient, high-yield approach to generate testable hypotheses that can be experimentally validated to identify novel mechanisms of action and molecular indicators of TBI. PMID:25399920

  3. A Visual Screen of a Gfp-Fusion Library Identifies a New Type of Nuclear Envelope Membrane Protein

    PubMed Central

    Rolls, Melissa M.; Stein, Pascal A.; Taylor, Stephen S.; Ha, Edward; McKeon, Frank; Rapoport, Tom A.

    1999-01-01

    The nuclear envelope (NE) is a distinct subdomain of the ER, but few membrane components have been described that are specific to it. We performed a visual screen in tissue culture cells to identify proteins targeted to the NE. This approach does not require assumptions about the nature of the association with the NE or the physical separation of NE and ER. We confirmed that screening a library of fusions to the green fluorescent protein can be used to identify proteins targeted to various subcompartments of mammalian cells, including the NE. With this approach, we identified a new NE membrane protein, named nurim. Nurim is a multispanning membrane protein without large hydrophilic domains that is very tightly associated with the nucleus. Unlike the known NE membrane proteins, it is neither associated with nuclear pores, nor targeted like lamin-associated membrane proteins. Thus, nurim is a new type of NE membrane protein that is localized to the NE by a distinct mechanism. PMID:10402458

  4. Differential proteomics analysis of synaptic proteins identifies potential cellular targets and protein mediators of synaptic neuroprotection conferred by the slow Wallerian degeneration (Wlds) gene

    PubMed Central

    Wishart, Thomas M.; Paterson, Janet M.; Short, Duncan M.; Meredith, Sara; Robertson, Kevin A.; Sutherland, Calum; Cousin, Michael A.; Dutia, Mayank B.; Gillingwater, Thomas H.

    2007-01-01

    SUMMARY Non-somatic synaptic and axonal compartments of neurons are primary pathological targets in many neurodegenerative conditions, ranging from Alzheimer's disease through to motor neuron disease. Axons and synapses are protected from degeneration by the slow Wallerian degeneration (Wlds) gene. Significantly, the molecular mechanisms through which this spontaneous genetic mutation delays degeneration remain controversial and the downstream protein targets of Wlds resident in non-somatic compartments remain unknown. Here we have used differential proteomic analysis to identify proteins whose expression levels were significantly altered in isolated synaptic preparations from the striatum of Wlds mice. 8 of the 16 proteins we identified as having modified expression levels in Wlds synapses are known regulators of mitochondrial stability and degeneration (including VDAC1, Aralar1 and mitofilin). Subsequent analyses demonstrated that other key mitochondrial proteins, not identified in our initial screen, are also modified in Wlds synapses. Of the non-mitochondrial proteins identified, several have been implicated in neurodegenerative diseases where synapses and axons are primary pathological targets (including DRP-2 and Rab GDI beta). In addition, we show that downstream protein changes can be identified in pathways corresponding to both Ube4b (including UBE1) and Nmnat1 (including VDAC1 and Aralar1) components of the chimeric Wlds gene, suggesting that full-length Wlds protein is required to elicit maximal changes in synaptic proteins. We conclude that altered mitochondrial responses to degenerative stimuli are likely to play an important role in the neuroprotective Wlds phenotype and that targeting proteins identified in the current study may lead to novel therapies for the treatment of neurodegenerative diseases in humans. PMID:17470424

  5. Complementary cell-based high-throughput screens identify novel modulators of the unfolded protein response.

    PubMed

    Fribley, Andrew M; Cruz, Patricia G; Miller, Justin R; Callaghan, Michael U; Cai, Peter; Narula, Neha; Neubig, Richard R; Showalter, Hollis D; Larsen, Scott D; Kirchhoff, Paul D; Larsen, Martha J; Burr, Douglas A; Schultz, Pamela J; Jacobs, Renju R; Tamayo-Castillo, Giselle; Ron, David; Sherman, David H; Kaufman, Randal J

    2011-09-01

    Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than 2 decades, indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, the authors hypothesized that high-throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small-molecule activators of the apoptotic arm of the UPR to control or kill OSCC. They have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2α/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR subpathways. An 66 K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of prefractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80 µM. A series of citrinin derivatives was isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds, the authors examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, it was found that patulin at 2.5 to 10 µM induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34, and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis

  6. Substrates of the Arabidopsis thaliana protein isoaspartyl methyltransferasel identified using phage display and biopanning

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The role of PROTEIN ISOASPARTYL-METHYLTRANSFERASE (PIMT) in repairing a wide assortment of damaged proteins in a host of organisms has been inferred from the affinity of the enzyme for isoaspartyl residues in a plethora of amino acid contexts. The identification of specific PIMT target proteins in p...

  7. Identifying Human Kinase-Specific Protein Phosphorylation Sites by Integrating Heterogeneous Information from Various Sources

    PubMed Central

    Li, Tingting; Du, Pufeng; Xu, Nanfang

    2010-01-01

    Phosphorylation is an important type of protein post-translational modification. Identification of possible phosphorylation sites of a protein is important for understanding its functions. Unbiased screening for phosphorylation sites by in vitro or in vivo experiments is time consuming and expensive; in silico prediction can provide functional candidates and help narrow down the experimental efforts. Most of the existing prediction algorithms take only the polypeptide sequence around the phosphorylation sites into consideration. However, protein phosphorylation is a very complex biological process in vivo. The polypeptide sequences around the potential sites are not sufficient to determine the phosphorylation status of those residues. In the current work, we integrated various data sources such as protein functional domains, protein subcellular location and protein-protein interactions, along with the polypeptide sequences to predict protein phosphorylation sites. The heterogeneous information significantly boosted the prediction accuracy for some kinase families. To demonstrate potential application of our method, we scanned a set of human proteins and predicted putative phosphorylation sites for Cyclin-dependent kinases, Casein kinase 2, Glycogen synthase kinase 3, Mitogen-activated protein kinases, protein kinase A, and protein kinase C families (avaiable at http://cmbi.bjmu.edu.cn/huphospho). The predicted phosphorylation sites can serve as candidates for further experimental validation. Our strategy may also be applicable for the in silico identification of other post-translational modification substrates. PMID:21085571

  8. Sonochemical effect on size reduction of CaCO3 nanoparticles derived from waste eggshells.

    PubMed

    Hassan, Tarig A; Rangari, Vijay K; Rana, Rohit K; Jeelani, Shaik

    2013-09-01

    A novel combination of mechanochemical and sonochemical techniques was developed to produce high-surface-area, bio-based calcium carbonate (CaCO3) nanoparticles from eggshells. Size reduction of eggshell achieved via mechanochemical and followed by sonochemical method. First, eggshells were cleaned and ground, then ball milled in wet condition using polypropylene glycol for ten hours to produce fine particles. The ball milled eggshell particles were then irradiated with a high intensity ultrasonic horn (Ti-horn, 20 kHz, and 100 W/cm(2)) in the presence of N,N-dimethylformamide (DMF); decahydronaphthalene (Decalin); or tetrahydrofuran (THF). The ultrasonic irradiation times varied from 1 to 5 h. Transmission electron microscopic (TEM) studies showed that the resultant particle shapes and sizes were different from each solvent. The sonochemical effect of DMF is more pronounced and the particles were irregular platelets of ~10 nm. The BET surface area (43.687 m(2)/g) of these nanoparticles is much higher than that of other nanoparticles derived from eggshells. PMID:23473569

  9. Freestanding eggshell membrane-based electrodes for high-performance supercapacitors and oxygen evolution reaction.

    PubMed

    Geng, Jing; Wu, Hao; Al-Enizi, Abdullah M; Elzatahry, Ahmed A; Zheng, Gengfeng

    2015-09-14

    A type of freestanding, light-weight eggshell membrane-based electrode is demonstrated for supercapacitors and for oxygen evolution reaction (OER) catalysis. As a widely available daily waste, eggshell membranes have unique porous three-dimensional grid-like fibrous structures with relatively high surface area and abundant macropores, allowing for effective conjugation of carbon nanotubes and growth of NiCo2O4 nanowire arrays, an effective supercapacitor material and OER catalyst. The three-dimensional fibrous eggshell membrane frameworks with carbon nanotubes offer efficient pathways for charge transport, and the macropores between adjacent fibers are fully accessible for electrolytes and bubble evolution. As a supercapacitor, the eggshell membrane/carbon nanotube/NiCo2O4 electrode shows high specific capacitances at current densities from 1 to 20 A g(-1), with excellent capacitance retention (>90%) at 10 A g(-1) for over 10,000 cycles. When employed as an OER catalyst, this eggshell membrane-based electrode exhibits an OER onset potential of 1.53 V vs. the reversible hydrogen electrode (RHE), and a stable catalytic current density of 20 mA cm(-2) at 1.65 V vs. the RHE. PMID:26247312

  10. Increased collagen accumulation in eggshell membrane after feeding with dietary wood charcoal powder and vinegar.

    PubMed

    Yamauchi, Kohsyo; Manabe, Noboru; Matsumoto, Yoshiki; Yamauchi, Koh-En

    2013-01-01

    Collagen in an eggshell membrane is important for egg preservation, medical burn treatment and manufacturing of cosmetics. Because collagen in the membrane is little, it is a need to improve the accumulation in the membrane to develop these applications. Wood charcoal powder with vinegar (WCV) is a natural substance that improves poultry production. In hen fed with WCV, total collagen in the eggshell membrane increased with an increase in dietary WCV and significantly increased in the 1.0% WCV group (p < 0.05). Scanning and light microscopic images revealed that this group had thicker eggshell membranes and a fine mesh structure composed of finer and more densely distributed fibres than in the control. Eggs from WCV group showed slow Haugh unit decrease during egg storage and the decrease correlated with total collagen in eggshell membrane. In intact chicken, type I and type III collagens were found in different specific locations in the oviduct but not in the membrane. The finding that collagen accumulates in the eggshell membrane under WCV feeding suggests that feeding chicken with WCV will permit long-term storage of eggs in poultry production, and the increased volume of total collagen will facilitate its application in medicine and cosmetics. PMID:23972401

  11. Salmonella enterica isolates from layer farm environments are able to form biofilm on eggshell surfaces.

    PubMed

    Pande, Vivek V; McWhorter, Andrea R; Chousalkar, Kapil K

    2016-08-01

    This study examined the eggshell biofilm forming ability of Salmonella enterica isolates recovered from egg farms. Multicellular behaviour and biofilm production were examined at 22 and 37°C by Congo red morphology and the crystal violet staining assay. The results indicated that the biofilm forming behaviour of Salmonella isolates was dependent on temperature and associated with serovars. Significantly greater biofilm production was observed at 22°C compared with 37°C. The number of viable biofilm cells attached to eggshells after incubation for 48 h at 22°C was significantly influenced by serovar. Scanning electron microscopic examination revealed firm attachment of bacterial cells to the eggshell surface. The relative expression of csgD and adrA gene was significantly higher in eggshell biofilm cells of S. Mbandaka and S. Oranienburg. These findings demonstrate that Salmonella isolates are capable of forming biofilm on the eggshell surface and that this behaviour is influenced by temperature and serovar. PMID:27268931

  12. Inactivation of Salmonella on Eggshells by Chlorine Dioxide Gas.

    PubMed

    Kim, Hyobi; Yum, Bora; Yoon, Sung-Sik; Song, Kyoung-Ju; Kim, Jong-Rak; Myeong, Donghoon; Chang, Byungjoon; Choe, Nong-Hoon

    2016-01-01

    Microbiological contamination of eggs should be prevented in the poultry industry, as poultry is one of the major reservoirs of human Salmonella. ClO2 gas has been reported to be an effective disinfectant in various industry fields, particularly the food industry. The aims of this study were to evaluate the antimicrobial effect of chlorine dioxide gas on two strains of Salmonella inoculated onto eggshells under various experimental conditions including concentrations, contact time, humidity, and percentage organic matter. As a result, it was shown that chlorine dioxide gas under wet conditions was more effective in inactivating Salmonella Enteritidis and Salmonella Gallinarum compared to that under dry conditions independently of the presence of organic matter (yeast extract). Under wet conditions, a greater than 4 log reduction in bacterial populations was achieved after 30 min of exposure to ClO2 each at 20 ppm, 40 ppm, and 80 ppm against S. Enteritidis; 40 ppm and 80 ppm against S. Gallinarum. These results suggest that chlorine dioxide gas is an effective agent for controlling Salmonella, the most prevalent contaminant in the egg industry. PMID:27499670

  13. Thiolated eggshell membranes sorb and speciate inorganic selenium.

    PubMed

    Yang, Ting; Chen, Ming-Li; Hu, Xian-Wei; Wang, Zhao-Wen; Wang, Jian-Hua; Dasgupta, Purnendu K

    2011-01-01

    Eggshell membranes (ESMs) provide a unique, disulfide bond-rich surface. Thioglycolate reduction was used to generate thiol (-SH) groups on the ESM surface by S-S bond cleavage. The thiol-bearing ESMs (TESMs) were characterized by scanning electron microscopy and Raman spectroscopy. The fibrous network structure of the ESM is retained in the TESMs. TESMs adsorb both Se(IV) and Se(VI) but by different mechanisms: Se(VI) is retained reversibly, possibly via ionic interactions, while Se(IV) is reduced to Se(0) and deposited. We thus demonstrate speciation of selenium species, by using samples (a) as such and after prior oxidation to Se(VI), (b) preconcentration on a TESM microcolumn, (c) elution by 0.5 M HNO(3) that only elutes Se(VI) and (d) detection by graphite furnace atomic absorption spectrometry (GFAAS). The Se(IV) amount is determined by difference. For a 1.0 mL sample, the enrichment factor was 17.2, the S/N = 3 detection limit was 0.06 μg L(-1) and the precision was 3.3% at 0.50 μg L(-1). The linear range was 0.25-2.50 μg L(-1). The procedure was validated by analyzing selenium in certified reference materials of human hair (GBW 09101) and rice (GBW 10010). We further demonstrate utility by speciation of inorganic selenium in a series of water samples. PMID:20877887

  14. Inactivation of Salmonella on Eggshells by Chlorine Dioxide Gas

    PubMed Central

    Yum, Bora; Yoon, Sung-Sik; Song, Kyoung-Ju; Kim, Jong-Rak

    2016-01-01

    Microbiological contamination of eggs should be prevented in the poultry industry, as poultry is one of the major reservoirs of human Salmonella. ClO2 gas has been reported to be an effective disinfectant in various industry fields, particularly the food industry. The aims of this study were to evaluate the antimicrobial effect of chlorine dioxide gas on two strains of Salmonella inoculated onto eggshells under various experimental conditions including concentrations, contact time, humidity, and percentage organic matter. As a result, it was shown that chlorine dioxide gas under wet conditions was more effective in inactivating Salmonella Enteritidis and Salmonella Gallinarum compared to that under dry conditions independently of the presence of organic matter (yeast extract). Under wet conditions, a greater than 4 log reduction in bacterial populations was achieved after 30 min of exposure to ClO2 each at 20 ppm, 40 ppm, and 80 ppm against S. Enteritidis; 40 ppm and 80 ppm against S. Gallinarum. These results suggest that chlorine dioxide gas is an effective agent for controlling Salmonella, the most prevalent contaminant in the egg industry. PMID:27499670

  15. Human-chromatin-related protein interactions identify a demethylase complex required for chromosome segregation.

    PubMed

    Marcon, Edyta; Ni, Zuyao; Pu, Shuye; Turinsky, Andrei L; Trimble, Sandra Smiley; Olsen, Jonathan B; Silverman-Gavrila, Rosalind; Silverman-Gavrila, Lorelei; Phanse, Sadhna; Guo, Hongbo; Zhong, Guoqing; Guo, Xinghua; Young, Peter; Bailey, Swneke; Roudeva, Denitza; Zhao, Dorothy; Hewel, Johannes; Li, Joyce; Gräslund, Susanne; Paduch, Marcin; Kossiakoff, Anthony A; Lupien, Mathieu; Emili, Andrew; Wodak, Shoshana J; Greenblatt, Jack

    2014-07-10

    Chromatin regulation is driven by multicomponent protein complexes, which form functional modules. Deciphering the components of these modules and their interactions is central to understanding the molecular pathways these proteins are regulating, their functions, and their relation to both normal development and disease. We describe the use of affinity purifications of tagged human proteins coupled with mass spectrometry to generate a protein-protein interaction map encompassing known and predicted chromatin-related proteins. On the basis of 1,394 successful purifications of 293 proteins, we report a high-confidence (85% precision) network involving 11,464 protein-protein interactions among 1,738 different human proteins, grouped into 164 often overlapping protein complexes with a particular focus on the family of JmjC-containing lysine demethylases, their partners, and their roles in chromatin remodeling. We show that RCCD1 is a partner of histone H3K36 demethylase KDM8 and demonstrate that both are important for cell-cycle-regulated transcriptional repression in centromeric regions and accurate mitotic division. PMID:24981860

  16. Identifying initial molecular targets of PDT: protein and lipid oxidation products

    NASA Astrophysics Data System (ADS)

    Oleinick, Nancy L.; Kim, Junhwan; Rodriguez, Myriam E.; Xue, Liang-yan; Kenney, Malcolm E.; Anderson, Vernon E.

    2009-06-01

    Photodynamic Therapy (PDT) generates singlet oxygen (1O2) which oxidizes biomolecules in the immediate vicinity of its formation. The phthalocyanine photosensitizer Pc 4 localizes to mitochondria and endoplasmic reticulum, and the primary targets of Pc 4-PDT are expected to be lipids and proteins of those membranes. The initial damage then causes apoptosis in cancer cells via the release of cytochrome c (Cyt-c) from mitochondria into the cytosol, followed by the activation of caspases. That damage also triggers the induction of autophagy, an attempt by the cells to eliminate damaged organelles, or when damage is too extensive, to promote cell death. Cyt-c is bound to the cytosolic side of the mitochondrial inner membrane through association with cardiolipin (CL), a phospholipid containing four unsaturated fatty acids and thus easily oxidized by 1O2 or by other oxidizing agents. Increasing evidence suggests that oxidation of CL loosens its association with Cyt-c, and that the peroxidase activity of Cyt-c can oxidize CL. In earlier studies of Cyt-c in homogeneous medium by MALDI-TOF-MS and LC-ESI-MS, we showed that 1O2 generated by Pc 4-PDT oxidized histidine, methionine, tryptophan, and unexpectedly phenylalanine but not tyrosine. Most of the oxidation products were known to be formed by other oxidizing agents, such as hydroxyl radical, superoxide radical anion, and peroxynitrite. However, two products of histidine were unique to 1O2 and may be useful for reporting the action of 1O2 in cells and tissues. These products, as well as CL oxidation products, have now been identified in liposomes and mitochondria after Pc 4-PDT. In mitochondria, the PDT dose-dependent oxidations can be related to specific changes in mitochondrial function, Bcl-2 photodamage, and Cyt-c release. Thus, the role of PDT-generated 1O2 in oxidizing Cyt-c and CL and the interplay between protein and lipid targets may be highly relevant to understanding one mechanism for cell killing by PDT.

  17. LASP1, a Newly Identified Melanocytic Protein with a Possible Role in Melanin Release, but Not in Melanoma Progression

    PubMed Central

    Houben, Roland; Grunewald, Thomas G. P.; Goebeler, Matthias; Butt, Elke

    2015-01-01

    The LIM and SH3 protein 1 (LASP1) is a focal adhesion protein. Its expression is increased in many malignant tumors. However, little is known about the physiological role of the protein. In the present study, we investigated the expression and function of LASP1 in normal skin, melanocytic nevi and malignant melanoma. In normal skin, a distinct LASP1 expression is visible only in the basal epidermal layer while in nevi LASP1 protein is detected in all melanocytes. Melanoma exhibit no increase in LASP1 mRNA compared to normal skin. In melanocytes, the protein is bound to dynamin and mainly localized at late melanosomes along the edges and at the tips of the cell. Knockdown of LASP1 results in increased melanin concentration in the cells. Collectively, we identified LASP1 as a hitherto unknown protein in melanocytes and as novel partner of dynamin in the physiological process of membrane constriction and melanosome vesicle release. PMID:26061439

  18. DNA affinity labeling of adenovirus type 2 upstream promoter sequence-binding factors identifies two distinct proteins

    SciTech Connect

    Safer, B.; Cohen, R.B.; Garfinkel, S.; Thompson, J.A.

    1988-01-01

    A rapid affinity labeling procedure with enhanced specificity was developed to identify DNA-binding proteins. /sup 32/P was first introduced at unique phosphodiester bonds within the DNA recognition sequence. UV light-dependent cross-linking of pyrimidines to amino acid residues in direct contact at the binding site, followed by micrococcal nuclease digestion, resulted in the transfer of /sup 32/P to only those specific protein(s) which recognized the binding sequence. This method was applied to the detection and characterization of proteins that bound to the upstream promoter sequence (-50 to -66) of the human adenovirus type 2 major late promoter. We detected two distinct proteins with molecular weights of 45,000 and 116,000 that interacted with this promoter element. The two proteins differed significantly in their chromatographic and cross-linking behaviors.

  19. Unveiling the hidden function of long non-coding RNA by identifying its major partner-protein.

    PubMed

    Yang, Yongfang; Wen, Liwei; Zhu, Hongliang

    2015-01-01

    Tens of thousands of long non-coding RNAs (lncRNAs) have been discovered in eukarya, but their functions are largely unknown. Fortunately, lncRNA-protein interactions may offer details of how lncRNAs play important roles in various biological processes, thus identifying proteins associated with lncRNA is critical. Here we review progress of molecular archetypes that lncRNAs execute as guides, scaffolds, or decoys for protein, focusing on advantages, shortcomings and applications of various conventional and emerging technologies to probe lncRNAs and protein interactions, including protein-centric biochemistry approaches such as nRIP and CLIP, and RNA-centric biochemistry approaches such as ChIRP, CHART and RAP. Overall, this review provides strategies for probing interactions between lncRNAs and protein. PMID:26500759

  20. Utilization of calcium carbonate particles from eggshell waste as coating pigments for ink-jet printing paper.

    PubMed

    Yoo, Sukjoon; Hsieh, Jeffery S; Zou, Peter; Kokoszka, John

    2009-12-01

    The effective treatment and utilization of biowaste have been emphasized in our society for environmental and economic concerns. Recently, the eggshell waste in the poultry industry has been highlighted because of its reclamation potential. This study presents an economical treatment process to recover useful bioproducts from eggshell waste and their utilization in commercial products. We developed the dissolved air floatation (DAF) separation unit, which successfully recovered 96% of eggshell membrane and 99% of eggshell calcium carbonate (ECC) particles from eggshell waste within 2 h of operation. The recovered ECC particles were utilized as coating pigments for ink-jet printing paper and their impact on the ink density and paper gloss were investigated. The addition of the ECC particles as coating pigments enhances the optical density of cyan, magenta and yellow inks while decreasing the black ink density and the gloss of the coated paper. PMID:19665373

  1. topPTM: a new module of dbPTM for identifying functional post-translational modifications in transmembrane proteins.

    PubMed

    Su, Min-Gang; Huang, Kai-Yao; Lu, Cheng-Tsung; Kao, Hui-Ju; Chang, Ya-Han; Lee, Tzong-Yi

    2014-01-01

    Transmembrane (TM) proteins have crucial roles in various cellular processes. The location of post-translational modifications (PTMs) on TM proteins is associated with their functional roles in various cellular processes. Given the importance of PTMs in the functioning of TM proteins, this study developed topPTM (available online at http://topPTM.cse.yzu.edu.tw), a new dbPTM module that provides a public resource for identifying the functional PTM sites on TM proteins with structural topology. Experimentally verified TM topology data were integrated from TMPad, TOPDB, PDBTM and OPM. In addition to the PTMs obtained from dbPTM, experimentally verified PTM sites were manually extracted from research articles by text mining. In an attempt to provide a full investigation of PTM sites on TM proteins, all UniProtKB protein entries containing annotations related to membrane localization and TM topology were considered potential TM proteins. Two effective tools were then used to annotate the structural topology of the potential TM proteins. The TM topology of TM proteins is represented by graphical visualization, as well as by the PTM sites. To delineate the structural correlation between the PTM sites and TM topologies, the tertiary structure of PTM sites on TM proteins was visualized by Jmol program. Given the support of research articles by manual curation and the investigation of domain-domain interactions in Protein Data Bank, 1347 PTM substrate sites are associated with protein-protein interactions for 773 TM proteins. The database content is regularly updated on publication of new data by continuous surveys of research articles and available resources. PMID:24302577

  2. Two-Dimensional Differential Gel Electrophoresis to Identify Protein Biomarkers in Amniotic Fluid of Edwards Syndrome (Trisomy 18) Pregnancies

    PubMed Central

    Hsu, Te-Yao; Lin, Hao; Hung, Hsuan-Ning; Yang, Kuender D.; Ou, Chia-Yu; Tsai, Ching-Chang; Cheng, Hsin-Hsin; Chung, Su-Hai; Cheng, Bi-Hua; Wong, Yi-Hsun; Chou, An Kuo; Hsiao, Chang-Chun

    2016-01-01

    Background Edwards syndrome (ES) is a severe chromosomal abnormality with a prevalence of about 0.8 in 10,000 infants born alive. The aims of this study were to identify candidate proteins associated with ES pregnancies from amniotic fluid supernatant (AFS) using proteomics, and to explore the role of biological networks in the pathophysiology of ES. Methods AFS from six second trimester pregnancies with ES fetuses and six normal cases were included in this study. Fluorescence-based two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) were used for comparative proteomic analysis. The identified proteins were further validated by Western blotting and the role of biological networks was analyzed. Results Twelve protein spots were differentially expressed by more than 1.5-fold in the AFS of the ES pregnancies. MALDI-TOF/MS identified one up-regulated protein: apolipoprotein A1 (ApoA1), and four under-regulated proteins: vitamin D binding protein (VDBP), alpha-1-antitrypsin (A1AT), insulin-like growth factor-binding protein 1 (IGFBP-1), and transthyretin (TTR). Western blot and densitometric analysis of ApoA1, A1AT, IGFBP-1, and TTR confirmed the alteration of these proteins in the amniotic fluid samples. Biological network analysis revealed that the proteins of the ES AFS were involved mainly in lipid and hormone metabolism, immune response, and cardiovascular disease. Conclusions These five proteins may be involved in the pathogenesis of ES. Further studies are needed to explore. PMID:26752631

  3. Isotopic ordering in eggshells reflects body temperatures and suggests differing thermophysiology in two Cretaceous dinosaurs

    NASA Astrophysics Data System (ADS)

    Eagle, Robert A.; Enriquez, Marcus; Grellet-Tinner, Gerald; Pérez-Huerta, Alberto; Hu, David; Tütken, Thomas; Montanari, Shaena; Loyd, Sean J.; Ramirez, Pedro; Tripati, Aradhna K.; Kohn, Matthew J.; Cerling, Thure E.; Chiappe, Luis M.; Eiler, John M.

    2015-10-01

    Our understanding of the evolutionary transitions leading to the modern endothermic state of birds and mammals is incomplete, partly because tools available to study the thermophysiology of extinct vertebrates are limited. Here we show that clumped isotope analysis of eggshells can be used to determine body temperatures of females during periods of ovulation. Late Cretaceous titanosaurid eggshells yield temperatures similar to large modern endotherms. In contrast, oviraptorid eggshells yield temperatures lower than most modern endotherms but ~6 °C higher than co-occurring abiogenic carbonates, implying that this taxon did not have thermoregulation comparable to modern birds, but was able to elevate its body temperature above environmental temperatures. Therefore, we observe no strong evidence for end-member ectothermy or endothermy in the species examined. Body temperatures for these two species indicate that variable thermoregulation likely existed among the non-avian dinosaurs and that not all dinosaurs had body temperatures in the range of that seen in modern birds.

  4. Heavy metal removal from acid mine drainage by calcined eggshell and microalgae hybrid system.

    PubMed

    Choi, Hee-Jeong; Lee, Seung-Mok

    2015-09-01

    This study investigates the use of calcined eggshells and microalgae for the removal of heavy metals from acid mine drainage (AMD) and the simultaneous enhancement of biomass productivity. The experiment was conducted over a period of 6 days in a hybrid system containing calcined eggshells and the microalgae Chlorella vulgaris. The results show that the biomass productivity increased to ~8.04 times its initial concentration of 0.367 g/L as measured by an optical panel photobioreactor (OPPBR) and had a light transmittance of 95 % at a depth of 305 mm. On the other hand, the simultaneous percent removal of Fe, Cu, Zn, Mn, As, and Cd from the AMD effluent was found to be 99.47 to 100 %. These results indicate that the hybrid system with calcined eggshells and microalgae was highly effective for heavy metal removal in the AMD. PMID:25940497

  5. Biosorption of malachite green by eggshells: mechanism identification and process optimization.

    PubMed

    Podstawczyk, Daria; Witek-Krowiak, Anna; Chojnacka, Katarzyna; Sadowski, Zygmunt

    2014-05-01

    In the present work, eggshells were used to remove a dye (malachite green) from wastewater. The study was focused on identification and describing the binding mechanism of the dye by eggshells in a biosorption process optimized by Response Surface Methodology based on the Box-Behnken Design. The mechanism of biosorption was determined by characterization of the biosorbent before and after biosorption using scanning electron microscopy, X-ray diffraction analysis, the Brunauer-Emmett-Teller isotherm method, Fourier transform infrared spectroscopy. The second-order polynomial equation and 3D response surface plots were used to quantitatively determine the relationships between dependent and independent variables. The obtained results suggested the mechanism of wastewater treatment that included physical adsorption, alkaline fading phenomenon and microprecipitation. The results of the present study showed that waste eggshells have the potential to be used as an inexpensive but effective biosorbent useful in wastewater treatment. PMID:24507580

  6. A novel RNA-binding protein from Triturus carnifex identified by RNA-ligand screening with the newt hammerhead ribozyme.

    PubMed

    Denti, M A; Martínez de Alba, A E; Sägesser, R; Tsagris, M; Tabler, M

    2000-03-01

    The newt hammerhead ribozyme is transcribed from Satellite 2 DNA, which consists of tandemly repeated units of 330 bp. However, different transcripts are synthesized in different tissues. In all somatic tissues and in testes, dimeric and multimeric RNA transcripts are generated which, to some extent, self-cleave into monomers at the hammerhead domain. In ovaries, primarily a distinct monomeric unit is formed by transcription, which retains an intact hammerhead self-cleavage site. The ovarian monomeric RNA associates to form a 12S complex with proteins that are poorly characterised so far. In this work we identified NORA, a protein that binds the ovarian form of the newt ribozyme. We show that the newt ribozyme binds to the Escherichia coli -expressed protein, as well as to a protein of identical size that is found exclusively in newt ovaries. Also NORA mRNA was detectable only in ovary, but in neither somatic tissues nor testes. The tissue-specific expression of NORA is analogous to the ovary-specific transcription of the newt ribozyme. Although NORA was identified by its ability to bind to the newt ribozyme in the presence of a vast excess of carrier RNA, it was able to interact with certain other RNA probes. This novel RNA-binding protein does not contain any motif characteristic for RNA-binding proteins or any other known protein domain, but it shares a striking similarity with a rat resiniferatoxin-binding protein. PMID:10666442

  7. A novel RNA-binding protein from Triturus carnifex identified by RNA-ligand screening with the newt hammerhead ribozyme

    PubMed Central

    Denti, Michela A.; Alba, A. Emilio Martínez de; Sägesser, Rudolf; Tsagris, Mina; Tabler, Martin

    2000-01-01

    The newt hammerhead ribozyme is transcribed from Satellite 2 DNA, which consists of tandemly repeated units of 330 bp. However, different transcripts are synthesized in different tissues. In all somatic tissues and in testes, dimeric and multimeric RNA transcripts are generated which, to some extent, self-cleave into monomers at the hammerhead domain. In ovaries, primarily a distinct monomeric unit is formed by transcription, which retains an intact hammerhead self-cleavage site. The ovarian monomeric RNA associates to form a 12S complex with proteins that are poorly characterised so far. In this work we identified NORA, a protein that binds the ovarian form of the newt ribozyme. We show that the newt ribozyme binds to the Escherichia coli-expressed protein, as well as to a protein of identical size that is found exclusively in newt ovaries. Also NORA mRNA was detectable only in ovary, but in neither somatic tissues nor testes. The tissue-specific expression of NORA is analogous to the ovary-specific transcription of the newt ribozyme. Although NORA was identified by its ability to bind to the newt ribozyme in the presence of a vast excess of carrier RNA, it was able to interact with certain other RNA probes. This novel RNA-binding protein does not contain any motif characteristic for RNA-binding proteins or any other known protein domain, but it shares a striking similarity with a rat resiniferatoxin-binding protein. PMID:10666442

  8. PDB2Graph: A toolbox for identifying critical amino acids map in proteins based on graph theory.

    PubMed

    Niknam, Niloofar; Khakzad, Hamed; Arab, Seyed Shahriar; Naderi-Manesh, Hossein

    2016-05-01

    The integrative and cooperative nature of protein structure involves the assessment of topological and global features of constituent parts. Network concept takes complete advantage of both of these properties in the analysis concomitantly. High compatibility to structural concepts or physicochemical properties in addition to exploiting a remarkable simplification in the system has made network an ideal tool to explore biological systems. There are numerous examples in which different protein structural and functional characteristics have been clarified by the network approach. Here, we present an interactive and user-friendly Matlab-based toolbox, PDB2Graph, devoted to protein structure network construction, visualization, and analysis. Moreover, PDB2Graph is an appropriate tool for identifying critical nodes involved in protein structural robustness and function based on centrality indices. It maps critical amino acids in protein networks and can greatly aid structural biologists in selecting proper amino acid candidates for manipulating protein structures in a more reasonable and rational manner. To introduce the capability and efficiency of PDB2Graph in detail, the structural modification of Calmodulin through allosteric binding of Ca(2+) is considered. In addition, a mutational analysis for three well-identified model proteins including Phage T4 lysozyme, Barnase and Ribonuclease HI, was performed to inspect the influence of mutating important central residues on protein activity. PMID:27043857

  9. Penguin eggshell membranes reflect homogeneity of mercury in the marine food web surrounding the Antarctic Peninsula.

    PubMed

    Brasso, Rebecka L; Polito, Michael J; Lynch, Heather J; Naveen, R; Emslie, Steven D

    2012-11-15

    Remote regions such as the Antarctic have become increasingly important for investigations into far-reaching anthropogenic impacts on the environment, most recently in regard to the global mercury cycle. Spatial patterns of mercury availability in four regions of the Antarctic Peninsula were investigated using three species of sympatrically breeding Pygoscelis penguins as biomonitors. Eggshells with intact membranes from Adélie, Gentoo, and Chinstrap penguins were collected at 24 breeding colonies in the South Orkney Islands, South Shetland Islands, eastern Antarctic Peninsula, and western Antarctic Peninsula during the 2006/2007 austral summer. In addition, we compared eggshell membrane mercury concentrations with eggshell stable isotope values (δ(15)N and δ(13)C) to determine if species-specific trophic or foraging habitat preferences influenced female mercury exposure prior to breeding. With few exceptions, mercury concentrations were found to be fairly homogeneous throughout the Antarctic Peninsula suggesting little spatial variation in the risk of exposure to dietary mercury in this food web. Mercury concentrations in Gentoo and Adélie penguins were similar while Chinstrap penguins tended to have higher eggshell membrane mercury concentrations than their congeners. However, inter and intra-specific differences in eggshell membrane mercury concentration were not related to eggshell δ(15)N or δ(13)C values, a likely result of all three species foraging at similar trophic positions. The lack of regional-scale differences in mercury availability in this marine ecosystem may be a reflection of generally uniform atmospheric deposition and upwelling of regionally homogeneous deep water rather than from geographically distinct point sources. PMID:23069932

  10. Potential ecotoxicological significance of elevated concentrations of strontium in eggshells of passerine birds

    USGS Publications Warehouse

    Mora, Miguel A.; Taylor, Robert J.; Brattin, Bryan L.

    2007-01-01

    We investigated the occurrence and potential ecotoxicological significance of elevated concentrations of strontium (Sr) in eggshells of nine passerine birds from four regions in Arizona. Concentrations of Sr in eggshells ranged from 70 to 1360 µg g−1 dry weight (overall mean  =  684 ± 345 SD µg g−1 dw) for the four regions. 23% of the eggshells had Sr concentrations greater than 1000 µg g−1 dw. To our knowledge, these are among the highest levels of Sr that have been reported in bird eggshells in North America. Of the nine species, Brown-headed Cowbirds (Molothrus ater) had the greatest concentrations of Sr. There was a significant positive correlation between Sr and calcium (Ca), and between barium (Ba) and Ca. Ca, Sr, and Ba interact with each other and can exert similar chemical and pharmacological effects. Mean (n ≥ 3) eggshell∶egg ratios for Sr varied with species and ranged from 6.1∶1 to 40.2∶1; ratios for individual eggs reached 92.7∶1. Mean Sr/Ca values ranged from 1.3 × 10−3 to 3.0 × 10−3 and mean eggshell thickness ranged from 83 ± 6 to 120 ± 9 µm for all species. Eggshell thickness was not significantly correlated with Sr for any species but tended to increase with Sr concentrations. We postulate that high concentrations of Sr in the shell could affect later-stage embryos by possible interference with Ca metabolism and bone growth, resulting in reduced hatching success and potential minor beak deformities.

  11. Nest Bacterial Environment Affects Microbiome of Hoopoe Eggshells, but Not That of the Uropygial Secretion.

    PubMed

    Martínez-García, Ángela; Martín-Vivaldi, Manuel; Rodríguez-Ruano, Sonia M; Peralta-Sánchez, Juan Manuel; Valdivia, Eva; Soler, Juan J

    2016-01-01

    The study of associations between symbiotic bacterial communities of hosts and those of surrounding environments would help to understand how bacterial assemblages are acquired, and how they are transmitted from one to another location (i.e. symbiotic bacteria acquisition by hosts). Hoopoes (Upupa epops) smear their eggshells with uropygial secretion (oily secretion produced in their uropygial gland) that harbors antibiotic producing bacteria. Trying to elucidate a possible role of nest material and cloaca microbiota in determining the bacterial community of the uropygial gland and the eggshells of hoopoes, we characterized bacterial communities of nest material, cloaca, uropygial gland and eggshells by the ARISA fingerprinting. Further, by adding material with scarce bacteria and antimicrobial properties, we manipulated the bacterial community of nest material and thus tested experimentally its effects on the microbiomes of the uropygial secretion and of the eggshells. The experiment did not influence the microbiome of the uropygial secretion of females, but affected the community established on eggshells. This is the first experimental evidence indicating that nest material influences the bacterial community of the eggshells and, therefore, probability of embryo infection. Some of the bacterial strains detected in the secretion were also in the bacterial communities of the nest material and of cloaca, but their occurrence within nests was not associated, which suggests that bacterial environments of nest material and cloaca are not sources of symbiotic bacteria for the gland. These results do not support a role of nest environments of hoopoes as reservoirs of symbiotic bacteria. We discuss possible scenarios explaining bacterial acquisition by hoopoes that should be further explored. PMID:27409772

  12. Clumped isotope paleothermometry of eggshells as an indicator of vertebrate endothermy

    NASA Astrophysics Data System (ADS)

    Canavan, R. R.; Field, D. J.; Therrien, F.; Zelenitsky, D.; Affek, H. P.

    2014-12-01

    Isotopic analyses of the calcite or aragonite shells of aquatic organisms are often used in the study of the environmental conditions in which they grow; however, this approach is less straightforward in the terrestrial realm, where environments may be more heterogeneous. In such terrestrial localities, the bioapatite of vertebrate teeth comprises the typical archival material for isotopic analyses. The calcitic eggshells of birds and other reptiles may provide suitable material for isotopic analyses that are aimed at studying their physiology and ecology. Here we apply a novel thermometer, carbonate clumped isotopes (Δ47), to test for endothermy in extinct non-avian dinosaurs in the context provided by eggs of modern reptiles and birds. These Δ47-derived temperatures should reflect the temperature of shell formation, which in endothermic animals such as birds should represent the mother's internal body temperature. In ectothermic animals, the same is true although their body temperatures are more affected by the external environment and thus Δ47 temperatures could more accurately describe local environmental temperatures during eggshell formation. Fossil eggshells represent appropriate material for reconstructing internal body temperatures of extinct non-avian dinosaurs since they mineralized within the mother's body, and fragments of eggshell are commonly recovered from dinosaur-bearing fossil deposits. The dimensions of these fragments provide sufficient material for the relatively large sample required for clumped isotope analysis (~20mg). Fossil eggshell samples from several taxa of Late Cretaceous non-avian dinosaurs were analyzed using Δ47 paleothermometry. Textural inspection was used as a first test for diagenetic alteration of the original calcite, and histological indicators were used for broad taxonomic identifications. Preliminary results of Δ47-derived body temperature estimates from eggshells are consistent with previous body temperatures

  13. Nest Bacterial Environment Affects Microbiome of Hoopoe Eggshells, but Not That of the Uropygial Secretion

    PubMed Central

    Martínez-García, Ángela; Martín-Vivaldi, Manuel; Rodríguez-Ruano, Sonia M.; Peralta-Sánchez, Juan Manuel; Valdivia, Eva; Soler, Juan J.

    2016-01-01

    The study of associations between symbiotic bacterial communities of hosts and those of surrounding environments would help to understand how bacterial assemblages are acquired, and how they are transmitted from one to another location (i.e. symbiotic bacteria acquisition by hosts). Hoopoes (Upupa epops) smear their eggshells with uropygial secretion (oily secretion produced in their uropygial gland) that harbors antibiotic producing bacteria. Trying to elucidate a possible role of nest material and cloaca microbiota in determining the bacterial community of the uropygial gland and the eggshells of hoopoes, we characterized bacterial communities of nest material, cloaca, uropygial gland and eggshells by the ARISA fingerprinting. Further, by adding material with scarce bacteria and antimicrobial properties, we manipulated the bacterial community of nest material and thus tested experimentally its effects on the microbiomes of the uropygial secretion and of the eggshells. The experiment did not influence the microbiome of the uropygial secretion of females, but affected the community established on eggshells. This is the first experimental evidence indicating that nest material influences the bacterial community of the eggshells and, therefore, probability of embryo infection. Some of the bacterial strains detected in the secretion were also in the bacterial communities of the nest material and of cloaca, but their occurrence within nests was not associated, which suggests that bacterial environments of nest material and cloaca are not sources of symbiotic bacteria for the gland. These results do not support a role of nest environments of hoopoes as reservoirs of symbiotic bacteria. We discuss possible scenarios explaining bacterial acquisition by hoopoes that should be further explored. PMID:27409772

  14. Heavy metal immobilization in soil near abandoned mines using eggshell waste and rapeseed residue.

    PubMed

    Lee, Sang Soo; Lim, Jung Eun; El-Azeem, Samy A M Abd; Choi, Bongsu; Oh, Sang-Eun; Moon, Deok Hyun; Ok, Yong Sik

    2013-03-01

    Heavy metal contamination of agricultural soils has received great concern due to potential risk to human health. Cadmium and Pb are largely released from abandoned or closed mines in Korea, resulting in soil contamination. The objective of this study was to evaluate the effects of eggshell waste in combination with the conventional nitrogen, phosphorous, and potassium fertilizer (also known as NPK fertilizer) or the rapeseed residue on immobilization of Cd and Pb in the rice paddy soil. Cadmium and Pb extractabilities were tested using two methods of (1) the toxicity characteristics leaching procedure (TCLP) and (2) the 0.1 M HCl extraction. With 5 % eggshell addition, the values of soil pH were increased from 6.33 and 6.51 to 8.15 and 8.04 in combination with NPK fertilizer and rapeseed residue, respectively, compared to no eggshell addition. The increase in soil pH may contribute to heavy metal immobilization by altering heavy metals into more stable in soils. Concentrations of TCLP-extracted Cd and Pb were reduced by up to 67.9 and 93.2 % by addition of 5 % eggshell compared to control. For 0.1 M HCl extraction method, the concentration of 0.1 M HCl-Cd in soils treated with NPK fertilizer and rapeseed residue was significantly reduced by up to 34.01 and 46.1 %, respectively, with 5 % eggshell addition compared to control. A decrease in acid phosphatase activity and an increase in alkaline phosphatase activity at high soil pH were also observed. Combined application of eggshell waste and rapeseed residue can be cost-effective and beneficial way to remediate the soil contaminated with heavy metals. PMID:22864756

  15. Mechanism-based Proteomic Screening Identifies Targets of Thioredoxin-like Proteins*

    PubMed Central

    Nakao, Lia S.; Everley, Robert A.; Marino, Stefano M.; Lo, Sze M.; de Souza, Luiz E.; Gygi, Steven P.; Gladyshev, Vadim N.

    2015-01-01

    Thioredoxin (Trx)-fold proteins are protagonists of numerous cellular pathways that are subject to thiol-based redox control. The best characterized regulator of thiols in proteins is Trx1 itself, which together with thioredoxin reductase 1 (TR1) and peroxiredoxins (Prxs) comprises a key redox regulatory system in mammalian cells. However, there are numerous other Trx-like proteins, whose functions and redox interactors are unknown. It is also unclear if the principles of Trx1-based redox control apply to these proteins. Here, we employed a proteomic strategy to four Trx-like proteins containing CXXC motifs, namely Trx1, Rdx12, Trx-like protein 1 (Txnl1) and nucleoredoxin 1 (Nrx1), whose cellular targets were trapped in vivo using mutant Trx-like proteins, under conditions of low endogenous expression of these proteins. Prxs were detected as key redox targets of Trx1, but this approach also supported the detection of TR1, which is the Trx1 reductant, as well as mitochondrial intermembrane proteins AIF and Mia40. In addition, glutathione peroxidase 4 was found to be a Rdx12 redox target. In contrast, no redox targets of Txnl1 and Nrx1 could be detected, suggesting that their CXXC motifs do not engage in mixed disulfides with cellular proteins. For some Trx-like proteins, the method allowed distinguishing redox and non-redox interactions. Parallel, comparative analyses of multiple thiol oxidoreductases revealed differences in the functions of their CXXC motifs, providing important insights into thiol-based redox control of cellular processes. PMID:25561728

  16. Identifying the Proteins that Mediate the Ionizing Radiation Resistance of Deinococcus Radiodurans R1

    SciTech Connect

    Battista, John R

    2010-02-22

    The primary objectives of this proposal was to define the subset of proteins required for the ionizing radiation (IR) resistance of Deinococcus radiodurans R1, characterize the activities of those proteins, and apply what was learned to problems of interest to the Department of Energy.

  17. A predicted protein interactome identifies conserved global networks and disease resistance subnetworks in maize

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An interactome is the genome-wide roadmap of protein-protein interactions that occur within an organism. Interactomes for humans, the fruit fly, and now plants such as Arabidopsis thaliana and Oryza sativa have been generated using high throughput experimental methods. It is possible to use these ...

  18. Machine-learning scoring functions for identifying native poses of ligands docked to known and novel proteins

    PubMed Central

    2015-01-01

    Background Molecular docking is a widely-employed method in structure-based drug design. An essential component of molecular docking programs is a scoring function (SF) that can be used to identify the most stable binding pose of a ligand, when bound to a receptor protein, from among a large set of candidate poses. Despite intense efforts in developing conventional SFs, which are either force-field based, knowledge-based, or empirical, their limited docking power (or ability to successfully identify the correct pose) has been a major impediment to cost-effective drug discovery. Therefore, in this work, we explore a range of novel SFs employing different machine-learning (ML) approaches in conjunction with physicochemical and geometrical features characterizing protein-ligand complexes to predict the native or near-native pose of a ligand docked to a receptor protein's binding site. We assess the docking accuracies of these new ML SFs as well as those of conventional SFs in the context of the 2007 PDBbind benchmark dataset on both diverse and homogeneous (protein-family-specific) test sets. Further, we perform a systematic analysis of the performance of the proposed SFs in identifying native poses of ligands that are docked to novel protein targets. Results and conclusion We find that the best performing ML SF has a success rate of 80% in identifying poses that are within 1 Å root-mean-square deviation from the native poses of 65 different protein families. This is in comparison to a success rate of only 70% achieved by the best conventional SF, ASP, employed in the commercial docking software GOLD. In addition, the proposed ML SFs perform better on novel proteins that they were never trained on before. We also observed steady gains in the performance of these scoring functions as the training set size and number of features were increased by considering more protein-ligand complexes and/or more computationally-generated poses for each complex. PMID:25916860

  19. Eggshell permeability: a standard technique for determining interspecific rates of water vapor conductance.

    PubMed

    Portugal, Steven J; Maurer, Golo; Cassey, Phillip

    2010-01-01

    Typically, eggshell water vapor conductance is measured on whole eggs, freshly collected at the commencement of a study. At times, however, it may not be possible to obtain whole fresh eggs but rather egg fragments or previously blown eggs. Here we evaluate and describe in detail a technique for modern laboratory analysis of eggshell conductance that uses fragments from fresh and museum eggs to determine eggshell water vapor conductance. We used fresh unincubated eggs of domesticated chickens (Gallus gallus domesticus), ducks (Anas platyrhynchos domesticus), and guinea fowl (Numida meleagris) to investigate the reliability, validity, and repeatability of the technique. To assess the suitability of museum samples, museum and freshly collected black-headed gull eggs (Larus ridibundus) were used. Fragments were cut out of the eggshell from the blunt end (B), equator (E), and pointy end (P). Eggshell fragments were glued to the top of a 0.25-mL micro test tube (Eppendorf) filled with 200 μL of distilled water and placed in a desiccator at 25°C. Eppendorfs were weighed three times at 24-h intervals, and mass loss was assumed to be a result of water evaporation. We report the following results: (1) mass loss between weighing sessions was highly repeatable and consistent in all species; (2) the majority of intraspecific variability in eggshell water vapor conductance between different eggs of the same species was explained through the differences in water vapor conductance between the three eggshell parts of the same egg (B, E, and P); (3) the technique was sensitive enough to detect significant differences between the three domestic species; (4) there was no overall significant difference between water vapor conductance of museum and fresh black-headed gull eggs; (5) there was no significant difference in water vapor conductance for egg fragments taken from the same egg both between different trials and within the same trial. We conclude, therefore, that this technique

  20. Comment on "Ecological importance of the thermal emissivity of avian eggshells".

    PubMed

    Jiménez-Muñoz, Juan C; Sobrino, José A

    2012-07-01

    Eggshell emissivity must be known to determine accurately the cooling rate of avian eggs when the parent, after heating by conduction during the incubation, is temporarily absent. We estimate possible values of eggshell emissivities from in-situ measurements and spectral libraries. Emissivity is near to 1 (probably higher than 0.95) and therefore its effect on cooling rate may be negligible, with differences between the temperature of the egg assuming a value of ε=0.95 and that of a blackbody (ε=1) below 0.2 °C. PMID:22484000

  1. Eggshell thinning and contaminant levels in bald eagle eggs from Arizona, 1977 to 1985

    USGS Publications Warehouse

    Grubb, T.G.; Wiemeyer, Stanley N.; Kiff, L.F.

    1990-01-01

    The mean eggshell thickness of 11 eggs of bald eagles (Haliaeetus leuccocephalus) and 21 samples of eggshell fragments (n = 32) collected from 14 nests in Arizona during 1977 to 1985 was 0.539 mm, 8.8% thinner than the pre-1947 (pre-DDT) mean for eggs from southern California and Baja California, Mexico. Eleven eggs analyzed for contaminants contained slightly elevated concentrations of DDE (3.3pmm wet weight) and mercury (0.14 ppm). Productivity of sampled pairs was low but improving. The predictive equations of Wiemeyer et al. (1984) were also verified.

  2. Mem-ADSVM: A two-layer multi-label predictor for identifying multi-functional types of membrane proteins.

    PubMed

    Wan, Shibiao; Mak, Man-Wai; Kung, Sun-Yuan

    2016-06-01

    Identifying membrane proteins and their multi-functional types is an indispensable yet challenging topic in proteomics and bioinformatics. However, most of the existing membrane-protein predictors have the following problems: (1) they do not predict whether a given protein is a membrane protein or not; (2) they are limited to predicting membrane proteins with single-label functional types but ignore those with multi-functional types; and (3) there is still much room for improvement for their performance. To address these problems, this paper proposes a two-layer multi-label predictor, namely Mem-ADSVM, which can identify membrane proteins (Layer I) and their multi-functional types (Layer II). Specifically, given a query protein, its associated gene ontology (GO) information is retrieved by searching a compact GO-term database with its homologous accession number. Subsequently, the GO information is classified by a binary support vector machine (SVM) classifier to determine whether it is a membrane protein or not. If yes, it will be further classified by a multi-label multi-class SVM classifier equipped with an adaptive-decision (AD) scheme to determine to which functional type(s) it belongs. Experimental results show that Mem-ADSVM significantly outperforms state-of-the-art predictors in terms of identifying both membrane proteins and their multi-functional types. This paper also suggests that the two-layer prediction architecture is better than the one-layer for prediction performance. For reader׳s convenience, the Mem-ADSVM server is available online at http://bioinfo.eie.polyu.edu.hk/MemADSVMServer/. PMID:27000774

  3. Self-Organizing Feature Maps Identify Proteins Critical to Learning in a Mouse Model of Down Syndrome

    PubMed Central

    Higuera, Clara; Gardiner, Katheleen J.; Cios, Krzysztof J.

    2015-01-01

    Down syndrome (DS) is a chromosomal abnormality (trisomy of human chromosome 21) associated with intellectual disability and affecting approximately one in 1000 live births worldwide. The overexpression of genes encoded by the extra copy of a normal chromosome in DS is believed to be sufficient to perturb normal pathways and normal responses to stimulation, causing learning and memory deficits. In this work, we have designed a strategy based on the unsupervised clustering method, Self Organizing Maps (SOM), to identify biologically important differences in protein levels in mice exposed to context fear conditioning (CFC). We analyzed expression levels of 77 proteins obtained from normal genotype control mice and from their trisomic littermates (Ts65Dn) both with and without treatment with the drug memantine. Control mice learn successfully while the trisomic mice fail, unless they are first treated with the drug, which rescues their learning ability. The SOM approach identified reduced subsets of proteins predicted to make the most critical contributions to normal learning, to failed learning and rescued learning, and provides a visual representation of the data that allows the user to extract patterns that may underlie novel biological responses to the different kinds of learning and the response to memantine. Results suggest that the application of SOM to new experimental data sets of complex protein profiles can be used to identify common critical protein responses, which in turn may aid in identifying potentially more effective drug targets. PMID:26111164

  4. Self-Organizing Feature Maps Identify Proteins Critical to Learning in a Mouse Model of Down Syndrome.

    PubMed

    Higuera, Clara; Gardiner, Katheleen J; Cios, Krzysztof J

    2015-01-01

    Down syndrome (DS) is a chromosomal abnormality (trisomy of human chromosome 21) associated with intellectual disability and affecting approximately one in 1000 live births worldwide. The overexpression of genes encoded by the extra copy of a normal chromosome in DS is believed to be sufficient to perturb normal pathways and normal responses to stimulation, causing learning and memory deficits. In this work, we have designed a strategy based on the unsupervised clustering method, Self Organizing Maps (SOM), to identify biologically important differences in protein levels in mice exposed to context fear conditioning (CFC). We analyzed expression levels of 77 proteins obtained from normal genotype control mice and from their trisomic littermates (Ts65Dn) both with and without treatment with the drug memantine. Control mice learn successfully while the trisomic mice fail, unless they are first treated with the drug, which rescues their learning ability. The SOM approach identified reduced subsets of proteins predicted to make the most critical contributions to normal learning, to failed learning and rescued learning, and provides a visual representation of the data that allows the user to extract patterns that may underlie novel biological responses to the different kinds of learning and the response to memantine. Results suggest that the application of SOM to new experimental data sets of complex protein profiles can be used to identify common critical protein responses, which in turn may aid in identifying potentially more effective drug targets. PMID:26111164

  5. An integrated approach to identify protein complex based on best neighbour and modularity increment.

    PubMed

    Shen, Xianjun; Zhao, Yanli; Li, Yanan; Yi, Yang; He, Tingting; Yang, Jincai

    2015-01-01

    In order to overcome the limitations of global modularity and the deficiency of local modularity, we propose a hybrid modularity measure Local-Global Quantification (LGQ) which considers global modularity and local modularity together. LGQ adopts a suitable module feature adjustable parameter to control the balance of global detecting capability and local search capability in Protein-Protein Interactions (PPI) Network. Furthermore, we develop a new protein complex mining algorithm called Best Neighbour and Local-Global Quantification (BN-LGQ) which integrates the best neighbour node and modularity increment. BN-LGQ expands the protein complex by fast searching the best neighbour node of the current cluster and by calculating the modularity increment as a metric to determine whether the best neighbour node can join the current cluster. The experimental results show BN-LGQ performs a better accuracy on predicting protein complexes and has a higher match with the reference protein complexes than MCL and MCODE algorithms. Moreover, BN-LGQ can effectively discover protein complexes with better biological significance in the PPI network. PMID:26336669

  6. A Comparative Quantitative Proteomic Study Identifies New Proteins Relevant for Sulfur Oxidation in the Purple Sulfur Bacterium Allochromatium vinosum

    PubMed Central

    Weissgerber, Thomas; Sylvester, Marc; Kröninger, Lena

    2014-01-01

    In the present study, we compared the proteome response of Allochromatium vinosum when growing photoautotrophically in the presence of sulfide, thiosulfate, and elemental sulfur with the proteome response when the organism was growing photoheterotrophically on malate. Applying tandem mass tag analysis as well as two-dimensional (2D) PAGE, we detected 1,955 of the 3,302 predicted proteins by identification of at least two peptides (59.2%) and quantified 1,848 of the identified proteins. Altered relative protein amounts (≥1.5-fold) were observed for 385 proteins, corresponding to 20.8% of the quantified A. vinosum proteome. A significant number of the proteins exhibiting strongly enhanced relative protein levels in the presence of reduced sulfur compounds are well documented essential players during oxidative sulfur metabolism, e.g., the dissimilatory sulfite reductase DsrAB. Changes in protein levels generally matched those observed for the respective relative mRNA levels in a previous study and allowed identification of new genes/proteins participating in oxidative sulfur metabolism. One gene cluster (hyd; Alvin_2036-Alvin_2040) and one hypothetical protein (Alvin_2107) exhibiting strong responses on both the transcriptome and proteome levels were chosen for gene inactivation and phenotypic analyses of the respective mutant strains, which verified the importance of the so-called Isp hydrogenase supercomplex for efficient oxidation of sulfide and a crucial role of Alvin_2107 for the oxidation of sulfur stored in sulfur globules to sulfite. In addition, we analyzed the sulfur globule proteome and identified a new sulfur globule protein (SgpD; Alvin_2515). PMID:24487535

  7. A Novel Feature Extraction Method with Feature Selection to Identify Golgi-Resident Protein Types from Imbalanced Data

    PubMed Central

    Yang, Runtao; Zhang, Chengjin; Gao, Rui; Zhang, Lina

    2016-01-01

    The Golgi Apparatus (GA) is a major collection and dispatch station for numerous proteins destined for secretion, plasma membranes and lysosomes. The dysfunction of GA proteins can result in neurodegenerative diseases. Therefore, accurate identification of protein subGolgi localizations may assist in drug development and understanding the mechanisms of the GA involved in various cellular processes. In this paper, a new computational method is proposed for identifying cis-Golgi proteins from trans-Golgi proteins. Based on the concept of Common Spatial Patterns (CSP), a novel feature extraction technique is developed to extract evolutionary information from protein sequences. To deal with the imbalanced benchmark dataset, the Synthetic Minority Over-sampling Technique (SMOTE) is adopted. A feature selection method called Random Forest-Recursive Feature Elimination (RF-RFE) is employed to search the optimal features from the CSP based features and g-gap dipeptide composition. Based on the optimal features, a Random Forest (RF) module is used to distinguish cis-Golgi proteins from trans-Golgi proteins. Through the jackknife cross-validation, the proposed method achieves a promising performance with a sensitivity of 0.889, a specificity of 0.880, an accuracy of 0.885, and a Matthew’s Correlation Coefficient (MCC) of 0.765, which remarkably outperforms previous methods. Moreover, when tested on a common independent dataset, our method also achieves a significantly improved performance. These results highlight the promising performance of the proposed method to identify Golgi-resident protein types. Furthermore, the CSP based feature extraction method may provide guidelines for protein function predictions. PMID:26861308

  8. A Novel Feature Extraction Method with Feature Selection to Identify Golgi-Resident Protein Types from Imbalanced Data.

    PubMed

    Yang, Runtao; Zhang, Chengjin; Gao, Rui; Zhang, Lina

    2016-01-01

    The Golgi Apparatus (GA) is a major collection and dispatch station for numerous proteins destined for secretion, plasma membranes and lysosomes. The dysfunction of GA proteins can result in neurodegenerative diseases. Therefore, accurate identification of protein subGolgi localizations may assist in drug development and understanding the mechanisms of the GA involved in various cellular processes. In this paper, a new computational method is proposed for identifying cis-Golgi proteins from trans-Golgi proteins. Based on the concept of Common Spatial Patterns (CSP), a novel feature extraction technique is developed to extract evolutionary information from protein sequences. To deal with the imbalanced benchmark dataset, the Synthetic Minority Over-sampling Technique (SMOTE) is adopted. A feature selection method called Random Forest-Recursive Feature Elimination (RF-RFE) is employed to search the optimal features from the CSP based features and g-gap dipeptide composition. Based on the optimal features, a Random Forest (RF) module is used to distinguish cis-Golgi proteins from trans-Golgi proteins. Through the jackknife cross-validation, the proposed method achieves a promising performance with a sensitivity of 0.889, a specificity of 0.880, an accuracy of 0.885, and a Matthew's Correlation Coefficient (MCC) of 0.765, which remarkably outperforms previous methods. Moreover, when tested on a common independent dataset, our method also achieves a significantly improved performance. These results highlight the promising performance of the proposed method to identify Golgi-resident protein types. Furthermore, the CSP based feature extraction method may provide guidelines for protein function predictions. PMID:26861308

  9. Venom gland transcriptomics for identifying, cataloging, and characterizing venom proteins in snakes.

    PubMed

    Brahma, Rajeev Kungur; McCleary, Ryan J R; Kini, R Manjunatha; Doley, Robin

    2015-01-01

    Snake venoms are cocktails of protein toxins that play important roles in capture and digestion of prey. Significant qualitative and quantitative variation in snake venom composition has been observed among and within species. Understanding these variations in protein components is instrumental in interpreting clinical symptoms during human envenomation and in searching for novel venom proteins with potential therapeutic applications. In the last decade, transcriptomic analyses of venom glands have helped in understanding the composition of various snake venoms in great detail. Here we review transcriptomic analysis as a powerful tool for understanding venom profile, variation and evolution. PMID:25448392

  10. Novel Conserved Group A Streptococcal Proteins Identified by the Antigenome Technology as Vaccine Candidates for a Non-M Protein-Based Vaccine ▿

    PubMed Central

    Fritzer, Andrea; Senn, Beatrice M.; Minh, Duc Bui; Hanner, Markus; Gelbmann, Dieter; Noiges, Birgit; Henics, Tamás; Schulze, Kai; Guzman, Carlos A.; Goodacre, John; von Gabain, Alexander; Nagy, Eszter; Meinke, Andreas L.

    2010-01-01

    Group A streptococci (GAS) can cause a wide variety of human infections ranging from asymptomatic colonization to life-threatening invasive diseases. Although antibiotic treatment is very effective, when left untreated, Streptococcus pyogenes infections can lead to poststreptococcal sequelae and severe disease causing significant morbidity and mortality worldwide. To aid the development of a non-M protein-based prophylactic vaccine for the prevention of group A streptococcal infections, we identified novel immunogenic proteins using genomic surface display libraries and human serum antibodies from donors exposed to or infected by S. pyogenes. Vaccine candidate antigens were further selected based on animal protection in murine lethal-sepsis models with intranasal or intravenous challenge with two different M serotype strains. The nine protective antigens identified are highly conserved; eight of them show more than 97% sequence identity in 13 published genomes as well as in approximately 50 clinical isolates tested. Since the functions of the selected vaccine candidates are largely unknown, we generated deletion mutants for three of the protective antigens and observed that deletion of the gene encoding Spy1536 drastically reduced binding of GAS cells to host extracellular matrix proteins, due to reduced surface expression of GAS proteins such as Spy0269 and M protein. The protective, highly conserved antigens identified in this study are promising candidates for the development of an M-type-independent, protein-based vaccine to prevent infection by S. pyogenes. PMID:20624906

  11. An in vivo crosslinking system for identifying mycobacterial protein–protein interactions

    PubMed Central

    Lougheed, Kathryn E.A.; Bennett, Mark H.; Williams, Huw D.

    2014-01-01

    The analysis of protein–protein interactions in Mycobacterium tuberculosis has the potential to shed light on the functions of the large number of predicted open-reading frames annotated as conserved hypothetical proteins. We have developed a formaldehyde crosslinking system to detect in vivo interactions in mycobacteria. Our Gateway-adapted vector system uses three promoter strengths, including constitutive and regulatable versions, for the expression of target proteins with either an N- or C-terminal His–Strep–Strep tag. Tandem affinity purification using the His- and Strep-tags is well-suited to the isolation of protein complexes with a high purity and no detectable background. We have validated this approach using the well-described pyruvate dehydrogenase complex. PMID:25034228

  12. A method to identify and characterize Z-DNA binding proteins using a linear oligodeoxynucleotide

    NASA Technical Reports Server (NTRS)

    Herbert, A. G.; Rich, A.

    1993-01-01

    An oligodeoxynucleotide that readily flips to the Z-DNA conformation in 10mM MgCl2 was produced by using Klenow enzyme to incorporate 5-bromodeoxycytosine and deoxyguanosine into a (dC-dG)22 template. During synthesis the oligomer can be labeled with 32P to high specific activity. The labeled oligodeoxynucleotide can be used in bandshift experiment to detect proteins that bind Z-DNA. This allows the binding specificity of such proteins to be determined with high reliability using unlabeled linear and supercoiled DNA competitors. In addition, because the radioactive oligodeoxynucleotide contains bromine atoms, DNA-protein complexes can be readily crosslinked using UV light. This allows an estimate to be made of the molecular weight of the proteins that bind to the radioactive probe. Both techniques are demonstrated using a goat polyclonal anti-Z-DNA antiserum.

  13. Identify Secretory Protein of Malaria Parasite with Modified Quadratic Discriminant Algorithm and Amino Acid Composition.

    PubMed

    Feng, Yong-E

    2016-06-01

    Malaria parasite secretes various proteins in infected red blood cell for its growth and survival. Thus identification of these secretory proteins is important for developing vaccine or drug against malaria. In this study, the modified method of quadratic discriminant analysis is presented for predicting the secretory proteins. Firstly, 20 amino acids are divided into five types according to the physical and chemical characteristics of amino acids. Then, we used five types of amino acids compositions as inputs of the modified quadratic discriminant algorithm. Finally, the best prediction performance is obtained by using 20 amino acid compositions, the sensitivity of 96 %, the specificity of 92 % with 0.88 of Mathew's correlation coefficient in fivefold cross-validation test. The results are also compared with those of existing prediction methods. The compared results shown our method are prominent in the prediction of secretory proteins. PMID:26286010

  14. A Proteomics Approach to Identify New Putative Cardiac Intercalated Disk Proteins

    PubMed Central

    Soni, Siddarth; Raaijmakers, Antonia J. A.; Raaijmakers, Linsey M.; Damen, J. Mirjam A.; van Stuijvenberg, Leonie; Vos, Marc A.; Heck, Albert J. R.

    2016-01-01

    Aims Synchronous beating of the heart is dependent on the efficient functioning of the cardiac intercalated disk (ID). The ID is composed of a complex protein network enabling electrical continuity and chemical communication between individual cardiomyocytes. Recently, several different studies have shed light on increasingly prevalent cardiac diseases involving the ID. Insufficient knowledge of its composition makes it difficult to study these disease mechanisms in more detail and therefore here we aim expand the ID proteome. Here, using a combination of general membrane enrichment, in-depth quantitative proteomics and an intracellular location driven bioinformatics approach, we aim to discover new putative ID proteins in rat ventricular tissue. Methods and Results General membrane isolation, enriched amongst others also with ID proteins as based on presence of the established markers connexin-43 and n-cadherin, was performed using centrifugation. By mass spectrometry, we quantitatively evaluated the level of 3455 proteins in the enriched membrane fraction (EMF) and its counterpart, the soluble cytoplasmic fraction. These data were stringently filtered to generate a final set of 97 enriched, putative ID proteins. These included Cx43 and n-cadherin, but also many interesting novel candidates. We selected 4 candidates (Flotillin-2 (FLOT2), Nexilin (NEXN), Popeye-domain-containg-protein 2 (POPDC2) and thioredoxin-related-transmembrane-protein 2 (TMX2)) and confirmed their co-localization with n-cadherin in the ID of human and rat heart cryo-sections, and isolated dog cardiomyocytes. Conclusion The presented proteomics dataset of putative new ID proteins is a valuable resource for future research into this important molecular intersection of the heart. PMID:27148881

  15. A survey of PPR proteins identifies DYW domains like those of land plant RNA editing factors in diverse eukaryotes

    PubMed Central

    Schallenberg-Rüdinger, Mareike; Lenz, Henning; Polsakiewicz, Monika; Gott, Jonatha M; Knoop, Volker

    2013-01-01

    The pentatricopeptide repeat modules of PPR proteins are key to their sequence-specific binding to RNAs. Gene families encoding PPR proteins are greatly expanded in land plants where hundreds of them participate in RNA maturation, mainly in mitochondria and chloroplasts. Many plant PPR proteins contain additional carboxyterminal domains and have been identified as essential factors for specific events of C-to-U RNA editing, which is abundant in the two endosymbiotic plant organelles. Among those carboxyterminal domain additions to plant PPR proteins, the so-called DYW domain is particularly interesting given its similarity to cytidine deaminases. The frequency of organelle C-to-U RNA editing and the diversity of DYW-type PPR proteins correlate well in plants and both were recently identified outside of land plants, in the protist Naegleria gruberi. Here we present a systematic survey of PPR protein genes and report on the identification of additional DYW-type PPR proteins in the protists Acanthamoeba castellanii, Malawimonas jakobiformis, and Physarum polycephalum. Moreover, DYW domains were also found in basal branches of multi-cellular lineages outside of land plants, including the alga Nitella flexilis and the rotifers Adineta ricciae and Philodina roseola. Intriguingly, the well-characterized and curious patterns of mitochondrial RNA editing in the slime mold Physarum also include examples of C-to-U changes. Finally, we identify candidate sites for mitochondrial RNA editing in Malawimonas, further supporting a link between DYW-type PPR proteins and C-to-U editing, which may have remained hitherto unnoticed in additional eukaryote lineages. PMID:23899506

  16. A survey of PPR proteins identifies DYW domains like those of land plant RNA editing factors in diverse eukaryotes.

    PubMed

    Schallenberg-Rüdinger, Mareike; Lenz, Henning; Polsakiewicz, Monika; Gott, Jonatha M; Knoop, Volker

    2013-01-01

    The pentatricopeptide repeat modules of PPR proteins are key to their sequence-specific binding to RNAs. Gene families encoding PPR proteins are greatly expanded in land plants where hundreds of them participate in RNA maturation, mainly in mitochondria and chloroplasts. Many plant PPR proteins contain additional carboxyterminal domains and have been identified as essential factors for specific events of C-to-U RNA editing, which is abundant in the two endosymbiotic plant organelles. Among those carboxyterminal domain additions to plant PPR proteins, the so-called DYW domain is particularly interesting given its similarity to cytidine deaminases. The frequency of organelle C-to-U RNA editing and the diversity of DYW-type PPR proteins correlate well in plants and both were recently identified outside of land plants, in the protist Naegleria gruberi. Here we present a systematic survey of PPR protein genes and report on the identification of additional DYW-type PPR proteins in the protists Acanthamoeba castellanii, Malawimonas jakobiformis, and Physarum polycephalum. Moreover, DYW domains were also found in basal branches of multi-cellular lineages outside of land plants, including the alga Nitella flexilis and the rotifers Adineta ricciae and Philodina roseola. Intriguingly, the well-characterized and curious patterns of mitochondrial RNA editing in the slime mold Physarum also include examples of C-to-U changes. Finally, we identify candidate sites for mitochondrial RNA editing in Malawimonas, further supporting a link between DYW-type PPR proteins and C-to-U editing, which may have remained hitherto unnoticed in additional eukaryote lineages. PMID:23899506

  17. Difference gel electrophoresis identifies differentially expressed proteins in endoscopically collected pancreatic fluid.

    PubMed

    Paulo, Joao A; Lee, Linda S; Banks, Peter A; Steen, Hanno; Conwell, Darwin L

    2011-08-01

    Alterations in the pancreatic fluid proteome of individuals with chronic pancreatitis (CP) may offer insights into the development and progression of the disease. The endoscopic pancreatic function test (ePFT) can safely collect large volumes of pancreatic fluid that are potentially amenable to proteomic analyses using difference gel electrophoresis (DIGE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Pancreatic fluid was collected endoscopically using the ePFT method following secretin stimulation from three individuals with severe CP and three chronic abdominal pain (CAP) controls. The fluid was processed to minimize protein degradation and the protein profiles of each cohort, as determined by DIGE and LC-MS/MS, were compared. This DIGE-LC-MS/MS analysis reveals proteins that are differentially expressed in CP compared with CAP controls. Proteins with higher abundance in pancreatic fluid from CP individuals include: actin, desmoplankin, α-1-antitrypsin, SNC73, and serotransferrin. Those of relatively lower abundance include carboxypeptidase B, lipase, α-1-antichymotrypsin, α-2-macroglobulin, actin-related protein (Arp2/3) subunit 4, glyceraldehyde-3-phosphate dehydrogenase, and protein disulfide isomerase. Endoscopic collection (ePFT) in tandem with DIGE-LC-MS/MS is a suitable approach for pancreatic fluid proteome analysis; however, further optimization of our protocol, as outlined herein, may improve proteome coverage in future analyses. PMID:21792986

  18. Systematic Phenotypic Screen of Arabidopsis Peroxisomal Mutants Identifies Proteins Involved in β-Oxidation1[W][OPEN

    PubMed Central

    Cassin-Ross, Gaëlle; Hu, Jianping

    2014-01-01

    Peroxisomes are highly dynamic and multifunctional organelles essential to development. Plant peroxisomes accommodate a multitude of metabolic reactions, many of which are related to the β-oxidation of fatty acids or fatty acid-related metabolites. Recently, several dozens of novel peroxisomal proteins have been identified from Arabidopsis (Arabidopsis thaliana) through in silico and experimental proteomic analyses followed by in vivo protein targeting validations. To determine the functions of these proteins, we interrogated their transfer DNA insertion mutants with a series of physiological, cytological, and biochemical assays to reveal peroxisomal deficiencies. Sugar dependence and 2,4-dichlorophenoxybutyric acid and 12-oxo-phytodienoic acid response assays uncovered statistically significant phenotypes in β-oxidation-related processes in mutants for 20 of 27 genes tested. Additional investigations uncovered a subset of these mutants with abnormal seed germination, accumulation of oil bodies, and delayed degradation of long-chain fatty acids during early seedling development. Mutants for seven genes exhibited deficiencies in multiple assays, strongly suggesting the involvement of their gene products in peroxisomal β-oxidation and initial seedling growth. Proteins identified included isoforms of enzymes related to β-oxidation, such as acyl-CoA thioesterase2, acyl-activating enzyme isoform1, and acyl-activating enzyme isoform5, and proteins with functions previously unknown to be associated with β-oxidation, such as Indigoidine synthase A, Senescence-associated protein/B12D-related protein1, Betaine aldehyde dehydrogenase, and Unknown protein5. This multipronged phenotypic screen allowed us to reveal β-oxidation proteins that have not been discovered by single assay-based mutant screens and enabled the functional dissection of different isoforms of multigene families involved in β-oxidation. PMID:25253886

  19. Structural genomics analysis of uncharacterized protein families overrepresented in human gut bacteria identifies a novel glycoside hydrolase

    PubMed Central

    2014-01-01

    Background Bacteroides spp. form a significant part of our gut microbiome and are well known for optimized metabolism of diverse polysaccharides. Initial analysis of the archetypal Bacteroides thetaiotaomicron genome identified 172 glycosyl hydrolases and a large number of uncharacterized proteins associated with polysaccharide metabolism. Results BT_1012 from Bacteroides thetaiotaomicron VPI-5482 is a protein of unknown function and a member of a large protein family consisting entirely of uncharacterized proteins. Initial sequence analysis predicted that this protein has two domains, one on the N- and one on the C-terminal. A PSI-BLAST search found over 150 full length and over 90 half size homologs consisting only of the N-terminal domain. The experimentally determined three-dimensional structure of the BT_1012 protein confirms its two-domain architecture and structural analysis of both domains suggests their specific functions. The N-terminal domain is a putative catalytic domain with significant similarity to known glycoside hydrolases, the C-terminal domain has a beta-sandwich fold typically found in C-terminal domains of other glycosyl hydrolases, however these domains are typically involved in substrate binding. We describe the structure of the BT_1012 protein and discuss its sequence-structure relationship and their possible functional implications. Conclusions Structural and sequence analyses of the BT_1012 protein identifies it as a glycosyl hydrolase, expanding an already impressive catalog of enzymes involved in polysaccharide metabolism in Bacteroides spp. Based on this we have renamed the Pfam families representing the two domains found in the BT_1012 protein, PF13204 and PF12904, as putative glycoside hydrolase and glycoside hydrolase-associated C-terminal domain respectively. PMID:24742328

  20. Identifying novel protein interactions: Proteomic methods, optimisation approaches and data analysis pipelines.

    PubMed

    Carneiro, Daniel Gonçalves; Clarke, Thomas; Davies, Clare C; Bailey, Dalan

    2016-02-15

    The technological revolution in high-throughput nucleic acid and protein analysis in the last 15 years has launched the field of 'omics' and led to great advances in our understanding of cell biology. Consequently the study of the cellular proteome and protein dynamics, in particular interactomics, has been a matter of intense investigation, specifically the determination and description of complex protein interaction networks in the cell, not only with other proteins but also with RNA and DNA. The analysis of these interactions, beginning with their identification and ultimately resulting in structural level examination, is one of the cornerstones of modern biological science underpinning basic research and impacting on applied biology, biomedicine and drug discovery. In this review we summarise a selection of emerging and established techniques currently being applied in this field with a particular focus on affinity-based purification systems and their optimisation, including tandem affinity purification (TAP) tagging, isolation of proteins on nascent DNA (IPOND) and RNA-protein immunoprecipitation in tandem (RIPiT). The recent application of quantitative proteomics to improve stringency and specificity is also discussed, including the use of metabolic labelling by stable isotope labelling by amino acids in cell culture (SILAC), localization of organelle proteins by isotope tagging (LOPIT) and proximity-dependent biotin identification (BioID). Finally, we describe a range of software resources that can be applied to interactomics, both to handle raw data and also to scrutinise its broader biological context. In this section we focus especially on open-access online interactomic databases such as Reactome and IntAct. PMID:26320829

  1. Biosorption of multifold toxic heavy metal ions from aqueous water onto food residue eggshell membrane functionalized with ammonium thioglycolate.

    PubMed

    Wang, Sha; Wei, Minghong; Huang, Yuming

    2013-05-29

    A new biosorbent material from eggshell membrane was synthesized through thiol functionalization, which is based on the reduction of disulfide bonds in eggshell membrane by ammonium thioglycolate. The thiol-functionalized eggshell membrane was characterized, and its application as an adsorbent for removal of Cr(VI), Hg(II), Cu(II), Pb(II), Cd(II), and Ag(I) from aqueous water has been investigated. The experimental results revealed that the adsorption abilities of the thiol-functionalized eggshell membrane toward Cr(VI), Hg(II), Cu(II), Pb(II), Cd(II), and Ag(I) improved 1.6-, 5.5-, 7.7-, 12.4-, 12.7-, and 21.1-fold, respectively, compared with that of the eggshell membrane control. The adsorption mechanism and adsorption performance, including the adsorption capacity and the kinetics of the thiol-functionalized eggshell membrane for the target heavy metals, were investigated. The effects of solution pH, coexisting substances, and natural water matrices were studied. The thiol-functionalized eggshell membrane can be used as column packing to fabricate a column for real wastewater purification. PMID:23663117

  2. A Tyrosine-Rich Cell Surface Protein in the Diatom Amphora coffeaeformis Identified through Transcriptome Analysis and Genetic Transformation

    PubMed Central

    Buhmann, Matthias T.; Poulsen, Nicole; Klemm, Jennifer; Kennedy, Matthew R.; Sherrill, C. David; Kröger, Nils

    2014-01-01

    Diatoms are single-celled eukaryotic microalgae that are ubiquitously found in almost all aquatic ecosystems, and are characterized by their intricately structured SiO2 (silica)-based cell walls. Diatoms with a benthic life style are capable of attaching to any natural or man-made submerged surface, thus contributing substantially to both microbial biofilm communities and economic losses through biofouling. Surface attachment of diatoms is mediated by a carbohydrate- and protein- based glue, yet no protein involved in diatom underwater adhesion has been identified so far. In the present work, we have generated a normalized transcriptome database from the model adhesion diatom Amphora coffeaeformis. Using an unconventional bioinformatics analysis we have identified five proteins that exhibit unique amino acid sequences resembling the amino acid composition of the tyrosine-rich adhesion proteins from mussel footpads. Establishing the first method for the molecular genetic transformation of A. coffeaeformis has enabled investigations into the function of one of these proteins, AC3362, through expression as YFP fusion protein. Biochemical analysis and imaging by fluorescence microscopy revealed that AC3362 is not involved in adhesion, but rather plays a role in biosynthesis and/or structural stability of the cell wall. The methods established in the present study have paved the way for further molecular studies on the mechanisms of underwater adhesion and biological silica formation in the diatom A. coffeaeformis. PMID:25372470

  3. Proteomic Analysis of Rhizoctonia solani Identifies Infection-specific, Redox Associated Proteins and Insight into Adaptation to Different Plant Hosts.

    PubMed

    Anderson, Jonathan P; Hane, James K; Stoll, Thomas; Pain, Nicholas; Hastie, Marcus L; Kaur, Parwinder; Hoogland, Christine; Gorman, Jeffrey J; Singh, Karam B

    2016-04-01

    Rhizoctonia solaniis an important root infecting pathogen of a range of food staples worldwide including wheat, rice, maize, soybean, potato and others. Conventional resistance breeding strategies are hindered by the absence of tractable genetic resistance in any crop host. Understanding the biology and pathogenicity mechanisms of this fungus is important for addressing these disease issues, however, little is known about howR. solanicauses disease. This study capitalizes on recent genomic studies by applying mass spectrometry based proteomics to identify soluble, membrane-bound and culture filtrate proteins produced under wheat infection and vegetative growth conditions. Many of the proteins found in the culture filtrate had predicted functions relating to modification of the plant cell wall, a major activity required for pathogenesis on the plant host, including a number found only under infection conditions. Other infection related proteins included a high proportion of proteins with redox associated functions and many novel proteins without functional classification. The majority of infection only proteins tested were confirmed to show transcript up-regulation during infection including a thaumatin which increased susceptibility toR. solaniwhen expressed inNicotiana benthamiana In addition, analysis of expression during infection of different plant hosts highlighted how the infection strategy of this broad host range pathogen can be adapted to the particular host being encountered. Data are available via ProteomeXchange with identifier PXD002806. PMID:26811357

  4. Proteomic Analysis of Rhizoctonia solani Identifies Infection-specific, Redox Associated Proteins and Insight into Adaptation to Different Plant Hosts*

    PubMed Central

    Anderson, Jonathan P.; Hane, James K.; Stoll, Thomas; Pain, Nicholas; Hastie, Marcus L.; Kaur, Parwinder; Hoogland, Christine; Gorman, Jeffrey J.; Singh, Karam B.

    2016-01-01

    Rhizoctonia solani is an important root infecting pathogen of a range of food staples worldwide including wheat, rice, maize, soybean, potato and others. Conventional resistance breeding strategies are hindered by the absence of tractable genetic resistance in any crop host. Understanding the biology and pathogenicity mechanisms of this fungus is important for addressing these disease issues, however, little is known about how R. solani causes disease. This study capitalizes on recent genomic studies by applying mass spectrometry based proteomics to identify soluble, membrane-bound and culture filtrate proteins produced under wheat infection and vegetative growth conditions. Many of the proteins found in the culture filtrate had predicted functions relating to modification of the plant cell wall, a major activity required for pathogenesis on the plant host, including a number found only under infection conditions. Other infection related proteins included a high proportion of proteins with redox associated functions and many novel proteins without functional classification. The majority of infection only proteins tested were confirmed to show transcript up-regulation during infection including a thaumatin which increased susceptibility to R. solani when expressed in Nicotiana benthamiana. In addition, analysis of expression during infection of different plant hosts highlighted how the infection strategy of this broad host range pathogen can be adapted to the particular host being encountered. Data are available via ProteomeXchange with identifier PXD002806. PMID:26811357

  5. S100 and annexin proteins identify cell membrane damage as the Achilles heel of metastatic cancer cells

    PubMed Central

    Jaiswal, Jyoti K; Nylandsted, Jesper

    2015-01-01

    Mechanical activity of cells and the stress imposed on them by extracellular environment is a constant source of injury to the plasma membrane (PM). In invasive tumor cells, increased motility together with the harsh environment of the tumor stroma further increases the risk of PM injury. The impact of these stresses on tumor cell plasma membrane and mechanism by which tumor cells repair the PM damage are poorly understood. Ca2+ entry through the injured PM initiates repair of the PM. Depending on the cell type, different organelles and proteins respond to this Ca2+ entry and facilitate repair of the damaged plasma membrane. We recently identified that proteins expressed in various metastatic cancers including Ca2+-binding EF hand protein S100A11 and its binding partner annexin A2 are used by tumor cells for plasma membrane repair (PMR). Here we will discuss the involvement of S100, annexin proteins and their regulation of actin cytoskeleton, leading to PMR. Additionally, we will show that another S100 member – S100A4 accumulates at the injured PM. These findings reveal a new role for the S100 and annexin protein up regulation in metastatic cancers and identify these proteins and PMR as targets for treating metastatic cancers. PMID:25565331

  6. PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70-KILODALTON HEAT SHOCK PROTEIN HSPA2

    EPA Science Inventory

    THE PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM
    IS IDENTIFIED AS THE 70 kDa HEAT SHOCK PROTEIN HSPA2

    * Gabor Huszar1, Kathryn Stone2, David Dix3 and Lynne Vigue1
    1The Sperm Physiology Laboratory, Department of Obstetrics and Gynecology, 2 W.M. Keck Foundatio...

  7. An Integrative Method for Identifying the Over-Annotated Protein-Coding Genes in Microbial Genomes

    PubMed Central

    Yu, Jia-Feng; Xiao, Ke; Jiang, Dong-Ke; Guo, Jing; Wang, Ji-Hua; Sun, Xiao

    2011-01-01

    The falsely annotated protein-coding genes have been deemed one of the major causes accounting for the annotating errors in public databases. Although many filtering approaches have been designed for the over-annotated protein-coding genes, some are questionable due to the resultant increase in false negative. Furthermore, there is no webserver or software specifically devised for the problem of over-annotation. In this study, we propose an integrative algorithm for detecting the over-annotated protein-coding genes in microorganisms. Overall, an average accuracy of 99.94% is achieved over 61 microbial genomes. The extremely high accuracy indicates that the presented algorithm is efficient to differentiate the protein-coding genes from the non-coding open reading frames. Abundant analyses show that the predicting results are reliable and the integrative algorithm is robust and convenient. Our analysis also indicates that the over-annotated protein-coding genes can cause the false positive of horizontal gene transfers detection. The webserver of the proposed algorithm can be freely accessible from www.cbi.seu.edu.cn/RPGM. PMID:21903723

  8. Functional phylogenetic analysis of LGI proteins identifies an interaction motif crucial for myelination.

    PubMed

    Kegel, Linde; Jaegle, Martine; Driegen, Siska; Aunin, Eerik; Leslie, Kris; Fukata, Yuko; Watanabe, Masahiko; Fukata, Masaki; Meijer, Dies

    2014-04-01

    The cellular interactions that drive the formation and maintenance of the insulating myelin sheath around axons are only partially understood. Leucine-rich glioma-inactivated (LGI) proteins play important roles in nervous system development and mutations in their genes have been associated with epilepsy and amyelination. Their function involves interactions with ADAM22 and ADAM23 cell surface receptors, possibly in apposing membranes, thus attenuating cellular interactions. LGI4-ADAM22 interactions are required for axonal sorting and myelination in the developing peripheral nervous system (PNS). Functional analysis revealed that, despite their high homology and affinity for ADAM22, LGI proteins are functionally distinct. To dissect the key residues in LGI proteins required for coordinating axonal sorting and myelination in the developing PNS, we adopted a phylogenetic and computational approach and demonstrate that the mechanism of action of LGI4 depends on a cluster of three amino acids on the outer surface of the LGI4 protein, thus providing a structural basis for the mechanistic differences in LGI protein function in nervous system development and evolution. PMID:24715463

  9. Peptide aptamer identified by molecular docking targeting translationally controlled tumor protein in leukemia cells.

    PubMed

    Kadioglu, Onat; Efferth, Thomas

    2016-08-01

    Bioinformatics screening and molecular docking analyses were utilized to select high affinity peptides targeting translationally controlled tumor protein (TCTP). Selected peptide aptamers were tested towards cancer cell lines with different levels of TCTP expression. One peptide (WGQWPYHC) revealed specific cytotoxicity according to the TCTP expression in tumor cells without affecting normal cells. Western blot analysis showed peptide-induced down-regulation of TCTP as primary target as well as of cell-cycle related downstream proteins (CDK2, CDK6, Cyclin D3) in MOLT-4 leukemia cells. "WGQWPYHC" deserves further analysis for targeted therapy of TCTP-expressing tumor cells. Graphical abstract Molecular docking on TCTP, cytotoxicity toward MOLT-4 leukemia cell line and downregulation of CDK2, CDK6, CyclinD3 and TCTP proteins. PMID:26972431

  10. Results of a screening programme to identify plants or plant extracts that inhibit ruminal protein degradation.

    PubMed

    Selje, N; Hoffmann, E M; Muetzel, S; Ningrat, R; Wallace, R J; Becker, K

    2007-07-01

    One aim of the EC Framework V project, 'Rumen-up' (QLK5-CT-2001-00 992), was to find plants or plant extracts that would inhibit the nutritionally wasteful degradation of protein in the rumen. A total of 500 samples were screened in vitro using 14C-labelled casein in a 30-min incubation with ruminal digesta. Eight were selected for further investigation using a batch fermentation system and soya protein and bovine serum albumin as proteolysis substrates; proteolysis was monitored over 12 h by the disappearance of soluble protein and the production of branched SCFA and NH3. Freeze-dried, ground foliage of Peltiphyllum peltatum, Helianthemum canum, Arbutus unedo, Arctostaphylos uva-ursi and Knautia arvensis inhibited proteolysis (P < 0.05), while Daucus carota, Clematis vitalba and Erica arborea had little effect. Inhibition by the first four samples appeared to be caused by the formation of insoluble tannin-protein complexes. The samples were rich in phenolics and inhibition was reversed by polyethyleneglycol. In contrast, K. arvensis contained low concentrations of phenolics and no tannins, had no effect in the 30-min assay, yet inhibited the degradation rate of soluble protein (by 14 %, P < 0.0001) and the production of branched SCFA (by 17 %, P < 0.05) without precipitating protein in the 12-h batch fermentation. The effects showed some resemblance to those obtained in parallel incubations containing 3 mum-monensin, suggesting that K. arvensis may be a plant-derived feed additive that can suppress growth and activity of key proteolytic ruminal micro-organisms in a manner similar to that already well known for monensin. PMID:17445338

  11. A constitutive nucleolar protein identified as a member of the nucleoplasmin family.

    PubMed Central

    Schmidt-Zachmann, M S; Hügle-Dörr, B; Franke, W W

    1987-01-01

    Using monoclonal antibodies we have localized a polypeptide, appearing on gel electrophoresis with a Mr of approximately 38,000 and a pI of approximately 5.6, to the granular component of the nucleoli of Xenopus laevis oocytes and a broad range of cells from various species. The protein (NO38) also occurs in certain distinct nucleoplasmic particles but is not detected in ribosomes and other cytoplasmic components. During mitosis NO38-containing material dissociates from the nucleolar organizer region and distributes over the chromosomal surfaces and the perichromosomal cytoplasm; in telophase it re-populates the forming nucleoli. With these antibodies we have isolated from a X. laevis ovary lambda gt11 expression library a cDNA clone encoding a polypeptide which, on one- and two-dimensional gel electrophoresis, co-migrates with authentic NO38. The amino acid sequence deduced from this clone defines a polypeptide of 299 amino acids of mol. wt 33,531 which is characterized by the presence of two domains exceptionally rich in aspartic and glutamic acid, one of them flanked by two putative karyophilic signal heptapeptides. Comparison with other protein sequences shows that NO38 is closely related to the histone-binding, karyophilic protein nucleoplasmin: the first 124 amino acids have 58 amino acid positions in common. Protein NO38 also shows striking homologies to the phosphopeptide region of rat nucleolar protein B23 and the carboxyterminal region of human B23. We propose that protein NO38, which forms distinct homo-oligomers of approximately 7S and Mr of approximately 230,000, is a member of a family of karyophilic proteins, the 'nucleoplasmin family'. It is characterized by its specific association with the nucleolus and might be involved in nuclear accumulation, nucleolar storage and pre-rRNA assembly of ribosomal proteins in a manner similar to that discussed for the role of nucleoplasmin in histone storage and chromatin assembly. Images Fig. 1. Fig. 2. Fig. 3

  12. Developmental expression of calcium transport proteins in extraembryonic membranes of oviparous and viviparous Zootoca vivipara (Lacertilia, Lacertidae).

    PubMed

    Stewart, James R; Ecay, Tom W; Heulin, Benoit; Fregoso, Santiago P; Linville, Brent J

    2011-09-15

    The eggshell of oviparous lizards is a significant source of calcium for embryos, whereas the eggshell of viviparous lizards, when present, contains little calcium. In view of the potential cost to embryonic nutrition occasioned by the loss of eggshell calcium, the large number of independent origins of viviparity among lizards is surprising. Concomitant evolution of viviparity and calcium placentotrophy would ameliorate the loss of eggshell calcium, but a mechanism linking these events has yet to be discovered. Zootoca vivipara, a lizard with geographic variation in its mode of parity, is an excellent model for studying mechanisms of calcium transport to oviparous and viviparous embryos because each is highly dependent on calcium secreted by the uterus (eggshell or placenta) and ontogenetic patterns of embryonic calcium mobilization are similar. We compared developmental expression of the calcium transport protein calbindin-D(28K) in yolk splanchnopleure and chorioallantoic membranes of oviparous and viviparous embryos to test the hypothesis that the mechanism of calcium transport does not differ between modes of parity. We found that the ontogenetic pattern of protein expression is similar between reproductive modes and is correlated with calcium uptake from yolk and either eggshell or placenta. Calbindin-D(28K) is localized in the chorionic epithelium of embryos of both reproductive modes. These findings suggest that the embryonic calcium transport machinery is conserved in the transition between reproductive modes and that an adaptation of oviparous embryos for calcium uptake from eggshells functions similarly to transport calcium directly from uterine secretions. PMID:21865511

  13. Diversity in the matrix structure of eggshells in the Testudines (Reptilia).

    PubMed

    Kusuda, Satoshi; Yasukawa, Yuichirou; Shibata, Hiroki; Saito, Tomomi; Doi, Osamu; Ohya, Yutaka; Yoshizaki, Norio

    2013-05-01

    The eggshells of 56 chelonians were examined by electron microscopy and X-ray diffractometry. They were classified into six types in terms of the matrix structure of their calcareous layer; type I was composed of a thin calcareous layer with minerals in an amorphous structure; type II with shell units composed of mammillary cores calcified with aragonite crystals; type III with shell units composed of mammillary cores, plus a single palisade layer also calcified with aragonite crystals, and with each shell unit separated; type IV with shell units the same as type III, but tightly packed together; type V with shell units composed of mammillary cores plus two palisade layers; and type VI with a cuticle layer calcified with calcite crystals over the same structure as that of type V. X-ray diffraction analyses at the outer surface of eggshells showed a gradual change in crystal disposition from the random disposition of type II to the single direction-oriented disposition of type V. The shell height was approximately parallel to the development of the palisade-layer matrix. The limiting membrane of all eggshell types was perforated with canals and that of type I was partially missing. Type I had a parchment shell, types II and III had a pliable shell (some were rigid) and types IV to VI had rigid shells. The present study showed that the hardness of eggshells can be determined by the composition of the shell matrices, as shell matrices are the framework for mineralization. PMID:23646941

  14. Dinosaur eggshell geochemistry as an indicator of Central Asian Cretaceous paleoenvironments

    NASA Astrophysics Data System (ADS)

    Montanari, S.; Higgins, P.; Norell, M.

    2011-12-01

    The Late Cretaceous fossiliferous beds of Mongolia's Gobi Desert have yielded spectacular articulated remains of an extraordinary diversity of fossil mammals, reptiles, birds, and dinosaurs. Paleoenvironmental interpretations of the deposits at these localities have ranged from arid wind-blown dune fields to more mesic, moist environments. Among the diversity of fossils, dinosaur eggshells are commonly found at these localities and dinosaur embryos, although rare, are also encountered. Dinosaur (including modern bird) eggs are constructed of calcite (CaCO3) allowing carbon and oxygen stable isotopes to be quantified to provide information about the environment the egg-laying animals were living in. Here we show that dinosaur eggshell from the Djadokhta Formation at one locality has not been significantly altered and reflects an environment that of dry-climate adapted C3 plants and isolated, ephemeral water sources during the egg-laying season. Carbonate nodules from the same eggshell-bearing layers also independently reflects a similar environmental signal. This study represents the first geochemical analysis of dinosaur remains from the Cretaceous of Mongolia and illustrates the potential of utilizing dinosaur eggshell geochemistry to reconstruct Mesozoic environments.
    Mean, n, standard deviation, and range for both carbon and oxygen isotope values for all materials sampled

  15. Alternative mechanisms of increased eggshell hardness of avian brood parasites relative to host species

    PubMed Central

    Igic, Branislav; Braganza, Kim; Hyland, Margaret M.; Silyn-Roberts, Heather; Cassey, Phillip; Grim, Tomas; Rutila, Jarkko; Moskát, Csaba; Hauber, Mark E.

    2011-01-01

    Obligate brood parasitic birds lay their eggs in nests of other species and parasite eggs typically have evolved greater structural strength relative to host eggs. Increased mechanical strength of the parasite eggshell is an adaptation that can interfere with puncture ejection behaviours of discriminating hosts. We investigated whether hardness of eggshells is related to differences between physical and chemical traits from three different races of the parasitic common cuckoo Cuculus canorus, and their respective hosts. Using tools developed for materials science, we discovered a novel correlate of increased strength of parasite eggs: the common cuckoo's egg exhibits a greater microhardness, especially in the inner region of the shell matrix, relative to its host and sympatric non-host species. We then tested predictions of four potential mechanisms of shell strength: (i) increased relative thickness overall, (ii) greater proportion of the structurally harder shell layers, (iii) higher concentration of inorganic components in the shell matrix, and (iv) elevated deposition of a high density compound, MgCO3, in the shell matrix. We confirmed support only for hypothesis (i). Eggshell characteristics did not differ between parasite eggs sampled from different host nests in distant geographical sites, suggesting an evolutionarily shared microstructural mechanism of stronger parasite eggshells across diverse host-races of brood parasitic cuckoos. PMID:21561966

  16. Chlorinated hydrocarbons and eggshell changes in raptorial and fish-eating birds

    USGS Publications Warehouse

    Hickey, J.J.; Anderson, D.W.

    1968-01-01

    Catastrophic declines of three raptorial species in the United States have been accompanied by decreases in eggshell thickness that began in 1947, have amounted to 19 percent or more, and were identical to phenomena reported in Britain. In 1967, shell thickness in herring gull eggs from five states decreased with increases in chlorinated hydrocarbon residues.

  17. Eggshell surface and deep bacteria recovered from non-sanitized and sanitized broielr hatching eggs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study was conducted to evaluate the superficial and deep eggshell bacteriology of hatching eggs sanitized in a commercial hatchery. Two setting buggies of hatching eggs (5,040/buggy) were sanitized the day prior to placing the buggies into the setter for incubation. Eggs were either spray or ...

  18. Eggshell surface and deep bacteria recovered from non-sanitized and sanitized broiler hatching eggs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study was conducted to evaluate the superficial and deep eggshell bacteriology of hatching eggs sanitized in a commercial hatchery. Two setting buggies of hatching eggs (5,040/buggy) were sanitized the day prior to placing the buggies into the setter for incubation. Eggs were either spray or ...

  19. How important is the eggshell as a source for initial acquisition of Salmonella in hatchling turtles?

    PubMed

    Holgersson, Mikael C N; Nichols, Wade A; Paitz, Ryan T; Bowden, Rachel M

    2016-02-01

    How and when turtles first acquire gut microflora is largely speculative. In this study, the eggshell and hatching process were evaluated for their role in the initial acquisition of Salmonella, by red-eared slider turtles (Trachemys scripta elegans). First, we examined whether the eggshell is a viable substrate for bacterial persistence during incubation, and if internal egg components (i.e., albumen, yolk, and embryo) have detectable bacterial loads. Second, we experimentally manipulated Salmonella by treating eggs with combinations of Salmonella and gentamicin, an effective Gram-negative antibiotic. We found that the eggshell is a viable substrate for maintaining bacteria, as well as an effective barrier to Salmonella transmission as internal egg components were largely bacteria-free. Water samples collected 18 days post-hatch from individuals that were experimentally inoculated with a topical application of Salmonella as eggs had a higher prevalence of Salmonella than those from eggs inoculated with Salmonella but topically treated with gentamicin prior to hatching, control eggs, and eggs only treated with gentamicin, but by day 35 post-hatch there were no detectable differences among the treatment groups. Though it can also act as a barrier that prevents the bacteria from infecting the embryo prior to hatching these findings suggest that the eggshell is a likely source of Salmonella infection in turtle hatchlings. PMID:26817746

  20. Synthesis of three-dimensional calcium carbonate nanofibrous structure from eggshell using femtosecond laser ablation

    PubMed Central

    2011-01-01

    Background Natural biomaterials from bone-like minerals derived from avian eggshells have been considered as promising bone substitutes owing to their biodegradability, abundance, and lower price in comparison with synthetic biomaterials. However, cell adhesion to bulk biomaterials is poor and surface modifications are required to improve biomaterial-cell interaction. Three-dimensional (3D) nanostructures are preferred to act as growth support platforms for bone and stem cells. Although there have been several studies on generating nanoparticles from eggshells, no research has been reported on synthesizing 3D nanofibrous structures. Results In this study, we propose a novel technique to synthesize 3D calcium carbonate interwoven nanofibrous platforms from