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Sample records for identify eggshell proteins

  1. 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.

  2. The major structural proteins of cod (Gadus morhua) eggshells and protein crosslinking during teleost egg hardening.

    PubMed

    Oppen-Berntsen, D O; Helvik, J V; Walther, B T

    1990-02-01

    The highly hydrophobic protein aggregate which constitutes the fish eggshell has for the first time been quantitatively solubilized. This study shows that the nonactivated eggshell from cod is composed primarily of only three protein monomers, designated alpha (74 kDa) beta (54 kDa) and gamma (47 kDa). Protein extraction studies of the eggshells before and after egg activation demonstrate that egg hardening is accompanied by a 10-fold decline in total protein solubility, which is due to nonextraction of the alpha, beta, and gamma chains. When present during the egg activation process monodansylcadaverine (MDC-a fluorescent lysine analog) inhibits eggshell hardening and at the same time becomes covalently incorporated into the eggshell. This MDC incorporation is calcium-dependent and suggests the induction of a perivitelline transglutaminase activity after egg activation. (Transglutaminases catalyze the formation of an amide bond (isopeptide bond) between the gamma-carbonyl group of glutamine and the epsilon-amino group of lysine with release of ammonia. Crosslinks between proteins are generated when the two amino acid residues are located on different proteins.) Protein solubilization studies and NaDodSO4 gel analysis of the eggshell proteins from eggs subjected to 5 mM MDC during egg activation, reveal that when eggshell hardening is blocked by MDC, the three main eggshell proteins remain extractable even after egg activation. Simultaneously we observed a covalent incorporation of MDC into the gamma protein. PMID:2303164

  3. Ovotransferrin is a matrix protein of the hen eggshell membranes and basal calcified layer.

    PubMed

    Gautron, J; Hincke, M T; Panheleux, M; Garcia-Ruiz, J M; Boldicke, T; Nys, Y

    2001-01-01

    The eggshell is an highly ordered structure deposited in the distal oviduct and composed of calcium carbonate and an organic matrix which is believed to influence its fabric. We have identified ovotransferrin as an 80 kDa matrix protein observed at high concentration in the uterine fluid at the initial stage of shell mineralization, by N-terminal sequencing and western blotting using monoclonal and polyclonal antibodies. It is present in extracts from demineralized eggshell and was localized by immunofluorescence in the eggshell membranes and mammillae, which are the sites of calcite nucleation. Northern blotting and RT-PCR demonstrated that ovotransferrin message was expressed in the proximal oviduct (magnum and white isthmus), and at a lower magnitude in the distal oviduct (red isthmus and uterus). Ovotransferrin was revealed by immunofluorescence in the tubular gland cells of the uterus. Calcium carbonate crystals grown in vitro in the presence of purified ovotransferrin showed large modifications of the calcite morphology. These observations and its presence in eggshell and membranes suggest a dual role for ovotransferrin, as a protein influencing nucleation and growth of calcite crystals and as a bacteriostatic filter to reinforce its inhibition of Salmonella growth in egg albumen.

  4. Fabrication of a biocomposite reinforced with hydrophilic eggshell proteins.

    PubMed

    Kim, GeunHyung; Min, Taijin; Park, Su A; Kim, Wan Doo; 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(epsilon-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. PMID:18458482

  5. 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.

  6. 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

  7. 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.

  8. The proteome of the insoluble Schistosoma mansoni eggshell skeleton.

    PubMed

    Dewalick, Saskia; Bexkens, Michiel L; van Balkom, Bas W M; Wu, Ya-Ping; Smit, Cornelis H; Hokke, Cornelis H; de Groot, Philip G; Heck, Albert J R; Tielens, Aloysius G M; van Hellemond, Jaap J

    2011-04-01

    In schistosomiasis, the majority of symptoms of the disease is caused by the eggs that are trapped in the liver. These eggs elicit an immune reaction that leads to the formation of granulomas. The eggshell, which is a rigid insoluble structure built from cross-linked proteins, is the site of direct interaction between the egg and the immune system. However, the exact protein composition of the insoluble eggshell was previously unknown. To identify the proteins of the eggshell of Schistosoma mansoni we performed LC-MS/MS analysis, immunostaining and amino acid analysis on eggshell fragments. For this, eggshell protein skeleton was prepared by thoroughly cleaning eggshells in a four-step stripping procedure of increasing strength including urea and SDS to remove all material that is not covalently linked to the eggshell itself, but is part of the inside of the egg, such as Reynold's layer, von Lichtenberg's envelope and the miracidium. We identified 45 proteins of which the majority are non-structural proteins and non-specific for eggs, but are house-keeping proteins that are present in large quantities in worms and miracidia. Some of these proteins are known to be immunogenic, such as HSP70, GST and enolase. In addition, a number of schistosome-specific proteins with unknown function and no homology to any known annotated protein were found to be incorporated in the eggshell. Schistosome-specific glycoconjugates were also shown to be present on the eggshell protein skeleton. This study also confirmed that the putative eggshell protein p14 contributes largely to the eggshell. Together, these results give new insights into eggshell composition as well as eggshell formation. Those proteins that are present at the site and time of eggshell formation are incorporated in the cross-linked eggshell and this cross-linking does no longer occur when the miracidium starts secreting proteins. PMID:21236260

  9. Structural proteins in the egg-shell of the oriental garden cricket, Gryllus mitratus

    PubMed Central

    Kawasaki, Hiroya; Sato, Hitoshi; Suzuki, Motoko

    1971-01-01

    1. The egg-shell of the oriental garden cricket, Gryllus mitratus, contained at least two different types of structural protein in an approximate ratio of 5:1. The major fraction was extracted in a solvent containing dithiothreitol, EDTA and 8m-urea, and was purified to apparent homogeneity as judged by free-boundary electrophoresis and ultracentrifugation. This was designated SH-fraction and its S-carboxymethyl derivative (CM-fraction) was also prepared. The minor fraction, insoluble in the solvent, was designated insoluble residue. 2. The major fraction was a phosphoprotein, rich in serine (29.8mol% of the total amino acids) and phosphate (nearly equimolar to serine), and O-phosphoserine was identified in its partial acid hydrolysate. The content of cystine was rather low (0.9mol%) in spite of the importance of this amino acid residue in the native form of the protein. The insoluble residue contained only a small amount of phosphorus, and its amino acid composition was clearly different from the major fraction. 3. CM-fraction, a fibrous protein with an average molecular weight of 57500, behaved as a typical polyanion owing to the high content of phosphate. SH-fraction and CM-fraction were precipitable from their aqueous solutions by the addition of bivalent metal cations, and the precipitation of CM-fraction by Ca2+ and Mg2+ was studied in detail. 4. When SH-fraction was exposed to air, intermolecular disulphide linkages were formed, yielding a net-like gel that changed its volume with changes in Ca2+, Mg2+ and Na+. 5. The possible role of this protein fraction in maintaining the integrity of the egg-shell, and a comparison of its composition and properties with other egg-shell proteins and other phosphoproteins, are discussed. ImagesFig. 2.PLATE 1 PMID:5004198

  10. Dating pleistocene archeological sites by protein diagenesis in ostrich eggshell.

    PubMed

    Brooks, A S; Hare, P E; Kokis, J E; Miller, G H; Ernst, R D; Wendorf, F

    1990-04-01

    Eggshells of the African ostrich (Struthio camelus), ubiquitous in archeological sites in Africa, have been shown by laboratory simulation experiments to retain their indigenous organic matrix residues during diagenesis far better than any other calcified tissue yet studied. The rate of L-isoleucine epimerization to D-alloisoleucine follows reversible first-order kinetics and has been calibrated for local temperature effects and used to estimate the age range of stratified archeological sites. Age estimates are consistent with radiocarbon dates from several stratified archeological sites. With adequate calibration, this technique can provide accurate ages to within 10 to 15 percent for strata deposited within the last 200,000 years in the tropics and the last 1,000,000 years in colder regions such as China.

  11. Ovocalyxin-36 Is a Pattern Recognition Protein in Chicken Eggshell Membranes

    PubMed Central

    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

  12. 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.

  13. 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

  14. Integrated proteomic and transcriptomic analysis of the Aedes aegypti eggshell

    PubMed Central

    2014-01-01

    Background Mosquito eggshells show remarkable diversity in physical properties and structure consistent with adaptations to the wide variety of environments exploited by these insects. We applied proteomic, transcriptomic, and hybridization in situ techniques to identify gene products and pathways that participate in the assembly of the Aedes aegypti eggshell. Aedes aegypti population density is low during cold and dry seasons and increases immediately after rainfall. The survival of embryos through unfavorable periods is a key factor in the persistence of their populations. The work described here supports integrated vector control approaches that target eggshell formation and result in Ae. aegypti drought-intolerant phenotypes for public health initiatives directed to reduce mosquito-borne diseases. Results A total of 130 proteins were identified from the combined mass spectrometric analyses of eggshell preparations. Conclusions Classification of proteins according to their known and putative functions revealed the complexity of the eggshell structure. Three novel Ae. aegypti vitelline membrane proteins were discovered. Odorant-binding and cysteine-rich proteins that may be structural components of the eggshell were identified. Enzymes with peroxidase, laccase and phenoloxidase activities also were identified, and their likely involvements in cross-linking reactions that stabilize the eggshell structure are discussed. PMID:24707823

  15. Structure of synthetic peptide analogues of an eggshell protein of Schistosoma mansoni.

    PubMed Central

    Middaugh, C. R.; Thomson, J. A.; Burke, C. J.; Mach, H.; Naylor, A. M.; Bogusky, M. J.; Ryan, J. A.; Pitzenberger, S. M.; Ji, H.; Cordingley, J. S.

    1993-01-01

    The peptide (Gly-L-Tyr-L-Asp-L-Lys-L-Tyr)6, referred to as F4-6, was synthesized as a model for a schistosome eggshell protein in which the Gly-Tyr-Asp-Lys-Tyr consensus sequence is repeated over 40 times. Analysis by CD, Fourier transform infrared spectroscopy, potentiometric and spectrophotomertric titrations, NMR, and molecular modeling suggests that F4-6 forms some type of left-handed structure. A likely possibility appears to be a left-handed alpha-helix stabilized by Lysi-Aspi +4 salt bridges and possibly Aspi-Tyri +4 hydrogen bonding and Tyr-Tyr interactions. Spectroscopic studies of a number of F4-6 analogues support this conclusion. For example, substitution of D-Ala for Gly produces a peptide with enhanced left-handed helical spectral characteristics, whereas an L-Ala substitution results in a peptide with minimal structure. These studies suggest that the F4 protein from Schistosoma mansoni may be the first example of a naturally occurring protein devoid of proline and carbohydrate that forms a left-handed helix composed of L-amino acids, although alternative forms of other left-handed structures have yet to be rigorously excluded. PMID:8318895

  16. 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

  17. 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

  18. Eggshell and egg yolk proteins in fish: hepatic proteins for the next generation: oogenetic, population, and evolutionary implications of endocrine disruption

    PubMed Central

    Arukwe, Augustine; Goksøyr, Anders

    2003-01-01

    The oocyte is the starting point for a new generation. Most of the machinery for DNA and protein synthesis needed for the developing embryo is made autonomously by the fertilized oocyte. However, in fish and in many other oviparous vertebrates, the major constituents of the egg, i.e. yolk and eggshell proteins, are synthesized in the liver and transported to the oocyte for uptake. Vitellogenesis, the process of yolk protein (vitellogenin) synthesis, transport, and uptake into the oocyte, and zonagenesis, the synthesis of eggshell zona radiata proteins, their transport and deposition by the maturing oocyte, are important aspects of oogenesis. The many molecular events involved in these processes require tight, coordinated regulation that is under strict endocrine control, with the female sex steroid hormone estradiol-17β in a central role. The ability of many synthetic chemical compounds to mimic this estrogen can lead to unscheduled hepatic synthesis of vitellogenin and zona radiata proteins, with potentially detrimental effects to the adult, the egg, the developing embryo and, hence, to the recruitment to the fish population. This has led to the development of specific and sensitive assays for these proteins in fish, and the application of vitellogenin and zona radiata proteins as informative biomarkers for endocrine disrupting effects of chemicals and effluents using fish as test organisms. The genes encoding these important reproductive proteins are conserved in the animal kingdom and are products of several hundred million years of evolution. PMID:12685931

  19. Recent patents on eggshell: shell and membrane applications.

    PubMed

    Cordeiro, Cristianne M M; Hincke, Maxwell T

    2011-01-01

    The chicken eggshell and its membranes are an inexpensive and abundant waste material which exhibit interesting characteristics for many potential applications. The eggshell is formed mainly of calcium carbonate (CaCO3) and is used widely as an animal feed, lime (Ca(OH)2) substitute or a fertilizer. Moreover, the associated eggshell membranes have a high content of bioactive components, as well as properties of moisture retention and biodegradability which have potential use for clinical, cosmetic, nutraceutical and nanotechnology applications. The eggshell membranes have been also used for biosorption of heavy metals and dyes and as a template to synthesize metal nanoparticles. The combination of nanosized calcium phosphate (Ca3(PO4)2) biomaterials synthesized from eggshell and eggshell membrane show promise to develop drug delivery system and nanowires for electronic devices. In addition, a derived product, the soluble eggshell membrane protein (SEP) has applications in tissue engineering. This review discusses the patented applications of eggshell membrane waste: shell and membrane for the last 10 years as well as their future applications. PMID:21114472

  20. 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.

  1. 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.

  2. A long-term increase in eggshell thickness of Greenlandic Peregrine Falcons Falco peregrinus tundrius.

    PubMed

    Falk, Knud; Møller, Søren; Mattox, William G

    2006-02-15

    Thickness of eggshell fragments and whole eggs from the Peregrine Falcon Falco peregrinus collected in South and West Greenland between 1972 and 2003 was measured and compared to shell thickness of pre-DDT eggs, also collected in Greenland. Linear regression yields a significant increase in the average thickness of eggshells over the period of 0.19% per year, corresponding to a change in eggshell thinning from 13.9% in 1972 to 7.8% in 2003. Backwards extrapolation of the data, suggests that the Greenlandic Peregrine population probably was never critically affected by DDT-induced eggshell thinning. By sampling eggshell fragments in many nests the spatial and temporal sample distribution was enlarged, allowing the detection of a significant long-term decrease in pollutant-induced eggshell thinning--a trend that could not have been identified if only the rarer whole, addled eggs had been sampled.

  3. Mapping of quantitative trait loci affecting eggshell quality on chromosome 9 in an F(2) intercross between two chicken lines divergently selected for eggshell strength.

    PubMed

    Takahashi, H; Yang, D; Sasaki, O; Furukawa, T; Nirasawa, K

    2009-10-01

    Broken and cracked eggshells are major causes of significant economic losses to the egg production industry. The quantitative trait loci (QTL) on chromosome 9 influencing the quality of eggshells were identified by analysing an intercross between two parent lines developed from the same founder population by a two-way selection for eggshell strength with non-destructive deformation conducted over 14 generations. Chromosome-wide highly significant (P < 0.01) QTL associated with egg weight (EW), short length of egg (SLE), long length of egg (LLE) and eggshell weight were mapped to the distal region of chromosome 9. Among the QTL affecting EW, SLE and LLE, ovocalyxin-32 was identified as a potential candidate gene influencing eggshell traits. Marker-assisted selection based on these QTL could be used to develop strategies for reducing the breakage and cracking of eggs in commercial layer houses. PMID:19780721

  4. Chemical Proteomic Platform To Identify Citrullinated Proteins

    PubMed Central

    2015-01-01

    Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA) and are routinely used for disease diagnosis. Protein citrullination is also increased in cancer and other autoimmune disorders, suggesting that citrullinated proteins may serve as biomarkers for diseases beyond RA. To identify these citrullinated proteins, we developed biotin-conjugated phenylglyoxal (biotin-PG). Using this probe and our platform technology, we identified >50 intracellular citrullinated proteins. More than 20 of these are involved in RNA splicing, suggesting, for the first time, that citrullination modulates RNA biology. Overall, this chemical proteomic platform will play a key role in furthering our understanding of protein citrullination in rheumatoid arthritis and potentially a wider spectrum of inflammatory diseases. PMID:26360112

  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. Ontology integration to identify protein complex in protein interaction networks

    PubMed Central

    2011-01-01

    Background Protein complexes can be identified from the protein interaction networks derived from experimental data sets. However, these analyses are challenging because of the presence of unreliable interactions and the complex connectivity of the network. The integration of protein-protein interactions with the data from other sources can be leveraged for improving the effectiveness of protein complexes detection algorithms. Methods We have developed novel semantic similarity method, which use Gene Ontology (GO) annotations to measure the reliability of protein-protein interactions. The protein interaction networks can be converted into a weighted graph representation by assigning the reliability values to each interaction as a weight. Following the approach of that of the previously proposed clustering algorithm IPCA which expands clusters starting from seeded vertices, we present a clustering algorithm OIIP based on the new weighted Protein-Protein interaction networks for identifying protein complexes. Results The algorithm OIIP is applied to the protein interaction network of Sacchromyces cerevisiae and identifies many well known complexes. Experimental results show that the algorithm OIIP has higher F-measure and accuracy compared to other competing approaches. PMID:22165991

  8. Elemental analysis of the eggshell of the Karner Blue butterfly ( Lycaeides melissa samuelis) using a nuclear microprobe

    NASA Astrophysics Data System (ADS)

    Nickles, Elizabeth P.; Bakhru, Hassaram; Ghiradella, Helen T.; Haberl, Arthur

    1995-05-01

    The University at Albany nuclear microprobe was used to determine the location of trace elements in the eggshell of the Karner Blue butterfly ( Lycaeides melissa samuelis). Both PIXE and RBS spectra were used to identify the elements present in various areas of the eggshell. Maps showing the distribution of specific elements were compared with micrographs of the eggshell taken with a scanning electron microscope (SEM). The location of several of the trace elements may have structural and/or functional significance. The SEM was also used to assess structural damage to the eggshell following microbeam irradiation.

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. Association between ovocalyxin-32 gene haplotypes and eggshell quality traits in an F2 intercross between two chicken lines divergently selected for eggshell strength.

    PubMed

    Takahashi, H; Sasaki, O; Nirasawa, K; Furukawa, T

    2010-10-01

    Broken and cracked eggshells contribute significantly to economic losses in the egg production industry. We previously identified ovocalyxin-32 as a potential gene influencing eggshell traits, by analysing an intercross between two parent lines developed from the same founder population by a two-way selection for eggshell strength with non-destructive deformation (DEF) conducted over 14 generations. We determined the nucleotide sequences of six ovocalyxin-32 exons in the parent individuals and analysed the association between ovocalyxin-32 and eggshell traits in the F2 individuals. We identified three haplotypes (W, M and S) of ovocalyxin-32 in the parent individuals. A mismatch amplification mutation assay was performed to distinguish six diplotype individuals (WW, MM, SS, WM, MS and WS) inthe F2 population. The egg weight (EW) of SS-diplotype individuals was significantly higher than that of WW-, WM- and WS-diplotypes. Short length of the egg (SLE) of SS-diplotype individuals was significantly higher than that of WW-, WM- and MS-diplotypes. Long length of the egg (LLE) of SS-diplotype individuals was significantly higher than that of WM and WS-diplotypes. DEF of WW-diplotype individuals was significantly higher than that ofSS-, WM, MS and WM-diplotypes. Haplotypic effect analyses showed significant differences between the W-haplotype and the S-haplotypes in the EW, SLE, LLE and DEF. The DEF of M-haplotype was significantly lower than that of W- and S-haplotypes. These results suggest that S- and M-haplotypes are critical for high quality of eggshells in the F2 population. In conclusion, ovocalyxin-32 is a useful marker of eggshell traits and can be used to develop strategies for improving eggshell traits in commercial layer houses. PMID:20331600

  15. 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

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

    PubMed Central

    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.

    2011-01-01

    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

  17. 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

  18. The Drosophila BMPRII, wishful thinking, is required for eggshell patterning.

    PubMed

    Marmion, Robert A; Jevtic, Milica; Springhorn, Alexander; Pyrowolakis, George; Yakoby, Nir

    2013-03-01

    The Drosophila eggshell is an elaborate structure that is derived from a monolayer of follicular epithelium surrounding the developing oocyte within the female ovary. The bone morphogenetic protein (BMP) signaling pathway is essential for controlling the patterning and morphogenesis of the eggshell. During oogenesis, the roles of patterning and morphogenesis by the BMP type I receptor thickveins (tkv) have been studied extensively. However, signaling through this pathway requires both type I and II receptors, and the latter has yet to be established in oogenesis. We focus on wishful thinking (wit), the Drosophila homolog to the mammalian BMP type II receptor, BMPRII. We found that wit is expressed dynamically in the FCs of D. melanogaster in an evolutionary conserved pattern. The expression patterns are highly correlated with the dynamics of the BMP signaling, which is consistent with our finding that wit is a target of BMP signaling. Furthermore, we established that WIT is necessary for BMP signaling, and loss of WIT is associated with cell autonomous loss of BMP responses. Of importance, we demonstrated that perturbations in WIT led to changes in eggshell morphologies in domains that are patterned by BMP signaling. Previous studies have shown a role for WIT in BMP signaling during neurogenesis; however, our results reveal a role for WIT in epithelial cells' development.

  19. 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.

  20. 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

  1. Interaction Trap/Two-Hybrid System to Identify Interacting Proteins

    PubMed Central

    Golemis, Erica A.; Serebriiskii, Ilya; Finley, Russell L.; Kolonin, Mikhail G.; Gyuris, Jeno; Brent, Roger

    2014-01-01

    The yeast two-hybrid method (or interaction trap) is a powerful technique for detecting protein interactions. The procedure is performed using transcriptional activation of a dual reporter system in yeast to identify interactions between a protein of interest (the bait protein) and the candidate proteins for interaction. The method can be used to screen a protein library for interactions with a bait protein or to test for association between proteins that are expected to interact based on prior evidence. Interaction mating facilitates the screening of a library with multiple bait proteins. PMID:18228339

  2. 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.

  3. 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.

  4. 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...

  5. 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

  6. 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.

  7. Eggshell conductance--Fick's or Stefan's law?

    PubMed

    Simkiss, K

    1986-08-01

    The diffusion of gases through the avian eggshell has usually been interpreted as an example of Fick's law, and this appears to be the best explanation for gas exchange through narrow long pores. Many eggshells, however, contain more elaborate pores, of which the funnel-shaped type is the most common. It is suggested that most of the resistance to diffusion through these types of pores is best explained by Stefan's law of diffusion through apertures. The implications of this theory to the understanding of pore functions are discussed.

  8. 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

  9. 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.

  10. 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

  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

    2015-05-11

    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.

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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.

  18. Improvements in the Protein Identifier Cross-Reference service.

    PubMed

    Wein, Samuel P; Côté, Richard G; Dumousseau, Marine; Reisinger, Florian; Hermjakob, Henning; Vizcaíno, Juan A

    2012-07-01

    The Protein Identifier Cross-Reference (PICR) service is a tool that allows users to map protein identifiers, protein sequences and gene identifiers across over 100 different source databases. PICR takes input through an interactive website as well as Representational State Transfer (REST) and Simple Object Access Protocol (SOAP) services. It returns the results as HTML pages, XLS and CSV files. It has been in production since 2007 and has been recently enhanced to add new functionality and increase the number of databases it covers. Protein subsequences can be Basic Local Alignment Search Tool (BLAST) against the UniProt Knowledgebase (UniProtKB) to provide an entry point to the standard PICR mapping algorithm. In addition, gene identifiers from UniProtKB and Ensembl can now be submitted as input or mapped to as output from PICR. We have also implemented a 'best-guess' mapping algorithm for UniProt. In this article, we describe the usefulness of PICR, how these changes have been implemented, and the corresponding additions to the web services. Finally, we explain that the number of source databases covered by PICR has increased from the initial 73 to the current 102. New resources include several new species-specific Ensembl databases as well as the Ensembl Genome ones. PICR can be accessed at http://www.ebi.ac.uk/Tools/picr/. PMID:22544604

  19. Identifying the singleplex and multiplex proteins based on transductive learning for protein subcellular localization prediction.

    PubMed

    Cao, Junzhe; Liu, Wenqi; He, Jianjun; Gu, Hong

    2013-07-01

    A new method is proposed to identify whether a query protein is singleplex or multiplex for improving the quality of protein subcellular localization prediction. Based on the transductive learning technique, this approach utilizes the information from the both query proteins and known proteins to estimate the subcellular location number of every query protein so that the singleplex and multiplex proteins can be recognized and distinguished. Each query protein is then dealt with by a targeted single-label or multi-label predictor to achieve a high-accuracy prediction result. We assess the performance of the proposed approach by applying it to three groups of protein sequences datasets. Simulation experiments show that the proposed approach can effectively identify the singleplex and multiplex proteins. Through a comparison, the reliably of this method for enhancing the power of predicting protein subcellular localization can also be verified.

  20. 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

  1. Using ensemble classifier to identify membrane protein types.

    PubMed

    Shen, H-B; Chou, K-C

    2007-01-01

    Predicting membrane protein type is both an important and challenging topic in current molecular and cellular biology. This is because knowledge of membrane protein type often provides useful clues for determining, or sheds light upon, the function of an uncharacterized membrane protein. With the explosion of newly-found protein sequences in the post-genomic era, it is in a great demand to develop a computational method for fast and reliably identifying the types of membrane proteins according to their primary sequences. In this paper, a novel classifier, the so-called "ensemble classifier", was introduced. It is formed by fusing a set of nearest neighbor (NN) classifiers, each of which is defined in a different pseudo amino acid composition space. The type for a query protein is determined by the outcome of voting among these constituent individual classifiers. It was demonstrated through the self-consistency test, jackknife test, and independent dataset test that the ensemble classifier outperformed other existing classifiers widely used in biological literatures. It is anticipated that the idea of ensemble classifier can also be used to improve the prediction quality in classifying other attributes of proteins according to their sequences.

  2. 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.

  3. 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.

  4. Monitoring of eggshell breakage and eggshell strength in different production chains of consumption eggs.

    PubMed

    Mertens, K; Bamelis, F; Kemps, B; Kamers, B; Verhoelst, E; De Ketelaere, B; Bain, M; Decuypere, E; De Baerdemaeker, J

    2006-09-01

    We first tried to monitor the critical points for eggshell breakage in different logistic chains. Second, we examined whether there was a difference in eggshell strength among eggs produced in different housing systems. Finally, we developed a model to investigate the relation between eggshell strength and the likelihood of an egg cracking during handling and grading. Four logistic chains with different housing systems (battery cages, furnished cages, aviary, and free-range), all housing Bovans Goldline chickens in their mid-lay (45 wk), were compared. In every chain, a randomized set of 1,500 eggs was sampled, and the strength was defined. At every critical point in every logistic chain, the eggs were reexamined for breakage. The classic and furnished cage systems showed the highest percentage of breakage directly at point of lay (6.73 and 10.72%), whereas the other systems showed lower breakage (1.94% in the aviary and 1.99% in the free-range system). Further, in the logistic chain, grading and packing of the eggs generated the second highest percentage of breakage (from 1.50 to 2.65%). Breakage due to transportation ranged from 0.16 to 2.65%. There was a significant difference among the eggshell strength (shell stiffness and damping ratio) of eggs from chickens in different housing systems, showing eggs from chickens in the aviary system to be stronger than cage eggs (classic and furnished) and free-range eggs to be weaker than the other eggs. A significant correlation was found between eggshell strength and the likelihood of breakage in the production chains. In conclusion, it was first shown that, besides the laying, packing of the eggs is a critical point in the logistic chain of consumption eggs; second, the strength of the eggs in the different housing systems differed, and, finally, the eggshell stiffness and damping ratio of consumption eggs are an acceptable measure for rapid eggshell quality assessment and could provide a good predictive value for

  5. An oxysterol-binding protein family identified in the mouse.

    PubMed

    Anniss, Angela M; Apostolopoulos, Jim; Dworkin, Sebastian; Purton, Louise E; Sparrow, Rosemary L

    2002-08-01

    Oxysterols are oxygenated derivatives of cholesterol. They have been shown to influence a variety of biological functions including sterol metabolism, lipid trafficking, and apoptosis. Recently, 12 human OSBP-related genes have been identified. In this study, we have identified a family of 12 oxysterol-binding protein (OSBP)-related proteins (ORPs) in the mouse. A high level of amino acid identity (88-97%) was determined between mouse and human ORPs, indicating a very high degree of evolutionary conservation. All proteins identified contained the conserved OSBP amino acid sequence signature motif "EQVSHHPP," and most contained a pleckstrin homology (PH) domain. Using RT-PCR, each mouse ORP gene was found to exhibit a unique tissue distribution with many showing high expression in testicular, brain, and heart tissues. Interestingly, the tissue distribution of ORP-4 and ORP-10 were the most selective within the family. Expression of the various ORP genes was also investigated, specifically in highly purified populations of hemopoietic precursor cells defined by the lin(-) c-kit(+) Sca-1(+) (LKS(+)) and lin(-) c-kit(+) Sca-1(-) (LKS(-)) immunophenotype. Most ORP genes were expressed in both LKS(+) and LKS(-) populations, although ORP-4 appeared to be more highly expressed in the primitive, stem-cell enriched LKS(+) population, whereas ORP-10 was more highly expressed by maturing LKS(-) cells. The identification of a family of ORP proteins in the mouse, the frequently preferred animal model for in vivo studies, should further our understanding of the function of these proteins and their interactions with each other.

  6. 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

  7. 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

  8. 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

  9. 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

  10. Identifying and quantifying orphan protein sequences in fungi.

    PubMed

    Ekman, Diana; Elofsson, Arne

    2010-02-19

    For large regions of many proteins, and even entire proteins, no homology to known domains or proteins can be detected. These sequences are often referred to as orphans. Surprisingly, it has been reported that the large number of orphans is sustained in spite of a rapid increase of available genomic sequences. However, it is believed that de novo creation of coding sequences is rare in comparison to mechanisms such as domain shuffling and gene duplication; hence, most sequences should have homologs in other genomes. To investigate this, the sequences of 19 complete fungi genomes were compared. By using the phylogenetic relationship between these genomes, we could identify potentially de novo created orphans in Saccharomyces cerevisiae. We found that only a small fraction, <2%, of the S. cerevisiae proteome is orphan, which confirms that de novo creation of coding sequences is indeed rare. Furthermore, we found it necessary to compare the most closely related species to distinguish between de novo created sequences and rapidly evolving sequences where homologs are present but cannot be detected. Next, the orphan proteins (OPs) and orphan domains (ODs) were characterized. First, it was observed that both OPs and ODs are short. In addition, at least some of the OPs have been shown to be functional in experimental assays, showing that they are not pseudogenes. Furthermore, in contrast to what has been reported before and what is seen for older orphans, S. cerevisiae specific ODs and proteins are not more disordered than other proteins. This might indicate that many of the older, and earlier classified, orphans indeed are fast-evolving sequences. Finally, >90% of the detected ODs are located at the protein termini, which suggests that these orphans could have been created by mutations that have affected the start or stop codons.

  11. 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

  12. 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

  13. Organic solvents identify specific ligand binding sites on protein surfaces.

    PubMed

    Liepinsh, E; Otting, G

    1997-03-01

    Enzymes frequently recognize substrates and pharmaceutical drugs through specific binding interactions in deep pockets on the protein surface. We show how the specificity-determining substrate binding site of hen egg-white lysozyme (HEWL) can be readily identified in aqueous solution by nuclear magnetic resonance spectroscopy using small organic solvent molecules as detection probes. Exchange of magnetization between the 1H nuclei of the protein and the ligands through dipole-dipole interactions is observed which allows the modeling of their position and orientation at the binding site. Combined with site-specific binding constants measured by titration experiments with different organic solvents, the method can provide important information for rational drug design. In addition, the lifetime of nonspecific interactions of HEWL with organic solvents is shown to be in the sub-nanosecond time range. PMID:9062927

  14. Identifying subcellular protein localization with fluorescent protein fusions after transient expression in onion epidermal cells.

    PubMed

    Nebenführ, Andreas

    2014-01-01

    Most biochemical functions of plant cells are carried out by proteins which act at very specific places within these cells, for example, within different organelles. Identifying the subcellular localization of proteins is therefore a useful tool to narrow down the possible functions that a novel or unknown protein may carry out. The discovery of genetically encoded fluorescent markers has made it possible to tag specific proteins and visualize them in vivo under a variety of conditions. This chapter describes a simple method to use transient expression of such fluorescently tagged proteins in onion epidermal cells to determine their subcellular localization relative to known markers.

  15. Ratite eggshells from lanzarote, canary islands.

    PubMed

    Franz Sauer, E G; Rothe, P

    1972-04-01

    Struthious and aepyornithoid eggshells from Tertiary calcareous sediments on Lanzarote prove the presence, until about 12 million years ago, of large flightless birds. The calcarenite horizon is recognized as an old land surface. Mesozoic sedimentary rocks in the basement of the volcanic islands of Lanzarote and neighboring Fuerteventura indicate that at least part of the Canary Archipelago is underlain by continental crust. Separation of the eastern Canaries from Africa raight have been by rifting, and a land connection might still have existed in the lower Pliocene.

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

    PubMed Central

    Kim, Dae In; Raida, Manfred; Burke, Brian

    2012-01-01

    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

  17. Transcriptome-wide ribonuclease-mediated protein footprinting to identify RNA-protein interaction sites.

    PubMed

    Silverman, Ian M; Gregory, Brian D

    2015-01-15

    RNA-binding proteins (RBPs) are intimately involved in all aspects of RNA processing and regulation and are linked to neurodegenerative diseases and cancer. Therefore, investigating the relationship between RBPs and their RNA targets is critical for a broader understanding of post-transcriptional regulation in normal and disease processes. The majority of approaches to study RNA-protein interactions interrogate only individual RBPs. However, there are hundreds of these proteins encoded in the human genome, and each cell type expresses a different repertoire, greatly limiting the ability of current methods to capture the global landscape of RNA-protein interactions. To address this gap, we and others have recently developed methods to globally identify regions of RNAs that are bound by proteins in an unbiased manner. Here, we describe a detailed protocol for performing our ribonuclease-mediated protein footprint sequencing approach, termed protein interaction profile sequencing (PIP-seq). In this protocol, RNA-protein interactions are stabilized by cross-linking, and unbound regions are digested with ribonucleases (RNases), leaving only the protein-bound regions intact. To control for RNase insensitive regions, proteins are first denatured and degraded, then protein-depleted RNAs are subjected to RNase treatment. After high-throughput sequencing of the remaining fragments, peak calling is performed to identify protein-protected sites (PPSs). We describe the application of this protocol to a human embryonic kidney cell line (HEK293T) and perform basic quality control, reproducibility, and benchmarking analyses. Finally, we delineate the landscape of protein-interactions in HEK293T cells, underscoring the value of this approach. Future applications of this method to study the dynamics of RNA-protein interactions in developmental and disease processes will help to further uncover the role of RBPs in post-transcriptional regulation.

  18. Transcriptome-wide ribonuclease-mediated protein footprinting to identify RNA-protein interaction sites.

    PubMed

    Silverman, Ian M; Gregory, Brian D

    2015-01-15

    RNA-binding proteins (RBPs) are intimately involved in all aspects of RNA processing and regulation and are linked to neurodegenerative diseases and cancer. Therefore, investigating the relationship between RBPs and their RNA targets is critical for a broader understanding of post-transcriptional regulation in normal and disease processes. The majority of approaches to study RNA-protein interactions interrogate only individual RBPs. However, there are hundreds of these proteins encoded in the human genome, and each cell type expresses a different repertoire, greatly limiting the ability of current methods to capture the global landscape of RNA-protein interactions. To address this gap, we and others have recently developed methods to globally identify regions of RNAs that are bound by proteins in an unbiased manner. Here, we describe a detailed protocol for performing our ribonuclease-mediated protein footprint sequencing approach, termed protein interaction profile sequencing (PIP-seq). In this protocol, RNA-protein interactions are stabilized by cross-linking, and unbound regions are digested with ribonucleases (RNases), leaving only the protein-bound regions intact. To control for RNase insensitive regions, proteins are first denatured and degraded, then protein-depleted RNAs are subjected to RNase treatment. After high-throughput sequencing of the remaining fragments, peak calling is performed to identify protein-protected sites (PPSs). We describe the application of this protocol to a human embryonic kidney cell line (HEK293T) and perform basic quality control, reproducibility, and benchmarking analyses. Finally, we delineate the landscape of protein-interactions in HEK293T cells, underscoring the value of this approach. Future applications of this method to study the dynamics of RNA-protein interactions in developmental and disease processes will help to further uncover the role of RBPs in post-transcriptional regulation. PMID:25448484

  19. Ovarian dual oxidase (Duox) activity is essential for insect eggshell hardening and waterproofing.

    PubMed

    Dias, Felipe A; Gandara, Ana Caroline P; Queiroz-Barros, Fernanda G; Oliveira, Raquel L L; Sorgine, Marcos H F; Braz, Glória R C; Oliveira, Pedro L

    2013-12-01

    In insects, eggshell hardening involves cross-linking of chorion proteins via their tyrosine residues. This process is catalyzed by peroxidases at the expense of H2O2 and confers physical and biological protection to the developing embryo. Here, working with Rhodnius prolixus, the insect vector of Chagas disease, we show that an ovary dual oxidase (Duox), a NADPH oxidase, is the source of the H2O2 that supports dityrosine-mediated protein cross-linking and eggshell hardening. RNAi silencing of Duox activity decreased H2O2 generation followed by a failure in embryo development caused by a reduced resistance to water loss, which, in turn, caused embryos to dry out following oviposition. Phenotypes of Duox-silenced eggs were reversed by incubation in a water-saturated atmosphere, simultaneous silencing of the Duox and catalase genes, or H2O2 injection into the female hemocoel. Taken together, our results show that Duox-generated H2O2 fuels egg chorion hardening and that this process plays an essential role during eggshell waterproofing.

  20. [Identifying phosphopeptide by searching a site annotated protein database].

    PubMed

    Cheng, Kai; Wang, Fangjun; Bian, Yangyang; Ye, Mingling; Zou, Hanfa

    2015-01-01

    Phosphoproteome analysis is one of the important research fields in proteomics. In shotgun proteomics, phosphopeptides could be identified directly by setting phosphorylation as variable modifications in database search. However, search space increases significantly when variable modifications are set in post-translation modifications (PTMs) analysis, which will decrease the identification sensitivity. Because setting a variable modification on a specific type of amino acid residue means all of this amino acid residues in the database might be modified, which is not consistent with actual conditions. Phosphorylation and dephosphorylation are regulated by protein kinases and phosphatases, which can only occur on particular substrates. Therefore only residues within specific sequence are potential sites which may be modified. To address this issue, we extracted the characteristic sequence from the identified phosphorylation sites and created an annotated database containing phosphorylation site information, which allowed the searching engine to set variable modifications only on the serine, threonine and tyrosine residues that were identified to be phosphorylated previously. In this database only annotated serine, threonine and tyrosine can be modified. This strategy significantly reduced the search space. The performance of this new database searching strategy was evaluated by searching different types of data with Mascot, and higher sensitivity for phosphopeptide identification was achieved with high reliability.

  1. 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

  2. Identifying Protein-Protein Associations at the Nuclear Envelope with BioID.

    PubMed

    Kim, Dae In; Jensen, Samuel C; Roux, Kyle J

    2016-01-01

    The nuclear envelope (NE) is a critical cellular structure whose constituents and roles in a myriad of cellular processes seem ever expanding. To determine the underlying mechanisms by which the NE constituents participate in various cellular events, it is necessary to understand the nature of their protein-protein associations. BioID (proximity-dependent biotin identification) is a recently established method to generate a history of protein-protein associations as they occur over time in living cells. BioID is based on fusion of a bait protein to a promiscuous biotin ligase. Expression of the BioID fusion protein in a relevant cellular environment enables biotinylation of vicinal and interacting proteins of the bait protein, permitting isolation and identification by conventional biotin-affinity capture and mass-spec analysis. In this way, BioID provides unique capabilities to identify protein-protein associations at the NE. In this chapter we provide a detailed protocol for the application of BioID to the study of NE proteins.

  3. 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

  4. Diffusive resistance of avian eggshell pores.

    PubMed

    Tøien, O; Paganelli, C V; Rahn, H; Johnson, R R

    1988-12-01

    Resistance to gas diffusion through the avian eggshell resides in the microscopic pores which penetrate the shell. We calculated the resistance to water vapor diffusion of individual pores in the shells of 23 species of avian eggs, based on measurements of pore dimensions taken from drawings of 321 pore casts published by Tyler (1962, 1964, 1965) and Tyler and Simkiss (1959). Diffusive resistances were calculated from Fick's first law, using a 100-segment model of each pore. In addition, we added 2 series resistances, calculated from Stefan's law, to account for boundary layer resistances at the inner and outer pore apertures. Convective resistances for the same 100-segment model were computed from Poiseuille's law. A special, symmetrically branching model is presented for the diffusive resistance of the branched pores of ostrich eggshells, based on the drawings of Tyler and Simkiss (1959). The total aperture resistance was less than 6.2% of total pore resistance, while the outside aperture effect was on average only 1.5%. The calculated average pore conductance for all species was 5.4 micrograms (day Torr)-1, about three times higher than the average value of 1.6 micrograms (day Torr)-1 obtained by dividing measured shell conductance by the number of pores (Ar and Rahn, 1985). A possible explanation for this discrepancy is advanced. However, it is to be noted that in spite of the discrepancy, both calculated and functional values of pore conductance appear to be independent of egg mass.

  5. FunMod: a Cytoscape plugin for identifying functional modules in undirected protein-protein networks.

    PubMed

    Natale, Massimo; Benso, Alfredo; Di Carlo, Stefano; Ficarra, Elisa

    2014-08-01

    The characterization of the interacting behaviors of complex biological systems is a primary objective in protein-protein network analysis and computational biology. In this paper we present FunMod, an innovative Cytoscape version 2.8 plugin that is able to mine undirected protein-protein networks and to infer sub-networks of interacting proteins intimately correlated with relevant biological pathways. This plugin may enable the discovery of new pathways involved in diseases. In order to describe the role of each protein within the relevant biological pathways, FunMod computes and scores three topological features of the identified sub-networks. By integrating the results from biological pathway clustering and topological network analysis, FunMod proved to be useful for the data interpretation and the generation of new hypotheses in two case studies.

  6. Anticytolytic screen identifies inhibitors of mycobacterial virulence protein secretion.

    PubMed

    Rybniker, Jan; Chen, Jeffrey M; Sala, Claudia; Hartkoorn, Ruben C; Vocat, Anthony; Benjak, Andrej; Boy-Röttger, Stefanie; Zhang, Ming; Székely, Rita; Greff, Zoltán; Orfi, László; Szabadkai, István; Pató, János; Kéri, György; Cole, Stewart T

    2014-10-01

    Mycobacterium tuberculosis (Mtb) requires protein secretion systems like ESX-1 for intracellular survival and virulence. The major virulence determinant and ESX-1 substrate, EsxA, arrests phagosome maturation and lyses cell membranes, resulting in tissue damage and necrosis that promotes pathogen spread. To identify inhibitors of Mtb protein secretion, we developed a fibroblast survival assay exploiting this phenotype and selected molecules that protect host cells from Mtb-induced lysis without being bactericidal in vitro. Hit compounds blocked EsxA secretion and promoted phagosome maturation in macrophages, thus reducing bacterial loads. Target identification studies led to the discovery of BTP15, a benzothiophene inhibitor of the histidine kinase MprB that indirectly regulates ESX-1, and BBH7, a benzyloxybenzylidene-hydrazine compound. BBH7 affects Mtb metal-ion homeostasis and revealed zinc stress as an activating signal for EsxA secretion. This screening approach extends the target spectrum of small molecule libraries and will help tackle the mounting problem of antibiotic-resistant mycobacteria.

  7. Molecular preservation in Late Cretaceous sauropod dinosaur eggshells

    PubMed Central

    Schweitzer, M.H; Chiappe, L; Garrido, A.C; Lowenstein, J.M; Pincus, S.H

    2005-01-01

    Exceptionally preserved sauropod eggshells discovered in Upper Cretaceous (Campanian) deposits in Patagonia, Argentina, contain skeletal remains and soft tissues of embryonic Titanosaurid dinosaurs. To preserve these labile embryonic remains, the rate of mineral precipitation must have superseded post-mortem degradative processes, resulting in virtually instantaneous mineralization of soft tissues. If so, mineralization may also have been rapid enough to retain fragments of original biomolecules in these specimens. To investigate preservation of biomolecular compounds in these well-preserved sauropod dinosaur eggshells, we applied multiple analytical techniques. Results demonstrate organic compounds and antigenic structures similar to those found in extant eggshells. PMID:15888409

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

    DOE PAGES

    Shatsky, Maxim; Dong, Ming; Liu, Haichuan; Yang, Lee Lisheng; Choi, Megan; Singer, Mary; Geller, Jil; Fisher, Susan; Hall, Steven; Hazen, Terry C.; et al

    2016-04-20

    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 ofmore » 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.« less

  9. 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

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

    SciTech Connect

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

    2016-01-01

    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.

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

    PubMed Central

    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.

    2016-01-01

    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

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. A chitin-like component in Aedes aegypti eggshells, eggs and ovaries.

    PubMed

    Moreira, Mônica F; Dos Santos, Amanda S; Marotta, Humberto R; Mansur, Juliana F; Ramos, Isabela B; Machado, Ednildo A; Souza, Gustavo H M F; Eberlin, Marcos N; Kaiser, Carlos R; Kramer, Karl J; Muthukrishnan, Subbaratnam; Vasconcellos, Ana Maria H

    2007-12-01

    An insoluble white substance was prepared from extracts of eggshells of Aedes aegypti, the yellow fever mosquito and dengue vector. Its infrared and proton NMR spectra were similar to that of standard commercial chitin. This putative chitin-like material, also obtained from ovaries, newly laid and dark eggs, was hydrolyzed in acid and a major product was identified by HPLC to be glucosamine. The eggshell acid hydrolysate was also analyzed by ESI-MS and an ion identical to a glucosamine monoprotonated species was detected. The presence of chitin was also analyzed during different developmental stages of the ovary using a fluorescent microscopy technique and probes specific for chitin. The results showed that a chitin-like material accumulates in oocytes during oogenesis. Streptomyces griseus chitinase pre-treatment of oocytes greatly reduced the chitin-derived fluorescence. Chitinase activity was detected in newborn larvae and eggs prior to hatching. Feeding experiments indicated that the chitin synthesis inhibitor lufenuron inhibited chitin synthesis, either when mosquitoes were allowed to feed directly on lufenuron-treated chickens or when an artificial feeding system was used. Lufenuron inhibited egg hatch, larval development and reduced mosquito viability. These data demonstrate for the first time that (1) a chitin-like material is present in A. aegypti eggs, ovaries and eggshells; (2) a chitin synthesis inhibitor can be used to inhibit mosquito oogenesis; and (3) chitin synthesis inhibitors have potential for controlling mosquito populations. PMID:17967344

  18. 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... Unique Identifier SYN-IR67B-1 in cotton; exemption from the requirement of a tolerance. Residues of... exempt from the requirement of a tolerance when used as a plant-incorporated protectant in cotton;...

  19. 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... Unique Identifier SYN-IR67B-1 in cotton; exemption from the requirement of a tolerance. Residues of... exempt from the requirement of a tolerance when used as a plant-incorporated protectant in cotton;...

  20. 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... Unique Identifier SYN-IR67B-1 in cotton; exemption from the requirement of a tolerance. Residues of... exempt from the requirement of a tolerance when used as a plant-incorporated protectant in cotton;...

  1. 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

    ... protein as identified under OECD Unique Identifier SYN-IR67B-1 in cotton; exemption from the requirement... Unique Identifier SYN-IR67B-1 in cotton; exemption from the requirement of a tolerance. Residues of... exempt from the requirement of a tolerance when used as a plant-incorporated protectant in cotton;...

  2. 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... Unique Identifier SYN-IR67B-1 in cotton; exemption from the requirement of a tolerance. Residues of... exempt from the requirement of a tolerance when used as a plant-incorporated protectant in cotton;...

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

    DOE PAGES

    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

  4. The Use of Multimeric Protein Scaffolds for Identifying Multi-SUMO Binding Proteins.

    PubMed

    Aguilar-Martínez, Elisa; Sharrocks, Andrew D

    2016-01-01

    The use of in vitro assays, such as glutathione S-transferase (GST) pull-downs, enables the study of complex cellular processes in a simplified form. Pull-down assays facilitate the discovery and detailed study of protein-protein interactions, which can then be extrapolated to the cellular environment. Here, we describe the expression, purification and use of a multi-SUMO platform to identify SUMO-interacting proteins. This SUMO-platform can be easily expressed and purified from bacterial cells for use as baits in pull-down assays. This methodology facilitates the discovery of novel SUMO-binding proteins or further characterization of SUMO with known binding partners. PMID:27631807

  5. Eggshell thickness and DDE residue levels in vlulture eggs

    USGS Publications Warehouse

    Kiff, L.F.; Peakall, D.B.; Morrison, M.L.; Wilbur, S.R.; Wilbur, Sanford R.; Jackson, Jerome A.

    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.

  6. 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

  7. Resorption kinetics of eggshell: an in vivo study.

    PubMed

    Dupoirieux, L; Pourquier, D; Neves, M; Téot, L

    2001-01-01

    Eggshell has been recently introduced as a bone substitute candidate in reconstructive surgery. The aim of this experimental study study was to determine its degradation rate in both a skeletal and extraskeletal site. In experiment 1, eggshell particles with four different sizes (50, 75, 150, and 300 microns in diameter) were implanted in subcutaneous pouches of 30 rats. In experiment 2, a fragment of ostrich eggshell was implanted on the nasal dorsum of 10 rats. Animals were sacrificed at 1 (N = 10), 2 (N = 10), and 4 months (N = 10) during the first stage of the study, and at 1 year during the second stage of the study. The results were assessed by X-ray examination and routine histological techniques. In experiment 1, all animals healed uneventfully. At 1 month, only 50-micron particles had undergone resorption. At 2 months, both 50- and 75-micron particles had undergone resorption. At 4 months, the 150- and 300-micron particles were resorbed incompletely. Histologically, the eggshell elicited a mild inflammatory reaction at 1 month that decreased progressively at further stages. In experiment 2, all animals except one healed uneventfully. Radiologically, the eggshell implant displayed a noticeable stability. Histologically, seven of nine implants were encapsulated, but two of them were surrounded by a bony rim. In conclusion, eggshell is a resorbable implant, but the degradation kinetic is size dependent. Large ostrich grafts are also suitable as onlay graft, but a complementary osteosynthesis is recommended to enhance osteointegration. PMID:11314188

  8. Global protein profiling studies of chikungunya virus infection identify different proteins but common biological processes.

    PubMed

    Smith, Duncan R

    2015-01-01

    Chikungunya fever (CHIKF) caused by the mosquito-transmitted chikungunya virus (CHIKV) swept into international prominence from late 2005 as an epidemic of CHIKF spread around countries surrounding the Indian Ocean. Although significant advances have been made in understanding the pathobiology of CHIKF, numerous questions still remain. In the absence of commercially available specific drugs to treat the disease, or a vaccine to prevent the diseases, the questions have particular significance. A number of studies have used global proteome analysis to increase our understanding of the process of CHIKV infection using a number of different experimental techniques and experimental systems. In all, over 700 proteins have been identified in nine different analyses by five different groups as being differentially regulated. Remarkably, only a single protein, eukaryotic elongation factor 2, has been identified by more than two different groups as being differentially regulated during CHIKV infection. This review provides a critical overview of the studies that have used global protein profiling to understand CHIKV infection and shows that while a broad consensus is emerging on which biological processes are altered during CHIKV infection, this consensus is poorly supported in terms of consistent identification of any key proteins mediating those biological processes.

  9. RNA-protein complexes identified by crosslinking of polysomes.

    PubMed

    Sköld, S E

    1981-01-01

    The bifunctional cleavable reagent diepoxybutane was used to investigate the crosslinking of proteins to the 16S and 23S RNA in Escherichia coli ribosomes. The crosslinking patterns from polysomes, accumulated in the absence and presence of oxytetracycline, as well as reassociated 70S ribosomes were compared. The 30S proteins: S3, S4, S5, S7, S8, S9, S12, S13, S14 and S18 were recovered crosslinked to the 16S RNA and the 50S: proteins L1, L2, L4, L13, L14-L21, L15, L16, L17, L18-L23, L19-22-24, L27 and L28 were recovered crosslinked to the 23S RNA, in all three associated states. Proteins crosslinked to the RNA of the heterologous subunit and therefore considered to be at or near the ribosomal subunit interface were, for all three states, proteins S1, S4, S6, S9, S12, S13, S14 and S18 from the small subunit and proteins L16, L17, L20 and L27 from the large subunit. Finally, the recovery of intrasubunit crosslinks was measured for the isolated subunits. Additional crosslinked complexes were observed between 16S RNA and S1, S2 as well as S6 from the 30S subunit; and between 23S RNA and L10, L11, L7/12 from the 50S subunit.

  10. Trans-acting amplification mutants and other eggshell mutants of the third chromosome in Drosophila melanogaster.

    PubMed Central

    Snyder, P B; Galanopoulos, V K; Kafatos, F C

    1986-01-01

    We report on the characterization of five third chromosome mutations with strong effects on the formation of the eggshell or chorion. Three mutations, defining two loci, result in substantially reduced follicle cell-specific amplification of the major chorion structural genes and, hence, in underproduction of the corresponding mRNAs and proteins. The other two mutations, though displaying structural chorion abnormalities, appear to have no significant effect on amplification and to express normally the major chorion structural genes. The possible nature of these mutations is discussed. Images PMID:3085087

  11. 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

  12. Factors influencing bacterial eggshell contamination in conventional cages, furnished cages and free-range systems for laying hens under commercial conditions.

    PubMed

    Huneau-Salaün, A; Michel, V; Huonnic, D; Balaine, L; Le Bouquin, S

    2010-04-01

    1. The aim was to assess eggshell contamination in various laying hen-housing systems and to identify factors influencing this contamination. 2. Fifty-eight laying hen farms in France were studied, including 21 flocks housed in conventional cages, 7 in furnished cages and 30 kept on-floor. 3. Sixty eggs per flock were analysed to obtain counts of the total mesophilic flora. Data on equipment and hen management were collected. 4. Mean bacterial count on eggshells tended to be higher in on-floor systems (4.82 +/- 0.51 log CFU/eggshell) than in cage systems (4.57 +/- 0.58 log CFU/eggshell, P = 0.09). 5. Contamination increased with age of the hens, airborne dust concentration, manual packing of the eggs, and packing in plastic rather than in recycled-pulp egg-flats. 6. The effect of the housing system on eggshell contamination, previously described in experimental assays, was confirmed under production conditions.

  13. Tandem affinity purification to identify cytosolic and nuclear gβγ-interacting proteins.

    PubMed

    Campden, Rhiannon; Pétrin, Darlaine; Robitaille, Mélanie; Audet, Nicolas; Gora, Sarah; Angers, Stéphane; Hébert, Terence E

    2015-01-01

    It has become clear in recent years that the Gβγ subunits of heterotrimeric proteins serve broad roles in the regulation of cellular activity and interact with many proteins in different subcellular locations including the nucleus. Protein affinity purification is a common method to identify and confirm protein interactions. When used in conjugation with mass spectrometry it can be used to identify novel protein interactions with a given bait protein. The tandem affinity purification (TAP) technique identifies partner proteins bound to tagged protein bait. Combined with protocols to enrich the nuclear fraction of whole cell lysate through sucrose cushions, TAP allows for purification of interacting proteins found specifically in the nucleus. Here we describe the use of the TAP technique on cytosolic and nuclear lysates to identify candidate proteins, through mass spectrometry, that bind to Gβ1 subunits.

  14. A candidate plasma protein classifier to identify Alzheimer's disease.

    PubMed

    Zhao, Xuemei; Lejnine, Serguei; Spond, Jeffrey; Zhang, Chunsheng; Ramaraj, T C; Holder, Daniel J; Dai, Hongyue; Weiner, Russell; Laterza, Omar F

    2015-01-01

    Biomarkers currently used in the aid for the diagnosis of Alzheimer's disease (AD) are cerebrospinal fluid (CSF) protein markers and brain neuroimaging markers. These biomarkers, however, either involve semi-invasive procedures or are costly to measure. Thus, AD biomarkers from more easily accessible body fluids, such as plasma, are very enticing. Using an aptamer-based proteomic technology, we profiled 1,129 plasma proteins of AD patients and non-demented control individuals. A 5-protein classifier for AD identification was constructed in the discovery study with excellent 10-fold cross-validation performance (90.1% sensitivity, 84.2% specificity, 87.9% accuracy, and AUC as 0.94). In an independent validation study, the classifier was applied and correctly predicted AD with 100.0% sensitivity, 80.0% specificity, and 90.0% accuracy, matching or outperforming the CSF Aβ42 and tau biomarkers whose performance were assessed in individual-matched CSF samples obtained at the same visit as plasma sample collection. Moreover, the classifier also correctly predicted mild cognitive impairment, an early pre-dementia state of the disease, with 96.7% sensitivity, 80.0% specificity, and 92.5% accuracy. These studies demonstrate that plasma proteins could be used effectively and accurately to contribute to the clinical diagnosis of AD. Although additional and more diverse cohorts are needed for further validation of the robustness, including the support of postmortem diagnosis, the 5-protein classifier appears to be a promising blood test to contribute diagnosis of AD. PMID:25114072

  15. A candidate plasma protein classifier to identify Alzheimer's disease.

    PubMed

    Zhao, Xuemei; Lejnine, Serguei; Spond, Jeffrey; Zhang, Chunsheng; Ramaraj, T C; Holder, Daniel J; Dai, Hongyue; Weiner, Russell; Laterza, Omar F

    2015-01-01

    Biomarkers currently used in the aid for the diagnosis of Alzheimer's disease (AD) are cerebrospinal fluid (CSF) protein markers and brain neuroimaging markers. These biomarkers, however, either involve semi-invasive procedures or are costly to measure. Thus, AD biomarkers from more easily accessible body fluids, such as plasma, are very enticing. Using an aptamer-based proteomic technology, we profiled 1,129 plasma proteins of AD patients and non-demented control individuals. A 5-protein classifier for AD identification was constructed in the discovery study with excellent 10-fold cross-validation performance (90.1% sensitivity, 84.2% specificity, 87.9% accuracy, and AUC as 0.94). In an independent validation study, the classifier was applied and correctly predicted AD with 100.0% sensitivity, 80.0% specificity, and 90.0% accuracy, matching or outperforming the CSF Aβ42 and tau biomarkers whose performance were assessed in individual-matched CSF samples obtained at the same visit as plasma sample collection. Moreover, the classifier also correctly predicted mild cognitive impairment, an early pre-dementia state of the disease, with 96.7% sensitivity, 80.0% specificity, and 92.5% accuracy. These studies demonstrate that plasma proteins could be used effectively and accurately to contribute to the clinical diagnosis of AD. Although additional and more diverse cohorts are needed for further validation of the robustness, including the support of postmortem diagnosis, the 5-protein classifier appears to be a promising blood test to contribute diagnosis of AD.

  16. 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

  17. 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

  18. 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

  19. 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.

  20. Constant photoperiods and eggshell quality in broiler breeder pullets.

    PubMed

    Backhouse, D; Lewis, P D; Gous, R M

    2005-04-01

    Broiler breeder pullets were exposed to constant 10-, 11-, 12-, 13-, 14- or 16-h photoperiods from 3 d of age. Egg weight, eggshell weight and shell thickness index were determined at 52 weeks of age. Egg weight increased by 0.31 g, shell weight decreased by 30 mg and shell thickness index decreased by 0.57 mg/cm2 for each one-hour increase in photoperiod. Whilst the changes in egg weight and eggshell thickness index might be overstated because eggs were collected at the same chronological time, the effect of time of egg-laying within the day was minimal in comparison, and did not negate the conclusion that egg weight increases, and shell weight and thickness index decrease with lengthening photoperiods. The effect of photoperiod on eggshell quality was not due to differences in the rate of lay between treatments. Shell weight was unaffected by time of lay.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. The eggshell features and clutch viability of the broad-snouted caiman (Caiman latirostris) are associated with the egg burden of organochlorine compounds.

    PubMed

    Stoker, C; Zayas, M A; Ferreira, M A; Durando, M; Galoppo, G H; Rodríguez, H A; Repetti, M R; Beldoménico, H R; Caldini, E G; Luque, E H; Muñoz-de-Toro, M

    2013-12-01

    Organochlorine compounds (OCCs) are toxic and have been identified as endocrine-disrupting chemicals (EDCs). The broad-snouted Caiman (Caiman latirostris) is an oviparous species widely distributed in South America with potential to accumulate OCCs. The eggshell is formed during passage of the eggs through the oviduct. Since the oviduct is a target of hormone actions, exposure to OCCs could modify eggshell quality, thus affecting clutch viability. Eight clutches were collected from wetlands of Parana River tributaries, in north-eastern Argentina. Two to four eggs per clutch were used to establish the burden of OCCs, eggshell thickness and eggshell porosity. The remaining eggs were incubated in controlled conditions. Ten days after hatching, hatchling survival was assessed. Organochlorine pesticide residues (OCPs) were found in all clutches, while polychlorinated biphenyls (PCBs) were present in all but one clutch. The principal contributors to the OCP burden were members of the DDT family and oxychlordane. Eggshell thickness was 400.9±6.0 μm and, unexpectedly, no association between eggshell thickness and the OCC burden was found. The number of pores in the outer surface was 25.3±4.3 pores/cm². A significant inverse correlation between porosity and OCC burden was found (Pearson r= -0.81, p= 0.01). Furthermore, a decrease in caiman survival with decreased pore density was observed (Pearson r= 0.73, p= 0.04). Our findings highlight another potential negative impact of current and past use of OCCs on wildlife species.

  10. Identifying dynamic protein complexes based on gene expression profiles and PPI networks.

    PubMed

    Li, Min; Chen, Weijie; Wang, Jianxin; Wu, Fang-Xiang; Pan, Yi

    2014-01-01

    Identification of protein complexes from protein-protein interaction networks has become a key problem for understanding cellular life in postgenomic era. Many computational methods have been proposed for identifying protein complexes. Up to now, the existing computational methods are mostly applied on static PPI networks. However, proteins and their interactions are dynamic in reality. Identifying dynamic protein complexes is more meaningful and challenging. In this paper, a novel algorithm, named DPC, is proposed to identify dynamic protein complexes by integrating PPI data and gene expression profiles. According to Core-Attachment assumption, these proteins which are always active in the molecular cycle are regarded as core proteins. The protein-complex cores are identified from these always active proteins by detecting dense subgraphs. Final protein complexes are extended from the protein-complex cores by adding attachments based on a topological character of "closeness" and dynamic meaning. The protein complexes produced by our algorithm DPC contain two parts: static core expressed in all the molecular cycle and dynamic attachments short-lived. The proposed algorithm DPC was applied on the data of Saccharomyces cerevisiae and the experimental results show that DPC outperforms CMC, MCL, SPICi, HC-PIN, COACH, and Core-Attachment based on the validation of matching with known complexes and hF-measures. PMID:24963481

  11. 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.

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

    PubMed Central

    Lin, Peng-Lin; Yu, Ya-Wen

    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

  13. 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

  14. Identifying functions of protein complexes based on topology similarity with random forest.

    PubMed

    Li, Zhan-Chao; Lai, Yan-Hua; Chen, Li-Li; Xie, Yun; Dai, Zong; Zou, Xiao-Yong

    2014-03-01

    Elucidating the functions of protein complexes is critical for understanding disease mechanisms, diagnosis and therapy. In this study, based on the concept that protein complexes with similar topology may have similar functions, we firstly model protein complexes as weighted graphs with nodes representing the proteins and edges indicating interaction between proteins. Secondly, we use topology features derived from the graphs to characterize protein complexes based on the graph theory. Finally, we construct a predictor by using random forest and topology features to identify the functions of protein complexes. Effectiveness of the current method is evaluated by identifying the functions of mammalian protein complexes. And then the predictor is also utilized to identify the functions of protein complexes retrieved from human protein-protein interaction networks. We identify some protein complexes with significant roles in the occurrence of tumors, vesicles and retinoblastoma. It is anticipated that the current research has an important impact on pathogenesis and the pharmaceutical industry. The source code of Matlab and the dataset are freely available on request from the authors. PMID:24389559

  15. A Topology Potential-Based Method for Identifying Essential Proteins from PPI Networks.

    PubMed

    Li, Min; Lu, Yu; Wang, Jianxin; Wu, Fang-Xiang; Pan, Yi

    2015-01-01

    Essential proteins are indispensable for cellular life. It is of great significance to identify essential proteins that can help us understand the minimal requirements for cellular life and is also very important for drug design. However, identification of essential proteins based on experimental approaches are typically time-consuming and expensive. With the development of high-throughput technology in the post-genomic era, more and more protein-protein interaction data can be obtained, which make it possible to study essential proteins from the network level. There have been a series of computational approaches proposed for predicting essential proteins based on network topologies. Most of these topology based essential protein discovery methods were to use network centralities. In this paper, we investigate the essential proteins' topological characters from a completely new perspective. To our knowledge it is the first time that topology potential is used to identify essential proteins from a protein-protein interaction (PPI) network. The basic idea is that each protein in the network can be viewed as a material particle which creates a potential field around itself and the interaction of all proteins forms a topological field over the network. By defining and computing the value of each protein's topology potential, we can obtain a more precise ranking which reflects the importance of proteins from the PPI network. The experimental results show that topology potential-based methods TP and TP-NC outperform traditional topology measures: degree centrality (DC), betweenness centrality (BC), closeness centrality (CC), subgraph centrality (SC), eigenvector centrality (EC), information centrality (IC), and network centrality (NC) for predicting essential proteins. In addition, these centrality measures are improved on their performance for identifying essential proteins in biological network when controlled by topology potential.

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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

  1. Combination of MS protein identification and bioassay of chromatographic fractions to identify biologically active substances from complex protein sources.

    PubMed

    Kuromitsu, Sadao; Yokota, Hiroyuki; Hiramoto, Masashi; Yuri, Masatoshi; Naitou, Masanori; Nakamura, Naoto; Kawabata, Shigeki; Kobori, Masato; Katoh, Masao; Furuchi, Kiyoshi; Mita, Haruhisa; Yamada, Tetsuo

    2009-06-01

    Purification of biologically active proteins from complex biological sources is a difficult task, usually requiring large amounts of sample and many separation steps. We found an active substance in a serum response element-dependent luciferase reporter gene bioassay in interstitial cystitis urine that we attempted to purify with column chromatography and the bioassay. With anion-exchange Mono Q and C4 reversed-phase columns, apparently sharp active peaks were obtained. However, more than 20 kinds of proteins were identified from the active fractions with MS, indicating that the purification was not complete. As further purification was difficult, we chose a candidate molecule by means of studying the correlation between MS protein identification scores and bioassay responses of chromatographic fractions near the active peaks. As a result, epidermal growth factor (EGF) was nominated as a candidate molecule among the identified proteins because the elution profile of EGF was consistent with that of the bioassay, and the correlation coefficient of EGF between MS protein identification scores and bioassay responses was the highest among all the identified proteins. With recombinant EGF and anti-EGF and anti-EGF receptor antibodies, EGF was confirmed to be the desired substance in interstitial cystitis urine. This approach required only 20 ml of urine sample and two column chromatographic steps. The combination of MS protein identification and bioassay of chromatographic fractions may be useful for identifying biologically active substances from complex protein sources.

  2. 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

  3. 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

  4. 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

  5. 9 CFR 147.13 - Procedure for bacteriological culturing of eggshells for colon bacilli organisms.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... bacteriological culturing of eggshells for colon bacilli organisms. Proper precautions to avoid environmental contamination of the samples during the collection and laboratory process, and proper handling of the...

  6. 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.

  7. Transgelin: an androgen-dependent protein identified in the seminal vesicles of three Saharan rodents.

    PubMed

    Kaci-Ouchfoun, Naïma; Izemrane, Djamila; Boudrissa, Abdelkrim; Gernigon, Thérèse; Khammar, Farida; Exbrayat, Jean Marie

    2013-10-15

    During the breeding season, a major androgen-dependent protein with an apparent molecular weight of 21 kDa was isolated and purified from the seminal vesicles of three Saharan rodents (MLVSP21 from Meriones libycus, MSVSP21 from Meriones shawi, and MCVSP21 from Meriones crassus). The 21-kDa protein was isolated and purified from soluble seminal vesicle proteins of homogenate by one-dimensional polyacrylamide gel electrophoresis (SDS-PAGE). Using polyclonal antibodies directed against POSVP21 (Psammomys obesus seminal vesicles protein of 21 kDa), a major androgen-dependent secretory protein from sand rat seminal vesicles, identified previously as transgelin, we showed an immunological homology with POSVP21 by immunoblotting. These three major androgen-dependent proteins with a same apparent molecular weight of 21 kDa designated as MLVSP21 (Meriones libycus seminal vesicles protein of 21 kDa), MSVSP21 (Meriones shawi seminal vesicles protein of 21 kDa), and MCVSP21 (Meriones crassus seminal vesicles protein of 21 kDa) were localized by immunohistochemistry and identified by applying a proteomic approach. Our results indicated that the isolated proteins MLSVP21, MSSVP21, and MCSVP21 seem to correspond to the same protein: the transgelin. So that transgelin can be used as a specific marker of these rodent physiological reproduction mechanisms.

  8. 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.

  9. Identifying specific protein-DNA interactions using SILAC-based quantitative proteomics.

    PubMed

    Spruijt, Cornelia G; Baymaz, H Irem; Vermeulen, Michiel

    2013-01-01

    A comprehensive identification of protein-DNA interactions that drive processes such as transcription and replication, both in prokaryotic and eukaryotic organisms, remains a major technical challenge. In this chapter, we present a SILAC-based DNA affinity purification method that can be used to identify specific interactions between proteins and functional DNA elements in an unbiased manner.

  10. Secretomics identifies Fusarium graminearum proteins involved in the interaction with barley and wheat.

    PubMed

    Yang, Fen; Jensen, Jens D; Svensson, Birte; Jørgensen, Hans J L; Collinge, David B; Finnie, Christine

    2012-06-01

    Fusarium graminearum is a phytopathogenic fungus primarily infecting small grain cereals, including barley and wheat. Secreted enzymes play important roles in the pathogenicity of many fungi. In order to access the secretome of F. graminearum, the fungus was grown in liquid culture with barley or wheat flour as the sole nutrient source to mimic the host-pathogen interaction. A gel-based proteomics approach was employed to identify the proteins secreted into the culture medium. Sixty-nine unique fungal proteins were identified in 154 protein spots, including enzymes involved in the degradation of cell walls, starch and proteins. Of these proteins, 35% had not been identified in previous in planta or in vitro studies, 70% were predicted to contain signal peptides and a further 16% may be secreted in a nonclassical manner. Proteins identified in the 72 spots showing differential appearance between wheat and barley flour medium were mainly involved in fungal cell wall remodelling and the degradation of plant cell walls, starch and proteins. The in planta expression of corresponding F. graminearum genes was confirmed by quantitative reverse transcriptase-polymerase chain reaction in barley and wheat spikelets harvested at 2-6 days after inoculation. In addition, a clear difference in the accumulation of fungal biomass and the extent of fungal-induced proteolysis of plant β-amylase was observed in barley and wheat. The present study considerably expands the current database of F. graminearum secreted proteins which may be involved in Fusarium head blight.

  11. 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).

  12. Proteomic study identifies proteins involved in brassinosteroid regulation of rice growth.

    PubMed

    Wang, Fengru; Bai, Ming-Yi; Deng, Zhiping; Oses-Prieto, Juan A; Burlingame, Alma L; Lu, Tiegang; Chong, Kang; Wang, Zhi-Yong

    2010-12-01

    Brassinosteroids (BRs) are essential hormones for growth and development of plant. In rice, BRs regulate multiple developmental processes and affect many important traits such as height, leaf angle, fertility and seed filling. We identified brassinosteroid-regulated proteins in rice using proteomic approaches and performed functional analysis of some BR-regulated proteins by overexpression experiments. Using two-dimensional difference gel electrophoresis (2-D DIGE) followed by protein identification by mass spectrometry, we compared proteomic differences in the shoots and roots of the BR-insensitive mutant d61-4 and BR-deficient mutant brd1-3. We identified a large number of proteins differentially expressed in the mutants compared with wild type control. These include a glycine-rich RNA-binding protein (OsGRP1) and a DREPP2 protein, which showed reduced levels in the BR mutants. Overexpression of these two proteins partially suppressed the dwarf phenotype of the Arabidopsis BR-insensitive mutant bri1-5. In contrast to the reduced protein level, the RNA level of OsGRP1 was not significantly affected in the BR mutants or by BR treatment, suggesting BR regulation of OsGRP1 at the posttranslational level. This study identifies many BR-regulated proteins and demonstrates that OsGRP1 functions downstream in the BR signal transduction pathway to promote cell expansion.

  13. Using an Isolated Rat Kidney Model to Identify Kidney Origin Proteins in Urine

    PubMed Central

    Jia, Lulu; Li, Xundou; Shao, Chen; Wei, Lilong; Li, Menglin; Guo, Zhengguang; Liu, Zhihong; Gao, Youhe

    2013-01-01

    The use of targeted proteomics to identify urinary biomarkers of kidney disease in urine can avoid the interference of serum proteins. It may provide better sample throughput, higher sensitivity, and specificity. Knowing which urinary proteins to target is essential. By analyzing the urine from perfused isolated rat kidneys, 990 kidney origin proteins with human analogs were identified in urine. Of these proteins, 128 were not found in normal human urine and may become biomarkers with zero background. A total of 297 proteins were not found in normal human plasma. These proteins will not be influenced by other normal organs and will be kidney specific. The levels of 33 proteins increased during perfusion with an oxygen-deficient solution compared to those perfused with oxygen. The 75 proteins in the perfusion-driven urine have a significantly increased abundance ranking compared to their ranking in normal human urine. When compared with existing candidate biomarkers, over ninety percent of the kidney origin proteins in urine identified in this study have not been examined as candidate biomarkers of kidney diseases. PMID:23825584

  14. 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

  15. 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

  16. 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.

  17. Incorporating significant amino acid pairs to identify O-linked glycosylation sites on transmembrane proteins and non-transmembrane proteins

    PubMed Central

    2010-01-01

    Background While occurring enzymatically in biological systems, O-linked glycosylation affects protein folding, localization and trafficking, protein solubility, antigenicity, biological activity, as well as cell-cell interactions on membrane proteins. Catalytic enzymes involve glycotransferases, sugar-transferring enzymes and glycosidases which trim specific monosaccharides from precursors to form intermediate structures. Due to the difficulty of experimental identification, several works have used computational methods to identify glycosylation sites. Results By investigating glycosylated sites that contain various motifs between Transmembrane (TM) and non-Transmembrane (non-TM) proteins, this work presents a novel method, GlycoRBF, that implements radial basis function (RBF) networks with significant amino acid pairs (SAAPs) for identifying O-linked glycosylated serine and threonine on TM proteins and non-TM proteins. Additionally, a membrane topology is considered for reducing the false positives on glycosylated TM proteins. Based on an evaluation using five-fold cross-validation, the consideration of a membrane topology can reduce 31.4% of the false positives when identifying O-linked glycosylation sites on TM proteins. Via an independent test, GlycoRBF outperforms previous O-linked glycosylation site prediction schemes. Conclusion A case study of Cyclic AMP-dependent transcription factor ATF-6 alpha was presented to demonstrate the effectiveness of GlycoRBF. Web-based GlycoRBF, which can be accessed at http://GlycoRBF.bioinfo.tw, can identify O-linked glycosylated serine and threonine effectively and efficiently. Moreover, the structural topology of Transmembrane (TM) proteins with glycosylation sites is provided to users. The stand-alone version of GlycoRBF is also available for high throughput data analysis. PMID:21034461

  18. Novel proteins identified in the insoluble byssal matrix of the freshwater zebra mussel.

    PubMed

    Gantayet, Arpita; Rees, David J; Sone, Eli D

    2014-04-01

    The freshwater zebra mussel, Dreissena polymorpha, is an invasive, biofouling species that adheres to a variety of substrates underwater, using a proteinaceous anchor called the byssus. The byssus consists of a number of threads with adhesive plaques at the tips. It contains the unusual amino acid 3, 4-dihydroxyphenylalanine (DOPA), which is believed to play an important role in adhesion, in addition to providing structural integrity to the byssus through cross-linking. Extensive DOPA cross-linking, however, renders the zebra mussel byssus highly resistant to protein extraction, and therefore limits byssal protein identification. We report here on the identification of seven novel byssal proteins in the insoluble byssal matrix following protein extraction from induced, freshly secreted byssal threads with minimal cross-linking. These proteins were identified by LC-MS/MS analysis of tryptic digests of the matrix proteins by spectrum matching against a zebra mussel cDNA library of genes unique to the mussel foot, the organ that secretes the byssus. All seven proteins were present in both the plaque and thread. Comparisons of the protein sequences revealed common features of zebra mussel byssal proteins, and several recurring sequence motifs. Although their sequences are unique, many of the proteins display similarities to marine mussel byssal proteins, as well as to adhesive and structural proteins from other species. The large expansion of the byssal proteome reported here represents an important step towards understanding zebra mussel adhesion. PMID:24057171

  19. 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

  20. 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

  1. Lsa63, a newly identified surface protein of Leptospira interrogans binds laminin and collagen IV.

    PubMed

    Vieira, Monica L; de Morais, Zenaide M; Gonçales, Amane P; Romero, Eliete C; Vasconcellos, Silvio A; Nascimento, Ana L T O

    2010-01-01

    Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease that affects populations worldwide. We have identified in proteomic studies a protein that is encoded by the gene LIC10314 and expressed in virulent strain of L. interrogans serovar Pomona. This protein was predicted to be surface exposed by PSORT program and contains a p83/100 domain identified by BLAST analysis that is conserved in protein antigens of several strains of Borrelia and Treponema spp. The proteins containing this domain have been claimed antigen candidates for serodiagnosis of Lyme borreliosis. Thus, we have cloned the LIC10314 and expressed the protein in Escherichia coli BL21-SI strain by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. This protein is conserved among several species of pathogenic Leptospira and absent in the saprophytic strain L. biflexa. We confirm by liquid-phase immunofluorescence assays with living organisms that this protein is most likely a new surface leptospiral protein. The ability of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC10314, named Lsa63 (Leptospiral surface adhesin of 63kDa), binds strongly to laminin and collagen IV in a dose-dependent and saturable fashion. In addition, Lsa63 is probably expressed during infection since it was recognized by antibodies of serum samples of confirmed-leptospirosis patients in convalescent phase of the disease. Altogether, the data suggests that this novel identified surface protein may be involved in leptospiral pathogenesis.

  2. 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

  3. 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.

  4. Proteomic Analysis of Oral Cavity Squamous Cell Carcinoma Specimens Identifies Patient Outcome–Associated Proteins

    PubMed Central

    Harris, Thomas M.; Du, Peicheng; Kawachi, Nicole; Belbin, Thomas J.; Wang, Yanhua; Schlecht, Nicolas F.; Ow, Thomas J.; Keller, Christian E.; Childs, Geoffrey J.; Smith, Richard V.; Angeletti, Ruth Hogue; Prystowsky, Michael B.; Lim, Jihyeon

    2015-01-01

    Context Global proteomic analysis of oral cavity squamous cell carcinoma was performed to identify changes that reflect patient outcomes. Objectives To identify differentially expressed proteins associated with patient outcomes and to explore the use of imaging mass spectrometry as a clinical tool to identify clinically relevant proteins. Design Two-dimensional separation of digested peptides generated from 43 specimens with high-resolution mass spectrometry identified proteins associated with disease-specific death, distant metastasis, and loco-regional recurrence. RNA expressions had been correlated to protein levels to test transcriptional regulation of clinically relevant proteins. Imaging mass spectrometry explored an alternative platform for assessing clinically relevant proteins that would complement surgical pathologic diagnosis. Results Seventy-two peptide features were found to be associated with 3 patient outcomes: disease-specific death (9), distant metastasis (16), and loco-regional recurrence (39); 8 of them were associated with multiple outcomes. Functional ontology revealed major changes in cell adhesion and calcium binding. Thirteen RNAs showed strong correlation with their encoded proteins, implying transcriptional control. Reduction of DSP, PKP1, and TRIM29 was associated with significantly shorter time to onset of distant metastasis. Reduction of PKP1 and TRIM29 correlated with poorer disease-specific survival. Additionally, S100A8 and S100A9 reductions were verified for their association with poor prognosis using imaging mass spectrometry, a platform more adaptable for use with surgical pathology. Conclusions Using global proteomic analysis, we have identified proteins associated with clinical outcomes. The list of clinically relevant proteins observed will provide a means to develop clinical assays for prognosis and optimizing treatment selection. PMID:25295583

  5. 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

  6. 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

  7. Dissecting protein function: an efficient protocol for identifying separation-of-function mutations that encode structurally stable proteins.

    PubMed

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

    2013-03-01

    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.

  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. A high-throughput method to examine protein-nucleotide interactions identifies targets of the bacterial transcriptional regulatory protein fur.

    PubMed

    Yu, Chunxiao; Lopez, Carlos A; Hu, Han; Xia, Yu; Freedman, David S; Reddington, Alexander P; Daaboul, George G; Unlü, M Selim; Genco, Caroline Attardo

    2014-01-01

    The Ferric uptake regulatory protein (Fur) is a transcriptional regulatory protein that functions to control gene transcription in response to iron in a number of pathogenic bacteria. In this study, we applied a label-free, quantitative and high-throughput analysis method, Interferometric Reflectance Imaging Sensor (IRIS), to rapidly characterize Fur-DNA interactions in vitro with predicted Fur binding sequences in the genome of Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea. IRIS can easily be applied to examine multiple protein-protein, protein-nucleotide and nucleotide-nucleotide complexes simultaneously and demonstrated here that seventy percent of the predicted Fur boxes in promoter regions of iron-induced genes bound to Fur in vitro with a range of affinities as observed using this microarray screening technology. Combining binding data with mRNA expression levels in a gonococcal fur mutant strain allowed us to identify five new gonococcal genes under Fur-mediated direct regulation.

  11. A structural bioinformatics approach for identifying proteins predisposed to bind linear epitopes on pre-selected target proteins.

    PubMed

    Choi, Eun Jung; Jacak, Ron; Kuhlman, Brian

    2013-04-01

    We have developed a protocol for identifying proteins that are predisposed to bind linear epitopes on target proteins of interest. The protocol searches through the protein database for proteins (scaffolds) that are bound to peptides with sequences similar to accessible, linear epitopes on the target protein. The sequence match is considered more significant if residues calculated to be important in the scaffold-peptide interaction are present in the target epitope. The crystal structure of the scaffold-peptide complex is then used as a template for creating a model of the scaffold bound to the target epitope. This model can then be used in conjunction with sequence optimization algorithms or directed evolution methods to search for scaffold mutations that further increase affinity for the target protein. To test the applicability of this approach we targeted three disease-causing proteins: a tuberculosis virulence factor (TVF), the apical membrane antigen (AMA) from malaria, and hemagglutinin from influenza. In each case the best scoring scaffold was tested, and binders with Kds equal to 37 μM and 50 nM for TVF and AMA, respectively, were identified. A web server (http://rosettadesign.med.unc.edu/scaffold/) has been created for performing the scaffold search process with user-defined target sequences.

  12. 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

  13. Identifying Gastric Cancer Related Genes Using the Shortest Path Algorithm and Protein-Protein Interaction Network

    PubMed Central

    Shi, Ying; Li, Li-Peng; Ren, Hui

    2014-01-01

    Gastric cancer, as one of the leading causes of cancer related deaths worldwide, causes about 800,000 deaths per year. Up to now, the mechanism underlying this disease is still not totally uncovered. Identification of related genes of this disease is an important step which can help to understand the mechanism underlying this disease, thereby designing effective treatments. In this study, some novel gastric cancer related genes were discovered based on the knowledge of known gastric cancer related ones. These genes were searched by applying the shortest path algorithm in protein-protein interaction network. The analysis results suggest that some of them are indeed involved in the biological process of gastric cancer, which indicates that they are the actual gastric cancer related genes with high probability. It is hopeful that the findings in this study may help promote the study of this disease and the methods can provide new insights to study various diseases. PMID:24729971

  14. Urine Proteins Identified by Two-Dimensional Differential Gel Electrophoresis Facilitate the Differential Diagnoses of Scrapie

    PubMed Central

    Lamoureux, Lise; Simon, Sharon L. R.; Plews, Margot; Ruddat, Viola; Brunet, Simone; Graham, Catherine; Czub, Stefanie; Knox, J. David

    2013-01-01

    The difficulty in developing a diagnostic assay for Creutzfeldt - Jakob disease (CJD) and other transmissible spongiform encephalopathies (TSEs) stems in part from the fact that the infectious agent is an aberrantly folded form of an endogenous cellular protein. This precludes the use of the powerful gene based technologies currently applied to the direct detection of other infectious agents. To circumvent this problem our research objective has been to identify a set of proteins exhibiting characteristic differential abundance in response to TSE infection. The objective of the present study was to assess the disease specificity of differentially abundant urine proteins able to identify scrapie infected mice. Two-dimensional differential gel electrophoresis was used to analyze longitudinal collections of urine samples from both prion-infected mice and a transgenic mouse model of Alzheimer's disease. The introduction of fluorescent dyes, that allow multiple samples to be co-resolved and visualized on one two dimensional gel, have increased the accuracy of this methodology for the discovery of robust protein biomarkers for disease. The accuracy of a small panel of differentially abundant proteins to correctly classify an independent naïve sample set was determined. The results demonstrated that at the time of clinical presentation the differential abundance of urine proteins were capable of identifying the prion infected mice with 87% sensitivity and 93% specificity. The identity of the diagnostic differentially abundant proteins was investigated by mass spectrometry. PMID:23704971

  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.

  16. 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.

  17. 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.

  18. 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

  19. Novel method for identifying sequence-specific DNA-binding proteins.

    PubMed Central

    Levens, D; Howley, P M

    1985-01-01

    We developed a general method for the enrichment and identification of sequence-specific DNA-binding proteins. A well-characterized protein-DNA interaction is used to isolate from crude cellular extracts or fractions thereof proteins which bind to specific DNA sequences; the method is based solely on this binding property of the proteins. The DNA sequence of interest, cloned adjacent to the lac operator DNA segment is incubated with a lac repressor-beta-galactosidase fusion protein which retains full operator and inducer binding properties. The DNA fragment bound to the lac repressor-beta-galactosidase fusion protein is precipitated by the addition of affinity-purified anti-beta-galactosidase immobilized on beads. This forms an affinity matrix for any proteins which might interact specifically with the DNA sequence cloned adjacent to the lac operator. When incubated with cellular extracts in the presence of excess competitor DNA, any protein(s) which specifically binds to the cloned DNA sequence of interest can be cleanly precipitated. When isopropyl-beta-D-thiogalactopyranoside is added, the lac repressor releases the bound DNA, and thus the protein-DNA complex consisting of the specific restriction fragment and any specific binding protein(s) is released, permitting the identification of the protein by standard biochemical techniques. We demonstrate the utility of this method with the lambda repressor, another well-characterized DNA-binding protein, as a model. In addition, with crude preparations of the yeast mitochondrial RNA polymerase, we identified a 70,000-molecular-weight peptide which binds specifically to the promoter region of the yeast mitochondrial 14S rRNA gene. Images PMID:3016526

  20. 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

  1. 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.

  2. 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

  3. Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases.

    PubMed

    Henzel, W J; Billeci, T M; Stults, J T; Wong, S C; Grimley, C; Watanabe, C

    1993-06-01

    A rapid method for the identification of known proteins separated by two-dimensional gel electrophoresis is described in which molecular masses of peptide fragments are used to search a protein sequence database. The peptides are generated by in situ reduction, alkylation, and tryptic digestion of proteins electroblotted from two-dimensional gels. Masses are determined at the subpicomole level by matrix-assisted laser desorption/ionization mass spectrometry of the unfractionated digest. A computer program has been developed that searches the protein sequence database for multiple peptides of individual proteins that match the measured masses. To ensure that the most recent database updates are included, a theoretical digest of the entire database is generated each time the program is executed. This method facilitates simultaneous processing of a large number of two-dimensional gel spots. The method was applied to a two-dimensional gel of a crude Escherichia coli extract that was electroblotted onto poly(vinylidene difluoride) membrane. Ten randomly chosen spots were analyzed. With as few as three peptide masses, each protein was uniquely identified from over 91,000 protein sequences. All identifications were verified by concurrent N-terminal sequencing of identical spots from a second blot. One of the spots contained an N-terminally blocked protein that required enzymatic cleavage, peptide separation, and Edman degradation for confirmation of its identity.

  4. 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

  5. Identifying proteins in zebrafish embryos using spectral libraries generated from dissected adult organs and tissues.

    PubMed

    van der Plas-Duivesteijn, Suzanne J; Mohammed, Yassene; Dalebout, Hans; Meijer, Annemarie; Botermans, Anouk; Hoogendijk, Jordy L; Henneman, Alex A; Deelder, André M; Spaink, Herman P; Palmblad, Magnus

    2014-03-01

    Spectral libraries provide a sensitive and accurate method for identifying peptides from tandem mass spectra, complementary to searching genome-derived databases or sequencing de novo. Their application requires comprehensive libraries including peptides from low-abundant proteins. Here we describe a method for constructing such libraries using biological differentiation to "fractionate" the proteome by harvesting adult organs and tissues and build comprehensive libraries for identifying proteins in zebrafish (Danio rerio) embryos and larvae (an important and widely used model system). Hierarchical clustering using direct comparison of spectra was used to prioritize organ selection. The resulting and publicly available library covers 14,164 proteins, significantly improved the number of peptide-spectrum matches in zebrafish developmental stages, and can be used on data from different instruments and laboratories. The library contains information on tissue and organ expression of these proteins and is also applicable for adult experiments. The approach itself is not limited to zebrafish but would work for any model system.

  6. 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.

  7. A 700-year record of mercury in avian eggshells of Guangjin Island, South China Sea.

    PubMed

    Xu, Li-Qiang; Liu, Xiao-Dong; Sun, Li-guang; Chen, Qian-Qian; Yan, Hong; Liu, Yi; Luo, Yu-Han; Huang, Jing

    2011-04-01

    Ancient eggshells over the past 700 years were extracted from an ornithogenic sediment profile on Guangjin Island, South China Sea. Based on SEM and nitrogen isotope analyses, we determined that neither post-depositional processes nor seabirds' dietary changes had a large influence on eggshell Hg levels. The historical change of Hg in these eggshells was reconstructed. Eggshell Hg was a marker for past Hg deposition in marine environment. The eggshell Hg showed three small peaks at around 1300AD, 1600 AD and 1700-1750AD and rapid increase since 1800 AD. Before 1970 AD the Hg deposition in the Xisha area had global distribution characteristics, with increased Hg emissions due to global anthropogenic activities in industrial times. However, after 1970 AD, a further sharp increase up to present day occurred, implying that the Hg production center had gradually shifted from Europe and America to Asia.

  8. 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

  9. 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

  10. 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

  11. 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

  12. Proteomic analysis identifies differentially expressed proteins after red propolis treatment in Hep-2 cells.

    PubMed

    Frozza, Caroline Olivieri da Silva; Ribeiro, Tanara da Silva; Gambato, Gabriela; Menti, Caroline; Moura, Sidnei; Pinto, Paulo Marcos; Staats, Charley Christian; Padilha, Francine Ferreira; Begnini, Karine Rech; de Leon, Priscila Marques Moura; Borsuk, Sibele; Savegnago, Lucielli; Dellagostin, Odir; Collares, Tiago; Seixas, Fabiana Kömmling; Henriques, João Antonio Pêgas; Roesch-Ely, Mariana

    2014-01-01

    Here we investigated alterations in the protein profile of Hep-2 treated with red propolis using two-dimensional electrophoresis associated to mass spectrometry and apoptotic rates of cells treated with and without red propolis extracts through TUNEL and Annexin-V assays. A total of 325 spots were manually excised from the two-dimensional gel electrophoresis and 177 proteins were identified using LC-MS-MS. Among all proteins identified that presented differential expression, most were down-regulated in presence of red propolis extract at a concentration of 120 μg/mL (IC50): GRP78, PRDX2, LDHB, VIM and TUBA1A. Only two up-regulated proteins were identified in this study in the non-cytotoxic (6 μg/mL) red propolis treated group: RPLP0 and RAD23B. TUNEL staining assay showed a markedly increase in the mid- to late-stage apoptosis of Hep-2 cells induced by red propolis at concentrations of 60 and 120 μg/mL when compared with non-treated cells. The increase of late apoptosis was confirmed by in situ Annexin-V analysis in which red propolis extract induced late apoptosis in a dose-dependent manner. The differences in tumor cell protein profiles warrant further investigations including isolation of major bioactive compounds of red propolis in different cell lines using proteomics and molecular tests to validate the protein expression here observed.

  13. 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

  14. 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.

  15. Identifying Schistosoma japonicum excretory/secretory proteins and their interactions with host immune system.

    PubMed

    Liao, Qi; Yuan, Xiongying; Xiao, Hui; Liu, Changning; Lv, Zhiyue; Zhao, Yi; Wu, Zhongdao

    2011-01-01

    Schistosoma japonicum is a major infectious agent of schistosomiasis. It has been reported that large number of proteins excreted and secreted by S. japonicum during its life cycle are important for its infection and survival in definitive hosts. These proteins can be used as ideal candidates for vaccines or drug targets. In this work, we analyzed the protein sequences of S. japonicum and found that compared with other proteins in S. japonicum, excretory/secretory (ES) proteins are generally longer, more likely to be stable and enzyme, more likely to contain immune-related binding peptides and more likely to be involved in regulation and metabolism processes. Based on the sequence difference between ES and non-ES proteins, we trained a support vector machine (SVM) with much higher accuracy than existing approaches. Using this SVM, we identified 191 new ES proteins in S. japonicum, and further predicted 7 potential interactions between these ES proteins and human immune proteins. Our results are useful to understand the pathogenesis of schistosomiasis and can serve as a new resource for vaccine or drug targets discovery for anti-schistosome.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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

  3. Coevolutionary Analysis Identifies Protein–Protein Interaction Sites between HIV-1 Reverse Transcriptase and Integrase

    PubMed Central

    Arachchilage, Madara Hetti; Piontkivska, Helen

    2016-01-01

    The replication of human immunodeficiency virus-1 (HIV-1) requires reverse transcription of the viral RNA genome and integration of newly synthesized pro-viral DNA into the host genome. This is mediated by the viral proteins reverse transcriptase (RT) and integrase (IN). The formation and stabilization of the pre-integration complex (PIC), which is an essential step for reverse transcription, nuclear import, chromatin targeting, and subsequent integration, involves direct and indirect modes of interaction between RT and IN proteins. While epitope-based treatments targeting IN–viral DNA and IN–RT complexes appear to be a promising combination for an anti-HIV treatment, the mechanisms of IN-RT interactions within the PIC are not well understood due to the transient nature of the protein complex and the intrinsic flexibility of its components. Here, we identify potentially interacting regions between the IN and RT proteins within the PIC through the coevolutionary analysis of amino acid sequences of the two proteins. Our results show that specific regions in the two proteins have strong coevolutionary signatures, suggesting that these regions either experience direct and prolonged interactions between them that require high affinity and/or specificity or that the regions are involved in interactions mediated by dynamic conformational changes and, hence, may involve both direct and indirect interactions. Other regions were found to exhibit weak, but positive correlations, implying interactions that are likely transient and/or have low affinity. We identified a series of specific regions of potential interactions between the IN and RT proteins (e.g., specific peptide regions within the C-terminal domain of IN were identified as potentially interacting with the Connection domain of RT). Coevolutionary analysis can serve as an important step in predicting potential interactions, thus informing experimental studies. These studies can be integrated with structural data

  4. Systematic phenotypic screen of Arabidopsis peroxisomal mutants identifies proteins involved in β-oxidation.

    PubMed

    Cassin-Ross, Gaëlle; Hu, Jianping

    2014-11-01

    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

  5. Systematic phenotypic screen of Arabidopsis peroxisomal mutants identifies proteins involved in β-oxidation.

    PubMed

    Cassin-Ross, Gaëlle; Hu, Jianping

    2014-11-01

    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.

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. Protein array–based profiling of CSF identifies RBPJ as an autoantigen in multiple sclerosis

    PubMed Central

    Querol, Luis; Clark, Pamela L.; Bailey, Mary A.; Cotsapas, Chris; Cross, Anne H.; Hafler, David A.; Kleinstein, Steven H.; Lee, Jae-Yun; Yaari, Gur

    2013-01-01

    Objective: To profile the reactivity of CSF-derived immunoglobulin from patients with multiple sclerosis (MS) against a large panel of antigens, to identify disease-specific reactivities. Methods: CSF from subjects with MS with elevated immunoglobulin G and CSF from control subjects presenting with other inflammatory neurologic disease were screened against a protein array consisting of 9,393 proteins. Reactivity to a candidate protein identified using these arrays was confirmed with ELISA and immunocytochemistry. Results: Autoantibodies against one protein on the array, recombination signal binding protein for immunoglobulin kappa J region (RBPJ), discriminated between patients with MS and controls (p = 0.0052). Using a large validation cohort, we found a higher prevalence of autoantibodies against RBPJ in the CSF of patients with MS (12.5%) compared with the CSF of patients with other neurologic diseases (1.6%; p = 0.02) by ELISA. This difference in reactivity was restricted to the CSF as serum reactivity against RBPJ did not differ between patients and controls. The presence of CSF autoantibodies against RBPJ was further confirmed by immunocytochemistry. Conclusions: These data indicate that RBPJ, a ubiquitous protein of the Notch signaling pathway that plays an important role in Epstein-Barr virus infection, is a novel MS autoantigen candidate that is recognized by CSF-derived immunoglobulin G in a subset of patients with MS. PMID:23921886

  11. Functional characterization of candidate effector proteins identified from the wheat scab fungus Fusarium graminearum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fungal pathogens often produce certain small secreted cysteine-rich proteins (SSCPs) during pathogenesis that may function in triggering resistance or susceptibility in specific host plants. We have recently identified a total of 190 SSCPs encoded in the genome of the wheat scab fungus Fusarium gra...

  12. 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.

  13. 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...

  14. 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

  15. 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

  16. 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

  17. 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

  18. An ensemble method with hybrid features to identify extracellular matrix proteins.

    PubMed

    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.

  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.

  20. 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.

  1. 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

  2. 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.

  3. Synthesis of nano-textured biocompatible scaffolds from chicken eggshells

    NASA Astrophysics Data System (ADS)

    Asghar, Waseem; Kim, Young-Tae; Ilyas, Azhar; Sankaran, Jeyantt; Wan, Yuan; Iqbal, Samir M.

    2012-11-01

    Cell adhesion, morphology and growth are influenced by surface topography at nano and micrometer scales. Nano-textured surfaces are prepared using photolithography, plasma etching and long polymer chemical etching which are cost prohibitive and require specialized equipment. This article demonstrates a simple approach to synthesize nano-textured scaffolds from chicken eggshells. Varieties of pattern are made on the eggshells like micro-needle forests and nanopores, giving very uniform nano-textures to the surfaces. The surfaces are characterized for chemical composition and crystal phase. The novel patterns are transferred to PDMS surfaces and the nano-textured PDMS surfaces are used to study the effect of texturing on human fibroblast cell growth and attachment. The effects of surface topographies, along with laminin coating on cell cultures, are also studied. We find an exciting phenomenon that the initial seeding density of the fibroblast cells affects the influence of the nano-texturing on cell growth. These nano-textured surfaces give 16 times more fibroblast growth when compared to flat PDMS surfaces. The novel nano-textured patterns also double the laminin adsorption on PDMS.

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

    USGS Publications Warehouse

    Clark, D.R.; 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.

  5. Protein kinase C phosphorylates a recently identified membrane skeleton-associated calmodulin-binding protein in human erythrocytes.

    PubMed

    Ling, E; Gardner, K; Bennett, V

    1986-10-25

    A membrane skeleton-associated protein with calmodulin-binding activity recently has been purified and characterized from human erythrocytes (Gardner, K. and Bennett, V. (1986) J. Biol. Chem. 261, 1339-1348). This new protein (CaM-BP103/97) has now been identified as a major substrate for protein kinase C in erythrocytes since phosphorylation of both of its subunits (Mr = 103,000 and 97,000) is elevated 3-15-fold in the presence of the phorbol ester, 12-O-tetradecanoylphorbol beta-acetate (TPA), under the following conditions: ghost membranes incubated with protein kinase C purified from rat brain, ghost membranes from erythrocytes pretreated with TPA, and intact erythrocytes metabolically labeled with 32PO4 and stimulated by TPA. The sites of phosphorylation of this protein by exogenous and endogenous protein kinase C are identical since two-dimensional 32P-peptide maps of both subunits labeled by either endogenous or exogenous enzyme are indistinguishable. Each subunit of CaM-BP103/97 accepts up to 3 mol of phosphate/polypeptide chain. In the presence of low calcium concentrations and in the absence of cytosol, the phosphorylation of CaM-BP103/97 is, on a molar basis, equal to or greater than that of proteins 4.1 and 4.9. As a target for both calmodulin and protein kinase C, CaM-BP103/97 is likely to play a key role in the effect of calcium on erythrocyte membrane shape and stability.

  6. 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

  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. 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

  9. Proteomic analysis identifies interleukin 11 regulated plasma membrane proteins in human endometrial epithelial cells in vitro

    PubMed Central

    2011-01-01

    Background During the peri-implantation period, the embryo adheres to an adequately prepared or receptive endometrial surface epithelium. Abnormal embryo adhesion to the endometrium results in embryo implantation failure and infertility. Endometrial epithelial cell plasma membrane proteins critical in regulating adhesion may potentially be infertility biomarkers or targets for treating infertility. Interleukin (IL) 11 regulates human endometrial epithelial cells (hEEC) adhesion. Its production is abnormal in women with infertility. The objective of the study was to identify IL11 regulated plasma membrane proteins in hEEC in vitro using a proteomic approach. Methods Using a 2D-differential in-gel electrophoresis (DIGE) electrophoresis combined with LCMS/MS mass spectrometry approach, we identified 20 unique plasma membrane proteins differentially regulated by IL11 in ECC-1 cells, a hEEC derived cell line. Two IL11 regulated proteins with known roles in cell adhesion, annexin A2 (ANXA2) and flotillin-1 (FLOT1), were validated by Western blot and immunocytochemistry in hEEC lines (ECC-1 and an additional cell line, Ishikawa) and primary hEEC. Flotilin-1 was further validated by immunohistochemistry in human endometrium throughout the menstrual cycle (n = 6-8/cycle). Results 2D-DIGE analysis identified 4 spots that were significantly different between control and IL11 treated group. Of these 4 spots, there were 20 proteins that were identified with LCMS/MS. Two proteins; ANXA2 and FLOT1 were chosen for further analyses and have found to be significantly up-regulated following IL11 treatment. Western blot analysis showed a 2-fold and a 2.5-fold increase of ANXA2 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. Similarly, a 1.8-fold and a 2.3/2.4-fold increase was also observed for FLOT1 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. In vitro, IL11 induced stronger ANXA2 expression on cell surface of primary hEEC and ECC-1 whilst

  10. 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

  11. 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

  12. 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

  13. Early life stress shapes female reproductive strategy through eggshell pigmentation in Japanese quail.

    PubMed

    Duval, Camille; Zimmer, Cédric; Mikšík, Ivan; Cassey, Phillip; Spencer, Karen A

    2014-11-01

    Physiological constraints on colouration have been widely reported; especially in birds, which trade-off antioxidant responses against colourful costly signals. One female extended phenotypic trait, which might also highlight important physiological trade-offs, is the pigmentation of their eggshells. In ground-nesting species, producing eggs that are visually undetectable by predators is the best camouflage strategy. However, the condition-dependence of eggshell pigmentation, and the pigments role in oxidative stress, may constrain females to trade-off between their antioxidant capacity and maximising the camouflage of their eggs when they deposit eggshell pigments. Developmental stress is one factor that influences female antioxidant capacity, and could lead to variations in eggshell pigmentation that might have crucial consequences on individual fitness if egg crypsis is compromised especially under stressful conditions. We investigated the interaction between developmental and breeding conditions with respect to eggshell pigmentation in Japanese quail. We studied 30 females that bred under both control and stressful conditions, and were exposed to pre- and/or post-natal stress, or neither. Pre- and post-natal stress independently influenced eggshell pigmentation strategies under stressful breeding conditions. Under stressful reproduction, eggshell protoporphyrin concentration and maculation were affected by pre-natal stress, whereas eggshell reflectance and biliverdin concentration were influenced by post-natal stress. These changes may reflect potential adaptive strategies shaped by developmental stress, but additional data on the benefit of egg crypsis in quail, combined with studies on the role of both pigments on chick survival, will help to clarify whether early life stress can enhance fitness through eggshell pigmentation when developmental and reproductive environments match.

  14. Mass spectrometric approach for identifying putative plasma membrane proteins of Arabidopsis leaves associated with cold acclimation.

    PubMed

    Kawamura, Yukio; Uemura, Matsuo

    2003-10-01

    Although enhancement of freezing tolerance in plants during cold acclimation is closely associated with an increase in the cryostability of plasma membrane, the molecular mechanism for the increased cryostability of plasma membrane is still to be elucidated. In Arabidopsis, enhanced freezing tolerance was detectable after cold acclimation at 2 degrees C for as short as 1 day, and maximum freezing tolerance was attained after 1 week. To identify the plasma membrane proteins that change in quantity in response to cold acclimation, a highly purified plasma membrane fraction was isolated from leaves before and during cold acclimation, and the proteins in the fraction were separated with gel electrophoresis. We found that there were substantial changes in the protein profiles after as short as 1 day of cold acclimation. Subsequently, using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS), we identified 38 proteins that changed in quantity during cold acclimation. The proteins that changed in quantity during the first day of cold acclimation include those that are associated with membrane repair by membrane fusion, protection of the membrane against osmotic stress, enhancement of CO2 fixation, and proteolysis.

  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. A Novel Assay to Identify the Trafficking Proteins that Bind to Specific Vesicle Populations.

    PubMed

    Bentley, Marvin; Banker, Gary

    2015-01-01

    Here we describe a method capable of identifying interactions between candidate trafficking proteins and a defined vesicle population in intact cells. The assay involves the expression of an FKBP12-rapamycin binding domain (FRB)-tagged candidate vesicle-binding protein that can be inducibly linked to an FKBP-tagged molecular motor. If the FRB-tagged candidate protein binds the labeled vesicles, then linking the FRB and FKBP domains recruits motors to the vesicles and causes a predictable, highly distinctive change in vesicle trafficking. We describe two versions of the assay: a general protocol for use in cells with a typical microtubule-organizing center and a specialized protocol designed to detect protein-vesicle interactions in cultured neurons. We have successfully used this assay to identify kinesins and Rabs that bind to a variety of different vesicle populations. In principle, this assay could be used to investigate interactions between any category of vesicle trafficking proteins and any vesicle population that can be specifically labeled. PMID:26621371

  17. Identifying DNA-binding proteins using structural motifs and the electrostatic potential.

    PubMed

    Shanahan, Hugh P; Garcia, Mario A; Jones, Susan; Thornton, Janet M

    2004-01-01

    Robust methods to detect DNA-binding proteins from structures of unknown function are important for structural biology. This paper describes a method for identifying such proteins that (i) have a solvent accessible structural motif necessary for DNA-binding and (ii) a positive electrostatic potential in the region of the binding region. We focus on three structural motifs: helix-turn-helix (HTH), helix-hairpin-helix (HhH) and helix-loop-helix (HLH). We find that the combination of these variables detect 78% of proteins with an HTH motif, which is a substantial improvement over previous work based purely on structural templates and is comparable to more complex methods of identifying DNA-binding proteins. Similar true positive fractions are achieved for the HhH and HLH motifs. We see evidence of wide evolutionary diversity for DNA-binding proteins with an HTH motif, and much smaller diversity for those with an HhH or HLH motif. PMID:15356290

  18. Quantitative Proteomic Analysis of Ovarian Cancer Cells Identified Mitochondrial Proteins Associated with Paclitaxel Resistance

    PubMed Central

    Tian, Yuan; Tan, Aik-Choon; Sun, Xiaer; Olson, Matthew T; Xie, Zhi; Jinawath, Natini; Chan, Daniel W.; Shih, Ie-Ming; Zhang, Zhen; Zhang, Hui

    2010-01-01

    Paclitaxel has been widely used as an anti-mitotic agent in chemotherapy for a variety of cancers and adds substantial efficacy as the first-line chemotherapeutic regimen for ovarian cancers. However, the frequent occurrence of paclitaxel resistance limits its function in long-term management. Despite abundant clinical and cellular demonstration of paclitaxel resistant tumors, the molecular mechanisms leading to paclitaxel resistance are poorly understood. Using genomic approaches, we have previously identified an association between a BTB/POZ gene, Nac1, and paclitaxel resistance in ovarian cancer. The experiments presented here have applied multiple quantitative proteomic methods to identify protein changes associated with paclitaxel resistance and Nac1 function. The SKOV-3 ovarian serous carcinoma cell line, which has inducible expression of dominant negative Nac1, was used to determine the paclitaxel treatment associated changes in the presence and absence of functional Nac1. Quantitative proteomic analyses were performed using iTRAQ labeling and mass spectrometry. Two label-free quantitative proteomic methods: LC-MS and spectral count were used to increase confidence of proteomic quantification. A total of 1371 proteins were quantified by at least one of the quantitative proteomic methods. Candidate proteins related to paclitaxel and NAC1 function were identified in this study. Go analysis of the protein changes identified upon paclitaxel resistance revealed that cell component enrichment related to mitochondria. Moreover, tubulin and mitochondrial proteins were the major cellular components with changes associated with paclitaxel treatment. This suggests that mitochondria may play a role in paclitaxel resistance. PMID:21113235

  19. 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

  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 sequence-based hybrid predictor for identifying conformationally ambivalent regions in proteins

    PubMed Central

    2009-01-01

    Background Proteins are dynamic macromolecules which may undergo conformational transitions upon changes in environment. As it has been observed in laboratories that protein flexibility is correlated to essential biological functions, scientists have been designing various types of predictors for identifying structurally flexible regions in proteins. In this respect, there are two major categories of predictors. One category of predictors attempts to identify conformationally flexible regions through analysis of protein tertiary structures. Another category of predictors works completely based on analysis of the polypeptide sequences. As the availability of protein tertiary structures is generally limited, the design of predictors that work completely based on sequence information is crucial for advances of molecular biology research. Results In this article, we propose a novel approach to design a sequence-based predictor for identifying conformationally ambivalent regions in proteins. The novelty in the design stems from incorporating two classifiers based on two distinctive supervised learning algorithms that provide complementary prediction powers. Experimental results show that the overall performance delivered by the hybrid predictor proposed in this article is superior to the performance delivered by the existing predictors. Furthermore, the case study presented in this article demonstrates that the proposed hybrid predictor is capable of providing the biologists with valuable clues about the functional sites in a protein chain. The proposed hybrid predictor provides the users with two optional modes, namely, the high-sensitivity mode and the high-specificity mode. The experimental results with an independent testing data set show that the proposed hybrid predictor is capable of delivering sensitivity of 0.710 and specificity of 0.608 under the high-sensitivity mode, while delivering sensitivity of 0.451 and specificity of 0.787 under the high

  2. A High-Throughput Method to Examine Protein-Nucleotide Interactions Identifies Targets of the Bacterial Transcriptional Regulatory Protein Fur

    PubMed Central

    Hu, Han; Xia, Yu; Freedman, David S.; Reddington, Alexander P.; Daaboul, George G.; Ünlü, M. Selim; Genco, Caroline Attardo

    2014-01-01

    The Ferric uptake regulatory protein (Fur) is a transcriptional regulatory protein that functions to control gene transcription in response to iron in a number of pathogenic bacteria. In this study, we applied a label-free, quantitative and high-throughput analysis method, Interferometric Reflectance Imaging Sensor (IRIS), to rapidly characterize Fur-DNA interactions in vitro with predicted Fur binding sequences in the genome of Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea. IRIS can easily be applied to examine multiple protein-protein, protein-nucleotide and nucleotide-nucleotide complexes simultaneously and demonstrated here that seventy percent of the predicted Fur boxes in promoter regions of iron-induced genes bound to Fur in vitro with a range of affinities as observed using this microarray screening technology. Combining binding data with mRNA expression levels in a gonococcal fur mutant strain allowed us to identify five new gonococcal genes under Fur-mediated direct regulation. PMID:24811061

  3. 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.

  4. 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

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

    PubMed Central

    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

    2016-01-01

    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

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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.

  15. A proteomic approach to identify proteins from Trichuris trichiura extract with immunomodulatory effects.

    PubMed

    Santos, L N; Gallo, M B C; Silva, E S; Figueiredo, C A V; Cooper, P J; Barreto, M L; Loureiro, S; Pontes-de-Carvalho, L C; Alcantara-Neves, N M

    2013-01-01

    Infections with Trichuris trichiura and other trichurid nematodes have been reported to display protective effects against atopy, allergic and autoimmune diseases. The aims of the present study were to investigate the immunomodulatory properties of T. trichiura adult worm extract (TtE) and its fractions (TtEFs) on the production of cytokines by peripheral blood mononuclear cells and to identify their proteinaceous components. Fourteen TtEFs were obtained by ion exchange chromatography and tested for effects on cytokine production by peripheral blood mononuclear cells. The molecular constituents of the six most active fractions were evaluated using nano-LC/mass spectrometry. The homology between T. trichiura and the related nematode Trichinella spiralis was used to identify 12 proteins in TtEFs. Among those identified, fructose biphosphate aldolase, a homologue of macrophage migration inhibitory factor and heat-shock protein 70 may contribute to the immunomodulatory effects of TtEFs. The identification of such proteins could lead to the development of novel drugs for the therapy of allergic and other inflammatory diseases.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

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

    PubMed Central

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

    2015-01-01

    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

  3. 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.

  4. 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

  5. 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...

  6. Pathogen specificity of Treponema pallidum subsp. pallidum integral membrane proteins identified by phase partitioning with Triton X-114.

    PubMed

    Radolf, J D; Norgard, M V

    1988-07-01

    The antigenically conserved proteins of Treponema pallidum subsp. pallidum and four nonpathogenic cultivatable treponemes were investigated by phase partitioning with the nonionic detergent Triton X-114 and immunoblot analysis. None of the T. pallidum integral membrane proteins identified by phase partitioning (detergent-phase proteins) appeared to be antigenically related to proteins of the nonpathogens. Protease-resistant material similar to lipopolysaccharide was identified in the detergent phase from T. phagedenis biotype Reiter but was not detected in T. pallidum.

  7. 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.

  8. 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

  9. 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

  10. 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

  11. 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

  12. Laying date, incubation and egg breakage as determinants of bacterial load on bird eggshells: experimental evidence.

    PubMed

    Soler, Juan José; Ruiz-Rodríguez, Magdalena; Martín-Vivaldi, Manuel; Peralta-Sánchez, Juan Manuel; Ruiz-Castellano, Cristina; Tomás, Gustavo

    2015-09-01

    Exploring factors guiding interactions of bacterial communities with animals has become of primary importance for ecologists and evolutionary biologists during the last years because of their likely central role in the evolution of animal life history traits. We explored the association between laying date and eggshell bacterial load (mesophilic bacteria, Enterobacteriaceae, Staphylococci, and Enterococci) in natural and artificial magpie (Pica pica) nests containing fresh commercial quail (Coturnix coturnix) eggs. We manipulated hygiene conditions by spilling egg contents on magpie and artificial nests and explored experimental effects during the breeding season. Egg breakage is a common outcome of brood parasitism by great spotted cuckoos (Clamator glandarius) on the nests of magpie, one of its main hosts. We found that the treatment increased eggshell bacterial load in artificial nests, but not in magpie nests with incubating females, which suggests that parental activity prevents the proliferation of bacteria on the eggshells in relation to egg breakage. Moreover, laying date was positively related to eggshell bacterial load in active magpie nests, but negatively in artificial nests. The results suggest that variation in parental characteristics of magpies rather than climatic variation during the breeding season explained the detected positive association. Because the eggshell bacterial load is a proxy of hatching success, the detected positive association between eggshell bacterial loads and laying date in natural, but not in artificial nests, suggests that the generalized negative association between laying date and avian breeding success can be, at least partially, explained by differential bacterial effects.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. Identifying Plasmodium falciparum merozoite surface protein-10 human erythrocyte specific binding regions.

    PubMed

    Puentes, Alvaro; Ocampo, Marisol; Rodríguez, Luis Eduardo; Vera, Ricardo; Valbuena, John; Curtidor, Hernando; García, Javier; López, Ramsés; Tovar, Diana; Cortes, Jimena; Rivera, Zuly; Patarroyo, Manuel Elkin

    2005-05-01

    Receptor-ligand interactions between synthetic peptides and normal human erythrocytes were studied to determine P. falciparum merozoite surface protein-10 (MSP-10) regions specifically binding to membrane surface receptors on human erythrocytes. Three MSP-10 protein High Activity Binding Peptides (HABPs) were identified, whose binding to erythrocytes became saturable and sensitive on being treated with neuraminidase, trypsin and chymotrypsin. Some of them specifically recognised a 50 kDa erythrocyte membrane protein. Some HABPs inhibited in vitro P. falciparum merozoite invasion of erythrocytes by 70%, suggesting that MSP-10 protein's possible role in the invasion process probably functions by using similar mechanisms to those described for other MSP family antigens. In addition to above results, the high homology in amino-acid sequence and superimposition of both MSP-10, MSP-8 and MSP-1 EGF-like domains and HABPs 31132, 26373 and 5501 suggest that tridimensional structure could be playing an important role in the invasion process and in designing synthetic multi-stage anti-malarial vaccines.

  18. 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).

  19. Optimisation of Downscaled Tandem Affinity Purifications to Identify Core Protein Complexes

    PubMed Central

    Haura, Eric B.; Sacco, Roberto; Li, Jiannong; Müller, André C.; Grebien, Florian; Superti-Furga, Giulio; Bennett, Keiryn L.

    2013-01-01

    In this study we show that via stable, retroviral-expression of tagged EGFR del (L747-S752 deletion mutant) in the PC9 lung cancer cell line and stable doxycycline-inducible expression of tagged Grb2 using a Flp-mediated recombination HEK293 cell system, the SH-TAP can be downscaled to 5 to 12.5 mg total protein input (equivalent to 0.5 - 1 × 15 cm culture plate or 4 - 8 × 106 cells). The major constituents of the EGFR del complex (USB3B, GRB2, ERRFI, HSP7C, GRP78, HSP71) and the Grb2 complex (ARHG5, SOS1, ARG35, CBL, CBLB, PTPRA, SOS2, DYN2, WIPF2, IRS4) were identified. Adjustment of the quantity of digested protein injected into the mass spectrometer reveals that optimisation is required as high quantities of material led to a decrease in protein sequence coverage and the loss of some interacting proteins. This investigation should aid other researchers in performing tandem affinity purifications in general, and in particular, from low quantities of input material. PMID:24077984

  20. 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.

  1. 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

  2. A high throughput screening strategy to identify protein-protein interaction inhibitors that block the Fanconi anemia DNA repair pathway

    PubMed Central

    Voter, Andrew F.; Manthei, Kelly A.

    2016-01-01

    Induction of the Fanconi anemia (FA) DNA repair pathway is a common mechanism by which tumors evolve resistance to DNA crosslinking chemotherapies. Proper execution of the FA pathway requires interaction between the FA complementation group M protein (FANCM) and the RecQ-mediated genome instability protein (RMI) complex, and mutations that disrupt FANCM/RMI interactions sensitize cells to DNA crosslinking agents. Inhibitors that block FANCM/RMI complex formation could be useful therapeutics for re-sensitizing tumors that have acquired chemotherapeutic resistance. To identify such inhibitors, we have developed and validated high-throughput fluorescence polarization and proximity assays that are sensitive to inhibitors that disrupt interactions between the RMI complex and its binding site on FANCM (a peptide referred to as MM2). A pilot screen of 74,807 small molecules was performed using the fluorescence polarization assay. Hits from the primary screen were further tested using the proximity assay and an orthogonal proximity assay was used to assess inhibitor selectivity. Direct physical interaction between the RMI complex and the most selective inhibitor identified through the screening process was measured by surface plasmon resonance and isothermal titration calorimetry. Observation of direct binding by this small molecule validates the screening protocol. PMID:26962873

  3. Analysis of evolutionary conservation patterns and their influence on identifying protein functional sites.

    PubMed

    Fang, Chun; Noguchi, Tamotsu; Yamana, Hayato

    2014-10-01

    Evolutionary conservation information included in position-specific scoring matrix (PSSM) has been widely adopted by sequence-based methods for identifying protein functional sites, because all functional sites, whether in ordered or disordered proteins, are found to be conserved at some extent. However, different functional sites have different conservation patterns, some of them are linear contextual, some of them are mingled with highly variable residues, and some others seem to be conserved independently. Every value in PSSMs is calculated independently of each other, without carrying the contextual information of residues in the sequence. Therefore, adopting the direct output of PSSM for prediction fails to consider the relationship between conservation patterns of residues and the distribution of conservation scores in PSSMs. In order to demonstrate the importance of combining PSSMs with the specific conservation patterns of functional sites for prediction, three different PSSM-based methods for identifying three kinds of functional sites have been analyzed. Results suggest that, different PSSM-based methods differ in their capability to identify different patterns of functional sites, and better combining PSSMs with the specific conservation patterns of residues would largely facilitate the prediction.

  4. 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

  5. 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

  6. Protein Profiling of Human Breast Tumor Cells Identifies Novel Biomarkers Associated with Molecular Subtypes*S⃞

    PubMed Central

    Gonçalves, Anthony; Charafe-Jauffret, Emmanuelle; Bertucci, François; Audebert, Stéphane; Toiron, Yves; Esterni, Benjamin; Monville, Florence; Tarpin, Carole; Jacquemier, Jocelyne; Houvenaeghel, Gilles; Chabannon, Christian; Extra, Jean-Marc; Viens, Patrice; Borg, Jean-Paul; Birnbaum, Daniel

    2008-01-01

    Molecular subtypes of breast cancer with relevant biological and clinical features have been defined recently, notably ERBB2-overexpressing, basal-like, and luminal-like subtypes. To investigate the ability of mass spectrometry-based proteomics technologies to analyze the molecular complexity of human breast cancer, we performed a SELDI-TOF MS-based protein profiling of human breast cell lines (BCLs). Triton-soluble proteins from 27 BCLs were incubated with ProteinChip arrays and subjected to SELDI analysis. Unsupervised global hierarchical clustering spontaneously discriminated two groups of BCLs corresponding to “luminal-like” cell lines and to “basal-like” cell lines, respectively. These groups of BCLs were also different in terms of estrogen receptor status as well as expression of epidermal growth factor receptor and other basal markers. Supervised analysis revealed various protein biomarkers with differential expression in basal-like versus luminal-like cell lines. We identified two of them as a carboxyl terminus-truncated form of ubiquitin and S100A9. In a small series of frozen human breast tumors, we confirmed that carboxyl terminus-truncated ubiquitin is observed in primary breast samples, and our results suggest its higher expression in luminal-like tumors. S100A9 up-regulation was found as part of the transcriptionally defined basal-like cluster in DNA microarrays analysis of human tumors. S100A9 association with basal subtypes as well as its poor prognosis value was demonstrated on a series of 547 tumor samples from early breast cancer deposited in a tissue microarray. Our study shows the potential of integrated genomics and proteomics profiling to improve molecular knowledge of complex tumor phenotypes and identify biomarkers with valuable diagnostic or prognostic values. PMID:18426791

  7. 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.

  8. High content screening biosensor assay to identify disruptors of p53-hDM2 protein-protein interactions.

    PubMed

    Hua, Yun; Strock, Christopher J; Johnston, Paul A

    2015-01-01

    This chapter describes the implementation of the p53-hDM2 protein-protein interaction (PPI) biosensor (PPIB) HCS assay to identify disruptors of p53-hDM2 PPIs. Recombinant adenovirus expression constructs were generated bearing the individual p53-GFP and hDM2-RFP PPI partners. The N-terminal p53 transactivating domain that contains the binding site for hDM2 is expressed as a GFP fusion protein that is targeted and anchored in the nucleolus of infected cells by a nuclear localization (NLS) sequence. The p53-GFP biosensor is localized to the nucleolus to enhance and facilitate the image acquisition and analysis of the PPIs. The N-terminus of hDM2 encodes the domain for binding to the transactivating domain of p53, and is expressed as a RFP fusion protein that includes both an NLS and a nuclear export sequence (NES). In U-2 OS cells co-infected with both adenovirus constructs, the binding interactions between hDM2 and p53 result in both biosensors becoming co-localized within the nucleolus. Upon disruption of the p53-hDM2 PPIs, the p53-GFP biosensor remains in the nucleolus while the shuttling hDM2-RFP biosensor redistributes into the cytoplasm. p53-hDM2 PPIs are measured by acquiring fluorescent images of cells co-infected with both adenovirus biosensors on an automated HCS imaging platform and using an image analysis algorithm to quantify the relative distribution of the hDM2-RFP shuttling component of the biosensor between the cytoplasm and nuclear regions of compound treated cells.

  9. 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

  10. 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.

  11. Evaluating the automatic mapping of human gene and protein mentions to unique identifiers.

    PubMed

    Morgan, Alexander A; Wellner, Benjamin; Colombe, Jeffrey B; Arens, Robert; Colosimo, Marc E; Hirschman, Lynette

    2007-01-01

    We have developed a challenge task for the second BioCreAtIvE (Critical Assessment of Information Extraction in Biology) that requires participating systems to provide lists of the EntrezGene (formerly LocusLink) identifiers for all human genes and proteins mentioned in a MEDLINE abstract. We are distributing 281 annotated abstracts and another 5,000 noisily annotated abstracts along with a gene name lexicon to participants. We have performed a series of baseline experiments to better characterize this dataset and form a foundation for participant exploration.

  12. Beta atomic contacts: identifying critical specific contacts in protein binding interfaces.

    PubMed

    Liu, Qian; Kwoh, Chee Keong; Hoi, Steven C H

    2013-01-01

    Specific binding between proteins plays a crucial role in molecular functions and biological processes. Protein binding interfaces and their atomic contacts are typically defined by simple criteria, such as distance-based definitions that only use some threshold of spatial distance in previous studies. These definitions neglect the nearby atomic organization of contact atoms, and thus detect predominant contacts which are interrupted by other atoms. It is questionable whether such kinds of interrupted contacts are as important as other contacts in protein binding. To tackle this challenge, we propose a new definition called beta (β) atomic contacts. Our definition, founded on the β-skeletons in computational geometry, requires that there is no other atom in the contact spheres defined by two contact atoms; this sphere is similar to the van der Waals spheres of atoms. The statistical analysis on a large dataset shows that β contacts are only a small fraction of conventional distance-based contacts. To empirically quantify the importance of β contacts, we design βACV, an SVM classifier with β contacts as input, to classify homodimers from crystal packing. We found that our βACV is able to achieve the state-of-the-art classification performance superior to SVM classifiers with distance-based contacts as input. Our βACV also outperforms several existing methods when being evaluated on several datasets in previous works. The promising empirical performance suggests that β contacts can truly identify critical specific contacts in protein binding interfaces. β contacts thus provide a new model for more precise description of atomic organization in protein quaternary structures than distance-based contacts.

  13. 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

  14. Trace element concentrations in eggshells and egg contents of black-tailed gull (Larus crassirostris) from Korea.

    PubMed

    Kim, Jungsoo; Oh, Jong-Min

    2014-09-01

    Concentrations of trace elements (cadmium, lead, copper, manganese and zinc) were examined in eggs of black-tailed gulls (Larus crassirostris) from Hongdo Island, Korea to determine the difference and distribution of trace elements in eggshells and egg contents. Cadmium, lead and manganese concentrations were greater in eggshells than in egg contents. In contrast, zinc concentrations were higher in egg contents than in eggshells. Trace element concentrations followed the order: zinc > lead = manganese = copper > cadmium (eggshells) and zinc > copper > manganese > lead > cadmium (egg contents). Cadmium concentrations were relatively low (<1 μg/g dw) in egg contents and eggshells. Concentrations of cadmium, lead and copper were significantly correlated between egg contents and eggshells. This indicates that cadmium, lead and copper levels in the eggshell can reflect their levels in the egg contents. There was also a high ratio (3.2) of eggshell/egg content for lead. These results indicate that the eggshell might be useful as a bio-indicator for monitoring cadmium, lead and copper in the egg content.

  15. Candidate serological biomarkers for cancer identified from the secretomes of 23 cancer cell lines and the human protein atlas.

    PubMed

    Wu, Chih-Ching; Hsu, Chia-Wei; Chen, Chi-De; Yu, Chia-Jung; Chang, Kai-Ping; Tai, Dar-In; Liu, Hao-Ping; Su, Wen-Hui; Chang, Yu-Sun; Yu, Jau-Song

    2010-06-01

    Although cancer cell secretome profiling is a promising strategy used to identify potential body fluid-accessible cancer biomarkers, questions remain regarding the depth to which the cancer cell secretome can be mined and the efficiency with which researchers can select useful candidates from the growing list of identified proteins. Therefore, we analyzed the secretomes of 23 human cancer cell lines derived from 11 cancer types using one-dimensional SDS-PAGE and nano-LC-MS/MS performed on an LTQ-Orbitrap mass spectrometer to generate a more comprehensive cancer cell secretome. A total of 31,180 proteins was detected, accounting for 4,584 non-redundant proteins, with an average of 1,300 proteins identified per cell line. Using protein secretion-predictive algorithms, 55.8% of the proteins appeared to be released or shed from cells. The identified proteins were selected as potential marker candidates according to three strategies: (i) proteins apparently secreted by one cancer type but not by others (cancer type-specific marker candidates), (ii) proteins released by most cancer cell lines (pan-cancer marker candidates), and (iii) proteins putatively linked to cancer-relevant pathways. We then examined protein expression profiles in the Human Protein Atlas to identify biomarker candidates that were simultaneously detected in the secretomes and highly expressed in cancer tissues. This analysis yielded 6-137 marker candidates selective for each tumor type and 94 potential pan-cancer markers. Among these, we selectively validated monocyte differentiation antigen CD14 (for liver cancer), stromal cell-derived factor 1 (for lung cancer), and cathepsin L1 and interferon-induced 17-kDa protein (for nasopharyngeal carcinoma) as potential serological cancer markers. In summary, the proteins identified from the secretomes of 23 cancer cell lines and the Human Protein Atlas represent a focused reservoir of potential cancer biomarkers.

  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. A chemical screen identifies class A G-protein coupled receptors as regulators of cilia

    PubMed Central

    Avasthi, Prachee; Marley, Aaron; Lin, Henry; Gregori-Puigjane, Elisabet; Shoichet, Brian K.; von Zastrow, Mark; Marshall, Wallace F.

    2012-01-01

    Normal cilia length and motility are critical for proper cellular function. Prior studies of the regulation of ciliary structure and length have primarily focused on the intraflagellar transport machinery and motor proteins required for ciliary assembly and disassembly. However, several mutants with abnormal length flagella highlight the importance of signaling proteins as well. In this study, an unbiased chemical screen was performed to uncover signaling pathways that are critical for ciliogenesis and length regulation using flagella of the green alga Chlamydomonas reinhardtii as a model. The annotated Sigma LOPAC1280 chemical library was screened for effects on flagellar length, motility and severing as well as cell viability. Assay data were clustered to identify pathways regulating flagella. The most frequently target found to be involved in flagellar length regulation was the family of dopamine binding G-protein coupled receptors (GPCRs). In mammalian cells, cilium length could indeed be altered with expression of the dopamine D1 receptor. Our screen thus reveals signaling pathways that are potentially critical for ciliary formation, resorption, and length maintenance, which represent candidate targets for therapeutic intervention of disorders involving ciliary malformation and malfunction. PMID:22375814

  18. Modified yeast-two-hybrid system to identify proteins interacting with the growth factor progranulin.

    PubMed

    Tian, Qing-Yun; Zhao, Yun-Peng; Liu, Chuan-ju

    2012-01-17

    Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing, inflammation, and host defense. PGRN also functions as a neurotrophic factor, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel, extracellular matrix protein, and growth factor. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor.

  19. Alternate dissociation pathways identified in charge-reduced protein complex ions.

    PubMed

    Pagel, Kevin; Hyung, Suk-Joon; Ruotolo, Brandon T; Robinson, Carol V

    2010-06-15

    Tandem mass spectrometry (MS) of large protein complexes has proven to be capable of assessing the stoichiometry, connectivity, and structural details of multiprotein assemblies. While the utility of tandem MS is without question, a deeper understanding of the mechanism of protein complex dissociation will undoubtedly drive the technology into new areas of enhanced utility and information content. We present here the systematic analysis of the charge state dependent decay of the noncovalently associated complex of human transthyretin, generated by collision-induced dissociation (CID). A crown ether based charge reduction approach was applied to generate intact transthyretin tetramers with charge states ranging from 15+ to 7+. These nine charge states were subsequently analyzed by means of tandem MS and ion mobility spectrometry. Three different charge-dependent mechanistic regimes were identified: (1) common asymmetric dissociation involving ejection of unfolded monomers, (2) expulsion of folded monomers from the intact tetramer, and (3) release of C-terminal peptide fragments from the intact complex. Taken together, the results presented highlight the potential of charge state modulation as a method for directing the course of gas-phase dissociation and unfolding of protein complexes.

  20. Immunohistochemical expression pattern of MMR protein can specifically identify patients with colorectal cancer microsatellite instability.

    PubMed

    Amira, Arfaoui Toumi; Mouna, Trabelsi; Ahlem, Blel; Raoudha, Aloui; Majid, Ben Hmida; Amel, Hamza; Rachida, Zermani; Nadia, Kourdaa

    2014-07-01

    The microsatellite instability (MSI) pathway is found in most cases of hereditary nonpolyposis colorectal cancer (HNPCC) and in 12 % of sporadic colorectal cancer (CRC). It involves inactivation of deoxyribonucleic acid mismatch repair (MMR) genes MLH1, MSH2, PMS2, and MSH6. MMR germline mutation detections are an important supplement to HNPCC clinical diagnosis. It enables at-risk and mutation-positive relatives to be informed about their cancer risks and to benefit from intensive surveillance programs that have been proven to reduce the incidence of CRC. In this study, we analyzed for the first time in Tunisia the potential value of immunohistochemical assessment of MMR protein to identify microsatellite instability in CRC. We evaluate by immunohistochemistry MMR protein expression loss in tumoral tissue compared to positive expression in normal mucosa. Immunohistochemistry revealed loss of expression for MLH1, MSH2, MSH6, and PMS2 in 15, 21, 13, and 15 % of cases, respectively. Here, we report a more elevated frequency of MSI compared to data of the literature. In fact, by immunohistochemistry, 70 % of cases were shown to be MSS phenotype, whereas 30 % of cases, in our set, were instable. Moreover, according to molecular investigation, 71 % of cases were instable (MSI-H) and remaining cases were stable (29 %). Thus, we found a perfect association between MMR immunohistochemical analyses and MSI molecular investigation. Immunohistochemical analysis of MMR gene product expression may allow one to specifically identify MSI phenotype of patients with colorectal carcinomas.

  1. 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

  2. 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

  3. Altered protein S-glutathionylation identifies a potential mechanism of resistance to acetaminophen-induced hepatotoxicity.

    PubMed

    McGarry, David J; Chakravarty, Probir; Wolf, C Roland; Henderson, Colin J

    2015-11-01

    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

  4. Chemical Screens Identify Drugs that Enhance or Mitigate Cellular Responses to Antibody-Toxin Fusion Proteins.

    PubMed

    Antignani, Antonella; Mathews Griner, Lesley; 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

  5. Protein misfolding is the molecular mechanism underlying MCADD identified in newborn screening.

    PubMed

    Maier, Esther M; Gersting, Søren W; Kemter, Kristina F; Jank, Johanna M; Reindl, Maria; Messing, Dunja D; Truger, Marietta S; Sommerhoff, Christian P; Muntau, Ania C

    2009-05-01

    Newborn screening (NBS) for medium-chain acyl-CoA dehydrogenase deficiency (MCADD) revealed a higher birth prevalence and genotypic variability than previously estimated, including numerous novel missense mutations in the ACADM gene. On average, these mutations are associated with milder biochemical phenotypes raising the question about their pathogenic relevance. In this study, we analyzed the impact of 10 ACADM mutations identified in NBS (A27V, Y42H, Y133H, R181C, R223G, D241G, K304E, R309K, I331T and R388S) on conformation, stability and enzyme kinetics of the corresponding proteins. Partial to total rescue of aggregation by co-overexpression of GroESL indicated protein misfolding. This was confirmed by accelerated thermal unfolding in all variants, as well as decreased proteolytic stability and accelerated thermal inactivation in most variants. Catalytic function varied from high residual activity to markedly decreased activity or substrate affinity. Mutations mapping to the beta-domain of the protein predisposed to severe destabilization. In silico structural analyses of the affected amino acid residues revealed involvement in functionally relevant networks. Taken together, our results substantiate the hypothesis of protein misfolding with loss-of-function being the common molecular basis in MCADD. Moreover, considerable structural alterations in all analyzed variants do not support the view that novel mutations found in NBS bear a lower risk of metabolic decompensation than that associated with mutations detected in clinically ascertained patients. Finally, the detailed insight into how ACADM missense mutations induce loss of MCAD function may provide guidance for risk assessment and counseling of patients, and in future may assist delineation of novel pharmacological strategies.

  6. Efficient Isothermal Titration Calorimetry Technique Identifies Direct Interaction of Small Molecule Inhibitors with the Target Protein.

    PubMed

    Gal, Maayan; Bloch, Itai; Shechter, Nelia; Romanenko, Olga; Shir, Ofer M

    2016-01-01

    Protein-protein interactions (PPI) play a critical role in regulating many cellular processes. Finding novel PPI inhibitors that interfere with specific binding of two proteins is considered a great challenge, mainly due to the complexity involved in characterizing multi-molecular systems and limited understanding of the physical principles governing PPIs. Here we show that the combination of virtual screening techniques, which are capable of filtering a large library of potential small molecule inhibitors, and a unique secondary screening by isothermal titration calorimetry, a label-free method capable of observing direct interactions, is an efficient tool for finding such an inhibitor. In this study we applied this strategy in a search for a small molecule capable of interfering with the interaction of the tumor-suppressor p53 and the E3-ligase MDM2. We virtually screened a library of 15 million small molecules that were filtered to a final set of 80 virtual hits. Our in vitro experimental assay, designed to validate the activity of mixtures of compounds by isothermal titration calorimetry, was used to identify an active molecule against MDM2. At the end of the process the small molecule (4S,7R)-4-(4-chlorophenyl)-5-hydroxy-2,7-dimethyl-N-(6-methylpyridin-2-yl)-4,6,7,8 tetrahydrIoquinoline-3-carboxamide was found to bind MDM2 with a dissociation constant of ~2 µM. Following the identification of this single bioactive compound, spectroscopic measurements were used to further characterize the interaction of the small molecule with the target protein. 2D NMR spectroscopy was used to map the binding region of the small molecule, and fluorescence polarization measurement confirmed that it indeed competes with p53.

  7. A novel protein tyrosine kinase Tec identified in lamprey, Lampetra japonica.

    PubMed

    Li, Ranran; Su, Peng; Liu, Chang; Zhang, Qiong; Zhu, Ting; Pang, Yue; Liu, Xin; Li, Qingwei

    2015-08-01

    Protein tyrosine kinase Tec, a kind of non-receptor tyrosine kinase, is primarily found to be expressed in T cells, B cells, hematopoietic cells, and liver cells as a cytoplasmic protein. Tec has been proved to be a critical modulator of T cell receptor signaling pathway. In the present study, a homolog of Tec was identified in the lamprey, Lampetra japonica. The full-length Tec cDNA of L. japonica (Lja-Tec) contains a 1923 bp open reading frame that encodes a 641-amino acid protein. The multi-alignment of the deduced amino acid sequence of Lja-Tec with typical vertebrate Tecs showed that it possesses all conserved domains of the Tec family proteins, indicating that an ortholog of Tec exists in the extant jawless vertebrate. In the phylogenetic tree that was reconstructed with 24 homologs of jawless and jawed vertebrates, the Tecs from lampreys and hagfish were clustered as a single clade. The genetic distance between the outgroup and agnathan Tecs' group is closer than that between outgroup and gnathostome Tecs' group, indicating that its origin was far earlier than any of the jawed vertebrates. The mRNA levels of Lja-Tec in lymphocyte-like cells and gills were detected by real-time quantitative polymerase chain reaction. Results showed that it was significantly upregulated under stimulation with mixed pathogens. This result was further confirmed by western blot analysis. All these results indicated that Lja-Tec plays an important role in immune response. Our data will provide a reference for the further study of lamprey Tec and its immunological function in jawless vertebrates. PMID:26079172

  8. Quantitative Proteomics Identifies Vasopressin-Responsive Nuclear Proteins in Collecting Duct Cells

    PubMed Central

    Schenk, Laura K.; Bolger, Steven J.; Luginbuhl, Kelli; Gonzales, Patricia A.; Rinschen, Markus M.; Yu, Ming-Jiun; Hoffert, Jason D.; Pisitkun, Trairak

    2012-01-01

    Vasopressin controls transport in the renal collecting duct, in part, by regulating transcription. This complex process, which can involve translocation and/or modification of transcriptional regulators, is not completely understood. Here, we applied a method for large-scale profiling of nuclear proteins to quantify vasopressin-induced changes in the nuclear proteome of cortical collecting duct (mpkCCD) cells. Using stable isotope labeling and tandem mass spectrometry, we quantified 3987 nuclear proteins and identified significant changes in the abundance of 65, including previously established targets of vasopressin signaling in the collecting duct. Vasopressin-induced changes in the abundance of the transcription factors JunB, Elf3, Gatad2b, and Hmbox1; transcriptional co-regulators Ctnnb1 (β-catenin) and Crebbp; subunits of the Mediator complex; E3 ubiquitin ligase Nedd4; nuclear transport regulator RanGap1; and several proteins associated with tight junctions and adherens junctions. Bioinformatic analysis showed that many of the quantified transcription factors have putative binding sites in the 5′-flanking regions of genes coding for the channel proteins Aqp2, Aqp3, Scnn1b (ENaCβ), and Scnn1g (ENaCγ), which are known targets of vasopressin. Immunoblotting demonstrated that the increase in β-catenin in nuclear fractions was accompanied by an even larger increase in its phosphorylated form (pSer552). The findings provide a new online database resource for nuclear proteomics (http://helixweb.nih.gov/ESBL/Database/mNPD/) and generate new hypotheses regarding vasopressin-mediated transcriptional regulation in the collecting duct. PMID:22440904

  9. Quantitative proteomics identifies vasopressin-responsive nuclear proteins in collecting duct cells.

    PubMed

    Schenk, Laura K; Bolger, Steven J; Luginbuhl, Kelli; Gonzales, Patricia A; Rinschen, Markus M; Yu, Ming-Jiun; Hoffert, Jason D; Pisitkun, Trairak; Knepper, Mark A

    2012-06-01

    Vasopressin controls transport in the renal collecting duct, in part, by regulating transcription. This complex process, which can involve translocation and/or modification of transcriptional regulators, is not completely understood. Here, we applied a method for large-scale profiling of nuclear proteins to quantify vasopressin-induced changes in the nuclear proteome of cortical collecting duct (mpkCCD) cells. Using stable isotope labeling and tandem mass spectrometry, we quantified 3987 nuclear proteins and identified significant changes in the abundance of 65, including previously established targets of vasopressin signaling in the collecting duct. Vasopressin-induced changes in the abundance of the transcription factors JunB, Elf3, Gatad2b, and Hmbox1; transcriptional co-regulators Ctnnb1 (β-catenin) and Crebbp; subunits of the Mediator complex; E3 ubiquitin ligase Nedd4; nuclear transport regulator RanGap1; and several proteins associated with tight junctions and adherens junctions. Bioinformatic analysis showed that many of the quantified transcription factors have putative binding sites in the 5'-flanking regions of genes coding for the channel proteins Aqp2, Aqp3, Scnn1b (ENaCβ), and Scnn1g (ENaCγ), which are known targets of vasopressin. Immunoblotting demonstrated that the increase in β-catenin in nuclear fractions was accompanied by an even larger increase in its phosphorylated form (pSer552). The findings provide a new online database resource for nuclear proteomics (http://helixweb.nih.gov/ESBL/Database/mNPD/) and generate new hypotheses regarding vasopressin-mediated transcriptional regulation in the collecting duct. PMID:22440904

  10. A novel protein tyrosine kinase Tec identified in lamprey, Lampetra japonica.

    PubMed

    Li, Ranran; Su, Peng; Liu, Chang; Zhang, Qiong; Zhu, Ting; Pang, Yue; Liu, Xin; Li, Qingwei

    2015-08-01

    Protein tyrosine kinase Tec, a kind of non-receptor tyrosine kinase, is primarily found to be expressed in T cells, B cells, hematopoietic cells, and liver cells as a cytoplasmic protein. Tec has been proved to be a critical modulator of T cell receptor signaling pathway. In the present study, a homolog of Tec was identified in the lamprey, Lampetra japonica. The full-length Tec cDNA of L. japonica (Lja-Tec) contains a 1923 bp open reading frame that encodes a 641-amino acid protein. The multi-alignment of the deduced amino acid sequence of Lja-Tec with typical vertebrate Tecs showed that it possesses all conserved domains of the Tec family proteins, indicating that an ortholog of Tec exists in the extant jawless vertebrate. In the phylogenetic tree that was reconstructed with 24 homologs of jawless and jawed vertebrates, the Tecs from lampreys and hagfish were clustered as a single clade. The genetic distance between the outgroup and agnathan Tecs' group is closer than that between outgroup and gnathostome Tecs' group, indicating that its origin was far earlier than any of the jawed vertebrates. The mRNA levels of Lja-Tec in lymphocyte-like cells and gills were detected by real-time quantitative polymerase chain reaction. Results showed that it was significantly upregulated under stimulation with mixed pathogens. This result was further confirmed by western blot analysis. All these results indicated that Lja-Tec plays an important role in immune response. Our data will provide a reference for the further study of lamprey Tec and its immunological function in jawless vertebrates.

  11. The role of python eggshell permeability dynamics in a respiration-hydration trade-off.

    PubMed

    Stahlschmidt, Zachary R; Heulin, Benoit; DeNardo, Dale F

    2010-01-01

    Parental care is taxonomically widespread because it improves developmental conditions and thus fitness of offspring. Although relatively simplistic compared with parental behaviors of other taxa, python egg-brooding behavior exemplifies parental care because it mediates a trade-off between embryonic respiration and hydration. However, because egg brooding increases gas-exchange resistance between embryonic and nest environments and because female pythons do not adjust their brooding behavior in response to the increasing metabolic requirements of developing offspring, python egg brooding imposes hypoxic costs on embryos during the late stages of incubation. We conducted a series of experiments to determine whether eggshells coadapted with brooding behavior to minimize the negative effects of developmental hypoxia. We tested the hypotheses that python eggshells (1) increase permeability over time to accommodate increasing embryonic respiration and (2) exhibit permeability plasticity in response to chronic hypoxia. Over incubation, we serially measured the atomic and structural components of Children's python (Antaresia childreni) eggshells as well as in vivo and in vitro gas exchange across eggshells. In support of our first hypothesis, A. childreni eggshells exhibited a reduced fibrous layer, became more permeable, and facilitated greater gas exchange as incubation progressed. Our second hypothesis was not supported, as incubation O(2) concentration did not affect the shells' permeabilities to O(2) and H(2)O vapor. Our results suggest that python eggshell permeability changes during incubation but that the alterations over time are fixed and independent of environmental conditions. These findings are of broad evolutionary interest because they demonstrate that, even in relatively simple parental-care models, successful parent-offspring relationships depend on adjustments made by both the parent (i.e., egg-brooding behavioral shifts) and the offspring (i

  12. 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.

  13. 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

  14. e-Driver: a novel method to identify protein regions driving cancer

    PubMed Central

    Porta-Pardo, Eduard; Godzik, Adam

    2014-01-01

    Motivation: Most approaches used to identify cancer driver genes focus, true to their name, on entire genes and assume that a gene, treated as one entity, has a specific role in cancer. This approach may be correct to describe effects of gene loss or changes in gene expression; however, mutations may have different effects, including their relevance to cancer, depending on which region of the gene they affect. Except for rare and well-known exceptions, there are not enough data for reliable statistics for individual positions, but an intermediate level of analysis, between an individual position and the entire gene, may give us better statistics than the former and better resolution than the latter approach. Results: We have developed e-Driver, a method that exploits the internal distribution of somatic missense mutations between the protein’s functional regions (domains or intrinsically disordered regions) to find those that show a bias in their mutation rate as compared with other regions of the same protein, providing evidence of positive selection and suggesting that these proteins may be actual cancer drivers. We have applied e-Driver to a large cancer genome dataset from The Cancer Genome Atlas and compared its performance with that of four other methods, showing that e-Driver identifies novel candidate cancer drivers and, because of its increased resolution, provides deeper insights into the potential mechanism of cancer driver genes identified by other methods. Availability and implementation: A Perl script with e-Driver and the files to reproduce the results described here can be downloaded from https://github.com/eduardporta/e-Driver.git Contact: adam@godziklab.org or eppardo@sanfordburnham.org Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25064568

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. Beyond BLASTing: Tertiary and Quaternary Structure Analysis Helps Identify Major Vault Proteins

    PubMed Central

    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

  1. Identifying protein interaction subnetworks by a bagging Markov random field-based method.

    PubMed

    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.

  2. 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.

  3. p53 protein overexpression identifies a group of central primitive neuroectodermal tumours with poor prognosis.

    PubMed

    Jaros, E; Lunec, J; Perry, R H; Kelly, P J; Pearson, A D

    1993-10-01

    Primitive neuroectodermal tumours (PNET's) or medulloblastomas are common primary brain tumours of childhood. Current treatment protocols achieve 50-60% cures. However, it has proved difficult to develop better treatment for the remaining patients because prognostic factors are not established. We have investigated the prognostic value of p53 protein expression in 87 PNET's using immunohistochemistry with DO-7 and CM-1 antibodies on biopsy paraffin sections. Eight patients (9%) had intensely reactive tumour cell nuclei, and a significantly reduced survival (P = 0.002); only one survives and this with a recurrent tumour 50 months following diagnosis. Sixty eight per cent of patients had faintly reactive tumour cell nuclei, a reduced survival up to 4 years but a long term survival not significantly different (P = 0.41) from 23% of patients with p53 negative PNET's; the 10 year survival rates were 37% and 40%, respectively. Males had a reduced survival (P = 0.04) with a 2-fold relative risk of death compared to females. Multivariate analysis showed that intense overexpression of p53 protein identifies a group of PNET patients with a 7-fold relative risk of death compared to all other cases, irrespective of sex. This marked difference suggests the involvement of p53 in the pathogenesis of PNET's which have a particularly poor response to treatment, and should help to develop new therapies for this group of patients.

  4. 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

  5. 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

  6. Sodium chloride concentration in drinking water and eggshell quality.

    PubMed

    Damron, B L

    1998-10-01

    Two experiments with White Leghorn hens (36 and 49 wk old, respectively) were conducted to examine the effects of varying NaCl concentrations provided through drinking water upon eggshell quality. Experiments were of 6 and 5 wk duration, respectively. Seven replicate pens of five individually caged hens received each water treatment. Treatments were the same in both studies and consisted of low-Na (9 ppm) tap water supplemented with 0, 200, 400, 600, or 800 ppm NaCl. Solutions were prepared weekly. All eggs produced were examined visually for shell defects, and egg weight and specific gravity were determined. In the second experiment, eggs from 2 consecutive d were also broken out each week for Haugh Unit determinations. In both studies, hen-day egg production, daily feed and water intake, egg weight, and body weight change over the experimental period were not influenced by any level of waterborne NaCl. Haugh Units were also not affected in Experiment 2. In contrast to other literature reports, visually determined shell defects and egg specific gravity were not adversely affected by NaCl supplementation of layer drinking water.

  7. Hydrolysate from eggshell membrane ameliorates intestinal inflammation in mice.

    PubMed

    Shi, Yaning; Rupa, Prithy; Jiang, Bo; Mine, Yoshinori

    2014-12-09

    Inflammatory bowel diseases (IBD) comprises of ulcerative colitis (UC) and Cohn's disease (CD) as two main idiopathic pathologies resulting in immunologically mediated chronic inflammatory conditions. Several bioactive peptides and hydro lysates from natural sources have now been tested in animal models of human diseases for potential anti-inflammatory effects. Eggshell membrane (ESM) is a well-known natural bioactive material. In this study, we aim to study the anti-inflammatory activity of ESM hydro lysate (AL-PS) in vitro and in vivo. In vitro, AL-PS was shown to inhibit pro-inflammatory cytokine IL-8 secretion. In vivo treatment with AL-PS was shown to reduce dextran sodium sulphate (DSS)-induced weight loss, clinical signs of colitis and secretion of interleukin (IL)-6 (p < 0.05). In addition, treatment with AL-PS also attenuated the severity of intestinal inflammation via down-regulation of IL-10 an anti-inflammatory cytokine. This validates potential benefits of AL-PS as a novel preventative target molecule for treatment of IBD.

  8. The eggshell of Drosophila melanogaster. I. Fine structure of the layers and regions of the wild-type eggshell.

    PubMed

    Margaritis, L H; Kafatos, F C; Petri, W H

    1980-06-01

    The fine structure of the several layers and regional specializations in the Drosophila melanogaster eggshell has been studied by a combination of shell isolation procedures and ultrastructural techniques (conventional TEM, whole-mount TEM, SEM, HVEM, freeze-fracture electron microscopy utilizing rotary replication, shadow casting, optical diffraction and stereo imaging). The main shell consists of 5 layers: the vitelline membrane (300 nm thick), the wax layer, the innermost chorionic layer (40-50 nm), the endochorion (500-700 nm), and the exochorion (300-500 nm). The vitelline membrane consists of irregularly organized particles. The wax layer appears to contain multilayered hydrophobic plates which split tangenitally upon freeze fracturing. The innermost chorionic layer is composed of a crystalling lattice. The endochorion is made of a thin (40 nm) fenestrated floor composed of 40-nm fibres and an outer solid (200 nm) roof covered with a network of 40-nm strands. Intermittently spaced pillar connect these 2 parts. Similarities in the substructure of the floor, pillars and roof suggest that they may be composed of similar or identical structural elements. The specialized regions of the shell are the 2 respiratory appendages, the operculum area and the posterior pole. The appendages exhibit 2 sharply distinct surfaces, a dorsal side with isolated 1.5-micrometer plaques and a ventral side with strands of 40-50 nm connected in a network with openings of 70-80 nm. The operculum area, which includes the micropoyle and the collar, is distinguished by 3 unique types of cell imprints. The posterior pole contains 2 distinctive populations of cell imprints: the central area has very thin intercellular ridges and a thin, perforated, endochorionic roof, while the peripheral area contains mixed, thick and thin, intercellular ridges and serves as a transition zone to the main shell pattern. The pillars in the central area of the posterior pole have a distinct arrangement

  9. 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

  10. 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.

  11. [Powdered eggshell in the repair of cystic cavities of the jaw. Preliminary study].

    PubMed

    Baliga, M; Davies, P; Dupoirieux, L

    1998-07-01

    This paper is a clinical report on the use of eggshell as a bone substitute in oral surgery. Five volunteers who presented with a cystic cavity of the jaw with a mean size of 2 x 1 cm, were enrolled in the study. After enucleation of the cyst, the cavity was packed with eggshell powder. The results were assessed by clinical examination and repeated radiological examination at 2, 4, and 6 weeks. Two operative sites were checked at 2 months for histological examination. Clinically, the eggshell implants were very well tolerated. In 3 out of 5 patients, a progressive opacification of the cavities with a centripetal ossification occurred within 6 weeks. In one patient, a generalized resorption was noticed. Histological examination confirmed that eggshell powder was not osteoinductive, but it enhanced the bone regeneration from the margins. In conclusions, eggshell powder is a worth-while bone substitute because it is a safe and easily available material. However, only a modest beneficial effect can be expected on bone regeneration. PMID:9697237

  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

    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. Dual activators of Protein Kinase R (PKR) and Protein Kinase R Like Kinase (PERK) Identify Common and Divergent Catalytic Targets

    PubMed Central

    Ming, Jie; Sun, Hong; Cao, Peng; Fusco, Dahlene N.; Chung, Raymond T.; Chorev, Michael; Jin, Qi; Aktas, Bertal H.

    2013-01-01

    Chemical genetics has evolved into a powerful tool for studying gene function in normal- and patho-biology. PKR and PERK, two eukaryotic translation initiation factor 2 alpha (eIF2α) kinases, play critical roles in maintenance of cellular hemostasis, metabolic stability, and anti-viral defenses. Both kinases interact with and phosphorylate additional substrates including tumor suppressor p53 and nuclear protein 90. Loss of function of both kinases has been studied by reverse genetics and recently identified inhibitors. In contrast, activating probes for studying the role of catalytic activity of these kinases are not available. We identified a 3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5,7-dihydroxy-4H-chromen-4-one (DHBDC) as specific dual activator of PKR and PERK by screening a chemical library of 20,000 small molecules in a dual luciferase surrogate eIF2α phosphorylation assay. We present here extensive biological characterization and preliminary structure-activity relationship of DHBDC, which phosphorylate eIF2α by activating PKR and PERK but no other eIF2α kinases. These agents also activate downstream effectors of eIF2α phosphorylation; inducing CHOP and suppressing cyclin D1 expression and inhibiting cancer cell proliferation, all in a manner dependent on PKR and PERK. Consistent with the role of eIF2α phosphorylation in viral infection, DHBDC inhibits proliferation of human hepatitis C virus. Finally, DHBDC induces phosphorylation of Ikβα, and activates NF-κB pathway. Surprisingly, activation of NF-κB pathway is dependent on PERK but independent of PKR activity. These data indicate that DHBDC is an invaluable probe for elucidating the role of PKR and PERK in normal- and patho-biology. PMID:23784735

  15. Comprehensive Antigen Screening Identifies Moraxella catarrhalis Proteins That Induce Protection in a Mouse Pulmonary Clearance Model

    PubMed Central

    Verhaegh, Suzanne J. C.; Niebisch, Axel; Hanner, Markus; Selak, Sanja; Schüler, Wolfgang; Morfeldt, Eva; Hellberg, Christel; Nagy, Eszter; Lundberg, Urban; Hays, John P.; Meinke, Andreas; Henriques-Normark, Birgitta

    2013-01-01

    Moraxella catarrhalis is one of the three most common causative bacterial pathogens of otitis media, however no effective vaccine against M. catarrhalis has been developed so far. To identify M. catarrhalis vaccine candidate antigens, we used carefully selected sera from children with otitis media and healthy individuals to screen small-fragment genomic libraries that are expressed to display frame-selected peptides on a bacterial cell surface. This ANTIGENome technology led to the identification of 214 antigens, 23 of which were selected by in vitro or in vivo studies for additional characterization. Eight of the 23 candidates were tested in a Moraxella mouse pulmonary clearance model, and 3 of these antigens induced significantly faster bacterial clearance compared to adjuvant or to the previously characterized antigen OmpCD. The most significant protection data were obtained with the antigen MCR_1416 (Msp22), which was further investigated for its biological function by in vitro studies suggesting that Msp22 is a heme binding protein. This study comprises one of the most exhaustive studies to identify potential vaccine candidate antigens against the bacterial pathogen M. catarrhalis. PMID:23671716

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. The evolution of host-specific variation in cuckoo eggshell strength.

    PubMed

    Spottiswoode, C N

    2010-08-01

    Cuckoo eggs are renowned for their mimicry of different host species, leading to the evolution of host-specific races (or 'gentes') defined by egg colour and pattern. This study aims to test the prediction that another property of parasitic eggs, namely shell strength, might also have experienced divergent selection within cuckoo species. Host races of the common cuckoo Cuculus canorus encountering stronger host rejection have thicker-shelled eggs than those parasitising less discriminating species, as expected if egg strengthening discourages host rejection. Moreover, in the diederik cuckoo Chrysococcyx caprius, eggshell thickness was correlated across cuckoo gentes and host species, as expected if eggshell strength has been involved in coevolutionary interactions. This is the first report of host-specific differences in cuckoo egg properties other than colour and pattern and lends correlational support to the hypothesis that the strong eggshells of brood parasites are an adaptation to reduce host rejection.

  4. 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

  5. An integrated transcriptomic and proteomic analysis of sea star epidermal secretions identifies proteins involved in defense and adhesion.

    PubMed

    Hennebert, Elise; Leroy, Baptiste; Wattiez, Ruddy; Ladurner, Peter

    2015-10-14

    Sea stars rely on epidermal secretions to cope with their benthic life. Their integument produces a mucus, which represents the first barrier against invaders; and their tube feet produce adhesive secretions to pry open mussels and attach strongly but temporarily to rocks. In this study, we combined high-throughput sequencing of expressed mRNA and mass-spectrometry-based identification of proteins to establish the first proteome of mucous and adhesive secretions from the sea star Asterias rubens. We show that the two secretions differ significantly, the major adhesive proteins being only present in trace amounts in the mucus secretion. Except for 41 proteins which were present in both secretions, a total of 34 and 244 proteins were identified as specific of adhesive secretions and mucus, respectively. We discuss the role of some of these proteins in the adhesion of sea stars as well as in their protection against oxygen reactive species and microorganisms. In addition, 58% of the proteins identified in adhesive secretions did not present significant similarity to other known proteins, revealing a list of potential novel sea star adhesive proteins uncharacterized so far. The panel of proteins identified in this study offers unprecedented opportunities for the development of sea star-inspired biomimetic materials.

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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

  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. TRIBE: Hijacking an RNA-Editing Enzyme to Identify Cell-Specific Targets of RNA-Binding Proteins.

    PubMed

    McMahon, Aoife C; Rahman, Reazur; Jin, Hua; Shen, James L; Fieldsend, Allegra; Luo, Weifei; Rosbash, Michael

    2016-04-21

    RNA transcripts are bound and regulated by RNA-binding proteins (RBPs). Current methods for identifying in vivo targets of an RBP are imperfect and not amenable to examining small numbers of cells. To address these issues, we developed TRIBE (targets of RNA-binding proteins identified by editing), a technique that couples an RBP to the catalytic domain of the Drosophila RNA-editing enzyme ADAR and expresses the fusion protein in vivo. RBP targets are marked with novel RNA editing events and identified by sequencing RNA. We have used TRIBE to identify the targets of three RBPs (Hrp48, dFMR1, and NonA). TRIBE compares favorably to other methods, including CLIP, and we have identified RBP targets from as little as 150 specific fly neurons. TRIBE can be performed without an antibody and in small numbers of specific cells.

  14. A novel method to detect proteins evolving at correlated rates: identifying new functional relationships between coevolving proteins.

    PubMed

    Clark, Nathaniel L; Aquadro, Charles F

    2010-05-01

    Interacting proteins evolve at correlated rates, possibly as the result of evolutionary pressures shared by functional groups and/or coevolution between interacting proteins. This evolutionary signature can be exploited to learn more about protein networks and to infer functional relationships between proteins on a genome-wide scale. Multiple methods have been introduced that detect correlated evolution using amino acid distances. One assumption made by these methods is that the neutral rate of nucleotide substitution is uniform over time; however, this is unlikely and such rate heterogeneity would adversely affect amino acid distance methods. We explored alternative methods that detect correlated rates using protein-coding nucleotide sequences in order to better estimate the rate of nonsynonymous substitution at each branch (d(N)) normalized by the underlying synonymous substitution rate (d(S)). Our novel likelihood method, which was robust to realistic simulation parameters, was tested on Drosophila nuclear pore proteins, which form a complex with well-documented physical interactions. The method revealed significantly correlated evolution between nuclear pore proteins, where members of a stable subcomplex showed stronger correlations compared with those proteins that interact transiently. Furthermore, our likelihood approach was better able to detect correlated evolution among closely related species than previous methods. Hence, these sequence-based methods are a complementary approach for detecting correlated evolution and could be applied genome-wide to provide candidate protein-protein interactions and functional group assignments using just coding sequences.

  15. 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

  16. Identifying recommended dietary allowances for protein and amino acids: a critique of the 2007 WHO/FAO/UNU report.

    PubMed

    Millward, D Joe

    2012-08-01

    The WHO/FAO/UNU (2007) report examines dietary protein and amino acid requirements for all age groups, protein requirements during pregnancy, lactation and catch-up growth in children, the implications of these requirements for developing countries and protein quality evaluation. Requirements were defined as the minimum dietary intake which satisfies the metabolic demand and achieves nitrogen equilibrium and maintenance of the body protein mass, plus the needs for growth in children and pregnancy and lactation in healthy women. Insufficient evidence was identified to enable recommendations for specific health outcomes. A meta analysis of nitrogen balance studies identifies protein requirements for adults 10 % higher than previous values with no influence of gender or age, consistent with a subsequently published comprehensive study. A new factorial model for infants and children, validated on the basis of the adequacy of breast milk protein intakes and involving a lower maintenance requirement value, no provision for saltatory growth and new estimates of protein deposition identifies lower protein requirements than in previous reports. Higher values for adult amino acid requirements, derived from a re-evaluation of nitrogen balance studies and new stable isotope studies, identify some cereal-based diets as being inadequate for lysine. The main outstanding issues relate to the biological implausibility of the very low efficiencies of protein utilisation used in the factorial models for protein requirements for all population groups especially pregnancy when requirements may be overestimated. Also considerable uncertainty remains about the design and interpretation of most of the studies used to identify amino acid requirement values.

  17. 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

  18. 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.

  19. 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.

  20. A method for systematic mapping of protein lysine methylation identifies new functions for HP1β in DNA damage repair

    PubMed Central

    Liu, Huadong; Galka, Marek; Liu, Xuguang; Lin, Yu-fen; Pittock, Paula; Voss, Courtney; Dhami, Gurpreet; Li, Xing; Miyaji, Masaaki; Lajoie, Gilles; Chen, Benjamin; Li, Shawn S.-C.

    2014-01-01

    SUMMARY Lysine methylation occurs on both histone and non-histone proteins. However, our knowledge on the prevalence and function of non-histone protein methylation is poor. We describe here an approach that combines peptide array, bioinformatic and mass spectrometric analyses to systematically identify lysine methylation sites in proteins and methyllysine-mediated protein-protein interactions. We demonstrate the utility of this approach by identifying a methyllysine-driven interactome of the heterochromatin protein (HP) 1β and uncovering, simultaneously, numerous methyllysine sites on non-histone proteins. The HP1β interactome is enriched with proteins involved in DNA damage repair and RNA splicing. We showed that lysine methylation played a pivotal role in the function of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and its interaction with HP1β during DNA damage response. Moreover, by combining heavy methyl SILAC with Multiple Reaction Monitoring (MRM) mass spectrometry (MS), we showed that lysine methylation underwent widespread and large changes in response to DNA damage. Our work indicates that lysine methylation is a highly dynamic post-translational modification occurring frequently on non-histone proteins and that the approach presented herein may be extended to many methyllysine-binding modules to systematically uncover lysine methylation events in the cell. PMID:23707759

  1. 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

  2. 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.

  3. 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

  4. Proteomic analysis of chicory root identifies proteins typically involved in cold acclimation.

    PubMed

    Degand, Hervé; Faber, Anne-Marie; Dauchot, Nicolas; Mingeot, Dominique; Watillon, Bernard; Cutsem, Pierre Van; Morsomme, Pierre; Boutry, Marc

    2009-05-01

    Chicory (Cichorium intybus) roots contain high amounts of inulin, a fructose polymer used as a storage carbohydrate by the plant and as a human dietary and prebiotic compound. We performed 2-D electrophoretic analysis of proteins from root material before the first freezing period. The proteins were digested with trypsin and the peptides analyzed by MS (MALDI-TOF/TOF). From the 881 protein spots analyzed, 714 proteins corresponded to a database accession, 619 of which were classified into functional categories. Besides expected proteins (e.g. related to metabolism, energy, protein synthesis, or cell structure), other well-represented categories were proteins related to folding and stability (49 spots), proteolysis (49 spots), and the stress response (67 spots). The importance of abiotic stress response was confirmed by the observation that 7 of the 21 most intense protein spots are known to be involved in cold acclimation. These results suggest a major effect of the low temperature period that preceded root harvesting.

  5. Identifying an ovarian cancer cell hierarchy regulated by bone morphogenetic protein 2

    PubMed Central

    Choi, Yun-Jung; Ingram, Patrick N.; Yang, Kun; Coffman, Lan; Iyengar, Mangala; Bai, Shoumei; Thomas, Dafydd G.; Yoon, Euisik; Buckanovich, Ronald J.

    2015-01-01

    Whether human cancer follows a hierarchical or stochastic model of differentiation is controversial. Furthermore, the factors that regulate cancer stem-like cell (CSC) differentiation potential are largely unknown. We used a novel microfluidic single-cell culture method to directly observe the differentiation capacity of four heterogeneous ovarian cancer cell populations defined by the expression of the CSC markers aldehyde dehydrogenase (ALDH) and CD133. We evaluated 3,692 progeny from 2,833 cells. We found that only ALDH+CD133+ cells could generate all four ALDH+/−CD133+/− cell populations and identified a clear branched differentiation hierarchy. We also observed a single putative stochastic event. Within the hierarchy of cells, bone morphologenetic protein 2 (BMP2) is preferentially expressed in ALDH−CD133− cells. BMP2 promotes ALDH+CD133+ cell expansion while suppressing the proliferation of ALDH−CD133− cells. As such, BMP2 suppressed bulk cancer cell growth in vitro but increased tumor initiation rates, tumor growth, and chemotherapy resistance in vivo whereas BMP2 knockdown reduced CSC numbers, in vivo growth, and chemoresistance. These data suggest a hierarchical differentiation pattern in which BMP2 acts as a feedback mechanism promoting ovarian CSC expansion and suppressing progenitor proliferation. These results explain why BMP2 suppresses growth in vitro and promotes growth in vivo. Together, our results support BMP2 as a therapeutic target in ovarian cancer. PMID:26621735

  6. Identifying Druggable Targets by Protein Microenvironments Matching: Application to Transcription Factors

    PubMed Central

    Liu, T; Altman, R B

    2014-01-01

    Druggability of a protein is its potential to be modulated by drug-like molecules. It is important in the target selection phase. We hypothesize that: (i) known drug-binding sites contain advantageous physicochemical properties for drug binding, or “druggable microenvironments” and (ii) given a target, the presence of multiple druggable microenvironments similar to those seen previously is associated with a high likelihood of druggability. We developed DrugFEATURE to quantify druggability by assessing the microenvironments in potential small-molecule binding sites. We benchmarked DrugFEATURE using two data sets. One data set measures druggability using NMR-based screening. DrugFEATURE correlates well with this metric. The second data set is based on historical drug discovery outcomes. Using the DrugFEATURE cutoffs derived from the first, we accurately discriminated druggable and difficult targets in the second. We further identified novel druggable transcription factors with implications for cancer therapy. DrugFEATURE provides useful insight for drug discovery, by evaluating druggability and suggesting specific regions for interacting with drug-like molecules. PMID:24452614

  7. 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

  8. Comparative proteomic analysis of Streptococcus suis biofilms and planktonic cells that identified biofilm infection-related immunogenic proteins.

    PubMed

    Wang, Yang; Yi, Li; Wu, Zongfu; Shao, Jing; Liu, Guangjin; Fan, Hongjie; Zhang, Wei; Lu, Chengping

    2012-01-01

    Streptococcus suis (SS) is a zoonotic pathogen that causes severe disease symptoms in pigs and humans. Biofilms of SS bind to extracellular matrix proteins in both endothelial and epithelial cells and cause persistent infections. In this study, the differences in the protein expression profiles of SS grown either as planktonic cells or biofilms were identified using comparative proteomic analysis. The results revealed the existence of 13 proteins of varying amounts, among which six were upregulated and seven were downregulated in the Streptococcus biofilm compared with the planktonic controls. The convalescent serum from mini-pig, challenged with SS, was applied in a Western blot assay to visualize all proteins from the biofilm that were grown in vitro and separated by two-dimensional gel electrophoresis. A total of 10 immunoreactive protein spots corresponding to nine unique proteins were identified by MALDI-TOF/TOF-MS. Of these nine proteins, five (Manganese-dependent superoxide dismutase, UDP-N-acetylglucosamine 1-carboxyvinyltransferase, ornithine carbamoyltransferase, phosphoglycerate kinase, Hypothetical protein SSU05_0403) had no previously reported immunogenic properties in SS to our knowledge. The remaining four immunogenic proteins (glyceraldehyde-3-phosphate dehydrogenase, hemolysin, pyruvate dehydrogenase and DnaK) were identified under both planktonic and biofilm growth conditions. In conclusion, the protein expression pattern of SS, grown as biofilm, was different from the SS grown as planktonic cells. These five immunogenic proteins that were specific to SS biofilm cells may potentially be targeted as vaccine candidates to protect against SS biofilm infections. The four proteins common to both biofilm and planktonic cells can be targeted as vaccine candidates to protect against both biofilm and acute infections.

  9. Comparative Proteomic Analysis of Streptococcus suis Biofilms and Planktonic Cells That Identified Biofilm Infection-Related Immunogenic Proteins

    PubMed Central

    Wang, Yang; Yi, Li; Wu, Zongfu; Shao, Jing; Liu, Guangjin; Fan, Hongjie; Zhang, Wei; Lu, Chengping

    2012-01-01

    Streptococcus suis (SS) is a zoonotic pathogen that causes severe disease symptoms in pigs and humans. Biofilms of SS bind to extracellular matrix proteins in both endothelial and epithelial cells and cause persistent infections. In this study, the differences in the protein expression profiles of SS grown either as planktonic cells or biofilms were identified using comparative proteomic analysis. The results revealed the existence of 13 proteins of varying amounts, among which six were upregulated and seven were downregulated in the Streptococcus biofilm compared with the planktonic controls. The convalescent serum from mini-pig, challenged with SS, was applied in a Western blot assay to visualize all proteins from the biofilm that were grown in vitro and separated by two-dimensional gel electrophoresis. A total of 10 immunoreactive protein spots corresponding to nine unique proteins were identified by MALDI-TOF/TOF-MS. Of these nine proteins, five (Manganese-dependent superoxide dismutase, UDP-N-acetylglucosamine 1-carboxyvinyltransferase, ornithine carbamoyltransferase, phosphoglycerate kinase, Hypothetical protein SSU05_0403) had no previously reported immunogenic properties in SS to our knowledge. The remaining four immunogenic proteins (glyceraldehyde-3-phosphate dehydrogenase, hemolysin, pyruvate dehydrogenase and DnaK) were identified under both planktonic and biofilm growth conditions. In conclusion, the protein expression pattern of SS, grown as biofilm, was different from the SS grown as planktonic cells. These five immunogenic proteins that were specific to SS biofilm cells may potentially be targeted as vaccine candidates to protect against SS biofilm infections. The four proteins common to both biofilm and planktonic cells can be targeted as vaccine candidates to protect against both biofilm and acute infections. PMID:22514606

  10. 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...

  11. 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.

  12. Sequence Analysis of Hypothetical Proteins from Helicobacter pylori 26695 to Identify Potential Virulence Factors

    PubMed Central

    Naqvi, Ahmad Abu Turab; Anjum, Farah; Khan, Faez Iqbal; Islam, Asimul; Ahmad, Faizan

    2016-01-01

    Helicobacter pylori is a Gram-negative bacteria that is responsible for gastritis in human. Its spiral flagellated body helps in locomotion and colonization in the host environment. It is capable of living in the highly acidic environment of the stomach with the help of acid adaptive genes. The genome of H. pylori 26695 strain contains 1,555 coding genes that encode 1,445 proteins. Out of these, 340 proteins are characterized as hypothetical proteins (HP). This study involves extensive analysis of the HPs using an established pipeline which comprises various bioinformatics tools and databases to find out probable functions of the HPs and identification of virulence factors. After extensive analysis of all the 340 HPs, we found that 104 HPs are showing characteristic similarities with the proteins with known functions. Thus, on the basis of such similarities, we assigned probable functions to 104 HPs with high confidence and precision. All the predicted HPs contain representative members of diverse functional classes of proteins such as enzymes, transporters, binding proteins, regulatory proteins, proteins involved in cellular processes and other proteins with miscellaneous functions. Therefore, we classified 104 HPs into aforementioned functional groups. During the virulence factors analysis of the HPs, we found 11 HPs are showing significant virulence. The identification of virulence proteins with the help their predicted functions may pave the way for drug target estimation and development of effective drug to counter the activity of that protein. PMID:27729842

  13. Newly Identified Phosphorylation Site in the Vesicular Stomatitis Virus P Protein Is Required for Viral RNA Synthesis

    PubMed Central

    Mondal, Arindam; Victor, Ken G.; Pudupakam, R. S.; Lyons, Charles E.

    2014-01-01

    The vesicular stomatitis virus (VSV) RNA-dependent RNA polymerase consists of two viral proteins; the large (L) protein is the main catalytic subunit, and the phosphoprotein (P) is an essential cofactor for polymerase function. The P protein interacts with the L protein and the N-RNA template, thus connecting the polymerase to the template. P protein also binds to free N protein to maintain it in a soluble, encapsidation-competent form. Previously, five sites of phosphorylation were identified on the P protein and these sites were reported to be differentially important for mRNA synthesis or genomic replication. The previous studies were carried out by biochemical analysis of portions of the authentic viral P protein or by analysis of bacterium-expressed, exogenously phosphorylated P protein by mutagenesis. However, there has been no systematic biochemical search for phosphorylation sites on authentic, virus-expressed P protein. In this study, we analyzed the P protein isolated from VSV-infected cells for sites of phosphorylation by mass spectrometry. We report the identification of Tyr14 as a previously unidentified phosphorylation site of VSV P and show that it is essential for viral transcription and replication. However, our mass spectral analysis failed to observe the phosphorylation of previously reported C-terminal residues Ser226 and Ser227 and mutagenic analyses did not demonstrate a role for these sites in RNA synthesis. PMID:24257610

  14. 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.

  15. 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.

  16. 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

  17. Heterologous expression of Translocated promoter region protein, Tpr, identified as a transcription factor from Rattus norvegicus.

    PubMed

    Agarwal, Shivani; Yadav, Sunita Kumari; Dixit, Aparna

    2011-05-01

    Our earlier studies have demonstrated that the 35 kDa isoform of Translocated promoter region protein (Tpr) of Rattus norvegicus was able to augment c-jun transcription efficiently. Identification of direct targets that may in part downregulate c-jun transcription might prove to be an ideal target to curtail the proliferation of normal cells under pathophysiological conditions. In order to evaluate its potential as a pharmaceutical target, the protein must be produced and purified in sufficiently high yields. In the present study, we report the high level expression of Tpr protein of R. norvegicus employing heterologous host, Escherichia coli, to permit its structural characterization in great detail. We here demonstrate that the Tpr protein was expressed in soluble form and approximately 90 mg/L of the purified protein at the shake flask level could be achieved to near homogeneity using single step-metal chelate affinity chromatography. The amino acid sequence of the protein was confirmed by mass spectroscopic analysis. The highly unstable and disordered Tpr protein was imparted structural and functional stability by the addition of glycerol and it has been shown that the natively unfolded Tpr protein retains DNA binding ability under these conditions only. Thus, the present study emphasizes the significance of an efficient prokaryotic system, which results in a high level soluble expression of a DNA binding protein of eukaryotic origin. Thus, the present strategy employed for purification of the R. norvegicus Tpr protein bypasses the need for the tedious expression strategies associated with the eukaryotic expression systems.

  18. Bioorthogonal labeling cell-surface proteins expressed in pancreatic cancer cells to identify potential diagnostic/therapeutic biomarkers

    PubMed Central

    Haun, Randy S; Quick, Charles M; Siegel, Eric R; Raju, Ilangovan; Mackintosh, Samuel G; Tackett, Alan J

    2015-01-01

    To develop new diagnostic and therapeutic tools to specifically target pancreatic tumors, it is necessary to identify cell-surface proteins that may serve as potential tumor-specific targets. In this study we used an azido-labeled bioorthogonal chemical reporter to metabolically label N-linked glycoproteins on the surface of pancreatic cancer cell lines to identify potential targets that may be exploited for detection and/or treatment of pancreatic cancer. Labeled glycoproteins were tagged with biotin using click chemistry, purified by streptavidin-coupled magnetic beads, separated by gel electrophoresis, and identified by liquid chromatography-tandem mass spectrometry (MS). MS/MS analysis of peptides from 3 cell lines revealed 954 unique proteins enriched in the azido sugar samples relative to control sugar samples. A comparison of the proteins identified in each sample indicated 20% of these proteins were present in 2 cell lines (193 of 954) and 17 of the proteins were found in all 3 cell lines. Five of the 17 proteins identified in all 3 cell lines have not been previously reported to be expressed in pancreatic cancer; thus indicating that novel cell-surface proteins can be revealed through glycoprotein profiling. Western analysis of one of these glycoproteins, ecto-5′-nucleotidase (NT5E), revealed it is expressed in 8 out of 8 pancreatic cancer cell lines examined. Further, immunohistochemical analysis of human pancreatic tissues indicates NT5E is significantly overexpressed in pancreatic tumors compared to normal pancreas. Thus, we have demonstrated that metabolic labeling with bioorthogonal chemical reporters can be used to selectively enrich and identify novel cell-surface glycoproteins expressed in pancreatic ductal adenocarcinomas. PMID:26176765

  19. 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

  20. Proteomic and genetic approaches to identifying defence-related proteins in rice challenged with the fungal pathogen Rhizoctonia solani.

    PubMed

    Lee, Joohyun; Bricker, Terry M; Lefevre, Michael; Pinson, Shannon R M; Oard, James H

    2006-09-01

    SUMMARY Sheath blight, caused by the fungus Rhizoctonia solani, is a major disease of rice world-wide, but little is known about the host response to infection. The objective of this study was to identify proteins and DNA markers in resistant and susceptible rice associated with response to infection by R. solani. Replicated two-dimensional polyacrylamide gel electrophoresis experiments were conducted to detect proteins differentially expressed under inoculated and non-inoculated conditions. Tandem mass spectra analysis using electrospray ionization quadrupole-time of flight mass spectrometry (ESI Q-TOF MS) was carried out for protein identification with the NCBI non-redundant protein database. Seven proteins were increased after inoculation in both susceptible and resistant plants. Six of the seven proteins were identified with presumed antifungal, photosynthetic and proteolytic activities. An additional 14 proteins were detected in the response of the resistant line. Eleven of the 14 proteins were identified with presumed functions relating to antifungal activity, signal transduction, energy metabolism, photosynthesis, molecular chaperone, proteolysis and antioxidation. The induction of 3-beta-hydroxysteroid dehydrogenase/isomerase was detected for the first time in resistant rice plants after pathogen challenge, suggesting a defensive role of this enzyme in rice against attack by R. solani. The chromosomal locations of four induced proteins were found to be in close physical proximity to genetic markers for sheath blight resistance in two genetic mapping populations. The proteomic and genetic results from this study indicate a complex response of rice to challenge by R. solani that involves simultaneous induction of proteins from multiple defence pathways.

  1. Rapid evolution of outer egg membrane proteins in the Drosophila melanogaster subgroup: a case of ecologically driven evolution of female reproductive traits.

    PubMed

    Jagadeeshan, Santosh; Singh, Rama S

    2007-04-01

    Although sexual selection has been predominantly used to explain the rapid evolution of sexual traits, eggs of oviparous organisms directly face both the challenges of sexual selection as well as natural selection (environmental challenges, survival in niches, etc.). Being the outermost membrane in most insect eggs, the chorion layer is the interface between the embryo and the environment, thereby serving to protect the egg. Adaptive ecological radiations such as divergence in ovipositional substrate usage and host-plant specializations can therefore influence the evolution of eggshell proteins. We can hypothesize that proteins localized on the outer eggshell may be affected to a greater degree by ecological challenges compared with inner eggshell proteins, and therefore, proteins localized in the outer eggshell (chorion membrane) may evolve differently (faster) than proteins localized in the inner egg membrane (vitelline membrane). We compared the evolutionary divergence of vitelline with chorion membrane proteins in species of the melanogaster subgroup and found that chorion proteins as a group are indeed evolving faster than vitelline membrane proteins. At least one vitelline membrane protein (Vm32E), specifically localized on the outer eggshell, is also evolving faster than other vitelline membrane proteins suggesting that all proteins localized on the outer eggshell may be evolving rapidly. We also found evidence that specific codons in chorion proteins cp15 and cp16 are evolving under positive selection. Polymorphism surveys of cp16 revealed inflated levels of divergence relative to polymorphism in specific regions of the gene, indicating that these regions are under strong selection. At the morphological level, we found notable difference in eggshell surface morphologies between specialist (Drosophila sechellia and Drosophila erecta) and generalist species of Drosophila. We do not know if any of the chorion proteins actually interact with spermatozoids

  2. 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

  3. Oxygen isotope fractionation between bird eggshell calcite and body water: application to fossil eggs from Lanzarote (Canary Islands)

    NASA Astrophysics Data System (ADS)

    Lazzerini, Nicolas; Lécuyer, Christophe; Amiot, Romain; Angst, Delphine; Buffetaut, Eric; Fourel, François; Daux, Valérie; Betancort, Juan Francisco; Flandrois, Jean-Pierre; Marco, Antonio Sánchez; Lomoschitz, Alejandro

    2016-10-01

    Oxygen and carbon isotope compositions of fossil bird eggshell calcite (δ18Ocalc and δ13Ccalc) are regularly used to reconstruct paleoenvironmental conditions. However, the interpretation of δ18Ocalc values of fossil eggshells has been limited to qualitative variations in local climatic conditions as oxygen isotope fractionations between calcite, body fluids, and drinking water have not been determined yet. For this purpose, eggshell, albumen water, and drinking water of extant birds have been analyzed for their oxygen and carbon isotope compositions. Relative enrichments in 18O relative to 16O between body fluids and drinking water of +1.6 ± 0.9 ‰ for semi-aquatic birds and of +4.4 ± 1.9 ‰ for terrestrial birds are observed. Surprisingly, no significant dependence to body temperature on the oxygen isotope fractionation between eggshell calcite and body fluids is observed, suggesting that bird eggshells precipitate out of equilibrium. Two empirical equations relating the δ18Ocalc value of eggshell calcite to the δ18Ow value of ingested water have been established for terrestrial and semi-aquatic birds. These equations have been applied to fossil eggshells from Lanzarote in order to infer the ecologies of the Pleistocene marine bird Puffinus sp. and of the enigmatic giant birds from the Pliocene. Both δ13Ccalc and δ18Ocalc values of Puffinus eggshells point to a semi-aquatic marine bird ingesting mostly seawater, whereas low δ13Ccalc and high δ18Ocalc values of eggshells from the Pliocene giant bird suggest a terrestrial lifestyle. This set of equations can help to quantitatively estimate the origin of waters ingested by extinct birds as well as to infer either local environmental or climatic conditions.

  4. Oxygen isotope fractionation between bird eggshell calcite and body water: application to fossil eggs from Lanzarote (Canary Islands).

    PubMed

    Lazzerini, Nicolas; Lécuyer, Christophe; Amiot, Romain; Angst, Delphine; Buffetaut, Eric; Fourel, François; Daux, Valérie; Betancort, Juan Francisco; Flandrois, Jean-Pierre; Marco, Antonio Sánchez; Lomoschitz, Alejandro

    2016-10-01

    Oxygen and carbon isotope compositions of fossil bird eggshell calcite (δ(18)Ocalc and δ(13)Ccalc) are regularly used to reconstruct paleoenvironmental conditions. However, the interpretation of δ(18)Ocalc values of fossil eggshells has been limited to qualitative variations in local climatic conditions as oxygen isotope fractionations between calcite, body fluids, and drinking water have not been determined yet. For this purpose, eggshell, albumen water, and drinking water of extant birds have been analyzed for their oxygen and carbon isotope compositions. Relative enrichments in (18)O relative to (16)O between body fluids and drinking water of +1.6 ± 0.9 ‰ for semi-aquatic birds and of +4.4 ± 1.9 ‰ for terrestrial birds are observed. Surprisingly, no significant dependence to body temperature on the oxygen isotope fractionation between eggshell calcite and body fluids is observed, suggesting that bird eggshells precipitate out of equilibrium. Two empirical equations relating the δ(18)Ocalc value of eggshell calcite to the δ(18)Ow value of ingested water have been established for terrestrial and semi-aquatic birds. These equations have been applied to fossil eggshells from Lanzarote in order to infer the ecologies of the Pleistocene marine bird Puffinus sp. and of the enigmatic giant birds from the Pliocene. Both δ(13)Ccalc and δ(18)Ocalc values of Puffinus eggshells point to a semi-aquatic marine bird ingesting mostly seawater, whereas low δ(13)Ccalc and high δ(18)Ocalc values of eggshells from the Pliocene giant bird suggest a terrestrial lifestyle. This set of equations can help to quantitatively estimate the origin of waters ingested by extinct birds as well as to infer either local environmental or climatic conditions.

  5. Oxygen isotope fractionation between bird eggshell calcite and body water: application to fossil eggs from Lanzarote (Canary Islands).

    PubMed

    Lazzerini, Nicolas; Lécuyer, Christophe; Amiot, Romain; Angst, Delphine; Buffetaut, Eric; Fourel, François; Daux, Valérie; Betancort, Juan Francisco; Flandrois, Jean-Pierre; Marco, Antonio Sánchez; Lomoschitz, Alejandro

    2016-10-01

    Oxygen and carbon isotope compositions of fossil bird eggshell calcite (δ(18)Ocalc and δ(13)Ccalc) are regularly used to reconstruct paleoenvironmental conditions. However, the interpretation of δ(18)Ocalc values of fossil eggshells has been limited to qualitative variations in local climatic conditions as oxygen isotope fractionations between calcite, body fluids, and drinking water have not been determined yet. For this purpose, eggshell, albumen water, and drinking water of extant birds have been analyzed for their oxygen and carbon isotope compositions. Relative enrichments in (18)O relative to (16)O between body fluids and drinking water of +1.6 ± 0.9 ‰ for semi-aquatic birds and of +4.4 ± 1.9 ‰ for terrestrial birds are observed. Surprisingly, no significant dependence to body temperature on the oxygen isotope fractionation between eggshell calcite and body fluids is observed, suggesting that bird eggshells precipitate out of equilibrium. Two empirical equations relating the δ(18)Ocalc value of eggshell calcite to the δ(18)Ow value of ingested water have been established for terrestrial and semi-aquatic birds. These equations have been applied to fossil eggshells from Lanzarote in order to infer the ecologies of the Pleistocene marine bird Puffinus sp. and of the enigmatic giant birds from the Pliocene. Both δ(13)Ccalc and δ(18)Ocalc values of Puffinus eggshells point to a semi-aquatic marine bird ingesting mostly seawater, whereas low δ(13)Ccalc and high δ(18)Ocalc values of eggshells from the Pliocene giant bird suggest a terrestrial lifestyle. This set of equations can help to quantitatively estimate the origin of waters ingested by extinct birds as well as to infer either local environmental or climatic conditions. PMID:27639729

  6. Kinetic and equilibrium studies of adsorptive removal of phenol onto eggshell waste.

    PubMed

    Daraei, H; Mittal, A; Noorisepehr, M; Daraei, F

    2013-07-01

    The aim of the present research is to develop economic, fast, and versatile method for the removal of toxic organic pollutant phenol from wastewater using eggshell. The batch experiments are conducted to evaluate the effect of pH, phenol concentration, dosage of adsorbent, and contact time on the removal of phenol. The paper includes in-depth kinetic studies of the ongoing adsorption process. Attempts have also been made to verify Langmuir and Freundlich adsorption isotherms. The morphology and characteristics of eggshell have also been studied using scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and X-ray fluorescence analysis. At ambient temperature, the maximum adsorption of phenol onto eggshells has been achieved at pH 9 and the contact time, 90 min. The experimental data give best-fitted straight lines for pseudo-first-order as well as pseudo-second-order kinetic models. Furthermore, the adsorption process verifies Freundlich and Langmuir adsorption isotherms, and on the basis of mathematical expressions of these models, various necessary adsorption constants have been calculated. Using adsorption data, various thermodynamic parameters like change in enthalpy (∆H(0)), change in entropy (∆S(0)), and change in free energy ∆G(0) have also been evaluated. Results clearly reveal that the solid waste material eggshell acts as an effective adsorbent for the removal of phenol from aqueous solutions. PMID:23274804

  7. 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

  8. 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

  9. 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.

  10. 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 eggshells using high repetition femtosecond laser irradiation. The eggshell waste is value engineered to calcium carbonate nanofibrous layer in a single step under ambient conditions. Our striking results demonstrate that by controlling the laser pulse repetition, nanostructures with different nanofiber density can be achieved. This approach presents an important step towards synthesizing 3D interwoven nanofibrous platforms from natural biomaterials. Conclusion The synthesized 3D nanofibrous structures can promote biomaterial interfacial properties to improve cell-platform surface interaction and develop new functional biomaterials for a variety of biomedical applications. PMID:21251288

  11. 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

  12. 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.

  13. An Integrated Bioinformatics and Computational Biology Approach Identifies New BH3-Only Protein Candidates.

    PubMed

    Hawley, Robert G; Chen, Yuzhong; Riz, Irene; Zeng, Chen

    2012-05-01

    In this study, we utilized an integrated bioinformatics and computational biology approach in search of new BH3-only proteins belonging to the BCL2 family of apoptotic regulators. The BH3 (BCL2 homology 3) domain mediates specific binding interactions among various BCL2 family members. It is composed of an amphipathic α-helical region of approximately 13 residues that has only a few amino acids that are highly conserved across all members. Using a generalized motif, we performed a genome-wide search for novel BH3-containing proteins in the NCBI Consensus Coding Sequence (CCDS) database. In addition to known pro-apoptotic BH3-only proteins, 197 proteins were recovered that satisfied the search criteria. These were categorized according to α-helical content and predictive binding to BCL-xL (encoded by BCL2L1) and MCL-1, two representative anti-apoptotic BCL2 family members, using position-specific scoring matrix models. Notably, the list is enriched for proteins associated with autophagy as well as a broad spectrum of cellular stress responses such as endoplasmic reticulum stress, oxidative stress, antiviral defense, and the DNA damage response. Several potential novel BH3-containing proteins are highlighted. In particular, the analysis strongly suggests that the apoptosis inhibitor and DNA damage response regulator, AVEN, which was originally isolated as a BCL-xL-interacting protein, is a functional BH3-only protein representing a distinct subclass of BCL2 family members.

  14. A set of descriptors for identifying the protein-drug interaction in cellular networking.

    PubMed

    Nanni, Loris; Lumini, Alessandra; Brahnam, Sheryl

    2014-10-21

    The study of protein-drug interactions is a significant issue for drug development. Unfortunately, it is both expensive and time-consuming to perform physical experiments to determine whether a drug and a protein are interacting with each other. Some previous attempts to design an automated system to perform this task were based on the knowledge of the 3D structure of a protein, which is not always available in practice. With the availability of protein sequences generated in the post-genomic age, however, a sequence-based solution to deal with this problem is necessary. Following other works in this area, we propose a new machine learning system based on several protein descriptors extracted from several protein representations, such as, variants of the position specific scoring matrix (PSSM) of proteins, the amino-acid sequence, and a matrix representation of a protein. The prediction engine is operated by an ensemble of support vector machines (SVMs), with each SVM trained on a specific descriptor and the results of each SVM combined by sum rule. The overall success rate achieved by our final ensemble is notably higher than previous results obtained on the same datasets using the same testing protocols reported in the literature. MATLAB code and the datasets used in our experiments are freely available for future comparison at http://www.dei.unipd.it/node/2357.

  15. 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 ...

  16. Application of the chromatin immunoprecipitation method to identify in vivo protein-DNA associations in fission yeast.

    PubMed

    Takahashi, K; Saitoh, S; Yanagida, M

    2000-10-31

    The chromatin immunoprecipitation (ChIP) method provides an ideal tool for detecting direct or indirect interactions between proteins of interest and DNAs with known sequences. Here, we introduce the ChIP protocol used in our laboratory to identify in vivo protein-DNA association in the fission yeast Schizosaccharomyces pombe. The cytological and genetic merits of the fission yeast for studying control of the eukaryotic cell cycle and chromosome dynamics are reinforced by application of this ChIP method.

  17. Correlation of eggshell strength and Salmonella enteritidis contamination of commercial shell eggs.

    PubMed

    Jones, D R; Musgrove, M T

    2005-10-01

    Shell quality has been identified as a heritable trait that can be manipulated by genetic selection. Previous research has concluded that many methods of determining shell quality produce variable results. With the development of newer, more precise measuring technologies, shell strength can now be assessed in a consistent, objective fashion. A research project was conducted to determine what role shell strength might play in affecting external Salmonella Enteritidis contamination of egg contents. Visibly clean eggs were collected from an in-line shell egg-processing facility at the accumulator. Eggs were inoculated by dipping in a concentrated suspension of nalidixic acid-resistant Salmonella Enteritidis. After storage, eggs were assessed for shell strength and both external and internal Salmonella Enteritidis contamination. In the first study, there was a significant difference (P < 0.05) in shell strength among the three replicates. No differences between treatments were found for shell strength or Salmonella Enteritidis contamination of contents. In the second study, there were no replicate differences for any of the monitored factors. When rinsate and content samples were enriched, 100% of the rinsates were positive for Salmonella Enteritidis. No content samples were shown to be contaminated with Salmonella Enteritidis during direct plating, but 3 to 5% of the samples from each replicate were positive after enrichment. Correlation analysis of the results from each study found only weak correlations between shell strength and Salmonella Enteritidis contamination on eggshell surface or contents. Within the range of shell strengths recorded in this study, the correlation analysis suggests that shell strength does not play a major role in Salmonella Enteritidis contamination. Further work with eggs that represent a greater range of shell strengths could provide a clearer indication of the interaction of shell strength and Salmonella Enteritidis contamination.

  18. 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

  19. 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.

  20. 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

  1. 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

  2. Eggshell penetration of various types of hens' eggs by Salmonella enterica serovar Enteritidis.

    PubMed

    Messens, Winy; Grijspeerdt, Koen; De Reu, Koen; De Ketelaere, Bart; Mertens, Kristof; Bamelis, Flip; Kemps, Bart; De Baerdemaeker, Josse; Decuypere, Eddy; Herman, Lieve

    2007-03-01

    Egg weight, shell thickness, number of pores, cuticle deposition, eggshell strength (dynamic stiffness and damping ratio), and the ability of Salmonella enterica serovar Enteritidis (SE) to penetrate the eggshell were determined. Penetration was assessed by filling the eggs with a selective medium that allowed viewing of Salmonella growth on the inside of the shell and membrane complex. After inoculation of each shell with on average 2.71 log CFU, the eggs were stored for up to 14 days at 20 degrees C and 60% relative humidity. Commercially available eggs were used. At 14 days of storage, only 6.0% of the eggs from free-range hens and 16.0% of the generic (i.e., eggs from hens in conventional battery cages that were given standard feed) white eggs were penetrated. The generic brown, organic, and omega-3-enriched eggs were penetrated at a frequency of 30 to 34%. In a second experiment it was shown that the layer strains of the hen (ISA-Brown Warren versus Bovans Goldline), which were kept in furnished cages, did not affect eggshell penetration by SE. For Bovans Goldline hens, the housing system (furnished cage versus aviary) did not affect penetration, while a trend was visible toward a higher fraction of penetrated eggshells when hens were fed corncob mix rather than standard feed. Eggshell penetration was observed more frequently in the absence of cuticle spots and for eggs having lower dynamic stiffness values. Shell contamination at the end of storage was highly correlated with SE penetration.

  3. Eggshell pigmentation pattern in relation to breeding performance of blue tits Cyanistes caeruleus.

    PubMed

    Sanz, Juan José; García-Navas, Vicente

    2009-01-01

    1. We test the consequences, in terms of breeding success and parental effort, of eggshell pigmentation pattern in a hole-nesting bird, the blue tit Cyanistes caeruleus that lays eggs asymmetrically speckled with reddish spots (maculated eggs). 2. We assess the effect of distribution of spots (pigment 'spread') and spot size and pigment intensity (pigment 'darkness') on eggshell physical properties and breeding parameters concerning nestling condition, investment of parents in offspring care and reproductive output in two different habitat types: a deciduous oakwoodland and an evergreen forest. 3. Blue tit clutches with more widely distributed spots showed a thicker eggshell, a shorter incubation period, a lesser amount of mass loss per day and a higher hatching probability than those with spots forming a 'corona' ring. While eggs with larger and darker (more pigment intensity) spots showed a thicker eggshell and a shorter incubation period. In the light of 'signal function hypothesis', these egg traits may reflect female health status and, consequently, this could affect male parental effort. 4. Here we show supports for some of the necessary assumptions of this hypothesis. We found a positive relationship between egg pigment 'spread' and male but not female provisioning rates per day. On the other hand, pigment 'darkness' of blue tits' clutches was positively related to female tarsus length, while pigment 'spread' was positively related to clutch size, male body mass and nestling tarsus length. Our study shows that eggshell pigment 'spread' can be used as an indicator of clutch quality. Further investigations are needed to understand the role of calcium availability as possible causal agent of deviant eggs and its relation to the maculation phenomenon.

  4. Use of protein-engineered fabrics to identify design rules for integrin ligand clustering in biomaterials.

    PubMed

    Benitez, Patrick L; Mascharak, Shamik; Proctor, Amy C; Heilshorn, Sarah C

    2016-01-01

    While ligand clustering is known to enhance integrin activation, this insight has been difficult to apply to the design of implantable biomaterials because the local and global ligand densities that enable clustering-enhanced integrin signaling were unpredictable. Here, two general design principles for biomaterial ligand clustering are elucidated. First, clustering ligands enhances integrin-dependent signals when the global ligand density, i.e., the ligand density across the cellular length scale, is near the ligand's effective dissociation constant (KD,eff). Second, clustering ligands enhances integrin activation when the local ligand density, i.e., the ligand density across the length scale of individual focal adhesions, is less than an overcrowding threshold. To identify these principles, we fabricated a series of elastin-like, electrospun fabrics with independent control over the local (0 to 122 000 ligands μm(-2)) and global (0 to 71 000 ligand μm(-2)) densities of an arginine-glycine-aspartate (RGD) ligand. Antibody blocking studies confirmed that human umbilical vein endothelial cell adhesion to these protein-engineered biomaterials was primarily due to αVβ3 integrin binding. Clustering ligands enhanced cell proliferation, focal adhesion number, and focal adhesion kinase expression near the ligand's KD,eff of 12 000 RGD μm(-2). Near this global ligand density, cells on ligand-clustered fabrics behaved similarly to cells grown on fabrics with significantly larger global ligand densities but without clustering. However, this enhanced ligand-clustering effect was not observed above a threshold cut-off concentration. At a local ligand density of 122 000 RGD μm(-2), cell division, focal adhesion number, and focal adhesion kinase expression were significantly reduced relative to fabrics with identical global ligand density and lesser local ligand densities. Thus, when clustering results in overcrowding of ligands, integrin receptors are no longer

  5. Xp11.2 translocation renal cell carcinoma with egg-shell calcification mimicking a benign renal tumour: A case report

    PubMed Central

    LIANG, WENJIE; XU, SHUNLIANG

    2015-01-01

    The present study reports the case of a 20-year-old female who was identified to have a left renal angiomyolipoma (AML) with hemorrhage. Following temporary conservative observation, the patient received continuous ultrasonic follow-up. Due to the rapid growth of the lesion, further examinations were performed. Computed tomography (CT) plain scans revealed a partly high-density mass with marginal egg-shell calcification. Enhanced CT revealed a solid tumor with a rich blood supply. Since no fats were detected, the possibility of a typical AML was excluded, but the diagnoses of epithelioid AML or renal cancer were considered. Finally, the left kidney was partially excised laparoscopically. The intraoperative frozen section indicated a diagnosis of renal cell carcinoma (RCC). The left kidney was subsequently radically excised. Routine histopathological and immunohistochemical tests confirmed that the lesion was an RCC with an Xp11.2 translocation. The present study introduces the pitfalls in the diagnosis of Xp11.2 translocation RCC, which is a rare RCC subtype accompanied with uncommon imaging manifestations. The study suggests that when a rapidly-growing AML is detected by ultrasound, renal cancer with marginal calcification should be considered. Moreover, although egg-shell calcification mostly occurs in benign renal lesions, further examinations, such as enhanced CT, are recommended for identifying the nature of the masses and excluding the possibility of malignant tumors. PMID:26722310

  6. 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.

  7. 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

  8. A proteomics approach to identifying key protein targets involved in VEGF inhibitor mediated attenuation of bleomycin-induced pulmonary fibrosis.

    PubMed

    Kulkarni, Yogesh M; Dutta, Sucharita; Iyer, Anand Krishnan V; Venkatadri, Rajkumar; Kaushik, Vivek; Ramesh, Vani; Wright, Clayton A; Semmes, Oliver John; Yakisich, Juan S; Azad, Neelam

    2016-01-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a life expectancy of less than 5 years post diagnosis for most patients. Poor molecular characterization of IPF has led to insufficient understanding of the pathogenesis of the disease, resulting in lack of effective therapies. In this study, we have integrated a label-free LC-MS based approach with systems biology to identify signaling pathways and regulatory nodes within protein interaction networks that govern phenotypic changes that may lead to IPF. Ingenuity Pathway Analysis of proteins modulated in response to bleomycin treatment identified PI3K/Akt and Wnt signaling as the most significant profibrotic pathways. Similar analysis of proteins modulated in response to vascular endothelial growth factor (VEGF) inhibitor (CBO-P11) treatment identified natural killer cell signaling and PTEN signaling as the most significant antifibrotic pathways. Mechanistic/mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase (ERK) were identified to be key mediators of pro- and antifibrotic response, where bleomycin (BLM) treatment resulted in increased expression and VEGF inhibitor treatment attenuated expression of mTOR and ERK. Using a BLM mouse model of pulmonary fibrosis and VEGF inhibitor CBO-P11 as a therapeutic measure, we identified a comprehensive set of signaling pathways and proteins that contribute to the pathogenesis of pulmonary fibrosis that can be targeted for therapy against this fatal disease.

  9. 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

  10. 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.

  11. SERF: in vitro election of random RNA fragments to identify protein binding sites within large RNAs.

    PubMed

    Stelzl, U; Nierhaus, K H

    2001-11-01

    In vitro selection experiments have various goals depending on the composition of the initial pool and the selection method applied. We developed an in vitro selection variant (SERF, selection of random RNA fragments) that is useful for the identification of short RNA fragments originating from large RNAs that bind specifically to a protein. A pool of randomly fragmented RNA is constructed from a large RNA, which is the natural binding partner for a protein. Such a pool contains all the potential binding sites and is therefore used as starting material for affinity selection with the purified protein to find its natural target. Here we provide a detailed experimental protocol of the method. SERF has been developed for ribosomal systems and is a general approach providing a basis for functional and structural characterization of RNA-protein interactions in large ribonucleoprotein particles.

  12. 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

  13. Purification and characterization of a vaterite-inducing peptide, pelovaterin, from the eggshells of Pelodiscus sinensis (Chinese soft-shelled turtle).

    PubMed

    Lakshminarayanan, Rajamani; Chi-Jin, Emma Ooi; Loh, Xian Jun; Kini, R Manjunatha; Valiyaveettil, Suresh

    2005-01-01

    Proteins play a crucial role in the biomineralization of hard tissues such as eggshells. We report here the purification, characterization, and in vitro mineralization studies of a peptide, pelovaterin, extracted from eggshells of a soft-shelled turtle. It is a glycine-rich peptide with 42 amino acid residues and three disulfide bonds. When tested in vitro, the peptide induced the formation of a metastable vaterite phase. The floret-shaped morphology formed at a lower concentration ( approximately 1 microM) was transformed into spherical particles at higher concentrations (>500 microM). The solution properties of the peptide are investigated by circular dichroism (CD), fluorescence emission spectroscopy, and dynamic light scattering (DLS) experiments. The conformation of pelovaterin changed from an unordered state at a low concentration to a beta-sheet structure at high concentrations. Fluorescence emission studies indicated that the quantum yield is significantly decreased at higher concentrations, accompanied by a blue shift in the emission maximum. At higher concentrations a red-edge excitation shift was observed, indicating the restricted mobility of the peptide. On the basis of these observations, we discuss the presence of a peptide concentration-dependent monomer-multimer equilibrium in solution and its role in controlling the nucleation, growth, and morphology of CaCO(3) crystals. This is the first peptide known to induce the nucleation and stabilization of the vaterite phase in solution.

  14. 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.

  15. 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

  16. 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

  17. Transient changes in intercellular protein variability identify sources of noise in gene expression.

    PubMed

    Singh, Abhyudai

    2014-11-01

    Protein levels differ considerably between otherwise identical cells, and these differences significantly affect biological function and phenotype. Previous work implicated various noise mechanisms that drive variability in protein copy numbers across an isogenic cell population. For example, transcriptional bursting of mRNAs has been shown to be a major source of noise in the expression of many genes. Additional expression variability, referred to as extrinsic noise, arises from intercellular variations in mRNA transcription and protein translation rates attributed to cell-to-cell differences in cell size, abundance of ribosomes, etc. We propose a method to determine the magnitude of different noise sources in a given gene of interest. The method relies on blocking transcription and measuring changes in protein copy number variability over time. Our results show that this signal has sufficient information to quantify both the extent of extrinsic noise and transcription bursting in gene expression. Moreover, if the mean mRNA count is known, then the relative contributions of transcription versus translation rate fluctuations to extrinsic noise can also be determined. In summary, our study provides an easy-to-implement method for characterizing noisy protein expression that complements existing techniques for studying stochastic dynamics of genetic circuits.

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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

  3. Characterization of RAB-like4, the first identified RAB-like protein from Trypanosoma cruzi with GTPase activity.

    PubMed

    Ramos, Fabiane Pereira; Araripe, Júlia Rolão; Urményi, Turán Péter; Silva, Rosane; Cunha e Silva, Narcisa Leal; Leite Fontes, Carlos Frederico; da Silveira, José Franco; Rondinelli, Edson

    2005-08-01

    RAB proteins, which belong to the RAS superfamily, regulate exocytic and endocytic pathways of eukaryotic cells, controlling vesicle docking and fusion. Few RAB proteins have been identified in parasites. Molecular markers for cellular compartments are important to studies concerning about the protein traffic in Trypanosoma cruzi, the causal agent of Chagas disease. In this work, we describe the characterization of TcRABL4, the first RAB-like gene identified in T. cruzi (GenBank Accession No.: ), present as a single-copy gene. TcRABL4 contains all five consensus RAB motifs but lacks cysteine residues at the C terminus, which are essential to isoprenylation, an absolute prerequisite for membrane association of these proteins. TcRABL4 is a functional GTPase that is able to bind and hydrolyze GTP, and its gene is transcribed as a single 1.2 kb mRNA in epimastigotes. TcRABL4 appears to be differentially regulated in the three cell forms of the parasite, and the protein is not associated to membranes, unlike other RAB proteins. It is possible that TcRABL4 may be a member of a novel family of small GTPases.

  4. 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...

  5. Data mining of protein-binding profiling data identifies structural modifications that distinguish selective and promiscuous compounds.

    PubMed

    Yongye, Austin B; Medina-Franco, José L

    2012-09-24

    Activity profiling of compound collections across multiple targets is increasingly being used in probe and drug discovery. Herein, we discuss an approach to systematically analyzing the structure-activity relationships of a large screening profile data with emphasis on identifying structural changes that have a significant impact on the number of proteins to which a compound binds. As a case study, we analyzed a recently released public data set of more than 15 000 compounds screened across 100 sequence-unrelated proteins. The screened compounds have different origins and include natural products, synthetic molecules from academic groups, and commercial compounds. Similar synthetic structures from academic groups showed, overall, greater promiscuity differences than do natural products and commercial compounds. The method implemented in this work readily identified structural changes that differentiated highly specific from promiscuous compounds. This approach is general and can be applied to analyze any other large-scale protein-binding profile data.

  6. PROMOTIF--a program to identify and analyze structural motifs in proteins.

    PubMed Central

    Hutchinson, E. G.; Thornton, J. M.

    1996-01-01

    We describe a suite of programs, PROMOTIF, that analyzes a protein coordinate file and provides details about the structural motifs in the protein. The program currently analyzes the following structural features: secondary structure; beta-and gamma-turns; helical geometry and interactions; beta-strands and beta-sheet topology; beta-bulges; beta-hairpins; beta-alpha-beta units and psi-loops; disulphide bridges; and main-chain hydrogen bonding patterns. PROMOTIF creates postscript files showing the examples of each type of motif in the protein, and a summary page. The program can also be used to compare motifs in a group of related structures, such as an ensemble of NMR structures. PMID:8745398

  7. 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

  8. Eggshell Appearance Does Not Signal Maternal Corticosterone Exposure in Japanese Quail: An Experimental Study with Brown-Spotted Eggs

    PubMed Central

    Duval, Camille; Cassey, Phillip; Lovell, Paul G.; Mikšík, Ivan; Reynolds, S. James; Spencer, Karen A.

    2013-01-01

    Reproduction is a critical period for birds as they have to cope with many stressful events. One consequence of an acute exposure to stress is the release of corticosterone, the avian stress hormone. Prolonged stress can have negative impacts on the immune system, resulting in, for example, increased oxidative stress. Through maternal effects, females are known to modulate their investment in eggs content according to their own physiological condition. Less is known about maternal investment in eggshells, especially in pigments. The two main eggshell pigments may possess opposite antioxidant properties: protoporphyrin (brown) is a pro-oxidant, whereas biliverdin (blue-green) is an antioxidant. In Japanese quail, we know that the deposition of both pigments is related to female body condition. Thus, a chronic stress response may be reflected in eggshell coloration. Using female Japanese quails that lay brown-spotted eggs, we explored whether physiological exposure to corticosterone induces a change in female basal stress and antioxidant factors, and eggshell pigment concentration, spectrophotometric reflectance, and maculation coverage. We supplemented adult females over a 2 week period with either peanut oil (control) or corticosterone (treatment). We collected pre- and post-supplementation eggs and analysed the effect of corticosterone treatment on female physiology and eggshell appearance parameters. Except for corticosterone-fed birds which laid eggs with brighter spots, supplementation had no significant effect on female physiology or eggshell pigment concentration, reflectance and maculation. The change in eggshell spot brightness was not detected by a photoreceptor noise-limited color opponent model of avian visual perception. Our data confirms that eggshell reflectance in spotted eggs varies over the laying sequence, and spot reflectance may be a key factor that is affected by females CORT exposure, even if the changes are not detected by an avian visual

  9. Eggshell appearance does not signal maternal corticosterone exposure in Japanese quail: an experimental study with brown-spotted eggs.

    PubMed

    Duval, Camille; Cassey, Phillip; Lovell, Paul G; Mikšík, Ivan; Reynolds, S James; Spencer, Karen A

    2013-01-01

    Reproduction is a critical period for birds as they have to cope with many stressful events. One consequence of an acute exposure to stress is the release of corticosterone, the avian stress hormone. Prolonged stress can have negative impacts on the immune system, resulting in, for example, increased oxidative stress. Through maternal effects, females are known to modulate their investment in eggs content according to their own physiological condition. Less is known about maternal investment in eggshells, especially in pigments. The two main eggshell pigments may possess opposite antioxidant properties: protoporphyrin (brown) is a pro-oxidant, whereas biliverdin (blue-green) is an antioxidant. In Japanese quail, we know that the deposition of both pigments is related to female body condition. Thus, a chronic stress response may be reflected in eggshell coloration. Using female Japanese quails that lay brown-spotted eggs, we explored whether physiological exposure to corticosterone induces a change in female basal stress and antioxidant factors, and eggshell pigment concentration, spectrophotometric reflectance, and maculation coverage. We supplemented adult females over a 2 week period with either peanut oil (control) or corticosterone (treatment). We collected pre- and post-supplementation eggs and analysed the effect of corticosterone treatment on female physiology and eggshell appearance parameters. Except for corticosterone-fed birds which laid eggs with brighter spots, supplementation had no significant effect on female physiology or eggshell pigment concentration, reflectance and maculation. The change in eggshell spot brightness was not detected by a photoreceptor noise-limited color opponent model of avian visual perception. Our data confirms that eggshell reflectance in spotted eggs varies over the laying sequence, and spot reflectance may be a key factor that is affected by females CORT exposure, even if the changes are not detected by an avian visual

  10. Can You Solve the Crime? Using Agarose Electrophoresis To Identify an Unknown Colored Protein.

    ERIC Educational Resources Information Center

    Wiltfong, Cynthia L.; Chester, Emily; Albertin, Faith; Smith, Julia; Hall, Judith C.; Arth, Emily C.; Martin, Stephanie

    2003-01-01

    Describes a lab that introduces agarose electrophoresis techniques and basic information on proteins to middle school and high school students. Insists that, built around a scenario in which students must solve a crime, the lab has real-world applications that should spark student interest. (KHR)

  11. RIPSeeker: a statistical package for identifying protein-associated transcripts from RIP-seq experiments

    PubMed Central

    Li, Yue; Zhao, Dorothy Yanling; Greenblatt, Jack F.; Zhang, Zhaolei

    2013-01-01

    RIP-seq has recently been developed to discover genome-wide RNA transcripts that interact with a protein or protein complex. RIP-seq is similar to both RNA-seq and ChIP-seq, but presents unique properties and challenges. Currently, no statistical tool is dedicated to RIP-seq analysis. We developed RIPSeeker (http://www.bioconductor.org/packages/2.12/bioc/html/RIPSeeker.html), a free open-source Bioconductor/R package for de novo RIP peak predictions based on HMM. To demonstrate the utility of the software package, we applied RIPSeeker and six other published programs to three independent RIP-seq datasets and two PAR-CLIP datasets corresponding to six distinct RNA-binding proteins. Based on receiver operating curves, RIPSeeker demonstrates superior sensitivity and specificity in discriminating high-confidence peaks that are consistently agreed on among a majority of the comparison methods, and dominated 9 of the 12 evaluations, averaging 80% area under the curve. The peaks from RIPSeeker are further confirmed based on their significant enrichment for biologically meaningful genomic elements, published sequence motifs and association with canonical transcripts known to interact with the proteins examined. While RIPSeeker is specifically tailored for RIP-seq data analysis, it also provides a suite of bioinformatics tools integrated within a self-contained software package comprehensively addressing issues ranging from post-alignments’ processing to visualization and annotation. PMID:23455476

  12. Identifying the adaptive mechanism in globular proteins: Fluctuations in densely packed regions manipulate flexible parts

    NASA Astrophysics Data System (ADS)

    Yilmaz, Lutfu Safak; Atilgan, Ali Rana

    2000-09-01

    A low-resolution structural model based on the packing geometry of α-carbons is utilized to establish a connection between the flexible and rigid parts of a folded protein. The former commonly recognizes a complementing molecule for making a complex, while the latter manipulates the necessary conformational change for binding. We attempt analytically to distinguish this control architecture that intrinsically exists in globular proteins. First with two-dimensional simple models, then for a native protein, bovine pancreatic trypsin inhibitor, we explicitly demonstrate that inserting fluctuations in tertiary contacts supported by the stable core, one can regulate the displacement of residues on loop regions. The positional fluctuations of the flexible regions are annihilated by the rest of the protein in conformity with the Le Chatelier-Braun principle. The results indicate that the distortion of the principal nonbonded contacts between highly packed residues is accompanied by that of the slavery fluctuations that are widely distributed over the native structure. These positional arrangements do not appear in a reciprocal relation between a perturbation and the associated response; the effect of a movement of residue i on residue j is not equal to that of the same movement of residue j on residue i.

  13. R4 regulators of G protein signaling (RGS) identify an ancient MHC-linked synteny group

    PubMed Central

    Suurväli, Jaanus; Robert, Jacques; Boudinot, Pierre; Boudinot, Sirje Rüütel

    2012-01-01

    Regulators of G Protein Signaling (RGS) are key regulators of G protein signaling. RGS proteins of the R4 RGS group are composed of a mere RGS domain and are mainly involved in immune response modulation. In both human and mouse, most genes encoding the R4 RGS proteins are located in the same region of chromosome 1. We show here that the RGS1/RGS16 neighborhood constitutes a synteny group well conserved across tetrapods, and closely linked to the MHC paralogon of chromosome 1. Genes located in the RGS1/RGS16 region have paralogs close to the MHC on chromosome 6 or close to the other MHC paralogons. In amphioxus, a cephalochordate, these genes possess orthologs that are located in the same scaffolds as a number of markers defining the proto-MHC in this species (Abi-Rached et al. 2002). We therefore propose that the RGS1/RGS16 region provides useful markers to investigate the origins and the evolution of the MHC. In addition, we show that some genes of the region appear to have immune functions not only in human, but also in Xenopus. PMID:23129146

  14. Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action.

    PubMed

    Wang, Pengcheng; Xue, Liang; Batelli, Giorgia; Lee, Shinyoung; Hou, Yueh-Ju; Van Oosten, Michael J; Zhang, Huiming; Tao, W Andy; Zhu, Jian-Kang

    2013-07-01

    Sucrose nonfermenting 1 (SNF1)-related protein kinase 2s (SnRK2s) are central components of abscisic acid (ABA) signaling pathways. The snrk2.2/2.3/2.6 triple-mutant plants are nearly completely insensitive to ABA, suggesting that most of the molecular actions of ABA are triggered by the SnRK2s-mediated phosphorylation of substrate proteins. Only a few substrate proteins of the SnRK2s are known. To identify additional substrate proteins of the SnRK2s and provide insight into the molecular actions of ABA, we used quantitative phosphoproteomics to compare the global changes in phosphopeptides in WT and snrk2.2/2.3/2.6 triple mutant seedlings in response to ABA treatment. Among the 5,386 unique phosphorylated peptides identified in this study, we found that ABA can increase the phosphorylation of 166 peptides and decrease the phosphorylation of 117 peptides in WT seedlings. In the snrk2.2/2.3/2.6 triple mutant, 84 of the 166 peptides, representing 58 proteins, could not be phosphorylated, or phosphorylation was not increased under ABA treatment. In vitro kinase assays suggest that most of the 58 proteins can serve as substrates of the SnRK2s. The SnRK2 substrates include proteins involved in flowering time regulation, RNA and DNA binding, miRNA and epigenetic regulation, signal transduction, chloroplast function, and many other cellular processes. Consistent with the SnRK2 phosphorylation of flowering time regulators, the snrk2.2/2.3/2.6 triple mutant flowered significantly earlier than WT. These results shed new light on the role of the SnRK2 protein kinases and on the downstream effectors of ABA action, and improve our understanding of plant responses to adverse environments.

  15. Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action

    PubMed Central

    Wang, Pengcheng; Xue, Liang; Batelli, Giorgia; Lee, Shinyoung; Hou, Yueh-Ju; Van Oosten, Michael J.; Zhang, Huiming; Tao, W. Andy; Zhu, Jian-Kang

    2013-01-01

    Sucrose nonfermenting 1 (SNF1)-related protein kinase 2s (SnRK2s) are central components of abscisic acid (ABA) signaling pathways. The snrk2.2/2.3/2.6 triple-mutant plants are nearly completely insensitive to ABA, suggesting that most of the molecular actions of ABA are triggered by the SnRK2s-mediated phosphorylation of substrate proteins. Only a few substrate proteins of the SnRK2s are known. To identify additional substrate proteins of the SnRK2s and provide insight into the molecular actions of ABA, we used quantitative phosphoproteomics to compare the global changes in phosphopeptides in WT and snrk2.2/2.3/2.6 triple mutant seedlings in response to ABA treatment. Among the 5,386 unique phosphorylated peptides identified in this study, we found that ABA can increase the phosphorylation of 166 peptides and decrease the phosphorylation of 117 peptides in WT seedlings. In the snrk2.2/2.3/2.6 triple mutant, 84 of the 166 peptides, representing 58 proteins, could not be phosphorylated, or phosphorylation was not increased under ABA treatment. In vitro kinase assays suggest that most of the 58 proteins can serve as substrates of the SnRK2s. The SnRK2 substrates include proteins involved in flowering time regulation, RNA and DNA binding, miRNA and epigenetic regulation, signal transduction, chloroplast function, and many other cellular processes. Consistent with the SnRK2 phosphorylation of flowering time regulators, the snrk2.2/2.3/2.6 triple mutant flowered significantly earlier than WT. These results shed new light on the role of the SnRK2 protein kinases and on the downstream effectors of ABA action, and improve our understanding of plant responses to adverse environments. PMID:23776212

  16. Differentially Expressed RNA from Public Microarray Data Identifies Serum Protein Biomarkers for Cross-Organ Transplant Rejection and Other Conditions

    PubMed Central

    Chen, Rong; Sigdel, Tara K.; Li, Li; Kambham, Neeraja; Dudley, Joel T.; Hsieh, Szu-chuan; Klassen, R. Bryan; Chen, Amery; Caohuu, Tuyen; Morgan, Alexander A.; Valantine, Hannah A.; Khush, Kiran K.; Sarwal, Minnie M.; Butte, Atul J.

    2010-01-01

    Serum proteins are routinely used to diagnose diseases, but are hard to find due to low sensitivity in screening the serum proteome. Public repositories of microarray data, such as the Gene Expression Omnibus (GEO), contain RNA expression profiles for more than 16,000 biological conditions, covering more than 30% of United States mortality. We hypothesized that genes coding for serum- and urine-detectable proteins, and showing differential expression of RNA in disease-damaged tissues would make ideal diagnostic protein biomarkers for those diseases. We showed that predicted protein biomarkers are significantly enriched for known diagnostic protein biomarkers in 22 diseases, with enrichment significantly higher in diseases for which at least three datasets are available. We then used this strategy to search for new biomarkers indicating acute rejection (AR) across different types of transplanted solid organs. We integrated three biopsy-based microarray studies of AR from pediatric renal, adult renal and adult cardiac transplantation and identified 45 genes upregulated in all three. From this set, we chose 10 proteins for serum ELISA assays in 39 renal transplant patients, and discovered three that were significantly higher in AR. Interestingly, all three proteins were also significantly higher during AR in the 63 cardiac transplant recipients studied. Our best marker, serum PECAM1, identified renal AR with 89% sensitivity and 75% specificity, and also showed increased expression in AR by immunohistochemistry in renal, hepatic and cardiac transplant biopsies. Our results demonstrate that integrating gene expression microarray measurements from disease samples and even publicly-available data sets can be a powerful, fast, and cost-effective strategy for the discovery of new diagnostic serum protein biomarkers. PMID:20885780

  17. Using networks to identify fine structural differences between functionally distinct protein states.

    PubMed

    Swint-Kruse, Liskin

    2004-08-31

    The vast increase in available data from the "-omics" revolution has enabled the fields of structural proteomics and structure prediction to make great progress in assigning realistic three-dimensional structures to each protein molecule. The challenge now lies in determining the fine structural details that endow unique functions to sequences that assume a common fold. Similar problems are encountered in understanding how distinct conformations contribute to different phases of a single protein's dynamic function. However, efforts are hampered by the complexity of these large, three-dimensional molecules. To overcome this limitation, structural data have been recast as two-dimensional networks. This analysis greatly reduces visual complexity but retains information about individual residues. Such diagrams are very useful for comparing multiple structures, including (1) homologous proteins, (2) time points throughout a dynamics simulation, and (3) functionally different conformations of a given protein. Enhanced structural examination results in new functional hypotheses to test experimentally. Here, network representations were key to discerning a difference between unliganded and inducer-bound lactose repressor protein (LacI), which were previously presumed to be identical structures. Further, the interface of unliganded LacI was surprisingly similar to that of the K84L variant and various structures generated by molecular dynamics simulations. Apo-LacI appears to be poised to adopt the conformation of either the DNA- or inducer-bound structures, and the K84L mutation appears to freeze the structure partway through the conformational transition. Additional examination of the effector binding pocket results in specific hypotheses about how inducer, anti-inducer, and neutral sugars exert their effects on repressor function. PMID:15323549

  18. Integrated Translatomics with Proteomics to Identify Novel Iron–Transporting Proteins in Streptococcus pneumoniae

    PubMed Central

    Yang, Xiao-Yan; He, Ke; Du, Gaofei; Wu, Xiaohui; Yu, Guangchuang; Pan, Yunlong; Zhang, Gong; Sun, Xuesong; He, Qing-Yu

    2016-01-01

    Streptococcus pneumoniae (S.pneumoniae) is a major human pathogen causing morbidity and mortality worldwide. Efficiently acquiring iron from the environment is critical for S. pneumoniae to sustain growth and cause infection. There are only three known iron-uptake systems in Streptococcal species responsible for iron acquisition from the host, including ABC transporters PiaABC, PiuABC, and PitABC. Besides, no other iron-transporting system has been suggested. In this work, we employed our newly established translating mRNA analysis integrated with proteomics to evaluate the possible existence of novel iron transporters in the bacterium. We simultaneously deleted the iron-binding protein genes of the three iron-uptake systems to construct a piaA/piuA/pitA triple mutant (Tri-Mut) of S. pneumoniae D39, in which genes and proteins related to iron transport should be regulated in response to the deletion. With ribosome associated mRNA sequencing-based translatomics focusing on translating mRNA and iTRAQ quantitative proteomics based on the covalent labeling of peptides with tags of varying mass, we indeed observed a large number of genes and proteins representing various coordinated biological pathways with significantly altered expression levels in the Tri-Mut mutant. Highlighted in this observation is the identification of several new potential iron-uptake ABC transporters participating in iron metabolism of Streptococcus. In particular, putative protein SPD_1609 in operon 804 was verified to be a novel iron-binding protein with similar function to PitA in S. pneumoniae. These data derived from the integrative translatomics and proteomics analyses provided rich information and insightful clues for further investigations on iron-transporting mechanism in bacteria and the interplay between Streptococcal iron availability and the biological metabolic pathways. PMID:26870030

  19. Discovery Proteomics Identifies a Molecular Link between the Coatomer Protein Complex I and Androgen Receptor-dependent Transcription*

    PubMed Central

    Hsiao, Jordy J.; Smits, Melinda M.; Ng, Brandon H.; Lee, Jinhee; Wright, Michael E.

    2016-01-01

    Aberrant androgen receptor (AR)-dependent transcription is a hallmark of human prostate cancers. At the molecular level, ligand-mediated AR activation is coordinated through spatial and temporal protein-protein interactions involving AR-interacting proteins, which we designate the “AR-interactome.” Despite many years of research, the ligand-sensitive protein complexes involved in ligand-mediated AR activation in prostate tumor cells have not been clearly defined. Here, we describe the development, characterization, and utilization of a novel human LNCaP prostate tumor cell line, N-AR, which stably expresses wild-type AR tagged at its N terminus with the streptavidin-binding peptide epitope (streptavidin-binding peptide-tagged wild-type androgen receptor; SBP-AR). A bioanalytical workflow involving streptavidin chromatography and label-free quantitative mass spectrometry was used to identify SBP-AR and associated ligand-sensitive cytosolic proteins/protein complexes linked to AR activation in prostate tumor cells. Functional studies verified that ligand-sensitive proteins identified in the proteomic screen encoded modulators of AR-mediated transcription, suggesting that these novel proteins were putative SBP-AR-interacting proteins in N-AR cells. This was supported by biochemical associations between recombinant SBP-AR and the ligand-sensitive coatomer protein complex I (COPI) retrograde trafficking complex in vitro. Extensive biochemical and molecular experiments showed that the COPI retrograde complex regulates ligand-mediated AR transcriptional activation, which correlated with the mobilization of the Golgi-localized ARA160 coactivator to the nuclear compartment of prostate tumor cells. Collectively, this study provides a bioanalytical strategy to validate the AR-interactome and define novel AR-interacting proteins involved in ligand-mediated AR activation in prostate tumor cells. Moreover, we describe a cellular system to study how compartment-specific AR

  20. Activity-based protein profiling identifies a host enzyme, carboxylesterase 1, which is differentially active during hepatitis C virus replication.

    PubMed

    Blais, David R; Lyn, Rodney K; Joyce, Michael A; Rouleau, Yanouchka; Steenbergen, Rineke; Barsby, Nicola; Zhu, Lin-Fu; Pegoraro, Adrian F; Stolow, Albert; Tyrrell, David L; Pezacki, John Paul

    2010-08-13

    Hepatitis C virus (HCV) relies on many interactions with host cell proteins for propagation. Successful HCV infection also requires enzymatic activity of host cell enzymes for key post-translational modifications. To identify such enzymes, we have applied activity-based protein profiling to examine the activity of serine hydrolases during HCV replication. Profiling of hydrolases in Huh7 cells replicating HCV identified CES1 (carboxylesterase 1) as a differentially active enzyme. CES1 is an endogenous liver protein involved in processing of triglycerides and cholesterol. We observe that CES1 expression and activity were altered in the presence of HCV. The knockdown of CES1 with siRNA resulted in lower levels of HCV replication, and up-regulation of CES1 was observed to favor HCV propagation, implying an important role for this host cell protein. Experiments in HCV JFH1-infected cells suggest that CES1 facilitates HCV release because less intracellular HCV core protein was observed, whereas HCV titers remained high. CES1 activity was observed to increase the size and density of lipid droplets, which are necessary for the maturation of very low density lipoproteins, one of the likely vehicles for HCV release. In transgenic mice containing human-mouse chimeric livers, HCV infection also correlates with higher levels of endogenous CES1, providing further evidence that CES1 has an important role in HCV propagation. PMID:20530478

  1. Bacterial beta-lactamase fragmentation complementation strategy can be used as a method for identifying interacting protein pairs.

    PubMed

    Park, Jong-Hwa; Back, Jung Ho; Hahm, Soo Hyun; Shim, Hye-Young; Park, Min Ju; Ko, Sung Il; Han, Ye Sun

    2007-10-01

    We investigated the applicability of the TEM-1 beta- lactamase fragment complementation (BFC) system to develop a strategy for the screening of protein-protein interactions in bacteria. A BFC system containing a human Fas-associated death domain (hFADD) and human Fas death domain (hFasDD) was generated. The hFADD-hFasDD interaction was verified by cell survivability in ampicillin-containing medium and the colorimetric change of nitrocefin. It was also confirmed by His pull-down assay using cell lysates obtained in selection steps. A coiled-coil helix coiled-coil domain-containing protein 5 (CHCH5) was identified as an interacting protein of human uracil DNA glycosylase (hUNG) from the bacterial BFC cDNA library strategy. The interaction between hUNG and CHCH5 was further confirmed with immunoprecipitation using a mammalian expression system. CHCH5 enhanced the DNA glycosylase activity of hUNG to remove uracil from DNA duplexes containing a U/G mismatch pair. These results suggest that the bacterial BFC cDNA library strategy can be effectively used to identify interacting protein pairs.

  2. Application of a proteomic approach to identify proteins associated with primary graft non-function after liver transplantation.

    PubMed

    Kornasiewicz, Oskar; Bojarczuk, Kamil; Bugajski, Marek; Golab, Jakub; Krawczyk, Marek

    2012-10-01

    Primary graft non-function (PNF) is a rare, life-threatening complication of liver transplantation. Increasing use of extended criteria donor pools and high-risk recipients seem to influence the incidence of PNF. Primary failure is associated with high patient morbidity and inferior graft survival. The only available treatment for PNF is emergency hepatic retransplantation, which is also correlated with significant morbidity and mortality. Therefore, researchers are working to identify risk factors of diagnostic value to prevent PNF. The current study attempted to explore liver proteomic patterns in patients with PNF. Using two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry (LC-MS), we compared liver protein homogenates from 3 patients with PNF to those obtained from 6 healthy liver samples to identify potential new biomarkers of PNF. Our comparisons revealed 21 proteins with differential expression (13 upregulated and 8 downregulated). Most of these proteins are involved in energy metabolism, lipid metabolism, peptide cleavage, cell differentiation, and apoptosis. Although none of these proteins appeared more than once in separate analyses, this preliminary study shows that two-dimensional gel electrophoresis and LC-MS may allow identification of characteristic proteins to be used as biomarkers of a life-threatening complication of liver transplantation. Larger-scale analyses could improve patient care by finding suitable prognostic and therapeutic options. These data represent the first global proteomic approach to study PNF.

  3. ASSESSMENT OF MARKER PROTEINS IDENTIFIED IN WHOLE CELL EXTRACTS FOR BACTERIAL SPECIATION USING LIQUID CHROMATOGRAPHY ELECTROSPRAY IONIZATION TANDEM MASS SPECTROMETRY

    PubMed Central

    Kooken, Jennifer; Fox, Karen; Fox, Alvin; Wunschel, David

    2014-01-01

    Staphylococcal strains (CoNS) were speciated in this study. Digests of proteins released from whole cells were converted to tryptic peptides for analysis. Liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI MS/MS, Orbitrap) was employed for peptide analysis. Data analysis was performed employing the open-source software X!Tandem which uses sequenced genomes to generate a virtual peptide database for comparison to experimental data. The search database was modified to include the genomes of the 11 Staphylococcus species most commonly isolated from man. The number of total peptides matching each protein along with the number of peptides specifically matching to the homologue (or homologues) for strains of the same species were assesed. Any peptides not matching to the species examined were considered conflict peptides. The proteins typically identified with the largest percentage of sequence coverage, number of matched peptides and number of peptides corresponding to only the correct species were elongation factor Tu (EF Tu) and enolase (Enol). Additional proteins with consistently observed peptides as well as peptides matching only homologues from the same species were citrate synthase (CS) and 1-pyrroline-5-carboxylate dehydrogenase (1P5CD). Protein markers, previously identified from gel slices, (aconitate hydratase and oxoglutarate dehydrogenase) were found to provide low confidence scores when employing whole cell digests. The methodological approach described here provides a simple yet elegant way of identification of staphylococci. However, perhaps more importantly the technology should be applicable universally for identification of any bacterial species. PMID:23994725

  4. Comparative Genomics Identifies Epidermal Proteins Associated with the Evolution of the Turtle Shell

    PubMed Central

    Holthaus, Karin Brigit; Strasser, Bettina; Sipos, Wolfgang; Schmidt, Heiko A.; Mlitz, Veronika; Sukseree, Supawadee; Weissenbacher, Anton; Tschachler, Erwin; Alibardi, Lorenzo; Eckhart, Leopold

    2016-01-01

    The evolution of reptiles, birds, and mammals was associated with the origin of unique integumentary structures. Studies on lizards, chicken, and humans have suggested that the evolution of major structural proteins of the outermost, cornified layers of the epidermis was driven by the diversification of a gene cluster called Epidermal Differentiation Complex (EDC). Turtles have evolved unique defense mechanisms that depend on mechanically resilient modifications of the epidermis. To investigate whether the evolution of the integument in these reptiles was associated with specific adaptations of the sequences and expression patterns of EDC-related genes, we utilized newly available genome sequences to determine the epidermal differentiation gene complement of turtles. The EDC of the western painted turtle (Chrysemys picta bellii) comprises more than 100 genes, including at least 48 genes that encode proteins referred to as beta-keratins or corneous beta-proteins. Several EDC proteins have evolved cysteine/proline contents beyond 50% of total amino acid residues. Comparative genomics suggests that distinct subfamilies of EDC genes have been expanded and partly translocated to loci outside of the EDC in turtles. Gene expression analysis in the European pond turtle (Emys orbicularis) showed that EDC genes are differentially expressed in the skin of the various body sites and that a subset of beta-keratin genes within the EDC as well as those located outside of the EDC are expressed predominantly in the shell. Our findings give strong support to the hypothesis that the evolutionary innovation of the turtle shell involved specific molecular adaptations of epidermal differentiation. PMID:26601937

  5. Hammock: a hidden Markov model-based peptide clustering algorithm to identify protein-interaction consensus motifs in large datasets

    PubMed Central

    Krejci, Adam; Hupp, Ted R.; Lexa, Matej; Vojtesek, Borivoj; Muller, Petr

    2016-01-01

    Motivation: Proteins often recognize their interaction partners on the basis of short linear motifs located in disordered regions on proteins’ surface. Experimental techniques that study such motifs use short peptides to mimic the structural properties of interacting proteins. Continued development of these methods allows for large-scale screening, resulting in vast amounts of peptide sequences, potentially containing information on multiple protein-protein interactions. Processing of such datasets is a complex but essential task for large-scale studies investigating protein-protein interactions. Results: The software tool presented in this article is able to rapidly identify multiple clusters of sequences carrying shared specificity motifs in massive datasets from various sources and generate multiple sequence alignments of identified clusters. The method was applied on a previously published smaller dataset containing distinct classes of ligands for SH3 domains, as well as on a new, an order of magnitude larger dataset containing epitopes for several monoclonal antibodies. The software successfully identified clusters of sequences mimicking epitopes of antibody targets, as well as secondary clusters revealing that the antibodies accept some deviations from original epitope sequences. Another test indicates that processing of even much larger datasets is computationally feasible. Availability and implementation: Hammock is published under GNU GPL v. 3 license and is freely available as a standalone program (from http://www.recamo.cz/en/software/hammock-cluster-peptides/) or as a tool for the Galaxy toolbox (from https://toolshed.g2.bx.psu.edu/view/hammock/hammock). The source code can be downloaded from https://github.com/hammock-dev/hammock/releases. Contact: muller@mou.cz Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26342231

  6. Synthesis and evaluation of radioactive and fluorescent residualizing labels for identifying sites of plasma protein catabolism

    SciTech Connect

    Maxwell, J.L.; Baynes, J.W.; Thorpe, S.R.

    1986-05-01

    Inulin and lactose were each coupled to tyramine by reductive amination with NaBH/sub 3/CN and the tyramine then labeled with /sup 125/I. Dilactitol-/sup 125/I-tyramine (DLT) and inulin-/sup 125/I-tyramine (InTn) were coupled by reductive amination and cyanuric chloride, respectively, to asialofetuin (ASF), fetuin and rat serum albumin (RSA). Attachment of either label had no effect on the circulating half-lives of the proteins. Radioactivity from labeled ASF was recovered in rat liver (> 90%) by 1 h post-injection and remained in liver with half-lives of 2 and 6 days, respectively, for the DLT and InTn labels. Whole body recoveries of radioactivity from DLT- and InTn labels. Whole body recoveries of radioactivity from DLT- and InTn-labeled RSA were 5 and 6.5 days, respectively, again indicating that the larger glycoconjugate label residualized more efficiently in cells following protein degradation. (Lactitol)/sub 2/-N-CH/sub 2/-CH/sub 2/-NH-fluroescein (DLF) was also coupled to ASF by reductive amination and recovered quantitatively in liver at 1 h post-injection. Native ASF was an effective competitor for clearance of DLF-ASF from the circulation. Fluorescent degradation products were retained in liver with a half-life of 1.2 days. Residualizing fluorescent labels should be useful for identification and sorting of cells active in the degradation of plasma proteins.

  7. Direct Metal Transfer Between Periplasmic Proteins Identifies a Bacterial Copper Chaperone

    SciTech Connect

    Bagai, I.; Rensing, C.; Blackburn, N.; McEvoy, M.M.

    2009-05-11

    Transition metals require exquisite handling within cells to ensure that cells are not harmed by an excess of free metal species. In Gram-negative bacteria, cop