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Sample records for proline rich region

  1. Structure and function of the proline-rich region of myelin basic protein.

    PubMed

    Fraser, P E; Deber, C M

    1985-08-13

    Myelin basic protein (MBP)--the major extrinsic membrane protein of central nervous system myelin--from several species contains a rarely encountered highly conserved triproline segment as residues 99-101 of its 170-residue sequence. Cis peptide bonds are known to arise at X-Pro junctions in proteins and may be of functional significance in protein folding, chain reversal, and/or maintenance of tertiary structure. We have examined the conformation of this proline-rich region using principally 13C nuclear magnetic resonance spectroscopy (125 MHz) both in intact bovine MBP and in several MBP fragment peptides which we synthesized, including octapeptide 97-104 (Arg-Thr-Pro-Pro-Pro-Ser-Gln-Gly). Results suggested an all-trans conformation in aqueous solution for the triproline segment in MBP hexapeptide (99-104), heptapeptide (98-104), and octapeptide. Comparison with the 13C spectrum of intact MBP (125 MHz) suggested that the proline-rich region, as well as all other X-Pro MBP peptide junctures, was also essentially all trans in aqueous solution. Although experiments in which octapeptide 97-104 was bound to a lipid preparation (4:1 dipalmitoylphosphatidylcholine/dimyristoylphosphatidic acid) demonstrated that cis-proline bonds do arise (to the extent of ca. 5%) in the membrane environment, a role of linear chain propagation is suggested for the triproline segment of myelin basic protein.

  2. Proline-rich Sequence Recognition

    PubMed Central

    Schlundt, Andreas; Sticht, Jana; Piotukh, Kirill; Kosslick, Daniela; Jahnke, Nadin; Keller, Sandro; Schuemann, Michael; Krause, Eberhard; Freund, Christian

    2009-01-01

    The tumor maintenance protein Tsg101 has recently gained much attention because of its involvement in endosomal sorting, virus release, cytokinesis, and cancerogenesis. The ubiquitin-E2-like variant (UEV) domain of the protein interacts with proline-rich sequences of target proteins that contain P(S/T)AP amino acid motifs and weakly binds to the ubiquitin moiety of proteins committed to sorting or degradation. Here we performed peptide spot analysis and phage display to refine the peptide binding specificity of the Tsg101 UEV domain. A mass spectrometric proteomics approach that combines domain-based pulldown experiments, binding site inactivation, and stable isotope labeling by amino acids in cell culture (SILAC) was then used to delineate the relative importance of the peptide and ubiquitin binding sites. Clearly “PTAP” interactions dominate target recognition, and we identified several novel binders as for example the poly(A)-binding protein 1 (PABP1), Sec24b, NFκB2, and eIF4b. For PABP1 and eIF4b the interactions were confirmed in the context of the corresponding full-length proteins in cellular lysates. Therefore, our results strongly suggest additional roles of Tsg101 in cellular regulation of mRNA translation. Regulation of Tsg101 itself by the ubiquitin ligase TAL (Tsg101-associated ligase) is most likely conferred by a single PSAP binding motif that enables the interaction with Tsg101 UEV. Together with the results from the accompanying article (Kofler, M., Schuemann, M., Merz, C., Kosslick, D., Schlundt, A., Tannert, A., Schaefer, M., Lührmann, R., Krause, E., and Freund, C. (2009) Proline-rich sequence recognition: I. Marking GYF and WW domain assembly sites in early spliceosomal complexes. Mol. Cell. Proteomics 8, 2461–2473) on GYF and WW domain pathways our work defines major proline-rich sequence-mediated interaction networks that contribute to the modular assembly of physiologically relevant protein complexes. PMID:19542561

  3. Structural insights into the specific binding of huntingtin proline-rich region with the SH3 and WW domains.

    PubMed

    Gao, Yong-Guang; Yan, Xian-Zhong; Song, Ai-Xin; Chang, Yong-Gang; Gao, Xue-Chao; Jiang, Nan; Zhang, Qi; Hu, Hong-Yu

    2006-12-01

    The interactions of huntingtin (Htt) with the SH3 domain- or WW domain-containing proteins have been implicated in the pathogenesis of Huntington's disease (HD). We report the specific interactions of Htt proline-rich region (PRR) with the SH3GL3-SH3 domain and HYPA-WW1-2 domain pair by NMR. The results show that Htt PRR binds with the SH3 domain through nearly its entire chain, and that the binding region on the domain includes the canonical PxxP-binding site and the specificity pocket. The C terminus of PRR orients to the specificity pocket, whereas the N terminus orients to the PxxP-binding site. Htt PRR can also specifically bind to WW1-2; the N-terminal portion preferentially binds to WW1, while the C-terminal portion binds to WW2. This study provides structural insights into the specific interactions between Htt PRR and its binding partners as well as the alteration of these interactions that involve PRR, which may have implications for the understanding of HD.

  4. A conserved proline-rich region of the Saccharomyces cerevisiae cyclase-associated protein binds SH3 domains and modulates cytoskeletal localization.

    PubMed Central

    Freeman, N L; Lila, T; Mintzer, K A; Chen, Z; Pahk, A J; Ren, R; Drubin, D G; Field, J

    1996-01-01

    Saccharomyces cerevisiae cyclase-associated protein (CAP or Srv2p) is multifunctional. The N-terminal third of CAP binds to adenylyl cyclase and has been implicated in adenylyl cyclase activation in vivo. The widely conserved C-terminal domain of CAP binds to monomeric actin and serves an important cytoskeletal regulatory function in vivo. In addition, all CAP homologs contain a centrally located proline-rich region which has no previously identified function. Recently, SH3 (Src homology 3) domains were shown to bind to proline-rich regions of proteins. Here we report that the proline-rich region of CAP is recognized by the SH3 domains of several proteins, including the yeast actin-associated protein Abp1p. Immunolocalization experiments demonstrate that CAP colocalizes with cortical actin-containing structures in vivo and that a region of CAP containing the SH3 domain binding site is required for this localization. We also demonstrate that the SH3 domain of yeast Abp1p and that of the yeast RAS protein guanine nucleotide exchange factor Cdc25p complex with adenylyl cyclase in vitro. Interestingly, the binding of the Cdc25p SH3 domain is not mediated by CAP and therefore may involve direct binding to adenylyl cyclase or to an unidentified protein which complexes with adenylyl cyclase. We also found that CAP homologous from Schizosaccharomyces pombe and humans bind SH3 domains. The human protein binds most strongly to the SH3 domain from the abl proto-oncogene. These observations identify CAP as an SH3 domain-binding protein and suggest that CAP mediates interactions between SH3 domain proteins and monomeric actin. PMID:8552082

  5. A conserved proline-rich region of the Saccharomyces cerevisiae cyclase-associated protein binds SH3 domains and modulates cytoskeletal localization.

    PubMed

    Freeman, N L; Lila, T; Mintzer, K A; Chen, Z; Pahk, A J; Ren, R; Drubin, D G; Field, J

    1996-02-01

    Saccharomyces cerevisiae cyclase-associated protein (CAP or Srv2p) is multifunctional. The N-terminal third of CAP binds to adenylyl cyclase and has been implicated in adenylyl cyclase activation in vivo. The widely conserved C-terminal domain of CAP binds to monomeric actin and serves an important cytoskeletal regulatory function in vivo. In addition, all CAP homologs contain a centrally located proline-rich region which has no previously identified function. Recently, SH3 (Src homology 3) domains were shown to bind to proline-rich regions of proteins. Here we report that the proline-rich region of CAP is recognized by the SH3 domains of several proteins, including the yeast actin-associated protein Abp1p. Immunolocalization experiments demonstrate that CAP colocalizes with cortical actin-containing structures in vivo and that a region of CAP containing the SH3 domain binding site is required for this localization. We also demonstrate that the SH3 domain of yeast Abp1p and that of the yeast RAS protein guanine nucleotide exchange factor Cdc25p complex with adenylyl cyclase in vitro. Interestingly, the binding of the Cdc25p SH3 domain is not mediated by CAP and therefore may involve direct binding to adenylyl cyclase or to an unidentified protein which complexes with adenylyl cyclase. We also found that CAP homologous from Schizosaccharomyces pombe and humans bind SH3 domains. The human protein binds most strongly to the SH3 domain from the abl proto-oncogene. These observations identify CAP as an SH3 domain-binding protein and suggest that CAP mediates interactions between SH3 domain proteins and monomeric actin.

  6. Molecular Basis of Interactions Between SH3 Domain-Containing Proteins and the Proline-Rich Region of the Ubiquitin Ligase Itch.

    PubMed

    Desrochers, Guillaume; Cappadocia, Laurent; Lussier-Price, Mathieu; Ton, Anh-Tien; Ayoubi, Riham; Serohijos, Adrian; Omichinski, James G; Angers, Annie

    2017-02-24

    The ligase Itch plays major roles in signalling pathways by inducing ubiquitylation-dependent degradation of several substrates. Substrate recognition and binding is critical for the regulation of this reaction. Like closely related ligases, Itch can interact with proteins containing a PPxY motif via its WW domains. In addition to these WW domains, Itch possesses a proline-rich region (PRR) that has been shown to interact with several Src Homology 3 (SH3) domain-containing proteins. We have previously established that despite the apparent surface uniformity and conserved fold of SH3 domains, they display different binding mechanisms and affinities for their interaction with the PRR of Itch. Here, we attempt to determine the molecular bases underlying the wide range of binding properties of the Itch PRR. Using pull-down assays combined with mass spectrometry analysis, we show that the Itch PRR preferentially forms complexes with Endophilins, Amphyphisins and Pacsins, but can also target a variety of other SH3 domain-containing proteins. In addition, we map the binding sites of these proteins using a combination of PRR sub-sequences and mutants. We find that different SH3 domains target distinct proline-rich sequences overlapping significantly. We also structurally analyze these protein complexes using crystallography and molecular modelling. These structures depict the position of Itch PRR engaged in a 1:2 protein complex with β-PIX and a 1:1 complex with the other SH3 domain-containing proteins. Taken together, these results reveal the binding preferences of the Itch PRR towards its most common SH3 domain-containing partners, and demonstrate that the PRR region is sufficient for binding.

  7. The proline-rich region of glyceraldehyde-3-phosphate dehydrogenase from human sperm may bind SH3 domains, as revealed by a bioinformatic study of low-complexity protein segments.

    PubMed

    Tatjewski, Marcin; Gruca, Aleksandra; Plewczynski, Dariusz; Grynberg, Marcin

    2016-02-01

    Glyceraldehyde-3-phosphate dehydrogenase from human sperm (GAPDHS) provides energy to the sperm flagellum, and is therefore essential for sperm motility and male fertility. This isoform is distinct from somatic GAPDH, not only in being specific for the testis but also because it contains an additional amino-terminal region that encodes a proline-rich motif that is known to bind to the fibrous sheath of the sperm tail. By conducting a large-scale sequence comparison on low-complexity sequences available in databases, we identified a strong similarity between the proline-rich motif from GAPDHS and the proline-rich sequence from Ena/vasodilator-stimulated phosphoprotein-like (EVL), which is known to bind an SH3 domain of dynamin-binding protein (DNMBP). The putative binding partners of the proline-rich GAPDHS motif include SH3 domain-binding protein 4 (SH3BP4) and the IL2-inducible T-cell kinase/tyrosine-protein kinase ITK/TSK (ITK). This result implies that GAPDHS participates in specific signal-transduction pathways. Gene Ontology category-enrichment analysis showed several functional classes shared by both proteins, of which the most interesting ones are related to signal transduction and regulation of hydrolysis. Furthermore, a mutation of one EVL proline to leucine is known to cause colorectal cancer, suggesting that mutation of homologous amino acid residue in the GAPDHS motif may be functionally deleterious.

  8. Association of elongation factor 1 alpha and ribosomal protein L3 with the proline-rich region of yeast adenylyl cyclase-associated protein CAP.

    PubMed

    Yanagihara, C; Shinkai, M; Kariya, K; Yamawaki-Kataoka, Y; Hu, C D; Masuda, T; Kataoka, T

    1997-03-17

    CAP is a multifunctional protein; the N-terminal region binds adenylyl cyclase and controls its response to Ras while the C-terminal region is involved in cytoskeletal regulation. In between the two regions, CAP possesses two proline-rich segments, P1 and P2, resembling a consensus sequence for binding SH3 domains. We have identified two yeast proteins with molecular sizes of 48 and 46 kDa associated specifically with P2. Determination of partial protein sequences demonstrated that the 48-kDa and 46-kDa proteins correspond to EF1 alpha and rL3, respectively, neither of which contains any SH3-domain-like sequence. Deletion of P2 from CAP resulted in loss of the activity to bind the two proteins either in vivo or in vitro. Yeast cells whose chromosomal CAP was replaced by the P2-deletion mutant displayed an abnormal phenotype represented by dissociated localizations of CAP and F-actin, which were colocalized in wild-type cells. These results suggest that these associations may have functional significance.

  9. Importance of the proline-rich region for the regulatory function of RcsF, an outer membrane lipoprotein component of the Escherichia coli Rcs signal transduction system.

    PubMed

    Umekawa, Mitsuru; Miyagawa, Hiroyoshi; Kondo, Daitetsu; Matsuoka, Satoshi; Matsumoto, Kouji; Hara, Hiroshi

    2013-09-01

    The outer membrane lipoprotein RcsF is an essential component of the Rcs phosphorelay signal transduction system in Escherichia coli. It senses stresses imposed on the cell envelope and conveys the information to histidine kinase RcsC in the cytoplasmic membrane. Mislocalization of RcsF to the periplasm, effected by fusing it to the periplasmic maltose-binding protein, or to the cytoplasmic membrane, brought about by changing the lipoprotein sorting signal, leads to high activation of the Rcs system, suggesting that RcsF functions as a ligand for RcsC in activating the system. Here, we focus on the proline-rich region (PRR) in the N-terminal half of RcsF, a region which also contains many basic amino acid residues. Deletion of the PRR in the mislocalized RcsF resulted in even higher activation of the Rcs system. The same deletion in wild-type RcsF lipoprotein that is correctly localized to the outer membrane, however, blocked activation of the system under stresses that normally should activate it. It is highly likely that the PRR plays an important role in the regulation of the function of RcsF in activating the Rcs system.

  10. Proline-rich cell wall proteins accumulate in growing regions and phloem tissue in response to water deficit in common bean seedlings.

    PubMed

    Battaglia, Marina; Solórzano, Rosa M; Hernández, Magdalena; Cuéllar-Ortiz, Sonia; García-Gómez, Blanca; Márquez, Judith; Covarrubias, Alejandra A

    2007-04-01

    Plant cell walls undergo dynamic changes in response to different environmental stress conditions. In response to water deficit, two related proline-rich glycoproteins, called p33 and p36, accumulate in the soluble fraction of the cell walls in Phaseolus vulgaris (Covarrubias et al. in Plant Physiol 107:1119-1128, 1995). In this work, we show that p33 and p36 are able to form a 240 kDa oligomer, which is found in the cell wall soluble fraction. We present evidence indicating that the highest accumulation of these proteins in response to water deficit occurs in the growing regions of common bean seedlings, particularly in the phloem tissues. These proteins were detected in P. vulgaris cell suspension cultures, where the p33/p36 ratio was higher under hyperosmotic conditions than in bean seedlings subjected to the same treatment. The results support a role for these proteins during the plant cell response to changes in its water status, and suggest that cell wall modifications are induced in active growing cells of common bean in response to water limitation.

  11. Autoinhibitory structure of the WW domain of HYPB/SETD2 regulates its interaction with the proline-rich region of huntingtin.

    PubMed

    Gao, Yong-Guang; Yang, Hui; Zhao, Jian; Jiang, Ya-Jun; Hu, Hong-Yu

    2014-03-04

    Huntington's disease (HD) is an autosomally dominant neurodegenerative disorder caused by expansion of polyglutamine (polyQ) in the huntingtin (Htt) protein. Htt yeast two-hybrid protein B (HYPB/SETD2), a histone methyltransferase, directly interacts with Htt and is involved in HD pathology. Using NMR techniques, we characterized a polyproline (polyP) stretch at the C terminus of HYPB, which directly interacts with the following WW domain and leads this domain predominantly to be in a closed conformational state. The solution structure shows that the polyP stretch extends from the back and binds to the WW core domain in a typical binding mode. This autoinhibitory structure regulates interaction between the WW domain of HYPB and the proline-rich region (PRR) of Htt, as evidenced by NMR and immunofluorescence techniques. This work provides structural and mechanistic insights into the intramolecular regulation of the WW domain in Htt-interacting partners and will be helpful for understanding the pathology of HD.

  12. A Synthetic Virus-Like Particle Streptococcal Vaccine Candidate Using B-Cell Epitopes from the Proline-Rich Region of Pneumococcal Surface Protein A.

    PubMed

    Tamborrini, Marco; Geib, Nina; Marrero-Nodarse, Aniebrys; Jud, Maja; Hauser, Julia; Aho, Celestine; Lamelas, Araceli; Zuniga, Armando; Pluschke, Gerd; Ghasparian, Arin; Robinson, John A

    2015-10-16

    Alternatives to the well-established capsular polysaccharide-based vaccines against Streptococcus pneumoniae that circumvent limitations arising from limited serotype coverage and the emergence of resistance due to capsule switching (serotype replacement) are being widely pursued. Much attention is now focused on the development of recombinant subunit vaccines based on highly conserved pneumococcal surface proteins and virulence factors. A further step might involve focusing the host humoral immune response onto protective protein epitopes using as immunogens structurally optimized epitope mimetics. One approach to deliver such epitope mimetics to the immune system is through the use of synthetic virus-like particles (SVLPs). SVLPs are made from synthetic coiled-coil lipopeptides that are designed to spontaneously self-assemble into 20-30 nm diameter nanoparticles in aqueous buffer. Multivalent display of epitope mimetics on the surface of SVLPs generates highly immunogenic nanoparticles that elicit strong epitope-specific humoral immune responses without the need for external adjuvants. Here, we set out to demonstrate that this approach can yield vaccine candidates able to elicit a protective immune response, using epitopes derived from the proline-rich region of pneumococcal surface protein A (PspA). These streptococcal SVLP-based vaccine candidates are shown to elicit strong humoral immune responses in mice. Following active immunization and challenge with lethal doses of streptococcus, SVLP-based immunogens are able to elicit significant protection in mice. Furthermore, a mimetic-specific monoclonal antibody is shown to mediate partial protection upon passive immunization. The results show that SVLPs combined with synthetic epitope mimetics may have potential for the development of an effective vaccine against Streptococcus pneumoniae.

  13. A Synthetic Virus-Like Particle Streptococcal Vaccine Candidate Using B-Cell Epitopes from the Proline-Rich Region of Pneumococcal Surface Protein A

    PubMed Central

    Tamborrini, Marco; Geib, Nina; Marrero-Nodarse, Aniebrys; Jud, Maja; Hauser, Julia; Aho, Celestine; Lamelas, Araceli; Zuniga, Armando; Pluschke, Gerd; Ghasparian, Arin; Robinson, John A.

    2015-01-01

    Alternatives to the well-established capsular polysaccharide-based vaccines against Streptococcus pneumoniae that circumvent limitations arising from limited serotype coverage and the emergence of resistance due to capsule switching (serotype replacement) are being widely pursued. Much attention is now focused on the development of recombinant subunit vaccines based on highly conserved pneumococcal surface proteins and virulence factors. A further step might involve focusing the host humoral immune response onto protective protein epitopes using as immunogens structurally optimized epitope mimetics. One approach to deliver such epitope mimetics to the immune system is through the use of synthetic virus-like particles (SVLPs). SVLPs are made from synthetic coiled-coil lipopeptides that are designed to spontaneously self-assemble into 20–30 nm diameter nanoparticles in aqueous buffer. Multivalent display of epitope mimetics on the surface of SVLPs generates highly immunogenic nanoparticles that elicit strong epitope-specific humoral immune responses without the need for external adjuvants. Here, we set out to demonstrate that this approach can yield vaccine candidates able to elicit a protective immune response, using epitopes derived from the proline-rich region of pneumococcal surface protein A (PspA). These streptococcal SVLP-based vaccine candidates are shown to elicit strong humoral immune responses in mice. Following active immunization and challenge with lethal doses of streptococcus, SVLP-based immunogens are able to elicit significant protection in mice. Furthermore, a mimetic-specific monoclonal antibody is shown to mediate partial protection upon passive immunization. The results show that SVLPs combined with synthetic epitope mimetics may have potential for the development of an effective vaccine against Streptococcus pneumoniae. PMID:26501327

  14. The Proline Rich Tetramerization Peptides in Equine Serum Butyrylcholinesterase

    PubMed Central

    Biberoglu, Kevser; Schopfer, Lawrence M.; Tacal, Ozden; Lockridge, Oksana

    2012-01-01

    Summary Soluble, tetrameric, plasma butyrylcholinesterase from horse has previously been shown to include a non-covalently attached polyproline peptide in its structure. The polyproline peptide matched the polyproline rich region of human lamellipodin. Our goal was to examine the tetramer organizing peptides of horse butyrylcholinesterase in more detail. Horse butyrylcholinesterase was denatured by boiling, thus releasing a set of polyproline peptides ranging in mass from 1173 to 2098 Da. The peptide sequences were determined by fragmentation in the MALDI-TOF-TOF and LTQ-Orbitrap mass spectrometers. Twenty-seven polyproline peptides grouped into 13 families were identified. Peptides contained a minimum of 11 consecutive proline residues and as many as 21. Many of the peptides had a non-proline amino acid at the N-terminus. A search of the protein databanks matched peptides to 9 proteins, though not all peptides matched a known protein. It is concluded that polyproline peptides of various lengths and sequences are included in the tetramer structure of horse BChE. The function of these polyproline peptides is to serve as tetramer organizing peptides. PMID:22889087

  15. Patterns of soybean proline-rich protein gene expression.

    PubMed Central

    Wyatt, R E; Nagao, R T; Key, J L

    1992-01-01

    The expression patterns of three members of a gene family that encodes proline-rich proteins in soybean (SbPRPs) were examined using in situ hybridization experiments. In most instances, the expression of SbPRP genes was intense in a limited number of cell types of a particular organ. SbPRP1 RNA was localized in several cell types of soybean hypocotyls, including cells within the phloem and xylem. SbPRP1 expression increased within epidermal cells in the elongating and mature regions of the hypocotyl; expression was detected also in lignified cells surrounding the hilum of mature seeds. SbPRP2 RNA was present in cortical cells and in the vascular tissue of the hypocotyl, especially cells of the phloem. This gene was expressed also in the inner integuments of the mature seed coat. SbPRP3 RNA was localized specifically to the endodermoid layer of cells surrounding the stele in the elongating region of the hypocotyl, as well as in the epidermal cells of leaves and cotyledons. These data show that members of this gene family exhibit cell-specific expression. The members of the SbPRP gene family are expressed in different types of cells and in some cell types that also express the glycine-rich protein or hydroxyproline-rich glycoprotein classes of genes. PMID:1525563

  16. Oyster hemocytes express a proline-rich peptide displaying synergistic antimicrobial activity with a defensin.

    PubMed

    Gueguen, Yannick; Bernard, Romestand; Julie, Fievet; Paulina, Schmitt; Delphine, Destoumieux-Garzón; Franck, Vandenbulcke; Philippe, Bulet; Evelyne, Bachère

    2009-02-01

    A cDNA sequence that encodes a 61-amino acid polypeptide precursor with homologies to proline-rich antimicrobial peptides (AMPs) was identified in the oyster Crassostrea gigas. After release of a hydrophobic signal peptide, the resulting 37-amino acid peptide, Cg-Prp, is composed of an acidic region and a cationic proline-rich region. To evaluate the biological properties of Cg-Prp, multiple proline-rich peptides corresponding to putative processing of the full-length Cg-Prp were synthesized. A limited antimicrobial activity was observed for two of them, which also showed strong synergistic antimicrobial activity with Cg-Def, a defensin from C. gigas. To our knowledge, this is the first evidence of synergy between a defensin and another AMP in an invertebrate. By in situ hybridization, the expression of Cg-prp was found to be restricted to hemocytes and induced following bacterial challenge. Cg-prp transcripts were also detected in hemocytes infiltrating mantle, where Cg-Def is expressed. Additionally, by immunocytochemistry, we showed that Cg-Prp or one of its variants is present in some hemocytes together with defensins. In conclusion, we described here the first proline-rich AMP from mollusk. From our study, it is likely to provide a first line of defense against bacterial invasion by acting through synergy with defensins.

  17. Oxidative modification of a proline-rich gliadin peptide.

    PubMed

    Huang, Xin; Kanerva, Päivi; Salovaara, Hannu; Loponen, Jussi; Sontag-Strohm, Tuula

    2013-12-01

    Prolamins are proline-rich proteins occurring in cereal grains. Prolamins of wheat, barley and rye, or gluten protein, can cause coeliac disease in individuals not tolerating gluten. Degrading harmful prolamins can reduce their toxicity. A model peptide sequenced in α-gliadin, 33-mer (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF), was chosen for our study. The metal-catalysed oxidation of 33-mer was studied, instead of enzymatic hydrolysis. Peptide 33-mer was treated in several oxidative systems. Iron-catalysed hydrogen peroxide-induced oxidation showed the greatest modification of 33-mer. Carbonyl groups and dityrosine cross-links were readily formed. At best, the immunological activity of 33-mer was reduced to 18% of its initial level after 24h of oxidation. Oxidative treatment can be further applied for the modification of cereal prolamin proteins, since it appears to be a potential alternative for reduction of coeliac immunological activities in gluten proteins.

  18. Identification of polyproline II regions derived from the proline-rich nuclear receptor coactivators PNRC and PNRC2: new insights for ERα coactivator interactions.

    PubMed

    Byrne, C; Miclet, E; Broutin, I; Gallo, D; Pelekanou, V; Kampa, M; Castanas, E; Leclercq, G; Jacquot, Y

    2013-10-01

    Protein-protein interactions are crucial for signal transductions required for cell differentiation and proliferation. Their modulation is therefore key to the development of therapeutic alternatives, particularly in the context of cancer. According to literature data, the polyproline-rich nuclear receptor coactivators PNRC and PNRC2 interact with estrogen receptor (ERα) through their PxxP SH3-binding motifs. In a search to identify the molecular features governing this interaction, we explored using electronic circular dichroism (ECD) spectroscopy and molecular dynamics (MD) calculations, the capacity of a range of putative biologically active peptides derived from these proteins and containing this PxxP motif(s) to form polyproline II (PPII) domains. An additional more exhaustive structural study on a lead PPII peptide was also performed using 2D nuclear magnetic resonance (NMR) spectroscopy. With the exception of one of all the investigated peptides (PNRC-D), binding assays failed to detect any affinity for Grb2 SH3 domains, suggesting that PPII motifs issued from Grb2 antagonists have a binding mode distinct from those derived from Grb2 agonists. Instead, the peptides revealed a competitive binding ability against a synthetic peptide (ERα17p) with a putative PPII-cognate domain located within a coregulator recruitment region of ERα (AF-2 site). Our work, which constitutes the first structure-related interaction study concerning PNRC and PNRC2, supports not only the existence of PxxP-induced PPII sequences in these coregulators, but also confirms the presence of a PPII recognition site in the AF-2 of the steroid receptor ERα, a region important for transcription regulation.

  19. Polyproline II helix conformation in a proline-rich environment: a theoretical study.

    PubMed

    Vila, Jorge A; Baldoni, Héctor A; Ripoll, Daniel R; Ghosh, Avijit; Scheraga, Harold A

    2004-02-01

    Interest centers here on whether a polyproline II helix can propagate through adjacent non-proline residues, and on shedding light on recent experimental observations suggesting the presence of significant PP(II) structure in a short alanine-based peptide with no proline in the sequence. For this purpose, we explored the formation of polyproline II helices in proline-rich peptides with the sequences Ac-(Pro)(3)-X-(Pro)(3)-Gly-Tyr-NH(2), with X = Pro (PPP), Ala (PAP), Gln (PQP), Gly (PGP), and Val (PVP), and Ac-(Pro)(3)-Ala-Ala-(Pro)(3)-Gly-Tyr-NH(2) (PAAP), by using a theoretical approach that includes a solvent effect as well as cis <--> trans isomerization of the peptide groups and puckering conformations of the pyrrolidine ring of the proline residues. Since (13)C chemical shifts have proven to be useful for identifying secondary-structure preferences in proteins and peptides, and because values of the dihedral angles (phi,psi) are the main determinants of their magnitudes, we have, therefore, computed the Boltzmann-averaged (13)C chemical shifts for the guest residues in the PXP peptide (X = Pro, Ala, Gln, Gly, and Val) with a combination of approaches, involving molecular mechanics, statistical mechanics, and quantum mechanics. In addition, an improved procedure was used to carry out the conformational searches and to compute the solvent polarization effects faster and more accurately than in previous work. The current theoretical work and additional experimental evidence show that, in short proline-rich peptides, alanine decreases the polyproline II helix content. In particular, the theoretical evidence accumulated in this work calls into question the proposal that alanine has a strong preference to adopt conformations in the polyproline II region of the Ramachandran map.

  20. Polyproline II Helix Conformation in a Proline-Rich Environment: A Theoretical Study

    PubMed Central

    Vila, Jorge A.; Baldoni, Héctor A.; Ripoll, Daniel R.; Ghosh, Avijit; Scheraga, Harold A.

    2004-01-01

    Interest centers here on whether a polyproline II helix can propagate through adjacent non-proline residues, and on shedding light on recent experimental observations suggesting the presence of significant PPII structure in a short alanine-based peptide with no proline in the sequence. For this purpose, we explored the formation of polyproline II helices in proline-rich peptides with the sequences Ac-(Pro)3-X-(Pro)3-Gly-Tyr-NH2, with X = Pro (PPP), Ala (PAP), Gln (PQP), Gly (PGP), and Val (PVP), and Ac-(Pro)3-Ala-Ala-(Pro)3-Gly-Tyr-NH2 (PAAP), by using a theoretical approach that includes a solvent effect as well as cis ↔ trans isomerization of the peptide groups and puckering conformations of the pyrrolidine ring of the proline residues. Since 13C chemical shifts have proven to be useful for identifying secondary-structure preferences in proteins and peptides, and because values of the dihedral angles (φ,ψ) are the main determinants of their magnitudes, we have, therefore, computed the Boltzmann-averaged 13C chemical shifts for the guest residues in the PXP peptide (X = Pro, Ala, Gln, Gly, and Val) with a combination of approaches, involving molecular mechanics, statistical mechanics, and quantum mechanics. In addition, an improved procedure was used to carry out the conformational searches and to compute the solvent polarization effects faster and more accurately than in previous work. The current theoretical work and additional experimental evidence show that, in short proline-rich peptides, alanine decreases the polyproline II helix content. In particular, the theoretical evidence accumulated in this work calls into question the proposal that alanine has a strong preference to adopt conformations in the polyproline II region of the Ramachandran map. PMID:14747311

  1. Direct interaction between Tks proteins and the N-terminal proline-rich region (PRR) of NoxA1 mediates Nox1-dependent ROS generation.

    PubMed

    Gianni, Davide; DerMardirossian, Céline; Bokoch, Gary M

    2011-01-01

    NADPH oxidase (Nox) family enzymes are one of the main sources of cellular reactive oxygen species (ROS), which have been implicated in several physiological and pathophysiological processes. To date seven members of this family have been reported, including Nox1-5 and Duox1 and 2. With the exception of Nox2, the regulation of the Nox enzymes is still poorly understood. Nox1 is highly expressed in the colon, and requires two cytosolic regulators, the organizer subunit NoxO1 and the activator subunit NoxA1, as well as the binding of Rac1 GTPase, for its activity. Recently, we identified the c-Src substrate proteins Tks4 and Tks5 as functional members of a p47(phox)-related organizer superfamily. As a functional consequence of this interaction, Nox1 localizes to invadopodia, actin-rich membrane protrusions of cancer cells which facilitate pericellular proteolysis and invasive behavior. Here, we report that Tks4 and Tks5 directly bind to NoxA1. Moreover, the integrity of the N-terminal PRR of NoxA1 is essential for this direct interaction with the Tks proteins. When the PRR in NoxA1 is disrupted, Tks proteins cannot bind NoxA1 and lose their ability to support Nox1-dependent ROS generation. Consistent with this, Tks4 and Tks5 are unable to act as organizers for Nox2 because of their inability to interact with p67(phox), which lacks the N-terminal PRR, thus conferring a unique specificity to Tks4 and 5. Taken together, these results clarify the molecular basis for the interaction between NoxA1 and the Tks proteins and may provide new insights into the pharmacological design of a more effective anti-metastatic strategy.

  2. Proline-rich polypeptides in Alzheimer's disease and neurodegenerative disorders -- therapeutic potential or a mirage?

    PubMed

    Gladkevich, A; Bosker, F; Korf, J; Yenkoyan, K; Vahradyan, H; Aghajanov, M

    2007-10-01

    The development of effective and safe drugs for a growing Alzheimer disease population is an increasing need at present. Both experimental and clinical evidence support a beneficial effect of proline-rich polypeptides in a number of neurodegenerative diseases, including Alzheimer disease. Experimental data have shown that proline-rich polypeptides isolated from bovine neurohypophisis possess neuroprotective and neuromodulatory properties in mice with aluminum neurotoxicosis or neuronal damage caused by venoms and toxins. Proline-rich polypeptides from ovine colostrums, so called Colostrinin, have been shown to produce cognitive improvement in an experimental model and in patients with Alzheimer disease. However, the precise mechanism underlying the neuroprotective action of proline-rich polypeptides is not very well established. Moreover, studies pointing at a neuroprotective effect of proline-rich polypeptides from bovine neurohypophisis in humans have not been reported thus far. The authors conclude that more detailed information on the mode of action of proline-rich polypeptides is needed as well as confirmation of their efficacy in broad clinical trials before this approach can really show its potential in the treatment of neurodegenerative disorders.

  3. Role of the Escherichia coli SbmA in the antimicrobial activity of proline-rich peptides.

    PubMed

    Mattiuzzo, Maura; Bandiera, Antonella; Gennaro, Renato; Benincasa, Monica; Pacor, Sabrina; Antcheva, Nikolinka; Scocchi, Marco

    2007-10-01

    In contrast to many antimicrobial peptides, members of the proline-rich group of antimicrobial peptides inactivate Gram-negative bacteria by a non-lytic mechanism. Several lines of evidence indicate that they are internalized into bacteria and their activity mediated by interaction with unknown cellular components. With the aim of identifying such interactors, we selected mutagenized Escherichia coli clones resistant to the proline-rich Bac7(1-35) peptide and analysed genes responsible for conferring resistance, whose products may thus be involved in the peptide's mode of action. We isolated a number of genomic regions bearing such genes, and one in particular coding for SbmA, an inner membrane protein predicted to be part of an ABC transporter. An E. coli strain carrying a point mutation in sbmA, as well as other sbmA-null mutants, in fact showed resistance to several proline-rich peptides but not to representative membranolytic peptides. Use of fluorescently labelled Bac7(1-35) confirmed that resistance correlated with a decreased ability to internalize the peptide, suggesting that a bacterial protein, SbmA, is necessary for the transport of, and for susceptibility to, proline-rich antimicrobial peptides of eukaryotic origin.

  4. The mammalian profilin isoforms display complementary affinities for PIP2 and proline-rich sequences.

    PubMed

    Lambrechts, A; Verschelde, J L; Jonckheere, V; Goethals, M; Vandekerckhove, J; Ampe, C

    1997-02-03

    We present a study on the binding properties of the bovine profilin isoforms to both phosphatidylinositol 4,5-bisphosphate (PIP2) and proline-rich peptides derived from vasodilator-stimulated phosphoprotein (VASP) and cyclase-associated protein (CAP). Using microfiltration, we show that compared with profilin II, profilin I has a higher affinity for PIP2. On the other hand, fluorescence spectroscopy reveals that proline-rich peptides bind better to profilin II. At micromolar concentrations, profilin II dimerizes upon binding to proline-rich peptides. Circular dichroism measurements of profilin II reveal a significant conformational change in this protein upon binding of the peptide. We show further that PIP2 effectively competes for binding of profilin I to poly-L-proline, since this isoform, but not profilin II, can be eluted from a poly-L-proline column with PIP2. Using affinity chromatography on either profilin isoform, we identified profilin II as the preferred ligand for VASP in bovine brain extracts. The complementary affinities of the profilin isoforms for PIP2 and the proline-rich peptides offer the cell an opportunity to direct actin assembly at different subcellular localizations through the same or different signal transduction pathways.

  5. The Binding of Syndapin SH3 Domain to Dynamin Proline-rich Domain Involves Short and Long Distance Elements.

    PubMed

    Luo, Lin; Xue, Jing; Kwan, Ann; Gamsjaeger, Roland; Wielens, Jerome; von Kleist, Lisa; Cubeddu, Liza; Guo, Zhong; Stow, Jennifer L; Parker, Michael W; Mackay, Joel P; Robinson, Phillip J

    2016-04-29

    Dynamin is a GTPase that mediates vesicle fission during synaptic vesicle endocytosis. Its long C-terminal proline-rich domain contains 13 PXXP motifs, which orchestrate its interactions with multiple proteins. The SH3 domains of syndapin and endophilin bind the PXXP motifs called Site 2 and 3 (Pro-786-Pro-793) at the N-terminal end of the proline-rich domain, whereas the amphiphysin SH3 binds Site 9 (Pro-833-Pro-836) toward the C-terminal end. In some proteins, SH3/peptide interactions also involve short distance elements, which are 5-15 amino acid extensions flanking the central PXXP motif for high affinity binding. Here we found two previously unrecognized elements in the central and the C-terminal end of the dynamin proline-rich domain that account for a significant increase in syndapin binding affinity compared with a previously reported Site 2 and Site 3 PXXP peptide alone. The first new element (Gly-807-Gly-811) is short distance element on the C-terminal side of Site 2 PXXP, which might contact a groove identified under the RT loop of the SH3 domain. The second element (Arg-838-Pro-844) is located about 50 amino acids downstream of Site 2. These two elements provide additional specificity to the syndapin SH3 domain outside of the well described polyproline-binding groove. Thus, the dynamin/syndapin interaction is mediated via a network of multiple contacts outside the core PXXP motif over a previously unrecognized extended region of the proline-rich domain. To our knowledge this is the first example among known SH3 interactions to involve spatially separated and extended long-range elements that combine to provide a higher affinity interaction.

  6. Post-Translational Modification and Secretion of Azelaic Acid Induced 1 (AZI1), a Hybrid Proline-Rich Protein from Arabidopsis

    PubMed Central

    Pitzschke, Andrea; Xue, Hui; Persak, Helene; Datta, Sneha; Seifert, Georg J.

    2016-01-01

    Arabidopsis EARLI-type hybrid proline-rich proteins (HyPRPs) consist of a putative N-terminal secretion signal, a proline-rich domain (PRD), and a characteristic eight-cysteine-motif (8-CM). They have been implicated in biotic and abiotic stress responses. AZI1 is required for systemic acquired resistance and it has recently been identified as a target of the stress-induced mitogen-activated protein kinase MPK3. AZI1 gel migration properties strongly indicate AZI1 to undergo major post-translational modifications. These occur in a stress-independent manner and are unrelated to phosphorylation by MAPKs. As revealed by transient expression of AZI1 in Nicotiana benthamiana and Tropaeolum majus, the Arabidopsis protein is similarly modified in heterologous plant species. Proline-rich regions, resembling arabinogalactan proteins point to a possible proline hydroxylation and subsequent O-glycosylation of AZI1. Consistently, inhibition of prolyl hydroxylase reduces its apparent protein size. AZI1 secretion was examined using Arabidopsis protoplasts and seedling exudates. Employing Agrobacterium-mediated leaf infiltration of N. benthamiana, we attempted to assess long-distance movement of AZI1. In summary, the data point to AZI1 being a partially secreted protein and a likely new member of the group of hydroxyproline-rich glycoproteins. Its dual location suggests AZI1 to exert both intra- and extracellular functions. PMID:26771603

  7. Characterization of the cell penetrating properties of a human salivary proline-rich peptide.

    PubMed

    Radicioni, Giorgia; Stringaro, Annarita; Molinari, Agnese; Nocca, Giuseppina; Longhi, Renato; Pirolli, Davide; Scarano, Emanuele; Iavarone, Federica; Manconi, Barbara; Cabras, Tiziana; Messana, Irene; Castagnola, Massimo; Vitali, Alberto

    2015-11-01

    Saliva contains hundreds of small proline-rich peptides most of which derive from the post-translational and post-secretory processing of the acidic and basic salivary proline-rich proteins. Among these peptides we found that a 20 residue proline-rich peptide (p1932), commonly present in human saliva and patented for its antiviral activity, was internalized within cells of the oral mucosa. The cell-penetrating properties of p1932 have been studied in a primary gingival fibroblast cell line and in a squamous cancer cell line, and compared to its retro-inverso form. We observed by mass-spectrometry, flow cytometry and confocal microscopy that both peptides were internalized in the two cell lines on a time scale of minutes, being the natural form more efficient than the retro-inverso one. The cytosolic localization was dependent on the cell type: both peptide forms were able to localize within nuclei of tumoral cells, but not in the nuclei of gingival fibroblasts. The uptake was shown to be dependent on the culture conditions used: peptide internalization was indeed effective in a complete medium than in a serum-free one allowing the hypothesis that the internalization could be dependent on the cell cycle. Both peptides were internalized likely by a lipid raft-mediated endocytosis mechanism as suggested by the reduced uptake in the presence of methyl-ß-cyclodextrin. These results suggest that the natural peptide may play a role within the cells of the oral mucosa after its secretion and subsequent internalization. Furthermore, lack of cytotoxicity of both peptide forms highlights their possible application as novel drug delivery agents.

  8. Natural Proline-Rich Cyclopolypeptides from Marine Organisms: Chemistry, Synthetic Methodologies and Biological Status

    PubMed Central

    Fang, Wan-Yin; Dahiya, Rajiv; Qin, Hua-Li; Mourya, Rita; Maharaj, Sandeep

    2016-01-01

    Peptides have gained increased interest as therapeutics during recent years. More than 60 peptide drugs have reached the market for the benefit of patients and several hundreds of novel therapeutic peptides are in preclinical and clinical development. The key contributor to this success is the potent and specific, yet safe, mode of action of peptides. Among the wide range of biologically-active peptides, naturally-occurring marine-derived cyclopolypeptides exhibit a broad range of unusual and potent pharmacological activities. Because of their size and complexity, proline-rich cyclic peptides (PRCPs) occupy a crucial chemical space in drug discovery that may provide useful scaffolds for modulating more challenging biological targets, such as protein-protein interactions and allosteric binding sites. Diverse pharmacological activities of natural cyclic peptides from marine sponges, tunicates and cyanobacteria have encouraged efforts to develop cyclic peptides with well-known synthetic methods, including solid-phase and solution-phase techniques of peptide synthesis. The present review highlights the natural resources, unique structural features and the most relevant biological properties of proline-rich peptides of marine-origin, focusing on the potential therapeutic role that the PRCPs may play as a promising source of new peptide-based novel drugs. PMID:27792168

  9. First evidences of interaction between pyranoanthocyanins and salivary proline-rich proteins.

    PubMed

    García-Estévez, Ignacio; Cruz, Luís; Oliveira, Joana; Mateus, Nuno; de Freitas, Victor; Soares, Susana

    2017-08-01

    The contribution of other classes of polyphenol compounds besides tannins to the overall perception of astringency is still poorly understood. So, this work aimed to study the interaction between a family of salivary proline-rich proteins (aPRPs) and representative pyranoanthocyanins in red wines [pyranomalvidin-3-glucoside (vitisin B), pyranomalvidin-3-glucoside-catechol, and pyranomalvidin-3-glucoside-epicatechin] using saturation transfer difference-NMR and MALDI-TOF. For vitisin B KD was of 1.74mM; for pyranomalvidin-3-glucoside-catechol was 1.17mM and for pyranomalvidin-3-glucoside-epicatechin it was 0.87mM. The presence of the flavanol structural unit in the pyranoanthocyanins led to an increase in their interaction with aPRPs. Further, it is also interesting that the values obtained were in the range of KD obtained previously reported for the interaction between the human saliva proline-rich peptides (IB714 and IB937) and procyanidins. Overall, the results obtained suggest that, along with tannins, other polyphenols present in red wine, namely pyranoanthocyanins, could actively contribute to red wine global astringency.

  10. Identification of a Novel Proline-Rich Antimicrobial Peptide from Brassica napus.

    PubMed

    Cao, Huihui; Ke, Tao; Liu, Renhu; Yu, Jingyin; Dong, Caihua; Cheng, Mingxing; Huang, Junyan; Liu, Shengyi

    2015-01-01

    Proline-rich antimicrobial peptides (PR-AMPs) are a group of cationic host defense peptides that are characterized by a high content of proline residues. Up to now, they have been reported in some insects, vertebrate and invertebrate animals, but are not found in plants. In this study, we performed an in silico screening of antimicrobial peptides, which led to discovery of a Brassica napus gene encoding a novel PR-AMP. This gene encodes a 35-amino acid peptide with 13 proline residues, designated BnPRP1. BnPRP1 has 40.5% identity with a known proline-rich antimicrobial peptide SP-B from the pig. BnPRP1 was artificially synthetized and cloned into the prokaryotic expression vector pET30a/His-EDDIE-GFP. Recombinant BnPRP1 was produced in Escherichia coli and has a predicted molecular mass of 3.8 kDa. Analysis of its activity demonstrated that BnPRP1 exhibited strong antimicrobial activity against Gram-positive bacterium, Gram-negative bacterium, yeast and also had strong antifungal activity against several pathogenic fungi, such as Sclerotinia sclerotiorum, Mucor sp., Magnaporthe oryzae and Botrytis cinerea. Circular dichroism (CD) revealed the main secondary structure of BnPRP1 was the random coil. BnPRP1 gene expression detected by qRT-PCR is responsive to pathogen inoculation. At 48 hours after S. sclerotiorum inoculation, the expression of BnPRP1 increased significantly in the susceptible lines while slight decrease occurred in resistant lines. These suggested that BnPRP1 might play a role in the plant defense response against S. sclerotiorum. BnPRP1 isolated from B. napus was the first PR-AMP member that was characterized in plants, and its homology sequences were found in some other Brassicaceae plants by the genome sequences analysis. Compared with the known PR-AMPs, BnPRP1 has the different primary sequences and antimicrobial activity. Above all, this study gives a chance to cast a new light on further understanding about the AMPs' mechanism and application.

  11. Cotton Effect in Copper-Proline Complexes in the Visible Region

    ERIC Educational Resources Information Center

    Volkov, Victor; Pfister, Rolf

    2005-01-01

    The electronic properties of Cu(II) complex with proline are considered to demonstrate the Cotton effect in the visible region. A series of experiments in optical rotatory dispersion spectroscopy with free D- and L-proline and their complexes with the Cu(II) ion in aqueous solution is suggested.

  12. Salivary proline-rich proteins and gluten: Do structural similarities suggest a role in celiac disease?

    PubMed Central

    Tian, Na; Messana, Irene; Leffler, Daniel A.; Kelly, Ciaran P.; Hansen, Joshua; Cabras, Tiziana; D'Alessandro, Alfredo; Schuppan, Detlef; Castagnola, Massimo; Helmerhorst, Eva J.

    2015-01-01

    Purpose Gluten proteins, the culprits in celiac disease (CD), show striking similarities in primary structure with human salivary proline-rich proteins (PRPs). Both are enriched in pro-line and glutamine residues that often occur consecutively in their sequences. We investigated potential differences in the spectrum of salivary PRPs in health and CD. Experimental design Stimulated salivary secretions were collected from CD patients, patients with refractory CD, patients with gastrointestinal complaints but no CD, and healthy controls. PRP isoforms/peptides were characterized by anionic and SDS-PAGE, PCR, and LC-ESI-MS. Results The gene frequencies of the acidic PRP isoforms PIF, Db, Pa, PRP1, and PRP2 did not differ between groups. At the protein level, PRPs peptides showed minor group differences, but these could not differentiate the CD and/or refractory CDs groups from the controls. Conclusions and clinical relevance This extensive study established that salivary PRPs, despite similarity to gluten proteins, show no apparent correlation with CD and thus will not serve as diagnostic markers for the disease. The structural basis for the tolerance to the gluten-like PRP proteins in CD is worthy of further exploration and may lead to the development of gluten-like analogs lacking immunogenicity that could be used therapeutically. PMID:25726832

  13. A modular toolkit to inhibit proline-rich motif–mediated protein–protein interactions

    PubMed Central

    Opitz, Robert; Müller, Matthias; Reuter, Cédric; Barone, Matthias; Soicke, Arne; Roske, Yvette; Piotukh, Kirill; Huy, Peter; Beerbaum, Monika; Wiesner, Burkhard; Beyermann, Michael; Schmieder, Peter; Freund, Christian; Volkmer, Rudolf; Oschkinat, Hartmut; Schmalz, Hans-Günther; Kühne, Ronald

    2015-01-01

    Small-molecule competitors of protein–protein interactions are urgently needed for functional analysis of large-scale genomics and proteomics data. Particularly abundant, yet so far undruggable, targets include domains specialized in recognizing proline-rich segments, including Src-homology 3 (SH3), WW, GYF, and Drosophila enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Here, we present a modular strategy to obtain an extendable toolkit of chemical fragments (ProMs) designed to replace pairs of conserved prolines in recognition motifs. As proof-of-principle, we developed a small, selective, peptidomimetic inhibitor of Ena/VASP EVH1 domain interactions. Highly invasive MDA MB 231 breast-cancer cells treated with this ligand showed displacement of VASP from focal adhesions, as well as from the front of lamellipodia, and strongly reduced cell invasion. General applicability of our strategy is illustrated by the design of an ErbB4-derived ligand containing two ProM-1 fragments, targeting the yes-associated protein 1 (YAP1)-WW domain with a fivefold higher affinity. PMID:25848013

  14. Binding effect of proline-rich-proteins (PRPs) on in vitro antimicrobial activity of the flavonoids

    PubMed Central

    Ansari, Jawaad Ahmed; Naz, Shahina; Tarar, Omer Mukhtar; Siddiqi, Rahmanullah; Haider, Muhammad Samee; Jamil, Khalid

    2015-01-01

    The interaction of the cyanidin, pelargonidin, catechin, myrecetin and kaempferol with casein and gelatin, as proline rich proteins (PRPs), was studied. The binding constants calculated for both flavonoid-casein and flavonoid-gelatin were fairly large (10 5 –10 7 M −1 ) indicating strong interaction. Due to higher proline content in gelatin, the binding constants of flavonoid-gelatin (2.5 × 10 5 –6.2 × 10 7 M −1 ) were found to be higher than flavonoid-casein (1.2 × 10 5 –5.0 × 10 7 M −1 ). All the flavonoids showed significant antibacterial activity against the tested strains. Significant loss in activity was observed due to the complexation with PRPs confirming that binding effectively reduced the concentration of the free flavonoids to be available for antibacterial activity. The decline in activity was corresponding to the values of the binding constants. Though the activities of free catechin and myrecetin were higher compared to pelargonidin, cyanidin and kaempferol yet the decline in activity of catechin and myrecetin due to complexation with casein and gelatin was more pronounced. PMID:26221106

  15. RNAi-based functional elucidation of PtrPRP, a gene encoding a hybrid proline rich protein, in cold tolerance of Poncirus trifoliata.

    PubMed

    Peng, Ting; Jia, Mao-Mao; Liu, Ji-Hong

    2015-01-01

    Hybrid proline-rich proteins (HyPRPs) have been suggested to play important roles in various plant development and stress response. In this study, we report the cloning and functional analysis of PtrPRP, a HyPRP-encoding gene of Poncirus trifoliata. PtrPRP contains 176 amino acids, among which 21% are proline residues, and has an 8-cysteine motif (8 CM) domain at the C terminal, a signal peptide and a proline-rich region at the N terminal. PtrPRP is constitutively expressed in root, stem and leaf, with the highest expression levels in leaf. It was progressively induced by cold, but transiently upregulated by salt and ABA. Transgenic P. trifoliata plants with knock-down PtrPRP by RNA interference (RNAi) were generated to investigate the role of PtrPRP in cold tolerance. When challenged by low temperature, the PtrPRP-RNAi plants displayed more sensitive performance compared with wild type (WT), as shown by higher electrolyte leakage and malondialdehyde content. In addition, the RNAi lines accumulated more reactive oxygen species (ROS) and lower levels of proline relative to WT. These results suggested that PtrPRP might be positively involved in cold tolerance by maintaining membrane integrity and ROS homeostasis.

  16. Epigallocatechin-3-gallate inhibits lactase but is alleviated by salivary proline-rich proteins.

    PubMed

    Naz, Shahina; Siddiqi, Rahmanullah; Dew, Tristan P; Williamson, Gary

    2011-03-23

    Lactase phlorizin hydrolase is a small intestinal brush border enzyme that catalyzes the hydrolysis of the milk sugar, lactose, and also many flavonoid glucosides. We demonstrate that epigallocatechin-3-gallate (EGCG), the principal flavonoid from green tea, inhibits in vitro hydrolysis of lactose by intestinal lactase. We then tested the hypothesis that salivary proline-rich proteins (PRPs) could modulate this inhibition and stabilize EGCG. Inhibition by EGCG of digestive enzymes (α-amylase>chymotrypsin>trypsin>lactase≫pepsin) was alleviated ∼2-6-fold by PRPs. Furthermore, PRPs appeared stable to proteolysis and also stabilized EGCG under digestive conditions in vitro. This is the first report on EGCG inhibition of lactase, and it quantifies the protective role of PRPs against EGCG inhibition of digestive enzymes.

  17. Ste20-related proline/alanine-rich kinase: A novel regulator of intestinal inflammation

    PubMed Central

    Yan, Yutao; Merlin, Didier

    2008-01-01

    Recently, inflammatory bowel disease (IBD) has been the subject of considerable research, with increasing attention being paid to the loss of intestinal epithelial cell barrier function as a mechanism of pathogenesis. Ste20-related proline/alanine-rich kinase (SPAK) is involved in regulating barrier function. SPAK is known to interact with inflammation-related kinases (such as p38, JNK, NKCC1, PKCtheta;, WNK and MLCK), and with transcription factor AP-1, resulting in diverse biological phenomena, including cell differentiation, cell transformation and proliferation, cytoskeleton rearrangement, and regulation of chloride transport. This review examines the involvement of Ste20-like kinases and downstream mitogen-activated protein kinases (MAPKs) pathways in the pathogenesis and control of intestinal inflammation. The primary focus will be on the molecular features of intestinal inflammation, with an emphasis on the interaction between SPAK and other molecules, and the effect of these interactions on homeostatic maintenance, cell volume regulation and increased cell permeability in intestinal inflammation. PMID:18985800

  18. Abscisic acid- and stress-induced highly proline-rich glycoproteins regulate root growth in rice.

    PubMed

    Tseng, I-Chieh; Hong, Chwan-Yang; Yu, Su-May; Ho, Tuan-Hua David

    2013-09-01

    In the root of rice (Oryza sativa), abscisic acid (ABA) treatment, salinity, or water deficit stress induces the expression of a family of four genes, REPETITIVE PROLINE-RICH PROTEIN (RePRP). These genes encode two subclasses of novel proline-rich glycoproteins with highly repetitive PX₁PX₂ motifs, RePRP1 and RePRP2. RePRP orthologs exist only in monocotyledonous plants, and their functions are virtually unknown. Rice RePRPs are heavily glycosylated with arabinose and glucose on multiple hydroxyproline residues. They are significantly different from arabinogalactan proteins that have glycan chains composed of arabinose and galactose. Transient and stable expressions of RePRP-green fluorescent protein reveal that a fraction of this protein is localized to the plasma membrane. In rice roots, ABA treatment increases RePRP expression preferentially in the elongation zone. Overexpression of RePRP in transgenic rice reduces root cell elongation in the absence of ABA, similar to the effect of ABA on wild-type roots. Conversely, simultaneous knockdown of the expression of RePRP1 and RePRP2 reduces the root sensitivity to ABA, indicating that RePRP proteins play an essential role in ABA/stress regulation of root growth and development. Moreover, rice RePRPs specifically interact with a polysaccharide, arabinogalactan, in a dosage-dependent manner. It is suggested that RePRP1 and RePRP2 are functionally redundant suppressors of root cell expansion and probably act through interactions with cell wall components near the plasma membrane.

  19. A Novel Topology of Proline-rich Transmembrane Protein 2 (PRRT2)

    PubMed Central

    Rossi, Pia; Sterlini, Bruno; Castroflorio, Enrico; Marte, Antonella; Onofri, Franco; Valtorta, Flavia; Maragliano, Luca; Corradi, Anna; Benfenati, Fabio

    2016-01-01

    Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including epilepsy, paroxysmal movement disorders, and migraine. On the basis of topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the two-transmembrane domain topology with a large N terminus and short C terminus oriented toward the outside of the cell. Because PRRT2 plays a role at the synapse, it is important to confirm the exact orientation of its N and C termini with respect to the plasma membrane to get clues regarding its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein in which only the second hydrophobic segment spans the plasma membrane, whereas the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space but is localized intracellularly, and only the short C terminus is extracellular (Ncyt/Cexo topology). Accordingly, we show that PRRT2 interacts with the Src homology 3 domain-bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to the clarification of the role of PRRT2 at the synapse and the understanding of pathogenic mechanisms on the basis of PRRT2-related neurological disorders. PMID:26797119

  20. A Novel Vasoactive Proline-Rich Oligopeptide from the Skin Secretion of the Frog Brachycephalus ephippium

    PubMed Central

    Arcanjo, Daniel Dias Rufino; Vasconcelos, Andreanne Gomes; Comerma-Steffensen, Simón Gabriel; Jesus, Joilson Ramos; Silva, Luciano Paulino; Pires, Osmindo Rodrigues; Costa-Neto, Claudio Miguel; Oliveira, Eduardo Brandt; Migliolo, Ludovico; Franco, Octávio Luiz; Restini, Carolina Baraldi Araújo; Paulo, Michele; Bendhack, Lusiane Maria; Bemquerer, Marcelo Porto; Oliveira, Aldeidia Pereira; Simonsen, Ulf; Leite, José Roberto de Souza de Almeida

    2015-01-01

    Proline-rich oligopeptides (PROs) are a large family which comprises the bradykinin-potentiating peptides (BPPs). They inhibit the activity of the angiotensin I-converting enzyme (ACE) and have a typical pyroglutamyl (Pyr)/proline-rich structure at the N- and C-terminus, respectively. Furthermore, PROs decrease blood pressure in animals. In the present study, the isolation and biological characterization of a novel vasoactive BPP isolated from the skin secretion of the frog Brachycephalus ephippium is described. This new PRO, termed BPP-Brachy, has the primary structure WPPPKVSP and the amidated form termed BPP-BrachyNH2 inhibits efficiently ACE in rat serum. In silico molecular modeling and docking studies suggest that BPP-BrachyNH2 is capable of forming a hydrogen bond network as well as multiple van der Waals interactions with the rat ACE, which blocks the access of the substrate to the C-domain active site. Moreover, in rat thoracic aorta BPP-BrachyNH2 induces potent endothelium-dependent vasodilatation with similar magnitude as captopril. In DAF-FM DA-loaded aortic cross sections examined by confocal microscopy, BPP-BrachyNH2 was found to increase the release of nitric oxide (NO). Moreover, BPP-BrachyNH2 was devoid of toxicity in endothelial and smooth muscle cell cultures. In conclusion, the peptide BPP-BrachyNH2 has a novel sequence being the first BPP isolated from the skin secretion of the Brachycephalidae family. This opens for exploring amphibians as a source of new biomolecules. The BPP-BrachyNH2 is devoid of cytotoxicity and elicits endothelium-dependent vasodilatation mediated by NO. These findings open for the possibility of potential application of these peptides in the treatment of endothelial dysfunction and cardiovascular diseases. PMID:26661890

  1. Blood-brain barrier transport of short proline-rich antimicrobial peptides.

    PubMed

    Stalmans, Sofie; Wynendaele, Evelien; Bracke, Nathalie; Knappe, Daniel; Hoffmann, Ralf; Peremans, Kathelijne; Polis, Ingeborgh; Burvenich, Christian; De Spiegeleer, Bart

    2014-04-01

    Infections by antibiotic-resistant bacteria are becoming a great risk for human health, leading to an urgent need for new efficient antibacterial therapies. The short, proline-rich antimicrobial peptides from insects gained a lot of interest as a potential antibacterial treatment, having a low toxicity profile and being mainly active against Gram-negative bacteria. To know whether these antimicrobial peptides can be used for the treatment of cerebral infections, the blood-brain barrier transport characteristics of these peptides were investigated. This study describes the results of the in vivo blood-brain barrier experiments in mice, as well as the in vitro metabolic stability in mouse plasma and brain of apidaecin Api137, oncocin, drosocin and drosocin Pro5Hyp. The four investigated peptides showed a significant influx into the brain with a K(in) ranging between 0.37 and 0.86 µL/g x min and brain distribution volumes of 19.6 to 25.8 µL/g. Only for drosocin, a significant efflux was determined, with a k(out) of 0.22 min(-1). After entering the brain, oncocin was for approximately 80% trapped in the endothelial cells, while the other peptides reached the brain parenchyma for about 70%. All peptides were stable in plasma and brain during the experiments, with estimated metabolic half-lives ranging between 47 min and 637 min. We conclude that the investigated short, proline-rich antimicrobial peptides show an influx into the brain, which make them a promising antibacterial treatment of cerebral infections.

  2. A Novel Vasoactive Proline-Rich Oligopeptide from the Skin Secretion of the Frog Brachycephalus ephippium.

    PubMed

    Arcanjo, Daniel Dias Rufino; Vasconcelos, Andreanne Gomes; Comerma-Steffensen, Simón Gabriel; Jesus, Joilson Ramos; Silva, Luciano Paulino; Pires Júnior, Osmindo Rodrigues; Costa-Neto, Claudio Miguel; Oliveira, Eduardo Brandt; Migliolo, Ludovico; Franco, Octávio Luiz; Restini, Carolina Baraldi Araújo; Paulo, Michele; Bendhack, Lusiane Maria; Bemquerer, Marcelo Porto; Oliveira, Aldeidia Pereira; Simonsen, Ulf; Leite, José Roberto de Souza de Almeida

    2015-01-01

    Proline-rich oligopeptides (PROs) are a large family which comprises the bradykinin-potentiating peptides (BPPs). They inhibit the activity of the angiotensin I-converting enzyme (ACE) and have a typical pyroglutamyl (Pyr)/proline-rich structure at the N- and C-terminus, respectively. Furthermore, PROs decrease blood pressure in animals. In the present study, the isolation and biological characterization of a novel vasoactive BPP isolated from the skin secretion of the frog Brachycephalus ephippium is described. This new PRO, termed BPP-Brachy, has the primary structure WPPPKVSP and the amidated form termed BPP-BrachyNH2 inhibits efficiently ACE in rat serum. In silico molecular modeling and docking studies suggest that BPP-BrachyNH2 is capable of forming a hydrogen bond network as well as multiple van der Waals interactions with the rat ACE, which blocks the access of the substrate to the C-domain active site. Moreover, in rat thoracic aorta BPP-BrachyNH2 induces potent endothelium-dependent vasodilatation with similar magnitude as captopril. In DAF-FM DA-loaded aortic cross sections examined by confocal microscopy, BPP-BrachyNH2 was found to increase the release of nitric oxide (NO). Moreover, BPP-BrachyNH2 was devoid of toxicity in endothelial and smooth muscle cell cultures. In conclusion, the peptide BPP-BrachyNH2 has a novel sequence being the first BPP isolated from the skin secretion of the Brachycephalidae family. This opens for exploring amphibians as a source of new biomolecules. The BPP-BrachyNH2 is devoid of cytotoxicity and elicits endothelium-dependent vasodilatation mediated by NO. These findings open for the possibility of potential application of these peptides in the treatment of endothelial dysfunction and cardiovascular diseases.

  3. The role of the proline-rich domain of Ssdp1 in the modular architecture of the vertebrate head organizer

    PubMed Central

    Enkhmandakh, Badam; Makeyev, Alexandr V.; Bayarsaihan, Dashzeveg

    2006-01-01

    Lim1, Ssdp1, and Ldb1 proteins are components of the Ldb1-associated transcriptional complex, which is important in the head-organizing activity during early mouse development. Depletion of each individual protein alone causes a headless phenotype. To explore in more detail the modular architecture of the complex, we have generated two different gene-trapped mouse lines that express truncated forms of Ssdp1. Embryos derived from the gene-trapped line that encodes a truncated Ssdp1 lacking the proline-rich sequence exhibit a lethal abnormal head-development phenotype, resembling mouse embryos deficient for Lim1, Ssdp1, or Otx2 genes. Embryos derived from the second gene-trapped line, in which most of the proline-rich domain of Ssdp1 is retained, did not show abnormalities in head development. Our data demonstrate that components of the Ldb1-dependent module can be subdivided further into discrete functional domains and that the proline-rich stretch of Ssdp1 is critical for embryonic head development. Furthermore, phylogenetic comparisons revealed that in Caenorhabditis elegans, a similar proline-rich sequence is absent in Ssdp but present in Ldb1. We conclude that although the overall architecture of the Ldb1-dependent module has been preserved, the genetic specification of its individual components has diversified during evolution, without compromising the function of the module. PMID:16864769

  4. Antimicrobial proline-rich peptides from the hemolymph of marine snail Rapana venosa.

    PubMed

    Dolashka, Pavlina; Moshtanska, Vesela; Borisova, Valika; Dolashki, Aleksander; Stevanovic, Stefan; Dimanov, Tzvetan; Voelter, Wolfgang

    2011-07-01

    Hemolymph of Rapana venosa snails is a complex mixture of biochemically and pharmacologically active components such as peptides and proteins. Antimicrobial peptides are gaining attention as antimicrobial alternatives to chemical food preservatives and commonly used antibiotics. Therefore, for the first time we have explored the isolation, identification and characterisation of 11 novel antimicrobial peptides produced by the hemolymph of molluscs. The isolated peptides from the hemolymph applying ultrafiltration and reverse-phase high-performance liquid chromatography (RP-HPLC) have molecular weights between 3000 and 9500 Da, determined by mass spectrometric analysis. The N-terminal sequences of the peptides identified by Edman degradation matched no peptides in the MASCOT search database, indicating novel proline-rich peptides. UV spectra revealed that these substances possessed the characteristics of protein peptides with acidic isoelectric points. However, no Cotton effects were observed between 190 and 280 nm by circular dichroism spectroscopy. Four of the pro-rich peptides also showed strong antimicrobial activities against tested microorganisms including Gram-positive and Gram-negative bacteria.

  5. The proline-rich region of 18.5 kDa myelin basic protein binds to the SH3-domain of Fyn tyrosine kinase with the aid of an upstream segment to form a dynamic complex in vitro.

    PubMed

    De Avila, Miguel; Vassall, Kenrick A; Smith, Graham S T; Bamm, Vladimir V; Harauz, George

    2014-12-08

    The intrinsically disordered 18.5 kDa classic isoform of MBP (myelin basic protein) interacts with Fyn kinase during oligodendrocyte development and myelination. It does so primarily via a central proline-rich SH3 (Src homology 3) ligand (T92-R104, murine 18.5 kDa MBP sequence numbering) that is part of a molecular switch due to its high degree of conservation and modification by MAP (mitogen-activated protein) and other kinases, especially at residues T92 and T95. Here, we show using co-transfection experiments of an early developmental oligodendroglial cell line (N19) that an MBP segment upstream of the primary ligand is involved in MBP-Fyn-SH3 association in cellula. Using solution NMR spectroscopy in vitro, we define this segment to comprise MBP residues (T62-L68), and demonstrate further that residues (V83-P93) are the predominant SH3-target, assessed by the degree of chemical shift change upon titration. We show by chemical shift index analysis that there is no formation of local poly-proline type II structure in the proline-rich segment upon binding, and by NOE (nuclear Overhauser effect) and relaxation measurements that MBP remains dynamic even while complexed with Fyn-SH3. The association is a new example first of a non-canonical SH3-domain interaction and second of a fuzzy MBP complex.

  6. Delayed skin wound repair in proline-rich protein tyrosine kinase 2 knockout mice.

    PubMed

    Koppel, Aaron C; Kiss, Alexi; Hindes, Anna; Burns, Carole J; Marmer, Barry L; Goldberg, Gregory; Blumenberg, Miroslav; Efimova, Tatiana

    2014-05-15

    Proline-rich protein tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family. We used Pyk2 knockout (Pyk2-KO) mice to study the role of Pyk2 in cutaneous wound repair. We report that the rate of wound closure was delayed in Pyk2-KO compared with control mice. To examine whether impaired wound healing of Pyk2-KO mice was caused by a keratinocyte cell-autonomous defect, the capacities of primary keratinocytes from Pyk2-KO and wild-type (WT) littermates to heal scratch wounds in vitro were compared. The rate of scratch wound repair was decreased in Pyk2-KO keratinocytes compared with WT cells. Moreover, cultured human epidermal keratinocytes overexpressing the dominant-negative mutant of Pyk2 failed to heal scratch wounds. Conversely, stimulation of Pyk2-dependent signaling via WT Pyk2 overexpression induced accelerated scratch wound closure and was associated with increased expression of matrix metalloproteinase (MMP)-1, MMP-9, and MMP-10. The Pyk2-stimulated increase in the rate of scratch wound repair was abolished by coexpression of the dominant-negative mutant of PKCδ and by GM-6001, a broad-spectrum inhibitor of MMP activity. These results suggest that Pyk2 is essential for skin wound reepithelialization in vivo and in vitro and that it regulates epidermal keratinocyte migration via a pathway that requires PKCδ and MMP functions.

  7. A proline-rich polypeptide from ovine colostrum: colostrinin with immunomodulatory activity.

    PubMed

    Zimecki, Michal

    2008-01-01

    A proline-rich polypeptide (PRP), later called colostrinin (CLN), was originally found as a fraction accompanying sheep colostral immunoglobulins. Extensive in vitro and in vivo studies in mice revealed its interesting T cell-tropic activities. The polypeptide promoted T cell maturation from early thymic precursors that acquired the phenotype and function of mature, helper cells; on the other hand, it also affected the phenotype and function of mature T cells. In particular, PRP was shown to recruit suppressor T cells in a model of T cell-independent humoral immune response and suppressed autoimmune hemolytic anemia in New Zealand Black mice. Subsequent in vitro studies in the human model revealed that CLN regulated mitogen-induced cytokine production in whole blood cultures. A discovery that CLN promoted procognitive functions in experimental animal models, supported by other laboratory findings, indicating prevention of pathological processes in the central nervous system, led to application of CLN in multicenter clinical trials. The trials demonstrated the therapeutic benefit of CLN in Alzheimer's disease (AD) patients by delaying progress of the disease.

  8. Modulation of 4HNE-mediated signaling by proline-rich peptides from ovine colostrum.

    PubMed

    Boldogh, Istvan; Liebenthal, Daniel; Hughes, T Kley; Juelich, Terry L; Georgiades, Jerzy A; Kruzel, Marian L; Stanton, G John

    2003-04-01

    In previous studies we showed that colostrinin (CLN), a complex of proline-rich polypeptides derived from ovine colostrum, induces mitogenic stimulation, as well as a variety of cytokines in human peripheral blood leukocytes, and possesses antioxidant activity in pheochromocytoma (PC12) cells. In this study we investigated the effects of CLN on 4-hydroxynonenal (4HNE)-mediated adduct formation, generation of reactive oxygen species (ROS), glutathione (GSH) metabolism, and the modification of signal transduction cascade that leads to activation of c-Jun N-terminal kinase (JNK) in PC12 cells. Here we demonstrate that CLN (1) reduced the abundance of 4HNE-protein adducts, as shown by fluorescent microscopy and Western blot analysis; (2) reduced intracellular levels of ROS, as shown by a decrease in 2',7'-dichlorodihydro-fluorescein-mediated fluorescence; (3) inhibited 4HNE-mediated GSH depletion, as determined fluorimetrically; and (4) inhibited 4HNE-induced activation of JNKs. Together, these findings suggest that CLN appears to down-regulate 4HNE-mediated lipid peroxidation and its product-induced signaling that otherwise may lead to pathological changes at the cellular and organ level. These findings also suggest further that CLN could be useful in the treatment of diseases such as Alzheimer's, as well as those in which ROS are implicated in pathogenesis.

  9. Supramolecular properties of the proline-rich gamma-Zein N-terminal domain.

    PubMed Central

    Kogan, Marcelo J; Dalcol, Ionara; Gorostiza, Pau; Lopez-Iglesias, Carmen; Pons, Ramon; Pons, Miquel; Sanz, Fausto; Giralt, Ernest

    2002-01-01

    Zeins are maize storages proteins that accumulate inside large vesicles called protein bodies. gamma-Zein lines the inner face of the protein body membrane, and its N-terminal proline-rich repetitive domain with the sequence (VHLPPP)(8) appears to be necessary for the accumulation of the protein within the organelle. Synthetic (VHLPPP)(8) adopts an amphipathic polyproline II conformation. In a preliminary recent work we used atomic force microscopy to study the surface organization of the octamer and transmission electron microscopy to visualize aggregates of the peptide from aqueous solution. We previously envisioned two self-assembly models (i.e., the geometric and the micellar) that take into account the observed features. In the present work we studied in detail the self-assembly of the peptide in solution and found that the peptide is able to form cylindrical micelles. Fibrils formed on graphite are generated by assembly of solution micelles. Based on the results of these studies, we focused exclusively on the micellar model. To our knowledge we have characterized for the first time supramolecular aggregates of polyproline structures other than collagen. The spontaneous arrangement of (VHLPPP)(8) suggests a role for the N-terminal domain of gamma-zein in the process of the whole protein deposition in protein bodies. PMID:12124299

  10. Clinical Manifestations in Paroxysmal Kinesigenic Dyskinesia Patients with Proline-Rich Transmembrane Protein 2 Gene Mutation

    PubMed Central

    Youn, Jinyoung; Kim, Ji Sun; Lee, Munhyang; Lee, Jeehun; Roh, Hakjae

    2014-01-01

    Background and Purpose Given the diverse phenotypes including combined non-dyskinetic symptoms in patients harboring mutations of the gene encoding proline-rich transmembrane protein 2 (PRRT2), the clinical significance of these mutations in paroxysmal kinesigenic dyskinesia (PKD) is questionable. In this study, we investigated the clinical characteristics of PKD patients with PRRT2 mutations. Methods Familial and sporadic PKD patients were enrolled and PRRT2 gene sequencing was performed. Demographic and clinical data were compared between PKD patients with and without a PRRT2 mutation. Results Among the enrolled PKD patients (8 patients from 5 PKD families and 19 sporadic patients), PRRT2 mutations were detected in 3 PKD families (60%) and 2 sporadic cases (10.5%). All familial patients with a PRRT2 gene mutation had the c.649dupC mutation, which is the most commonly reported mutation. Two uncommon mutations (c.649delC and c.629dupC) were detected only in the sporadic cases. PKD patients with PRRT2 mutation were younger at symptom onset and had more non-dyskinetic symptoms than those without PRRT2 mutation. However, the characteristics of dyskinetic movement did not differ between the two groups. Conclusions This is the first study of PRRT2 mutations in Korea. The presence of a PRRT2 mutation was more strongly related to familial PKD, and was clinically related with earlier age of onset and common non-dyskinetic symptoms in PKD patients. PMID:24465263

  11. Distribution of the SynDIG4/proline-rich transmembrane protein 1 in rat brain.

    PubMed

    Kirk, Lyndsey M; Ti, Shu W; Bishop, Hannah I; Orozco-Llamas, Mayra; Pham, Michelle; Trimmer, James S; Díaz, Elva

    2016-08-01

    The modulation of AMPA receptor (AMPAR) content at synapses is thought to be an underlying molecular mechanism of memory and learning. AMPAR content at synapses is highly plastic and is regulated by numerous AMPAR accessory transmembrane proteins such as TARPs, cornichons, and CKAMPs. SynDIG (synapse differentiation-induced gene) defines a family of four genes (SynDIG1-4) expressed in distinct and overlapping patterns in the brain. SynDIG1 was previously identified as a novel transmembrane AMPAR-associated protein that regulates synaptic strength. The related protein SynDIG4 [also known as Prrt1 (proline-rich transmembrane protein 1)] has recently been identified as a component of AMPAR complexes. In this study, we show that SynDIG1 and SynDIG4 have distinct yet overlapping patterns of expression in the central nervous system, with SynDIG4 having especially prominent expression in the hippocampus and particularly within CA1. In contrast to SynDIG1 and other traditional AMPAR auxiliary subunits, SynDIG4 is de-enriched at the postsynaptic density and colocalizes with extrasynaptic GluA1 puncta in primary dissociated neuron culture. These results indicate that, although SynDIG4 shares sequence similarity with SynDIG1, it might act through a unique mechanism as an auxiliary factor for extrasynaptic GluA1-containing AMPARs. J. Comp. Neurol. 524:2266-2280, 2016. © 2015 Wiley Periodicals, Inc.

  12. Characterization of the human salivary basic proline-rich protein complex by a proteomic approach.

    PubMed

    Messana, Irene; Cabras, Tiziana; Inzitari, Rosanna; Lupi, Alessandro; Zuppi, Cecilia; Olmi, Chiara; Fadda, Maria Benedetta; Cordaro, Massimo; Giardina, Bruno; Castagnola, Massimo

    2004-01-01

    Thirteen samples of human normal whole saliva were analyzed by RP-HPLC-ESI-MS and MALDI-TOF-MS to investigate the basic proline-rich protein complex. Between known basic-PRPs the P-B, P-C (or IB-8b), P-D (or IB-5), P-E (or IB-9), P-F (or IB-8c), P-H (or IB-4), IB-6, II-2, IB-1, and IB-8a glucosylated were identified, whereas the II-1, IB-7, PA, and D1-A peptides were not detected. Some detected masses not attributable to known basic-PRPs were putatively ascribed to II-2 and IB-1 nonphosphorylated, II-2 and IB-1 missing the C-terminal arginine residue, and the 1-62 fragment of IB-6, named P-J peptide. A correlation matrix analysis revealed a cluster of correlation among all the basic PRPs (apart from the P-B peptide) which is in agreement with their common parotid origin.

  13. Cell wall-associated ROOT HAIR SPECIFIC 10, a proline-rich receptor-like kinase, is a negative modulator of Arabidopsis root hair growth

    PubMed Central

    Hwang, Youra; Lee, Hyodong; Lee, Young-Sook; Cho, Hyung-Taeg

    2016-01-01

    Plant cell growth is restricted by the cell wall, and cell wall dynamics act as signals for the cytoplasmic and nuclear events of cell growth. Among various receptor kinases, ROOT HAIR SPECIFIC 10 (RHS10) belongs to a poorly known receptor kinase subfamily with a proline-rich extracellular domain. Here, we report that RHS10 defines the root hair length of Arabidopsis thaliana by negatively regulating hair growth. RHS10 modulates the duration of root hair growth rather than the growth rate. As poplar and rice RHS10 orthologs also showed a root hair-inhibitory function, this receptor kinase-mediated function appears to be conserved in angiosperms. RHS10 showed a strong association with the cell wall, most probably through its extracellular proline-rich domain (ECD). Deletion analysis of the ECD demonstrated that a minimal extracellular part, which includes a few proline residues, is required for RHS10-mediated root hair inhibition. RHS10 suppressed the accumulation of reactive oxygen species (ROS) in the root, which are necessary for root hair growth. A yeast two-hybrid screening identified an RNase (RNS2) as a putative downstream target of RHS10. Accordingly, RHS10 overexpression decreased and RHS10 loss increased RNA levels in the hair-growing root region. Our results suggest that RHS10 mediates cell wall-associated signals to maintain proper root hair length, at least in part by regulating RNA catabolism and ROS accumulation. PMID:26884603

  14. Can proline-rich polypeptide complex mimic the effect of nerve growth factor?

    PubMed

    Zabłocka, Agnieszka; Urbaniak, Anna; Kuropatwa, Marianna; Zyzak, Joanna; Rossowska, Joanna; Janusz, Maria

    2014-01-01

    Naturally occurring compounds that can act as prosurvival factors and neurite formation stimulants in the conditions of reduced neurotrophins production are important both in neuronal protection and therapy of neurodegenerative disorders. Therefore, the role of proline-rich polypeptide complex (PRP) and its nonapeptide fragment (NP) in the promotion of pheochromocytoma cell line (PC12) survival and neurite outgrowth pathway is presented. It was shown that PRP/NP did not affect the neuronal nitric oxide synthase (nNOS) at the transcriptional and protein level. However, the activity of nNOS and intracellular nitric oxide (NO) concentration was markedly increased after treatment of PC12 cells with peptides. This reaction was inhibited by L-NAME-nNOS inhibitor. It was shown that PRP and NP induce the soluble guanylyl cyclase to release higher amount of cyclic GMP (cGMP), and subsequently, the increased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) is observed. This effect was abolished by both U0126 (inhibitor of ERK1/2) and also by L-NAME. Reduction of ERK1/2 activity observed in the presence of nNOS inhibitor suggests that its activation is NO-dependent. The presented results shed some light on the mechanism of action of PRP complex. PRP and NP can activate NO/cGMP/ERK1/2 signaling pathway, similarly to nerve growth factor (NGF). The prosurvival action and short fibers formation suggest the role of PRP and NP in neuroprotection and the initiation of neuritogenesis. They can also participate in the amplification of signals controlling the survival and differentiation of neurons effect when the deficit of NGF takes place.

  15. C-Terminal Modification and Multimerization Increase the Efficacy of a Proline-Rich Antimicrobial Peptide.

    PubMed

    Li, Wenyi; O'Brien-Simpson, Neil M; Yao, Shenggen; Tailhades, Julien; Reynolds, Eric C; Dawson, Raymond M; Otvos, Laszlo; Hossain, Mohammed Akhter; Separovic, Frances; Wade, John D

    2017-01-05

    Two series of branched tetramers of the proline-rich antimicrobial peptide (PrAMP), Chex1-Arg20, were prepared to improve antibacterial selectivity and potency against a panel of Gram-negative nosocomial pathogens including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. First, tetramerization was achieved by dithiomaleimide (DTM) conjugation of two C-terminal-cysteine bearing dimers that also incorporated C-terminal peptide chemical modification. DTM-linked tetrameric peptides containing a C-terminal hydrazide moiety on each dimer exhibited highly potent activities in the minimum inhibitory concentration (MIC) range of 0.49-2.33 μm. A second series of tetrameric analogues with C-terminal hydrazide modification was prepared by using alternative conjugation linkers including trans-1,4-dibromo-2-butene, α,α'-dibromo-p-xylene, or 6-bismaleimidohexane to determine the effect of length on activity. Each displayed potent and broadened activity against Gram-negative nosocomial pathogens, particularly the butene-linked tetrameric hydrazide. Remarkably, the greatest MIC activity is against P. aeruginosa (0.77 μm/8 μg mL(-1) ) where the monomer is inactive. None of these peptides showed any cytotoxicity to mammalian cells up to 25 times the MIC. A diffusion NMR study of the tetrameric hydrazides showed that the more active antibacterial analogues were those with a more compact structure having smaller hydrodynamic radii. The results show that C-terminal PrAMP hydrazidation together with its rational tetramerization is an effective means for increasing both diversity and potency of PrAMP action.

  16. Thermodynamic contribution of backbone conformational entropy in the binding between SH3 domain and proline-rich motif.

    PubMed

    Zeng, Danyun; Shen, Qingliang; Cho, Jae-Hyun

    2017-02-26

    Biological functions of intrinsically disordered proteins (IDPs), and proteins containing intrinsically disordered regions (IDRs) are often mediated by short linear motifs, like proline-rich motifs (PRMs). Upon binding to their target proteins, IDPs undergo a disorder-to-order transition which is accompanied by a large conformational entropy penalty. Hence, the molecular mechanisms underlying control of conformational entropy are critical for understanding the binding affinity and selectivity of IDPs-mediated protein-protein interactions (PPIs). Here, we investigated the backbone conformational entropy change accompanied by binding of the N-terminal SH3 domain (nSH3) of CrkII and PRM derived from guanine nucleotide exchange factor 1 (C3G). In particular, we focused on the estimation of conformational entropy change of disordered PRM upon binding to the nSH3 domain. Quantitative characterization of conformational dynamics of disordered peptides like PRMs is limited. Hence, we combined various methods, including NMR model-free analysis, δ2D, DynaMine, and structure-based calculation of entropy loss. This study demonstrates that the contribution of backbone conformational entropy change is significant in the PPIs mediated by IDPs/IDRs.

  17. The rad9 gene of Coprinus cinereus encodes a proline-rich protein required for meiotic chromosome condensation and synapsis

    SciTech Connect

    Seitz, L.C.; Tang, Keliang; Cummings, W.J.; Zolan, M.E.

    1996-04-01

    The rad9 gene of Coprinus cinereus is essential for the normal completion of meiosis. We examined surface-spread preparations of wild-type and rad9-1 nuclei from the meiotic stages of karyogamy through metaphase I, and we determined the primary sequence, structure, and meiotic expression of the rad9 gene. In wild-type C. cinereus, karyogamy is followed by condensation and alignment of homologous chromosomes. Condensation and axial core development largely precede synapsis, which often initiates at telomeres. A diffuse diplotene phase coincides with dissolution of the synaptonemal complex, and subsequently chromosomes further condense as the cells progress into metaphase I. In contrast, although karyogamy and nucleolar fusion are apparently normal in rad9-1 basidia, only short stretches of synaptonemal complex form. These correlate with stretches of condensed chromatin, mostly at apparent chromosome ends, and regions of presumptive triple synapsis are numerous. rad9-1 basidia enter the diffuse stages of early diplotene, and then 50% of these cells enter metaphase I by the criteria of nucleolar elimination and at least some chromatin condensation. rad9 gene expression is induced after gamma irradiation and during meiosis. The gene has 27 exons and encodes a predicted protein of 2157 amino acids, with a proline-rich amino terminus. 62 refs., 10 figs.

  18. Subcellular localization of proline-rich tyrosine kinase 2 during oocyte fertilization and early-embryo development in mice

    PubMed Central

    MENG, Xiao-qian; DAI, Yuan-yuan; JING, Lai-dong; BAI, Jing; LIU, Shu-zhen; ZHENG, Ke-gang; PAN, Jie

    2016-01-01

    Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase, is a member of the focal adhesion kinase family and is highly expressed in oocytes. Using a combination of confocal microscopy and RNAi, we localized and studied the function of both Pyk2 and tyrosine-phosphorylated Pyk2 (p-Pyk2) during mouse oocyte fertilization and early embryo development. At the onset of fertilization, Pyk2 and p-Pyk2 were detected predominantly in sperm heads and the oocyte cytoplasm. Upon formation of male and female pronuclei, Pyk2 and its activated form leave the cytoplasm and accumulate in the two pronuclei. We detected Pyk2 in blastomere nuclei and found both Pyk2 and p-Pyk2 in the pre-blastula cytoplasm. Pyk2 and its activated form then disappeared from the blastula nuclei and localized to the perinuclear regions, where blastula cells come into contact with each other. Pyk2 knockdown via microinjection of siRNA into the zygote did not inhibit early embryo development. Our results suggest that Pyk2 plays multiple functional roles in mouse oocyte fertilization as well as throughout early embryo development. PMID:27086609

  19. Isolation of a mRNA encoding a glycine-proline-rich beta-keratin expressed in the regenerating epidermis of lizard.

    PubMed

    Dalla Valle, Luisa; Toffolo, Vania; Belvedere, Paola; Alibardi, Lorenzo

    2005-12-01

    During scale regeneration in lizard tail, an active differentiation of beta-keratin synthesizing cells occurs. The cDNA and amino acid sequence of a lizard beta-keratin has been obtained from mRNA isolated from regenerating epidermis. Degenerate oligonucleotides, selected from the translated amino acid sequence of a lizard claw protein, were used to amplify a specific lizard keratin cDNA fragment from the mRNA after reverse transcription with poly dT primer and subsequent polymerase chain reaction (3'-rapid amplification of cDNA ends analysis, 3'-RACE). The new sequence was used to design specific primers to obtain the complete cDNA sequence by 5'-RACE. The 835-nucleotide cDNA sequence encodes a glycine-proline-rich protein containing 163 amino acids with a molecular mass of 15.5 kDa; 4.3% of its amino acids is represented by cysteine, 4.9% by tyrosine, 8.0% by proline, and 29.4% by glycine. Tyrosine is linked to glycine, and proline is present mainly in the central region of the protein. Repeated glycine-glycine-X and glycine-X amino acid sequences are localized near the N-amino and C-terminal regions. The protein has the central amino acid region similar to that of claw-feather, whereas the head and tail regions are similar to glycine-tyrosine-rich proteins of mammalian hairs. In situ hybridization analysis at light and electron microscope reveals that the corresponding mRNA is expressed in cells of the differentiating beta-layers of the regenerating scales. The synthesis of beta-keratin from its mRNA occurs among ribosomes or is associated with the surface of beta-keratin filaments.

  20. Effect of cytostatic proline rich polypeptide-1 on tumor suppressors of inflammation pathway signaling in chondrosarcoma

    PubMed Central

    Galoian, Karina; Luo, Shihua; Qureshi, Amir; Patel, Parthik; Price, Rachel; Morse, Ashlyn S.; Chailyan, Gor; Abrahamyan, Silva; Temple, H. T.

    2016-01-01

    Cytokines produced in the tumour microenvironment exert an important role in cancer pathogenesis and in the inhibition of disease progression. Cancer of the cartilage is termed metastatic chondrosarcoma; however, the signaling events resulting in mesenchymal cell transformation to sarcoma have yet to be fully elucidated. The present study aimed to characterize the cytokine expression profile in the human JJ012 chondrosarcoma cell line, as well as the effect of cytostatic proline-rich polypeptide-1 (PRP-1). Western blot experiments demonstrated that the levels of suppressor of cytokine signaling 3 (SOCS3) were upregulated in chondrocytes compared with chondrosarcoma cells. Addition of PRP-1 restored the expression of the tumor suppressors, SOCS3 and ten-eleven-translocation methylcytosine dioxygenase 1 and 2 (TET1/2), in a dose-responsive manner. It is known that methylation of histone H3K9 was eliminated from the promoters of the inflammation-associated genes. PRP-1 inhibited H3K9 demethylase activity with an IC50 (concentration required to give half-maximal inhibition) value of 3.72 µg/ml in the chondrosarcoma cell line. Data obtained from ELISA experiments indicated that the expression of interleukin-6 (IL-6) in chondrosarcoma cells was 86-fold lower compared with that in C28 chondrocytes. In the present study, a 53-fold downregulation of IL-6 expression in co-culture of chondrosarcoma cells and C28 chondrocytes was identified as well. Downregulation of IL-6 expression has been documented in numerous other tumor types, although the reasons for this have not been fully established. In chondrosarcoma, IL-6 manifests itself as an anti-inflammatory agent and, possibly, as an anti-tumorigenic factor. To explore protein-DNA interactions leading to such differences, a gel-shift chemiluminescent assay was performed. Gel shifts were observed for chondrosarcoma and chondrocytes in the lanes that contained nuclear cell extract and oligo-IL-6 DNA. Notably, the DNA

  1. Differential Utilization of Basic Proline-Rich Glycoproteins during Growth of Oral Bacteria in Saliva

    PubMed Central

    Zhou, Yuan; Zhang, Luxia; Zhou, Xuedong; Cisar, John O.

    2016-01-01

    ABSTRACT Although saliva is widely recognized as a primary source of carbon and nitrogen for growth of the dental plaque biofilm community, little is known about how different oral bacteria utilize specific salivary components. To address this question, 32 strains representing 16 genera commonly isolated from early plaque biofilms were compared for growth over two transfers in stimulated (by chewing Parafilm) whole saliva that was stabilized by heat treatment and dialysis. The cell densities, measured by quantitative PCR (qPCR), ranged from ∼1 × 106 to 1 × 107/ml for strains of Streptococcus gordonii, Streptococcus oralis, and Streptococcus mitis and one strain of Streptococcus sanguinis. Strains of Streptococcus mutans, Gemella haemolysans, and Granulicatella adiacens reached ∼1 × 105 to 1 × 106/ml. In contrast, little or no growth was noted for three other strains of S. sanguinis, as well as for strains of Streptococcus parasanguinis, Streptococcus salivarius, Streptococcus vestibularis, Streptococcus sobrinus, Actinomyces spp., Abiotrophia defectiva, and Rothia dentocariosa. SDS-PAGE, lectin blotting, and two-dimensional gel electrophoresis of saliva from cultures of S. gordonii, S. oralis, and S. mitis revealed species-specific differences in the degradation of basic proline-rich glycoproteins (PRG). In contrast, saliva from cultures of other bacteria was indistinguishable from control saliva. Species-dependent differences in the utilization of individual host sugars were minor. Thus, differences in salivary glycan foraging between oral species may be important to cross-feeding and cooperation between organisms in dental plaque biofilm development. IMPORTANCE Bacteria in the mouth use saliva for nutrition. How each of the many types of bacteria uses saliva is not clear. We show that a major protein in saliva, called PRG, is an important nutrition source for certain bacteria but not for others. PRG has many sugar molecules linked in chains, but the sugar

  2. Effect of distal sugar and interglycosidic linkage of disaccharides on the activity of proline rich antimicrobial glycopeptides.

    PubMed

    Lele, Deepti S; Dwivedi, Rohini; Kumari, Saroj; Kaur, Kanwal J

    2015-11-01

    The effect of glycosylation on protein structure and function depends on a variety of intrinsic factors including glycan chain length. We have analyzed the effect of distal sugar and interglycosidic linkage of disaccharides on the properties of proline-rich antimicrobial glycopeptides, formaecin I and drosocin. Their glycosylated analogs-bearing lactose, maltose and cellobiose, as a glycan side chain on their conserved threonine residue, were synthesized where these disaccharides possess identical proximal sugar and vary in the nature of distal sugar and/or interglycosidic linkage. The structural and functional properties of these disaccharide-containing formaecin I and drosocin analogs were compared with their corresponding monoglycosylated forms, β-D-glucosyl-formaecin I and β-D-glucosyl-drosocin, respectively. We observed neither major secondary structural alterations studied by circular dichroism nor substantial differences in the toxicity with mammalian cells among all of these analogs. The comparative analyses of antibacterial activities of these analogs of formaecin I and drosocin displayed that β-D-maltosyl-formaecin I and β-D-maltosyl-drosocin were more potent than that of respective β-D-Glc-analog, β-D-cellobiosyl-analog and β-D-lactosyl-analog. Despite the differences in their antibacterial activity, all the analogs exhibited comparable binding affinity to DnaK that has been reported as one of the targets for proline-rich class of antibacterial peptides. The comparative-quantitative internalization studies of differentially active analogs revealed the differences in their uptake into bacterial cells. Our results exhibit that the sugar chain length as well as interglycosidic linkage of disaccharide may influence the antibacterial activity of glycosylated analogs of proline-rich antimicrobial peptides and the magnitude of variation in antibacterial activity depends on the peptide sequence.

  3. Isolation and characterization of the messenger RNA and the gene coding for a proline-rich zein from corn endosperm

    SciTech Connect

    Wang, S.Z.

    1985-01-01

    Gamma-zein, a proline-rich protein from corn endosperm, was investigated at the molecular level. Immunological and electrophoretic data indicated that gamma-zein was deposited into protein bodies in corn endosperm. Both isolated polysomes and poly(A)/sup +/ mRNA were found to direct into vitro synthesis of gamma-zein in a wheat germ system. In vitro synthesized gamma-zein was immunoprecipitated from the total in vitro translation products. A cDNA expression library was constructed by reverse transcription of total poly(A)/sup +/ mRNA using pUC8 plasmid as vector and E. coli strain DH1 as host. The library was screened for the expression of gamma-zein and alpha-zein by specific antibodies. The library was also screened with /sup 32/P-labeled gamma-zein and alpha-zein cDNA probes. The results indicated that gamma-zein and its fragments were readily expressed in E. coli while alpha-zein was not. Seven independently selected clones, six of which were selected by antibody and one by a cDNA probe, were sequenced. A comparison of sequence information from seven clones revealed that their overlapping regions were identical. This suggests that gamma-zein is encoded by a single gene. This finding is in conflict with what was expected on the basis of extensive charge heterogeneity of gamma-zein in isoelectric focusing. Individual bands cut from an IEF gel were rerun and shown to give several bands suggesting that the charge heterogeneity of gamma-zein may be an artifact. Sequence information of gamma-zein indicated that the gene encodes a mature protein whose primary structure includes 204 amino acids and has a molecular weight of 21,824 daltons.

  4. Structure of the dimeric exonuclease TREX1 in complex with DNA displays a proline-rich binding site for WW Domains.

    PubMed

    Brucet, Marina; Querol-Audí, Jordi; Serra, Maria; Ramirez-Espain, Ximena; Bertlik, Kamila; Ruiz, Lidia; Lloberas, Jorge; Macias, Maria J; Fita, Ignacio; Celada, Antonio

    2007-05-11

    TREX1 is the most abundant mammalian 3' --> 5' DNA exonuclease. It has been described to form part of the SET complex and is responsible for the Aicardi-Goutières syndrome in humans. Here we show that the exonuclease activity is correlated to the binding preferences toward certain DNA sequences. In particular, we have found three motifs that are selected, GAG, ACA, and CTGC. To elucidate how the discrimination occurs, we determined the crystal structures of two murine TREX1 complexes, with a nucleotide product of the exonuclease reaction, and with a single-stranded DNA substrate. Using confocal microscopy, we observed TREX1 both in nuclear and cytoplasmic subcellular compartments. Remarkably, the presence of TREX1 in the nucleus requires the loss of a C-terminal segment, which we named leucine-rich repeat 3. Furthermore, we detected the presence of a conserved proline-rich region on the surface of TREX1. This observation points to interactions with proline-binding domains. The potential interacting motif "PPPVPRPP" does not contain aromatic residues and thus resembles other sequences that select SH3 and/or Group 2 WW domains. By means of nuclear magnetic resonance titration experiments, we show that, indeed, a polyproline peptide derived from the murine TREX1 sequence interacted with the WW2 domain of the elongation transcription factor CA150. Co-immunoprecipitation studies confirmed this interaction with the full-length TREX1 protein, thereby suggesting that TREX1 participates in more functional complexes than previously thought.

  5. Allotides: Proline-Rich Cystine Knot α-Amylase Inhibitors from Allamanda cathartica.

    PubMed

    Nguyen, Phuong Q T; Luu, Thuy T; Bai, Yang; Nguyen, Giang K T; Pervushin, Konstantin; Tam, James P

    2015-04-24

    Cystine knot α-amylase inhibitors belong to a knottin family of peptidyl inhibitors of 30-32 residues and contain two to four prolines. Thus far, only four members of the group of cystine knot α-amylase inhibitors have been characterized. Herein, the discovery and characterization of five cystine knot α-amylase inhibitors, allotides C1-C5 (Ac1-Ac5) (1-5), from the medicinal plant Allamanda cathartica are reported using both proteomic and genomic methods. Proteomic analysis showed that 1-5 are 30 amino acids in length with three or four proline residues. NMR determination of 4 revealed that it has two cis- and one trans-proline residues and adopts two equally populated conformations in solution. Determination of disulfide connectivity of 2 by differential S-reduction and S-alkylation provided clues of its unfolding process. Genomic analysis showed that allotide precursors contain a three-domain arrangement commonly found in plant cystine knot peptides with conserved residues flanking the processing sites of the mature allotide domain. This work expands the number of known cystine knot α-amylase inhibitors and furthers the understanding of both the structural and biological diversity of this type of knottin family.

  6. Antagonistic Effect of a Salivary Proline-Rich Peptide on the Cytosolic Ca2+ Mobilization Induced by Progesterone in Oral Squamous Cancer Cells.

    PubMed

    Palmerini, Carlo Alberto; Mazzoni, Michela; Radicioni, Giorgia; Marzano, Valeria; Granieri, Letizia; Iavarone, Federica; Longhi, Renato; Messana, Irene; Cabras, Tiziana; Sanna, Maria Teresa; Castagnola, Massimo; Vitali, Alberto

    2016-01-01

    A salivary proline-rich peptide of 1932 Da showed a dose-dependent antagonistic effect on the cytosolic Ca2+ mobilization induced by progesterone in a tongue squamous carcinoma cell line. Structure-activity studies showed that the activity of the peptide resides in the C-terminal region characterized by a proline stretch flanked by basic residues. Furthermore, lack of activity of the retro-inverso peptide analogue suggested the involvement of stereospecific recognition. Mass spectrometry-based shotgun analysis, combined with Western blotting tests and biochemical data obtained with the Progesterone Receptor Membrane Component 1 (PGRMC1) inhibitor AG205, showed strong evidence that p1932 performs its modulatory action through an interaction with the progesterone receptor PGRMC1, which is predominantly expressed in this cell line and, clearly, plays a role in progesterone induced Ca2+ response. Thus, our results point to p1932 as a modulator of the transduction signal pathway mediated by this protein and, given a well-established involvement of PGRMC1 in tumorigenesis, highlight a possible therapeutic potential of p1932 for the treatment of oral cancer.

  7. Antagonistic Effect of a Salivary Proline-Rich Peptide on the Cytosolic Ca2+ Mobilization Induced by Progesterone in Oral Squamous Cancer Cells

    PubMed Central

    Palmerini, Carlo Alberto; Mazzoni, Michela; Radicioni, Giorgia; Marzano, Valeria; Granieri, Letizia; Iavarone, Federica; Longhi, Renato; Messana, Irene; Cabras, Tiziana; Sanna, Maria Teresa; Castagnola, Massimo; Vitali, Alberto

    2016-01-01

    A salivary proline-rich peptide of 1932 Da showed a dose-dependent antagonistic effect on the cytosolic Ca2+ mobilization induced by progesterone in a tongue squamous carcinoma cell line. Structure-activity studies showed that the activity of the peptide resides in the C-terminal region characterized by a proline stretch flanked by basic residues. Furthermore, lack of activity of the retro-inverso peptide analogue suggested the involvement of stereospecific recognition. Mass spectrometry-based shotgun analysis, combined with Western blotting tests and biochemical data obtained with the Progesterone Receptor Membrane Component 1 (PGRMC1) inhibitor AG205, showed strong evidence that p1932 performs its modulatory action through an interaction with the progesterone receptor PGRMC1, which is predominantly expressed in this cell line and, clearly, plays a role in progesterone induced Ca2+ response. Thus, our results point to p1932 as a modulator of the transduction signal pathway mediated by this protein and, given a well-established involvement of PGRMC1 in tumorigenesis, highlight a possible therapeutic potential of p1932 for the treatment of oral cancer. PMID:26814504

  8. Importance of the proline-rich multimerization domain on the oligomerization and nucleic acid binding properties of HIV-1 Vif.

    PubMed

    Bernacchi, Serena; Mercenne, Gaëlle; Tournaire, Clémence; Marquet, Roland; Paillart, Jean-Christophe

    2011-03-01

    The HIV-1 viral infectivity factor (Vif) is required for productive infection of non-permissive cells, including most natural HIV-1 targets, where it counteracts the antiviral activities of the cellular cytosine deaminases APOBEC-3G (A3G) and A3F. Vif is a multimeric protein and the conserved proline-rich domain (161)PPLP(164) regulating Vif oligomerization is crucial for its function and viral infectivity. Here, we expressed and purified wild-type Vif and a mutant protein in which alanines were substituted for the proline residues of the (161)PPLP(164) domain. Using dynamic light scattering, circular dichroism and fluorescence spectroscopy, we established the impact of these mutations on Vif oligomerization, secondary structure content and nucleic acids binding properties. In vitro, wild-type Vif formed oligomers of five to nine proteins, while Vif AALA formed dimers and/or trimers. Up to 40% of the unbound wild-type Vif protein appeared to be unfolded, but binding to the HIV-1 TAR apical loop promoted formation of β-sheets. Interestingly, alanine substitutions did not significantly affect the secondary structure of Vif, but they diminished its binding affinity and specificity for nucleic acids. Dynamic light scattering showed that Vif oligomerization, and interaction with folding-promoting nucleic acids, favor formation of high molecular mass complexes. These properties could be important for Vif functions involving RNAs.

  9. Bio-mimicking of proline-rich motif applied to carbon nanotube reveals unexpected subtleties underlying nanoparticle functionalization.

    PubMed

    Zhang, Yuanzhao; Jimenez-Cruz, Camilo A; Wang, Jian; Zhou, Bo; Yang, Zaixing; Zhou, Ruhong

    2014-11-27

    Here, we report computational studies of the SH3 protein domain interacting with various single-walled carbon nanotubes (SWCNT) either bare or functionalized by mimicking the proline-rich motif (PRM) ligand (PPPVPPRR) and compare it to the SH3-PRM complex binding. With prolines or a single arginine attached, the SWCNT gained slightly on specificity when compared with the bare control, whereas with multi-arginine systems the specificity dropped dramatically to our surprise. Although the electrostatic interaction provided by arginines is crucial in the recognition between PRM and SH3 domain, our results suggest that attaching multiple arginines to the SWCNT has a detrimental effect on the binding affinity. Detailed analysis of the MD trajectories found two main factors that modulate the specificity of the binding: the existence of competing acidic patches at the surface of SH3 that leads to "trapping and clamping" by the arginines, and the rigidity of the SWCNT introducing entropic penalties in the proper binding. Further investigation revealed that the same "clamping" phenomenon exits in the PRM-SH3 system, which has not been reported in previous literature. The competing effects between nanoparticle and its functionalization components revealed by our model system should be of value to current and future nanomedicine designs.

  10. Bio-mimicking of Proline-Rich Motif Applied to Carbon Nanotube Reveals Unexpected Subtleties Underlying Nanoparticle Functionalization

    NASA Astrophysics Data System (ADS)

    Zhang, Yuanzhao; Jimenez-Cruz, Camilo A.; Wang, Jian; Zhou, Bo; Yang, Zaixing; Zhou, Ruhong

    2014-11-01

    Here, we report computational studies of the SH3 protein domain interacting with various single-walled carbon nanotubes (SWCNT) either bare or functionalized by mimicking the proline-rich motif (PRM) ligand (PPPVPPRR) and compare it to the SH3-PRM complex binding. With prolines or a single arginine attached, the SWCNT gained slightly on specificity when compared with the bare control, whereas with multi-arginine systems the specificity dropped dramatically to our surprise. Although the electrostatic interaction provided by arginines is crucial in the recognition between PRM and SH3 domain, our results suggest that attaching multiple arginines to the SWCNT has a detrimental effect on the binding affinity. Detailed analysis of the MD trajectories found two main factors that modulate the specificity of the binding: the existence of competing acidic patches at the surface of SH3 that leads to ``trapping and clamping'' by the arginines, and the rigidity of the SWCNT introducing entropic penalties in the proper binding. Further investigation revealed that the same ``clamping'' phenomenon exits in the PRM-SH3 system, which has not been reported in previous literature. The competing effects between nanoparticle and its functionalization components revealed by our model system should be of value to current and future nanomedicine designs.

  11. The epidermis of scales in gecko lizards contains multiple forms of beta-keratins including basic glycine-proline-serine-rich proteins.

    PubMed

    Toni, M; Dalla Valle, L; Alibardi, L

    2007-05-01

    The epidermis of scales of gecko lizards comprises alpha- and beta-keratins. Using bidimensional electrophoresis and immunoblotting, we have characterized keratins of corneous layers of scales in geckos, especially beta-keratins in digit pad lamellae. In the latter, the formation of thin bristles (setae) allow for the adhesion and climbing vertical or inverted surfaces. alpha-Keratins of 55-66 kDa remain in the acidic and neutral range of pI, while beta-keratins of 13-18 kDa show a broader variation of pI (4-10). Some protein spots for beta-keratins correspond to previously sequenced, basic glycine-proline-serine-rich beta-keratins of 169-191 amino acids. The predicted secondary structure shows that a large part of the molecule has a random-coiled conformation, small alpha helix regions, and a central region with 2-3 strands (beta-folding). The latter, termed core-box, shows homology with feather-scale-claw keratins of birds and is involved in the formation of beta-keratin filaments. Immunolocalization of beta-keratins indicates that these proteins are mainly present in the beta-layer and oberhautchen layer, including setae. The sequenced proteins of setae form bundles of keratins that determine their elongation. This process resembles that of feather-keratin on the elongation of barbule cells in feathers. It is suggested that small proteins rich in glycine, serine, and proline evolved in reptiles and birds to reinforce the mechanical resistance of the cytokeratin cytoskeleton initially present in the epidermis of scales and feathers.

  12. N- and O-linked glycosylation site profiling of the human basic salivary proline-rich protein 3M.

    PubMed

    Manconi, Barbara; Cabras, Tiziana; Sanna, Monica; Piras, Valentina; Liori, Barbara; Pisano, Elisabetta; Iavarone, Federica; Vincenzoni, Federica; Cordaro, Massimo; Faa, Gavino; Castagnola, Massimo; Messana, Irene

    2016-05-01

    In the present study, we show that the heterogeneous mixture of glycoforms of the basic salivary proline-rich protein 3M, encoded by PRB3-M locus, is a major component of the acidic soluble fraction of human whole saliva in the first years of life. Reversed-phase high-performance liquid chromatography with high-resolution electrospray ionization mass spectrometry analysis of the intact proteoforms before and after N-deglycosylation with Peptide-N-Glycosidase F and tandem mass spectrometry sequencing of peptides obtained after Endoproteinase GluC digestion allowed the structural characterization of the peptide backbone and identification of N- and O-glycosylation sites. The heterogeneous mixture of the proteoforms derives from the combination of 8 different neutral and sialylated glycans O-linked to Threonine 50, and 33 different glycans N-linked to Asparagine residues at positions 66, 87, 108, 129, 150, 171, 192, and 213.

  13. A Novel Topology of Proline-rich Transmembrane Protein 2 (PRRT2): HINTS FOR AN INTRACELLULAR FUNCTION AT THE SYNAPSE.

    PubMed

    Rossi, Pia; Sterlini, Bruno; Castroflorio, Enrico; Marte, Antonella; Onofri, Franco; Valtorta, Flavia; Maragliano, Luca; Corradi, Anna; Benfenati, Fabio

    2016-03-18

    Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including epilepsy, paroxysmal movement disorders, and migraine. On the basis of topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the two-transmembrane domain topology with a large N terminus and short C terminus oriented toward the outside of the cell. Because PRRT2 plays a role at the synapse, it is important to confirm the exact orientation of its N and C termini with respect to the plasma membrane to get clues regarding its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein in which only the second hydrophobic segment spans the plasma membrane, whereas the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space but is localized intracellularly, and only the short C terminus is extracellular (N cyt/C exo topology). Accordingly, we show that PRRT2 interacts with the Src homology 3 domain-bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to the clarification of the role of PRRT2 at the synapse and the understanding of pathogenic mechanisms on the basis of PRRT2-related neurological disorders.

  14. The role of proline substitutions within flexible regions on thermostability of luciferase.

    PubMed

    Yu, Haoran; Zhao, Yang; Guo, Chao; Gan, Yiru; Huang, He

    2015-01-01

    Improving the stability of firefly luciferase has been a critical issue for its wider industrial applications. Studies about hyperthermophile proteins show that flexibility could be an effective indicator to find out weak spots to engineering thermostability of proteins. However, the relationship among flexibility, activity and stability in most of proteins is unclear. Proline is the most rigid residue and can be introduced to rigidify flexible regions to enhance thermostability of proteins. We firstly apply three different methods, molecular dynamics (MD) simulation, B-FITTER and framework rigidity optimized dynamics algorithm (FRODA) to determine the flexible regions of Photinus pyralis luciferase: Fragment 197-207; Fragment 471-481 and Fragment 487-495. Then, introduction of proline is used to rigidify these flexible regions. Two mutants D476P and H489P within most flexible regions are finally designed. In the results, H489P mutant shows improved thermostability while maintaining its catalytic efficiency compared to that of wild type luciferase. Flexibility analysis confirms that the overall rigidity and local rigidity of H489P mutant are greatly strengthened. D476P mutant shows decreased thermosatbility and the reason for this is elucidated at the molecular level. S307P mutation is randomly chosen outside the flexible regions as a control. Thermostability analysis shows that S307P mutation has decreased kinetic stability and enhanced thermodynamic stability.

  15. The mouse proline-rich protein MP6 promoter binds isoprenaline-inducible parotid nuclear proteins via a highly conserved NFkB/rel-like site.

    PubMed

    Roberts, S G; Layfield, R; McDonald, C J

    1991-10-11

    Proline-rich protein (PRP) gene MP6 was isolated from a mouse BALB/c genomic DNA library in lambda EMBL3, characterised by hybridisation and restriction mapping and the promoter region, from -162 to +72 around the PRP consensus cap-site, was sequenced. In gel shift assays this region formed complexes C1 and C2 with parotid nuclear proteins which were induced by the beta-adrenergic agonist isoprenaline. DNA competition studies and direct binding assays of promoter subfragments showed that it was the sequence from -157 to -91 that was forming the isoprenaline-dependent complexes. All PRP genes conserve a 23bp. sequence, termed PRP Box1, with ets and NFkB/rel binding site-like elements, upstream of their promoters. In the MP6 promoter, PRP Box1 was within the region forming the complexes. Further gel shift assays using PRP Box1 oligonucleotides as competitors and targets indicated that the NFkB/rel binding site-like element was important in formation of the isoprenaline-inducible complexes. HeLa nuclear extracts also formed complexes with PRP Box1 similar to C1 and C2 but nuclear extracts from spleen, submandibular gland and liver did not. These complexes are thus candidate regulators for the isoprenaline-dependent and tissue-specific transcription of PRP genes.

  16. Ginkgotides: Proline-Rich Hevein-Like Peptides from Gymnosperm Ginkgo biloba

    PubMed Central

    Wong, Ka H.; Tan, Wei Liang; Serra, Aida; Xiao, Tianshu; Sze, Siu Kwan; Yang, Daiwen; Tam, James P.

    2016-01-01

    Hevein and hevein-like peptides belong to the family of chitin-binding cysteine-rich peptides. They are classified into three subfamilies, the prototypic 8C- and the 6C- and 10C-hevein-like peptides. Thus far, only five 8C-hevein-like peptides have been characterized from three angiosperms and none from gymnosperm. To determine their occurrence and distribution in the gymnosperm, Ginkgo biloba leaves were examined. Here, we report the discovery and characterization of 11 novel 8C-hevein-like peptides, namely ginkgotides gB1–gB11. Proteomic analysis showed that the ginkgotides contain 41–44 amino acids (aa), a chitin-binding domain and are Pro-rich, a distinguishing feature that differs from other hevein-like peptides. Solution NMR structure determination revealed that gB5 contains a three β-stranded structure shaped by a cystine knot with an additional disulfide bond at the C-terminus. Transcriptomic analysis showed that the ginkgotide precursors contain a three-domain architecture, comprised of a C-terminal tail (20 aa) that is significantly shorter than those of other 8C- and 10C-hevein-like peptides, which generally contain a protein cargo such as a Barwin-like protein (126 aa) or class I chitinase (254 aa). Transcriptomic data mining found an additional 48 ginkgotide homologs in 39 different gymnosperms. Phylogenetic analysis revealed that ginkgotides and their homologs belong to a new class of 8C-hevein-like peptides. Stability studies showed that ginkgotides are highly resistant to thermal, acidic and endopeptidase degradation. Ginkgotides flanked at both the N- and C-terminal ends by Pro were resistant to exopeptidase degradation by carboxypeptidase A and aminopeptidase. Antifungal assays showed that ginkgotides inhibit the hyphal growth of phyto-pathogenic fungi. Taken together, ginkgotides represent the first suite of hevein-like peptides isolated and characterized from gymnosperms. As a group, they represent a novel class of 8C-hevein-like peptides

  17. Role of Interfacial Water Molecules in Proline-rich Ligand Recognition by the Src Homology 3 Domain of Abl*

    PubMed Central

    Palencia, Andres; Camara-Artigas, Ana; Pisabarro, M. Teresa; Martinez, Jose C.; Luque, Irene

    2010-01-01

    The interaction of Abl-Src homology 3 domain (SH3) with the high affinity peptide p41 is the most notable example of the inconsistency existing between the currently accepted description of SH3 complexes and their binding thermodynamic signature. We had previously hypothesized that the presence of interfacial water molecules is partially responsible for this thermodynamic behavior. We present here a thermodynamic, structural, and molecular dynamics simulation study of the interaction of p41 with Abl-SH3 and a set of mutants designed to alter the water-mediated interaction network. Our results provide a detailed description of the dynamic properties of the interfacial water molecules and a molecular interpretation of the thermodynamic effects elicited by the mutations in terms of the modulation of the water-mediated hydrogen bond network. In the light of these results, a new dual binding mechanism is proposed that provides a better description of proline-rich ligand recognition by Abl-SH3 and that has important implications for rational design. PMID:19906645

  18. Copper(II) interaction with peptide fragments of histidine-proline-rich glycoprotein: Speciation, stability and binding details.

    PubMed

    La Mendola, Diego; Magrì, Antonio; Santoro, Anna Maria; Nicoletti, Vincenzo G; Rizzarelli, Enrico

    2012-06-01

    GHHPH is the peptide repeat present in histidine-proline rich glycoprotein (HPRG), a plasma glycoprotein involved in angiogenesis process. The copper(II) ions interaction with mono (Ac-GHHPHG-NH(2)) and its bis-repeat (Ac-GHHPHGHHPHG-NH(2)) was investigated by means of potentiometric and spectroscopic techniques. To single out the copper(II) coordination environments of different species formed with Ac-GHHPHG-NH(2), three single point mutated peptides were also synthesized and their ability to coordinate Cu(2+) investigated. Ac-GHHPHG-NH(2) binds Cu(2+) by the imidazole side chain and the amide nitrogen deprotonation that takes place towards the N-terminus. The bis-repeat is able to bind Cu(2+) more efficiently than Ac-GHHPHG-NH(2). This difference is not only due to the number of His residues in the sequence but also to the different binding sites. In fact, the comparison of the potentiometric and spectroscopic data of the copper(II) complexes with a bis-repeatPeg construct Ac-(GHHPHG)-Peg-(GHHPHG)-NH(2) and those of the metal complexes with Ac-HGHH-NH(2), indicates that the central HGHH amino acid sequence is the main copper(II) binding site.

  19. Cell-cycle specific expression of a small proline-rich protein in Chinese hamster ovary cells

    SciTech Connect

    Tesfaigzi, J.

    1994-11-01

    Squamous metaplasia of the bronchial epithelium is generally believed to be involved in the neoplastic progression toward squamous cell carcinomas. Thus, it is important to understand the mechanisms controlling this type of differentiation. The induction of two families of cDNAs encoding a small proline-rich protein (sPRP), sprI and sprII, was first identified in human keratinocytes exhibiting squamous differentiation. cDNAs similar to sprI have also been identified in cultured tracheal epithelial cells undergoing squamous differentiation. The first step during the squamous differentiation process is the inhibition of cell growth; it has also been noted that a sPRP mRNA in Chinese hamster ovary (CHO) cells is induced 10-fold just before the cultures reach confluence. Thus, sPRP may stop cell division in cells undergoing squamous differentation. In support of this possibility are the recent investigations correlating expression of sPRP with cell morphology. Specific immunoreactivity to sPRP, using affinity-purified antibodies, showed a strong immunostaining in cells with a round configuration, while less staining was observed in other cells. The major part of the CHO population showed no immunoreactivity. One interpretation of this observation is that the expression of sPRP may be cell-cyle regulated. The purpose of this investigation was to determine the phase of the cell cycle where induced synthesis of sPRP mRNA occurs.

  20. The Proline-Rich Sequence of CD3ε as an amplifier of low-avidity TCR signaling

    PubMed Central

    Tailor, Pankaj; Tsai, Sue; Shameli, Afshin; Serra, Pau; Wang, Jinguo; Robbins, Stephen; Nagata, Masao; Szymczak-Workman, Andrea L.; Vignali, Dario A. A.; Santamaria, Pere

    2009-01-01

    Engagement of peptide-MHC (pMHC) by the T-cell receptor induces a conformational change in CD3ε that exposes a proline-rich sequence (PRS) and recruits the cytoskeletal adaptor Nck. This event, which precedes phosphorylation of the CD3ε ITAM, has been implicated in synapse formation and T-cell function. However, there is compelling evidence that responsiveness to TCR ligation is CD3ε PRS-independent. Here we show that the CD3ε PRS is necessary for pMHC-induced phosphorylation of CD3ε and for recruitment of PKCθ to the immune synapse in differentiated CD8+ T-lymphocytes. However, whereas these two events are dispensable for functional T-cell responsiveness to high-avidity ligands, they are required for responsiveness to low-avidity ones. Thus, in at least certain T-cell clonotypes, the CD3ε PRS amplifies weak TCR signals by promoting synapse formation and CD3ε phosphorylation. PMID:18566390

  1. Detection of proline-rich proteins for the identification of saliva by enzyme-linked immunosorbent assay.

    PubMed

    Igoh, Akihisa; Tomotake, Sho; Doi, Yusuke

    2015-05-01

    Saliva is one of the most common body fluids found at a crime scene. Therefore, identifying saliva is important in forensic science. However, the current protein marker assays used to identify saliva are not sufficiently specific. Although proline-rich proteins (PRPs) are highly specific for saliva, their forensic potential has not yet been investigated. In this study, we developed enzyme-linked immunosorbent assays (ELISAs) to detect acidic salivary PRP HaeIII subfamily 1/2 (PRH1/2) and basic salivary PRP 2 (PRB2). The specificity, sensitivity, and efficiency of the ELISAs for PRH1/2 and PRB2 were compared with those of the ELISA for statherin (STATH), a known protein marker for saliva. The levels of PRH1/2 were significantly higher in saliva and saliva stains than in other body fluids (nasal secretions, urine, semen, vaginal fluid, blood, and sweat). PRB2 and STATH were detected in both nasal secretions and saliva. The PRH1/2 ELISA showed sensitivity similar to that of STATH ELISA. The detection rate of PRH1/2 ELISA was almost similar to that of STATH ELISA, followed by the ELISA for PRB2. The PRH1/2 ELISA had higher specificity for saliva than STATH ELISA. Therefore, the PRH1/2 ELISA has potential as a method to identify saliva for forensic investigation.

  2. Structures of proline-rich peptides bound to the ribosome reveal a common mechanism of protein synthesis inhibition

    PubMed Central

    Gagnon, Matthieu G.; Roy, Raktim N.; Lomakin, Ivan B.; Florin, Tanja; Mankin, Alexander S.; Steitz, Thomas A.

    2016-01-01

    With bacterial resistance becoming a serious threat to global public health, antimicrobial peptides (AMPs) have become a promising area of focus in antibiotic research. AMPs are derived from a diverse range of species, from prokaryotes to humans, with a mechanism of action that often involves disruption of the bacterial cell membrane. Proline-rich antimicrobial peptides (PrAMPs) are instead actively transported inside the bacterial cell where they bind and inactivate specific targets. Recently, it was reported that some PrAMPs, such as Bac71–35, oncocins and apidaecins, bind and inactivate the bacterial ribosome. Here we report the crystal structures of Bac71–35, Pyrrhocoricin, Metalnikowin and two oncocin derivatives, bound to the Thermus thermophilus 70S ribosome. Each of the PrAMPs blocks the peptide exit tunnel of the ribosome by simultaneously occupying three well characterized antibiotic-binding sites and interferes with the initiation step of translation, thereby revealing a common mechanism of action used by these PrAMPs to inactivate protein synthesis. Our study expands the repertoire of PrAMPs and provides a framework for designing new-generation therapeutics. PMID:26809677

  3. Pigeonpea Hybrid-Proline-Rich Protein (CcHyPRP) Confers Biotic and Abiotic Stress Tolerance in Transgenic Rice

    PubMed Central

    Mellacheruvu, Sunitha; Tamirisa, Srinath; Vudem, Dashavantha Reddy; Khareedu, Venkateswara Rao

    2016-01-01

    In this study, we report the overexpression of Cajanus cajan hybrid-proline-rich protein encoding gene (CcHyPRP) in rice which resulted in increased tolerance to both abiotic and biotic stresses. Compared to the control plants, the transgenic rice lines, expressing CcHyPRP, exhibited high-level tolerance against major abiotic stresses, viz., drought, salinity, and heat, as evidenced by increased biomass, chlorophyll content, survival rate, root, and shoot growth. Further, transgenic rice lines showed increased panicle size and grain number compared to the control plants under different stress conditions. The CcHyPRP transgenics, as compared to the control, revealed enhanced activities of catalase and superoxide dismutase (SOD) enzymes and reduced malondialdehyde (MDA) levels. Expression pattern of CcHyPRP::GFP fusion-protein confirmed its predominant localization in cell walls. Moreover, the CcHyPRP transgenics, as compared to the control, exhibited increased resistance to the fungal pathogen Magnaporthe grisea which causes blast disease in rice. Higher levels of bZIP and endochitinase transcripts as well as endochitinase activity were observed in transgenic rice compared to the control plants. The overall results demonstrate the intrinsic role of CcHyPRP in conferring multiple stress tolerance at the whole-plant level. The multipotent CcHyPRP seems promising as a prime candidate gene to fortify crop plants for enhanced tolerance/resistance to different stress factors. PMID:26834756

  4. Structures of proline-rich peptides bound to the ribosome reveal a common mechanism of protein synthesis inhibition

    DOE PAGES

    Gagnon, Matthieu G.; Roy, Raktim N.; Lomakin, Ivan B.; ...

    2016-01-24

    Here, with bacterial resistance becoming a serious threat to global public health, antimicrobial peptides (AMPs) have become a promising area of focus in antibiotic research. AMPs are derived from a diverse range of species, from prokaryotes to humans, with a mechanism of action that often involves disruption of the bacterial cell membrane. Proline-rich antimicrobial peptides (PrAMPs) are instead actively transported inside the bacterial cell where they bind and inactivate specific targets. Recently, it was reported that some PrAMPs, such as Bac71–35, oncocins and apidaecins, bind and inactivate the bacterial ribosome. Here we report the crystal structures of Bac71–35, Pyrrhocoricin, Metalnikowinmore » and two oncocin derivatives, bound to the Thermus thermophilus 70S ribosome. Each of the PrAMPs blocks the peptide exit tunnel of the ribosome by simultaneously occupying three well characterized antibioticbinding sites and interferes with the initiation step of translation, thereby revealing a common mechanism of action used by these PrAMPs to inactivate protein synthesis. Our study expands the repertoire of PrAMPs and provides a framework for designing new-generation therapeutics.« less

  5. Structures of proline-rich peptides bound to the ribosome reveal a common mechanism of protein synthesis inhibition

    SciTech Connect

    Gagnon, Matthieu G.; Roy, Raktim N.; Lomakin, Ivan B.; Florin, Tanja; Mankin, Alexander S.; Steitz, Thomas A.

    2016-01-24

    Here, with bacterial resistance becoming a serious threat to global public health, antimicrobial peptides (AMPs) have become a promising area of focus in antibiotic research. AMPs are derived from a diverse range of species, from prokaryotes to humans, with a mechanism of action that often involves disruption of the bacterial cell membrane. Proline-rich antimicrobial peptides (PrAMPs) are instead actively transported inside the bacterial cell where they bind and inactivate specific targets. Recently, it was reported that some PrAMPs, such as Bac71–35, oncocins and apidaecins, bind and inactivate the bacterial ribosome. Here we report the crystal structures of Bac71–35, Pyrrhocoricin, Metalnikowin and two oncocin derivatives, bound to the Thermus thermophilus 70S ribosome. Each of the PrAMPs blocks the peptide exit tunnel of the ribosome by simultaneously occupying three well characterized antibioticbinding sites and interferes with the initiation step of translation, thereby revealing a common mechanism of action used by these PrAMPs to inactivate protein synthesis. Our study expands the repertoire of PrAMPs and provides a framework for designing new-generation therapeutics.

  6. The Proline Rich Homeodomain Protein PRH/Hhex Forms Stable Oligomers That Are Highly Resistant to Denaturation

    PubMed Central

    Shukla, Anshuman; Burton, Nicholas M.; Jayaraman, Padma-Sheela; Gaston, Kevin

    2012-01-01

    Background Many transcription factors control gene expression by binding to specific DNA sequences at or near the genes that they regulate. However, some transcription factors play more global roles in the control of gene expression by altering the architecture of sections of chromatin or even the whole genome. The ability to form oligomeric protein assemblies allows many of these proteins to manipulate extensive segments of DNA or chromatin via the formation of structures such as DNA loops or protein-DNA fibres. Principal Findings Here we show that the proline rich homeodomain protein PRH/Hhex forms predominantly octameric and/or hexadecameric species in solution as well as larger assemblies. We show that these assemblies are highly stable resisting denaturation by temperature and chemical denaturants. Conclusion These data indicate that PRH is functionally and structurally related to the Lrp/AsnC family of proteins, a group of proteins that are known to act globally to control gene expression in bacteria and archaea. PMID:22540015

  7. Immunolocalisation and oestrogen regulation of small proline-rich protein 2a protein in the mouse uterus.

    PubMed

    Lee, Hyang-Ah; Kim, Hye-Ryun; Lee, Young Jin; Lee, Seung-Joon; Kim, Woo Jin; Han, Seon-Sook; Yang, Se-Ran; Woo, Heung-Myong; Na, Sunghun; Song, Haengseok; Hong, Seok-Ho

    2014-06-01

    Small proline-rich protein 2a (Sprr2a) is one of the structural components of the cornified keratinocyte cell envelope that contributes to form a protective barrier in the skin against dehydration and environmental stress. Interestingly, Sprr2a mRNA is detected in the mouse uterus and is regulated by 17β-oestradiol (E2). In the present study, we investigated the effects of E2 and oestrogenic compounds on the regulation and localisation of Sprr2a protein in the mouse uterus. Immunohistochemical staining revealed that Sprr2a protein is detected only in the adult uterus, and not in the ovary, oviduct or testis. We also demonstrated that Sprr2a protein is tightly regulated by E2 in the mouse uterus and exclusively detected in luminal and glandular epithelial cells. Furthermore, Sprr2a is dose-dependently induced by oestrogenic compounds such as bisphenol A and 4-tert-octylphenol. Collectively, our studies suggest that Sprr2a protein may have a unique function in physiological events in the mouse uterus and can be used as an indicator to detect compounds with oestrogenic activity in the mouse uterus.

  8. Binding of the Grb2 SH2 domain to phosphotyrosine motifs does not change the affinity of its SH3 domains for Sos proline-rich motifs.

    PubMed

    Cussac, D; Frech, M; Chardin, P

    1994-09-01

    Phosphotyrosine peptide binding to Grb2 induces tryptophan fluorescence changes in the Src homology 2 (SH2) domain. Affinities are in the nanomolar range, the Shc peptide having the highest affinity, followed by peptides mimicking Grb2 binding sites on EGF and HGF receptors, the putative sites on insulin and IGF-1 receptors having much lower affinities. Proline-rich peptide binding to the SH3 domains induces fluorescence changes mainly in the C-terminal SH3. Affinities are in the micromolar range, the highest affinity peptides mimicking the first proline-rich motif of the Sos C-terminus. Additional residues before this PVPPPVPP motif provide a minor contribution to the binding, but the two residues after this motif are important and may contribute to specificity. The affinity of each SH3 for each proline-rich motif is too low to account for the high stability of the Grb2-Sos complex, suggesting that Grb2 recognizes other structural features in the Sos C-terminus. Binding of a phosphotyrosine peptide to the SH2 has no effect on the SH3s. Thus the binding of Grb2 to a receptor or to an associated protein phosphorylated on tyrosines is unlikely to activate the exchange factor activity of Sos through a conformational change transmitted from the SH2 to the SH3 domains.

  9. Proline/arginine-rich end leucine-rich repeat protein N-terminus is a novel osteoclast antagonist that counteracts bone loss.

    PubMed

    Rucci, Nadia; Capulli, Mattia; Ventura, Luca; Angelucci, Adriano; Peruzzi, Barbara; Tillgren, Viveka; Muraca, Maurizio; Heinegård, Dick; Teti, Anna

    2013-09-01

    (hbd) PRELP is a peptide corresponding to the N-terminal heparin binding domain of the matrix protein proline/arginine-rich end leucine-rich repeat protein (PRELP). (hbd) PRELP inhibits osteoclastogenesis entering pre-fusion osteoclasts through a chondroitin sulfate- and annexin 2-dependent mechanism and reducing the nuclear factor-κB transcription factor activity. In this work, we hypothesized that (hbd) PRELP could have a pharmacological relevance, counteracting bone loss in a variety of in vivo models of bone diseases induced by exacerbated osteoclast activity. In healthy mice, we demonstrated that the peptide targeted the bone and increased trabecular bone mass over basal level. In mice treated with retinoic acid to induce an acute increase of osteoclast formation, the peptide consistently antagonized osteoclastogenesis and prevented the increase of the serum levels of the osteoclast-specific marker tartrate-resistant acid phosphatase. In ovariectomized mice, in which osteoclast activity was chronically enhanced by estrogen deficiency, (hbd) PRELP counteracted exacerbated osteoclast activity and bone loss. In mice carrying osteolytic bone metastases, in which osteoclastogenesis and bone resorption were enhanced by tumor cell-derived factors, (hbd) PRELP reduced the incidence of osteolytic lesions, both preventively and curatively, with mechanisms involving impaired tumor cell homing to bone and tumor growth in the bone microenvironment. Interestingly, in tumor-bearing mice, (hbd) PRELP also inhibited breast tumor growth in orthotopic sites and development of metastatic disease in visceral organs, reducing cachexia and improving survival especially when administered preventively. (hbd) PRELP was retained in the tumor tissue and appeared to affect tumor growth by interacting with the microenvironment rather than by directly affecting the tumor cells. Because safety studies and high-dose treatments revealed no adverse effects, (hbd) PRELP could be employed as a

  10. Characterization of human reflex tear proteome reveals high expression of lacrimal proline-rich protein 4 (PRR4).

    PubMed

    Perumal, Natarajan; Funke, Sebastian; Wolters, Dominik; Pfeiffer, Norbert; Grus, Franz H

    2015-10-01

    In-depth studies on the proteome of reflex tears are still inadequate. Hence, further studies on this subject will unravel the key proteins which are conjectured to possess vital functions in the protection of the ocular surface. Therefore, this study investigated the differences in the expression levels in proteome of reflex compared to basal tears. Basal (n = 10) and reflex (n = 10) tear samples from healthy subjects were collected employing the capillary method, subsequently pooled and the proteomes were characterized employing 1DE combined with LC-ESI-MS/MS strategy for label-free quantitative (LFQ) analysis. The differentially expressed proteins were validated by 2DE combined with LC-ESI-MS/MS and targeted-MS approach called accurate inclusion mass screening (AIMS) strategies. The analysis of the reflex tear proteome demonstrated increased abundance in proline-rich protein 4 (PRR4) and zymogen granule protein 16 homolog B (ZG16B) for the first time. Other abundant lacrimal proteins, e.g. lactotransferrin and lysozyme remained constant. Predominantly, the lacrimal gland-specific PRR4 represents the major increased protein in reflex tears in an attempt to wash out irritants that come into contact with the eye. Conversely, decreased abundance in Ig alpha-1 chain C, polymeric immunoglobulin receptor, cystatin S/SN, clusterin and mammaglobin were observed. This study had further unraveled the intricate proteome regulation during reflex tearing, especially the potential role of PRR4, which may be the key player in the protection and maintenance of dynamic balance of the ocular surface.

  11. S-Nitrosylation of proline-rich tyrosine kinase 2 involves its activation induced by oxygen-glucose deprivation.

    PubMed

    Yan, Xian-Liang; Liu, Dong-Hai; Zhang, Gong-Liang; Hu, Shu-Qun; Chen, Yu-Guo; Xu, Tie

    2015-06-15

    Previous studies have demonstrated that activation of proline-rich tyrosine kinase 2 (PYK2) in cerebral ischemia is involved in the modulation of N-methyl-d-aspartate-type (NMDA) glutamate receptor activity and Ca(2+) dynamics, resulting in ischemic neuron death ultimately. A number of reports indicate that PYK2 is a redox sensitive kinase that must be activated by an estrogen-induced reactive oxygen species (ROS). However, the mechanism of PYK2 activation remains incompletely illustrated. Accumulating attention is focused on nitric oxide (NO, a free radical) which plays a critical role in cellular signal transduction through stimulus-coupled S-nitrosylation of cysteine residues. Here we reported that PYK2 over-expressed in human embryonic kidney (HEK293) cells was S-nitrosylated (forming SNO-PYK2) by reacting with GSNO, an exogenous NO donor, at one critical cysteine residue (Cys534) with a biotin switch assay. Moreover, our results showed that S-nitrosylation and phosphorylation of PYK2 over-expressed in SH-SY5Y cells was significantly increased after oxygen-glucose deprivation (OGD). We further investigated whether the activation (phosphorylation) of PYK2 was associated with S-nitrosylation following SH-SY5Y cells OGD. Our results showed that the cysteine534 residue (site of S-nitrosylation) mutant PYK2 over-expressed in SH-SY5Y cells diminished S-nitrosylation of PYK2 and inhibited its phosphorylation induced by OGD. In addition, overexpression of the mutant PYK2 protein could prevent nuclear accumulation and abrogate neuronal cell death compared to wild type PYK2 in SH-SY5Y cells induced by OGD. These data suggest that the activation of PYK2 following OGD may be modulated by S-nitrosylation, which provides a new avenue for stroke therapy by targeting the post-translational modification machinery.

  12. cDNA cloning and chromosomal mapping of a predicted coiled-coil proline-rich protein immunogenic in meningioma patients.

    PubMed

    Heckel, D; Brass, N; Fischer, U; Blin, N; Steudel, I; Türeci, O; Fackler, O; Zang, K D; Meese, E

    1997-11-01

    There is increasing evidence that tumor expressed genes induce immune responses in cancer patients. To identify meningioma expressed antigens, we established a meningioma expression library which was screened with autologous serum. Out of 20 positive cDNA clones eight share high sequence homologies as determined by sequence analysis. These eight clones can be grouped into three classes which differ in length and which are characterized by specific sequence variations. The longest open reading frame was found to be 2412 bp encoding an immunoreactive antigen termed meningioma expressed antigen 6 (MEA6). Using five sequence specific primer pairs, somatic hybrid panel mapping revealed locations of the three classes on several human chromosomes including chromosomes 2, 3, 6, 7, 9, 13 and 14. The mapping results were confirmed by fluorescence in situ hybridization. RT-PCR showed consistent expression of all classes in several meningiomas and additional tissues using the same set of primer pairs as for chromosomal mapping. The expression data were confirmed by northern blot analysis. For the predicted amino acid sequence BLASTX revealed a homology to a human C219-reactive peptide which was previously isolated by an antibody directed against p-glycoprotein. Sequence properties of the MEA protein include an acidic activation domain, a proline-rich region and two coiled-coil domains indicating protein binding and activation functions.

  13. Intracellular Toxicity of Proline-Rich Antimicrobial Peptides Shuttled into Mammalian Cells by the Cell-Penetrating Peptide Penetratin

    PubMed Central

    Hansen, Anne; Schäfer, Ingo; Knappe, Daniel; Seibel, Peter

    2012-01-01

    The health threat caused by multiresistant bacteria has continuously increased and recently peaked with pathogens resistant to all current drugs. This has triggered intense research efforts to develop novel compounds to overcome the resistance mechanisms. Thus, antimicrobial peptides (AMPs) have been intensively studied, especially the family of proline-rich AMPs (PrAMPs) that was successfully tested very recently in murine infection models. PrAMPs enter bacteria and inhibit chaperone DnaK. Here, we studied the toxicity of intracellular PrAMPs in HeLa and SH-SY5Y cells. As PrAMPs cannot enter most mammalian cells, we coupled the PrAMPs with penetratin (residues 43 to 58 in the antennapedia homeodomain) via a C-terminally added cysteine utilizing a thioether bridge. The resulting construct could transport the covalently linked PrAMP into mammalian cells. Penetratin ligation reduced the MIC for Gram-negative Escherichia coli only slightly (1 to 8 μmol/liter) but increased the activity against the Gram-positive Micrococcus luteus up to 32-fold (MIC ≈ 1 μmol/liter), most likely due to more effective penetration through the bacterial membrane. In contrast to native PrAMPs, the penetratin-PrAMP constructs entered the mammalian cells, aligned around the nucleus, and associated with the Golgi apparatus. At higher concentrations, the constructs reduced the cell viability (50% inhibitory concentration [IC50] ≈ 40 μmol/liter) and changed the morphology of the cells. No toxic effects or morphological changes were observed at concentrations of 10 μmol/liter or below. Thus, the IC50 values were around 5 to 40 times higher than the MIC values. In conclusion, PrAMPs are in general not toxic to mammalian cells, as they do not pass through the membrane. When shuttled into mammalian cells, however, PrAMPs are only slightly cross-reactive to mammalian chaperones or other intracellular mammalian proteins, providing a second layer of safety for in vivo applications, even if they

  14. Gene sequence of mouse B-type proline-rich protein MP4. Transcriptional start point and an upstream phylogenetic footprint with ets-like and rel/NFkB-like elements.

    PubMed

    Roberts, S G; Layfield, R; Bannister, A J; McDonald, C J

    1991-12-18

    A mouse genomic B-type proline-rich protein (PRP) cosmid clone was isolated by cDNA hybridisation and mapped, the gene region was subcloned and 3770 bp were sequenced. This gene (MP4) contained three introns and encoded a 1020-nt (nt, nucleotide) mRNA for a PRP precursor 300 amino acids long arranged with 11 imperfect 18-residue proline-rich repeats. The transcriptional start point was determined by S1 nuclease mapping and primer extension to be 26 bp downstream of a TATAA sequence. Sequence comparisons revealed that only two regions from positions -650 bp - -30 bp were highly conserved in all other PRP genes, PRP boxes 1 and 2. Box 1 at positions -112 to -135 contained ets-like and rel/NFkB-like elements and was 74% conserved over 23 bp. Box 2 at positions -33 - -51 was 53% conserved over 19 bp. A search of the EMBL and GenBank sequence libraries indicated that PRP box 1 was only present upstream of the known mammalian PRP gene sequences and was absent from other genes. These conserved sequences may thus be relevant to the tissue-specific and beta-adrenergic regulation of PRP gene transcription.

  15. The Proline-Rich Motif of the proDer p 3 Allergen Propeptide Is Crucial for Protease-Protease Interaction

    PubMed Central

    Dumez, Marie-Eve; Herman, Julie; Campisi, Vincenzo; Bouaziz, Ahlem; Rosu, Frédéric; Luxen, André; Vandenberghe, Isabel; de Pauw, Edwin; Frère, Jean-Marie; Matagne, André; Chevigné, Andy; Galleni, Moreno

    2013-01-01

    The majority of proteases are synthesized in an inactive form, termed zymogen, which consists of a propeptide and a protease domain. The propeptide is commonly involved in the correct folding and specific inhibition of the enzyme. The propeptide of the house dust mite allergen Der p 3, NPILPASPNAT, contains a proline-rich motif (PRM), which is unusual for a trypsin-like protease. By truncating the propeptide or replacing one or all of the prolines in the non-glycosylated zymogen with alanine(s), we demonstrated that the full-length propeptide is not required for correct folding and thermal stability and that the PRM is important for the resistance of proDer p 3 to undesired proteolysis when the protein is expressed in Pichia pastoris. Additionally, we followed the maturation time course of proDer p 3 by coupling a quenched-flow assay to mass spectrometry analysis. This approach allowed to monitor the evolution of the different species and to determine the steady-state kinetic parameters for activation of the zymogen by the major allergen Der p 1. This experiment demonstrated that prolines 5 and 8 are crucial for proDer p 3-Der p 1 interaction and for activation of the zymogen. PMID:24073192

  16. COORDINATED ACTIVATION OF THE RAC-GAP β2-CHIMAERIN BY AN ATYPICAL PROLINE-RICH DOMAIN AND DIACYLGLYCEROL

    PubMed Central

    Gutierrez-Uzquiza, Alvaro; Colon-Gonzalez, Francheska; Leonard, Thomas A.; Canagarajah, Bertram J.; Wang, HongBin; Mayer, Bruce J.; Hurley, James H.; Kazanietz, Marcelo G.

    2013-01-01

    Chimaerins, a family of GTPase activating proteins (GAPs) for the small G-protein Rac, have been implicated in development, neuritogenesis, and cancer. These Rac-GAPs are regulated by the lipid second messenger diacylglycerol (DAG) generated by tyrosine-kinases such as the epidermal growth factor receptor (EGFR). Here we identify an atypical Pro-rich motif in chimaerins that binds to the adaptor protein Nck1. Unlike most Nck1 partners, chimaerins bind to the third SH3 domain of Nck1. This association is mediated by electrostatic interactions of basic residues within the Pro-rich motif with acidic clusters in the SH3 domain. EGF promotes the binding of β2-chimaerin to Nck1 in the cell periphery in a DAG-dependent manner. Moreover, β2-chimaerin translocation to the plasma membrane and its peripheral association with Rac1 requires Nck1. Our studies underscore a coordinated mechanism for β2-chimaerin activation that involves lipid interactions via the C1 domain and protein-protein interactions via the N-terminal Pro-rich region. PMID:23673634

  17. First Total Synthesis and Biological Screening of a Proline-Rich Cyclopeptide from a Caribbean Marine Sponge

    PubMed Central

    Dahiya, Rajiv; Singh, Sunil; Sharma, Ajay; Chennupati, Suresh V.; Maharaj, Sandeep

    2016-01-01

    A natural heptacyclopeptide, stylissamide G (7), previously isolated from the Bahamian marine sponge Stylissa caribica from the Caribbean Sea, was synthesized via coupling of the tetrapeptide l-phenylalanyl-l-prolyl-l-phenylalanyl-l-proline methyl ester with the tripeptide Boc-l-leucyl-l-isoleucyl-l-proline, followed by cyclization of the linear heptapeptide fragment. The structure of the synthesized cyclooligopeptide was confirmed using quantitative elemental analysis, FT-IR, 1H NMR, 13C NMR and mass spectrometry. Results of pharmacological activity studies indicated that the newly synthesized cycloheptapeptide displayed good anthelmintic potential against Megascoplex konkanensis, Pontoscotex corethruses and Eudrilus eugeniea at 2 mg/mL and in addition, potent antifungal activity against pathogenic Candida albicans and dermatophytes Trichophyton mentagrophytes and Microsporum audouinii at a concentration of 6 μg/mL. PMID:27983681

  18. Penultimate proline in neuropeptides.

    PubMed

    Glover, Matthew S; Bellinger, Earl P; Radivojac, Predrag; Clemmer, David E

    2015-08-18

    A recent ion mobility spectrometry-mass spectrometry (IMS-MS) study revealed that tryptic peptide ions containing a proline residue at the second position from the N-terminus (i.e., penultimate proline) frequently adopt multiple conformations, owing to the cis-trans isomerization of Xaa(1)-Pro(2) peptide bonds [J. Am. Soc. Mass Spectrom. 2015, 26, 444]. Here, we present a statistical analysis of a neuropeptide database that illustrates penultimate proline residues are frequently found in neuropeptides. In order to probe the effect of penultimate proline on neuropeptide conformations, IMS-MS experiments were performed on two model peptides in which penultimate proline residues were known to be important for biological activity: the N-terminal region of human neuropeptide Y (NPY1-9, Tyr(1)-Pro(2)-Ser(3)-Lys(4)-Pro(5)-Asp(6)-Asn(7)-Pro(8)-Gly(9)-NH2) and a tachykinin-related peptide (CabTRP Ia, Ala(1)-Pro(2)-Ser(3)-Gly(4)-Phe(5)-Leu(6)-Gly(7)-Met(8)-Arg(9)-NH2). From these studies, it appears that penultimate prolines allow neuropeptides to populate multiple conformations arising from the cis-trans isomerization of Xaa(1)-Pro(2) peptide bonds. Although it is commonly proposed that the role of penultimate proline residues is to protect peptides from enzymatic degradation, the present results indicate that penultimate proline residues also are an important means of increasing the conformational heterogeneity of neuropeptides.

  19. The role of glandular kallikrein in the formation of a salivary proline-rich protein A by cleavage of a single bond in salivary protein C.

    PubMed Central

    Wong, R S; Madapallimattam, G; Bennick, A

    1983-01-01

    An enzyme was purified from human parotid saliva that can cleave a single arginine-glycine peptide bond between residues 106 and 107 in human salivary proline-rich protein C, hereby giving rise to another proline-rich protein A, which is also found in saliva. The enzyme was purified 2400-fold. It cleaved salivary protein C at the rate of 59 micrograms of protein/h per microgram of enzyme and had amino acid composition, molecular weight and inhibition characteristics similar to those reported for human salivary kallikrein. Confirmation that the enzyme was kallikrein was demonstrated by its kinin-generating ability. Histochemical evidence indicates that a post-synthetic cleavage of protein C by kallikrein would have to take place during passage of saliva through the secretory ducts. In secreted saliva, cleavage of salivary protein C can only be observed after 72 h incubation. In addition, there is no effect of salivary flow rate on the relative amounts of proteins A and C in saliva. On the basis of the experimental observations, it is proposed that in vivo it is unlikely that kallikrein secreted from ductal cells plays a significant role in converting protein C into protein A. PMID:6553499

  20. Epigenetic regulation of embryonic stem cell marker miR302C in human chondrosarcoma as determinant of antiproliferative activity of proline-rich polypeptide 1.

    PubMed

    Galoian, Karina; Qureshi, Amir; D'Ippolito, Gianluca; Schiller, Paul C; Molinari, Marco; Johnstone, Andrea L; Brothers, Shaun P; Paz, Ana C; Temple, H T

    2015-08-01

    Metastatic chondrosarcoma of mesenchymal origin is the second most common bone malignancy and does not respond either to chemotherapy or radiation; therefore, the search for new therapies is relevant and urgent. We described recently that tumor growth inhibiting cytostatic proline-rich polypeptide 1, (PRP-1) significantly upregulated tumor suppressor miRNAs, downregulated onco-miRNAs in human chondrosarcoma JJ012 cell line, compared to chondrocytes culture. In this study we hypothesized the existence and regulation of a functional marker in cancer stem cells, correlated to peptides antiproliferative activity. Experimental results indicated that among significantly downregulated miRNA after PRP-1treatment was miRNAs 302c*. This miRNA is a part of the cluster miR302‑367, which is stemness regulator in human embryonic stem cells and in certain tumors, but is not expressed in adult hMSCs and normal tissues. PRP-1 had strong inhibitory effect on viability of chondrosarcoma and multilineage induced multipotent adult cells (embryonic primitive cell type). Unlike chondrosarcoma, in glioblastoma, PRP-1 does not have any inhibitory activity on cell proliferation, because in glioblastoma miR-302-367 cluster plays an opposite role, its expression is sufficient to suppress the stemness inducing properties. The observed correlation between the antiproliferative activity of PRP-1 and its action on downregulation of miR302c explains the peptides opposite effects on the upregulation of proliferation of adult mesenchymal stem cells, and the inhibition of the proliferation of human bone giant-cell tumor stromal cells, reported earlier. PRP-1 substantially downregulated the miR302c targets, the stemness markers Nanog, c-Myc and polycomb protein Bmi-1. miR302c expression is induced by JMJD2-mediated H3K9me2 demethylase activity in its promoter region. JMJD2 was reported to be a positive regulator for Nanog. Our experimental results proved that PRP-1 strongly inhibited H3K9 activity

  1. The aberrant cell walls of boron-deficient bean root nodules have no covalently bound hydroxyproline-/proline-rich proteins.

    PubMed Central

    Bonilla, I; Mergold-Villaseñor, C; Campos, M E; Sánchez, N; Pérez, H; López, L; Castrejón, L; Sánchez, F; Cassab, G I

    1997-01-01

    B-deficient bean (Phaseolus vulgaris L.) nodules examined by light microscopy showed dramatic anatomical changes, mainly in the parenchyma region. Western analysis of total nodule extracts examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that one 116-kD polypeptide was recognized by antibodies raised against hydroxyproline-rich glycoproteins (HRGPs) from the soybean (Glycine max) seed coat. A protein with a comparable molecular mass of 116 kD was purified from the cell walls of soybean root nodules. The amino acid composition of this protein is similar to the early nodulin (ENOD2) gene. Immunoprecipitation of the soybean ENOD2 in vitro translation product showed that the soybean seed coat anti-HRGP antibodies recognized this early nodulin. Furthermore, we used these antibodies to localize the ENOD2 homolog in bean nodules. Immunocytochemistry revealed that in B-deficient nodules ENOD2 was absent in the walls of the nodule parenchyma. The absence of ENOD2 in B-deficient nodules was corroborated by performing hydroxyproline assays. Northern analysis showed that ENOD2 mRNA is present in B-deficient nodules; therefore, the accumulation of ENOD2 is not affected by B deficiency, but its assembly into the cell wall is. B-deficient nodules fix much less N2 than control nodules, probably because the nodule parenchyma is no longer an effective O2 barrier. PMID:9414547

  2. Histidine-proline-rich glycoprotein as a plasma pH sensor. Modulation of its interaction with glycosaminoglycans by ph and metals.

    PubMed

    Borza, D B; Morgan, W T

    1998-03-06

    The middle domain of plasma histidine-proline-rich glycoprotein (HPRG) contains unusual tandem pentapeptide repeats (consensus G(H/P)(H/P)PH) and binds heparin and transition metals. Unlike other proteins that interact with heparin via lysine or arginine residues, HPRG relies exclusively on histidine residues for this interaction. To assess the consequences of this unusual requirement, we have studied the interaction between human plasma HPRG and immobilized glycosaminoglycans (GAGs) using resonant mirror biosensor techniques. HPRG binding to immobilized heparin was strikingly pH-sensitive, producing a titration curve with a midpoint at pH 6.8. There was little binding of HPRG to heparin at physiological pH in the absence of metals, but the interaction was promoted by nanomolar concentrations of free zinc and copper, and its pH dependence was shifted toward alkaline pH by zinc. The affinity of HPRG for various GAGs measured in a competition assay decreased in the following order: heparin > dermatan sulfate > heparan sulfate > chondroitin sulfate A. Binding of HPRG to immobilized dermatan sulfate had a midpoint at pH 6.5, was less influenced by zinc, and exhibited cooperativity. Importantly, plasminogen interacted specifically with GAG-bound HPRG. We propose that HPRG is a physiological pH sensor, interacting with negatively charged GAGs on cell surfaces only when it acquires a net positive charge by protonation and/or metal binding. This provides a mechanism to regulate the function of HPRG (the local pH) and rationalizes the role of its unique, conserved histidine-proline-rich domain. Thus, under conditions of local acidosis (e.g. ischemia or hypoxia), HPRG can co-immobilize plasminogen at the cell surface as well as compete for heparin with other proteins such as antithrombin.

  3. Proline puckering parameters for collagen structure simulations

    SciTech Connect

    Wu, Di

    2015-03-15

    Collagen is made of triple helices rich in proline residues, and hence is influenced by the conformational motions of prolines. Because the backbone motions of prolines are restricted by the helical structures, the only side chain motion—proline puckering—becomes an influential factor that may affect the stability of collagen structures. In molecular simulations, a proper proline puckering population is desired so to yield valid results of the collagen properties. Here we design the proline puckering parameters in order to yield suitable proline puckering populations as demonstrated in the experimental results. We test these parameters in collagen and the proline dipeptide simulations. Compared with the results of the PDB and the quantum calculations, we propose the proline puckering parameters for the selected collagen model simulations.

  4. Proline puckering parameters for collagen structure simulations

    NASA Astrophysics Data System (ADS)

    Wu, Di

    2015-03-01

    Collagen is made of triple helices rich in proline residues, and hence is influenced by the conformational motions of prolines. Because the backbone motions of prolines are restricted by the helical structures, the only side chain motion—proline puckering—becomes an influential factor that may affect the stability of collagen structures. In molecular simulations, a proper proline puckering population is desired so to yield valid results of the collagen properties. Here we design the proline puckering parameters in order to yield suitable proline puckering populations as demonstrated in the experimental results. We test these parameters in collagen and the proline dipeptide simulations. Compared with the results of the PDB and the quantum calculations, we propose the proline puckering parameters for the selected collagen model simulations.

  5. StGCPRP, a Potato Gene Strongly Expressed in Stomatal Guard Cells, Defines a Novel Type of Repetitive Proline-Rich Proteins1

    PubMed Central

    Menke, Ulrich; Renault, Nathalie; Mueller-Roeber, Bernd

    2000-01-01

    Guard cells represent a highly differentiated cell type within the epidermis of plant leaves and stems. They respond to many endogenous and environmental signals and thereby modify the size of the stomatal pore they surround. We identified a novel gene that is highly expressed in guard cells of potato (Solanum tuberosum). It encodes a repetitive proline (Pro)-rich protein of 54 kD (491 amino acids) and was named StGCPRP (S. tuberosum guard cell Pro-rich protein). StGCPRP has a bipartite structure. The C-terminal part of StGCPRP contains a high percentage (46%) of Pro residues organized in distinct repetitive sequence motifs, whereas its extended N terminus is essentially free of Pros. StGCPRP represents the first member of a novel class of hybrid Pro-rich proteins that we designated NHyPRPs. In young but not in mature leaves, StGCPRP transcripts were also present at high levels in mesophyll cells (in addition to guard cells), indicating developmental regulation of StGCPRP gene expression. In addition, StGCPRP expression is regulated by environmental factors, as shown by a decrease in StGCPRP transcript levels under drought stress. Two proteins similar to StGCPRP were found to be encoded by the Arabidopsis genome, indicating that NHyPRPs are more widely distributed in higher plants. PMID:10712530

  6. Transcriptional activation by the sexual pheromone and wounding: a new gene family from Volvox encoding modular proteins with (hydroxy)proline-rich and metalloproteinase homology domains.

    PubMed

    Hallmann, A; Amon, P; Godl, K; Heitzer, M; Sumper, M

    2001-06-01

    The green alga Volvox represents the simplest kind of multicellular organism: it is composed of only two cell types, somatic and reproductive, making it suitable as a model system. The sexual development of males and females of Volvox carteri is triggered by a sex-inducing pheromone at a concentration of < 10-16 M. Early biochemical responses to the pheromone involve structural modifications within the extracellular matrix (ECM). By differential screenings of cDNA libraries made from mRNAs of pheromone-treated Volvox, four novel genes were identified that encode four closely related Volvox metalloproteinases that we use to define a new protein family, the VMPs. The existence of several features common to matrix glycoproteins, such as signal peptides, a (hydroxy)proline content of 12-25%, and Ser(Pro)2-4 repeats, suggest an extracellular localization of the VMPs within the ECM. Synthesis of VMP cDNAs is triggered not only by the sex-inducing pheromone, but also by wounding, and is restricted to the somatic cell type. Sequence comparisons suggest that the VMPs are members of the MB clan of zinc-dependent matrix metalloproteinases, although the putative zinc binding site of all VMPs is QEXXHXXGXXH rather than HEXXHXXGXXH. The presence of glutamine instead of histidine in the zinc binding motif suggests a novel family, or even clan, of peptidases. Like the matrixin family of human collagenases, Volvox VMPs exhibit a modular structure: they possess a metalloproteinase homology domain and a (hydroxy)proline-rich domain, and one of them, VMP4, also has two additional domains. Metalloproteinases seem to be crucial for biochemical modifications of the ECM during development or after wounding in the lower eukaryote Volvox with only two cell types, just as in higher organisms.

  7. Structural Characterization of Proline-rich Tyrosine Kinase 2 (PYK2) Reveals a Unique (DFG-out) Conformation and Enables Inhibitor Design

    SciTech Connect

    Han, Seungil; Mistry, Anil; Chang, Jeanne S.; Cunningham, David; Griffor, Matt; Bonnette, Peter C.; Wang, Hong; Chrunyk, Boris A.; Aspnes, Gary E.; Walker, Daniel P.; Brosius, Arthur D.; Buckbinder, Leonard; Pfizer

    2009-05-21

    Proline-rich tyrosine kinase 2 (PYK2) is a cytoplasmic, non-receptor tyrosine kinase implicated in multiple signaling pathways. It is a negative regulator of osteogenesis and considered a viable drug target for osteoporosis treatment. The high-resolution structures of the human PYK2 kinase domain with different inhibitor complexes establish the conventional bilobal kinase architecture and show the conformational variability of the DFG loop. The basis for the lack of selectivity for the classical kinase inhibitor, PF-431396, within the FAK family is explained by our structural analyses. Importantly, the novel DFG-out conformation with two diarylurea inhibitors (BIRB796, PF-4618433) reveals a distinct subclass of non-receptor tyrosine kinases identifiable by the gatekeeper Met-502 and the unique hinge loop conformation of Leu-504. This is the first example of a leucine residue in the hinge loop that blocks the ATP binding site in the DFG-out conformation. Our structural, biophysical, and pharmacological studies suggest that the unique features of the DFG motif, including Leu-504 hinge-loop variability, can be exploited for the development of selective protein kinase inhibitors.

  8. A proline-rich polypeptide complex (PRP) isolated from ovine colostrum. Modulation of H2O2 and cytokine induction in human leukocytes.

    PubMed

    Zabłocka, Agnieszka; Janusz, Maria; Macała, Józefa; Lisowski, Józef

    2007-07-01

    A proline-rich polypeptide complex (PRP) has immunoregulatory properties and also shows beneficial effects in Alzheimer's disease (AD). It is known that the unregulated activation of microglial cells in AD may result in chronic inflammatory response. There is a link between the activation of immune cells on the periphery and in the central nervous system (CNS). Therefore, we studied the effect of the PRP on human peripheral blood mononuclear cells (PBMCs) stimulated by LPS with PHA (LP) or PMA as proinflammatory activators. PRP and its nonapeptide fragment (NP) inhibited by 40-60% production of H(2)O(2) induced by PMA. The peptides also inhibited activity of superoxide dismutase. Both peptide preparations showed differential effects on the secretion of cytokines. NP induced TNF-alpha only while PRP induced IL-6, IL-10 and TNF-alpha. On the other hand, the release of TNF-alpha and IL-10 induced by LP in PBMCs was inhibited by PRP while NP inhibited the release of IFN-gamma and IL-10. The results obtained showed that PRP may affect not only adaptive immunity but also innate immunity and thus may regulate secretions of mediators of inflammation. The regulatory effect of the PRP on the innate immunity may shed some light on understanding the beneficial effects of this polypeptide complex in AD patients.

  9. The colloidal state of tannins impacts the nature of their interaction with proteins: the case of salivary proline-rich protein/procyanidins binding.

    PubMed

    Cala, Olivier; Dufourc, Erick J; Fouquet, Eric; Manigand, Claude; Laguerre, Michel; Pianet, Isabelle

    2012-12-18

    While the definition of tannins has been historically associated with its propensity to bind proteins in a nonspecific way, it is now admitted that specific interaction also occurs. The case of the astringency perception is a good example to illustrate this phenomenon: astringency is commonly described as a tactile sensation induced by the precipitation of a complex composed of proline-rich proteins present in the human saliva and tannins present in beverages such as tea or red wines. In the present work, the interactions between a human saliva protein segment and three different procyanidins (B1, B3, and C2) were investigated at the atomic level by NMR and molecular dynamics. The data provided evidence for (i) an increase in affinity compared to shortest human saliva peptides, which is accounted for by protein "wraping around" the tannin, (ii) a specificity in the interaction below tannin critical micelle concentration (CMC) of ca. 10 mM, with an affinity scale such that C2 > B1 > B3, and (iii) a nonspecific binding above tannin CMC that conducts irremediably to the precipitation of the tannins/protein complex. Such physicochemical findings describe in accurate terms saliva protein-tannin interactions and provide support for a more subtle description by oenologists of wine astringency perception in the mouth.

  10. Isolation and characterization of cDNA clones for carrot extensin and a proline-rich 33-kDa protein

    SciTech Connect

    Chen, J.; Varner, J.E.

    1985-07-01

    Extensins are hydroxyproline-rich glycoproteins associated with most dicotyledonous plant cell walls. To isolate cDNA clones encoding extensin, the authors started by isolating poly(A) RNA from carrot root tissue, and then translating the RNA in vitro, in the presence of tritiated leucine or proline. A 33-kDa peptide was identified in the translation products as a putative extensin precursor. From a cDNA library constructed with poly(A) RNA from wounded carrots, one cDNA clone (pDC5) was identified that specifically hybridized to poly(A) RNA encoding this 33-kDa peptide. They isolated three cDNA clones (pDC11, pDC12, and pDC16) from another cDNA library using pCD5 as a probe. DNA sequence data, RNA hybridization analysis, and hybrid released in vitro translation indicate that the cDNA clones pDC11 encodes extensin and that cDNA clones pDC12 and pDC16 encode the 33-kDa peptide, which as yet has an unknown identity and function. The assumption that the 33-kDa peptide was an extensin precursor was invalid. RNA hybridization analysis showed that RNA encoded by both clone types is accumulated upon wounding.

  11. Proline-rich tyrosine kinase 2 via enhancing signal transducer and activator of transcription 3-dependent cJun expression mediates retinal neovascularization

    PubMed Central

    Kumar, Raj; Singh, Nikhlesh K.; Rao, Gadiparthi N.

    2016-01-01

    Despite the involvement of proline-rich tyrosine kinase 2 (Pyk2) in endothelial cell angiogenic responses, its role in pathological retinal angiogenesis is not known. In the present study, we show that vascular endothelial growth factor A (VEGFA) induces Pyk2 activation in mediating human retinal microvascular endothelial cell (HRMVEC) migration, sprouting and tube formation. Downstream to Pyk2, VEGFA induced signal transducer and activator of transcription 3 (STAT3) activation and cJun expression in the modulation of HRMVEC migration, sprouting and tube formation. Consistent with these observations, hypoxia induced activation of Pyk2-STAT3-cJun signaling axis and siRNA-mediated downregulation of Pyk2, STAT3 or cJun levels substantially inhibited hypoxia-induced retinal endothelial cell proliferation, tip cell formation and neovascularization. Together, these observations suggest that activation of Pyk2-mediated STAT3-cJun signaling is required for VEGFA-induced HRMVEC migration, sprouting and tube formation in vitro and hypoxia-induced retinal endothelial cell proliferation, tip cell formation and neovascularization in vivo. PMID:27210483

  12. Asparagine-linked carbohydrate chains of inducible rat parotid proline-rich glycoprotein contain terminal beta-linked N-acetylgalactosamine.

    PubMed

    Bedi, G S

    1997-12-01

    Rats treated with daily injection of DL-isoproterenol for 10 consecutive days (25 mg kg(-1) body weight) showed marked induction of a proline-rich glycoprotein (GPRP) of 220 kDa. Proteinase K digestion of GPRP produced a homogeneous glycopeptide with an average chemical composition as follows (residues per mol): Pro4, Glx3, Asx2, Gly1, His1, Thr1, Arg1, GlcNAc5, GalNac1, Man3, Gal2-3, and Fuc1. The structural analysis of the asparagine-linked carbohydrate unit was performed by methylation, periodate oxidation and enzymatic degradation. Methylation studies indicated that the three mannosyl residues were substituted at 1,2-, 1,2,4-, and 1,3,6-positions. Fucose, N-acetylgalactosamine, 1.5 residues of galactose and 0.35 residues of N-acetylglucosamine were terminally located and one galactose residue was 1,4-substituted. Approximately four of the 5 N-acetylglucosamine residues were substituted at 1,4-position and approximately 1 residue of N-acetylglucosamine was substituted at 1,4,6-positions. Periodate oxidation studies and exoglycosidase results were consistent with the methylation data. Based on the results of Smith degradation, methylation and sequential exoglycosidase digestions a triantennary oligosaccharide structure having terminal N-acetylgalactosamine in one of the branches is proposed for the major Asn-linked carbohydrate moiety of GPRP.

  13. The SNF5 protein of Saccharomyces cerevisiae is a glutamine- and proline-rich transcriptional activator that affects expression of a broad spectrum of genes.

    PubMed Central

    Laurent, B C; Treitel, M A; Carlson, M

    1990-01-01

    The Saccharomyces cerevisiae SNF5 gene affects expression of both glucose- and phosphate-regulated genes and appears to function in transcription. We report the nucleotide sequence, which predicts that SNF5 encodes a 102,536-dalton protein. The N-terminal third of the protein is extremely rich in glutamine and proline. Mutants carrying a deletion of the coding sequence were viable but grew slowly, indicating that the SNF5 gene is important but not essential. Evidence that SNF5 affects expression of the cell type-specific genes MF alpha 1 and BAR1 at the RNA level extends the known range of SNF5 function. SNF5 is apparently required for expression of a wide variety of differently regulated genes. A bifunctional SNF5-beta-galactosidase fusion protein was localized in the nucleus by immunofluorescence. No DNA-binding activity was detected for SNF5. A LexA-SNF5 fusion protein, when bound to a lexA operator, functioned as a transcriptional activator. Images PMID:2233708

  14. The Effect of Selective D- or Nα-Methyl Arginine Substitution on the Activity of the Proline-Rich Antimicrobial Peptide, Chex1-Arg20

    PubMed Central

    Li, Wenyi; Sun, Zhe; O'Brien-Simpson, Neil M.; Otvos, Laszlo; Reynolds, Eric C.; Hossain, Mohammed A.; Separovic, Frances; Wade, John D.

    2017-01-01

    In vivo pharmacokinetics studies have shown that the proline-rich antimicrobial peptide, A3-APO, which is a discontinuous dimer of the peptide, Chex1-Arg20, undergoes degradation to small fragments at positions Pro6-Arg7 and Val19-Arg20. With the aim of minimizing or abolishing this degradation, a series of Chex1-Arg20 analogs were prepared via Fmoc/tBu solid phase peptide synthesis with D-arginine or, in some cases, peptide backbone Nα-methylated arginine, substitution at these sites. All the peptides were tested for antibacterial activity against the Gram-negative bacterium Klebsiella pneumoniae. The resulting activity of position-7 substitution of Chex1-Arg20 analogs showed that arginine-7 is a crucial residue for maintaining activity against K. pneumoniae. However, arginine-20 substitution had a much less deleterious effect on the antibacterial activity of the peptide. Moreover, none of these peptides displayed any cytotoxicity to HEK and H-4-II-E mammalian cells. These results will aid the development of more effective and stable PrAMPs via judicious amino acid substitutions. PMID:28154813

  15. Metallofullerenol Gd@C82(OH)22 distracts the proline-rich-motif from putative binding on the SH3 domain

    NASA Astrophysics Data System (ADS)

    Kang, Seung-Gu; Huynh, Tien; Zhou, Ruhong

    2013-03-01

    Biocompatibility is often regarded as one important aspect of de novo designed nanomaterials for biosafety. However, the toxicological effect, appearing along with its latency, is much more difficult to address by linearly mapping physicochemical properties of related nanomaterials with biological effects such as immune or cellular regulatory responses due to the complicated protein-protein interactions. Here, we investigate a potential interference of a metallofullerenol, Gd@C82(OH)22, on the function of SH3 domain, a highly promiscuous protein-protein interaction mediator involved in signaling and regulatory pathways through its binding with the proline-rich motif (PRM) peptides, using the atomistic molecular dynamics simulation. Our study shows that when only Gd@C82(OH)22 and the SH3 domain are present (without the PRM ligand), Gd@C82(OH)22 can interact with the SH3 domain by either directly blocking the hydrophobic active site or binding with a hydrophilic off-site with almost equal probability, which can be understood from its intrinsic amphiphilic nature. In a binding competition with the PRM onto the SH3 domain, however, the on-site binding mode is depleted while Gd@C82(OH)22 effectively intercepts the PRM from the putative binding site of the SH3 domain, implying that Gd@C82(OH)22 can disturb protein-protein interactions mediated by the SH3 domain. Despite a successful surface modification in an aqueous biological medium and a more recent demonstration as potential de novo cancer therapeutics, our study indicates that greater attention is needed in assessing the potential cytotoxicity of these nanomaterials.Biocompatibility is often regarded as one important aspect of de novo designed nanomaterials for biosafety. However, the toxicological effect, appearing along with its latency, is much more difficult to address by linearly mapping physicochemical properties of related nanomaterials with biological effects such as immune or cellular regulatory responses

  16. A proline-rich polypeptide complex (PRP) from ovine colostrum. Studies on the effect of PRP on nitric oxide (NO) production induced by LPS in THP-1 cells.

    PubMed

    Mikulska, Joanna Elzbieta; Lisowski, Józef

    2003-11-01

    A proline-rich polypeptide complex (PRP) isolated from ovine colostrum shows immunoregulatory activity. Similar activity was observed when PRP was replaced with a nonapeptide (NP) isolated from chymotryptic digest of PRP. The polypeptide complex also shows procognitive activity. In the form of orally administered tablets called Colostrinin, containing 100 microg of PRP, it improves the outcome of Alzheimer's disease (AD) patients. The mechanism of action of PRP/Colostrinin in AD is not yet clarified. Microglial cells involvement in AD has been related to amyloid beta (Abeta) internalization, the release of inflammatory cytokines, overproduction of nitrogen oxide (NO) and superoxide anion (O2-), and the development of neuritic plaques. It has been previously found in our laboratory that PRP regulates the secretion of an array of cytokines. It also was shown, in preliminary experiments using human blood cells and murine macrophages, that PRP inhibits production of NO and O2- induced by LPS. In the present work, to study the effect of PRP and NP on the release of NO and O2-induced by LPS we applied THP-1 cells. The human monocyte/macrophage THP-1 cell line has been widely used as a model of human microglial cells. The results obtained showed that THP-1 cells release NO when activated with LPS. However, neither PRP nor NP induced production of NO. Although the nonapeptide, at higher concentration (100 microg/mL), showed an inhibitory activity on the release of NO induced by LPS, no inhibition was observed when PRP was used. THP-1 cells treated with LPS, PRP or NP did not release O2-.

  17. Despite sequence homologies to gluten, salivary proline-rich proteins do not elicit immune responses central to the pathogenesis of celiac disease.

    PubMed

    Tian, Na; Leffler, Daniel A; Kelly, Ciaran P; Hansen, Joshua; Marietta, Eric V; Murray, Joseph A; Schuppan, Detlef; Helmerhorst, Eva J

    2015-12-01

    Celiac disease (CD) is an inflammatory disorder triggered by ingested gluten, causing immune-mediated damage to the small-intestinal mucosa. Gluten proteins are strikingly similar in amino acid composition and sequence to proline-rich proteins (PRPs) in human saliva. On the basis of this feature and their shared destination in the gastrointestinal tract, we hypothesized that salivary PRPs may modulate gluten-mediated immune responses in CD. Parotid salivary secretions were collected from CD patients, refractory CD patients, non-CD patients with functional gastrointestinal complaints, and healthy controls. Structural similarities of PRPs with gluten were probed with anti-gliadin antibodies. Immune responses to PRPs were investigated toward CD patient-derived peripheral blood mononuclear cells and in a humanized transgenic HLA-DQ2/DQ8 mouse model for CD. Anti-gliadin antibodies weakly cross-reacted with the abundant salivary amylase but not with PRPs. Likewise, the R5 antibody, recognizing potential antigenic gluten epitopes, showed negligible reactivity to salivary proteins from all groups. Inflammatory responses in peripheral blood mononuclear cells were provoked by gliadins whereas responses to PRPs were similar to control levels, and PRPs did not compete with gliadins in immune stimulation. In vivo, PRP peptides were well tolerated and nonimmunogenic in the transgenic HLA-DQ2/DQ8 mouse model. Collectively, although structurally similar to dietary gluten, salivary PRPs were nonimmunogenic in CD patients and in a transgenic HLA-DQ2/DQ8 mouse model for CD. It is possible that salivary PRPs play a role in tolerance induction to gluten early in life. Deciphering the structural basis for the lack of immunogenicity of salivary PRPs may further our understanding of the toxicity of gluten.

  18. Arginine methylation and citrullination of splicing factor proline- and glutamine-rich (SFPQ/PSF) regulates its association with mRNA.

    PubMed

    Snijders, Ambrosius P; Hautbergue, Guillaume M; Bloom, Alex; Williamson, James C; Minshull, Thomas C; Phillips, Helen L; Mihaylov, Simeon R; Gjerde, Douglas T; Hornby, David P; Wilson, Stuart A; Hurd, Paul J; Dickman, Mark J

    2015-03-01

    Splicing factor proline- and glutamine-rich (SFPQ) also commonly known as polypyrimidine tract-binding protein-associated-splicing factor (PSF) and its binding partner non-POU domain-containing octamer-binding protein (NONO/p54nrb), are highly abundant, multifunctional nuclear proteins. However, the exact role of this complex is yet to be determined. Following purification of the endogeneous SFPQ/NONO complex, mass spectrometry analysis identified a wide range of interacting proteins, including those involved in RNA processing, RNA splicing, and transcriptional regulation, consistent with a multifunctional role for SFPQ/NONO. In addition, we have identified several sites of arginine methylation in SFPQ/PSF using mass spectrometry and found that several arginines in the N-terminal domain of SFPQ/PSF are asymmetrically dimethylated. Furthermore, we find that the protein arginine N-methyltransferase, PRMT1, catalyzes this methylation in vitro and that this is antagonized by citrullination of SFPQ. Arginine methylation and citrullination of SFPQ/PSF does not affect complex formation with NONO. However, arginine methylation was shown to increase the association with mRNA in mRNP complexes in mammalian cells. Finally we show that the biochemical properties of the endogenous complex from cell lysates are significantly influenced by the ionic strength during purification. At low ionic strength, the SFPQ/NONO complex forms large heterogeneous protein assemblies or aggregates, preventing the purification of the SFPQ/NONO complex. The ability of the SFPQ/NONO complex to form varying protein assemblies, in conjunction with the effect of post-translational modifications of SFPQ modulating mRNA binding, suggests key roles affecting mRNP dynamics within the cell.

  19. Responsiveness to 6-n-Propylthiouracil (PROP) Is Associated with Salivary Levels of Two Specific Basic Proline-Rich Proteins in Humans

    PubMed Central

    Cabras, Tiziana; Melis, Melania; Castagnola, Massimo; Padiglia, Alessandra; Tepper, Beverly J.; Messana, Irene; Tomassini Barbarossa, Iole

    2012-01-01

    Thiourea tasting can be predictive of individual differences in bitter taste responses, general food preferences and eating behavior, and could be correlated with saliva chemical composition. We investigated the possible relationship between PROP bitter taste responsiveness and the salivary proteome in subjects genotyped for TAS2R38 and gustin gene polymorphisms. Taste perception intensity evoked by PROP and NaCl solutions was measured in sixty-three volunteers (21 males, 42 females, age 25±3 y) to establish their PROP taster status, and 24 PROP super-tasters and 21 nontasters were selected to participate in the study. TAS2R38 and gustin gene molecular analysis were performed using PCR techniques. Qualitative and quantitative determination of salivary proteins was performed by HPLC-ESI-MS before and after PROP taste stimulation. PROP super-tastings was strongly associated with the ‘taster’ variant (PAV haplotype) of TAS2R38 and the A allele of rs2274333 polymorphism in the gustin gene and nontasting was associated with the minor alleles at both loci. ANOVA revealed that basal levels of II-2 and Ps-1 proteins, belonging to the basic proline-rich protein (bPRPs) family, were significantly higher in PROP super-taster than in nontaster un-stimulated saliva, and that PROP stimulation elicited a rapid increase in the levels of these same proteins only in PROP super-taster saliva. These data show for the first time that responsiveness to PROP is associated with salivary levels of II-2 peptide and Ps-1 protein, which are products of the PRB1 gene. These findings suggest that PRB1, in addition to TAS2R38 and gustin, could contribute to individual differences in thiourea sensitivity, and the expression of the PROP phenotype as a complex genetic trait. PMID:22312435

  20. Responsiveness to 6-n-propylthiouracil (PROP) is associated with salivary levels of two specific basic proline-rich proteins in humans.

    PubMed

    Cabras, Tiziana; Melis, Melania; Castagnola, Massimo; Padiglia, Alessandra; Tepper, Beverly J; Messana, Irene; Tomassini Barbarossa, Iole

    2012-01-01

    Thiourea tasting can be predictive of individual differences in bitter taste responses, general food preferences and eating behavior, and could be correlated with saliva chemical composition. We investigated the possible relationship between PROP bitter taste responsiveness and the salivary proteome in subjects genotyped for TAS2R38 and gustin gene polymorphisms. Taste perception intensity evoked by PROP and NaCl solutions was measured in sixty-three volunteers (21 males, 42 females, age 25±3 y) to establish their PROP taster status, and 24 PROP super-tasters and 21 nontasters were selected to participate in the study. TAS2R38 and gustin gene molecular analysis were performed using PCR techniques. Qualitative and quantitative determination of salivary proteins was performed by HPLC-ESI-MS before and after PROP taste stimulation. PROP super-tastings was strongly associated with the 'taster' variant (PAV haplotype) of TAS2R38 and the A allele of rs2274333 polymorphism in the gustin gene and nontasting was associated with the minor alleles at both loci. ANOVA revealed that basal levels of II-2 and Ps-1 proteins, belonging to the basic proline-rich protein (bPRPs) family, were significantly higher in PROP super-taster than in nontaster un-stimulated saliva, and that PROP stimulation elicited a rapid increase in the levels of these same proteins only in PROP super-taster saliva. These data show for the first time that responsiveness to PROP is associated with salivary levels of II-2 peptide and Ps-1 protein, which are products of the PRB1 gene. These findings suggest that PRB1, in addition to TAS2R38 and gustin, could contribute to individual differences in thiourea sensitivity, and the expression of the PROP phenotype as a complex genetic trait.

  1. Pyrrhocoricin, a proline-rich antimicrobial peptide derived from insect, inhibits the translation process in the cell-free Escherichia coli protein synthesis system.

    PubMed

    Taniguchi, Masayuki; Ochiai, Akihito; Kondo, Hiroshi; Fukuda, Shun; Ishiyama, Yohei; Saitoh, Eiichi; Kato, Tetsuo; Tanaka, Takaaki

    2016-05-01

    Previous studies have shown that pyrrhocoricin, a proline-rich antimicrobial peptide (PrAMP), killed sensitive species in a dose-dependent manner by specifically binding to DnaK. Here, on the basis of the finding that DnaK-deficient Escherichia coli strains are susceptible to PrAMPs, we used pyrrhocoricin to investigate internal targets other than DnaK. Using conventional antibiotics (bleomycin, streptomycin, and fosfomycin) that have known modes of action, first, we validated the availability of an assay using a cell-free rapid translation system (RTS), which is an in vitro protein synthesis system based on E. coli lysate, for evaluating inhibition of protein synthesis. We found that, similarly to bleomycin and streptomycin, pyrrhocoricin inhibited GFP synthesis in RTS in a concentration-dependent manner. In addition, blockage of transcription and translation steps in RTS was individually estimated using RT-PCR after gene expression to determine mRNA products and using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the amounts of GFP expressed from purified mRNA, respectively. The results demonstrated that this inhibition of GFP synthesis by pyrrhocoricin did not occur at the transcription step but rather at the translation step, in a manner similar to that of GFP synthesis by streptomycin, an inhibitor of the translation step by causing misreading of tRNA. These results suggest that RTS is a powerful assay system for determining if antimicrobial peptides inhibit protein synthesis and its transcription and/or translation steps. This is the first study to have shown that pyrrhocoricin inhibited protein synthesis by specifically repressing the translation step.

  2. Effect of enamel matrix derivative and of proline-rich synthetic peptides on the differentiation of human mesenchymal stem cells toward the osteogenic lineage.

    PubMed

    Ramis, Joana Maria; Rubert, Marina; Vondrasek, Jiri; Gayà, Antoni; Lyngstadaas, Staale Petter; Monjo, Marta

    2012-06-01

    With the aim of discovering new molecules for induction of bone formation and biomineralization, combination of bioinformatics and simulation methods were used to design the structure of artificial peptides based on proline-rich domains of enamel matrix proteins. In this study, the effect of such peptides on the differentiation toward the osteogenic lineage of human umbilical cord mesenchymal stem cells (hUCMSCs) was evaluated with or without osteogenic supplements (hydrocortisone, β-glycerol phosphate, and ascorbic acid) and compared to the effect of the commercially available enamel matrix derivative (EMD). It was hypothesized that the differentiation toward the osteogenic lineage of hUCMSCs would be promoted by the treatment with the synthetic peptides when combined with differentiation media, or it could even be directed exclusively by the synthetic peptides. Osteoinductivity was assessed by cell proliferation, bone morphogenetic protein-2 secretion, and gene expression of osteogenic markers after 1, 3, and 14 days of treatment. All peptides were safe with the dosages used, showing lower cell toxicity. P2, P4, and P6 reduced cell proliferation with growing media by 10%-15%. Higher expression of early osteoblast markers was found after 3 days of treatment with EMD in combination with osteogenic supplements, while after 14 days of treatment, cells treated by the different synthetic peptides in combination with osteogenic supplements showed higher osteocalcin mRNA levels. We can conclude that osteogenic differentiation of hUCMSCs is promoted by short-term EMD treatment in combination with osteogenic supplements and by long-term treatment by the synthetic peptides in combination with osteogenic supplements, showing similar results for all the peptide variants analyzed in this study.

  3. Structural basis of Robo proline-rich motif recognition by the srGAP1 Src homology 3 domain in the Slit-Robo signaling pathway.

    PubMed

    Li, Xiaofeng; Chen, Yushu; Liu, Yiwei; Gao, Jia; Gao, Feng; Bartlam, Mark; Wu, Jane Y; Rao, Zihe

    2006-09-22

    The Slit-Robo (sr) GTPase-activating protein (GAPs) are important components in the intracellular pathway mediating Slit-Robo signaling in axon guidance and cell migration. We report the first crystal structure of the srGAP1 SH3 domain at 1.8-A resolution. The unusual side chain conformation of the conserved Phe-13 in the P1 pocket renders the ligand binding pocket shallow and narrow, which contributes toward the low binding affinity. Moreover, the opposing electrostatic charge and the hydrophobic properties of the P3 specificity pocket are consistent with the observed binding characteristics of the srGAP1 SH3 domain to its ligand. Surface plasmon resonance experiments indicate that the srGAP1 SH3 domain interacts with its natural ligand inaCtoN orientation. The srGAP1 SH3 domain can bind to both the CC2 and CC3 motifs in vitro. The N-terminal two acidic residues in the CC3 motif recognition site are necessary for srGAP1 SH3 domain binding. A longer CC3 peptide (CC3-FL) binds with greater affinity than its shorter counterpart, suggesting that the residues surrounding the proline-rich core are important for protein-peptide interactions. Our study reveals previously unknown properties of the srGAP-Robo interaction. Our data provide a structural basis for the srGAP-Robo interaction, consistent with the role of the Robo intracellular domain in interacting with other downstream signaling molecules and mediating versatile and dynamic responses to axon guidance and cell migration cues.

  4. Despite sequence homologies to gluten, salivary proline-rich proteins do not elicit immune responses central to the pathogenesis of celiac disease

    PubMed Central

    Tian, Na; Leffler, Daniel A.; Kelly, Ciaran P.; Hansen, Joshua; Marietta, Eric V.; Murray, Joseph A.; Schuppan, Detlef

    2015-01-01

    Celiac disease (CD) is an inflammatory disorder triggered by ingested gluten, causing immune-mediated damage to the small-intestinal mucosa. Gluten proteins are strikingly similar in amino acid composition and sequence to proline-rich proteins (PRPs) in human saliva. On the basis of this feature and their shared destination in the gastrointestinal tract, we hypothesized that salivary PRPs may modulate gluten-mediated immune responses in CD. Parotid salivary secretions were collected from CD patients, refractory CD patients, non-CD patients with functional gastrointestinal complaints, and healthy controls. Structural similarities of PRPs with gluten were probed with anti-gliadin antibodies. Immune responses to PRPs were investigated toward CD patient-derived peripheral blood mononuclear cells and in a humanized transgenic HLA-DQ2/DQ8 mouse model for CD. Anti-gliadin antibodies weakly cross-reacted with the abundant salivary amylase but not with PRPs. Likewise, the R5 antibody, recognizing potential antigenic gluten epitopes, showed negligible reactivity to salivary proteins from all groups. Inflammatory responses in peripheral blood mononuclear cells were provoked by gliadins whereas responses to PRPs were similar to control levels, and PRPs did not compete with gliadins in immune stimulation. In vivo, PRP peptides were well tolerated and nonimmunogenic in the transgenic HLA-DQ2/DQ8 mouse model. Collectively, although structurally similar to dietary gluten, salivary PRPs were nonimmunogenic in CD patients and in a transgenic HLA-DQ2/DQ8 mouse model for CD. It is possible that salivary PRPs play a role in tolerance induction to gluten early in life. Deciphering the structural basis for the lack of immunogenicity of salivary PRPs may further our understanding of the toxicity of gluten. PMID:26505973

  5. HPLC-ESI-MS and MS/MS structural characterization of multifucosylated N-glycoforms of the basic proline-rich protein IB-8a CON1+ in human saliva.

    PubMed

    Cabras, Tiziana; Boi, Roberto; Pisano, Elisabetta; Iavarone, Federica; Fanali, Chiara; Nemolato, Sonia; Faa, Gavino; Castagnola, Massimo; Messana, Irene

    2012-05-01

    This study describes the characterization of the glycan moieties and the peptide backbone of six glycoforms of IB-8a CON1(+), a basic proline-rich protein present in human saliva. MS analyses on the intact glycoproteins before and after N-deglycosylation with PNGase F and high-resolution MS/MS sequencing by LTQ Orbitrap XL of peptides and glycopeptides from tryptic digests allowed the structural characterization of the glycan moieties and the polypeptide backbone, as well as to establish the glycosylation site at the asparagine residue at 98th position. Five of the glycoforms carry a biantennary N-linked glycan fucosylated in the innermost N-acetylglucosamine of the core and showing from zero to four additional fucoses in the antennal region. The sixth glycoform carries a monoantennary monofucosylated oligosaccharide. The glycoform cluster was detected on 28 of 71 adult saliva specimens. Level of fucosylation showed interindividual variability with the major relative abundance for the trifucosylated glycoform. Nonglycosylated IB-8a CON1(+) and the variant IB-8a CON1(-), lacking of the glycosylation site, have been also detected in human saliva.

  6. Regulatory region with putA gene of proline dehydrogenase that links to the lum and the lux operons in Photobacterium leiognathi.

    PubMed

    Lin, J W; Yu, K Y; Chen, H Y; Weng, S F

    1996-02-27

    Nucleotide sequence of regulatory region (R & R) with putA gene (EMBL Accession No. U39227) from Photobacterium leiognathi PL741 has been determined, and the putA gene encoded amino acid sequence of proline dehydrogenase is deduced. Alignment and comparison of proline dehydrogenase of P. leiognathi with the proline dehydrogenase domain in the PutA protein of Escherichia coli and Salmonella typhimurium show that they are homologous. Nucleotide sequence reveals that regulatory region with the putA gene is linked to the lum and lux operons in genome; the gene order is <--putA--R & R(I)<--ter-lumQ-lumP-R & R-luxC-luxD-luxA-luxB-luxE--> (R & R: regulatory region; ter:transcriptional terminator), whereas the R & R is the regulatory region for the lum and the lux operons, ter is the transcriptional terminator for the lum operon, and R & R(I) apparently is the regulatory region for the putA and related genes. Nucleotide sequence analysis illustrates the specific inverted repeat (SIR), cAMP-CRP consensus sequence, canonical -10/-35 promoter, putative operator and Shine-Dalgarno (SD) sequence on the regulatory region R & R(I) for the putA and related genes; it suggests that the putA and related genes are simply linked to the lum and the lux operons in genome, the regulatory region R & R(I) is independent for the putA and related genes.

  7. A conserved proline residue in the leucine zipper region of AtbZIP34 and AtbZIP61 in Arabidopsis thaliana interferes with the formation of homodimer

    SciTech Connect

    Shen Huaishun; Cao Kaiming; Wang Xiping

    2007-10-19

    Two putative Arabidopsis E group bZIP transcript factors, AtbZIP34 and AtbZIP61, are nuclear-localized and their transcriptional activation domain is in their N-terminal region. By searching GenBank, we found other eight plant homologues of AtbZIP34 and AtbZIP61. All of them have a proline residue in the third heptad of zipper region. Yeast two-hybrid assay and EMSA showed that AtbZIP34 and AtbZIP61 could not form homodimer while their mutant forms, AtbZIP34m and AtbZIP61m, which the proline residue was replaced by an alanine residue in the zipper region, could form homodimer and bind G-box element. These results suggest that the conserved proline residue interferes with the homodimer formation. However, both AtbZIP34 and AtbZIP61 could form heterodimers with members of I group and S group transcription factors in which some members involved in vascular development. So we speculate that AtbZIP34 and AtbZIP61 may participate in plant development via interacting with other group bZIP transcription factors.

  8. Involvement of proline-rich tyrosine kinase 2 in platelet activation: tyrosine phosphorylation mostly dependent on alphaIIbbeta3 integrin and protein kinase C, translocation to the cytoskeleton and association with Shc through Grb2.

    PubMed Central

    Ohmori, T; Yatomi, Y; Asazuma, N; Satoh, K; Ozaki, Y

    2000-01-01

    Proline-rich tyrosine kinase 2 (Pyk2) (also known as RAFTK, CAKbeta or CADTK) has been identified as a member of the focal adhesion kinase (FAK) family of protein-tyrosine kinases and it has been suggested that the mode of Pyk2 activation is distinct from that of FAK. In the present study we investigated the mode of Pyk2 activation in human platelets. When platelets were stimulated with thrombin, Pyk2, as well as FAK, was markedly tyrosine-phosphorylated, in a manner mostly dependent on alphaIIbbeta3 integrin-mediated aggregation. The residual Pyk2 tyrosine phosphorylation observed in the absence of platelet aggregation was completely abolished by pretreatment with BAPTA/AM [bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester]. The Pyk2 phosphorylation was inhibited by protein kinase C (PKC) inhibitors at concentrations that inhibited platelet aggregation. In contrast, direct activation of PKC with the active phorbol ester PMA induced the tyrosine phosphorylation of Pyk2 and FAK but only when platelets were fully aggregated with the exogenous addition of fibrinogen (the ligand for alphaIIbbeta3 integrin). Furthermore, PMA-induced Pyk2 (and FAK) tyrosine phosphorylation was also observed when platelets adhered to immobilized fibrinogen. The activation of the von Willebrand factor (vWF)--glycoprotein Ib pathway with botrocetin together with vWF failed to induce Pyk2 (and FAK) tyrosine phosphorylation. Most Pyk2 and FAK was present in the cytosol and membrane skeleton fractions in unstimulated platelets. When platelets were stimulated with thrombin, both Pyk2 and FAK were translocated to the cytoskeleton in an aggregation-dependent manner. In immunoprecipitation studies, Pyk2, as well as FAK, seemed to associate with Shc through Grb2. With the use of glutathione S-transferase fusion proteins containing Shc-SH2, Grb2-SH2, and Grb2 N-terminal and C-terminal SH3 domains, it was implied that the proline-rich region of Pyk2 (and FAK) binds to the N

  9. Inner membrane proteins YgdD and SbmA are required for the complete susceptibility of E. coli to the proline-rich antimicrobial peptide arasin 1(1-25).

    PubMed

    Paulsen, Victoria S; Mardirossian, Mario; Blencke, Hans-Matti; Benincasa, Monica; Runti, Giulia; Nepa, Matteo; Haug, Tor; Stensvåg, Klara; Scocchi, Marco

    2016-02-08

    Arasin 1 from the spider crab Hyas araneus is a proline-rich antimicrobial peptide, which kills target bacteria by a non-membranolytic mechanism. By using a fluorescent derivative of the peptide, we showed that arasin 1 rapidly penetrates into Escherichia coli cells without membrane damage. To unravel its mode of action, a knock-out gene library of E. coli was screened and two types of mutants with a less susceptible phenotype to the arasin 1 fragment (1-23) were found. The first bore the mutation of sbmA, a gene coding for an inner membrane protein involved in the uptake of different antibiotic peptides. The second one was located in the ygdD gene, coding for a conserved inner membrane protein of unknown function. Functional studies showed that YgdD is required for the full susceptibility to arasin 1(1-25), possibly by supporting its uptake and/or intracellular action. These results indicate that different bacterial proteins are exploited by arasin 1(1-25) to exert its antibacterial activity and add new insights in the complex mode of action of proline-rich antimicrobial peptides.

  10. Demarcating the gene-rich regions of the wheat genome

    PubMed Central

    Erayman, Mustafa; Sandhu, Devinder; Sidhu, Deepak; Dilbirligi, Muharrem; Baenziger, P. S.; Gill, Kulvinder S.

    2004-01-01

    By physically mapping 3025 loci including 252 phenotypically characterized genes and 17 quantitative trait loci (QTLs) relative to 334 deletion breakpoints, we localized the gene-containing fraction to 29% of the wheat genome present as 18 major and 30 minor gene-rich regions (GRRs). The GRRs varied both in gene number and density. The five largest GRRs physically spanning <3% of the genome contained 26% of the wheat genes. Approximate size of the GRRs ranged from 3 to 71 Mb. Recombination mainly occurred in the GRRs. Various GRRs varied as much as 128-fold for gene density and 140-fold for recombination rates. Except for a general suppression in 25–40% of the chromosomal region around centromeres, no correlation of recombination was observed with the gene density, the size, or chromosomal location of GRRs. More than 30% of the wheat genes are in recombination-poor regions thus are inaccessible to map-based cloning. PMID:15240829

  11. Star forming regions in gas-rich SO galaxies

    NASA Technical Reports Server (NTRS)

    Pogge, Richard W.; Eskridge, Paul B.

    1987-01-01

    The first results of an H alpha imaging survey of HI rich SO galaxies, which were searched for HII regions and other sources of emission, are presented. The charge coupled device H alpha interference filter images were made of 16 galaxies. Eight of these galaxies show evidence for on-going star formation, one has nuclear emission but no HII regions, and the remaining seven have no emissions detected within well defined upper limits. With the exception of one notably peculiar galaxy in which the emission from HII regions appears pervasive, the HII regions are either organized into inner-disk rings or randomly distributed throughout the disk. A few of these galaxies are found to be clearly not SO's; or peculiar objects atypical of the SO class. Using simple models star formation rates (SFRs) and gas depletion times from the observed H alpha fluxes were estimated. In general, the derived SFRs are much lower than those found in isolated field spiral galaxies and the corresponding gas depletion time scales are also longer.

  12. The human homolog of a mouse-imprinted gene, Peg3, maps to a zinc finger gene-rich region of human chromosome 19q13.4.

    PubMed

    Kim, J; Ashworth, L; Branscomb, E; Stubbs, L

    1997-05-01

    Peg3 (paternally expressed gene 3) is the first imprinted gene detected in the proximal region of mouse chromosome 7. Because imprinting is a trait that is generally conserved among mammals, and imprinted domains generally encompass several adjacent genes, expression patterns and chromosomal environment of the human counterpart of Peg3 are of special interest. In this study we have localized human PEG3 approximately 2 Mb proximal of the telomere of chromosome 19q, within a region known to carry large numbers of tandemly clustered Krüppel-type zinc finger-containing (ZNF) genes. Peg3 also encodes a Krüppel-type ZNF protein but one that is distinguished from other ZNF gene products by the fact that it carries two novel proline-rich motifs. Comparison between mouse Peg3 and partial human PEG3 gene sequences revealed a high level of conservation between the two species, despite the fact that one of the two proline-rich repeats is absent from the human gene. Our data demonstrate that the human gene is expressed at highest levels in ovary and placenta; mouse Peg3, by contrast, is transcribed at highest levels in the adult brain. These comparative mapping, sequencing, and expression data provide the first clues to the potential activities of PEG3, and generate new tools to aid in the analysis of structure and function of a potentially new imprinted domain located in human chromosome 19q13.4 and mouse chromosome 7.

  13. Two novel mutations in the glycine-rich region of human PAX6 gene: Implications for an association of cataracts and anosmia with aniridia

    SciTech Connect

    Martha, A.; Ferrel, R.E.; Hittner, H.M.; Saunders, G.F.

    1994-09-01

    Aniridia (iris hyplasia) is a autosomal dominant congenital disorder of the eye. Mutations in the human aniridia (PAX6) gene have now been identified in many patients from various ethnic groups. In the present study we describe new mutations in this gene. Out of four mutations found, three were novel mutations; the fourth one is identical to the previously reported mutations (C{yields}T transition at nt 240). The three novel mutations analyzed were in the glycine-rich region (two) and in the proline/serine/threonine-rich (PST) region (one). Previously no mutations were reported for the glycine-rich region in humans. One of the mutations found in this region is associated with cataracts in an aniridia family. The other splice mutation found in the PST domain is associated with anosmia (lack of sensation to smell) in a sporadic aniridia case. Two of the mutations presented here, one in the glycine-rich region and the other in the PST domain, were not detected by SSCR. These mutations could be detected by using MDE gel and heteroduplex information. All mutations found in the present study are similar in that 32 of 33 PAX6 mutations result in protein truncation and haploinsufficiency.

  14. Proline and lysine residues provide modulatory switches in amyloid formation: Insights from prion protein.

    PubMed

    Kraus, Allison

    2016-01-01

    Amyloidogenic proteins have an increased propensity to reorganize into the highly structured, β sheet rich structures that characterize amyloid. The probability of attaining these highly structured assemblies is influenced by multiple factors, including amino acid composition and environmental conditions. Evolutionary selection for amino acid sequences that prevent amyloid formation could further modulate amyloid-forming propensity. Indeed, we have recently identified specific proline and lysine residues, contained within a highly conserved central region of prion protein (PrP), that impede PrP amyloid formation in vitro. These prolines are mutated in certain forms of the human familial genetic disease, Gerstmann-Straüssler-Schneiker (GSS) syndrome. Here, I discuss the influence of these proline and lysine residues on PrP amyloid formation and how such anti-amyloidogenic primary amino acid sequences might be modulated to influence protein amyloidogenicity.

  15. Solar source regions of 3HE-rich particle events

    NASA Technical Reports Server (NTRS)

    Kahler, S. W.; Lin, R. P.; Reames, D. V.; Stone, R. G.; Liggett, M.

    1985-01-01

    Hydrogen alpha X-ray, and metric and kilometric radio data to examine the solar sources of energetic 3He-rich particle events observed near earth in association with impulsive 2 to 100 keV electron events were applied. Each 3He/electron event is associated with a kilometric type 3 burst belonging to a family of such bursts characterized by similar interplanetary propagation paths from the same solar active region. The 3He/electron events correlate very well with the interplanetary low frequency radio brightnesses of these events, but progressively worse with signatures from regions closer to the Sun. When H alpha brightnings can be associated with 3He/electron events, they have onsets coinciding to within 1 min of that of the associated metric type 3 burst but are often too small to be reported. The data are consistent with the earlier idea that many type 3 bursts, the 3He/electron events, are due to particle acceleration in the corona, well above the associated H alpha and X-ray flares.

  16. On the relationship between regional and local species richness: a test of saturation theory.

    PubMed

    Starzomski, Brian M; Parker, Raenelle L; Srivastava, Diane S

    2008-07-01

    What are the local community consequences of changes in regional species richness and composition? To answer this question we followed the assembly of microarthropod communities in defaunated areas of moss, embedded in a larger moss "region." Regions were created by combining moss from spatially distinct sites, resulting in regional species pools that differed in both microarthropod richness and composition, but not area. Regional effects were less important than seasonality for local richness. Initial differences in regional richness had no direct effect on local species richness at any time along a successional gradient of 0.5-16 months. The structure of the regional pool affected both local richness and local composition, but these effects were seasonally dependent. Local species richness differed substantially between dates along the successional gradient and continued to increase 16 months after assembly began. To the best of our knowledge, this is the first critical test of saturation theory that experimentally manipulates regional richness. Further, our results failed to support the most important mechanisms proposed to explain the local richness-regional richness relationship. The results demonstrate that complicated interactions between assembly time, seasonality, and regional species pools contribute to structuring local species richness and composition in this community.

  17. TCR engagement induces proline-rich tyrosine kinase-2 (Pyk2) translocation to the T cell-APC interface independently of Pyk2 activity and in an immunoreceptor tyrosine-based activation motif-mediated fashion.

    PubMed

    Sancho, David; Montoya, María C; Monjas, Alicia; Gordón-Alonso, Mónica; Katagiri, Takuya; Gil, Diana; Tejedor, Reyes; Alarcón, Balbino; Sánchez-Madrid, Francisco

    2002-07-01

    The relocation of kinases in T lymphocytes during their cognate interaction with APCs is essential for lymphocyte activation. We found that the proline-rich tyrosine kinase-2 (Pyk2) is rapidly translocated to the T cell-APC contact area upon T cell-specific recognition of superantigen-pulsed APCs. Stimulation with anti-CD3-coated latex microspheres was sufficient for Pyk2 reorientation, and the coengagement of CD28 boosted Pyk2 redistribution. Nevertheless, Pyk2 translocation did not result in its recruitment to lipid rafts. Two results support that Pyk2 translocation was independent of its kinase activity. First, Lck activity was required for TCR-induced Pyk2 translocation, but not for TCR-induced Pyk2 activation. Second, a kinase-dead Pyk2 mutant was equally translocated upon TCR triggering. In addition, Lck activity alone was insufficient to induce Pyk2 reorientation and activation, requiring the presence of at least one intact immunoreceptor tyrosine-based activation motif (ITAM). Despite the dependence on functional Lck and on phosphorylated ITAM for Pyk2 translocation, the ITAM-binding tyrosine kinase zeta-associated protein 70 (ZAP-70) was not essential. All these data suggest that, by translocating to the vicinity of the immune synapse, Pyk2 could play an essential role in T cell activation and polarized secretion of cytokines.

  18. High-resolution high-performance liquid chromatography with electrospray ionization mass spectrometry and tandem mass spectrometry characterization of a new isoform of human salivary acidic proline-rich proteins named Roma-Boston Ser22(Phos) → Phe variant

    PubMed Central

    Iavarone, Federica; D’Alessandro, Alfredo; Tian, Na; Cabras, Tiziana; Messana, Irene; Helmerhorst, Eva J.; Oppenheim, Frank G.; Castagnola, Massimo

    2015-01-01

    During a survey of human saliva by a top-down reversed-phase high-performance liquid chromatography with electrospray ionization mass spectrometry approach, two proteins eluting at 27.4 and 28.4 min, with average masses of 15 494 ± 1 and 11 142 ± 1 Da, were detected in a subject from Boston. The Δmass value (4352 Da) of the two proteins was similar to the difference in mass values between intact (150 amino acids, [a.a.]) and truncated acidic proline-rich proteins (aPRPs; 106 a.a.) suggesting an a.a. substitution in the first 106 residues resulting in a strong reduction in polarity, since under the same experimental conditions aPRPs eluted at ~22.5 min (intact) and 23.5 min (truncated forms). Manual inspection of the high-resolution high-performance liquid chromatography with electrospray ionization tandem mass spectra of the truncated isoform showed the replacement of the phosphorylated Ser-22 in PRP-3 with a Phe residue. Inspection of the tandem mass spectra of the intact isoform confirmed the substitution, which is allowed by the code transition TCT→TTT and is in agreement with the dramatic increase in elution time. The isoform was also detected in two other subjects, one from Boston (unrelated to the previous) and one from Rome. For this reason we propose to name this variant PRP-1 (PRP-3) RB (Roma-Boston) Ser22(phos)→Phe. PMID:24771659

  19. Regional and local species richness in an insular environment: Serpentine plants in California

    USGS Publications Warehouse

    Harrison, S.; Safford, H.D.; Grace, J.B.; Viers, J.H.; Davies, K.F.

    2006-01-01

    We asked how the richness of the specialized (endemic) flora of serpentine rock outcrops in California varies at both the regional and local scales. Our study had two goals: first, to test whether endemic richness is affected by spatial habitat structure (e.g., regional serpentine area, local serpentine outcrop area, regional and local measures of outcrop isolation), and second, to conduct this test in the context of a broader assessment of environmental influences (e.g., climate, soils, vegetation, disturbance) and historical influences (e.g., geologic age, geographic province) on local and regional species richness. We measured endemic and total richness and environmental variables in 109 serpentine sites (1000-m2 paired plots) in 78 serpentine-containing regions of the state. We used structural equation modeling (SEM) to simultaneously relate regional richness to regionalscale predictors, and local richness to both local-scale and regional-scale predictors. Our model for serpentine endemics explained 66% of the variation in local endemic richness based on local environment (vegetation, soils, rock cover) and on regional endemic richness. It explained 73% of the variation in regional endemic richness based on regional environment (climate and productivity), historical factors (geologic age and geographic province), and spatial structure (regional total area of serpentine, the only significant spatial variable in our analysis). We did not find a strong influence of spatial structure on species richness. However, we were able to distinguish local vs. regional influences on species richness to a novel extent, despite the existence of correlations between local and regional conditions. ?? 2006 by the Ecological Society of America.

  20. Influence of the yjiL-mdtM Gene Cluster on the Antibacterial Activity of Proline-Rich Antimicrobial Peptides Overcoming Escherichia coli Resistance Induced by the Missing SbmA Transporter System

    PubMed Central

    Krizsan, Andor; Knappe, Daniel

    2015-01-01

    In view of increasing health threats from multiresistant pathogens, antimicrobial peptides (AMPs) and, specifically, proline-rich AMPs (PrAMPs) have been investigated in animal models. PrAMPs enter bacteria via the ABC transporter SbmA and inhibit intracellular targets. We used phage transduction (Tn10 insertion) to screen by random mutagenesis for alternative uptake mechanisms for analogs of apidaecin 1b, a honeybee-derived PrAMP. All 24 apidaecin-resistant mutants had the Tn10 insertion in the sbmA gene. These sbmA::Tn10 insertion mutants and the Escherichia coli BW25113 ΔsbmA (JW0368) strain were still susceptible to the bactenecin PrAMP Bac7(1-35) and oncocin PrAMPs Onc18 and Onc112, as well as to Chex1-Arg20, despite significantly reduced internalizations. In a second round of random mutagenesis, the remaining susceptibility was linked to the yjiL-mdtM gene cluster. E. coli BW25113 and its ΔyjiL null mutant (JW5785) were equally susceptible to all PrAMPs tested, whereas the BW25113 ΔmdtM mutant was less susceptible to oncocins. The JW0368 yjiL::Tn10 transposon mutant (BS2) was resistant to all short PrAMPs and susceptible only to full-length Bac7 and A3-APO. Interestingly, PrAMPs appear to enter bacteria via MdtM, a multidrug resistance transporter (drug/H+ antiporter) of the major facilitator superfamily (MFS) that can efflux antibiotics, biocides, and bile salts. In conclusion, PrAMPs enter bacteria via ABC and MFS transporters that efflux antibiotics and cytotoxic compounds from the cytoplasm to the periplasm. PMID:26169420

  1. Influence of the yjiL-mdtM Gene Cluster on the Antibacterial Activity of Proline-Rich Antimicrobial Peptides Overcoming Escherichia coli Resistance Induced by the Missing SbmA Transporter System.

    PubMed

    Krizsan, Andor; Knappe, Daniel; Hoffmann, Ralf

    2015-10-01

    In view of increasing health threats from multiresistant pathogens, antimicrobial peptides (AMPs) and, specifically, proline-rich AMPs (PrAMPs) have been investigated in animal models. PrAMPs enter bacteria via the ABC transporter SbmA and inhibit intracellular targets. We used phage transduction (Tn10 insertion) to screen by random mutagenesis for alternative uptake mechanisms for analogs of apidaecin 1b, a honeybee-derived PrAMP. All 24 apidaecin-resistant mutants had the Tn10 insertion in the sbmA gene. These sbmA::Tn10 insertion mutants and the Escherichia coli BW25113 ΔsbmA (JW0368) strain were still susceptible to the bactenecin PrAMP Bac7(1-35) and oncocin PrAMPs Onc18 and Onc112, as well as to Chex1-Arg20, despite significantly reduced internalizations. In a second round of random mutagenesis, the remaining susceptibility was linked to the yjiL-mdtM gene cluster. E. coli BW25113 and its ΔyjiL null mutant (JW5785) were equally susceptible to all PrAMPs tested, whereas the BW25113 ΔmdtM mutant was less susceptible to oncocins. The JW0368 yjiL::Tn10 transposon mutant (BS2) was resistant to all short PrAMPs and susceptible only to full-length Bac7 and A3-APO. Interestingly, PrAMPs appear to enter bacteria via MdtM, a multidrug resistance transporter (drug/H(+) antiporter) of the major facilitator superfamily (MFS) that can efflux antibiotics, biocides, and bile salts. In conclusion, PrAMPs enter bacteria via ABC and MFS transporters that efflux antibiotics and cytotoxic compounds from the cytoplasm to the periplasm.

  2. A gene encoding a protein with a proline-rich domain (MtPPRD1), revealed by suppressive subtractive hybridization (SSH), is specifically expressed in the Medicago truncatula embryo axis during germination.

    PubMed

    Bouton, Sophie; Viau, Laure; Lelièvre, Eric; Limami, Anis M

    2005-03-01

    A gene MtPPRD1, encoding a protein of 132 amino acids containing a proline-rich domain (PRD), has been revealed by suppressive subtractive hybridization (SSH) with two mRNA populations of embryo axes harvested immediately before and after radicle emergence. Although at the protein level MtPPRD1 showed low homology with plant lipid transfer proteins (LTPs), it did exhibit the eight cysteine residues conserved in all plant LTPs, a characteristic signature that allows the formation of a hydrophobic cavity adapted for loading hydrophobic molecules. Expression studies of MtPPRD1 have been carried out by quantitative real time RT-PCR throughout germination and post-germination processes in control seeds and seeds in which germination was delayed by abscisic acid (ABA) or the glutamine synthetase inhibitor methionine sulphoximine (MSX) treatments. The results showed that MtPPRD1 expression is developmentally regulated, induced in the embryo axis immediately before radicle emergence, reaches its maximum expression and declines during the early post-germination phase. Organ specificity studies showed that, except for a low and probably constitutive expression in roots, MtPPRD1 is specifically expressed in the embryo axis. Based on both experimental and in silico studies several putative roles are proposed for MtPPRD1 in Medicago truncatula, this protein can intervene (i) as an LTP in membrane biogenesis and regulation of the intracellular fatty acid pool by binding and transferring fatty acids and phospholipids between membranes, (ii) in the control of a developmental process specific to late germination and to early phases of post-germination, and (iii) and/or pathogen defence.

  3. Proline Mechanisms of Stress Survival

    PubMed Central

    Liang, Xinwen; Zhang, Lu; Natarajan, Sathish Kumar

    2013-01-01

    Abstract Significance: The imino acid proline is utilized by different organisms to offset cellular imbalances caused by environmental stress. The wide use in nature of proline as a stress adaptor molecule indicates that proline has a fundamental biological role in stress response. Understanding the mechanisms by which proline enhances abiotic/biotic stress response will facilitate agricultural crop research and improve human health. Recent Advances: It is now recognized that proline metabolism propels cellular signaling processes that promote cellular apoptosis or survival. Studies have shown that proline metabolism influences signaling pathways by increasing reactive oxygen species (ROS) formation in the mitochondria via the electron transport chain. Enhanced ROS production due to proline metabolism has been implicated in the hypersensitive response in plants, lifespan extension in worms, and apoptosis, tumor suppression, and cell survival in animals. Critical Issues: The ability of proline to influence disparate cellular outcomes may be governed by ROS levels generated in the mitochondria. Defining the threshold at which proline metabolic enzyme expression switches from inducing survival pathways to cellular apoptosis would provide molecular insights into cellular redox regulation by proline. Are ROS the only mediators of proline metabolic signaling or are other factors involved? Future Directions: New evidence suggests that proline biosynthesis enzymes interact with redox proteins such as thioredoxin. An important future pursuit will be to identify other interacting partners of proline metabolic enzymes to uncover novel regulatory and signaling networks of cellular stress response. Antioxid. Redox Signal. 19, 998–1011. PMID:23581681

  4. Disentangling the determinants of species richness of vascular plants and mammals from national to regional scales.

    PubMed

    Xu, Haigen; Cao, Mingchang; Wu, Yi; Cai, Lei; Cao, Yun; Wu, Jun; Lei, Juncheng; Le, Zhifang; Ding, Hui; Cui, Peng

    2016-02-23

    Understanding the spatial patterns in species richness gets new implication for biodiversity conservation in the context of climate change and intensified human intervention. Here, we created a database of the geographical distribution of 30,519 vascular plant species and 565 mammal species from 2,376 counties across China and disentangled the determinants that explain species richness patterns both at national and regional scales using spatial linear models. We found that the determinants of species richness patterns varied among regions: elevational range was the most powerful predictor for the species richness of plants and mammals across China. However, species richness patterns in the Qinghai-Tibetan Plateau Region (QTR) are quite unique, where net primary productivity was the most important predictor. We also detected that elevational range was positively related to plant species richness when it is less than 1,900 m, whereas the relationship was not significant when elevational range is larger than 1,900 m. It indicated that elevational range often emerges as the predominant controlling factor within the regions where energy is sufficient. The effects of land use on mammal species richness should attract special attention. Our study suggests that region-specific conservation policies should be developed based on the regional features of species richness.

  5. Disentangling the determinants of species richness of vascular plants and mammals from national to regional scales

    PubMed Central

    Xu, Haigen; Cao, Mingchang; Wu, Yi; Cai, Lei; Cao, Yun; Wu, Jun; Lei, Juncheng; Le, Zhifang; Ding, Hui; Cui, Peng

    2016-01-01

    Understanding the spatial patterns in species richness gets new implication for biodiversity conservation in the context of climate change and intensified human intervention. Here, we created a database of the geographical distribution of 30,519 vascular plant species and 565 mammal species from 2,376 counties across China and disentangled the determinants that explain species richness patterns both at national and regional scales using spatial linear models. We found that the determinants of species richness patterns varied among regions: elevational range was the most powerful predictor for the species richness of plants and mammals across China. However, species richness patterns in the Qinghai-Tibetan Plateau Region (QTR) are quite unique, where net primary productivity was the most important predictor. We also detected that elevational range was positively related to plant species richness when it is less than 1,900 m, whereas the relationship was not significant when elevational range is larger than 1,900 m. It indicated that elevational range often emerges as the predominant controlling factor within the regions where energy is sufficient. The effects of land use on mammal species richness should attract special attention. Our study suggests that region-specific conservation policies should be developed based on the regional features of species richness. PMID:26902418

  6. Disentangling the determinants of species richness of vascular plants and mammals from national to regional scales

    NASA Astrophysics Data System (ADS)

    Xu, Haigen; Cao, Mingchang; Wu, Yi; Cai, Lei; Cao, Yun; Wu, Jun; Lei, Juncheng; Le, Zhifang; Ding, Hui; Cui, Peng

    2016-02-01

    Understanding the spatial patterns in species richness gets new implication for biodiversity conservation in the context of climate change and intensified human intervention. Here, we created a database of the geographical distribution of 30,519 vascular plant species and 565 mammal species from 2,376 counties across China and disentangled the determinants that explain species richness patterns both at national and regional scales using spatial linear models. We found that the determinants of species richness patterns varied among regions: elevational range was the most powerful predictor for the species richness of plants and mammals across China. However, species richness patterns in the Qinghai-Tibetan Plateau Region (QTR) are quite unique, where net primary productivity was the most important predictor. We also detected that elevational range was positively related to plant species richness when it is less than 1,900 m, whereas the relationship was not significant when elevational range is larger than 1,900 m. It indicated that elevational range often emerges as the predominant controlling factor within the regions where energy is sufficient. The effects of land use on mammal species richness should attract special attention. Our study suggests that region-specific conservation policies should be developed based on the regional features of species richness.

  7. Fibronectin Fragment Activation of Proline-rich Tyrosine Kinase PYK2 Mediates Integrin Signals Regulating Collagenase-3 Expression by Human Chondrocytes through a Protein Kinase C-dependent Pathway*

    PubMed Central

    Loeser, Richard F.; Forsyth, Christopher B.; Samarel, Allen M.; Im, Hee-Jeong

    2010-01-01

    Fibronectin fragments (FN-f), including the 110-kDa fragment that binds the α5β1 integrin, stimulate collagenase-3 (MMP-13) production and cartilage destruction. In the present study, treatment of chondrocytes with the 110-kDa FN-f or an activating antibody to the α5β1 integrin was found to increase tyrosine autophosphorylation (Tyr-402) of the proline-rich tyrosine kinase-2 (PYK2) without significant change in autophosphorylation (Tyr-397) of focal adhesion kinase (FAK). The tyrosine kinase inhibitor tyrphostin A9, shown previously to block a PYK2-dependent pathway, blocked the FN-f-stimulated increase in MMP-13, whereas tyrphostin A25 did not. FN-f-stimulated PYK2 phosphorylation and MMP-13 production was also blocked by reducing intracellular calcium levels. Adenovirally mediated overexpression of wild type but not mutant PYK2 resulted in increased MMP-13 production. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate stimulated PYK2 phosphorylation and MMP-13 production. MMP-13 expression stimulated by either phorbol 12-myristate 13-acetate or FN-f was blocked by PKC inhibitors including the PKCδ inhibitor rottlerin. Furthermore, PKCδ translocation from cytosol to membrane was noted within 5 min of stimulation with FN-f. Immortalized human chondrocytes, transiently transfected with MMP-13 promoter-luciferase reporter constructs, showed increased promoter activity after FN-f treatment that was inhibited by co-transfection with either of two dominant negative mutants of PYK2 (Y402F and K457A). No inhibition was seen after co-transfection with wild type PYK2, a dominant negative of FAK (FRNK) or empty vector plasmid. FN-f-stimulated MMP-13 promoter activity was also inhibited by chemical inhibitors of ERK, JNK, and p38 mitogen-activated protein (MAP) kinases or by co-transfection of dominant negative MAP kinase mutant constructs. These studies have identified a novel pathway for the MAP kinase regulation of MMP-13 production which involves

  8. Landscape connectivity strengthens local-regional richness relationships in successional plant communities.

    PubMed

    Damschen, Ellen I; Brudvig, Lars A

    2012-04-01

    Local species diversity is maintained over ecological time by a balance between dispersal and species interactions. Local-regional species richness relationships are often used to investigate the relative importance of these two processes and the scales at which they operate. For communities undergoing succession, theory predicts a temporal progression in local-regional species richness relationships: from no relationship to positive linear to saturating. However, observational tests have been mixed, and experiments have been rare. Using a replicated large-scale experiment, we evaluate the impact of two dispersal-governing processes at the regional scale, connectivity and shape of the region (i.e., patches), on the progression of local-regional species richness relationships for plant communities undergoing succession. Regional connectivity accelerates the transition from no relationship to a positive linear relationship, while the shape of the region has no consistent effect nine years post-disturbance. Our results experimentally demonstrate the importance of dispersal in structuring local-regional species richness relationships over time and suggest that conservation corridors among regions can increase local diversity through regional enrichment of plant communities undergoing reassembly.

  9. Substrate channeling in proline metabolism

    PubMed Central

    Arentson, Benjamin W.; Sanyal, Nikhilesh; Becker, Donald F.

    2012-01-01

    Proline metabolism is an important pathway that has relevance in several cellular functions such as redox balance, apoptosis, and cell survival. Results from different groups have indicated that substrate channeling of proline metabolic intermediates may be a critical mechanism. One intermediate is pyrroline-5-carboxylate (P5C), which upon hydrolysis opens to glutamic semialdehyde (GSA). Recent structural and kinetic evidence indicate substrate channeling of P5C/GSA occurs in the proline catabolic pathway between the proline dehydrogenase and P5C dehydrogenase active sites of bifunctional proline utilization A (PutA). Substrate channeling in PutA is proposed to facilitate the hydrolysis of P5C to GSA which is unfavorable at physiological pH. The second intermediate, gamma-glutamyl phosphate, is part of the proline biosynthetic pathway and is extremely labile. Substrate channeling of gamma-glutamyl phosphate is thought to be necessary to protect it from bulk solvent. Because of the unfavorable equilibrium of P5C/GSA and the reactivity of gamma-glutamyl phosphate, substrate channeling likely improves the efficiency of proline metabolism. Here, we outline general strategies for testing substrate channeling and review the evidence for channeling in proline metabolism. PMID:22201749

  10. OcculterCut: A Comprehensive Survey of AT-Rich Regions in Fungal Genomes

    PubMed Central

    Testa, Alison C.; Oliver, Richard P.; Hane, James K.

    2016-01-01

    We present a novel method to measure the local GC-content bias in genomes and a survey of published fungal species. The method, enacted as “OcculterCut” (https://sourceforge.net/projects/occultercut, last accessed April 30, 2016), identified species containing distinct AT-rich regions. In most fungal taxa, AT-rich regions are a signature of repeat-induced point mutation (RIP), which targets repetitive DNA and decreases GC-content though the conversion of cytosine to thymine bases. RIP has in turn been identified as a driver of fungal genome evolution, as RIP mutations can also occur in single-copy genes neighboring repeat-rich regions. Over time RIP perpetuates “two speeds” of gene evolution in the GC-equilibrated and AT-rich regions of fungal genomes. In this study, genomes showing evidence of this process are found to be common, particularly among the Pezizomycotina. Further analysis highlighted differences in amino acid composition and putative functions of genes from these regions, supporting the hypothesis that these regions play an important role in fungal evolution. OcculterCut can also be used to identify genes undergoing RIP-assisted diversifying selection, such as small, secreted effector proteins that mediate host-microbe disease interactions. PMID:27289099

  11. Genome-Wide Chromatin Remodeling Identified at GC-Rich Long Nucleosome-Free Regions

    PubMed Central

    Hochreiter, Sepp

    2012-01-01

    To gain deeper insights into principles of cell biology, it is essential to understand how cells reorganize their genomes by chromatin remodeling. We analyzed chromatin remodeling on next generation sequencing data from resting and activated T cells to determine a whole-genome chromatin remodeling landscape. We consider chromatin remodeling in terms of nucleosome repositioning which can be observed most robustly in long nucleosome-free regions (LNFRs) that are occupied by nucleosomes in another cell state. We found that LNFR sequences are either AT-rich or GC-rich, where nucleosome repositioning was observed much more prominently in GC-rich LNFRs — a considerable proportion of them outside promoter regions. Using support vector machines with string kernels, we identified a GC-rich DNA sequence pattern indicating loci of nucleosome repositioning in resting T cells. This pattern appears to be also typical for CpG islands. We found out that nucleosome repositioning in GC-rich LNFRs is indeed associated with CpG islands and with binding sites of the CpG-island-binding ZF-CXXC proteins KDM2A and CFP1. That this association occurs prominently inside and also prominently outside of promoter regions hints at a mechanism governing nucleosome repositioning that acts on a whole-genome scale. PMID:23144837

  12. Importance of SARS-CoV spike protein Trp-rich region in viral infectivity

    SciTech Connect

    Lu Yanning; Neo, T.L.; Liu, D.Xi.; Tam, James P.

    2008-07-04

    SARS-CoV entry is mediated by spike glycoprotein. During the viral and host cellular membrane fusion, HR1 and HR2 form 6-helix bundle, positioning the fusion peptide closely to the C-terminal region of ectodomain to drive apposition and subsequent membrane fusion. Connecting to the HR2 region is a Trp-rich region which is absolutely conserved in members of coronaviruses. To investigate the importance of Trp-rich region in SARS-CoV entry, we produced different mutated S proteins using Alanine scan strategy. SARS-CoV pseudotyped with mutated S protein was used to measure viral infectivity. To restore the aromaticity of Ala-mutants, we performed rescue experiments using phenylalanine substitutions. Our results show that individually substituted Ala-mutants substantially decrease infectivity by >90%, global Ala-mutants totally abrogated infectivity. In contrast, Phe-substituted mutants are able to restore 10-25% infectivity comparing to the wild-type. The results suggest that the Trp-rich region of S protein is essential for SARS-CoV infectivity.

  13. Biogeographic affinity helps explain productivity-richness relationships at regional and local scales

    USGS Publications Warehouse

    Harrison, S.; Grace, J.B.

    2007-01-01

    The unresolved question of what causes the observed positive relationship between large-scale productivity and species richness has long interested ecologists and evolutionists. Here we examine a potential explanation that we call the biogeographic affinity hypothesis, which proposes that the productivity-richness relationship is a function of species' climatic tolerances that in turn are shaped by the earth's climatic history combined with evolutionary niche conservatism. Using botanical data from regions and sites across California, we find support for a key prediction of this hypothesis, namely, that the productivity-species richness relationship differs strongly and predictably among groups of higher taxa on the basis of their biogeographic affinities (i.e., between families or genera primarily associated with north-temperate, semiarid, or desert zones). We also show that a consideration of biogeographic affinity can yield new insights on how productivity-richness patterns at large geographic scales filter down to affect patterns of species richness and composition within local communities. ?? 2007 by The University of Chicago. All rights reserved.

  14. Species richness and distribution of blackflies (Diptera: Simuliidae) in the Chapada Diamantina region, Bahia, Brazil.

    PubMed

    Landeiro, Victor L; Pepinelli, Mateus; Hamada, Neusa

    2009-01-01

    Taxonomically, blackflies (Simuliidae) are among the best-known aquatic insects in Brazil. However, information on their distribution is lacking for many regions. We sampled simuliids in 50 streams in the Chapada Diamantina region, State of Bahia, located in three distinct geographical areas, 20 at Lençóis, 18 at Mucugê, and 12 at Rio de Contas. We analyzed simuliid species richness and distribution in the Chapada Diamantina area as a whole, as well as in each of the three distinct geographical areas. We collected 20 species, three of which were not yet described and two were described after our sampling. The three areas are distinct in relation to environmental variables, mainly owing to differences in altitude and water pH. Four species were restricted to Rio de Contas, while four other species were restricted to Mucugê. Sixteen species were present in Mucugê and fifteen in Rio de Contas. Only twelve species were present in Lençóis, despite the higher sampling effort. Rio de Contas appears to be the area that harbors the highest species richness. Species richness was related to environmental factors only when evaluated on a small spatial scale (i.e. when each area was analyzed separately). Although we have not tested for causal relationships, our findings agree with other studies that showed that pH and altitude are important factors with which species richness and species distribution appear to be associated.

  15. Nuclear structure in the neutron-rich doubly magic sup 78 Ni region

    SciTech Connect

    Hill, J.C.; Wohn, F.K.; Winger, J.A.; Warburton, E.K.; Gill, R.L.; Schuhmann, R.B.; Brookhaven National Lab., Upton, NY; Clark Univ., Worcester, MA )

    1989-01-01

    The magic numbers Z=28 and N=50 imply that very neutron-rich {sup 78}Ni, which has not yet been observed, is doubly magic. The {sup 78}Ni region was investigated by studying the N=50 isotones and neutron-rich Zn isotopes. Results on the level structure of {sup 83}As, {sup 74}Zn, and {sup 76}Zn populated in the decays of {sup 83}Ge, {sup 74}Cu, and {sup 76}Cu are presented. The parent nuclides were produced and mass separated using the TRISTAN facility on-line to the High-Flux Beam Reactor at Brookhaven. The systematics of the N=50 isotones and even-A Zn isotopes are discussed and compared with shell-model calculations involving active nucleons outside of a {sup 78}Ni and {sup 66}Ni core, respectively. The extent to which the {sup 78}Ni region can be considered doubly magic is assessed. 43 refs., 7 figs.

  16. Landscape correlates of breeding bird richness across the United States mid-Atlantic region

    USGS Publications Warehouse

    Jones, K.B.; Neale, A.C.; Nash, M.S.; Riitters, K.H.; Wickham, J.D.; O'Neill, R. V.; Van Remortel, R. D.

    2000-01-01

    Using a new set of landscape indicator data generated by the U.S.EPA, and a comprehensive breeding bird database from the National Breeding Bird Survey, we evaluated associations between breeding bird richness and landscape characteristics across the entire mid-Atlantic region of the United States. We evaluated how these relationships varied among different groupings (guilds) of birds based on functional, structural, and compositional aspects of individual species demographics. Forest edge was by far the most important landscape attribute affecting the richness of the lumped specialist and generalist guilds; specialist species richness was negatively associated with forest edge and generalist richness was positively associated with forest edge. Landscape variables (indicators) explained a greater proportion of specialist species richness than the generalist guild (46% and 31%, respectively). The lower value in generalists may reflect freer-scale distributions of open habitat that go undetected by the Landsat satellite, open habitats created by roads (the areas from which breeding bird data are obtained), and the lumping of a wide variety of species into the generalist category. A further breakdown of species into 16 guilds showed considerable variation in the response of breeding birds to landscape conditions; forest obligate species had the strongest association with landscape indicators measured in this study (55% of the total variation explained) and forest generalists and open ground nesters the lowest (17% of the total variation explained). The variable response of guild species richness to landscape pattern suggests that one must consider species' demographics when assessing the consequences of landscape change on breeding birds.Using a new set of landscape indicator data generated by the U.S. EPA, and a comprehensive breeding bird database from the National Breeding Bird Survey, we evaluated associations between breeding bird richness and landscape

  17. Specific Mg 2+ binding to AT-rich regions of chromatin in the evolution of eukaryotes

    NASA Astrophysics Data System (ADS)

    Strissel, P. L.; Gavrilov, K. L.; Levi-Setti, R.; Strick, R.

    2006-07-01

    At SIMS XIV, we reported SIMS evidence of specific Mg 2+ binding to the AT-rich regions of human metaphase chromosomes represented by G-bands. Subsequent Mg 2+-depletion experiments supported a direct role for Mg 2+ in promoting and maintaining the higher order chromatin structure originating G-bands, possibly due to both Mg 2+-DNA and Mg 2+-protein interactions. An in-depth study, reported elsewhere, implicated also Ca 2+ in the maintenance of chromatin ultrastructure in the scaffold of mammalian chromosomes, in association with topoisomerase II. We examine here the association of Mg 2+ with AT-rich regions of chromatin in the chromosomes of the Indian muntjac deer (IMD), leading to conclusions similar to the above. To answer the question whether the presumed divalent cation role in the chromosomes of advanced eukaryotes had an evolutionary history to be traced back to earlier evolutionary stages, we have SIMS-mapped Ca 2+ and Mg 2+ in BrdU-labeled polytene chromosomes from the salivary gland of the Dipteran Drosophila melanogaster. Striking Ca 2+ and Mg 2+ SIMS banding patterns correlating with those of the Br label (a thymidine analogue) implicate unequivocally a close association of both these cations with the AT-rich regions of DNA for these primitive eukaryotes.

  18. Proline derivatives in fruits of bergamot (Citrus bergamia Risso et Poit): presence of N-methyl-L-proline and 4-hydroxy-L-prolinebetaine.

    PubMed

    Servillo, Luigi; Giovane, Alfonso; Balestrieri, Maria Luisa; Cautela, Domenico; Castaldo, Domenico

    2011-01-12

    The content of proline and various compounds deriving from its metabolism (4-hydroxy-L-proline, N-methyl-L-proline, N,N-dimethylproline, and 4-hydroxy-L-prolinebetaine) was determined in fruits and seeds of Bergamot (Citrus bergamia Risso et Poit), growing in the Calabria region (South Italy). A HPLC-ESI-tandem mass spectrometry method, which allowed rapid determination of L-proline, 4-hydroxy-L-proline, N-methyl-L-proline, N,N-dimethylproline, and 4-hydroxy-L-prolinebetaine in juice and extracts of bergamot fruit with minimum sample preparation and short analysis time (about 10 min), is presented. Proline and 4-hydroxy-L-proline levels in the samples were also determined by HPLC analysis with fluorescence detection and the results compared to those obtained with HPLC-ESI-tandem mass spectrometry. For the first time, the presence of N-methyl-L-proline and 4-hydroxy-L-prolinebetaine in the fruits of a plant of the Citrus genus is reported.

  19. Multiple Determinants of Anuran Richness and Occurrence in an Agricultural Region in South-Eastern Brazil

    NASA Astrophysics Data System (ADS)

    Prado, Vitor H. M.; Rossa-Feres, Denise de C.

    2014-04-01

    In agricultural landscapes, studies that identify factors driving species richness and occupancy are important because they can guide farmers to use conservation practices that minimize species loss. In this context, anurans are threatened by habitat loss because they depend on the characteristics of both local water bodies and adjacent landscapes. We used a model selection approach to evaluate the influence of local and landscape variables in determining anuran species richness and occurrence in 40 freshwater bodies in a heavily deforested region of semideciduous Atlantic Forest in southeastern Brazil. Our aim was to develop recommendations for conservation of anuran communities in rural areas. Pond hydroperiod and area were the most important variables for explaining anuran species richness and occupancy, with greatest species richness being found in water bodies with intermediate hydroperiod and area. Other important variables that reflected individual species occupancies were the number of vegetation types and pond isolation. In addition, recent studies evidenced that water bodies near forest fragments have higher anuran abundance or diversity. In conclusion, we suggest the maintenance of semi-permanent ponds, isolated from large rivers or reservoirs and near forest fragments, as an effective strategy to conserve anuran fauna in agricultural landscapes of southeastern Brazil. Brazilian government requires the maintenance of forests as legal reserve in each farm, and farmers need to maintain ponds as drinking water for cattle or crop irrigation. For this reason, the guidelines suggested in the present study can be easily adopted, without additional costs to rural productivity.

  20. Role of proline in cell wall synthesis and plant development and its implications in plant ontogeny

    PubMed Central

    Kavi Kishor, Polavarapu B.; Hima Kumari, P.; Sunita, M. S. L.; Sreenivasulu, Nese

    2015-01-01

    Proline is a proteogenic amino acid and accumulates both under stress and non-stress conditions as a beneficial solute in plants. Recent discoveries point out that proline plays an important role in plant growth and differentiation across life cycle. It is a key determinant of many cell wall proteins that plays important roles in plant development. The role of extensins, arabinogalactan proteins and hydroxyproline- and proline-rich proteins as important components of cell wall proteins that play pivotal roles in cell wall signal transduction cascades, plant development and stress tolerance is discussed in this review. Molecular insights are also provided here into the plausible roles of proline transporters modulating key events in plant development. In addition, the roles of proline during seed developmental transitions including storage protein synthesis are discussed. PMID:26257754

  1. Simulation Study of RICH Detector for Particle Identification in Forward Region at Electron-Ion Collider

    NASA Astrophysics Data System (ADS)

    Wong, Cheuk-Ping

    2015-04-01

    eRD11 R&D program is focusing on the technology exploration for hadron particle identification in the forward region of Electron-Ion Collider (EIC) for studying quark and gluon distributions inside the nucleon. A modular Ring Imaging Cherenkov (RICH) detector has been extensively studied in Geant4-based simulation. The detector consists of a block of aerogel, Fresnel lens, four side mirrors and a photosensor plane. The simulated performance of this detector will be presented in this talk. For the eRD11 Collaboration.

  2. A carbon-rich region in Miller Range 091004 and implications for ureilite petrogenesis

    NASA Astrophysics Data System (ADS)

    Day, James M. D.; Corder, Christopher A.; Cartigny, Pierre; Steele, Andrew M.; Assayag, Nelly; Rumble, Douglas; Taylor, Lawrence A.

    2017-02-01

    Ureilite meteorites are partially melted asteroidal-peridotite residues, or more rarely, cumulates that can contain greater than three weight percent carbon. Here we describe an exceptional C-rich lithology, composed of 34 modal % large (up to 0.8 mm long) crystalline graphite grains, in the Antarctic ureilite meteorite Miller Range (MIL) 091004. This C-rich lithology is embedded within a silicate region composed dominantly of granular olivine with lesser quantities of low-Ca pyroxene, and minor FeNi metal, high-Ca pyroxene, spinel, schreibersite and troilite. Petrological evidence indicates that the graphite was added after formation of the silicate region and melt depletion. Associated with graphite is localized reduction of host olivine (Fo88-89) to nearly pure forsterite (Fo99), which is associated with FeNi metal grains containing up to 11 wt.% Si. The main silicate region is typical of ureilite composition, with highly siderophile element (HSE) abundances ∼0.3 × chondrite, 187Os/188Os of 0.1260-0.1262 and Δ17O of -0.81 ± 0.16‰. Mineral trace-element analyses reveal that the rare earth elements (REE) and the HSE are controlled by pyroxene and FeNi metal phases in the meteorite, respectively. Modeling of bulk-rock REE and HSE abundances indicates that the main silicate region experienced ∼6% silicate and >50% sulfide melt extraction, which is at the lower end of partial melt removal estimated for ureilites. Miller Range 091004 demonstrates heterogeneous distribution of carbon at centimeter scales and a limited range in Mg/(Mg + Fe) compositions of silicate grain cores, despite significant quantities of carbon. These observations demonstrate that silicate rim reduction was a rapid disequilibrium process, and came after silicate and sulfide melt removal in MIL 091004. The petrography and mineral chemistry of MIL 091004 is permissive of the graphite representing late-stage C-rich melt that pervaded silicates, or carbon that acted as a lubricant during

  3. Cortical rich club regions can organize state-dependent functional network formation by engaging in oscillatory behavior.

    PubMed

    Senden, Mario; Reuter, Niels; van den Heuvel, Martijn P; Goebel, Rainer; Deco, Gustavo

    2017-02-01

    Cognition is hypothesized to require the globally coordinated, functionally relevant integration of otherwise segregated information processing carried out by specialized brain regions. Studies of the macroscopic connectome as well as recent neuroimaging and neuromodeling research have suggested a densely connected collective of cortical hubs, termed the rich club, to provide a central workspace for such integration. In order for rich club regions to fulfill this role they must dispose of a dynamic mechanism by which they can actively shape networks of brain regions whose information processing needs to be integrated. A potential candidate for such a mechanism comes in the form of oscillations which might be employed to establish communication channels among relevant brain regions. We explore this possibility using an integrative approach combining whole-brain computational modeling with neuroimaging, wherein we investigate the local dynamics model brain regions need to exhibit in order to fit (dynamic) network behavior empirically observed for resting as well as a range of task states. We find that rich club regions largely exhibit oscillations during task performance but not during rest. Furthermore, oscillations exhibited by rich club regions can harmonize a set of asynchronous brain regions thus supporting functional coupling among them. These findings are in line with the hypothesis that the rich club can actively shape integration using oscillations.

  4. Using species distribution modeling to delineate the botanical richness patterns and phytogeographical regions of China

    NASA Astrophysics Data System (ADS)

    Zhang, Ming-Gang; Slik, J. W. Ferry; Ma, Ke-Ping

    2016-03-01

    The millions of plant specimens that have been collected and stored in Chinese herbaria over the past ~110 years have recently been digitized and geo-referenced. Here we use this unique collection data set for species distribution modeling exercise aiming at mapping & explaining the botanical richness; delineating China’s phytogeographical regions and investigating the environmental drivers of the dissimilarity patterns. We modeled distributions of 6,828 woody plants using MaxEnt and remove the collection bias using null model. The continental China was divided into different phytogeographical regions based on the dissimilarity patterns. An ordination and Getis-Ord Gi* hotspot spatial statistics were used to analysis the environmental drivers of the dissimilarity patterns. We found that the annual precipitation and temperature stability were responsible for observed species diversity. The mechanisms causing dissimilarity pattern seems differ among biogeographical regions. The identified environmental drivers of the dissimilarity patterns for southeast, southwest, northwest and northeast are annual precipitation, topographic & temperature stability, water deficit and temperature instability, respectively. For effective conservation of China’s plant diversity, identifying the historical refuge and protection of high diversity areas in each of the identified floristic regions and their subdivisions will be essential.

  5. Using species distribution modeling to delineate the botanical richness patterns and phytogeographical regions of China

    PubMed Central

    Zhang, Ming-Gang; Slik, J. W. Ferry; Ma, Ke-Ping

    2016-01-01

    The millions of plant specimens that have been collected and stored in Chinese herbaria over the past ~110 years have recently been digitized and geo-referenced. Here we use this unique collection data set for species distribution modeling exercise aiming at mapping & explaining the botanical richness; delineating China’s phytogeographical regions and investigating the environmental drivers of the dissimilarity patterns. We modeled distributions of 6,828 woody plants using MaxEnt and remove the collection bias using null model. The continental China was divided into different phytogeographical regions based on the dissimilarity patterns. An ordination and Getis-Ord Gi* hotspot spatial statistics were used to analysis the environmental drivers of the dissimilarity patterns. We found that the annual precipitation and temperature stability were responsible for observed species diversity. The mechanisms causing dissimilarity pattern seems differ among biogeographical regions. The identified environmental drivers of the dissimilarity patterns for southeast, southwest, northwest and northeast are annual precipitation, topographic & temperature stability, water deficit and temperature instability, respectively. For effective conservation of China’s plant diversity, identifying the historical refuge and protection of high diversity areas in each of the identified floristic regions and their subdivisions will be essential. PMID:26928763

  6. Projected impacts of climate change on regional capacities for global plant species richness

    PubMed Central

    Sommer, Jan Henning; Kreft, Holger; Kier, Gerold; Jetz, Walter; Mutke, Jens; Barthlott, Wilhelm

    2010-01-01

    Climate change represents a major challenge to the maintenance of global biodiversity. To date, the direction and magnitude of net changes in the global distribution of plant diversity remain elusive. We use the empirical multi-variate relationships between contemporary water-energy dynamics and other non-climatic predictor variables to model the regional capacity for plant species richness (CSR) and its projected future changes. We find that across all analysed Intergovernmental Panel on Climate Change emission scenarios, relative changes in CSR increase with increased projected temperature rise. Between now and 2100, global average CSR is projected to remain similar to today (+0.3%) under the optimistic B1/+1.8°C scenario, but to decrease significantly (−9.4%) under the ‘business as usual’ A1FI/+4.0°C scenario. Across all modelled scenarios, the magnitude and direction of CSR change are geographically highly non-uniform. While in most temperate and arctic regions, a CSR increase is expected, the projections indicate a strong decline in most tropical and subtropical regions. Countries least responsible for past and present greenhouse gas emissions are likely to incur disproportionately large future losses in CSR, whereas industrialized countries have projected moderate increases. Independent of direction, we infer that all changes in regional CSR will probably induce on-site species turnover and thereby be a threat to native floras. PMID:20335215

  7. Searching for high-K isomers in the proton-rich A ˜ 80 mass region

    NASA Astrophysics Data System (ADS)

    Bai, Zhi-Jun; Jiao, Chang-Feng; Gao, Yuan; Xu, Fu-Rong

    2016-09-01

    Configuration-constrained potential-energy-surface calculations have been performed to investigate the K isomerism in the proton-rich A ˜ 80 mass region. An abundance of high-K states are predicted. These high-K states arise from two and four-quasi-particle excitations, with Kπ = 8+ and Kπ = 16+, respectively. Their excitation energies are comparatively low, making them good candidates for long-lived isomers. Since most nuclei under study are prolate spheroids in their ground states, the oblate shapes of the predicted high-K states may indicate a combination of K isomerism and shape isomerism. Supported by National Key Basic Research Program of China (2013CB834402) and National Natural Science Foundation of China (11235001, 11320101004 and 11575007)

  8. Mutations of a residue within the polyproline-rich region of Env alter the replication rate and level of cytopathic effects in chimeric avian retroviral vectors.

    PubMed

    Chang, Kevin W; Barsov, Eugene V; Ferris, Andrea L; Hughes, Stephen H

    2005-08-01

    Previous attempts to extend the host range of the avian sarcoma/leukosis virus (ASLV)-based RCASBP vectors produced two viral vectors, RCASBP M2C (4070A) and RCASBP M2C (797-8), which replicate using the amphotropic murine leukemia virus 4070A Env protein (2). Both viruses were adapted to replicate efficiently in the avian cell line DF-1, but RCASBP M2C (4070A) caused extensive cytopathic effects (CPE) in DF-1 cells whereas RCASBP M2C (797-8) induced low levels of CPE. The two viruses differed only at amino acid 242 of the polyproline-rich region in the surface (SU) subunit of the Env protein. In RCASBP M2C (4070A), an isoleucine replaced the wild-type proline residue, whereas a threonine residue was found in RCASBP M2C (797-8). In the present study, we show that other amino acid substitutions at position 242 strongly influence the CPE and replication rate of the chimeric viruses. There was a correlation between the amount of unintegrated linear retroviral DNA present in infected DF-1 cells and the level of CPE. This suggests that there may be a role for superinfection in the CPE. The treatment of RCASBP M2C (4070A)-infected cells with dantrolene, which inhibits the release of calcium from the endoplasmic reticulum (ER), reduced the amount of CPE seen during infection with the highly cytotoxic virus. Dantrolene treatment did not appear to affect virus production, suggesting that Ca2+ release from the ER had a role in the CPE caused by these viruses.

  9. Untangling human and environmental effects on geographical gradients of mammal species richness: a global and regional evaluation.

    PubMed

    Torres-Romero, Erik Joaquín; Olalla-Tárraga, Miguel Á

    2015-05-01

    Different hypotheses (geographical, ecological, evolutionary or a combination of them) have been suggested to account for the spatial variation in species richness. However, the relative importance of environment and human impacts in explaining these patterns, either globally or at the biogeographical region level, remains largely unexplored. Here, we jointly evaluate how current environmental conditions and human impacts shape global and regional gradients of species richness in terrestrial mammals. We processed IUCN global distributional data for 3939 mammal species and a set of seven environmental and two human impact variables at a spatial resolution of 96.5 × 96.5 km. We used simple, multiple and partial regression techniques to evaluate environmental and human effects on species richness. Actual evapotranspiration (AET) is the main driver of mammal species richness globally. Together with our results at the biogeographical realm level, this lends strong support for the water-energy hypothesis (i.e. global diversity gradients are best explained by the interaction of water and energy, with a latitudinal shift in the relative importance of ambient energy vs. water availability as we move from the poles to the equator). While human effects on species richness are not easily detected at a global scale due to the large proportion of shared variance with the environment, these effects significantly emerge at the regional level. In the Nearctic, Palearctic and Oriental regions, the independent contribution of human impacts is almost as important as current environmental conditions in explaining richness patterns. The intersection of human impacts with climate drives the geographical variation in mammal species richness in the Palearctic, Nearctic and Oriental regions. Using a human accessibility variable, we show, for the first time, that the zones most accessible to humans are often those where we find lower mammal species richness.

  10. Proline is a protein solubilizing solute.

    PubMed

    Samuel, D; Kumar, T K; Jayaraman, G; Yang, P W; Yu, C

    1997-02-01

    The effect of proline on the prevention of trichloroacetic acid (TCA)-induced protein precipitation is studied. It is found that proline at high concentrations (> 4.0 M) completely prevents TCA-induced precipitation of hen egg white lysozyme. Other osmolytes such as ethylene glycol, glycerol and sucrose fail to prevent the TCA-induced precipitation of lysozyme. Viscosity and 1-anilino-8-naphthalene sulphonic acid binding experiments suggest that proline at high concentration forms an ordered supramolecular assembly. Proline is shown to increase the solubility of protein due to formation of such higher order assemblies. A model of the supra-molecular assembly of proline is proposed and a possible in vivo role of the increased levels of proline under water stress is discussed.

  11. Bridging Between Proline Structure, Functions, Metabolism, and Involvement in Organism Physiology.

    PubMed

    Saibi, Walid; Feki, Kaouthar; Yacoubi, Ines; Brini, Faiçal

    2015-08-01

    Much is now known about proline multifunctionality and metabolism; some aspects of its biological functions are still unclear. Here, we discuss some cases in the proline, structure, definition, metabolism, compartmentalization, accumulation, plausible functions and also its implication in homeostasis and organism physiology. Indeed, we report the role of proline in cellular homeostasis, including redox balance and energy status and their implication as biocatalyst for aldolase activity. Proline can act as a signaling molecule to modulate mitochondrial functions, influence cell proliferation or cell death, and trigger specific gene expression, which can be essential for plant recovery from stresses. Although, the regulation and the function of proline accumulation, during abiotic stresses, are not yet completely understood. The engineering of proline metabolism could lead to new opportunities to improve plant tolerance against environmental stresses. This atypical amino acid has a potential role in the toxicity during growth of some microorganism, vegetal, and mammalian species. Furthermore, we note that the purpose through the work is to provide a rich, concise, and mostly cohesive source on proline, considered as a platform and an anchor between several disciplines and biological functions.

  12. Regional species richness of families and the distribution of abundance and rarity in a local community of forest Hymenoptera

    NASA Astrophysics Data System (ADS)

    Ulrich, Werner

    2005-09-01

    Recent investigations about the relationship between the number of species of taxonomic lineages and regional patterns of species abundances gave indecisive results. Here, it is shown that mean densities of species of a species-rich community of forest Hymenoptera (673 species out of 25 families) were positively related to the number of European species per family. The fraction of abundant species per family declined and the fraction of rare species increased with species richness. Species rich families contained relatively more species, which were present in only one study year (occasional species), and relatively fewer species present during the whole study period (frequent species).

  13. Variation and Evolution in the Glutamine-Rich Repeat Region of Drosophila Argonaute-2

    PubMed Central

    Palmer, William H.; Obbard, Darren J.

    2016-01-01

    RNA interference pathways mediate biological processes through Argonaute-family proteins, which bind small RNAs as guides to silence complementary target nucleic acids . In insects and crustaceans Argonaute-2 silences viral nucleic acids, and therefore acts as a primary effector of innate antiviral immunity. Although the function of the major Argonaute-2 domains, which are conserved across most Argonaute-family proteins, are known, many invertebrate Argonaute-2 homologs contain a glutamine-rich repeat (GRR) region of unknown function at the N-terminus . Here we combine long-read amplicon sequencing of Drosophila Genetic Reference Panel (DGRP) lines with publicly available sequence data from many insect species to show that this region evolves extremely rapidly and is hyper-variable within species. We identify distinct GRR haplotype groups in Drosophila melanogaster, and suggest that one of these haplotype groups has recently risen to high frequency in a North American population. Finally, we use published data from genome-wide association studies of viral resistance in D. melanogaster to test whether GRR haplotypes are associated with survival after virus challenge. We find a marginally significant association with survival after challenge with Drosophila C Virus in the DGRP, but we were unable to replicate this finding using lines from the Drosophila Synthetic Population Resource panel. PMID:27317784

  14. Structural studies of the metal-rich region in the ternary Ta-Nb-S system

    SciTech Connect

    Yao, Xiaoqiang.

    1991-10-07

    Six new solid solution type compounds have been prepared using high temperature techniques and characterized by means of single crystal x-ray techniques during a study of the metal-rich region of the ternary Ta-Nb-S system. The structures of Nb{sub x}Ta{sub 11-x}S{sub 4} are reminiscent of niobium-rich sulfides, rather than of tantalum-rich sulfides. The coordinations of sulfur are capped trigonal prismatic while the metal coordinations are capped distorted cubic prismatic for Nb{sub x}Ta{sub 11-x}S{sub 4}, and capped distorted cubic prismatic and pentagonal prismatic for Nb{sub 12-x}Ta{sub x}S{sub 4}. The structures of Nb{sub x}Ta{sub 5-x}S{sub 2} contain homoatomic layers sequenced S-M3-M2-M1-M2-M3-S (M is mixed Nb, Ta) generating six-layer sheets, respectively. Weak S-S interactions at 3.26 and 3.19{Angstrom} between sheets contrast with the M-M binding within and between the sheets in these two novel layered compounds. The former are presumably responsible for the observed graphitic slippage of the samples. Nb{sub 21-x}Ta{sub x}S{sub 8} and Nb{sub x}Ta{sub 2-x}S are isostructural with Nb{sub 21}S{sub 8} and Ta{sub 2}S, respectively. Extended Hueckel band calculations were carried out for two layered compounds, Nb{sub x}Ta{sub 5-x}S{sub 2} (x {approx} 1.72) and Nb{sub x}ta{sub 2-x}S (x {approx} 0.95). Based upon band calculations metallic properties can be expected for these two layered compounds. The relative preference of the metal sites for the two metal elements (Ta, Nb) in two layered compounds is explained by the results of the band calculations. 17 figs., 31 tabs., 80 refs.

  15. Proline transport and stress tolerance of ammonia-insensitive mutants of the PUT4-encoded proline-specific permease in yeast.

    PubMed

    Poole, Kate; E Walker, Michelle; Warren, Tristan; Gardner, Jennie; McBryde, Colin; de Barros Lopes, Miguel; Jiranek, Vladimir

    2009-12-01

    The imino amino acid, proline, has roles in both cellular nutrition and response to stress. Proline uptake in Saccharomyces cerevisiae is largely mediated by a high affinity, specific permease, Put4p, and a low affinity general amino acid permease, Gap1p. Both are subject to nitrogen catabolite repression (NCR) and nitrogen catabolite inactivation (NCI). In order for proline to be fully exploited, its transport must be derepressed, as occurs upon depletion of preferred nitrogen sources, and molecular oxygen must be present to allow the first step of catabolism via proline oxidase. This study focuses on the isolation of variants of Put4p, which are insensitive to repression by a preferred nitrogen source (ammonia) and their subsequent effect on proline transport and stress tolerance. Specific amino acid residues in the carboxy-terminal region of Put4p were targeted by site-directed mutagenesis. Substitution at Serine(605), a potential phosphorylation target, led to the amelioration of ammonia-induced down-regulation of Put4p. When combined with a promoter mutation (-160), the S(605)A mutation resulted in increased proline uptake and accumulation. This increase in proline accumulation was associated with increased cell viability in conditions of high temperature and osmotic stress raising possible benefits in industrial fermentation applications.

  16. Role of proline under changing environments

    PubMed Central

    Hayat, Shamsul; Hayat, Qaiser; Alyemeni, Mohammed Nasser; Wani, Arif Shafi; Pichtel, John; Ahmad, Aqil

    2012-01-01

    When exposed to stressful conditions, plants accumulate an array of metabolites, particularly amino acids. Amino acids have traditionally been considered as precursors to and constituents of proteins, and play an important role in plant metabolism and development. A large body of data suggests a positive correlation between proline accumulation and plant stress. Proline, an amino acid, plays a highly beneficial role in plants exposed to various stress conditions. Besides acting as an excellent osmolyte, proline plays three major roles during stress, i.e., as a metal chelator, an antioxidative defense molecule and a signaling molecule. Review of the literature indicates that a stressful environment results in an overproduction of proline in plants which in turn imparts stress tolerance by maintaining cell turgor or osmotic balance; stabilizing membranes thereby preventing electrolyte leakage; and bringing concentrations of reactive oxygen species (ROS) within normal ranges, thus preventing oxidative burst in plants. Reports indicate enhanced stress tolerance when proline is supplied exogenously at low concentrations. However, some reports indicate toxic effects of proline when supplied exogenously at higher concentrations. In this article, we review and discuss the effects of exogenous proline on plants exposed to various abiotic stresses. Numerous examples of successful application of exogenous proline to improve stress tolerance are presented. The roles played by exogenous proline under varying environments have been critically examined and reviewed. PMID:22951402

  17. Does the colonization of new biogeographic regions influence the diversification and accumulation of clade richness among the Corvides (Aves: Passeriformes)?

    PubMed

    Kennedy, Jonathan D; Borregaard, Michael K; Jønsson, Knud A; Holt, Ben; Fjeldså, Jon; Rahbek, Carsten

    2017-01-01

    Regional variation in clade richness can be vast, reflecting differences in the dynamics of historical dispersal and diversification among lineages. Although it has been proposed that dispersal into new biogeographic regions may facilitate diversification, to date there has been limited assessment of the importance of this process in the generation, and maintenance, of broad-scale biodiversity gradients. To address this issue, we analytically derive biogeographic regions for a global radiation of passerine birds (the Corvides, c. 790 species) that are highly variable in the geographic and taxonomic distribution of species. Subsequently, we determine rates of historical dispersal between regions, the dynamics of diversification following regional colonization, and spatial variation in the distribution of species that differ in their rates of lineage diversification. The results of these analyses reveal spatiotemporal differences in the build-up of lineages across regions. The number of regions occupied and the rate of transition between regions both predict family richness well, indicating that the accumulation of high clade richness is associated with repeated expansion into new geographic areas. However, only the largest family (the Corvidae) had significantly heightened rates of both speciation and regional transition, implying that repeated regional colonization is not a general mechanism promoting lineage diversification among the Corvides.

  18. A census of glutamine/asparagine-rich regions: Implications for their conserved function and the prediction of novel prions

    PubMed Central

    Michelitsch, Melissa D.; Weissman, Jonathan S.

    2000-01-01

    Glutamine/asparagine (Q/N)-rich domains have a high propensity to form self-propagating amyloid fibrils. This phenomenon underlies both prion-based inheritance in yeast and aggregation of a number of proteins involved in human neurodegenerative diseases. To examine the prevalence of this phenomenon, complete proteomic sequences of 31 organisms and several incomplete proteomic sequences were examined for Q/N-rich regions. We found that Q/N-rich regions are essentially absent from the thermophilic bacterial and archaeal proteomes. Moreover, the average Q/N content of the proteins in these organisms is markedly lower than in mesophilic bacteria and eukaryotes. Mesophilic bacterial proteomes contain a small number (0–4) of proteins with Q/N-rich regions. Remarkably, Q/N-rich domains are found in a much larger number of eukaryotic proteins (107–472 per proteome) with diverse biochemical functions. Analyses of these regions argue they have been evolutionarily selected perhaps as modular “polar zipper” protein–protein interaction domains. These data also provide a large pool of potential novel prion-forming proteins, two of which have recently been shown to behave as prions in yeast, thus suggesting that aggregation or prion-like regulation of protein function may be a normal regulatory process for many eukaryotic proteins with a wide variety of functions. PMID:11050225

  19. Selective vulnerability of Rich Club brain regions is an organizational principle of structural connectivity loss in Huntington's disease.

    PubMed

    McColgan, Peter; Seunarine, Kiran K; Razi, Adeel; Cole, James H; Gregory, Sarah; Durr, Alexandra; Roos, Raymund A C; Stout, Julie C; Landwehrmeyer, Bernhard; Scahill, Rachael I; Clark, Chris A; Rees, Geraint; Tabrizi, Sarah J

    2015-11-01

    Huntington's disease can be predicted many years before symptom onset, and thus makes an ideal model for studying the earliest mechanisms of neurodegeneration. Diffuse patterns of structural connectivity loss occur in the basal ganglia and cortex early in the disease. However, the organizational principles that underlie these changes are unclear. By understanding such principles we can gain insight into the link between the cellular pathology caused by mutant huntingtin and its downstream effect at the macroscopic level. The 'rich club' is a pattern of organization established in healthy human brains, where specific hub 'rich club' brain regions are more highly connected to each other than other brain regions. We hypothesized that selective loss of rich club connectivity might represent an organizing principle underlying the distributed pattern of structural connectivity loss seen in Huntington's disease. To test this hypothesis we performed diffusion tractography and graph theoretical analysis in a pseudo-longitudinal study of 50 premanifest and 38 manifest Huntington's disease participants compared with 47 healthy controls. Consistent with our hypothesis we found that structural connectivity loss selectively affected rich club brain regions in premanifest and manifest Huntington's disease participants compared with controls. We found progressive network changes across controls, premanifest Huntington's disease and manifest Huntington's disease characterized by increased network segregation in the premanifest stage and loss of network integration in manifest disease. These regional and whole brain network differences were highly correlated with cognitive and motor deficits suggesting they have pathophysiological relevance. We also observed greater reductions in the connectivity of brain regions that have higher network traffic and lower clustering of neighbouring regions. This provides a potential mechanism that results in a characteristic pattern of structural

  20. Optimising Regionalisation Techniques: Identifying Centres of Endemism in the Extraordinarily Endemic-Rich Cape Floristic Region.

    PubMed

    Bradshaw, Peter L; Colville, Jonathan F; Linder, H Peter

    2015-01-01

    We used a very large dataset (>40% of all species) from the endemic-rich Cape Floristic Region (CFR) to explore the impact of different weighting techniques, coefficients to calculate similarity among the cells, and clustering approaches on biogeographical regionalisation. The results were used to revise the biogeographical subdivision of the CFR. We show that weighted data (down-weighting widespread species), similarity calculated using Kulczinsky's second measure, and clustering using UPGMA resulted in the optimal classification. This maximized the number of endemic species, the number of centres recognized, and operational geographic units assigned to centres of endemism (CoEs). We developed a dendrogram branch order cut-off (BOC) method to locate the optimal cut-off points on the dendrogram to define candidate clusters. Kulczinsky's second measure dendrograms were combined using consensus, identifying areas of conflict which could be due to biotic element overlap or transitional areas. Post-clustering GIS manipulation substantially enhanced the endemic composition and geographic size of candidate CoEs. Although there was broad spatial congruence with previous phytogeographic studies, our techniques allowed for the recovery of additional phytogeographic detail not previously described for the CFR.

  1. Optimising Regionalisation Techniques: Identifying Centres of Endemism in the Extraordinarily Endemic-Rich Cape Floristic Region

    PubMed Central

    Bradshaw, Peter L.; Colville, Jonathan F.; Linder, H. Peter

    2015-01-01

    We used a very large dataset (>40% of all species) from the endemic-rich Cape Floristic Region (CFR) to explore the impact of different weighting techniques, coefficients to calculate similarity among the cells, and clustering approaches on biogeographical regionalisation. The results were used to revise the biogeographical subdivision of the CFR. We show that weighted data (down-weighting widespread species), similarity calculated using Kulczinsky’s second measure, and clustering using UPGMA resulted in the optimal classification. This maximized the number of endemic species, the number of centres recognized, and operational geographic units assigned to centres of endemism (CoEs). We developed a dendrogram branch order cut-off (BOC) method to locate the optimal cut-off points on the dendrogram to define candidate clusters. Kulczinsky’s second measure dendrograms were combined using consensus, identifying areas of conflict which could be due to biotic element overlap or transitional areas. Post-clustering GIS manipulation substantially enhanced the endemic composition and geographic size of candidate CoEs. Although there was broad spatial congruence with previous phytogeographic studies, our techniques allowed for the recovery of additional phytogeographic detail not previously described for the CFR. PMID:26147438

  2. Stereoselective Synthesis of Quaternary Proline Analogues

    PubMed Central

    Calaza, M. Isabel

    2009-01-01

    This review describes available methods for the diastereoselective and asymmetric synthesis of quaternary prolines. The focus is on the preparation of α-functionalized prolines with the pyrrolidine moiety not embedded in a polycyclic frame. The diverse synthetic approaches are classified according to the bond which is formed to complete the quaternary skeleton. PMID:19655047

  3. Proline accumulation in plants: a review.

    PubMed

    Verbruggen, Nathalie; Hermans, Christian

    2008-11-01

    Proline (Pro) accumulation is a common physiological response in many plants in response to a wide range of biotic and abiotic stresses. Controversy has surrounded the possible role(s) of proline accumulation. In this review, knowledge on the regulation of Pro metabolism during development and stress, results of genetic manipulation of Pro metabolism and current debate on Pro toxicity in plants are presented.

  4. Monoclonal antibody specific to a subclass of polyproline-Arg motif provides evidence for the presence of an snRNA-free spliceosomal Sm protein complex in vivo: implications for molecular interactions involving proline-rich sequences of Sm B/B' proteins.

    PubMed

    Filali, M; Qiu, J; Awasthi, S; Fischer, U; Monos, D; Kamoun, M

    1999-08-01

    The human spliceosomal Sm B/B' proteins are essential for the biogenesis of the snRNP particles. B/B' proteins contain several clusters of the PPPPGM/IR sequence, which occurs within the C-terminus of Sm B/B'. This sequence is very similar to the PPPPPGHR sequence of the cytoplasmic tail of the CD2 receptor and closely resembles the class II of SH3 ligands, suggesting a similarly important role. We report that a monoclonal antibody (3E10) against the PPPPPGHR sequence recognizes spliceosomal Sm B/B' proteins. Proteins that are specifically immunoprecipitated by 3E10 include Sm B, B', D1, D2, D3, E, F, and G. However, unlike Y12 and other anti-Sm immunoprecipitates, 3E10 immunoprecipitates appear to lack the U1 snRNP-specific proteins A and C and U snRNAs. These findings indicate that 3E10 recognizes a subset of Sm protein core and suggest the presence of snRNA-free Sm protein complex(es) in vivo. We propose that the epitope binding for 3E10 may become unaccessible upon interactions of Sm proteins and their subsequent incorporation into the core particles. The Sm proline-rich sequences may have an important role in mediating protein-protein interactions necessary for the proper snRNP core assembly or function, or both. To our knowledge, 3E10 is the first well characterized mAb specific for a subclass of polyproline-arg motif recognizing Sm B/B' and CD2 proteins. 3E10 antibody can be used to further characterize the nature of protein components in the snRNA-free Sm subcore protein complex(es) that are formed during the snRNP core assembly steps.

  5. High-resolution high-performance liquid chromatography with electrospray ionization mass spectrometry and tandem mass spectrometry characterization of a new isoform of human salivary acidic proline-rich proteins named Roma-Boston Ser₂₂ (Phos) → Phe variant.

    PubMed

    Iavarone, Federica; D'Alessandro, Alfredo; Tian, Na; Cabras, Tiziana; Messana, Irene; Helmerhorst, Eva J; Oppenheim, Frank G; Castagnola, Massimo

    2014-07-01

    During a survey of human saliva by a top-down reversed-phase high-performance liquid chromatography with electrospray ionization mass spectrometry approach, two proteins eluting at 27.4 and 28.4 min, with average masses of 15 494 ± 1 and 11 142 ± 1 Da, were detected in a subject from Boston. The Δmass value (4352 Da) of the two proteins was similar to the difference in mass values between intact (150 amino acids, [a.a.]) and truncated acidic proline-rich proteins (aPRPs; 106 a.a.) suggesting an a.a. substitution in the first 106 residues resulting in a strong reduction in polarity, since under the same experimental conditions aPRPs eluted at ∼22.5 min (intact) and 23.5 min (truncated forms). Manual inspection of the high-resolution high-performance liquid chromatography with electrospray ionization tandem mass spectra of the truncated isoform showed the replacement of the phosphorylated Ser-22 in PRP-3 with a Phe residue. Inspection of the tandem mass spectra of the intact isoform confirmed the substitution, which is allowed by the code transition TCT→TTT and is in agreement with the dramatic increase in elution time. The isoform was also detected in two other subjects, one from Boston (unrelated to the previous) and one from Rome. For this reason we propose to name this variant PRP-1 (PRP-3) RB (Roma-Boston) Ser22 (phos)→Phe.

  6. PCR amplification of GC-rich DNA regions using the nucleotide analog N4-methyl-2'-deoxycytidine 5'-triphosphate.

    PubMed

    Flores-Juárez, Cyntia R; González-Jasso, Eva; Antaramian, Anaid; Pless, Reynaldo C

    2016-10-01

    GC-rich DNA regions were PCR-amplified with Taq DNA polymerase using either the canonical set of deoxynucleoside triphosphates or mixtures in which the dCTP had been partially or completely replaced by its N4-methylated analog, N4-methyl-2'-deoxycytidine 5'-triphosphate (N4me-dCTP). In the case of a particularly GC-rich region (78.9% GC), the PCR mixtures containing N4me-dCTP produced the expected amplicon in high yield, while mixtures containing the canonical set of nucleotides produced numerous alternative amplicons. For another GC-rich DNA region (80.6% GC), the target amplicon was only generated by re-amplifying a gel-purified sample of the original amplicon with N4me-dCTP-containing PCR mixtures. In a direct PCR comparison on a highly GC-rich template, mixtures containing N4me-dCTP clearly performed better than did solutions containing the canonical set of nucleotides mixed with various organic additives (DMSO, betaine, or ethylene glycol) that have been reported to resolve or alleviate problems caused by secondary structures in the DNA. This nucleotide analog was also tested in PCR amplification of DNA regions with intermediate GC content, producing the expected amplicon in each case with a melting temperature (Tm) clearly below the Tm of the same amplicon synthesized exclusively with the canonical bases.

  7. Comparative genomics of Lupinus angustifolius gene-rich regions: BAC library exploration, genetic mapping and cytogenetics

    PubMed Central

    2013-01-01

    Background The narrow-leafed lupin, Lupinus angustifolius L., is a grain legume species with a relatively compact genome. The species has 2n = 40 chromosomes and its genome size is 960 Mbp/1C. During the last decade, L. angustifolius genomic studies have achieved several milestones, such as molecular-marker development, linkage maps, and bacterial artificial chromosome (BAC) libraries. Here, these resources were integratively used to identify and sequence two gene-rich regions (GRRs) of the genome. Results The genome was screened with a probe representing the sequence of a microsatellite fragment length polymorphism (MFLP) marker linked to Phomopsis stem blight resistance. BAC clones selected by hybridization were subjected to restriction fingerprinting and contig assembly, and 232 BAC-ends were sequenced and annotated. BAC fluorescence in situ hybridization (BAC-FISH) identified eight single-locus clones. Based on physical mapping, cytogenetic localization, and BAC-end annotation, five clones were chosen for sequencing. Within the sequences of clones that hybridized in FISH to a single-locus, two large GRRs were identified. The GRRs showed strong and conserved synteny to Glycine max duplicated genome regions, illustrated by both identical gene order and parallel orientation. In contrast, in the clones with dispersed FISH signals, more than one-third of sequences were transposable elements. Sequenced, single-locus clones were used to develop 12 genetic markers, increasing the number of L. angustifolius chromosomes linked to appropriate linkage groups by five pairs. Conclusions In general, probes originating from MFLP sequences can assist genome screening and gene discovery. However, such probes are not useful for positional cloning, because they tend to hybridize to numerous loci. GRRs identified in L. angustifolius contained a low number of interspersed repeats and had a high level of synteny to the genome of the model legume G. max. Our results showed that

  8. Production and identification of new, neutron-rich nuclei in the [sup 208]Pb region

    SciTech Connect

    Rykaczewski, K. ); Szerypo, J.; Evensen, A.-H.; Kugler, E.; Lettry, J.; Ravn, H. ); Kurpeta, J.; Pkochocki, A.; Karny, M.; Szerypo, J. ); Szerypo, J. ); Andreyev, H.; Huyse, M.; Wo uml; hr, A. ); Aystuml, J.; Nieminen, A.; Huhta, M. ); Walter, G. ) Hoff, P. )

    1998-12-01

    The recently developed methods allowing the experimental studies on new neutron-rich nuclei beyond doubly-magic [sup 208]Pb are briefly described. An identification of new neutron-rich isotopes [sup 215]Pb and [sup 217]Bi, and new decay properties of [sup 216]Bi studied by means of a pulsed release element selective technique at PS Booster-ISOLDE are reported. [copyright] [ital 1998 American Institute of Physics.

  9. Genetic analysis of proline concentration under osmotic stress in sunflower (Helianthus annuus L.)

    PubMed Central

    Khalil, Farghama; Rauf, Saeed; Monneveux, Philippe; Anwar, Shoaib; Iqbal, Zafar

    2016-01-01

    Proline concentration has been often suggested as an indicator of osmotic stress. A better understanding of the genetics of this trait is however needed. In the present study, proline concentration has been assessed, together with root and stem growth, potassium, calcium and total soluble sugars concentration and stress injury symptoms, in seedlings of sunflower hybrids and their parents grown under control and osmotic conditions. Proline strongly accumulated with osmotic stress. Its concentration exhibited a large variation among genotypes and was higher in hybrids than in parental lines. A positive association was noted between proline concentration and osmotic adjustment that was reflected in a reduction of osmotic stress induced injury, as showed by the reduced number of calli in the hybrids with higher proline concentration. Broad and narrow sense heritability was higher under osmotic stress suggesting applying the selection in osmotic stress condition. In the control treatment, dominance effects explained most of the genetic variation for proline concentration while under osmotic stress both dominance and additive variance were high. The importance of dominance and additive effects suggested that several genomic regions are controlling this trait. Good general combiners, presumably carrying positive additive alleles affecting proline concentration, were identified. PMID:27795671

  10. Exotic clusters in an unbound region of light neutron-rich systems

    NASA Astrophysics Data System (ADS)

    Ito, Makoto

    2009-10-01

    In light neutron-rich systems, many kinds of molecular structures are discussed from the view point of the clustering phenomena.In particular, much attention has been concentrated on Be isotopes. The molecular orbital (MO), such as &-circ; and &+circ; associated with the covalent binding of atomic molecules, have been shown to give a good description for the low-lying states of these isotopes. In their highly-excited states, furthermore, recent experiments revealed the existence of the interesting resonant states which dominantly decay to the ^6,8He fragments. In this report, we show the unified study of the exotic structures of ^12Be=α+α+4N in an unbound region and the α+^6,8He resonant scattering. We applied the generalized two-center cluster model in which the covalent MO and the atomic orbital (AO) configurations with ^xHe+^yHe could be described in a unified manner. First, we calculated the energy spectra below an α decay-threshold. The (π32^-)^2 (σ12^+)^2 configuration corresponding to ν(0p)^4(sd)^2 becomes the ground state, while (π3 2^-)^2(π12^-)^2 having a large overlap with ν(0p)^6 appears as the first excited state. The rotational band of the ground state reaches to the maximum spin of J^π = 8^+. This result means that the magicity of N=8 is broken in ^12Be due to the formation of (π3 2^-)^2(σ12^+)^2. Next, we solved the scattering problem of α+^8He and identified the several resonance poles. In the continuum region, we found the rotational bands having the AO configurations of α+^8He, ^6He+^6He, and ^5He+^7 He. Furthermore, a much more exotic band appears in the same energy region. In this band, two valence neutrons are localized at individual α-cores (the ^5He+^5He cluster), while the other two neutrons form the covalent &+circ;- bonding between two ^5He clusters; hence, it has a ``hybrid structure'' between the MO configuration and the AO one. In the J^π=0^+ state, it is strongly excited by the two-neutron transfer reaction, α+^8He

  11. Effective prevention of chill-haze in beer using an acid proline-specific endoprotease from Aspergillus niger.

    PubMed

    Lopez, Michel; Edens, Luppo

    2005-10-05

    Chill-haze formation during beer production is known to involve polyphenols that interact with proline-rich proteins. We hypothesized that incubating beer wort with a proline-specific protease would extensively hydrolyze these proline-rich proteins, yielding a peptide fraction that is unable to form a haze. Predigestion of the proline-rich wheat gliadin with different proteases pointed toward a strong haze-suppressing effect by a proline-specific enzyme. This finding was confirmed in small-scale brewing experiments using a recently identified proline-specific protease with an acidic pH optimum. Subsequent pilot plant trials demonstrated that, upon its addition during the fermentation phase of beer brewing, even low levels of this acidic enzyme effectively prevented chill-haze formation in bottled beer. Results of beer foam stability measurements indicated that the enzyme treatment leaves the beer foam almost unaffected. In combination with the enzyme's cost-effectiveness and regulatory status, these preliminary test results seem to favor further industrial development of this enzymatic beer stabilization method.

  12. A highly selective receptor for zwitterionic proline.

    PubMed

    Temprano, Álvaro G; Monleón, Laura M; Rubio, Omayra H; Rubio, Luis Simón; Pérez, Asunción B; Sanz, Francisca; Morán, Joaquín R

    2016-01-28

    A chiral chromane receptor has been synthesized which mimics the oxyanion hole of the enzymes. In this receptor H-bonds and cation-π interactions team up to generate an apolar host-guest complex with zwitterionic proline. This complex allows the extraction of only proline to a chloroform phase, while no other natural amino acids are extracted. Due to the chiral nature of the receptor, enantioselective extraction from the aqueous proline solution to a chloroform phase takes place. L-Proline provided an easy method to resolve the receptor racemic mixture, while anisotropic effects, NOE and CD studies revealed the absolute configuration of the receptor. Modelling studies also support the proposed structures. The presence of an oxyanion-hole motif in this structure was corroborated by X-ray diffraction studies.

  13. Metabolism of Proline, Glutamate, and Ornithine in Proline Mutant Root Tips of Zea mays (L.)

    PubMed Central

    Dierks-Ventling, Christa; Tonelli, Chiara

    1982-01-01

    In excised pro1-1 mutant and corresponding normal type roots of Zea mays L. the uptake and interconversion of [14C]proline, [14C]glutamic acid, [14C]glutamine, and [14C]ornithine and their utilization for protein synthesis was measured with the intention of finding an explanation for the proline requirement of the mutant. Uptake of these four amino acids, with the exception of proline, was the same in mutant and normal roots, but utilization differed. Higher than normal utilization rates for proline and glutamic acid were noted in mutant roots leading to increased CO2 production, free amino acid interconversion, and protein synthesis. Proline was synthesized from either glutamic acid (or glutamine) or ornithine in both mutant and normal roots; it did not accumulate but rather was used for protein synthesis. Ornithine was not a good precursor for proline in either system, but was preferentially converted to arginine and glutamine, particularly in mutant roots. The pro1-1 mutant was thus not deficient in its ability to make proline. Based on these findings, and on the fact that ornithine, arginine, glutamic acid and aspartic acid are elevated as free amino acids in mutant roots, it is suggested that in the pro1-1 mutant proline catabolism prevails over proline synthesis. PMID:16662144

  14. ESTIMATING REGIONAL SPECIES RICHNESS USING A LIMITED NUMBER OF SURVEY UNITS

    EPA Science Inventory

    The accurate and precise estimation of species richness at large spatial scales using a limited number of survey units is of great significance for ecology and biodiversity conservation. We used the distribution data of native fish and resident breeding bird species compiled for ...

  15. On the Split Personality of Penultimate Proline

    NASA Astrophysics Data System (ADS)

    Glover, Matthew S.; Shi, Liuqing; Fuller, Daniel R.; Arnold, Randy J.; Radivojac, Predrag; Clemmer, David E.

    2015-03-01

    The influence of the position of the amino acid proline in polypeptide sequences is examined by a combination of ion mobility spectrometry-mass spectrometry (IMS-MS), amino acid substitutions, and molecular modeling. The results suggest that when proline exists as the second residue from the N-terminus (i.e., penultimate proline), two families of conformers are formed. We demonstrate the existence of these families by a study of a series of truncated and mutated peptides derived from the 11-residue peptide Ser1-Pro2-Glu3-Leu4-Pro5-Ser6-Pro7-Gln8-Ala9-Glu10-Lys11. We find that every peptide from this sequence with a penultimate proline residue has multiple conformations. Substitution of Ala for Pro residues indicates that multiple conformers arise from the cis- trans isomerization of Xaa1-Pro2 peptide bonds as Xaa-Ala peptide bonds are unlikely to adopt the cis isomer, and examination of spectra from a library of 58 peptides indicates that ~80% of sequences show this effect. A simple mechanism suggesting that the barrier between the cis- and trans-proline forms is lowered because of low steric impedance is proposed. This observation may have interesting biological implications as well, and we note that a number of biologically active peptides have penultimate proline residues.

  16. Proline Conformation in a Functional Tau Fragment.

    PubMed

    Ahuja, Puneet; Cantrelle, François-Xavier; Huvent, Isabelle; Hanoulle, Xavier; Lopez, Juan; Smet, Caroline; Wieruszeski, Jean-Michel; Landrieu, Isabelle; Lippens, G

    2016-01-16

    The conformational state of distinct prolines can determine the folding of a protein but equally other biological processes when coupled to a conformation-sensitive secondary reaction. For the neuronal tau protein, the importance of proline conformation is underscored by its interaction with different prolyl cis/trans isomerases. The proline conformation would gain even further importance after phosphorylation of the preceding residue by various proline-directed kinases. A number of molecular diseases including Alzheimer's disease and traumatic brain injury were thereby recently qualified as "cistauosis", as they would imply a cis conformation for the pThr231-Pro232 prolyl bond. We here investigate by NMR spectroscopy the conformation of all prolines in a functional Tau fragment, Tau[208-324]. Although we can detect and identify some minor conformers in the cis form, we show that all prolines are for over 90% in the trans conformation. Phosphorylation by CDK2/CycA3, which notably leads to complete modification of the Thr231 residue, does not change this conclusion. Our data hence disagree with the notion that specific prolyl bonds in tau would adopt preferentially the cis conformation.

  17. On the split personality of penultimate proline.

    PubMed

    Glover, Matthew S; Shi, Liuqing; Fuller, Daniel R; Arnold, Randy J; Radivojac, Predrag; Clemmer, David E

    2015-03-01

    The influence of the position of the amino acid proline in polypeptide sequences is examined by a combination of ion mobility spectrometry-mass spectrometry (IMS-MS), amino acid substitutions, and molecular modeling. The results suggest that when proline exists as the second residue from the N-terminus (i.e., penultimate proline), two families of conformers are formed. We demonstrate the existence of these families by a study of a series of truncated and mutated peptides derived from the 11-residue peptide Ser(1)-Pro(2)-Glu(3)-Leu(4)-Pro(5)-Ser(6)-Pro(7)-Gln(8)-Ala(9)-Glu(10)-Lys(11). We find that every peptide from this sequence with a penultimate proline residue has multiple conformations. Substitution of Ala for Pro residues indicates that multiple conformers arise from the cis-trans isomerization of Xaa(1)-Pro(2) peptide bonds as Xaa-Ala peptide bonds are unlikely to adopt the cis isomer, and examination of spectra from a library of 58 peptides indicates that ~80% of sequences show this effect. A simple mechanism suggesting that the barrier between the cis- and trans-proline forms is lowered because of low steric impedance is proposed. This observation may have interesting biological implications as well, and we note that a number of biologically active peptides have penultimate proline residues.

  18. The highly recombinogenic bz locus lies in an unusually gene-rich region of the maize genome.

    PubMed

    Fu, H; Park, W; Yan, X; Zheng, Z; Shen, B; Dooner, H K

    2001-07-17

    The bronze (bz) locus exhibits the highest rate of recombination of any gene in higher plants. To investigate the possible basis of this high rate of recombination, we have analyzed the physical organization of the region around the bz locus. Two adjacent bacterial artificial chromosome clones, comprising a 240-kb contig centered around the Bz-McC allele, were isolated, and 60 kb of contiguous DNA spanning the two bacterial artificial chromosome clones was sequenced. We find that the bz locus lies in an unusually gene-rich region of the maize genome. Ten genes, at least eight of which are shown to be transcribed, are contained in a 32-kb stretch of DNA that is uninterrupted by retrotransposons. We have isolated nearly full length cDNAs corresponding to the five proximal genes in the cluster. The average intertranscript distance between them is just 1 kb, revealing a surprisingly compact packaging of adjacent genes in this part of the genome. At least 11 small insertions, including several previously described miniature inverted repeat transposable elements, were detected in the introns and 3' untranslated regions of genes and between genes. The gene-rich region is flanked at the proximal and distal ends by retrotransposon blocks. Thus, the maize genome appears to have scattered regions of high gene density similar to those found in other plants. The unusually high rate of intragenic recombination seen in bz may be related to the very high gene density of the region.

  19. Characteristics of solar coronal source regions producing He-3-rich particle events

    NASA Technical Reports Server (NTRS)

    Kahler, S. W.; Lin, R. P.; Reames, D. V.; Stone, R. G.; Liggett, M.

    1987-01-01

    H-alpha, X-ray, and kilometric radio data are used to examine solar coronal activity associated with energetic He-3-rich particle events observed near earth. The basis of the study is the 12 He-3-rich events observed in association with impulsive 2-100 keV electron events reported by Reames et al. (1985). In three or four events, associated H-alpha or X-ray flares were found, and in two events even the metric type III bursts were weak or absent. The measured low energy electron spectra for these events show no evidence of a flattening due to Coulomb collisional losses. These results and several other recent findings are consistent with the idea that the He-3/electron events are due to particle acceleration in the corona well above the associated H-alpha and X-ray flares.

  20. Proline Metabolism and Its Implications for Plant-Environment Interaction

    PubMed Central

    Verslues, Paul E.

    2010-01-01

    Proline has long been known to accumulate in plants experiencing water limitation and this has driven studies of proline as a beneficial solute allowing plants to increase cellular osmolarity during water limitation. Proline metabolism also has roles in redox buffering and energy transfer and is involved in plant pathogen interaction and programmed cell death. Some of these unique roles of proline depend on the properties of proline itself, whereas others depend on the “proline cycle” of coordinated proline synthesis in the chloroplast and cytoplasm with proline catabolism in the mitochondria. The regulatory mechanisms controlling proline metabolism, intercellular and intracellular transport and connections of proline to other metabolic pathways are all important to the in vivo functions of proline metabolism. Connections of proline metabolism to the oxidative pentose phosphate pathway and glutamate-glutamine metabolism are of particular interest. The N-acetyl glutamate pathway can also produce ornithine and, potentially, proline but its role and activity are unclear. Use of model systems such as Arabidopsis thaliana to better understand both these long studied and newly emerging functions of proline can help in the design of next-generation experiments testing whether proline metabolism is a promising metabolic engineering target for improving stress resistance of economically important plants. PMID:22303265

  1. Comparative analysis of chicken chromosome 28 provides new clues to the evolutionary fragility of gene-rich vertebrate regions

    PubMed Central

    Gordon, Laurie; Yang, Shan; Tran-Gyamfi, Mary; Baggott, Dan; Christensen, Mari; Hamilton, Aaron; Crooijmans, Richard; Groenen, Martien; Lucas, Susan; Ovcharenko, Ivan; Stubbs, Lisa

    2007-01-01

    The chicken genome draft sequence has provided a valuable resource for studies of an important agricultural and experimental model species and an important data set for comparative analysis. However, some of the most gene-rich segments are missing from chicken genome draft assemblies, limiting the analysis of a substantial number of genes and preventing a closer look at regions that are especially prone to syntenic rearrangements. To facilitate the functional and evolutionary analysis of one especially gene-rich, rearrangement-prone genomic region, we analyzed sequence from BAC clones spanning chicken microchromosome GGA28; as a complement we also analyzed a gene-sparse, stable region from GGA11. In these two regions we documented the conservation and lineage-specific gain and loss of protein-coding genes and precisely mapped the locations of 31 major human-chicken syntenic breakpoints. Altogether, we identified 72 lineage-specific genes, many of which are found at or near syntenic breaks, implicating evolutionary breakpoint regions as major sites of genetic innovation and change. Twenty-two of the 31 breakpoint regions have been reused repeatedly as rearrangement breakpoints in vertebrate evolution. Compared with stable GC-matched regions, GGA28 is highly enriched in CpG islands, as are break-prone intervals identified elsewhere in the chicken genome; evolutionary breakpoints are further enriched in GC content and CpG islands, highlighting a potential role for these features in genome instability. These data support the hypothesis that chromosome rearrangements have not occurred randomly over the course of vertebrate evolution but are focused preferentially within “fragile” regions with unusual DNA sequence characteristics. PMID:17921355

  2. Solid-Phase Synthesis of Amine/Carboxyl Substituted Prolines and Proline Homologues: Scope and Limitations.

    PubMed

    Zhou, Ziniu; Scott, William L; O'Donnell, Martin J

    2016-03-15

    A solid-phase procedure is used to synthesize racemic peptidomimetics based on the fundamental peptide unit. The peptidomimetics are constructed around proline or proline homologues variably substituted at the amine and carbonyl sites. The procedure expands the diversity of substituted peptidomimetic molecules available to the Distributed Drug Discovery (D3) project. Using a BAL-based solid-phase synthetic sequence the proline or proline homologue subunit is both constructed and incorporated into the peptidomimetic by an α-alkylation, hydrolysis and intramolecular cyclization sequence. Further transformations on solid-phase provide access to a variety of piperazine derivatives representing a class of molecules known to exhibit central nervous system activity. The procedure works well with proline cores, but with larger six- and seven-membered ring homologues the nature of the carboxylic acid acylating the cyclic amine can lead to side reactions and result in poor overall yields.

  3. The effect of proline on the network structure of major ampullate silks as inferred from their mechanical and optical properties.

    PubMed

    Savage, Ken N; Gosline, John M

    2008-06-01

    The silk that orb-weaving spiders produce for use as dragline and for the frame of the web is spun from the major ampullate (MA) glands, and it is renowned for its exceptional toughness. The fibroins that make up MA silk have previously been organized into two major groupings, spidroin-1 and spidroin-2, based largely on differences in amino acid sequence. The most apparent difference between spidroin-1 and spidroin-2 fibroins is the lack of proline in spidroin-1. The MA silk of Araneus diadematus comprises two spidroin-2 fibroins, and is therefore proline-rich, whereas spidroin-1 is preferentially expressed in Nephila clavipes MA silk, and so this silk is proline deficient. Together, these two silks provide a system for testing the consequences of proline-rich and proline-deficient fibroin networks. This study measures the mechanical and optical properties of dry and hydrated Araneus and Nephila MA silks. Since proline acts to disrupt secondary structure, it is hypothesized that the fibroin network of Araneus MA silk will contain less secondary structure than the network of Nephila MA silk. Mechanical and optical studies clearly support this hypothesis. Although the dry properties of these two silks are indistinguishable, there are large differences between the hydrated silks. Nephila silk does not swell upon hydration to the same degree as Araneus silk. In addition, upon hydration, Nephila MA silk retains more of its initial dry stiffness, and retains more molecular order, as indicated by birefringence measurements.

  4. Evolution of collectivity in neutron-rich nuclei in the Sn132 region

    NASA Astrophysics Data System (ADS)

    Kshetri, Ritesh; Sarkar, M. Saha; Sarkar, S.

    2006-09-01

    Motivated by the observed regularity in the energy spectra and the structure of the shell model wave functions for the levels of Te137 and I137, a few weakly and moderately deformed neutron-rich odd-A nuclei above the doubly magic nucleus Sn132 were studied using the particle rotor model (PRM). The calculated energy spectra and branching ratios agree reasonably well with the most recent experimental data. In a few cases ambiguity in level ordering was resolved and spin-parities were assigned to the levels. Observed octupole correlation in some of these nuclei is discussed in the light of the present results.

  5. Behavioral and neurochemical effects of proline.

    PubMed

    Wyse, Angela T S; Netto, Carlos Alexandre

    2011-09-01

    Proline is an amino acid with an essential role for primary metabolism and physiologic functions. Hyperprolinemia results from the deficiency of specific enzymes for proline catabolism, leading to tissue accumulation of this amino acid. Hyperprolinemic patients can present neurological symptoms and brain abnormalities, whose aetiopathogenesis is poorly understood. This review addresses some of the findings obtained, mainly from animal studies, indicating that high proline levels may be associated to neuropathophysiology of some disorders. In this context, it has been suggested that energy metabolism deficit, Na(+),K(+)-ATPase, kinase creatine, oxidative stress, excitotoxicity, lipid content, as well as purinergic and cholinergic systems are involved in the effect of proline on brain damage and spatial memory deficit. The discussion focuses on the relatively low antioxidant defenses of the brain and the vulnerability of neural tissue to reactive species. This offers new perspectives for potential therapeutic strategies for this condition, which may include the early use of appropriate antioxidants as a novel adjuvant therapy, besides the usual treatment based on special diets poor in proline.

  6. Latitudinal concordance between biogeographic regionalization, community structure, and richness patterns: a study on the reptiles of China

    NASA Astrophysics Data System (ADS)

    Chen, Youhua; Srivastava, Diane S.

    2015-02-01

    Latitudinal patterns in species richness may be affected by both continuous variations in macroecological factors as well as discrete change between biogeographic regions. We examined whether latitudinal reptilian richness and community structure in China were best explained by three macroecological patterns (mid-domain effects, Rapoport's rule effects, or environmental correlates) within or across the ranges of biogeographic realms. The results showed that (1) there was a weak mid-domain effect within the Oriental Realm. However, the mid-domain effect was detected neither at the overall regional scale nor in the Palaearctic Realm. (2) Rapoport's rule was only weakly supported for reptilian fauna in China at lower latitudinal areas. (3) Environmental variables were more strongly correlated with species' latitudinal community structure and richness patterns at the scale of biogeographic realms. Based on the faunal similarity of reptilian community across latitudinal bands, we proposed a latitudinal delineation scheme at 34° N for dividing East Asia into Oriental and Palaearctic biogeographic realms. At last, at the functional group level, we also evaluated the relevant ecological patterns for lizard and snake species across different latitudinal bins, showing that the distributions of lizards presented strong mid-domain effects at the latitudinal ranges within the Oriental Realm and over the whole range but did not support Rapoport's rule. In comparison, snake species supported Rapoport's rule at low latitudinal zones but did not present any remarkable mid-domain effects at any spatial extents. In conclusion, biogeographic realms are an appropriate scale for studying macroecological patterns. Reptilian latitudinal richness patterns of China were explained by a combination of environmental factors and geometric constraints, while the latitudinal community structure patterns were greatly affected by environmental gradients. Functional guilds present differentiated

  7. Recombining Low Homology, Functionally Rich Regions of Bacterial Subtilisins by Combinatorial Fragment Exchange

    PubMed Central

    Jones, D. Dafydd

    2011-01-01

    Combinatorial fragment exchange was utilised to recombine key structural and functional low homology regions of bacilli subtilisins to generate new active hybrid proteases with altered substrate profiles. Up to six different regions comprising mostly of loop residues from the commercially important subtilisin Savinase were exchanged with the structurally equivalent regions of six other subtilisins. The six additional subtilisins derive from diverse origins and included thermophilic and intracellular subtilisins as well as other academically and commercially relevant subtilisins. Savinase was largely tolerant to fragment exchange; rational replacement of all six regions with 5 of 6 donating subtilisin sequences preserved activity, albeit reduced compared to Savinase. A combinatorial approach was used to generate hybrid Savinase variants in which the sequences derived from all seven subtilisins at each region were recombined to generate new region combinations. Variants with different substrate profiles and with greater apparent activity compared to Savinase and the rational fragment exchange variants were generated with the substrate profile exhibited by variants dependent on the sequence combination at each region. PMID:21915310

  8. Structural and functional characterization of EIAV gp45 fusion peptide proximal region and asparagine-rich layer

    SciTech Connect

    Duan, Liangwei; Du, Jiansen; Wang, Xuefeng; Zhou, Jianhua; Wang, Xiaojun; Liu, Xinqi

    2016-04-15

    Equine infectious anaemia virus (EIAV) and human immunodeficiency virus (HIV) are members of the lentiviral genus. Similar to HIV gp41, EIAV gp45 is a fusogenic protein that mediates fusion between the viral particle and the host cell membrane. The crystal structure of gp45 reported reveals a different conformation in the here that includes the fusion peptide proximal region (FPPR) and neighboring asparagine-rich layer compared with previous HIV-1 gp41 structures. A complicated hydrogen-bond network containing a cluster of solvent molecules appears to be critical for the stability of the gp45 helical bundle. Interestingly, viral replication was relatively unaffected by site-directed mutagenesis of EIAV, in striking contrast to that of HIV-1. Based on these observations, we speculate that EIAV is more adaptable to emergent mutations, which might be important for the evolution of EIAV as a quasi-species, and could potentially contribute to the success of the EIAV vaccine. - Highlights: • The crystal structure of EIAV gp45 was determined. • The fusion peptide proximal region adopts a novel conformation different to HIV-1. • The asparagine-rich layer includes an extensive hydrogen-bond network. • These regions of EIAV are highly tolerant to mutations. • The results provide insight into the mechanism of gp41/gp45-mediated membrane fusion.

  9. Thermodynamic and conformational study of proline stereoisomers.

    PubMed

    Santos, Ana Filipa L O M; Notario, Rafael; Ribeiro da Silva, Manuel A V

    2014-08-28

    Amino acids play fundamental roles both as building blocks of proteins and as intermediates in metabolism. Proline, one of the 20 natural amino acids, has a primordial function in enzymes, peptide hormones, and proteins. The energetic characterization of these molecules provides information concerning stability and reactivity and has great importance in understanding the activity and behavior of larger molecules containing these structures as fragments. In the present work, parallel experimental and computational studies have been performed. The experimental studies have been based on calorimetric and effusion techniques, from which the enthalpy of formation in the crystalline phase and the enthalpy of sublimation of the sterioisomers L-, D-, and the DL-mixture of proline have been derived. Additionally, vapor pressure measurements have also enabled the determination of the entropies and Gibbs energies of sublimation, at T = 298.15 K. From the former results, the experimental standard (p(o) = 0.1 MPa) molar enthalpies of formation, in the gaseous phase, at T = 298.15 K, of L-proline, D-proline, and DL-proline have been calculated as -388.6 ± 2.3, -391.9 ± 2.0, and -391.5 ± 2.4 kJ·mol(-1), respectively. A computational study at the G3 and G4 levels has been carried out. Conformational analysis has been done and the enthalpy of formation of proline as well as other intrinsic properties such as acidity, basicity, adiabatic ionization enthalpy, electron and proton affinities, and bond dissociation enthalpies have been calculated. There is a very good agreement between calculated and experimental values, when they are available.

  10. Isomers of Pm Isotopes on the Neutron-Rich Frontier of the Deformed Z ~ 60 Region

    NASA Astrophysics Data System (ADS)

    Yokoyama, Rin; Ideguchi, Eiji; Simpson, Gary; Tanaka, Mana; Nishimura, Shunji; Doornnbal, Pieter; Söderström, Pär-Anders; Lorusso, Giuseppe; Xu, Zhengyu; Wu, Jin; Sumikama, Toshiyuki; Aoi, Nori; Baba, Hidetada; Bello, Frank; Browne, Frank; Daido, Rie; Fang, Yifan; Fukuda, Naoki; Gey, Guillaume; Go, Shintaro; Inabe, Naohiro; Isobe, Tadaaki; Kameda, Daisuke; Kobayashi, Kazuma; Kobayashi, Motoki; Komatsubara, Tetsuro; Kubo, Toshiyuki; Kuti, Istvan; Li, Zhihuan; Matsushita, Masafumi; Michimasa, Shin'ichiro; Moon, Chang-Bum; Nishibata, Hiroki; Nishizuka, Ippei; Odahara, Atsuko; Patel, Zena; Rice, Simon; Sahin, Eda; Sinclair, Laura; Suzuki, Hiroshi; Takeda, Hiroyuki; Taprogge, Jan; Vajta, Zsolt; Watanabe, Hiroshi; Yagi, Ayumi

    Neutron-rich Pm (Z = 61) isotopes were studied by delayed γ-ray spectroscopy at RIBF, Riken Nishina Center using the in-flight fission of 345 MeV/u 238U beam. An array of cluster-type Ge detectors, EURICA, was used to measure the delayed γ rays from stopped ions. Isomers were observed in 158Pm, 159Pm and 161Pm with half-lives of >16, 4.64(21), and 0.88(10) µs respectively. Preliminary level schemes for 159Pm and 161Pm were constructed in this study. They have ground-state bands similar to those of 153Pm and 155Pm. The isomeric state of 161Pm could be interpreted as a two quasi-particle excitation of neutrons with configuration of ν 7/2[633] otimes ν 1/2[521] as other Kπ = 4 - isomers observed systematically in other N = 100 isotones.

  11. Silica-rich deposits and hydrated minerals at Gusev Crater, Mars: Vis-NIR spectral characterization and regional mapping

    USGS Publications Warehouse

    Rice, M.S.; Bell, J.F.; Cloutis, E.A.; Wang, A.; Ruff, S.W.; Craig, M.A.; Bailey, D.T.; Johnson, J. R.; De Souza, P.A.; Farrand, W. H.

    2010-01-01

    The Mars Exploration Rover (MER) Spirit has discovered surprisingly high concentrations of amorphous silica in soil and nodular outcrops in the Inner Basin of the Columbia Hills. In Pancam multispectral observations, we find that an absorption feature at the longest Pancam wavelength (1009 nm) appears to be characteristic of these silica-rich materials; however, spectral analyses of amorphous silica suggest that the ???1009 nm spectral feature is not a direct reflection of their silica-rich nature. Based on comparisons with spectral databases, we hypothesize that the presence of H2O or OH, either free (as water ice), adsorbed or bound in a mineral structure, is responsible for the spectral feature observed by Pancam. The Gertrude Weise soil, which is nearly pure opaline silica, may have adsorbed water cold-trapped on mineral grains. The origin of the ???1009 nm Pancam feature observed in the silica-rich nodular outcrops may result from the presence of additional hydrated minerals (specific sulfates, halides, chlorides, sodium silicates, carbonates or borates). Using the ???1009 nm feature with other spectral parameters as a "hydration signature" we have mapped the occurrence of hydrated materials along the extent of Spirit's traverse across the Columbia Hills from West Spur to Home Plate (sols 155-1696). We have also mapped this hydration signature across large panoramic images to understand the regional distribution of materials that are spectrally similar to the silica-rich soil and nodular outcrops. Our results suggest that hydrated materials are common in the Columbia Hills. ?? 2009 Elsevier Inc.

  12. Proline Utilization by Bacillus subtilis: Uptake and Catabolism

    PubMed Central

    Moses, Susanne; Sinner, Tatjana; Zaprasis, Adrienne; Stöveken, Nadine; Hoffmann, Tamara; Belitsky, Boris R.; Sonenshein, Abraham L.

    2012-01-01

    l-Proline can be used by Bacillus subtilis as a sole source of carbon or nitrogen. We traced l-proline utilization genetically to the putBCP (ycgMNO) locus. The putBCP gene cluster encodes a high-affinity proline transporter (PutP) and two enzymes, the proline dehydrogenase PutB and the Δ1-pyrroline-5-carboxylate dehydrogenase PutC, which jointly catabolize l-proline to l-glutamate. Northern blotting, primer extension, and putB-treA reporter gene fusion analysis showed that the putBCP locus is transcribed as an l-proline-inducible operon. Its expression was mediated by a SigA-type promoter and was dependent on the proline-responsive PutR activator protein. Induction of putBCP expression was triggered by the presence of submillimolar concentrations of l-proline in the growth medium. However, the very large quantities of l-proline (up to several hundred millimolar) synthesized by B. subtilis as a stress protectant against high osmolarity did not induce putBCP transcription. Induction of putBCP transcription by external l-proline was not dependent on l-proline uptake via the substrate-inducible PutP or the osmotically inducible OpuE transporter. It was also not dependent on the chemoreceptor protein McpC required for chemotaxis toward l-proline. Our findings imply that B. subtilis can distinguish externally supplied l-proline from internal l-proline pools generated through de novo synthesis. The molecular basis of this regulatory phenomenon is not understood. However, it provides the B. subtilis cell with a means to avoid a futile cycle of de novo l-proline synthesis and consumption by not triggering the expression of the putBCP l-proline catabolic genes in response to the osmoadaptive production of the compatible solute l-proline. PMID:22139509

  13. Proline: Mother Nature;s cryoprotectant applied to protein crystallography

    SciTech Connect

    Pemberton, Travis A.; Still, Brady R.; Christensen, Emily M.; Singh, Harkewal; Srivastava, Dhiraj; Tanner, John J.

    2012-09-05

    L-Proline is one of Mother Nature's cryoprotectants. Plants and yeast accumulate proline under freeze-induced stress and the use of proline in the cryopreservation of biological samples is well established. Here, it is shown that L-proline is also a useful cryoprotectant for protein crystallography. Proline was used to prepare crystals of lysozyme, xylose isomerase, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase for low-temperature data collection. The crystallization solutions in these test cases included the commonly used precipitants ammonium sulfate, sodium chloride and polyethylene glycol and spanned the pH range 4.6-8.5. Thus, proline is compatible with typical protein-crystallization formulations. The proline concentration needed for cryoprotection of these crystals is in the range 2.0-3.0 M. Complete data sets were collected from the proline-protected crystals. Proline performed as well as traditional cryoprotectants based on the diffraction resolution and data-quality statistics. The structures were refined to assess the binding of proline to these proteins. As observed with traditional cryoprotectants such as glycerol and ethylene glycol, the electron-density maps clearly showed the presence of proline molecules bound to the protein. In two cases, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase, proline binds in the active site. It is concluded that L-proline is an effective cryoprotectant for protein crystallography.

  14. Proline: Mother Nature's cryoprotectant applied to protein crystallography.

    PubMed

    Pemberton, Travis A; Still, Brady R; Christensen, Emily M; Singh, Harkewal; Srivastava, Dhiraj; Tanner, John J

    2012-08-01

    L-Proline is one of Mother Nature's cryoprotectants. Plants and yeast accumulate proline under freeze-induced stress and the use of proline in the cryopreservation of biological samples is well established. Here, it is shown that L-proline is also a useful cryoprotectant for protein crystallography. Proline was used to prepare crystals of lysozyme, xylose isomerase, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase for low-temperature data collection. The crystallization solutions in these test cases included the commonly used precipitants ammonium sulfate, sodium chloride and polyethylene glycol and spanned the pH range 4.6-8.5. Thus, proline is compatible with typical protein-crystallization formulations. The proline concentration needed for cryoprotection of these crystals is in the range 2.0-3.0 M. Complete data sets were collected from the proline-protected crystals. Proline performed as well as traditional cryoprotectants based on the diffraction resolution and data-quality statistics. The structures were refined to assess the binding of proline to these proteins. As observed with traditional cryoprotectants such as glycerol and ethylene glycol, the electron-density maps clearly showed the presence of proline molecules bound to the protein. In two cases, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase, proline binds in the active site. It is concluded that L-proline is an effective cryoprotectant for protein crystallography.

  15. Sinorhizobium meliloti chemoreceptor McpU mediates chemotaxis toward host plant exudates through direct proline sensing.

    PubMed

    Webb, Benjamin A; Hildreth, Sherry; Helm, Richard F; Scharf, Birgit E

    2014-06-01

    Bacterial chemotaxis is an important attribute that aids in establishing symbiosis between rhizobia and their legume hosts. Plant roots and seeds exude a spectrum of molecules into the soil to attract their bacterial symbionts. The alfalfa symbiont Sinorhizobium meliloti possesses eight chemoreceptors to sense its environment and mediate chemotaxis toward its host. The methyl accepting chemotaxis protein McpU is one of the more abundant S. meliloti chemoreceptors and an important sensor for the potent attractant proline. We established a dominant role of McpU in sensing molecules exuded by alfalfa seeds. Mass spectrometry analysis determined that a single germinating seed exudes 3.72 nmol of proline, producing a millimolar concentration near the seed surface which can be detected by the chemosensory system of S. meliloti. Complementation analysis of the mcpU deletion strain verified McpU as the key proline sensor. A structure-based homology search identified tandem Cache (calcium channels and chemotaxis receptors) domains in the periplasmic region of McpU. Conserved residues Asp-155 and Asp-182 of the N-terminal Cache domain were determined to be important for proline sensing by evaluating mutant strains in capillary and swim plate assays. Differential scanning fluorimetry revealed interaction of the isolated periplasmic region of McpU (McpU40-284) with proline and the importance of Asp-182 in this interaction. Using isothermal titration calorimetry, we determined that proline binds with a Kd (dissociation constant) of 104 μM to McpU40-284, while binding was abolished when Asp-182 was substituted by Glu. Our results show that McpU is mediating chemotaxis toward host plants by direct proline sensing.

  16. Sinorhizobium meliloti Chemoreceptor McpU Mediates Chemotaxis toward Host Plant Exudates through Direct Proline Sensing

    PubMed Central

    Webb, Benjamin A.; Hildreth, Sherry; Helm, Richard F.

    2014-01-01

    Bacterial chemotaxis is an important attribute that aids in establishing symbiosis between rhizobia and their legume hosts. Plant roots and seeds exude a spectrum of molecules into the soil to attract their bacterial symbionts. The alfalfa symbiont Sinorhizobium meliloti possesses eight chemoreceptors to sense its environment and mediate chemotaxis toward its host. The methyl accepting chemotaxis protein McpU is one of the more abundant S. meliloti chemoreceptors and an important sensor for the potent attractant proline. We established a dominant role of McpU in sensing molecules exuded by alfalfa seeds. Mass spectrometry analysis determined that a single germinating seed exudes 3.72 nmol of proline, producing a millimolar concentration near the seed surface which can be detected by the chemosensory system of S. meliloti. Complementation analysis of the mcpU deletion strain verified McpU as the key proline sensor. A structure-based homology search identified tandem Cache (calcium channels and chemotaxis receptors) domains in the periplasmic region of McpU. Conserved residues Asp-155 and Asp-182 of the N-terminal Cache domain were determined to be important for proline sensing by evaluating mutant strains in capillary and swim plate assays. Differential scanning fluorimetry revealed interaction of the isolated periplasmic region of McpU (McpU40-284) with proline and the importance of Asp-182 in this interaction. Using isothermal titration calorimetry, we determined that proline binds with a Kd (dissociation constant) of 104 μM to McpU40-284, while binding was abolished when Asp-182 was substituted by Glu. Our results show that McpU is mediating chemotaxis toward host plants by direct proline sensing. PMID:24657863

  17. CO2 and CH4 emissions from streams in a lake-rich landscape: Patterns, controls, and regional significance

    USGS Publications Warehouse

    Crawford, John T.; Lottig, Noah R.; Stanley, Emily H.; Walker, John F.; Hanson, Paul C.; Finlay, Jacques C.; Striegl, Robert G.

    2014-01-01

    Aquatic ecosystems are important components of landscape carbon budgets. In lake-rich landscapes, both lakes and streams may be important sources of carbon gases (CO2 and CH4) to the atmosphere, but the processes that control gas concentrations and emissions in these interconnected landscapes have not been adequately addressed. We use multiple data sets that vary in their spatial and temporal extent during 2001–2012 to investigate the carbon gas source strength of streams in a lake-rich landscape and to determine the contribution of lakes, metabolism, and groundwater to stream CO2 and CH4. We show that streams emit roughly the same mass of CO2 (23.4 Gg C yr−1; 0.49 mol CO2 m−2 d−1) as lakes at a regional scale (27 Gg C yr−1) and that stream CH4 emissions (189 Mg C yr−1; 8.46 mmol CH4 m−2 d−1) are an important component of the regional greenhouse gas balance. Gas transfer velocity variability (range = 0.34 to 13.5 m d−1) contributed to the variability of gas flux in this landscape. Groundwater inputs and in-stream metabolism control stream gas supersaturation at the landscape scale, while carbon cycling in lakes and deep groundwaters does not control downstream gas emissions. Our results indicate the need to consider connectivity of all aquatic ecosystems (lakes, streams, wetlands, and groundwater) in lake-rich landscapes and their connections with the terrestrial environment in order to understand the full nature of the carbon cycle.

  18. The complete mitogenomes of Calameuta filiformis (Eversmann, 1847) and Calameuta idolon (Rossi, 1794) (Hymenoptera: Cephidae): The remarkable features of the elongated A+T rich region in Cephini.

    PubMed

    Korkmaz, E Mahir; Budak, Mahir; Ördek, Merve Nur; Başıbüyük, Hasan Hüseyin

    2016-01-15

    Two complete mitogenomes of the stem borers, Calameuta filiformis and Calameuta idolon, and the complete A+T-rich region of Trachelus iudaicus (Hymenoptera: Cephidae), are reported. The mitogenomes of these species are the longest reported from hymenopterans to date. A remarkable increase in length of the A+T-rich region, the longest for Hymenoptera, was found and compared across the tribe Cephini. The presence of the tRNA- and rRNA-like sequences were reported in the A+T-rich region of sawflies and they were suggested to play a role in replication and/or transcription. The long and short tandem repeats were orderly located in both sides of the A+T-rich region producing stable secondary structures. We suggest that the short tandem repeats are likely to function as a replication fork barrier.

  19. 21 CFR 582.5650 - Proline.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Proline. 582.5650 Section 582.5650 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  20. 21 CFR 582.5650 - Proline.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Proline. 582.5650 Section 582.5650 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  1. Harsh Corporal Punishment Is Associated With Increased T2 Relaxation Time in Dopamine-Rich Regions

    PubMed Central

    Sheu, Yi-Shin; Polcari, Ann; Anderson, Carl M.; Teicher, Martin H.

    2010-01-01

    Harsh corporal punishment (HCP) was defined as frequent parental administration of corporal punishment (CP) for discipline, with occasional use of objects such as straps, or paddles. CP is linked to increased risk for depression and substance abuse. We examine whether long-term exposure to HCP acts as sub-traumatic stressor that contributes to brain alterations, particularly in dopaminergic pathways, which may mediate their increased vulnerability to drug and alcohol abuse. Nineteen young adults who experienced early HCP but no other forms of maltreatment and twenty-three comparable controls were studied. T2 relaxation time (T2-RT) measurements were performed with an echo planar imaging TE stepping technique and T2 maps were calculated and analyzed voxel-by-voxel to locate regional T2-RT differences between groups. Previous studies indicated that T2-RT provides an indirect index of resting cerebral blood volume. Region of interest (ROI) analyses were also conducted in caudate, putamen, nucleus accumbens, anterior cingulate cortex, dorsolateral prefrontal cortex, thalamus, globus pallidus and cerebellar hemispheres. Voxel-based relaxometry showed that HCP was associated with increased T2-RT in right caudate and putamen. ROI analyses also revealed increased T2-RT in dorsolateral prefrontal cortex, substantia nigra, thalamus and accumbens but not globus pallidus or cerebellum. There were significant associations between T2-RT measures in dopamine target regions and use of drugs and alcohol, and memory performance. Alteration in the paramagnetic or hemodynamic properties of dopaminergic cell body and projection regions were observed in subjects with HCP, and these findings may relate to their increased risk for drug and alcohol abuse. PMID:20600981

  2. Harsh corporal punishment is associated with increased T2 relaxation time in dopamine-rich regions.

    PubMed

    Sheu, Yi-Shin; Polcari, Ann; Anderson, Carl M; Teicher, Martin H

    2010-11-01

    Harsh corporal punishment (HCP) was defined as frequent parental administration of corporal punishment (CP) for discipline, with occasional use of objects such as straps, or paddles. CP is linked to increased risk for depression and substance abuse. We examine whether long-term exposure to HCP acts as sub-traumatic stressor that contributes to brain alterations, particularly in dopaminergic pathways, which may mediate their increased vulnerability to drug and alcohol abuse. Nineteen young adults who experienced early HCP but no other forms of maltreatment and twenty-three comparable controls were studied. T2 relaxation time (T2-RT) measurements were performed with an echo planar imaging TE stepping technique and T2 maps were calculated and analyzed voxel-by-voxel to locate regional T2-RT differences between groups. Previous studies indicated that T2-RT provides an indirect index of resting cerebral blood volume. Region of interest (ROI) analyses were also conducted in caudate, putamen, nucleus accumbens, anterior cingulate cortex, dorsolateral prefrontal cortex, thalamus, globus pallidus and cerebellar hemispheres. Voxel-based relaxometry showed that HCP was associated with increased T2-RT in right caudate and putamen. ROI analyses also revealed increased T2-RT in dorsolateral prefrontal cortex, substantia nigra, thalamus and accumbens but not globus pallidus or cerebellum. There were significant associations between T2-RT measures in dopamine target regions and use of drugs and alcohol, and memory performance. Alteration in the paramagnetic or hemodynamic properties of dopaminergic cell body and projection regions were observed in subjects with HCP, and these findings may relate to their increased risk for drug and alcohol abuse.

  3. WHY IS THE GREAT SOLAR ACTIVE REGION 12192 FLARE-RICH BUT CME-POOR?

    SciTech Connect

    Sun, Xudong; Bobra, Monica G.; Hoeksema, J. Todd; Liu, Yang; Couvidat, Sebastien; Norton, Aimee A.; Li, Yan; Fisher, George H.; Shen, Chenglong

    2015-05-10

    Solar active region (AR) 12192 of 2014 October hosts the largest sunspot group in 24 years. It is the most prolific flaring site of Cycle 24 so far, but surprisingly produced no coronal mass ejection (CME) from the core region during its disk passage. Here, we study the magnetic conditions that prevented eruption and the consequences that ensued. We find AR 12192 to be “big but mild”; its core region exhibits weaker non-potentiality, stronger overlying field, and smaller flare-related field changes compared to two other major flare-CME-productive ARs (11429 and 11158). These differences are present in the intensive-type indices (e.g., means) but generally not the extensive ones (e.g., totals). AR 12192's large amount of magnetic free energy does not translate into CME productivity. The unexpected behavior suggests that AR eruptiveness is limited by some relative measure of magnetic non-potentiality over the restriction of background field, and that confined flares may leave weaker photospheric and coronal imprints compared to their eruptive counterparts.

  4. Imaging Fluid-Rich Zones by Magnetotelluric Method at South Marmara Region, Turkey

    NASA Astrophysics Data System (ADS)

    Cengiz, Özlem; Bülent Tank, Sabri; Tolak Çiftçi, Elif; Kaya, Tülay; Ogawa, Yasuo; Honkura, Yoshimori; Kemal Tunçer, Mustafa; Matsushima, Masaki; Oshiman, Naoto; Çelik, Cengiz

    2013-04-01

    After the mainshock of 1999 İzmit (Turkey) earthquake, scientists have undertaken various kinds of observations in the Marmara region in order to increase understanding of crustal properties of North Anatolian Fault Zone (NAFZ). Most of these studies focused on the east Marmara region since the destructive earthquakes were occurring there. Considering the westward migration of significant earthquakes on the North Anatolian Fault (NAF), the next destructive earthquake is likely to occur beyond the westernmost part of 1999 İzmit earthquake epicenter, in or around Marmara Sea. For this reason, south of Marmara Sea was chosen as the survey area. Wide-band (320-0.0005 Hz) magnetotelluric (MT) data at sixteen sounding locations along two parallel profiles at south Marmara region were collected to improve the understanding of the crustal electrical conductivity structure. Both profiles are crossing several branches of North Anatolian Fault. The results were achieved by performing two-dimensional (2D) inversions of MT data with the transverse electric (TE) and transverse magnetic (TM) modes. These results show a relatively complex structure down to 4 km depth. The electrical resistivity pattern below this depth indicates a simpler structure with conductors (10 Ωm) beneath the northern ends of both profiles. While these deep conductive zones are attributed to partially melting in the crust, the highly resistive zones are associated with low fluid condition and high rigidity. In addition, the features characterized in geo-electric models correlate well with known faults in the survey area. The South Marmara Fault (SMF) possibly corresponds to a lateral resistive-conductive interface between Manyas-Karacabey basin and Bandirma-Karada uplift on the west (PW) and Uluabat uplift and Mudanya uplift on the east (PE) profiles.

  5. Salt stress encourages proline accumulation by regulating proline biosynthesis and degradation in Jerusalem artichoke plantlets.

    PubMed

    Huang, Zengrong; Zhao, Long; Chen, Dandan; Liang, Mingxiang; Liu, Zhaopu; Shao, Hongbo; Long, Xiaohua

    2013-01-01

    Proline accumulation is an important mechanism for osmotic regulation under salt stress. In this study, we evaluated proline accumulation profiles in roots, stems and leaves of Jerusalem artichoke (Helianthus tuberosus L.) plantlets under NaCl stress. We also examined HtP5CS, HtOAT and HtPDH enzyme activities and gene expression patterns of putative HtP5CS1, HtP5CS2, HtOAT, HtPDH1, and HtPDH2 genes. The objective of our study was to characterize the proline regulation mechanisms of Jerusalem artichoke, a moderately salt tolerant species, under NaCl stress. Jerusalem artichoke plantlets were observed to accumulate proline in roots, stems and leaves during salt stress. HtP5CS enzyme activities were increased under NaCl stress, while HtOAT and HtPDH activities generally repressed. Transcript levels of HtP5CS2 increased while transcript levels of HtOAT, HtPDH1 and HtPDH2 generally decreased in response to NaCl stress. Our results supports that for Jerusalem artichoke, proline synthesis under salt stress is mainly through the Glu pathway, and HtP5CS2 is predominant in this process while HtOAT plays a less important role. Both HtPDH genes may function in proline degradation.

  6. The gene encoding proline dehydrogenase modulates sensorimotor gating in mice.

    PubMed

    Gogos, J A; Santha, M; Takacs, Z; Beck, K D; Luine, V; Lucas, L R; Nadler, J V; Karayiorgou, M

    1999-04-01

    Hemizygous cryptic deletions of the q11 band of human chromosome 22 have been associated with a number of psychiatric and behavioural phenotypes, including schizophrenia. Here we report the isolation and characterization of PRODH, a human homologue of Drosophila melanogaster sluggish-A (slgA), which encodes proline dehydrogenase responsible for the behavioural phenotype of the slgA mutant. PRODH is localized at chromosome 22q11 in a region deleted in some psychiatric patients. We also isolated the mouse homologue of slgA (Prodh), identified a mutation in this gene in the Pro/Re hyperprolinaemic mouse strain and found that these mice have a deficit in sensorimotor gating accompanied by regional neurochemical alterations in the brain. Sensorimotor gating is a neural filtering process that allows attention to be focused on a given stimulus, and is affected in patients with neuropsychiatric disorders. Furthermore, several lines of evidence suggest that proline may serve as a modulator of synaptic transmission in the mammalian brain. Our observations, in conjunction with the chromosomal location of PRODH, suggest a potential involvement of this gene in the 22q11-associated psychiatric and behavioural phenotypes.

  7. Proline: Mother Nature’s cryoprotectant applied to protein crystallography

    SciTech Connect

    Pemberton, Travis A.; Still, Brady R.; Christensen, Emily M.; Singh, Harkewal; Srivastava, Dhiraj; Tanner, John J.

    2012-08-01

    The amino acid l-proline is shown to be a good cryoprotectant for protein crystals. Four examples are provided; the range of proline used for cryoprotection is 2.0–3.0 M. l-Proline is one of Mother Nature’s cryoprotectants. Plants and yeast accumulate proline under freeze-induced stress and the use of proline in the cryopreservation of biological samples is well established. Here, it is shown that l-proline is also a useful cryoprotectant for protein crystallography. Proline was used to prepare crystals of lysozyme, xylose isomerase, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase for low-temperature data collection. The crystallization solutions in these test cases included the commonly used precipitants ammonium sulfate, sodium chloride and polyethylene glycol and spanned the pH range 4.6–8.5. Thus, proline is compatible with typical protein-crystallization formulations. The proline concentration needed for cryoprotection of these crystals is in the range 2.0–3.0 M. Complete data sets were collected from the proline-protected crystals. Proline performed as well as traditional cryoprotectants based on the diffraction resolution and data-quality statistics. The structures were refined to assess the binding of proline to these proteins. As observed with traditional cryoprotectants such as glycerol and ethylene glycol, the electron-density maps clearly showed the presence of proline molecules bound to the protein. In two cases, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase, proline binds in the active site. It is concluded that l-proline is an effective cryoprotectant for protein crystallography.

  8. North African petroleum geology: regional structure and stratigraphic overview of a hydrocarbon-rich cratonic area

    SciTech Connect

    O'Connor, T.E.; Kanes, W.H.

    1985-02-01

    North Africa, including Sinai, contains some of the most important hydrocarbon-producing basins in the world. The North African Symposium is devoted to examining the exploration potential of the North African margin in light of the most recent and promising exploration discoveries. The geologic variety of the region is extraordinary and can challenge any exploration philosophy. Of primary interest are the Sirte basin of Libya, which has produced several billion barrels of oil, and the Gulf of Suez, a narrow, evaporite-capped trough with five fields that will produce more than 5 billion bbl. Both are extensional basins with minimal lateral movement and with good source rocks in direct proximity to reservoirs. Structural models of these basins give firm leads for future exploration. More difficult to evaluate are the Tethyan realm basins of the northern Sinai, and the Western Desert of Egypt, the Cyrenaican Platform of Libya, and the Tunisia-Sicily shelf area, where there are only limited subsurface data. These basins are extensional in origin also, but have been influenced by lateral tectonics. Favorable reservoirs exist, but source rocks have been a problem locally. Structural models with strong stratigraphic response offer several favorable play concepts. The Paleozoic Ghadames basin in Libya, Tunisia, and Algeria has the least complex structural history, and production appears to be limited to small structures. A series of stratigraphic models indicates additional areas with exploration potential. The Paleozoic megabasin of Morocco, with its downfaulted Triassic grabens, remains an untested but attractive area.

  9. Simulation of mixing in the quick quench region of a rich burn-quick quench mix-lean burn combustor

    NASA Technical Reports Server (NTRS)

    Shih, Tom I.-P.; Nguyen, H. Lee; Howe, Gregory W.; Li, Z.

    1991-01-01

    A computer program was developed to study the mixing process in the quick quench region of a rich burn-quick quench mix-lean burn combustor. The computer program developed was based on the density-weighted, ensemble-averaged conservation equations of mass, momentum (full compressible Navier-Stokes), total energy, and species, closed by a k-epsilon turbulence model with wall functions. The combustion process was modeled by a two-step global reaction mechanism, and NO(x) formation was modeled by the Zeldovich mechanism. The formulation employed in the computer program and the essence of the numerical method of solution are described. Some results obtained for nonreacting and reacting flows with different main-flow to dilution-jet momentum flux ratios are also presented.

  10. Effect of copper-rich regions on tensile properties of VPPA weldments of 2219-T87 aluminum

    NASA Technical Reports Server (NTRS)

    Hartman, J. A.; Beil, R. J.; Hahn, G. T.

    1987-01-01

    This study examines the relations between tensile properties and microstructural features of variable polarity plasma arc (VPPA) weldments of 2219-T87 aluminum. Crack initiation and weld failure of transverse tensile specimens of single and multipass weldments were studied. The specimens fractured on the rising portion of the stress-strain curve prior to necking, signifying that an increase in strength would accompany an increase in ductility. Of particular interest is a shallow, typically 0.001-0.003-in. (0.03-0.08-mm) deep, copper-rich region located in the crown and root corners of the weld. This region is a primary source of crack initiation and growth, due to its brittle nature and highly strained location. The brittle regions were removed by electropolishing and machining to determine their effect on weld tensile properties. The removal increased the ductility of the weld specimens, and in the case of single pass welds, actually increased the load carrying capacity. Local strain measurements and metallographic and chemical analyses are presented.

  11. Evolution of proline biosynthesis: enzymology, bioinformatics, genetics, and transcriptional regulation.

    PubMed

    Fichman, Yosef; Gerdes, Svetlana Y; Kovács, Hajnalka; Szabados, László; Zilberstein, Aviah; Csonka, Laszlo N

    2015-11-01

    Proline is not only an essential component of proteins but it also has important roles in adaptation to osmotic and dehydration stresses, redox control, and apoptosis. Here, we review pathways of proline biosynthesis in the three domains of life. Pathway reconstruction from genome data for hundreds of eubacterial and dozens of archaeal and eukaryotic organisms revealed evolutionary conservation and variations of this pathway across different taxa. In the most prevalent pathway of proline synthesis, glutamate is phosphorylated to γ-glutamyl phosphate by γ-glutamyl kinase, reduced to γ-glutamyl semialdehyde by γ-glutamyl phosphate reductase, cyclized spontaneously to Δ(1)-pyrroline-5-carboxylate and reduced to proline by Δ(1)-pyrroline-5-carboxylate reductase. In higher plants and animals the first two steps are catalysed by a bi-functional Δ(1) -pyrroline-5-carboxylate synthase. Alternative pathways of proline formation use the initial steps of the arginine biosynthetic pathway to ornithine, which can be converted to Δ(1)-pyrroline-5-carboxylate by ornithine aminotransferase and then reduced to proline or converted directly to proline by ornithine cyclodeaminase. In some organisms, the latter pathways contribute to or could be fully responsible for the synthesis of proline. The conservation of proline biosynthetic enzymes and significance of specific residues for catalytic activity and allosteric regulation are analysed on the basis of protein structural data, multiple sequence alignments, and mutant studies, providing novel insights into proline biosynthesis in organisms. We also discuss the transcriptional control of the proline biosynthetic genes in bacteria and plants.

  12. Short-range interactions of concentrated proline in aqueous solution.

    PubMed

    Busch, Sebastian; Lorenz, Christian D; Taylor, Jonathan; Pardo, Luis Carlos; McLain, Sylvia E

    2014-12-11

    Molecular interactions for proline in a highly concentrated aqueous solution (up to 1:5 proline:water molecular ratio) have been investigated using a variety of experimental and computational techniques. Rather than the solution containing either small crystallites or large aggregates of proline, three-dimensional structural analysis reveals the presence of proline-proline dimers. These dimers appear to be formed by cyclic electrostatic interactions between CO2(-) and NH2(+) groups on neighboring proline molecules, which causes the ring motifs of proline to be roughly parallel to one another. In addition, water appears to aggregate around the electrostatic groups of the proline-proline dimers where it may in fact bridge these groups on different molecules. The observed short-range interactions for proline in solution may explain its function as a hydrotrope in vivo in which this observed dimerization might allow proline molecules to generate small pockets of a hydrophobic environment that can associate with nonpolar motifs of other molecules in solution. The results presented here emphasize the need for careful three-dimensional analysis to assess the short-range order of highly concentrated solutions.

  13. Volatile-rich Crater Interior Deposits in the Polar Regions of Mars: Evidence for Ice Cap Advance and Retreat

    NASA Technical Reports Server (NTRS)

    Russell, Patrick S.; Head, James W.; Hecht, Michael H.

    2003-01-01

    Many craters on Mars are partially filled by distinctive material emplaced by post-impact processes. This crater fill material is an interior mound which is generally separated from the walls of the crater by a trough that may be continuous along the crater circumference (i.e. a ring-shaped trough), or which may only partially contact the crater walls (i.e. a crescent-shaped trough). The fill deposit is frequently offset from the crater center and may be asymmetric in plan view. Populations of such craters include those in the circum-south polar cap region, in Arabia Terra, associated with the Medusae Fossae Formation, and in the northern lowlands proximal to the north polar cap. We focus on those craters in circumpolar regions and assess their relationship to polar cap advance and retreat, especially the possibility that fill material represents remnants of a formerly larger contiguous cap. Volatile-rich deposits have the property of being modifiable by the local stability of the solid volatile, which is governed by local energy balance. Here we test the hypothesis that asymmetries in volatile fill shape, profile, and center-location within a crater result from asymmetries in local energy balance within the crater, due mainly to variation of solar insolation and radiative effects of the crater walls over the crater interior. Model profiles of crater fill are compared with MOLA topographic profiles to assess this hypothesis. If asymmetry in morphology and location of crater fill are consistent with radiative-dominated asymmetries in energy budget within the crater, then 1) the volatile-rich composition of the fill is supported (this process should not be effective at shaping volcanic or sedimentary deposits), and 2) the dominant factor determining the observed shape of volatile-rich crater fill is the local radiative energy budget (and erosive processes such as eolian deflation are secondary or unnecessary). We also use a geographic and energy model approach to

  14. Proline affects brain function in 22q11DS children with the low activity COMT 158 allele.

    PubMed

    Vorstman, Jacob A S; Turetsky, Bruce I; Sijmens-Morcus, Monique E J; de Sain, Monique G; Dorland, Bert; Sprong, Mirjam; Rappaport, Eric F; Beemer, Frits A; Emanuel, Beverly S; Kahn, René S; van Engeland, Herman; Kemner, Chantal

    2009-02-01

    The association between the 22q11.2 deletion syndrome (22q11DS) and psychiatric disorders, particularly psychosis, suggests a causal relationship between 22q11DS genes and abnormal brain function. The genes catechol-O-methyl-transferase (COMT) and proline dehydrogenase both reside within the commonly deleted region of 22q11.2. COMT activity and proline levels may therefore be altered in 22q11DS individuals. Associations of both COMT(158) genotype and elevated serum proline levels with abnormal brain function have been reported. Fifty-six 22q11DS children and 75 healthy controls were assessed on physiological measures of brain function, including prepulse inhibition (PPI) of startle, P50 auditory sensory gating and smooth pursuit eye movements (SPEM). COMT(158) genotype and plasma proline levels were determined in the 22q11DS children. We hypothesized an interaction between the COMT(158) genotype and proline, predicting the strongest negative effect of high proline on brain function to occur in 22q11DS children who are carriers of the COMT(met) allele. Of the three physiological measures, only SPEM and PPI were abnormal in the patient sample. With regard to the SPEM performance, there was a significant interaction between the COMT(158) genotype and proline level with significantly decreased SPEM performance in children with high plasma proline levels and the low activity COMT(met) allele. A similar interaction effect was not observed with regard to PPI. These findings are consistent with a model in which elevated proline negatively affects brain function by an increase in dopamine in the prefrontal cortex. 22q11DS patients with low dopamine catabolic capacity are therefore especially vulnerable to this functional disruption.

  15. Stress-induced synthesis of proline confers tolerance to water deficit in transgenic wheat.

    PubMed

    Vendruscolo, Eliane Cristina Gruszka; Schuster, Ivan; Pileggi, Marcos; Scapim, Carlos Alberto; Molinari, Hugo Bruno Correa; Marur, Celso Jamil; Vieira, Luiz Gonzaga Esteves

    2007-10-01

    Water deficit is one of the main abiotic factors that affect spring wheat planted in subtropical regions. Accumulation of proline appears to be a promising approach to maintain the productivity of plants under stress condition. However, morphological alterations and growth reduction are observed in transgenic plants carrying genes coding for osmoprotectants controlled by constitutive promoters. We report here the effects of water deficit on wheat plants transformed with the Vigna aconitifolia Delta(1)-pyrroline-5-carboxylate synthetase (P5CS) cDNA that encodes the key regulatory enzyme in proline biosynthesis, under the control of a stress-induced promoter complex-AIPC. Transgenic wheat plants submitted to 15 days of water shortage presented a distinct response. We have found that drought resulted in the accumulation of proline. The tolerance to water deficit observed in transgenic plants was mainly due to protection mechanisms against oxidative stress and not caused by osmotic adjustment.

  16. Regional Variation in Parasite Species Richness and Abundance in the Introduced Range of the Invasive Lionfish, Pterois volitans.

    PubMed

    Sellers, Andrew J; Ruiz, Gregory M; Leung, Brian; Torchin, Mark E

    2015-01-01

    Parasites can play an important role in biological invasions. While introduced species often lose parasites from their native range, they can also accumulate novel parasites in their new range. The accumulation of parasites by introduced species likely varies spatially, and more parasites may shift to new hosts where parasite diversity is high. Considering that parasitism and disease are generally more prevalent at lower latitudes, the accumulation of parasites by introduced hosts may be greater in tropical regions. The Indo-Pacific lionfish (Pterois volitans) has become widely distributed across the Western Atlantic. In this study, we compared parasitism across thirteen locations in four regions, spanning seventeen degrees of latitude in the lionfish's introduced range to examine potential spatial variation in parasitism. In addition, as an initial step to explore how indirect effects of parasitism might influence interactions between lionfish and ecologically similar native hosts, we also compared parasitism in lionfish and two co-occurring native fish species, the graysby grouper, Cephalopholis cruentata, and the lizardfish, Synodus intermedius, in the southernmost region, Panama. Our results show that accumulation of native parasites on lionfish varies across broad spatial scales, and that colonization by ectoparasites was highest in Panama, relative to the other study sites. Endoparasite richness and abundance, on the other hand, were highest in Belize where lionfish were infected by twice as many endoparasite species as lionfish in other regions. The prevalence of all but two parasite species infecting lionfish was below 25%, and we did not detect an association between parasite abundance and host condition, suggesting a limited direct effect of parasites on lionfish, even where parasitism was highest. Further, parasite species richness and abundance were significantly higher in both native fishes compared to lionfish, and parasite abundance was negatively

  17. Regional Variation in Parasite Species Richness and Abundance in the Introduced Range of the Invasive Lionfish, Pterois volitans

    PubMed Central

    2015-01-01

    Parasites can play an important role in biological invasions. While introduced species often lose parasites from their native range, they can also accumulate novel parasites in their new range. The accumulation of parasites by introduced species likely varies spatially, and more parasites may shift to new hosts where parasite diversity is high. Considering that parasitism and disease are generally more prevalent at lower latitudes, the accumulation of parasites by introduced hosts may be greater in tropical regions. The Indo-Pacific lionfish (Pterois volitans) has become widely distributed across the Western Atlantic. In this study, we compared parasitism across thirteen locations in four regions, spanning seventeen degrees of latitude in the lionfish's introduced range to examine potential spatial variation in parasitism. In addition, as an initial step to explore how indirect effects of parasitism might influence interactions between lionfish and ecologically similar native hosts, we also compared parasitism in lionfish and two co-occurring native fish species, the graysby grouper, Cephalopholis cruentata, and the lizardfish, Synodus intermedius, in the southernmost region, Panama. Our results show that accumulation of native parasites on lionfish varies across broad spatial scales, and that colonization by ectoparasites was highest in Panama, relative to the other study sites. Endoparasite richness and abundance, on the other hand, were highest in Belize where lionfish were infected by twice as many endoparasite species as lionfish in other regions. The prevalence of all but two parasite species infecting lionfish was below 25%, and we did not detect an association between parasite abundance and host condition, suggesting a limited direct effect of parasites on lionfish, even where parasitism was highest. Further, parasite species richness and abundance were significantly higher in both native fishes compared to lionfish, and parasite abundance was negatively

  18. Mass Spectrometric Identification of the Arginine and Lysine deficient Proline Rich Glutamine Rich Wheat Storage Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tandem mass spectrometry (MS/MS) of enzymatic digest has made possible identification of a wide variety of proteins and complex samples prepared by such techniques as RP-HPLC or 2-D gel electrophoresis. Success requires peptide fragmentation to be indicative of the peptide amino acid sequence. The f...

  19. Endothelin-1 expression is strongly repressed by AU-rich elements in the 3'-untranslated region of the gene.

    PubMed

    Reimunde, Francisco M; Castañares, Cristina; Redondo-Horcajo, Mariano; Lamas, Santiago; Rodríguez-Pascual, Fernando

    2005-05-01

    The regulation of the synthesis of the endothelial-derived vasoconstrictor ET-1 (endothelin-1) is a complex process that occurs mainly at the mRNA level. Transcription of the gene accounts for an important part of the regulation of expression, as already described for different modulators such as the cytokine TGF-beta (transforming growth factor-beta). However, very little is known about mechanisms governing ET-1 expression at the post-transcriptional level. The aim of the present study was to investigate the regulation of the ET-1 expression at this level. Since the 3'-UTR (3'-untranslated region) of mRNAs commonly contains genetic determinants for the post-transcriptional control of gene expression, we focused on the potential role of the 3'-UTR of ET-1 mRNA. Experiments performed with luciferase reporter constructs containing the 3'-UTR showed that this region exerts a potent destabilizing effect. Deletional analyses allowed us to locate this activity within a region at positions 924-1127. Some (but not all) of the AREs (AU-rich elements) present in this region were found to be essential for this mRNA-destabilizing activity. We also present evidence that cytosolic proteins from endothelial cells interact specifically with these RNA elements, and that a close correlation exists between the ability of the AREs to destabilize ET-1 mRNA and the binding of proteins to these elements. Our results are compatible with the existence of a strong repressional control of ET-1 expression mediated by destabilization of the mRNA exerted through the interaction of specific cytosolic proteins with AREs present in the 3'-UTR of the gene.

  20. Geography of Alaska Lake Districts: Identification, Description, and Analysis of Lake-Rich Regions of a Diverse and Dynamic State

    USGS Publications Warehouse

    Arp, Christopher D.; Jones, Benjamin M.

    2009-01-01

    Lakes are abundant landforms and important ecosystems in Alaska, but are unevenly distributed on the landscape with expansive lake-poor regions and several lake-rich regions. Such lake-rich areas are termed lake districts and have landscape characteristics that can be considered distinctive in similar respects to mountain ranges. In this report, we explore the nature of lake-rich areas by quantitatively identifying Alaska's lake districts, describing and comparing their physical characteristics, and analyzing how Alaska lake districts are naturally organized and correspond to climatic and geophysical characteristics, as well as studied and managed by people. We use a digital dataset (National Hydrography Dataset) of lakes greater than 1 hectare, which includes 409,040 individual lakes and represents 3.3 percent of the land-surface area of Alaska. The selection criteria we used to identify lake districts were (1) a lake area (termed limnetic ratio, in percent) greater than the mean for the State, and (2) a lake density (number of lakes per unit area) greater than the mean for the State using a pixel size scaled to the area of interest and number of lakes in the census. Pixels meeting these criteria were grouped and delineated and all groups greater than 1,000 square kilometers were identified as Alaska's lake districts. These lake districts were described according to lake size-frequency metrics, elevation distributions, geology, climate, and ecoregions to better understand their similarities and differences. We also looked at where lake research and relevant ecological monitoring has occurred in Alaska relative to lake districts and how lake district lands and waters are currently managed. We identified and delineated 20 lake districts in Alaska representing 16 percent of the State, but including 65 percent of lakes and 75 percent of lake area. The largest lake districts identified are the Yukon-Kuskokwim Delta, Arctic Coastal Plain, and Iliamna lake districts with

  1. Mycorrhizal-mediated lower proline accumulation in Poncirus trifoliata under water deficit derives from the integration of inhibition of proline synthesis with increase of proline degradation.

    PubMed

    Zou, Ying-Ning; Wu, Qiang-Sheng; Huang, Yong-Ming; Ni, Qiu-Dan; He, Xin-Hua

    2013-01-01

    Proline accumulation was often correlated with drought tolerance of plants infected by arbuscular mycorrhizal fungi (AMF), whereas lower proline in some AM plants including citrus was also found under drought stress and the relevant mechanisms have not been fully elaborated. In this study proline accumulation and activity of key enzymes relative to proline biosynthesis (▵(1)-pyrroline-5-carboxylate synthetase, P5CS; ornithine-δ-aminotransferase, OAT) and degradation (proline dehydrogenase, ProDH) were determined in trifoliate orange (Poncirus trifoliata, a widely used citrus rootstock) inoculated with or without Funneliformis mosseae and under well-watered (WW) or water deficit (WD). AMF colonization significantly increased plant height, stem diameter, leaf number, root volume, biomass production of both leaves and roots and leaf relative water content, irrespectively of water status. Water deficit induced more tissue proline accumulation, in company with an increase of P5CS activity, but a decrease of OAT and ProDH activity, no matter whether under AM or no-AM. Compared with no-AM treatment, AM treatment resulted in lower proline concentration and content in leaf, root, and total plant under both WW and WD. The AMF colonization significantly decreased the activity of both P5CS and OAT in leaf, root, and total plant under WW and WD, except for an insignificant difference of root OAT under WD. The AMF inoculation also generally increased tissue ProDH activity under WW and WD. Plant proline content significantly positively correlated with plant P5CS activity, negatively with plant ProDH activity, but not with plant OAT activity. These results suggest that AM plants may suffer less from WD, thereby inducing lower proline accumulation, which derives from the integration of an inhibition of proline synthesis with an enhancement of proline degradation.

  2. Mycorrhizal-Mediated Lower Proline Accumulation in Poncirus trifoliata under Water Deficit Derives from the Integration of Inhibition of Proline Synthesis with Increase of Proline Degradation

    PubMed Central

    Zou, Ying-Ning; Wu, Qiang-Sheng; Huang, Yong-Ming; Ni, Qiu-Dan; He, Xin-Hua

    2013-01-01

    Proline accumulation was often correlated with drought tolerance of plants infected by arbuscular mycorrhizal fungi (AMF), whereas lower proline in some AM plants including citrus was also found under drought stress and the relevant mechanisms have not been fully elaborated. In this study proline accumulation and activity of key enzymes relative to proline biosynthesis (▵1-pyrroline-5-carboxylate synthetase, P5CS; ornithine-δ-aminotransferase, OAT) and degradation (proline dehydrogenase, ProDH) were determined in trifoliate orange (Poncirus trifoliata, a widely used citrus rootstock) inoculated with or without Funneliformis mosseae and under well-watered (WW) or water deficit (WD). AMF colonization significantly increased plant height, stem diameter, leaf number, root volume, biomass production of both leaves and roots and leaf relative water content, irrespectively of water status. Water deficit induced more tissue proline accumulation, in company with an increase of P5CS activity, but a decrease of OAT and ProDH activity, no matter whether under AM or no-AM. Compared with no-AM treatment, AM treatment resulted in lower proline concentration and content in leaf, root, and total plant under both WW and WD. The AMF colonization significantly decreased the activity of both P5CS and OAT in leaf, root, and total plant under WW and WD, except for an insignificant difference of root OAT under WD. The AMF inoculation also generally increased tissue ProDH activity under WW and WD. Plant proline content significantly positively correlated with plant P5CS activity, negatively with plant ProDH activity, but not with plant OAT activity. These results suggest that AM plants may suffer less from WD, thereby inducing lower proline accumulation, which derives from the integration of an inhibition of proline synthesis with an enhancement of proline degradation. PMID:24260421

  3. Regulation and Function of Proline Oxidase under Nutrient Stress

    PubMed Central

    Pandhare, Jui; Cooper, Sandra K.; Donald, Steven P.; Phang, James M.

    2009-01-01

    Under conditions of nutrient stress, cells switch to a survival mode catabolizing cellular and tissue constituents for energy. Proline metabolism is especially important in nutrient stress because proline is readily available from the breakdown of extracellular matrix (ECM), and the degradation of proline through the proline cycle initiated by proline oxidase (POX), a mitochondrial inner membrane enzyme, can generate ATP. This degradative pathway generates glutamate and α-ketoglutarate, products that can play an anaplerotic role for the TCA cycle. In addition the proline cycle is in a metabolic interlock with the pentose phosphate pathway providing another bioenergetic mechanism. Herein we have investigated the role of proline metabolism in conditions of nutrient stress in the RKO colorectal cancer cell line. The induction of stress either by glucose withdrawal or by treatment with rapamycin, stimulated degradation of proline and increased POX catalytic activity. Under these conditions POX was responsible, at least in part, for maintenance of ATP levels. Activation of AMP-activated protein kinase (AMPK), the cellular energy sensor, by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), also markedly upregulated POX and increased POX-dependent ATP levels, further supporting its role during stress. Glucose deprivation increased intracellular proline levels, and expression of POX activated the pentose phosphate pathway. Together, these results suggest that the induction of proline cycle under conditions of nutrient stress may be a mechanism by which cells switch to a catabolic mode for maintaining cellular energy levels. PMID:19415679

  4. Proline metabolism in N2-fixing root nodules: energy transfer and regulation of purine synthesis.

    PubMed Central

    Kohl, D H; Schubert, K R; Carter, M B; Hagedorn, C H; Shearer, G

    1988-01-01

    N2-fixing root nodules of soybean (Glycine max L. Merr.) convert atmospheric N2 to ammonia(um) in an energy-intensive enzymatic reaction. These nodules synthesize large quantities of purines because nitrogen fixed by bacteria contained within this tissue is transferred to the shoots in the form of ureides, which are degradation products of purines. In animal systems, it has been proposed that proline biosynthesis by pyrroline-5-carboxylate reductase (P5CR) is used to generate the NADP+ required for the synthesis of the purine precursor ribose 5-phosphate. We have examined the levels, properties, and location of P5CR and proline dehydrogenase (ProDH) in soybean nodules. Nodule P5CR was found in the plant cytosol. Its activity was substantially higher than that reported for other animal and plant tissues and is 4-fold higher than in pea (Pisum sativum) nodules (which export amides). The Km for NADPH was lower by a factor of 25 than the Km for NADH, while the Vmax with NADPH was one-third of that with NADH. P5CR activity was diminished by NADP+ but not by proline. These characteristics are consistent with a role for P5CR in supporting nodule purine biosynthesis rather than in producing proline for incorporation into protein. ProDH activity was divided between the bacteroids and plant cytosol, but less than 2% was in the mitochondria-rich fractions. The specific activity of ProDH in soybean nodule bacteroids was comparable to that in rat liver mitochondria. In addition, we propose that some of the proline synthesized in the plant cytosol by P5CR is catabolized within the bacteroids by ProDH and that this represents a novel mechanism for transferring energy from the plant to its endosymbiont. PMID:3353366

  5. L-Proline nutrition and catabolism in Staphylococcus saprophyticus.

    PubMed

    Deutch, Charles E

    2011-05-01

    Staphylococcus saprophyticus strains ATCC 15305, ATCC 35552, and ATCC 49907 were found to require L-proline but not L-arginine for growth in a defined culture medium. All three strains could utilize L-ornithine as a proline source and contained L-ornithine aminotransferase and Δ(1)-pyrroline-5-carboxylate reductase activities; strains ATCC 35552 and ATCC 49907 could use L-arginine as a proline source and had L-arginase activity. The proline requirement also could be met by L-prolinamide, L-proline methyl ester, and the dipeptides L-alanyl-L-proline and L-leucyl-L-proline. The bacteria exhibited L-proline degradative activity as measured by the formation of Δ(1)-pyrroline-5-carboxylate. The specific activity of proline degradation was not affected by addition of L-proline or NaCl but was highest in strain ATCC 49907 after growth in Mueller-Hinton broth. A membrane fraction from this strain had L-proline dehydrogenase activity as detected both by reaction of Δ(1)-pyrroline-5-carboxylate with 2-aminobenzaldehyde (0.79 nmol min(-1) mg(-1)) and by the proline-dependent reduction of p-iodonitrotetrazolium (20.1 nmol min(-1) mg(-1)). A soluble fraction from this strain had Δ(1)-pyrroline-5-carboxylate dehydrogenase activity (88.8 nmol min(-1) mg(-1)) as determined by the NAD(+)-dependent oxidation of DL-Δ(1)-pyrroline-5-carboxylate. Addition of L-proline to several culture media did not increase the growth rate or final yield of bacteria but did stimulate growth during osmotic stress. When grown with L: -ornithine as the proline source, S. saprophyticus was most susceptible to the proline analogues L-azetidine-2-carboylate, 3,4-dehydro-DL-proline, DL-thiazolidine-2-carboxylate, and L-thiazolidine-4-carboxylate. These results indicate that proline uptake and metabolism may be a potential target of antimicrobial therapy for this organism.

  6. Enzymatic production of trans-4-hydroxy-L-proline by regio- and stereospecific hydroxylation of L-proline.

    PubMed

    Shibasaki, T; Mori, H; Ozaki, A

    2000-04-01

    A proline 4-hydroxylase gene, which was cloned from Dactylosporangium sp. RH1, was overexpressed in Escherichia coli W1485 on a plasmid under a tryptophan tandem promoter after the codon usage of the 5' end of the gene was optimized. The proline 4-hydroxylase activity was l600-fold higher than that in Dactylosporangium sp. RH1. trans-4-Hydroxy-L-proline(Hyp) was produced and accumulated to 41 g/L (87% yield from L-proline) in 100 h when the recombinant E. coli was cultivated in a medium containing L-proline and glucose. 2-Oxoglutarate, which is necessary for the hydroxylation of L-proline by proline 4-hydroxylase, was apparently supplied from glucose through the cellular metabolic pathway. The putA mutant of W1485, which is not able to degrade L-proline, has allowed the quantitative conversion of L-proline to Hyp. The formation of other isomers of hydroxyproline was not observed. Productivity of Hyp was almost the same in a larger-scale culture. The method of manufacturing Hyp from L-proline was established.

  7. Gene-rich chromosomal regions are preferentially localized in the lamin B deficient nuclear blebs of atypical progeria cells.

    PubMed

    Bercht Pfleghaar, Katrin; Taimen, Pekka; Butin-Israeli, Veronika; Shimi, Takeshi; Langer-Freitag, Sabine; Markaki, Yolanda; Goldman, Anne E; Wehnert, Manfred; Goldman, Robert D

    2015-01-01

    More than 20 mutations in the gene encoding A-type lamins (LMNA) cause progeria, a rare premature aging disorder. The major pathognomonic hallmarks of progeria cells are seen as nuclear deformations or blebs that are related to the redistribution of A- and B-type lamins within the nuclear lamina. However, the functional significance of these progeria-associated blebs remains unknown. We have carried out an analysis of the structural and functional consequences of progeria-associated nuclear blebs in dermal fibroblasts from a progeria patient carrying a rare point mutation p.S143F (C428T) in lamin A/C. These blebs form microdomains that are devoid of major structural components of the nuclear envelope (NE)/lamina including B-type lamins and nuclear pore complexes (NPCs) and are enriched in A-type lamins. Using laser capture microdissection and comparative genomic hybridization (CGH) analyses, we show that, while these domains are devoid of centromeric heterochromatin and gene-poor regions of chromosomes, they are enriched in gene-rich chromosomal regions. The active form of RNA polymerase II is also greatly enriched in blebs as well as nascent RNA but the nuclear co-activator SKIP is significantly reduced in blebs compared to other transcription factors. Our results suggest that the p.S143F progeria mutation has a severe impact not only on the structure of the lamina but also on the organization of interphase chromatin domains and transcription. These structural defects are likely to contribute to gene expression changes reported in progeria and other types of laminopathies.

  8. Gene-rich chromosomal regions are preferentially localized in the lamin B deficient nuclear blebs of atypical progeria cells

    PubMed Central

    Bercht Pfleghaar, Katrin; Taimen, Pekka; Butin-Israeli, Veronika; Shimi, Takeshi; Langer-Freitag, Sabine; Markaki, Yolanda; Goldman, Anne E; Wehnert, Manfred; Goldman, Robert D

    2015-01-01

    More than 20 mutations in the gene encoding A-type lamins (LMNA) cause progeria, a rare premature aging disorder. The major pathognomonic hallmarks of progeria cells are seen as nuclear deformations or blebs that are related to the redistribution of A- and B-type lamins within the nuclear lamina. However, the functional significance of these progeria-associated blebs remains unknown. We have carried out an analysis of the structural and functional consequences of progeria-associated nuclear blebs in dermal fibroblasts from a progeria patient carrying a rare point mutation p.S143F (C428T) in lamin A/C. These blebs form microdomains that are devoid of major structural components of the nuclear envelope (NE)/lamina including B-type lamins and nuclear pore complexes (NPCs) and are enriched in A-type lamins. Using laser capture microdissection and comparative genomic hybridization (CGH) analyses, we show that, while these domains are devoid of centromeric heterochromatin and gene-poor regions of chromosomes, they are enriched in gene-rich chromosomal regions. The active form of RNA polymerase II is also greatly enriched in blebs as well as nascent RNA but the nuclear co-activator SKIP is significantly reduced in blebs compared to other transcription factors. Our results suggest that the p.S143F progeria mutation has a severe impact not only on the structure of the lamina but also on the organization of interphase chromatin domains and transcription. These structural defects are likely to contribute to gene expression changes reported in progeria and other types of laminopathies. PMID:25738644

  9. Significant Local-Scale Plant-Insect Species Richness Relationship Independent of Abiotic Effects in the Temperate Cape Floristic Region Biodiversity Hotspot.

    PubMed

    Kemp, Jurene E; Ellis, Allan G

    2017-01-01

    Globally plant species richness is a significant predictor of insect richness. Whether this is the result of insect diversity responding directly to plant diversity, or both groups responding in similar ways to extrinsic factors, has been much debated. Here we assess this relationship in the Cape Floristic Region (CFR), a biodiversity hotspot. The CFR has higher plant diversity than expected from latitude (i.e., abiotic conditions), but very little is known about the diversity of insects residing in this region. We first quantify diversity relationships at multiple spatial scales for one of the dominant plant families in the CFR, the Restionaceae, and its associated insect herbivore community. Plant and insect diversity are significantly positively correlated at the local scales (10-50 m; 0.1-3 km), but not at the regional scales (15-20 km; 50-70 km). The local scale relationship remains significantly positively correlated even when accounting for the influence of extrinsic variables and other vegetation attributes. This suggests that the diversity of local insect assemblages may be more strongly influenced by plant species richness than by abiotic variables. Further, vegetation age and plant structural complexity also influenced insect richness. The ratio of insect species per plant species in the CFR is comparable to other temperate regions around the world, suggesting that the insect diversity of the CFR is high relative to other areas of the globe with similar abiotic conditions, primarily as a result of the unusually high plant diversity in the region.

  10. Significant Local-Scale Plant-Insect Species Richness Relationship Independent of Abiotic Effects in the Temperate Cape Floristic Region Biodiversity Hotspot

    PubMed Central

    Kemp, Jurene E.; Ellis, Allan G.

    2017-01-01

    Globally plant species richness is a significant predictor of insect richness. Whether this is the result of insect diversity responding directly to plant diversity, or both groups responding in similar ways to extrinsic factors, has been much debated. Here we assess this relationship in the Cape Floristic Region (CFR), a biodiversity hotspot. The CFR has higher plant diversity than expected from latitude (i.e., abiotic conditions), but very little is known about the diversity of insects residing in this region. We first quantify diversity relationships at multiple spatial scales for one of the dominant plant families in the CFR, the Restionaceae, and its associated insect herbivore community. Plant and insect diversity are significantly positively correlated at the local scales (10–50 m; 0.1–3 km), but not at the regional scales (15–20 km; 50–70 km). The local scale relationship remains significantly positively correlated even when accounting for the influence of extrinsic variables and other vegetation attributes. This suggests that the diversity of local insect assemblages may be more strongly influenced by plant species richness than by abiotic variables. Further, vegetation age and plant structural complexity also influenced insect richness. The ratio of insect species per plant species in the CFR is comparable to other temperate regions around the world, suggesting that the insect diversity of the CFR is high relative to other areas of the globe with similar abiotic conditions, primarily as a result of the unusually high plant diversity in the region. PMID:28076412

  11. Unconventional N-H…N Hydrogen Bonds Involving Proline Backbone Nitrogen in Protein Structures.

    PubMed

    Deepak, R N V Krishna; Sankararamakrishnan, Ramasubbu

    2016-05-10

    Contrary to DNA double-helical structures, hydrogen bonds (H-bonds) involving nitrogen as the acceptor are not common in protein structures. We systematically searched N-H…N H-bonds in two different sets of protein structures. Data set I consists of neutron diffraction and ultrahigh-resolution x-ray structures (0.9 Å resolution or better) and the hydrogen atom positions in these structures were determined experimentally. Data set II contains structures determined using x-ray diffraction (resolution ≤ 1.8 Å) and the positions of hydrogen atoms were generated using a computational method. We identified 114 and 14,347 potential N-H…N H-bonds from these two data sets, respectively, and 56-66% of these were of the Ni+1-Hi+1…Ni type, with Ni being the proline backbone nitrogen. To further understand the nature of such unusual contacts, we performed quantum chemical calculations on the model compound N-acetyl-L-proline-N-methylamide (Ace-Pro-NMe) with coordinates taken from the experimentally determined structures. A potential energy profile generated by varying the ψ dihedral angle in Ace-Pro-NMe indicates that the conformation with the N-H…N H-bond is the most stable. An analysis of H-bond-forming proline residues reveals that more than 30% of the proline carbonyl groups are also involved in n → π(∗) interactions with the carbonyl carbon of the preceding residue. Natural bond orbital analyses demonstrate that the strength of N-H…N H-bonds is less than half of that observed for a conventional H-bond. This study clearly establishes the H-bonding capability of proline nitrogen and its prevalence in protein structures. We found many proteins with multiple instances of H-bond-forming prolines. With more than 15% of all proline residues participating in N-H…N H-bonds, we suggest a new, to our knowledge, structural role for proline in providing stability to loops and capping regions of secondary structures in proteins.

  12. The Cys-Rich Region of Hepatitis A Virus Cellular Receptor 1 Is Required for Binding of Hepatitis A Virus and Protective Monoclonal Antibody 190/4

    PubMed Central

    Thompson, Peter; Lu, Jinhua; Kaplan, Gerardo G.

    1998-01-01

    The hepatitis A virus cellular receptor 1 (HAVcr-1) cDNA codes for a class I integral membrane glycoprotein, termed havcr-1, of unknown natural function which serves as an African green monkey kidney (AGMK) cell receptor for HAV. The extracellular domain of havcr-1 has an N-terminal Cys-rich region that displays homology with sequences of members of the immunoglobulin superfamily, followed by a Thr/Ser/Pro (TSP)-rich region characteristic of mucin-like O-glycosylated proteins. The havcr-1 glycoprotein contains four putative N-glycosylation sites, two in the Cys-rich region and two in the TSP-rich region. To characterize havcr-1 and define region(s) involved in HAV receptor function, we expressed the TSP-rich region in Escherichia coli fused to glutathione S-transferase and generated antibodies (Ab) in rabbits (anti-GST2 Ab). Western blot analysis with anti-GST2 Ab detected 62- and 65-kDa bands in AGMK cells and 59-, 62-, and 65-kDa bands in dog cells transfected with the HAVcr-1 cDNA (cr5 cells) but not in dog cells transfected with the vector alone (DR2 cells). Treatment of AGMK and cr5 cell extracts with peptide-N-glycosidase F resulted in the collapse of the havcr-1-specific bands into a single band of 56 kDa, which indicated that different N-glycosylated forms of havcr-1 were expressed in these cells. Treatment of AGMK and cr5 cells with tunicamycin reduced binding of protective monoclonal Ab (MAb) 190/4, which suggested that N-glycans are required for binding of MAb 190/4 to havcr-1. To test this hypothesis, havcr-1 mutants lacking the N-glycosylation motif at the first site (mut1), second site (mut2), and both (mut3) sites were constructed and transfected into dog cells. Binding of MAb 190/4 and HAV to mut1 and mut3 cells was highly reduced, while binding to mut2 cells was not affected and binding to dog cells expressing an havcr-1 construct containing a deletion of the Cys-rich region (d1− cells) was undetectable. HAV-infected cr5 and mut2 cells but not

  13. The Cys-rich region of hepatitis A virus cellular receptor 1 is required for binding of hepatitis A virus and protective monoclonal antibody 190/4.

    PubMed

    Thompson, P; Lu, J; Kaplan, G G

    1998-05-01

    The hepatitis A virus cellular receptor 1 (HAVcr-1) cDNA codes for a class I integral membrane glycoprotein, termed havcr-1, of unknown natural function which serves as an African green monkey kidney (AGMK) cell receptor for HAV. The extracellular domain of havcr-1 has an N-terminal Cys-rich region that displays homology with sequences of members of the immunoglobulin superfamily, followed by a Thr/Ser/Pro (TSP)-rich region characteristic of mucin-like O-glycosylated proteins. The havcr-1 glycoprotein contains four putative N-glycosylation sites, two in the Cys-rich region and two in the TSP-rich region. To characterize havcr-1 and define region(s) involved in HAV receptor function, we expressed the TSP-rich region in Escherichia coli fused to glutathione S-transferase and generated antibodies (Ab) in rabbits (anti-GST2 Ab). Western blot analysis with anti-GST2 Ab detected 62- and 65-kDa bands in AGMK cells and 59-, 62-, and 65-kDa bands in dog cells transfected with the HAVcr-1 cDNA (cr5 cells) but not in dog cells transfected with the vector alone (DR2 cells). Treatment of AGMK and cr5 cell extracts with peptide-N-glycosidase F resulted in the collapse of the havcr-1-specific bands into a single band of 56 kDa, which indicated that different N-glycosylated forms of havcr-1 were expressed in these cells. Treatment of AGMK and cr5 cells with tunicamycin reduced binding of protective monoclonal Ab (MAb) 190/4, which suggested that N-glycans are required for binding of MAb 190/4 to havcr-1. To test this hypothesis, havcr-1 mutants lacking the N-glycosylation motif at the first site (mut1), second site (mut2), and both (mut3) sites were constructed and transfected into dog cells. Binding of MAb 190/4 and HAV to mut1 and mut3 cells was highly reduced, while binding to mut2 cells was not affected and binding to dog cells expressing an havcr-1 construct containing a deletion of the Cys-rich region (d1- cells) was undetectable. HAV-infected cr5 and mut2 cells but not mut1

  14. A versatile proline/alanine transporter in the unicellular pathogen Leishmania donovani regulates amino acid homoeostasis and osmotic stress responses.

    PubMed

    Inbar, Ehud; Schlisselberg, Doreen; Suter Grotemeyer, Marianne; Rentsch, Doris; Zilberstein, Dan

    2013-01-15

    Unlike all other organisms, parasitic protozoa of the family Trypanosomatidae maintain a large cellular pool of proline that, together with the alanine pool, serve as alternative carbon sources as well as reservoirs of organic osmolytes. These reflect adaptation to their insect vectors whose haemolymphs are exceptionally rich in the two amino acids. In the present study we identify and characterize a new neutral amino acid transporter, LdAAP24, that translocates proline and alanine across the Leishmania donovani plasma membrane. This transporter fulfils multiple functions: it is the sole supplier for the intracellular pool of proline and contributes to the alanine pool; it is essential for cell volume regulation after osmotic stress; and it regulates the transport and homoeostasis of glutamate and arginine, none of which are its substrates. Notably, we provide evidence that proline and alanine exhibit different roles in the parasitic response to hypotonic shock; alanine affects swelling, whereas proline influences the rate of volume recovery. On the basis of our data we suggest that LdAAP24 plays a key role in parasite adaptation to its varying environments in host and vector, a phenomenon essential for successful parasitism.

  15. Connecting proline metabolism and signaling pathways in plant senescence

    PubMed Central

    Zhang, Lu; Becker, Donald F.

    2015-01-01

    The amino acid proline has a unique biological role in stress adaptation. Proline metabolism is manipulated under stress by multiple and complex regulatory pathways and can profoundly influence cell death and survival in microorganisms, plants, and animals. Though the effects of proline are mediated by diverse signaling pathways, a common theme appears to be the generation of reactive oxygen species (ROS) due to proline oxidation being coupled to the respiratory electron transport chain. Considerable research has been devoted to understand how plants exploit proline metabolism in response to abiotic and biotic stress. Here, we review potential mechanisms by which proline metabolism influences plant senescence, namely in the petal and leaf. Recent studies of petal senescence suggest proline content is manipulated to meet energy demands of senescing cells. In the flower and leaf, proline metabolism may influence ROS signaling pathways that delay senescence progression. Future studies focusing on the mechanisms by which proline metabolic shifts occur during senescence may lead to novel methods to rescue crops under stress and to preserve post-harvest agricultural products. PMID:26347750

  16. Spectroscopy of Neutron-rich Nuclei of the A{approx_equal}60 region populated through binary heavy-ion collisions

    SciTech Connect

    Lunardi, S.

    2008-11-11

    Neutron-rich nuclei of the mass A = 60 region (from V to Fe) have been studied through multi-nucleon transfer reactions by bombarding a {sup 238}U target with beams of {sup 64}Ni and {sup 70}Zn. Unambiguous identification of prompt {gamma} rays belonging to each nucleus has been achieved by using the efficient gamma-array CLARA coupled to the large-acceptance magnetic spectrometer PRISMA installed at the Legnaro National Laboratories. With the new data, the existence of the N = 32 sub-shell closure has been corroborated through the study of odd V isotopes, whereas a new region of deformation appears for neutron-rich Fe nuclei close to N = 40. The results obtained for all these nuclei are compared with shell model calculations which reproduces quite well the experimental data also for the most neutron-rich nuclei when excitations from the fp shell into the upper g{sub 9/2} orbital are allowed.

  17. Proline metabolism and cancer: emerging links to glutamine and collagen

    PubMed Central

    Phang, James M.; Liu, Wei; Hancock, Chad N.; Fischer, Joseph W.

    2015-01-01

    Purpose of review Proline metabolism impacts a number of regulatory targets in both animals and plants and is especially important in cancer. Glutamine, a related amino acid, is considered second in importance only to glucose as a substrate for tumors. But proline and glutamine are interconvertible and linked in their metabolism. In animals, proline and glutamine have specific regulatory functions and their respective physiologic sources. A comparison of the metabolism of proline and glutamine would help us understand the importance of these two nonessential amino acids in cancer metabolism. Recent findings The regulatory functions of proline metabolism proposed 3 decades ago have found relevance in many areas. For cancer, these functions play a role in apoptosis, autophagy and in response to nutrient and oxygen deprivation. Importantly, proline-derived reactive oxygen species served as a driving signal for reprogramming. This model has been applied by others to metabolic regulation for the insulin-prosurvival axis, induction of adipose triglyceride lipase for lipid metabolism and regulation of embryonic stem cell development. Of special interest, modulatory proteins such as parkinson protein 7 and oral cancer overexpressed 1 interact with pyrroline-5-carboxylate reductase, a critical component of the proline regulatory axis. Although the interconvertibility of proline and glutamine has been long established, recent findings showed that the proto-oncogene, cellular myelocytomatosis oncogene, upregulates glutamine utilization (glutaminase) and routes glutamate to proline biosynthesis (pyrroline-5-carboxylate synthase, pyrroline-5-carboxylate reductases). Additionally, collagen, which contains large amounts of proline, may be metabolized to serve as a reservoir for proline. This metabolic relationship as well as the new regulatory targets of proline metabolism invites an elucidation of the differential effects of these nonessential amino acids and their production

  18. Proline and hydroxyproline metabolism: implications for animal and human nutrition.

    PubMed

    Wu, Guoyao; Bazer, Fuller W; Burghardt, Robert C; Johnson, Gregory A; Kim, Sung Woo; Knabe, Darrell A; Li, Peng; Li, Xilong; McKnight, Jason R; Satterfield, M Carey; Spencer, Thomas E

    2011-04-01

    Proline plays important roles in protein synthesis and structure, metabolism (particularly the synthesis of arginine, polyamines, and glutamate via pyrroline-5-carboxylate), and nutrition, as well as wound healing, antioxidative reactions, and immune responses. On a per-gram basis, proline plus hydroxyproline are most abundant in collagen and milk proteins, and requirements of proline for whole-body protein synthesis are the greatest among all amino acids. Therefore, physiological needs for proline are particularly high during the life cycle. While most mammals (including humans and pigs) can synthesize proline from arginine and glutamine/glutamate, rates of endogenous synthesis are inadequate for neonates, birds, and fish. Thus, work with young pigs (a widely used animal model for studying infant nutrition) has shown that supplementing 0.0, 0.35, 0.7, 1.05, 1.4, and 2.1% proline to a proline-free chemically defined diet containing 0.48% arginine and 2% glutamate dose dependently improved daily growth rate and feed efficiency while reducing concentrations of urea in plasma. Additionally, maximal growth performance of chickens depended on at least 0.8% proline in the diet. Likewise, dietary supplementation with 0.07, 0.14, and 0.28% hydroxyproline (a metabolite of proline) to a plant protein-based diet enhanced weight gains of salmon. Based on its regulatory roles in cellular biochemistry, proline can be considered as a functional amino acid for mammalian, avian, and aquatic species. Further research is warranted to develop effective strategies of dietary supplementation with proline or hydroxyproline to benefit health, growth, and development of animals and humans.

  19. Ornithine cyclodeaminase-based proline production by Corynebacterium glutamicum

    PubMed Central

    2013-01-01

    Background The soil bacterium Corynebacterium glutamicum, best known for its glutamate producing ability, is suitable as a producer of a variety of bioproducts. Glutamate is the precursor of the amino acid proline. Proline biosynthesis typically involves three enzymes and a spontaneous cyclisation reaction. Alternatively, proline can be synthesised from ornithine, an intermediate of arginine biosynthesis. The direct conversion of ornithine to proline is catalysed by ornithine cyclodeaminase. An ornithine overproducing platform strain with deletions of argR and argF (ORN1) has been employed for production of derived compounds such as putrescine. By heterologous expression of ocd this platform strain can be engineered further for proline production. Results Plasmid-based expression of ocd encoding the putative ornithine cyclodeaminase of C. glutamicum did not result in detectable proline accumulation in the culture medium. However, plasmid-based expression of ocd from Pseudomonas putida resulted in proline production with yields up to 0.31 ± 0.01 g proline/g glucose. Overexpression of the gene encoding a feedback-alleviated N-acetylglutamate kinase further increased proline production to 0.36 ± 0.01 g/g. In addition, feedback-alleviation of N-acetylglutamate kinase entailed growth-coupled production of proline and reduced the accumulation of by-products in the culture medium. Conclusions The product spectrum of the platform strain C. glutamicum ORN1 was expanded to include the amino acid L-proline. Upon further development of the ornithine overproducing platform strain, industrial production of amino acids of the glutamate family and derived bioproducts such as diamines might become within reach. PMID:23806148

  20. Efficacy of proline in the treatment of menopause

    PubMed Central

    Nam, Sun-Young; Yoou, Myoung-Schook

    2016-01-01

    The amino acids in the placenta have multiple functions; however, the therapeutic effects of proline remain poorly for relief postmenopausal symptoms. The aim of present study was to evaluate the effects of proline in the treatment of menopause using in vitro and in vivo models. We assessed the therapeutic effects and regulatory mechanisms of proline by using MCF-7 estrogen-dependent cells, MG63 osteoblast cells, and ovariectomized mice model. An in vivo study was carried out in eight-week-old sham and ovariectomized group. The ovariectomized mouse was further subdivided into two groups administered orally with 17β-estradiol or proline (10 mg/kg/day) for eight weeks. Proline significantly increased cell proliferation and Ki-67 levels in MCF-7 cells and enhanced cell proliferation, alkaline phosphatase activity, extracellular signal-regulated kinase phosphorylation, and glutamyl-prolyl-tRNA synthetase activation in MG63 cells. The estrogen receptor-β and estrogen-response elements luciferase activity were significantly increased by proline in MCF-7 and MG63 cells. In ovariectomized mice, oral administration of proline (10 mg/kg/day) for eight weeks significantly reduced body and vaginal weights. Proline also significantly increased serum estradiol and alkaline phosphatase levels, whereas serum luteinizing hormone was decreased by proline. In addition, detailed microcomputed tomography analysis showed that the proline notably enhanced bone mineral density, trabecular bone volume, and trabecular number in ovariectomized mice. Those findings implied that proline can be a promising candidate for the treatment of menopause. PMID:26830682

  1. Efficacy of proline in the treatment of menopause.

    PubMed

    Nam, Sun-Young; Yoou, Myoung-Schook; Kim, Hyung-Min; Jeong, Hyun-Ja

    2016-03-01

    The amino acids in the placenta have multiple functions; however, the therapeutic effects of proline remain poorly for relief postmenopausal symptoms. The aim of present study was to evaluate the effects of proline in the treatment of menopause using in vitro and in vivo models. We assessed the therapeutic effects and regulatory mechanisms of proline by using MCF-7 estrogen-dependent cells, MG63 osteoblast cells, and ovariectomized mice model. An in vivo study was carried out in eight-week-old sham and ovariectomized group. The ovariectomized mouse was further subdivided into two groups administered orally with 17β-estradiol or proline (10 mg/kg/day) for eight weeks. Proline significantly increased cell proliferation and Ki-67 levels in MCF-7 cells and enhanced cell proliferation, alkaline phosphatase activity, extracellular signal-regulated kinase phosphorylation, and glutamyl-prolyl-tRNA synthetase activation in MG63 cells. The estrogen receptor-β and estrogen-response elements luciferase activity were significantly increased by proline in MCF-7 and MG63 cells. In ovariectomized mice, oral administration of proline (10 mg/kg/day) for eight weeks significantly reduced body and vaginal weights. Proline also significantly increased serum estradiol and alkaline phosphatase levels, whereas serum luteinizing hormone was decreased by proline. In addition, detailed microcomputed tomography analysis showed that the proline notably enhanced bone mineral density, trabecular bone volume, and trabecular number in ovariectomized mice. Those findings implied that proline can be a promising candidate for the treatment of menopause.

  2. Interspecific differences in tannin intakes of forest-dwelling rodents in the wild revealed by a new method using fecal proline content.

    PubMed

    Shimada, Takuya; Nishii, Eriko; Saitoh, Takashi

    2011-12-01

    Mammalian herbivores adopt various countermeasures against dietary tannins, which are among the most widespread plant secondary metabolites. The large Japanese wood mouse Apodemus speciosus produces proline-rich salivary tannin-binding proteins in response to tannins. Proline-rich proteins (PRPs) react with tannins to form stable complexes that are excreted in the feces. Here, we developed a new method for estimating the tannin intake of free-living small rodents, by measuring fecal proline content, and applied the method to a field investigation. A feeding experiment with artificial diets containing various levels of tannic acid revealed that fecal proline content was clearly related to dietary tannin content in three species (A. speciosus, Apodemus argenteus, and Myodes rufocanus). We then used fecal proline content to estimate the tannin intakes of these three forest-dwelling species in a forest in Hokkaido. In the autumn, estimated tannin intakes increased significantly in the Apodemus species, but not in M. rufocanus. We speculated that an increase in tannin intake during autumn may result from consumption of tannin-rich acorns. This hypothesis was consistent with population fluctuation patterns of the three species, which were well-synchronized with acorn abundance for the Apodemus species but not for M. rufocanus.

  3. Comparative Aspects of Tissue Glutamine and Proline Metabolism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cellular metabolism of glutamine and proline are closely interrelated since they can be interconverted with glutamate and ornithine via the mitochondrial pathway involving pyrolline-5-carboxylate (P5C). In adults, glutamine and proline are converted via P5C to citrulline in the gut, then citrul...

  4. Role of proline and GABA in sexual reproduction of angiosperms

    PubMed Central

    Biancucci, Marco; Mattioli, Roberto; Forlani, Giuseppe; Funck, Dietmar; Costantino, Paolo; Trovato, Maurizio

    2015-01-01

    Two glutamate derivatives, proline and γ-aminobutyric acid (GABA), appear to play pivotal roles in different aspects of sexual reproduction in angiosperms, although their precise function in plant reproduction and the molecular basis of their action are not yet fully understood. Proline and GABA have long been regarded as pivotal amino acids in pollen vitality and fertility. Proline may constitute up to 70% of the free amino acid pool in pollen grains and it has been recently shown that Arabidopsis mutants affected in the first and rate-limiting step in proline synthesis produce aberrant and infertile pollen grains, indicating that proline synthesis is required for pollen development and fertility. Concerning GABA, a large body of evidence points to this glutamate derivative as a key determinant of post-pollination fertilization. Intriguingly, proline has also been associated with pollination, another aspect of sexual reproduction, since honeybees were reported to show a strong preference for proline-enriched nectars. In this review, we survey current knowledge on the roles of proline and GABA in plant fertility, and discuss future perspectives potentially capable to improve our understanding on the functions of these amino acids in pollen development, pollination, and pollen tube guidance. PMID:26388884

  5. Proline-Dependent Regulation of Clostridium difficile Stickland Metabolism

    PubMed Central

    Bouillaut, Laurent; Self, William T.

    2013-01-01

    Clostridium difficile, a proteolytic Gram-positive anaerobe, has emerged as a significant nosocomial pathogen. Stickland fermentation reactions are thought to be important for growth of C. difficile and appear to influence toxin production. In Stickland reactions, pairs of amino acids donate and accept electrons, generating ATP and reducing power in the process. Reduction of the electron acceptors proline and glycine requires the d-proline reductase (PR) and the glycine reductase (GR) enzyme complexes, respectively. Addition of proline in the medium increases the level of PR protein but decreases the level of GR. We report the identification of PrdR, a protein that activates transcription of the PR-encoding genes in the presence of proline and negatively regulates the GR-encoding genes. The results suggest that PrdR is a central metabolism regulator that controls preferential utilization of proline and glycine to produce energy via the Stickland reactions. PMID:23222730

  6. Proline Modulates the Trypanosoma cruzi Resistance to Reactive Oxygen Species and Drugs through a Novel D, L-Proline Transporter

    PubMed Central

    Sayé, Melisa; Miranda, Mariana R.; di Girolamo, Fabio; de los Milagros Cámara, María; Pereira, Claudio A.

    2014-01-01

    Trypanosoma cruzi, the etiological agent of Chagas' disease, has a metabolism largely based on the consumption of glucose and proline. This amino acid is essential for host cells infection and intracellular differentiation. In this work we identified a proline transporter (TcAAAP069) by yeasts complementation assays and overexpression in Trypanosoma cruzi epimastigotes. TcAAAP069 is mono-specific for proline but presents an unusual feature; the lack of stereospecificity, because it is competitively inhibited by the D- enantiomer. Parasites overexpressing TcAAAP069 have an increased intracellular proline concentration, 2.6-fold higher than controls, as a consequence of a higher proline transport rate. Furthermore, augmented proline concentration correlates with an improved resistance to trypanocidal drugs and also to reactive oxygen species including hydrogen peroxide and nitric oxide, emulating natural physiological situations. The IC50s for nifurtimox, benznidazole, H2O2 and NO. were 125%, 68%, 44% and 112% higher than controls, respectively. Finally, proline metabolism generates a higher concentration (48%) of ATP in TcAAAP069 parasites. Since proline participates on essential energy pathways, stress and drug resistance responses, these results provide a novel target for the development of new drugs for the treatments for Chagas' disease. PMID:24637744

  7. Gene Mining for Proline Based Signaling Proteins in Cell Wall of Arabidopsis thaliana.

    PubMed

    Ihsan, Muhammad Z; Ahmad, Samina J N; Shah, Zahid Hussain; Rehman, Hafiz M; Aslam, Zubair; Ahuja, Ishita; Bones, Atle M; Ahmad, Jam N

    2017-01-01

    The cell wall (CW) as a first line of defense against biotic and abiotic stresses is of primary importance in plant biology. The proteins associated with cell walls play a significant role in determining a plant's sustainability to adverse environmental conditions. In this work, the genes encoding cell wall proteins (CWPs) in Arabidopsis were identified and functionally classified using geneMANIA and GENEVESTIGATOR with published microarrays data. This yielded 1605 genes, out of which 58 genes encoded proline-rich proteins (PRPs) and glycine-rich proteins (GRPs). Here, we have focused on the cellular compartmentalization, biological processes, and molecular functioning of proline-rich CWPs along with their expression at different plant developmental stages. The mined genes were categorized into five classes on the basis of the type of PRPs encoded in the cell wall of Arabidopsis thaliana. We review the domain structure and function of each class of protein, many with respect to the developmental stages of the plant. We have then used networks, hierarchical clustering and correlations to analyze co-expression, co-localization, genetic, and physical interactions and shared protein domains of these PRPs. This has given us further insight into these functionally important CWPs and identified a number of potentially new cell-wall related proteins in A. thaliana.

  8. Gene Mining for Proline Based Signaling Proteins in Cell Wall of Arabidopsis thaliana

    PubMed Central

    Ihsan, Muhammad Z.; Ahmad, Samina J. N.; Shah, Zahid Hussain; Rehman, Hafiz M.; Aslam, Zubair; Ahuja, Ishita; Bones, Atle M.; Ahmad, Jam N.

    2017-01-01

    The cell wall (CW) as a first line of defense against biotic and abiotic stresses is of primary importance in plant biology. The proteins associated with cell walls play a significant role in determining a plant's sustainability to adverse environmental conditions. In this work, the genes encoding cell wall proteins (CWPs) in Arabidopsis were identified and functionally classified using geneMANIA and GENEVESTIGATOR with published microarrays data. This yielded 1605 genes, out of which 58 genes encoded proline-rich proteins (PRPs) and glycine-rich proteins (GRPs). Here, we have focused on the cellular compartmentalization, biological processes, and molecular functioning of proline-rich CWPs along with their expression at different plant developmental stages. The mined genes were categorized into five classes on the basis of the type of PRPs encoded in the cell wall of Arabidopsis thaliana. We review the domain structure and function of each class of protein, many with respect to the developmental stages of the plant. We have then used networks, hierarchical clustering and correlations to analyze co-expression, co-localization, genetic, and physical interactions and shared protein domains of these PRPs. This has given us further insight into these functionally important CWPs and identified a number of potentially new cell-wall related proteins in A. thaliana. PMID:28289422

  9. High abundance of Serine/Threonine-rich regions predicted to be hyper-O-glycosylated in the secretory proteins coded by eight fungal genomes

    PubMed Central

    2012-01-01

    Background O-glycosylation of secretory proteins has been found to be an important factor in fungal biology and virulence. It consists in the addition of short glycosidic chains to Ser or Thr residues in the protein backbone via O-glycosidic bonds. Secretory proteins in fungi frequently display Ser/Thr rich regions that could be sites of extensive O-glycosylation. We have analyzed in silico the complete sets of putatively secretory proteins coded by eight fungal genomes (Botrytis cinerea, Magnaporthe grisea, Sclerotinia sclerotiorum, Ustilago maydis, Aspergillus nidulans, Neurospora crassa, Trichoderma reesei, and Saccharomyces cerevisiae) in search of Ser/Thr-rich regions as well as regions predicted to be highly O-glycosylated by NetOGlyc (http://www.cbs.dtu.dk). Results By comparison with experimental data, NetOGlyc was found to overestimate the number of O-glycosylation sites in fungi by a factor of 1.5, but to be quite reliable in the prediction of highly O-glycosylated regions. About half of secretory proteins have at least one Ser/Thr-rich region, with a Ser/Thr content of at least 40% over an average length of 40 amino acids. Most secretory proteins in filamentous fungi were predicted to be O-glycosylated, sometimes in dozens or even hundreds of sites. Residues predicted to be O-glycosylated have a tendency to be grouped together forming hyper-O-glycosylated regions of varying length. Conclusions About one fourth of secretory fungal proteins were predicted to have at least one hyper-O-glycosylated region, which consists of 45 amino acids on average and displays at least one O-glycosylated Ser or Thr every four residues. These putative highly O-glycosylated regions can be found anywhere along the proteins but have a slight tendency to be at either one of the two ends. PMID:22994653

  10. Regio- and stereoselective oxygenation of proline derivatives by using microbial 2-oxoglutarate-dependent dioxygenases.

    PubMed

    Hara, Ryotaro; Uchiumi, Naoko; Okamoto, Naoko; Kino, Kuniki

    2014-01-01

    We evaluated the substrate specificities of four proline cis-selective hydroxylases toward the efficient synthesis of proline derivatives. In an initial evaluation, 15 proline-related compounds were investigated as substrates. In addition to l-proline and l-pipecolinic acid, we found that 3,4-dehydro-l-proline, l-azetidine-2-carboxylic acid, cis-3-hydroxy-l-proline, and l-thioproline were also oxygenated. Subsequently, the product structures were determined, revealing cis-3,4-epoxy-l-proline, cis-3-hydroxy-l-azetidine-2-carboxylic acid, and 2,3-cis-3,4-cis-3,4-dihydroxy-l-proline.

  11. Prolidase-proline dehydrogenase/proline oxidase-collagen biosynthesis axis as a potential interface of apoptosis/autophagy.

    PubMed

    Zareba, Ilona; Palka, Jerzy

    2016-07-08

    Prolidase is a cytosolic imidodipeptidase that specifically splits imidodipeptides with C-terminal proline or hydroxyproline. The enzyme plays an important role in the recycling of proline from imidodipeptides for resynthesis of collagen and other proline-containing proteins. The mechanism of prolidase-dependent regulation of collagen biosynthesis was found at both transcriptional and post-transcriptional level. The increase in the enzyme activity is due to its phosphorylation on serine/threonine residues. Prolidase-dependent transcriptional regulation of collagen biosynthesis was found at the level of NF-κB, known inhibitor of type I collagen gene expression. Proline dehydrogenase/proline oxidase (PRODH/POX) is flavin-dependent enzyme associated with the inner mitochondrial membrane. The enzyme catalyzes conversion of proline into Δ(1) -pyrroline-5-carboxylate (P5C), during which reactive oxygen species (ROS) are produced, inducing intrinsic and extrinsic apoptotic pathways. Alternatively, under low glucose stress, PRODH/POX activation produces ATP for energy supply and survival. Of special interest is that PRODH/POX gene is induced by P53 and peroxisome proliferator-activated gamma receptor (PPARγ). Among down-regulators of PRODH/POX is an oncogenic transcription factor c-MYC and miR-23b*. On the other hand, PRODH/POX suppresses HIF-1α transcriptional activity, the MAPK pathway, cyclooxygenase-2, epidermal growth factor receptor and Wnt/b-catenin signaling. PRODH/POX expression is often down-regulated in various tumors, limiting mitochondrial proline utilization to P5C. It is accompanied by increased cytoplasmic level of proline. Proline availability for PRODH/POX-dependent ATP or ROS generation depends on activity of prolidase and utilization of proline in process of collagen biosynthesis. Therefore, Prolidase-PRODH/POX-Collagen Biosynthesis axis may represent potential interface that regulate apoptosis and survival. © 2016 BioFactors, 42(4):341-348, 2016.

  12. Hydrogen rich gas generator

    NASA Technical Reports Server (NTRS)

    Houseman, J. (Inventor)

    1976-01-01

    A process and apparatus is described for producing a hydrogen rich gas by introducing a liquid hydrocarbon fuel in the form of a spray into a partial oxidation region and mixing with a mixture of steam and air that is preheated by indirect heat exchange with the formed hydrogen rich gas, igniting the hydrocarbon fuel spray mixed with the preheated mixture of steam and air within the partial oxidation region to form a hydrogen rich gas.

  13. Molecular insights into protein synthesis with proline residues.

    PubMed

    Melnikov, Sergey; Mailliot, Justine; Rigger, Lukas; Neuner, Sandro; Shin, Byung-Sik; Yusupova, Gulnara; Dever, Thomas E; Micura, Ronald; Yusupov, Marat

    2016-12-01

    Proline is an amino acid with a unique cyclic structure that facilitates the folding of many proteins, but also impedes the rate of peptide bond formation by the ribosome. As a ribosome substrate, proline reacts markedly slower when compared with other amino acids both as a donor and as an acceptor of the nascent peptide. Furthermore, synthesis of peptides with consecutive proline residues triggers ribosome stalling. Here, we report crystal structures of the eukaryotic ribosome bound to analogs of mono- and diprolyl-tRNAs. These structures provide a high-resolution insight into unique properties of proline as a ribosome substrate. They show that the cyclic structure of proline residue prevents proline positioning in the amino acid binding pocket and affects the nascent peptide chain position in the ribosomal peptide exit tunnel. These observations extend current knowledge of the protein synthesis mechanism. They also revise an old dogma that amino acids bind the ribosomal active site in a uniform way by showing that proline has a binding mode distinct from other amino acids.

  14. Proline synthesis in barley under iron deficiency and salinity.

    PubMed

    Arias-Baldrich, Cirenia; Bosch, Nadja; Begines, Digna; Feria, Ana B; Monreal, José A; García-Mauriño, Sofía

    2015-07-01

    This work investigates proline synthesis in six barley varieties subjected to iron deficiency, salinity or both stresses. The highest growth under Fe sufficiency corresponded to Belgrano and Shakira. A moderate augment of leaf phosphoenolpyruvate carboxylase (PEPC) activity was observed in all six varieties in response to Fe deficiency, consistently in leaves and sporadically in roots. All six varieties accumulated proline under Fe deficiency, to a higher extent in leaves than in roots. The decrease of Fe supply from 100 μM NaFe(III)-EDTA to 0.5 μM NaFe(III)-EDTA reduced growth and photosynthetic pigments similarly in the six barley varieties. On the contrary, differences between varieties could be observed with respect to increased or, conversely, decreased proline content as a function of the amount of NaFe(III)-EDTA supplied. These two opposite types were represented by Belgrano (higher proline under Fe deficiency) and Shakira (higher proline under Fe sufficiency). Time-course experiments suggested that leaf PEPC activity was not directly responsible for supplying C for proline synthesis under Fe deficiency. High proline levels in the leaves of Fe-deficient Belgrano plants in salinity were associated to a better performance of this variety under these combined stresses.

  15. Microbial diversity in deep-sea sediment from the cobalt-rich crust deposit region in the Pacific Ocean.

    PubMed

    Liao, Li; Xu, Xue-Wei; Jiang, Xia-Wei; Wang, Chun-Sheng; Zhang, Dong-Sheng; Ni, Jian-Yu; Wu, Min

    2011-12-01

    Cobalt-rich crusts are important metallic mineral resources with great economic potential, usually distributed on seamounts located in the Pacific Ocean. Microorganisms are believed to play a role in the formation of crusts as well as in metal cycling. To explore the microbial diversity related to cobalt-rich crusts, 16S ribosomal RNA gene clone libraries were constructed from three consecutive sediment layers. In total, 417 bacterial clones were obtained from three bacterial clone libraries, representing 17 distinct phylogenetic groups. Proteobacteria dominated in the bacterial communities, followed by Acidobacteria and Planctomycetes. Compared with high bacterial diversity, archaea showed a remarkably low diversity, with all 137 clones belonging to marine archaeal group I except one novel euryarchaeotal clone. The microbial communities were potentially involved in sulfur, nitrogen and metal cycling in the area of cobalt-rich crusts. Sulfur oxidation and metal oxidation were potentially major sources of energy for this ecosystem. This is the first reported investigation of microbial diversity in sediments associated with cobalt-rich crusts, and it casts fresh light on the microbial ecology of these important ecosystems.

  16. Anthocyanin-rich blueberry diets enhance protection of critical brain regions exposed to acute levels of 56Fe cosmic radiation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The protective effects of anthocyanin-rich blueberries on brain health are well documented and are particularly important under conditions of high oxidative stress which can lead to “accelerated aging”. One such scenario is exposure to space radiation, which consists of high-energy and -charge parti...

  17. Independent metal-thiolate cluster formation in C-terminal Cys-rich region of a rice type 1 metallothionein isoform.

    PubMed

    Malekzadeh, Rahim; Shahpiri, Azar

    2017-03-01

    In this study we examined the independent self assembly of metal-binding in C-terminal Cys- rich region of a type 1 metallothionein (MT) isoform from rice (OsMTI-1b). To this end the N-terminal of OsMTI-1b (C-OsMTI-1b) was heterologously expressed in Escherichia coli as fusion protein with glutathione-S-transferase (GST). As compared with control (The E. coli cells containing pET41a without gene), transgenic E. coli cells expressing GST-C-OsMTI-1b accumulated more Ni(2+), Cd(2+), and Zn(2+) from culture medium and showed increased tolerance against these metals. The recombinant GST-C-OsMTI-1b was purified using affinity chromatography. According to in vitro assays the protein GST-C-OsMTI-1b was able to form complexes with Ni(2+), Cd(2+) and Zn(2+). These results demonstrate the formation of independent metal-thiolate cluster at C-terminal Cys-rich region of OsMTI-1b without participation of N-terminal Cys-rich region.

  18. Relative contributions of local and regional factors to species richness and total density of butterflies and moths in semi-natural grasslands.

    PubMed

    Pöyry, Juha; Paukkunen, Juho; Heliölä, Janne; Kuussaari, Mikko

    2009-06-01

    Metapopulation theory predicts that species richness and total population density of habitat specialists increase with increasing area and regional connectivity of the habitat. To test these predictions, we examined the relative contributions of habitat patch area, connectivity of the regional habitat network and local habitat quality to species richness and total density of butterflies and day-active moths inhabiting semi-natural grasslands. We studied butterflies and moths in 48 replicate landscapes situated in southwest Finland, including a focal patch and the surrounding network of other semi-natural grasslands within a radius of 1.5 km from the focal patch. By applying the method of hierarchical partitioning, which can distinguish between independent and joint contributions of individual explanatory variables, we observed that variables of the local habitat quality (e.g. mean vegetation height and nectar plant abundance) generally showed the highest independent effect on species richness and total density of butterflies and moths. Habitat area did not show a significant independent contribution to species richness and total density of butterflies and moths. The effect of habitat connectivity was observed only for total density of the declining butterflies and moths. These observations indicate that the local habitat quality is of foremost importance in explaining variation in species richness and total density of butterflies and moths. In addition, declining butterflies and moths have larger populations in well-connected networks of semi-natural grasslands. Our results suggest that, while it is crucial to maintain high-quality habitats by management, with limited resources it would be appropriate to concentrate grassland management and restoration to areas with well-connected grassland networks in which the declining species currently have their strongest populations.

  19. Is proline accumulation per se correlated with stress tolerance or is proline homeostasis a more critical issue?

    PubMed

    Kavi Kishor, Polavarapu B; Sreenivasulu, Nese

    2014-02-01

    Proline has been recognized as a multi-functional molecule, accumulating in high concentrations in response to a variety of abiotic stresses. It is able to protect cells from damage by acting as both an osmotic agent and a radical scavenger. Proline accumulated during a stress episode is degraded to provide a supply of energy to drive growth once the stress is relieved. Proline homeostasis is important for actively dividing cells as it helps to maintain sustainable growth under long-term stress. It also underpins the importance of the expansion of the proline sink during the transition from vegetative to reproductive growth and the initiation of seed development. Its role in the reproductive tissue is to stabilize seed set and productivity. Thus, to cope with abiotic stress, it is important to develop strategies to increase the proline sink in the reproductive tissue. We give a holistic account of proline homeostasis, taking into account the regulation of proline synthesis, its catabolism, and intra- and intercellular transport, all of which are vital components of growth and development in plants challenged by stress.

  20. Very low sound velocities in iron-rich (Mg,Fe)O : Implications for the core-mantle boundary region.

    SciTech Connect

    Wicks, J. K.; Jackson, J. M.; Sturhahn, W.; X-Ray Science Division; California Inst. of Tech.; Jet Propulsion Lab.

    2010-08-10

    The sound velocities of (Mg{sub .16}Fe{sub .84})O have been measured to 121 GPa at ambient temperature using nuclear resonant inelastic x-ray scattering. The effect of electronic environment of the iron sites on the sound velocities were tracked in situ using synchrotron Moessbauer spectroscopy. We found the sound velocities of (Mg{sub .16}Fe{sub .84})O to be much lower than those in other presumed mantle phases at similar conditions, most notably at very high pressures. Conservative estimates of the effect of temperature and dilution on aggregate sound velocities show that only a small amount of iron-rich (Mg,Fe)O can greatly reduce the average sound velocity of an assemblage. We propose that iron-rich (Mg,Fe)O be a source of ultra-low velocity zones. Other properties of this phase, such as enhanced density and dynamic stability, strongly support the presence of iron-rich (Mg,Fe)O in localized patches above the core-mantle boundary.

  1. Method Dependence of Proline Ring Flexibility in the Poly-l-Proline Type II Polymer.

    PubMed

    Cutini, Michele; Corno, Marta; Ugliengo, Piero

    2017-01-10

    We studied the sensitivity of the energetic and geometrical features of the proline ring (pyrrolidine) to the quantum mechanical computational approach by adopting the proline monomer, trimer, and polymer, as simplified collagen protein models. Within the Density Functional Theory (DFT) approach, we tested the ability of different functionals (GGA PBE and the hybrid B3LYP), added with a posteriori empirical dispersion corrections (D), to predict the conformational potential energy surface of the five-membered heterocycle pyrrolidine ring for the above models, dictating the collagen main features. We also compared the DFT-D results with those from the recently proposed cost-effective HF-3c method and our variant HF-3c-027, both based on Hartree-Fock Hamiltonian and Gaussian minimal basis set properly corrected for basis set superposition error, structure deficiencies, and dispersion interactions. We found that dispersion interactions are essential to destabilize specific conformers. While the HF-3c and its HF-3c-027 variant are unreliable to predict accurately the energy of the ring conformers, structures are accurate. Indeed, the cost-effective DFT-D//HF-3c-027 approach in which the energetic is from the accurate DFT-D method on HF-3c-027 structures provides energetic in line with that derived by the costly DFT-D//DFT-D approach, paving the way to simulate more realistic collagen models of much larger size. The adoption of either PBE or B3LYP functional for the electronic part of the DFT-D method gives very similar results, recommending the first as the most cost-effective method for simulating large collagen models. The predicted most stable conformation computed for the periodic poly proline (type II) model allows for a higher flexibility, in agreement with experimental studies on collagen protein. The present results open the way to large-scale calculations of the collagen/hydroxyapatite system, crucial for understanding the atomistic details in bones and teeth.

  2. Supramolecular architectures of N-acetyl-L-proline monohydrate and N-benzyl-L-proline.

    PubMed

    Rajalakshmi, P; Srinivasan, N; Krishnakumar, R V; Razak, Ibrahim Abdul; Rosli, Mohd Mustaqim

    2013-11-01

    The title compounds, N-acetyl-L-proline monohydrate, C7H11NO3·H2O, (I), and N-benzyl-L-proline, C12H15NO2, (II), crystallize in the monoclinic space group P21 with Z' = 1 and Z' = 2, respectively. The conformation of C(γ) with respect to the carboxylic acid group in (I) is C(γ)-exo or UP pucker, with the pyrrolidine ring twisted, while in (II), it is C(γ)-endo or DOWN, with the pyrrolidine ring assuming an envelope conformation. The crystal packing interactions in (I) are composed of two substructures, one characterized by an R6(6)(24) motif through O-H...O hydrogen bonds and the other by an R4(4)(23) ring through C-H...O interactions. In (II), the crystal packing interactions consist of N-H...O and C-H...O hydrogen bonds. Proline (Pro) exists in its neutral form in (I) and is zwitterionic in (II). This difference in the ionization states of Pro is manifested through the absence of N-H...O and presence of O-H...O interactions in (I), and the presence of N-H...O and absence of O-H...O hydrogen bonds in (II). While C-H...O interactions are present in both (I) and (II), the geometry of the synthons formed by them and their mode of participation in intermolecular interactions is different. Though the title compounds differ significantly in terms of modifications in the Pro skeleton, the differences in their supramolecular structures may also be viewed as a result of the molecular recognition facilitated by the presence of a solvent water molecule in (I) and the zwitterionic state of the amino acid in (II).

  3. Immunity related genes in dipterans share common enrichment of AT-rich motifs in their 5' regulatory regions that are potentially involved in nucleosome formation

    PubMed Central

    Hernandez-Romano, Jesus; Carlos-Rivera, Francisco J; Salgado, Heladia; Lamadrid-Figueroa, Hector; Valverde-Garduño, Veronica; Rodriguez, Mario H; Martinez-Barnetche, Jesus

    2008-01-01

    Background Understanding the transcriptional regulation mechanisms in response to environmental challenges is of fundamental importance in biology. Transcription factors associated to response elements and the chromatin structure had proven to play important roles in gene expression regulation. We have analyzed promoter regions of dipteran genes induced in response to immune challenge, in search for particular sequence patterns involved in their transcriptional regulation. Results 5' upstream regions of D. melanogaster and A. gambiae immunity-induced genes and their corresponding orthologous genes in 11 non-melanogaster drosophilid species and Ae. aegypti share enrichment in AT-rich short motifs. AT-rich motifs are associated with nucleosome formation as predicted by two different algorithms. In A. gambiae and D. melanogaster, many immunity genes 5' upstream sequences also showed NFκB response elements, located within 500 bp from the transcription start site. In A. gambiae, the frequency of ATAA motif near the NFκB response elements was increased, suggesting a functional link between nucleosome formation/remodelling and NFκB regulation of transcription. Conclusion AT-rich motif enrichment in 5' upstream sequences in A. gambiae, Ae. aegypti and the Drosophila genus immunity genes suggests a particular pattern of nucleosome formation/chromatin organization. The co-occurrence of such motifs with the NFκB response elements suggests that these sequence signatures may be functionally involved in transcriptional activation during dipteran immune response. AT-rich motif enrichment in regulatory regions in this group of co-regulated genes could represent an evolutionary constrained signature in dipterans and perhaps other distantly species. PMID:18613977

  4. Conformational studies of proline-, thiaproline- and dimethylsilaproline-containing diketopiperazines.

    PubMed

    Cavelier, Florine; Marchand, Damien; Mbassi, Patrick; Martinez, Jean; Marraud, Michel

    2006-10-01

    As proline plays an important role in biologically active peptides, many analogues of this residue have been developed to modulate the proportion of cis and trans conformers. A correlation between the pyrrolidine ring shape and structural properties of proline has been established. Diketopiperazine (DKP) is the model of choice to study the influence of the proline ring modification. In this contribution, cyclo(Gly-Pro) and two analogues cyclo(Sip-Pro) and cyclo(Thz-Pro) have been studied with proton NMR. We showed that both analogues with heteroatoms in gamma position, silicon and sulfur respectively, display a more rigid five-member ring. The usual flexibility of proline ring is restrained in both cases and only the two C(beta)-exo and C(beta)-endo conformations are observed.

  5. How reactive oxygen species and proline face stress together.

    PubMed

    Ben Rejeb, Kilani; Abdelly, Chedly; Savouré, Arnould

    2014-07-01

    Reactive oxygen species (ROS) are continuously generated as a consequence of plant metabolic processes due to incomplete reduction of O2. Previously considered to be only toxic by-products of metabolism, ROS are now known to act as second messengers in intracellular signalling cascades to trigger tolerance of various abiotic and biotic stresses. The accumulation of proline is frequently observed during the exposure of plants to adverse environmental conditions. Interestingly proline metabolism may also contribute to ROS formation in mitochondria, which play notably a role in hypersensitive response in plants, life-span extension in worms and tumor suppression in animals. Here we review current knowledge about the regulation of proline metabolism in response to environmental constraints and highlight the key role of ROS in the regulation of this metabolism. The impact of proline on ROS generation is also investigated. Deciphering and integrating these relationships at the whole plant level will bring new perspectives on how plants adapt to environmental stresses.

  6. Coral diversity and the severity of disease outbreaks: a cross-regional comparison of Acropora white syndrome in a species-rich region (American Samoa) with a species-poor region (Northwestern Hawaiian Islands).

    USGS Publications Warehouse

    Aeby, G.S.; Bourne, D.G.; Wilson, B.; Work, Thierry M.

    2011-01-01

    The dynamics of the coral disease, Acropora white syndrome (AWS), was directly compared on reefs in the species-poor region of the Northwestern Hawaiian Islands (NWHI) and the species-rich region of American Samoa (AS) with results suggesting that biodiversity, which can affect the abundance of susceptible hosts, is important in influencing the impacts of coral disease outbreaks. The diversity-disease hypothesis predicts that decreased host species diversity should result in increased disease severity of specialist pathogens. We found that AWS was more prevalent and had a higher incidence within the NWHI as compared to AS. Individual Acropora colonies affected by AWS showed high mortality in both regions, but case fatality rate and disease severity was higher in the NWHI. The site within the NWHI had a monospecific stand of A. cytherea; a species that is highly susceptible to AWS. Once AWS entered the site, it spread easily amongst the abundant susceptible hosts. The site within AS contained numerous Acropora species, which differed in their apparent susceptibility to infection and disease severity, which in turn reduced disease spread. Manipulative studies showed AWS was transmissible through direct contact in three Acropora species. These results will help managers predict and respond to disease outbreaks.

  7. Enhanced translation by Nucleolin via G-rich elements in coding and non-coding regions of target mRNAs.

    PubMed

    Abdelmohsen, Kotb; Tominaga, Kumiko; Lee, Eun Kyung; Srikantan, Subramanya; Kang, Min-Ju; Kim, Mihee M; Selimyan, Roza; Martindale, Jennifer L; Yang, Xiaoling; Carrier, France; Zhan, Ming; Becker, Kevin G; Gorospe, Myriam

    2011-10-01

    RNA-binding proteins (RBPs) regulate gene expression at many post-transcriptional levels, including mRNA stability and translation. The RBP nucleolin, with four RNA-recognition motifs, has been implicated in cell proliferation, carcinogenesis and viral infection. However, the subset of nucleolin target mRNAs and the influence of nucleolin on their expression had not been studied at a transcriptome-wide level. Here, we globally identified nucleolin target transcripts, many of which encoded cell growth- and cancer-related proteins, and used them to find a signature motif on nucleolin target mRNAs. Surprisingly, this motif was very rich in G residues and was not only found in the 3'-untranslated region (UTR), but also in the coding region (CR) and 5'-UTR. Nucleolin enhanced the translation of mRNAs bearing the G-rich motif, since silencing nucleolin did not change target mRNA stability, but decreased the size of polysomes forming on target transcripts and lowered the abundance of the encoded proteins. In summary, nucleolin binds G-rich sequences in the CR and UTRs of target mRNAs, many of which encode cancer proteins, and enhances their translation.

  8. Vertebrate Dissimilarity Due to Turnover and Richness Differences in a Highly Beta-Diverse Region: The Role of Spatial Grain Size, Dispersal Ability and Distance

    PubMed Central

    Calderón-Patrón, Jaime M.; Moreno, Claudia E.; Pineda-López, Rubén; Sánchez-Rojas, Gerardo; Zuria, Iriana

    2013-01-01

    We explore the influence of spatial grain size, dispersal ability, and geographic distance on the patterns of species dissimilarity of terrestrial vertebrates, separating the dissimilarity explained by species replacement (turnover) from that resulting from richness differences. With data for 905 species of terrestrial vertebrates distributed in the Isthmus of Tehuantepec, classified into five groups according to their taxonomy and dispersal ability, we calculated total dissimilarity and its additive partitioning as two components: dissimilarity derived from turnover and dissimilarity derived from richness differences. These indices were compared using fine (10 x 10 km), intermediate (20 x 20 km) and coarse (40 x 40 km) grain grids, and were tested for any correlations with geographic distance. The results showed that total dissimilarity is high for the terrestrial vertebrates in this region. Total dissimilarity, and dissimilarity due to turnover are correlated with geographic distance, and the patterns are clearer when the grain is fine, which is consistent with the distance-decay pattern of similarity. For all terrestrial vertebrates tested on the Isthmus of Tehuantepec both the dissimilarity derived from turnover and the dissimilarity resulting from richness differences make important contributions to total dissimilarity, and dispersal ability does not seem to influence the dissimilarity patterns. These findings support the idea that conservation efforts in this region require a system of interconnected protected areas that embrace the environmental, climatic and biogeographic heterogeneity of the area. PMID:24324840

  9. Proline improves copper tolerance in chickpea (Cicer arietinum).

    PubMed

    Singh, Vijeta; Bhatt, Indu; Aggarwal, Anjali; Tripathi, Bhumi Nath; Munjal, Ashok Kumar; Sharma, Vinay

    2010-09-01

    The present study suggests the involvement of proline in copper tolerance of four genotypes of Cicer arietinum (chickpea). Based on the data of tolerance index and lipid peroxidation, the order for copper tolerance was as follows: RSG 888 > CSG 144 > CSG 104 > RSG 44 in the selected genotypes. The basis of differential copper tolerance in chickpea genotypes was characterized by analyzing, antioxidant enzymes (superoxide dismutase, ascorbated peroxidase and catalase), phytochelatins, copper uptake, and proline accumulation. Chickpea genotypes showed stimulated superoxide dismutase activity at all tested concentrations of copper, but H(2)O(2) decomposing enzymes especially; ascorbate peroxidase did not increase with 25 and 50 microM copper treatments. Catalase activity, however, increased at lower copper concentrations but failed to stimulate at 50 microM copper. Such divergence in responses of these enzymes minimizes their importance in protecting chickpea against copper stress. The sensitive genotypes showed greater enhancement of phytochelatins than that of tolerant genotypes. Hence, the possibility of phytochelatins in improving copper tolerance in the test plant is also excluded. Interestingly, the order of proline accumulation in the chickpea genotypes (RSG 888 > CSG 144 > CSG 104 > RSG 44) was exactly similar to the order of copper tolerance. Based on hyperaccumulation of proline in tolerant genotype (RSG 44) and the reduction and improvement of lipid peroxidation and tolerance index, respectively, by proline pretreatment, we conclude that hyperaccumulation of proline improves the copper tolerance in chickpea.

  10. Proline and Glycine Betaine Influence Protein Solvation 1

    PubMed Central

    Paleg, Leslie G.; Stewart, George R.; Bradbeer, Joseph W.

    1984-01-01

    Glutamine synthetase from barley (Hordeum distichum L.) is precipitated by polyethylene glycol (PEG). Proline, in a concentration-dependent manner, reduces the amount of enzyme precipitated by PEG, although the effect of the imino acid can be counteracted by raising the level of PEG. The effect of PEG is a function of mer number and concentration and the influence of both elements can be ameliorated by proline. PEG-induced enzyme precipitation is a function of pH, as is its interaction with both proline and betaine in the reaction. The lack of effect of amount of enzyme on the proline and PEG effects supports the conclusion that, in this system, proline and PEG do not function through interaction with the protein. Other compounds, such as glycine, glucose, and sucrose, can decrease the PEG-induced precipitation of the enzyme, although glycerol was not active under the conditions employed. The results are consistent with the proposition that a protein-containing system in which high concentrations of proline and/or betaine are present, is better `protected' against the biologically unfavorable consequences of dehydration-induced thermodynamic perturbation. PMID:16663771

  11. Position of Proline Mediates the Reactivity of S-Palmitoylation.

    PubMed

    Khanal, Neelam; Pejaver, Vikas; Li, Zhiyu; Radivojac, Predrag; Clemmer, David E; Mukhopadhyay, Suchetana

    2015-11-20

    Palmitoylation, a post-translational modification in which a saturated 16-carbon chain is added predominantly to a cysteine residue, participates in various biological functions. The position of proline relative to other residues being post-translationally modified has been previously reported as being important. We determined that proline is statistically enriched around cysteines known to be S-palmitoylated. The goal of this work was to determine how the position of proline influences the palmitoylation of the cysteine residue. We established a mass spectrometry-based approach to investigate time- and temperature-dependent kinetics of autopalmitoylation in vitro and to derive the thermodynamic parameters of the transition state associated with palmitoylation; to the best of our knowledge, our work is the first to study the kinetics and activation properties of the palmitoylation process. We then used these thermochemical parameters to determine if the position of proline relative to the modified cysteine is important for palmitoylation. Our results show that peptides with proline at the -1 position of cysteine in their sequence (PC) have lower enthalpic barriers and higher entropic barriers in comparison to the same peptides with proline at the +1 position of cysteine (CP); interestingly, the free-energy barriers for both pairs are almost identical. Molecular dynamics studies demonstrate that the flexibility of the cysteine backbone in the PC-containing peptide when compared to the CP-containing peptide explains the increased entropic barrier and decreased enthalpic barrier observed experimentally.

  12. Glioma Specific Extracellular Missense Mutations in the First Cysteine Rich Region of Epidermal Growth Factor Receptor (EGFR) Initiate Ligand Independent Activation.

    PubMed

    Ymer, Susie I; Greenall, Sameer A; Cvrljevic, Anna; Cao, Diana X; Donoghue, Jacqui F; Epa, V Chandana; Scott, Andrew M; Adams, Timothy E; Johns, Terrance G

    2011-04-18

    The epidermal growth factor receptor (EGFR) is overexpressed or mutated in glioma. Recently, a series of missense mutations in the extracellular domain (ECD) of EGFR were reported in glioma patients. Some of these mutations clustered within a cysteine-rich region of the EGFR targeted by the therapeutic antibody mAb806. This region is only exposed when EGFR activates and appears to locally misfold during activation. We expressed two of these mutations (R324L and E330K) in NR6 mouse fibroblasts, as they do not express any EGFR-related receptors. Both mutants were autophosphorylated in the absence of ligand and enhanced cell survival and anchorage-independent and xenograft growth. The ECD truncation that produces the de2-7EGFR (or EGFRvIII), the most common EGFR mutation in glioma, generates a free cysteine in this same region. Using a technique optimized for detecting disulfide-bonded dimers, we definitively demonstrated that the de2-7EGFR is robustly dimerized and that ablation of the free cysteine prevents dimerization and activation. Modeling of the R324L mutation suggests it may cause transient breaking of disulfide bonds, leading to similar disulfide-bonded dimers as seen for the de2-7EGFR. These ECD mutations confirm that the cysteine-rich region of EGFR around the mAb806 epitope has a significant role in receptor activation.

  13. Glioma Specific Extracellular Missense Mutations in the First Cysteine Rich Region of Epidermal Growth Factor Receptor (EGFR) Initiate Ligand Independent Activation

    PubMed Central

    Ymer, Susie I.; Greenall, Sameer A.; Cvrljevic, Anna; Cao, Diana X.; Donoghue, Jacqui F.; Epa, V. Chandana; Scott, Andrew M.; Adams, Timothy E.; Johns, Terrance G.

    2011-01-01

    The epidermal growth factor receptor (EGFR) is overexpressed or mutated in glioma. Recently, a series of missense mutations in the extracellular domain (ECD) of EGFR were reported in glioma patients. Some of these mutations clustered within a cysteine-rich region of the EGFR targeted by the therapeutic antibody mAb806. This region is only exposed when EGFR activates and appears to locally misfold during activation. We expressed two of these mutations (R324L and E330K) in NR6 mouse fibroblasts, as they do not express any EGFR-related receptors. Both mutants were autophosphorylated in the absence of ligand and enhanced cell survival and anchorage-independent and xenograft growth. The ECD truncation that produces the de2-7EGFR (or EGFRvIII), the most common EGFR mutation in glioma, generates a free cysteine in this same region. Using a technique optimized for detecting disulfide-bonded dimers, we definitively demonstrated that the de2-7EGFR is robustly dimerized and that ablation of the free cysteine prevents dimerization and activation. Modeling of the R324L mutation suggests it may cause transient breaking of disulfide bonds, leading to similar disulfide-bonded dimers as seen for the de2-7EGFR. These ECD mutations confirm that the cysteine-rich region of EGFR around the mAb806 epitope has a significant role in receptor activation. PMID:24212795

  14. Cigarette smoke enhances chemotaxis via acetylation of proline-glycine-proline.

    PubMed

    Hardison, Matthew Thomas; Brown, Michael David; Snelgrove, Robert James; Blalock, James Edwin; Jackson, Patricia

    2012-06-01

    Several chronic lung diseases have been linked to cigarette smoking (Chronic Obstructive Pulmonary Disease (COPD), and cancer are associated with increased tobacco use). We recently described a collagen fragment, proline-glycine-proline (PGP), chemotactic for neutrophils, that appears to play a role in COPD, cystic fibrosis, and bronchiolitis obliterans syndrome. PGP can exist in either its native or acetylated form (NAcPGP), although the mechanism of N-terminal-acetylation remains unknown. This work investigates the possibility that cigarette smoke (CS) and its components acetylate PGP, describing a possible mechanism for some of the chronic inflammation seen in tobacco-associated disease. CSE and CSC (3.56 and 12.38 ng/ml NAcPGP respectively, p less than 0.01) and its components (acrolein, acetaldehyde, and methyl glyoxal) acetylated PGP (0.51, 1.03, and 0.23 ng/ml NAcPGP, p less than 0.01). Both N-acetyl-cysteine and carbocysteine (scavengers of reactive aldehydes) blocked chemical acetylation of PGP by CS (100 percent and 97 percent inhibition, respectively, p less than 0.01). NAcPGP is more chemoattractive to neutrophils, and less susceptible to degradation by Leukotriene-A4-Hydrolase (detected in the lung). These experiments propose a mechanism for the increased neutrophil recruitment seen in smoking-associated lung diseases.

  15. Probing the "additive effect" in the proline and proline hydroxamic acid catalyzed asymmetric addition of nitroalkanes to cyclic enones.

    PubMed

    Hanessian, Stephen; Govindan, Subramaniyan; Warrier, Jayakumar S

    2005-11-01

    The effect of chirality and steric bulk of 2,5-disubstituted piperazines as additives in the conjugate addition of 2-nitropropane to cyclohexenone, catalyzed by l-proline, was investigated. Neither chirality nor steric bulk affects the enantioselectivity of addition, which gives 86-93% ee in the presence of achiral and chiral nonracemic 2,5-disubstituted piperazines. Proline hydroxamic acid is shown for the first time to be an effective organocatalyst in the same Michael reaction.

  16. A Conserved Active Site Tyrosine Residue of Proline Dehydrogenase Helps Enforce the Preference for Proline over Hydroxyproline as the Substrate

    SciTech Connect

    Ostrander, E.L.; Larson, J.D.; Schuermann, J.P.; Tanner, J.J.

    2009-03-02

    Proline dehydrogenase (PRODH) catalyzes the oxidation of L-proline to {Delta}-1-pyrroline-5-carboxylate. PRODHs exhibit a pronounced preference for proline over hydroxyproline (trans-4-hydroxy-L-proline) as the substrate, but the basis for specificity is unknown. The goal of this study, therefore, is to gain insight into the structural determinants of substrate specificity of this class of enzyme, with a focus on understanding how PRODHs discriminate between the two closely related molecules, proline and hydroxyproline. Two site-directed mutants of the PRODH domain of Escherichia coli PutA were created: Y540A and Y540S. Kinetics measurements were performed with both mutants. Crystal structures of Y540S complexed with hydroxyproline, proline, and the proline analogue L-tetrahydro-2-furoic acid were determined at resolutions of 1.75, 1.90, and 1.85 {angstrom}, respectively. Mutation of Tyr540 increases the catalytic efficiency for hydroxyproline 3-fold and decreases the specificity for proline by factors of 20 (Y540S) and 50 (Y540A). The structures show that removal of the large phenol side chain increases the volume of the substrate-binding pocket, allowing sufficient room for the 4-hydroxyl of hydroxyproline. Furthermore, the introduced serine residue participates in recognition of hydroxyproline by forming a hydrogen bond with the 4-hydroxyl. This result has implications for understanding the substrate specificity of the related enzyme human hydroxyproline dehydrogenase, which has serine in place of tyrosine at this key active site position. The kinetic and structural results suggest that Tyr540 is an important determinant of specificity. Structurally, it serves as a negative filter for hydroxyproline by clashing with the 4-hydroxyl group of this potential substrate.

  17. Methionine-rich repeat proteins: a family of membrane-associated proteins which contain unusual repeat regions.

    PubMed

    Weiss, Jamie L; Evans, Nicholas A; Ahmed, Tanweer; Wrigley, Jonathan D J; Khan, Shukria; Wright, Charles; Keen, Jeffrey N; Holzenburg, Andreas; Findlay, John B C

    2005-03-01

    We report the protein isolation, cloning and characterization of members of an unusual protein family, which comprise the most abundant proteins present in the squid eye. The proteins in this family have a range of molecular weights from 32 to 36 kDa. Electron microscopy and detergent solubilization demonstrate that these proteins are tightly associated with membrane structures where they may form tetramers. Despite this, these proteins have no stretches of hydrophobic residues that could form typical transmembrane domains. They share an unusual protein sequence rich in methionine, and contain multiple repeating motifs. We have therefore named these proteins Methionine-Rich Repeat Proteins (MRRPs). The use of structure prediction algorithms suggest very little recognized secondary structure elements. At the time of cloning no sequence or structural homologues have been found in any database. We have isolated three closely related cDNA clones from the MRRP family. Coupled in vitro transcription/translation of the MRRP clones shows that they encode proteins with molecular masses similar to components of native MRRPs. Immunoblot analysis of these proteins reveals that they are also present in squid brain, optic lobe, and heart, and also indicate that MRRP-like protein motifs may also exist in mammalian tissues. We propose that MRRPs define a family of important proteins that have an unusual mode of attachment or insertion into cell membranes and are found in evolutionarily diverse organisms.

  18. The Importance of the KR-Rich Region of the Coat Protein of Ourmia melon virus for Host Specificity, Tissue Tropism, and Interference With Antiviral Defense.

    PubMed

    Rossi, Marika; Vallino, Marta; Abbà, Simona; Ciuffo, Marina; Balestrini, Raffaella; Genre, Andrea; Turina, Massimo

    2015-01-01

    The N-terminal region of the Ourmia melon virus (OuMV) coat protein (CP) contains a short lysine/arginine-rich (KR) region. By alanine scanning mutagenesis, we showed that the KR region influences pathogenicity and virulence of OuMV without altering viral particle assembly. A mutant, called OuMV6710, with three basic residue substitutions in the KR region, was impaired in the ability to maintain the initial systemic infection in Nicotiana benthamiana and to infect both cucumber and melon plants systemically. The integrity of this protein region was also crucial for encapsidation of viral genomic RNA; in fact, certain mutations within the KR region partially compromised the RNA encapsidation efficiency of the CP. In Arabidopsis thaliana Col-0, OuMV6710 was impaired in particle accumulation; however, this phenotype was abolished in dcl2/dcl4 and dcl2/dcl3/dcl4 Arabidopsis mutants defective for antiviral silencing. Moreover, in contrast to CPwt, in situ immunolocalization experiments indicated that CP6710 accumulates efficiently in the spongy mesophyll tissue of infected N. benthamiana and A. thaliana leaves but only occasionally infects palisade tissues. These results provided strong evidence of a crucial role for OuMV CP during viral infection and highlighted the relevance of the KR region in determining tissue tropism, host range, pathogenicity, and RNA affinity, which may be all correlated with a possible CP silencing-suppression activity.

  19. The maturation rate of girls living in rich and poor rural regions of Poland before and after the transformation of 1989.

    PubMed

    Laska-Mierzejewska, T; Olszewska, E

    2004-01-01

    The aim of this study was to find out whether the political and economic system transformation in Poland in 1989 had an influence on the rate of maturation of girls from various categories of the rural population in two regions, differing in wealth. The stratification of the villagers was based on the source of the family income (farmers, farmer-workers and non-farmers), on parents' education, and on the number of children per family. The age at menarche (AM) was used as biological indicator of living conditions. Rural girls aged 9.5-18.5 years were studied in 1987 in Leszno (L), a rich region (n = 2049), and in 1989 in Suwałki (S), a poor region (n = 2077). The study was repeated in 2001 in the same villages, the numbers of subjects amounting to 2440 and 2122, respectively. In the 1980s, AM in regions L and S amounted to 13.18 and 13.88 years, respectively, and was strongly related to social stratification. The earliest AM was found in daughters of non-farmers and the latest in the girls from farmers' families. In 2001, the acceleration in AM amounted to 0.14 years in the rich region and to 0.46 years in the poor region. The percentage of families owning a car, freezer, colour TV and automatic washing machine markedly increased and the education of parents improved. Such results are indicative of an improvement in the living standard of the groups investigated, but the villagers regard themselves as the losers as a result of the system transformation in Poland. Since the Polish population is considered as ethnically homogenous, the great differences in AM within the rural population, as well as the differences between the inhabitants of villages and large cities should be regarded as biological effects of social inequalities. Such differences indicate that Polish rural girls are far below the genetic potential for menarcheal age.

  20. Effects of three stabilizing agents--proline, betaine, and trehalose--on membrane phospholipids.

    PubMed

    Rudolph, A S; Crowe, J H; Crowe, L M

    1986-02-15

    We have studied the interaction between three compounds which accumulate in organisms under hydration stress--proline, betaine, and trehalose--and the membrane phospholipids dimyristoylphosphatidylcholine (DMPC), palmitoyloleoylphosphatidylcholine (POPC), and dimyristoylphosphatidylethanolamine in bulk solution. Film balance studies reveal that these compounds increase the area/molecule of these lipids. Differential scanning calorimetry has been employed to investigate the effect these agents have on the gel-to-liquid crystalline phase transition of multilamellar and small unilamellar vesicles of DMPC, dipalmitoylphosphatidylcholine, and POPC:phosphatidylserine (90:10 mole ratio) in bulk solution. In the presence of 1 M proline, trehalose, or betaine, the midtransition temperature in small unilamellar vesicles is reduced (up to 7 degrees C in 1 M trehalose), and the transition broadened. In contrast, multilamellar vesicles of similar lipid composition show an increased transition temperature in the presence of the same concentration of these compounds. This result suggests that the inner lamellae in multilamellar vesicles may be dehydrated with only a few outer lamellae exposed to the protective compound. Finally, we have used stereomodels of phosphatidylcholine to investigate the mechanism of action of these agents. Hydrogen bonding of trehalose to the head group region results in an increase in the distance between head groups of 6.9 A. This amount of spreading compares well with data from the monolayer experiments which indicate that maximal spreading of DMPC monolayers by trehalose is 6.5 A. Molecular models of proline and betaine have also been constructed, and these models suggest potential interactions between these compounds and phosphatidylcholines. For the amphipath proline, this interaction may involve intercalation between phospholipid head groups.

  1. Evaluation of binding selectivity of a polyamide probe to single base-pair different DNA in A.T-rich region by electrospray ionization mass spectrometry.

    PubMed

    Li, Huihui; Yuan, Gu

    2006-12-01

    In this study, electrospray ionization mass spectrometry (ESI-MS) was used for the evaluation of the binding selectivity of a polyamide probe to single-base pair different DNA in an A.T-rich region. In this procedure, DeltaIr(dsn) was introduced as a parameter to compare the binding affinities of the polyamides with the duplex DNA. The results show that ESI-MS is a very useful tool for analysis of binding selectivity of a polyamide probe to single-base pair different DNA.

  2. Requirement of proline synthesis during Arabidopsis reproductive development

    PubMed Central

    2012-01-01

    Background Gamete and embryo development are crucial for successful reproduction and seed set in plants, which is often the determining factor for crop yield. Proline accumulation was largely viewed as a specific reaction to overcome stress conditions, while recent studies suggested important functions of proline metabolism also in reproductive development. Both the level of free proline and proline metabolism were proposed to influence the transition to flowering, as well as pollen and embryo development. Results In this study, we performed a detailed analysis of the contribution of individual proline biosynthetic enzymes to vegetative development and reproductive success in Arabidopsis. In contrast to previous reports, we found that pyrroline-5-carboxylate (P5C) synthetase 2 (P5CS2) is not essential for sexual reproduction although p5cs2 mutant plants were retarded in vegetative development and displayed reduced fertility under long-day conditions. Single mutant plants devoid of P5CS1 did not show any developmental defects. Simultaneous absence of both P5CS isoforms resulted in pollen sterility, while fertile egg cells could still be produced. Expression of P5C reductase (P5CR) was indispensable for embryo development but surprisingly not needed for pollen or egg cell fertility. The latter observation could be explained by an extreme stability of P5CR activity, which had a half-life time of greater than 3 weeks in vitro. Expression of P5CR-GFP under the control of the endogenous P5CR promoter was able to restore growth of homozygous p5cr mutant embryos. The analysis of P5CR-GFP-fluorescence in planta supported an exclusively cytoplasmatic localisation of P5CR. Conclusions Our results demonstrate that potential alternative pathways for proline synthesis or inter-generation transfer of proline are not sufficient to overcome a defect in proline biosynthesis from glutamate during pollen development. Proline biosynthesis through P5CS2 and P5CR is limiting for

  3. Catabolism of proline by procyclic culture forms of Trypanosoma congolense.

    PubMed

    Obungu, V H; Kiaira, J K; Njogu, R M; Olembo, N K

    1999-05-01

    The effect of various metabolic inhibitors on the rate of oxygen consumption by procyclic culture forms of Trypanosoma congolense utilizing proline as substrate was investigated. Cyanide inhibited the rate of oxygen consumption by 81.0 +/- 6.7%, malonate inhibited the rate by 51.6 +/- 1.6% and Antimycin A by 73.1 +/- 5.9%. A combination of cyanide and malonate inhibited the rate of oxygen consumption by 84.9 +/- 6.7% while a combination of antimycin A and malonate inhibited the rate by 81.6 +/- 7.6%. Rotenone had no effect on the rate of respiration except when the intact cells were first permeabilized by digitonin after which rotenone decreased the rate of respiration by 20-30%. Salicylhydroxamate (SHAM) did not have any effect on the rate of oxygen consumption. Enzymes involved in the catabolism of proline with high activities were: proline dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, fumarase, NADP-linked malic enzyme, alanine aminotransferase and malate dehydrogenase. Activities of 1-pyrroline-5 carboxylate dehydrogenase, glutamate dehydrogenase, aspartate aminotransferase and NAD-linked malic enzyme were detectable but lower. The end products of proline catabolism were alanine and glutamate. Unlike the case in Trypanosoma brucei brucei aspartate was not detected. Possible pathways of proline catabolism in procyclic culture forms of T. congolense and of electron transfer are proposed.

  4. Differences between high-affinity forskolin binding sites in dopamine-riche and other regions of rat brain

    SciTech Connect

    Poat, J.A.; Cripps, H.E.; Iversen, L.L.

    1988-05-01

    Forskolin labelled with (/sup 3/H) bound to high- and low-affinity sites in the rat brain. The high-affinity site was discretely located, with highest densities in the striatum, nucleus accumbens, olfactory tubercule, substantia nigra, hippocampus, and the molecular layers of the cerebellum. This site did not correlate well with the distribution of adenylate cyclase. The high-affinity striatal binding site may be associated with a stimulatory guanine nucleotide-binding protein. Thus, the number of sites was increased by the addition of Mg/sup 2 +/ and guanylyl imidodiphosphate. Cholera toxin stereotaxically injected into rat striatum increased the number of binding sites, and no further increase was noted following the subsequent addition of guanyl nucleotide. High-affinity forskolin binding sites in non-dopamine-rich brain areas (hippocampus and cerebullum) were modulated in a qualitatively different manner by guanyl nucleotides. In these areas the number of binding sites was significantly reduced by the addition of guanyl nucleotide. These results suggest that forskolin may have a potential role in identifying different functional/structural guanine nucleotide-binding proteins.

  5. Proline Sulfonamide-Based Organocatalysis: Better Late than Never

    PubMed Central

    Yang, Hua; Carter, Rich G.

    2011-01-01

    The history of proline sulfonamides for use in catalyzing highly enantioselective and diastereoselective C-C bond forming reactions is described. Highlighted is the development of N-(p-dodecylphenylsulfonyl)-2-pyrrolidinecarboxamide (“Hua Cat”) and N-(carboxy-p-dodecylphenylsulfonyl)-2-pyrrolidinecarboxamide (“Hua Cat-II”) by Yang and Carter. Specific reactions covered include the aldol reaction, Mannich reaction, formal aza-Diels-Alder reaction, tandem Michael/Mannich reaction and Yamada-Otani reaction. The ability of proline aryl sulfonamides to construct all-carbon quaternary stereocenters in high enantioselectivity and diastereoselectivity is a notable feature of the reported work. The practicality of this chemistry for large scale and industrial applications is also included. Finally, a discussion of the future directions of proline sulfonamide organocatalysis is provided. PMID:21461132

  6. Modeling Complex Groundwater Systems at a Regional Scale: from a "Data-poor" Paradigm to a "Data-rich" Paradigm

    NASA Astrophysics Data System (ADS)

    Li, S.; Liao, H.

    2008-05-01

    Data limitation is probably the most significant challenge in groundwater modeling, especially at a regional scale. In this paper, we explore the use of recently-assembled statewide groundwater databases by the Michigan Department of Environmental Quality for the purpose of groundwater modeling. The databases contain virtually all data needed for typical regional scale flow simulations. In particular, the databases provide data needed for defining physical/hydraulic model boundaries (e.g., watershed delineations, streams, lakes, static water levels, land systems, DEM, lithologies), aquifer elevations (e.g., DEMs, lithologies), aquifer properties (e.g., specific capacities, aquifer test data, lithologies), aquifer stresses (e.g., rainfall, estimated recharge, streams, lakes, wetlands, drains, and water wells). The databases also provide information that can be used to define existing and potential contamination sources (e.g., industrial sites, landfills, underground storage tanks) and data needed for regional scale model calibration (e.g., static water levels, aquifer tests, stream flows, estimated baseflows). One major advantage of these data lies in that they are of a high resolution and available for "free" virtually in any geographic areas in Michigan. The data on aquifer properties, static water levels, and lithologies are particularly detailed and accumulate over time. The databases are seamlessly integrated with the GIS-enabled, hierarchical Interactive Ground Water modeling software system and necessary modeling data can be extracted and processed through built-in statistical and geostatistical filters and utility tools. Here we introduce the GIS-enabled Michigan Groundwater modeling system, with emphasis on its statistical data analysis and iterative hierarchical downscaling and upscaling capabilities, and evaluate systematically the effectiveness of the new statewide groundwater databases for regional-scale groundwater modeling.

  7. Modeling Complex Groundwater Systems at a Regional Scale: From a "Data-poor" Paradigm to a "Data-rich" Paradigm

    NASA Astrophysics Data System (ADS)

    Oztan, M.; Abbas, H.; Li, S.

    2006-12-01

    Data limitation is probably the most significant challenge in groundwater modeling, especially at regional scale. In this paper, we explore the use of a recently-assembled statewide groundwater database by Michigan Department of Environmental Quality for the purpose of groundwater modeling. The database contains virtually all data needed for typical regional scale flow simulations. In particular, the database provides data needed for defining model domain (e.g., watershed delineations, DEM, lithologies), aquifer framework (e.g., DEM, lithologies), aquifer properties (e.g., specific capacities, lithologies), aquifer stresses (e.g., recharge, streams, lakes, wetlands, drains, and wells). The database also provides information that can be used to define existing and potential contamination sources (e.g., industrial sites, landfills, underground storage tanks) and data needed for regional scale model calibration (e.g., water levels and baseflows). The advantage of these data lies in that they are of high resolution and available now for "free" virtually in any geographic areas in Michigan. The data on aquifer properties, water levels, and lithologies are currently available at more than 300,000 well locations across the state, with a particularly good resolution in areas where there are more groundwater problems. The database is seamlessly integrated with the GIS-enabled Interactive Groundwater (IGW) modeling software. The disadvantage lies in that some of these data are of lower quality than traditional measured data and in some cases they are indirectly derived. In this paper, we evaluate systematically how effective this type of data is for groundwater modeling and at what scale it is effective. We demonstrate this through a systematic comparative modeling exercise at a well-characterized watershed where there exists significant amount of traditional monitoring data.

  8. Genetic and epigenetic regulation of AHR gene expression in MCF-7 breast cancer cells: role of the proximal promoter GC-rich region

    PubMed Central

    Englert, Neal A.; Turesky, Robert J.; Han, Weiguo; Bessette, Erin E.; Spivack, Simon D.; Caggana, Michele; Spink, David C.; Spink, Barbara C.

    2014-01-01

    The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, contributes to carcinogenesis through its role in the regulation of cytochrome P450 1 (CYP1)-catalyzed metabolism of carcinogens. Here, we investigated genetic and epigenetic mechanisms that affect AhR expression. Analyses of the human AHR proximal promoter in MCF-7 human breast cancer cells using luciferase assays and electrophoretic mobility shift assays revealed multiple specificity protein (Sp) 1 binding sequences that are transcriptional activators in vitro. The regulation of AhR expression was evaluated in long-term estrogen exposed (LTEE) MCF-7 cells, which showed increased AhR expression, enhanced CYP1 inducibility, and increased capacity to form DNA adducts when exposed to the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. The increased AhR expression in LTEE cells was found not to result from increased mRNA stability, differential RNA processing, or decreased DNA methylation. Analysis of the AHR proximal promoter region using chromatin immunoprecipitation confirmed that enhanced expression of AhR in LTEE cells involves changes in histone modifications, notably decreased trimethylation of histone 3, lysine 27. Upon further examination of the GC-rich Sp1-binding region, we confirmed that it contains a polymorphic (GGGGC)n repeat. In a population of newborns from New York State, the allele frequency of (GGGGC)n was n = 4>5≫6, 2. Circular dichroism spectroscopy revealed the ability of sequences of this GC-rich region to form guanine-quadruplex structures in vitro. These studies revealed multiple levels at which AhR expression may be controlled, and offer additional insights into mechanisms regulating AhR expression that can ultimately impact carcinogenesis. PMID:22728919

  9. Genetic and epigenetic regulation of AHR gene expression in MCF-7 breast cancer cells: role of the proximal promoter GC-rich region.

    PubMed

    Englert, Neal A; Turesky, Robert J; Han, Weiguo; Bessette, Erin E; Spivack, Simon D; Caggana, Michele; Spink, David C; Spink, Barbara C

    2012-09-01

    The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, contributes to carcinogenesis through its role in the regulation of cytochrome P450 1 (CYP1)-catalyzed metabolism of carcinogens. Here, we investigated genetic and epigenetic mechanisms that affect AhR expression. Analyses of the human AHR proximal promoter in MCF-7 human breast cancer cells using luciferase assays and electrophoretic mobility shift assays revealed multiple specificity protein (Sp) 1 binding sequences that are transcriptional activators in vitro. The regulation of AhR expression was evaluated in long-term estrogen exposed (LTEE) MCF-7 cells, which showed increased AhR expression, enhanced CYP1 inducibility, and increased capacity to form DNA adducts when exposed to the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. The increased AhR expression in LTEE cells was found not to result from increased mRNA stability, differential RNA processing, or decreased DNA methylation. Analysis of the AHR proximal promoter region using chromatin immunoprecipitation confirmed that enhanced expression of AhR in LTEE cells involves changes in histone modifications, notably decreased trimethylation of histone 3, lysine 27. Upon further examination of the GC-rich Sp1-binding region, we confirmed that it contains a polymorphic (GGGGC)(n) repeat. In a population of newborns from New York State, the allele frequency of (GGGGC)(n) was n = 4 > 5 ≫ 6, 2. Circular dichroism spectroscopy revealed the ability of sequences of this GC-rich region to form guanine-quadruplex structures in vitro. These studies revealed multiple levels at which AhR expression may be controlled, and offer additional insights into mechanisms regulating AhR expression that can ultimately impact carcinogenesis.

  10. The lipid-rich core region of human atherosclerotic fibrous plaques. Prevalence of small lipid droplets and vesicles by electron microscopy.

    PubMed Central

    Guyton, J. R.; Klemp, K. F.

    1989-01-01

    Abundant extracellular lipid deposits are associated with cell necrosis and tissue weakening in the core region of human atherosclerotic fibrous plaques. The ultrastructural morphology of the core region, previously undefined because of lipid extraction artifacts, was studied with the aid of new osmium-thiocarbohydrazide-osmium and osmium-tannic acid-paraphenylenediamine sequences for tissue processing. Small droplets of neutral lipid (30 to 400 nm profile diameter) and lipid vesicles with aqueous centers accounted for more than 90% of the area occupied by lipid-rich structures in the core region. No foam cells were present. Cholesterol crystals, lipid droplets of a size similar to those in foam cells (0.4 to 6 mu), and larger neutral lipid deposits (greater than 6 mu) together occupied less than 10% of the total area of lipid structures. Abundant lipid vesicles were associated with the nearby presence of cholesterol crystals, whereas small lipid droplets were predominant in areas without crystals. Many droplets had surface defects in the form of pits and vesicular blebs. These morphologic findings are explained most concisely by postulating direct accumulation of extracellular lipid from interstitial lipoproteins as a major process in core region formation. Moreover, a dynamic state of ongoing physical/metabolic transformation of extracellular lipid deposits is suggested. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 7 Figure 8 Figure 9 PMID:2646938

  11. High-throughput engineering of a mammalian genome reveals building principles of methylation states at CG rich regions.

    PubMed

    Krebs, Arnaud R; Dessus-Babus, Sophie; Burger, Lukas; Schübeler, Dirk

    2014-09-26

    The majority of mammalian promoters are CpG islands; regions of high CG density that require protection from DNA methylation to be functional. Importantly, how sequence architecture mediates this unmethylated state remains unclear. To address this question in a comprehensive manner, we developed a method to interrogate methylation states of hundreds of sequence variants inserted at the same genomic site in mouse embryonic stem cells. Using this assay, we were able to quantify the contribution of various sequence motifs towards the resulting DNA methylation state. Modeling of this comprehensive dataset revealed that CG density alone is a minor determinant of their unmethylated state. Instead, these data argue for a principal role for transcription factor binding sites, a prediction confirmed by testing synthetic mutant libraries. Taken together, these findings establish the hierarchy between the two cis-encoded mechanisms that define the DNA methylation state and thus the transcriptional competence of CpG islands.

  12. Deconvolution of mixtures with high plagioclase content for the remote interpretation of lunar plagioclase-rich regions

    NASA Astrophysics Data System (ADS)

    Serventi, Giovanna; Carli, Cristian; Sgavetti, Maria

    2016-07-01

    Anorthositic rocks are widespread on the lunar surface and have probably been formed by flotation of PL over a magma ocean. A large portion of pristine rocks are characterized by a low Mg/(Mg+Fe) ratio, and have been classified as ferroan anorthosite, and recently, after observation from SELENE Spectral Profiler,pure anorthosites regions with more than 98% PL have been recognized. In this paper, we analyze a set of mixtures with PL content similar to the ferroan anorthosites and to the pure anorthosite regions, using the Origin Software and the Modified Gaussian Model. We consider three plagioclases with varying FeOwt% contents (PL1, PL2 and PL3)andthree mafic end-members (1) 100% orthopyroxene, (2) 56% orthopyroxene and 44% clinopyroxene, and (3) 100% olivine (OL). The spectral parameters considered here are: band depth, band center, band width, c0 (the continuum intercept) and c1 (the continuum offset). Here we have shown that in pyroxene (PX)-bearing mixtures, the PX is distinguishable even in mixtures with only 1% PX and that PX band at ca. 900 nm is always deeper than PL1 band while PL2 and PL3 are deeperthan OPX 900 nm band from 95, 96% PL. In OL-bearing mixtures, OL detection limit is 2% when mixed with PL1, and 3% and 4% if mixed with PL2 and PL3. We also demonstrated how spectral parameters vary with PL%, and, generally, increasing the PL content: (1) 1250 nm band depth decreases when mixed with OL, while it deepens in mixtures with PX; (2) 1250 nm band centers generally move towards longer wavelength for PL1-bearing mixtures, while do not show significant variations considering PL2/PL3-mixtures; (3) 1250 nm band width of PL1 in E1 and E5-mixtures substantially widens while in other mixtures it only slightly varies. Here we also proposed an application to a real case, from Proclus crater, revealing how studying terrestrial analogues is fundamental to infer hypothesis on the mineralogical composition of a planetary surface, but also how the spectral

  13. The TES Hematite-Rich Region in Sinus Meridiani: A Proposed Landing Site for the 2003 Rover

    NASA Technical Reports Server (NTRS)

    Christensen, Philip R.; Bandfield, Joshua; Hamilton, Victoria; Ruff, Steven; Morris, Richard; Lane, Melissa; Malin, Michael

    2001-01-01

    The Thermal Emission Spectrometer (TES) instrument on the Mars Global Surveyor (MGS) mission has identified an accumulation of crystalline hematite (alpha-Fe2O3) that covers an area with very sharp boundaries approximately 350 by 750 km in size centered near 2 S latitude between 0 and 8 W longitude (Sinus Meridiani). The depth and shape of the hematite fundamental bands in the TES spectra show that the hematite is relatively coarse grained (greater than 5-10 micrometers). The spectrally-derived areal abundance of hematite varies with particle size from approximately 10% for particles greater than 30 micrometers in diameter to 40-60% for unpacked 10 micrometer powders. The hematite in Sinus Meridiani is thus distinct from the fine-grained (diameter less than 5-10 micrometers), red, crystalline hematite considered, on the basis of visible and near-IR data, to be a minor spectral component in Martian bright regions. A global map of the hematite abundance has been constructed using TES data from the MGS mapping mission.

  14. Randomized Subspace Learning for Proline Cis-Trans Isomerization Prediction.

    PubMed

    Al-Jarrah, Omar Y; Yoo, Paul D; Taha, Kamal; Muhaidat, Sami; Shami, Abdallah; Zaki, Nazar

    2015-01-01

    Proline residues are common source of kinetic complications during folding. The X-Pro peptide bond is the only peptide bond for which the stability of the cis and trans conformations is comparable. The cis-trans isomerization (CTI) of X-Pro peptide bonds is a widely recognized rate-limiting factor, which can not only induces additional slow phases in protein folding but also modifies the millisecond and sub-millisecond dynamics of the protein. An accurate computational prediction of proline CTI is of great importance for the understanding of protein folding, splicing, cell signaling, and transmembrane active transport in both the human body and animals. In our earlier work, we successfully developed a biophysically motivated proline CTI predictor utilizing a novel tree-based consensus model with a powerful metalearning technique and achieved 86.58 percent Q2 accuracy and 0.74 Mcc, which is a better result than the results (70-73 percent Q2 accuracies) reported in the literature on the well-referenced benchmark dataset. In this paper, we describe experiments with novel randomized subspace learning and bootstrap seeding techniques as an extension to our earlier work, the consensus models as well as entropy-based learning methods, to obtain better accuracy through a precise and robust learning scheme for proline CTI prediction.

  15. Purification and properties of proline reductase from Clostridium sticklandii.

    PubMed

    Seto, B; Stadtman, T C

    1976-04-25

    Proline reductase of Clostridium sticklandii is a membrane-bound protein and is released by treatment with detergents. The enzyme has been purified to homogeneity and is estimated by gel filtration and sedimentation equilibrium centrifugation to have a molecular weight of 298,000 to 327,000. A minimum molecular weight of 30,000 to 31,000 was calculated on the basis of sodium dodecyl sulfate-acrylamide gel electrophoresis and amino acid composition. Amino acid analysis showed a preponderance of acidic amino acids. No tryptophan was detected in the protein either spectrophotometrically or by amino acid analysis. A total of 20 sulfhydryl groups measured by titration of the reduced protein with 5,5'-dithiobis(2-nitrobenzoic acid) is in agreement with 20 cystic acid residues determined in hydrolysates of performic acid-oxidized protein. No molybdenum, iron, or selenium was found in the pure protein. Although NADH is the physiological electron donor for the proline reductase complex, the purified 300,000 molecular weight reductase component is inactive in the presence of NADH in vitro. Dithiothreitol, in contrast, can serve as electron donor both for unpurified (putative proline reductase complex) and purified proline reductase in vitro.

  16. SSR Markers Associated with Proline in Drought Tolerant Wheat Germplasm.

    PubMed

    Iqbal, Muhammad Javid; Maqsood, Yasir; Abdin, Zain Ul; Manzoor, Atif; Hassan, Muhammad; Jamil, Amer

    2016-03-01

    Water stress causes major agricultural loss throughout the world as survival of the crops remained under stress and loss in yield. Plants respond to drought stress by means of different adaptive mechanisms such as accumulation of osmoprotectants to counteract the water stress. Amino acid proline is known to occur widely in higher plants and normally accumulates in large quantities as an osmolyte in response to environmental stresses. Biochemical estimation of proline was done in the drought-affected wheat genotypes by spectrophotometric method. Proline promoted a positive effect as root/shoot ratio was enhanced in wheat germplasm under drought stress. SSR primer pairs (45) were tested for polymorphism among selected wheat genotypes. The dendrogram results have shown the wheat genotype association with the levels of proline during induced drought stress. The relationship between pattern of drought responsive biochemical attributes and DNA markers in the selected wheat genotypes was recognized to select drought tolerant genotypes for sowing in drought affected areas of the country.

  17. Novel proline-hydroxyproline glycopeptides from the dandelion (Taraxacum officinale Wigg.) flowers: de novo sequencing and biological activity.

    PubMed

    Astafieva, Alexandra A; Enyenihi, Atim A; Rogozhin, Eugene A; Kozlov, Sergey A; Grishin, Eugene V; Odintsova, Tatyana I; Zubarev, Roman A; Egorov, Tsezi A

    2015-09-01

    Two novel homologous peptides named ToHyp1 and ToHyp2 that show no similarity to any known proteins were isolated from Taraxacum officinale Wigg. flowers by multidimensional liquid chromatography. Amino acid and mass spectrometry analyses demonstrated that the peptides have unusual structure: they are cysteine-free, proline-hydroxyproline-rich and post-translationally glycosylated by pentoses, with 5 carbohydrates in ToHyp2 and 10 in ToHyp1. The ToHyp2 peptide with a monoisotopic molecular mass of 4350.3Da was completely sequenced by a combination of Edman degradation and de novo sequencing via top down multistage collision induced dissociation (CID) and higher energy dissociation (HCD) tandem mass spectrometry (MS(n)). ToHyp2 consists of 35 amino acids, contains eighteen proline residues, of which 8 prolines are hydroxylated. The peptide displays antifungal activity and inhibits growth of Gram-positive and Gram-negative bacteria. We further showed that carbohydrate moieties have no significant impact on the peptide structure, but are important for antifungal activity although not absolutely necessary. The deglycosylated ToHyp2 peptide was less active against the susceptible fungus Bipolaris sorokiniana than the native peptide. Unique structural features of the ToHyp2 peptide place it into a new family of plant defense peptides. The discovery of ToHyp peptides in T. officinale flowers expands the repertoire of molecules of plant origin with practical applications.

  18. Mutagenic analysis of herpes simplex virus type 1 glycoprotein L reveals the importance of an arginine-rich region for function

    SciTech Connect

    Klyachkin, Yuri M.; Geraghty, Robert J.

    2008-04-25

    Herpes simplex virus type 1 (HSV-1) glycoproteins H and L (gH and gL) are required for virus-induced membrane fusion. Expression of gH at the virion or infected cell surface is mediated by the chaperone-like activity of gL. We have previously shown that a region between amino acids 155 and 161 is critical for gL chaperone-like activity. Here, we conducted Ala substitution mutagenesis of residues in this region and found that substitution of Cys160, Arg156, Arg158, or Arg156/158/159 with Ala resulted in a gL mutant that bound gH but displayed a reduced ability in gH trafficking and membrane fusion. Substitution of Arg156 with another positively charged amino acid, Lys, restored function. Substitution of Arg158 with Lys restored function in gH trafficking and cell fusion but not virus entry. These results indicate that an arginine-rich region of gL is critical for function.

  19. The relationship of meteorological patterns with changes in floristic richness along a large elevational gradient in a seasonally dry region of southern Mexico.

    PubMed

    Salas-Morales, Silvia H; Meave, Jorge A; Trejo, Irma

    2015-12-01

    Globally, climate is a fundamental driver of plant species' geographical distributions, yet we still lack a good understanding of climatic variation on tropical mountains and its consequences for elevational floristic patterns. In a seasonally dry region of southern Mexico, we analysed meteorological patterns along a large elevational gradient (0-3670 m a.s.l.) and examined their relationship with changes in floristic richness. Meteorological patterns were characterised using two data sources. First, climatic information was extracted from cartography and records from a few existing meteorological stations. Additionally, air temperature and humidity were recorded hourly during 1 year with data loggers, at sites representing 200-m elevation increments. Floristic information was extracted from a database containing 10,124 records of plant collections, and organized in 200-m elevational belts. Climatic charts distinguished three climate types along the gradient, all with marked rainfall seasonality, but these bore little correspondence with the information obtained with the data loggers. Mean annual air temperature decreased with increasing elevation (lapse rate of 0.542 °C 100 m(-1)). Thermal oscillation was minimum around 1400 m and increased towards both extremes of the gradient. Relative humidity opposed this pattern, with maxima between 800 and 1800 m, decreasing towards the highest elevations. An analysis of temperature frequency distributions revealed meteorological features undetectable from the annual or monthly means of this variable; despite an overall gradual transition of the proportions of time recorded at different temperatures, some changes did not conform to this pattern. The first discontinuity occurred between 1000-1200 m, where dominant temperatures shifted abruptly; also noticeable was an abrupt increase of the proportion of time elapsed at 0.1-10 °C between 2400 and 2600 m. Air temperature appears to be the most influential climatic factor

  20. The relationship of meteorological patterns with changes in floristic richness along a large elevational gradient in a seasonally dry region of southern Mexico

    NASA Astrophysics Data System (ADS)

    Salas-Morales, Silvia H.; Meave, Jorge A.; Trejo, Irma

    2015-12-01

    Globally, climate is a fundamental driver of plant species' geographical distributions, yet we still lack a good understanding of climatic variation on tropical mountains and its consequences for elevational floristic patterns. In a seasonally dry region of southern Mexico, we analysed meteorological patterns along a large elevational gradient (0-3670 m a.s.l.) and examined their relationship with changes in floristic richness. Meteorological patterns were characterised using two data sources. First, climatic information was extracted from cartography and records from a few existing meteorological stations. Additionally, air temperature and humidity were recorded hourly during 1 year with data loggers, at sites representing 200-m elevation increments. Floristic information was extracted from a database containing 10,124 records of plant collections, and organized in 200-m elevational belts. Climatic charts distinguished three climate types along the gradient, all with marked rainfall seasonality, but these bore little correspondence with the information obtained with the data loggers. Mean annual air temperature decreased with increasing elevation (lapse rate of 0.542 °C 100 m-1). Thermal oscillation was minimum around 1400 m and increased towards both extremes of the gradient. Relative humidity opposed this pattern, with maxima between 800 and 1800 m, decreasing towards the highest elevations. An analysis of temperature frequency distributions revealed meteorological features undetectable from the annual or monthly means of this variable; despite an overall gradual transition of the proportions of time recorded at different temperatures, some changes did not conform to this pattern. The first discontinuity occurred between 1000-1200 m, where dominant temperatures shifted abruptly; also noticeable was an abrupt increase of the proportion of time elapsed at 0.1-10 °C between 2400 and 2600 m. Air temperature appears to be the most influential climatic factor

  1. Stabilization of an α/β-Hydrolase by Introducing Proline Residues: Salicylic Acid Binding Protein 2 from Tobacco.

    PubMed

    Huang, Jun; Jones, Bryan J; Kazlauskas, Romas J

    2015-07-21

    α/β-Hydrolases are important enzymes for biocatalysis, but their stability often limits their application. We investigated a plant esterase, salicylic acid binding protein 2 (SABP2), as a model α/β-hydrolase. SABP2 shows typical stability to urea (unfolding free energy 6.9 ± 1.5 kcal/mol) and to heat inactivation (T1/2 15min 49.2 ± 0.5 °C). Denaturation in urea occurs in two steps, but heat inactivation occurs in a single step. The first unfolding step in urea eliminates catalytic activity. Surprisingly, we found that the first unfolding likely corresponds to the unfolding of the larger catalytic domain. Replacing selected amino acid residues with proline stabilized SABP2. Proline restricts the flexibility of the unfolded protein, thereby shifting the equilibrium toward the folded conformation. Seven locations for proline substitution were chosen either by amino acid sequence alignment with a more stable homologue or by targeting flexible regions in SABP2. Introducing proline in the catalytic domain stabilized SABP2 to the first unfolding in urea for three of five cases: L46P (+0.2 M urea), S70P (+0.1), and E215P (+0.9). Introducing proline in the cap domain did not stabilize SABP2 (two of two cases), supporting the assignment that the first unfolding corresponds to the catalytic domain. Proline substitutions in both domains stabilized SABP2 to heat inactivation: L46P (ΔT1/2 15min = +6.4 °C), S70P (+5.4), S115P (+1.8), S141P (+4.9), and E215P (+4.2). Combining substitutions did not further increase the stability to urea denaturation, but dramatically increased resistance to heat inactivation: L46P−S70P ΔT1/2 15min = +25.7 °C. This straightforward proline substitution approach may also stabilize other α/β-hydrolases.

  2. Stabilization of an α/β-hydrolase by introducing proline residues: salicylic binding protein 2 from tobacco

    PubMed Central

    Huang, Jun; Jones, Bryan J.; Kazlauskas, Romas J.

    2015-01-01

    α/β-Hydrolases are important enzymes for biocatalysis, but their stability often limits their application. As a model α/β-hydrolase, we investigated a plant esterase, salicylic acid binding protein 2 (SABP2). SABP2 shows typical stability to urea (unfolding free energy 6.9±1.5 kcal/mol) and to heat inactivation (T1/215 min 49.2±0.5 °C). Denaturation in urea occurs in two steps, but heat inactivation occurs in a single step. The first unfolding step in urea eliminates catalytic activity. Surprisingly, we found that the first unfolding likely corresponds to the unfolding of the larger catalytic domain. Replacing selected amino acid residues with proline stabilized SABP2. Proline restricts the flexibility of the unfolded protein, thereby shifting the equilibrium toward the folded conformation. Seven locations for proline substitution were chosen either by amino acid sequence alignment with a more stable homolog or by targeting flexible regions in SABP2. Introducing proline in the catalytic domain stabilized SABP2 to the first unfolding in urea for three of five cases: L46P (+0.2 M urea), S70P (+0.1) and E215P (+0.9). Introducing proline in the cap domain did not (two of two cases), supporting the assignment that the first unfolding corresponds to the catalytic domain. Proline substitutions in both domains stabilized SABP2 to heat inactivation: L46P (ΔT1/215 min = +6.4 °C), S70P (+5.4), S115P (+1.8), S141P (+4.9), and E215P (+4.2). Combining substitutions did not further increase the stability to urea denaturation, but dramatically increased resistance to heat inactivation: L46P-S70P ΔT1/215 min = +25.7 °C. This straightforward proline substitution approach may also stabilize other α/β-hydrolases. PMID:26110207

  3. Analysis of Proline Reduction in the Nosocomial Pathogen Clostridium difficile▿

    PubMed Central

    Jackson, Sarah; Calos, Mary; Myers, Andrew; Self, William T.

    2006-01-01

    Clostridium difficile, a proteolytic strict anaerobe, has emerged as a clinically significant nosocomial pathogen in recent years. Pathogenesis is due to the production of lethal toxins, A and B, members of the large clostridial cytotoxin family. Although it has been established that alterations in the amino acid content of the growth medium affect toxin production, the molecular mechanism for this observed effect is not yet known. Since there is a paucity of information on the amino acid fermentation pathways used by this pathogen, we investigated whether Stickland reactions might be at the heart of its bioenergetic pathways. Growth of C. difficile on Stickland pairs yielded large increases in cell density in a limiting basal medium, demonstrating that these reactions are tied to ATP production. Selenium supplementation was required for this increase in cell yield. Analysis of genome sequence data reveals genes encoding the protein components of two key selenoenzyme reductases, glycine reductase and d-proline reductase (PR). These selenoenzymes were expressed upon the addition of the corresponding Stickland acceptor (glycine, proline, or hydroxyproline). Purification of the selenoenzyme d-proline reductase revealed a mixed complex of PrdA and PrdB (SeCys-containing) proteins. PR utilized only d-proline but not l-hydroxyproline, even in the presence of an expressed and purified proline racemase. PR was found to be independent of divalent cations, and zinc was a potent inhibitor of PR. These results show that Stickland reactions are key to the growth of C. difficile and that the mechanism of PR may differ significantly from that of previously studied PR from nonpathogenic species. PMID:17041035

  4. The Sodium/Proline Transporter PutP of Helicobacter pylori

    PubMed Central

    Rivera-Ordaz, Araceli; Bracher, Susanne; Sarrach, Sannia; Li, Zheng; Shi, Lei; Quick, Matthias; Hilger, Daniel; Haas, Rainer; Jung, Heinrich

    2013-01-01

    Helicobacter pylori is cause of chronic gastritis, duodenal ulcer and gastric carcinoma in humans. L-proline is a preferred energy source of the microaerophilic bacterium. Previous analyses revealed that HpputP and HpputA, the genes that are predicted to play a central role in proline metabolism as they encode for the proline transporter and proline dehydrogenase, respectively, are essential for stomach colonization. Here, the molecular basis of proline transport in H. pylori by HpPutP was investigated experimentally for the first time. Measuring radiolabeled substrate transport in H. pylori and E. coli heterologously expressing HpputP as well as in proteoliposomes reconstituted with HpPutP, we demonstrate that the observed proline transport in H. pylori is mediated by HpPutP. HpPutP is specific and exhibits a high affinity for L-proline. Notably, L-proline transport is exclusively dependent on Na+ as coupling ion, i.e., Na+/L-proline symport, reminiscent to the properties of PutP of E. coli even though H. pylori lives in a more acidic environment. Homology model-based structural comparisons and substitution analyses identified amino acids crucial for function. HpPutP-catalyzed proline uptake was efficiently inhibited by the known proline analogs 3,4-dehydro-D,L-proline and L-azetidine-2-carboxylic acid. PMID:24358297

  5. Proline modulates the intracellular redox environment and protects mammalian cells against oxidative stress.

    PubMed

    Krishnan, Navasona; Dickman, Martin B; Becker, Donald F

    2008-02-15

    The potential of proline to suppress reactive oxygen species (ROS) and apoptosis in mammalian cells was tested by manipulating intracellular proline levels exogenously and endogenously by overexpression of proline metabolic enzymes. Proline was observed to protect cells against H(2)O(2), tert-butyl hydroperoxide, and a carcinogenic oxidative stress inducer but was not effective against superoxide generators such as menadione. Oxidative stress protection by proline requires the secondary amine of the pyrrolidine ring and involves preservation of the glutathione redox environment. Overexpression of proline dehydrogenase (PRODH), a mitochondrial flavoenzyme that oxidizes proline, resulted in 6-fold lower intracellular proline content and decreased cell survival relative to control cells. Cells overexpressing PRODH were rescued by pipecolate, an analog that mimics the antioxidant properties of proline, and by tetrahydro-2-furoic acid, a specific inhibitor of PRODH. In contrast, overexpression of the proline biosynthetic enzymes Delta(1)-pyrroline-5-carboxylate (P5C) synthetase (P5CS) and P5C reductase (P5CR) resulted in 2-fold higher proline content, significantly lower ROS levels, and increased cell survival relative to control cells. In different mammalian cell lines exposed to physiological H(2)O(2) levels, increased endogenous P5CS and P5CR expression was observed, indicating that upregulation of proline biosynthesis is an oxidative stress response.

  6. γ-(S)-Trifluoromethyl proline: evaluation as a structural substitute of proline for solid state (19)F-NMR peptide studies.

    PubMed

    Kubyshkin, Vladimir; Afonin, Sergii; Kara, Sezgin; Budisa, Nediljko; Mykhailiuk, Pavel K; Ulrich, Anne S

    2015-03-21

    γ-(4S)-Trifluoromethyl proline was synthesised according to a modified literature protocol with improved yield on a multigram scale. Conformational properties of the amide bond formed by the amino acid were characterised using N-acetyl methyl ester model. The amide populations (s-trans vs. s-cis) and thermodynamic parameters of the isomerization were found to be similar to the corresponding values for intact proline. Therefore, the γ-trifluoromethyl proline was suggested as a structurally low-disturbing proline substitution in peptides for their structural studies by (19)F-NMR. Indeed, the exchange of native proline for γ-trifluoromethyl proline in the peptide antibiotic gramicidin S was shown to preserve the overall amphipathic peptide structure. The utility of the amino acid as a selective (19)F-NMR label was demonstrated by observing the re-alignment of the labelled gramicidin S in oriented lipid bilayers.

  7. Endothelin-1 expression is strongly repressed by AU-rich elements in the 3′-untranslated region of the gene

    PubMed Central

    2004-01-01

    The regulation of the synthesis of the endothelial-derived vasoconstrictor ET-1 (endothelin-1) is a complex process that occurs mainly at the mRNA level. Transcription of the gene accounts for an important part of the regulation of expression, as already described for different modulators such as the cytokine TGF-β (transforming growth factor-β). However, very little is known about mechanisms governing ET-1 expression at the post-transcriptional level. The aim of the present study was to investigate the regulation of the ET-1 expression at this level. Since the 3′-UTR (3′-untranslated region) of mRNAs commonly contains genetic determinants for the post-transcriptional control of gene expression, we focused on the potential role of the 3′-UTR of ET-1 mRNA. Experiments performed with luciferase reporter constructs containing the 3′-UTR showed that this region exerts a potent destabilizing effect. Deletional analyses allowed us to locate this activity within a region at positions 924–1127. Some (but not all) of the AREs (AU-rich elements) present in this region were found to be essential for this mRNA-destabilizing activity. We also present evidence that cytosolic proteins from endothelial cells interact specifically with these RNA elements, and that a close correlation exists between the ability of the AREs to destabilize ET-1 mRNA and the binding of proteins to these elements. Our results are compatible with the existence of a strong repressional control of ET-1 expression mediated by destabilization of the mRNA exerted through the interaction of specific cytosolic proteins with AREs present in the 3′-UTR of the gene. PMID:15595926

  8. Normal Development and Function of T Cells in Proline Rich 7 (Prr7) Deficient Mice

    PubMed Central

    Hrdinka, Matous; Sudan, Kritika; Just, Sissy; Drobek, Ales; Stepanek, Ondrej; Schlüter, Dirk; Reinhold, Dirk; Jordan, Bryen A.; Gintschel, Patricia; Schraven, Burkhart; Kreutz, Michael R.

    2016-01-01

    Transmembrane adaptor proteins (TRAPs) are important organisers for the transduction of immunoreceptor-mediated signals. Prr7 is a TRAP that regulates T cell receptor (TCR) signalling and potently induces cell death when overexpressed in human Jurkat T cells. Whether endogenous Prr7 has a similar functional role is currently unknown. To address this issue, we analysed the development and function of the immune system in Prr7 knockout mice. We found that loss of Prr7 partially impairs development of single positive CD4+ T cells in the thymus but has no effect on the development of other T cell subpopulations, B cells, NK cells, or NKT cells. Moreover, Prr7 does not affect the TCR signalling pathway as T cells derived from Prr7 knockout and wild-type animals and stimulated in vitro express the same levels of the activation marker CD69, and retain their ability to proliferate and activate induced cell death programs. Importantly, Prr7 knockout mice retained the capacity to mount a protective immune response when challenged with Listeria monocytogenes infection in vivo. In addition, T cell effector functions (activation, migration, and reactivation) were normal following induction of experimental autoimmune encephalomyelitis (EAE) in Prr7 knockout mice. Collectively, our work shows that loss of Prr7 does not result in a major immune system phenotype and suggests that Prr7 has a dispensable function for TCR signalling. PMID:27657535

  9. Proline-rich peptide from the coral pathogen Vibrio shiloi that inhibits photosynthesis of Zooxanthellae.

    PubMed

    Banin, E; Khare, S K; Naider, F; Rosenberg, E

    2001-04-01

    The coral-bleaching bacterium Vibrio shiloi biosynthesizes and secretes an extracellular peptide, referred to as toxin P, which inhibits photosynthesis of coral symbiotic algae (zooxanthellae). Toxin P was produced during the stationary phase when the bacterium was grown on peptone or Casamino Acids media at 29 degrees C. Glycerol inhibited the production of toxin P. Toxin P was purified to homogeneity, yielding the following 12-residue peptide: PYPVYAPPPVVP (molecular weight, 1,295.54). The structure of toxin P was confirmed by chemical synthesis. In the presence of 12.5 mM NH(4)Cl, pure natural or synthetic toxin P (10 microM) caused a 64% decrease in the photosynthetic quantum yield of zooxanthellae within 5 min. The inhibition was proportional to the toxin P concentration. Toxin P bound avidly to zooxanthellae, such that subsequent addition of NH(4)Cl resulted in rapid inhibition of photosynthesis. When zooxanthellae were incubated in the presence of NH(4)Cl and toxin P, there was a rapid decrease in the pH (pH 7.8 to 7.2) of the bulk liquid, suggesting that toxin P facilitates transport of NH(3) into the cell. It is known that uptake of NH(3) into cells can destroy the pH gradient and block photosynthesis. This mode of action of toxin P can help explain the mechanism of coral bleaching by V. shiloi.

  10. Structural basis of the transcriptional regulation of the proline utilization regulon by multifunctional PutA.

    PubMed

    Zhou, Yuzhen; Larson, John D; Bottoms, Christopher A; Arturo, Emilia C; Henzl, Michael T; Jenkins, Jermaine L; Nix, Jay C; Becker, Donald F; Tanner, John J

    2008-08-01

    The multifunctional Escherichia coli proline utilization A (PutA) flavoprotein functions both as a membrane-associated proline catabolic enzyme and as a transcriptional repressor of the proline utilization genes putA and putP. To better understand the mechanism of transcriptional regulation by PutA, we have mapped the put-regulatory region, determined a crystal structure of the PutA ribbon-helix-helix domain (PutA52, a polypeptide corresponding to residues 1-52 of E. coli PutA) complexed with DNA, and examined the thermodynamics of DNA binding to PutA52. Five operator sites, each containing the sequence motif 5'-GTTGCA-3', were identified using gel-shift analysis. Three of the sites are shown to be critical for repression of putA, whereas the two other sites are important for repression of putP. The 2.25-A-resolution crystal structure of PutA52 bound to one of the operators (operator 2; 21 bp) shows that the protein contacts a 9-bp fragment corresponding to the GTTGCA consensus motif plus three flanking base pairs. Since the operator sequences differ in flanking bases, the structure implies that PutA may have different affinities for the five operators. This hypothesis was explored using isothermal titration calorimetry. The binding of PutA52 to operator 2 is exothermic, with an enthalpy of -1.8 kcal/mol and a dissociation constant of 210 nM. Substitution of the flanking bases of operator 4 into operator 2 results in an unfavorable enthalpy of 0.2 kcal/mol and a 15-fold-lower affinity, showing that base pairs outside of the consensus motif impact binding. Structural and thermodynamic data suggest that hydrogen bonds between Lys9 and bases adjacent to the GTTGCA motif contribute to transcriptional regulation by fine-tuning the affinity of PutA for put control operators.

  11. Post-transcriptional regulation of cytokine genes in fish: A role for conserved AU-rich elements located in the 3'-untranslated region of their mRNAs.

    PubMed

    Roca, Francisco J; Cayuela, María L; Secombes, Chris J; Meseguer, José; Mulero, Victoriano

    2007-01-01

    The overproduction of cytokines, such us interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha), contributes to the pathological complications observed in many inflammatory diseases caused by bacterial endotoxins. The synthesis of these cytokines is tightly regulated at both transcriptional and post-transcriptional levels. Post-transcriptional regulation of gene expression depends on specific cis-acting sequences and trans-acting factors. Thus, the presence of adenylate- and uridylate-rich (AU-rich) elements (AREs) has been described in the 3'-untranslated regions (UTRs) of many unstable mammalian mRNAs. Although, it represents the most widespread, phylogenetically conserved and efficient determinant of mRNA stability among those so far characterized in mammalian cells, no studies are available on the functional relevance of this sequence in non-mammalian vertebrates. In this contribution, we study the enzymatic activity of various luciferase reporter constructs, containing or lacking the 3'UTR of IL-1beta and TNFalpha from different fish species, and report the finding that bony fish AREs are able to decrease luciferase activity but are less potent than their mammalian counterparts. Surprisingly, the 3'UTR of the IL-1beta from the cartilaginous fish small spotted catshark had the greatest ability to decrease luciferase activity. Lastly, the functional significance of the above was confirmed by measuring the half-life of IL-1beta and TNFalpha mRNAs in gilthead seabream leukocytes by blocking transcription with actinomycin D. Both cytokine mRNAs were unstable with an estimated half-life of about 45 min in control and activated cells.

  12. The role of proline in the prevention of aggregation during protein folding in vitro.

    PubMed

    Kumar, T K; Samuel, D; Jayaraman, G; Srimathi, T; Yu, C

    1998-10-01

    Proline effectively inhibits protein aggregation during the refolding of bovine carbonic anhydrase. Other osmolytes used such as glycine and ethylene glycol fail to exhibit the 'aggregation-blockade' role shown by proline. Results of viscosity and ANS fluorescence (1-anilino-8-naphthalene sulphonic acid) experiments suggest that proline at high concentrations forms an ordered supramolecular assembly. Based on these results, it is proposed that proline behaves as a protein folding chaperone due to the formation of an ordered, amphipathic supramolecular assembly. To our knowledge, this is the first report wherein proline is proposed as a protein folding aid.

  13. Remnants of the Legume Ancestral Genome Preserved in Gene-Rich Regions: Insights from Lupinus angustifolius Physical, Genetic, and Comparative Mapping.

    PubMed

    Książkiewicz, Michał; Zielezinski, Andrzej; Wyrwa, Katarzyna; Szczepaniak, Anna; Rychel, Sandra; Karlowski, Wojciech; Wolko, Bogdan; Naganowska, Barbara

    The narrow-leafed lupin (Lupinus angustifolius) was recently considered as a legume reference species. Genetic resources have been developed, including a draft genome sequence, linkage maps, nuclear DNA libraries, and cytogenetic chromosome-specific landmarks. Here, we used a complex approach, involving DNA fingerprinting, sequencing, genetic mapping, and molecular cytogenetics, to localize and analyze L. angustifolius gene-rich regions (GRRs). A L. angustifolius genomic bacterial artificial chromosome (BAC) library was screened with short sequence repeat (SSR)-based probes. Selected BACs were fingerprinted and assembled into contigs. BAC-end sequence (BES) annotation allowed us to choose clones for sequencing, targeting GRRs. Additionally, BESs were aligned to the scaffolds of the genome sequence. The genetic map was supplemented with 35 BES-derived markers, distributed in 14 linkage groups and tagging 37 scaffolds. The identified GRRs had an average gene density of 19.6 genes/100 kb and physical-to-genetic distance ratios of 11 to 109 kb/cM. Physical and genetic mapping was supported by multi-BAC-fluorescence in situ hybridization (FISH), and five new linkage groups were assigned to the chromosomes. Syntenic links to the genome sequences of five legume species (Medicago truncatula, Glycine max, Lotus japonicus, Phaseolus vulgaris, and Cajanus cajan) were identified. The comparative mapping of the two largest lupin GRRs provides novel evidence for ancient duplications in all of the studied species. These regions are conserved among representatives of the main clades of Papilionoideae. Furthermore, despite the complex evolution of legumes, some segments of the nuclear genome were not substantially modified and retained their quasi-ancestral structures. Cytogenetic markers anchored in these regions constitute a platform for heterologous mapping of legume genomes.

  14. Life-long norepinephrine transporter (NET) knock-out leads to the increase in the NET mRNA in brain regions rich in norepinephrine terminals.

    PubMed

    Solich, Joanna; Kolasa, Magdalena; Kusmider, Maciej; Pabian, Paulina; Faron-Gorecka, Agata; Zurawek, Dariusz; Szafran-Pilch, Kinga; Kedracka-Krok, Sylwia; Jankowska, Urszula; Swiderska, Bianka; Dziedzicka-Wasylewska, Marta

    2015-08-01

    These studies aimed to identify the genes differentially expressed in the frontal cortex of mice bearing a life-long norepinephrine transporter knock-out (NET-KO) and wild-type animals (WT). Differences in gene expression in the mouse frontal cortex were studied using a whole-genome microarray approach. Using an alternative approach, i.e. RT-PCR (reverse transcription polymerase chain reaction) with primers complementary to various exons of the NET gene, as well as TaqMan arrays, the level of mRNA encoding the NET in other brain regions of the NET-KO mice was also examined. The analyses revealed a group of 92 transcripts (27 genes) that differentiated the NET-KO mice from the WT mice. Surprisingly, the studies have shown that the mRNA encoding NET accumulated in the brain regions rich in norepinephrine nerve endings in the NET-KO mice. Because there is no other source of NET mRNA besides the noradrenergic terminals in the brain regions studied, these results might speak in favor of the presence of mRNA in axon terminals. RNA-Binding Protein Immunoprecipitation approach indicated that mRNA encoding NET was detected in the Ago2 protein/mRNA complex. In addition, the amount of Ago2 protein in the frontal cortex was significantly higher in NET-KO mice as compared with that of the WT animals. These results are important for further characterization of the NET-KO mice, which - besides other merits - might serve as a good model to study the fate of truncated mRNA in neurons.

  15. Regulation of proline accumulation in detached rice leaves exposed to excess copper.

    PubMed

    Chen, C T.; Chen, L -M.; Lin, C C.; Kao, C H.

    2001-01-05

    Accumulation of proline in response to excess Cu was studied in detached leaves of rice (Oryza sativa). CuSO(4) was effective in inducing proline accumulation in detached rice leaves under both light and dark conditions. CuSO(4) and CuCl(2) were equally effective in inducing proline accumulation, indicating that proline accumulation is induced by Cu. Sulfate salts of Mg, Mn, and Fe were ineffective in inducing proline accumulation in detached rice leaves. Excess Cu had no effect on relative water content of detached rice leaves, suggesting that Cu-induced proline accumulation is unlikely due to water deficit. Proline accumulation induced by excess Cu was related to proteolysis and an increase in Delta(1)-pyrroline-5-carboxylate reductase or ornithine-delta-aminotransferase activity and could not be explained by proline utilization or stress-induced modifications in proline dehydrogenase or Delta(1)-pyrroline-5-carboxylate dehydrogenase. The content of glutamic acid decreased by excess Cu. The increase in arginine but not ornithine was found to be associated with the increase in proline content in Cu-stressed detached rice leaves. CuSO(4) treatment resulted in an increase in abscisic acid content in detached rice leaves. The possibility that proline accumulation induced by excess Cu is mediated through abscisic acid is discussed.

  16. Proline metabolism in sepsis, cirrhosis and general surgery. The peripheral energy deficit.

    PubMed

    Cerra, F B; Caprioli, J; Siegel, J H; McMenamy, R R; Border, J R

    1979-11-01

    Proline metabolism was prospectively evaluated in patients with surgical sepsis, cirrhosis, and elective surgical procedures. Significant correlations were found in the septic patients. Proline levels were an excellent indicator of mortality and correlated positively with lactate levels. Lactate and proline were inversely related to total peripheral resistance and oxygen consumption. In septic patients who expired: the metabolites involved in the hepatic pathways of proline degradation were elevated in proportion to proline; lactate, glutamate and proline were directly related to pyruvate; lactate/pyruvate ratios were constant; proline, glutamate, ammonia, ornithine, lactate and pyruvate levels were inversely proportional to oxygen consumption and total peripheral resistance. The primary defects in sepsis seem to be metabolic; there are very strong correlations in time between physiology and metabolism; the metabolic abnormality seems to be a progressive energy-fuel deficit, possibly from a progressive inhibition of substrate entry into the Krebs cycle.

  17. Hydrogen rich gas generator

    NASA Technical Reports Server (NTRS)

    Houseman, J.; Rupe, J. H.; Kushida, R. O. (Inventor)

    1976-01-01

    A process and apparatus is described for producing a hydrogen rich gas by injecting air and hydrocarbon fuel at one end of a cylindrically shaped chamber to form a mixture and igniting the mixture to provide hot combustion gases by partial oxidation of the hydrocarbon fuel. The combustion gases move away from the ignition region to another region where water is injected to be turned into steam by the hot combustion gases. The steam which is formed mixes with the hot gases to yield a uniform hot gas whereby a steam reforming reaction with the hydrocarbon fuel takes place to produce a hydrogen rich gas.

  18. Cyclophilin A catalyzes proline isomerization by an electrostatic handle mechanism

    SciTech Connect

    Camilloni, Carlo; Sahakyan, Aleksander B.; Holliday, Michael; Isern, Nancy G.; Zhang, Fengli; Eisenmesser, Elan Z.; Vendruscolo, Michele

    2014-07-15

    Proline isomerization is a ubiquitous process that plays a key role in the folding of proteins and in the regulation of their functions1-3. Different families of enzymes, known as peptidyl-prolyl isomerases (PPIases), catalyse this reaction, which involves the interconversion between the cis and trans isomers of the Nterminal amide bond of the amino acid proline2,3. A complete description of the mechanisms by which these enzymes function, however, has remained elusive. Here, we show that cyclophilin A, one of the most common PPIases4, provides a catalytic environment that acts on the substrate through an electrostatic lever mechanism. In this mechanism, the electrostatic field in the catalytic site turns the electric dipole associated with the carboxylic group of the amino acid preceding the proline in the substrate, thus causing the rotation of the peptide bond between the two residues. This mechanism resulted from the analysis of an ensemble of conformations populated by cyclophilin A during the enzymatic reaction using a combination of NMR measurements, molecular dynamics simulations and density functional theory calculations. We anticipate that this approach will be helpful in elucidating whether the electrostatic lever mechanism that we describe is common to other PPIases, and more generally to characterise other enzymatic processes.

  19. Conformational Preferences of α-Substituted Proline Analogues

    PubMed Central

    Flores-Ortega, Alejandra; Jiménez, Ana I.; Cativiela, Carlos; Nussinov, Ruth; Alemán, Carlos; Casanovas, Jordi

    2009-01-01

    DFT calculations at the B3LYP/6-31+G(d,p) level have been used to investigate how the replacement of the α hydrogen by a more sterically demanding group affects the conformational preferences of proline. Specifically, the N-acetyl-N’-methylamide derivatives of L-proline, L-α-methylproline and L-α-phenylproline have been calculated, with both the cis/trans isomerism of the peptide bonds and the puckering of the pyrrolidine ring being considered. The effects of solvation have been evaluated using a Self Consistent Reaction Field model. As expected, tetrasubstitution at the α carbon destabilizes the conformers with one or more peptide bonds arranged in cis. The lowest energy minimum has been found to be identical for the three compounds investigated, but important differences are observed regarding other energetically accessible backbone conformations. The results obtained provide evidence that the distinct steric requirements of the substituent at Cα may play a significant role in modulating the conformational preferences of proline. PMID:18351745

  20. Cloning, purification and crystallization of Thermus thermophilus proline dehydrogenase

    SciTech Connect

    White, Tommi A.; Tanner, John J.

    2005-08-01

    Cloning, purification and crystallization of T. thermophilus proline dehydrogenase is reported. The detergent n-octyl β-d-glucopyranoside was used to reduce polydispersity, which enabled crystallization. Nature recycles l-proline by converting it to l-glutamate. This four-electron oxidation process is catalyzed by the two enzymes: proline dehydrogenase (PRODH) and Δ{sup 1}-pyrroline-5-carboxylate dehydrogenase. This note reports the cloning, purification and crystallization of Thermus thermophilus PRODH, which is the prototype of a newly discovered superfamily of bacterial monofunctional PRODHs. The results presented here include production of a monodisperse protein solution through use of the detergent n-octyl β-d-glucopyranoside and the growth of native crystals that diffracted to 2.3 Å resolution at Advanced Light Source beamline 4.2.2. The space group is P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 82.2, b = 89.6, c = 94.3 Å. The asymmetric unit is predicted to contain two protein molecules and 46% solvent. Molecular-replacement trials using a fragment of the PRODH domain of the multifunctional Escherichia coli PutA protein as the search model (24% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of T. thermophilus PRODH will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.

  1. A case study of proline isomerization in cell signaling.

    PubMed

    Min, Lie; Fulton, D Bruce; Andreotti, Amy H

    2005-01-01

    Protein-mediated interactions and enzymatic function provide the foundation upon which cellular signaling cascades control all of the activities of a cell. Post-translational modifications such as phosphorylation or ubiquitiation are well known means for modulating protein activity within the cell. These chemical modifications create new recognition motifs on proteins or shift conformational preferences such that protein catalytic and binding functions are altered in response to external stimuli. Moreover, detection of such modifications is often straightforward by conventional biochemical methods leading investigators toward mechanistic models of cell signaling involving post-translational modifications such as phosphorylation/dephosphorylation. While there is little doubt that such modifications play a significant role in transmission of information throughout the cell, there are certainly other mechanisms at work that are not as well understood at this time. Of particular interest in the context of this review is the intrinsic conformational switch afforded to a polypeptide by peptidyl prolyl cis/trans isomerization. Proline isomerization is emerging as a critical component of certain cell signaling cascades. In addition to serving as a conformational switch that enables a protein to adopt functionally distinct states, proline isomerization may serve as a recognition element for the ubiquitous peptidyl prolyl isomerases. This overview takes a close look at one particular signaling protein, the T cell specific tyrosine kinase Itk, and examines the role of proline isomerization and the peptidyl prolyl isomerase cyclophilin A in mediating Itk function following T cell receptor engagement.

  2. Expression in Escherichia coli of the catalytic domain of human proline oxidase.

    PubMed

    Tallarita, Elena; Pollegioni, Loredano; Servi, Stefano; Molla, Gianluca

    2012-04-01

    The human PRODH gene has been shown to have unique roles in regulating cell survival and apoptotic pathways and it has been related to velocardiofacial syndrome/DiGeorge syndrome and increased susceptibility to schizophrenia. It encodes for the flavoprotein proline oxidase (PO), which catalyzes the conversion of l-proline to Δ(1)-pyrroline-5-carboxylate. Despite the important physiological and medical interest in human PO, up to now only microbial homologues of PO have been expressed as recombinant protein and fully characterized. By using a bioinformatics analysis aimed at identifying the catalytic domain and the regions with a high intrinsic propensity to structural disorder, we designed deletion variants of human PO that were successfully expressed in Escherichia coli as soluble proteins in fairly high amounts (up to 10mg/L of fermentation broth). The His-tagged PO-barrelN protein was isolated as an active (the specific activity is 0.032U/mg protein), dimeric holoenzyme showing the typical spectral properties of FAD-containing flavoprotein oxidases. These results pave the way for elucidating structure-function relationships of this human flavoenzyme and clarifying the effect of the reported polymorphisms associated with disease states.

  3. High Pressure Cosmochemistry of Major Planetary Interiors: Laboratory Studies of the Water-rich Region of the System Ammonia-water

    NASA Technical Reports Server (NTRS)

    Nicol, M.; Johnson, M.; Koumvakalis, A. S.

    1985-01-01

    The behavior of gas-ice mixtures in major planets at very high pressures was studied. Some relevant pressure-temperature-composition (P-T-X) regions of the hydrogen (H2)-helium (He)-water (H2O-ammonia (NH3)-methane (CH4) phase diagram were determined. The studies, and theoretical model, of the relevant phases, are needed to interpret the compositions of ice-gas systems at conditions of planetary interest. The compositions and structures of a multiphase, multicomponent system at very high pressures care characterized, and the goal is to characterize this system over a wide range of low and high temperatures. The NH3-H2O compositions that are relevant to planetary problems yet are easy to prepare were applied. The P-T surface of water was examined and the corresponding surface for NH3 was determined. The T-X diagram of ammonia-water at atmospheric pressure was studied and two water-rich phases were found, NH3-2H2O (ammonia dihydrate), which melts incongruently, and NH3.H2O (ammonia monohydrate), which is nonstoichiometric and melts at a higher temperature than the dihydrate. It is suggested that a P-T surface at approximately the monohydrate composition and the P-X surface at room temperature is determined.

  4. sup 40 Ar- sup 39 Ar and K-Ar dating of K-rich rocks from the Roccamonfina volcano, Roman Comagmatic Region, Italy

    SciTech Connect

    Di Brozolo, F.R.; Di Girolamo, P.; Turi, B.; Oddone, M. )

    1988-06-01

    Roccamonfina is the northernmost Volcano of the Campanian area of the K-rich Roman comagmatic Region of Italy. It erupted a huge amount of pyroclastics and lavas belonging to both the Leucite-Basanite and Leucitite Series (LBLS) and the Shoshonite Series (SS), spread over an area of about 300 km{sup 2}. The above series correspond to the High-K Series (HKS) and Low-K Series (LKS) of Appleton (1971), respectively. {sup 40}Ar-{sup 39}Ar and K-Ar dating of samples from both series gave ages ranging from 0.656 to 0.096 Ma for the SS and from 1.03( ) to 0.053 Ma for the LBLS. These results indicate that the products of the two series were outpoured together at least between 0.7 and 0.1 Ma age, i.e. during both the so-called pre-caldera phase and the post-caldera phase of activity. The latest products of the volcanism at Roccamonfina were erupted just before the deposition of the Grey Campanian Ignimbrite, which erupted from vents located about 50 km to the south in the Phlegrean Fields near Naples and has an age of about 33,000 years. Taking into account all the available all the available radiometric data the authors conclude that Roccamonfina was active between 1.5 and 0.05 Ma ago, in excellent agreement with the stratigraphic evidence. In this same time span is concentrated the activity of all the centers of the Roman Region north of Naples.

  5. The 3'-untranslated region length and AU-rich RNA location modulate RNA-protein interaction and translational control of β2-adrenergic receptor mRNA.

    PubMed

    Subramaniam, Kothandharaman; Kandasamy, Karthikeyan; Joseph, Kusumam; Spicer, Eleanor K; Tholanikunnel, Baby G

    2011-06-01

    Posttranscriptional controls play a major role in β(2)-adrenergic receptor (β(2)-AR) expression. We recently reported that β(2)-AR mRNA translation is suppressed by elements in its 3'-untranslated region (UTR). We also identified T-cell-restricted intracellular antigen-related protein (TIAR) and HuR as prominent AU-rich (ARE) RNA-binding proteins that associate with β(2)-AR mRNA 3'-UTR. In this study, we identified a poly(U) region at the distal end of the 3'-UTR as critical for TIAR binding to β(2)-AR mRNA and for translational suppression. Here, we also report that the locations of the poly(U) and ARE sequences within the 3'-UTR are important determinants that control the translation of β(2)-AR mRNA. Consistent with this finding, a 20-nucleotide ARE RNA from the proximal 3'-UTR that did not inhibit mRNA translation in its native position was able to suppress translation when re-located to the distal 3'-UTR of the receptor mRNA. Immunoprecipitation and polysome profile analysis demonstrated the importance of 3'-UTR length and the ARE RNA location within the 3'-UTR, as key determinants of RNA/protein interactions and translational control of β(2)-AR mRNA. Further, the importance of 3'-UTR length and ARE location in TIAR and HuR association with mRNA and translational suppression was demonstrated using a chimeric luciferase reporter gene.

  6. Proline is required for male gametophyte development in Arabidopsis

    PubMed Central

    2012-01-01

    Background In crosses between the proline-deficient mutant homozygous for p5cs1 and heterozygous for p5cs2 (p5cs1 p5cs2/P5CS2), used as male, and different Arabidopsis mutants, used as females, the p5cs2 mutant allele was rarely transmitted to the outcrossed progeny, suggesting that the fertility of the male gametophyte carrying mutations in both P5CS1 and P5CS2 is severely compromised. Results To confirm the fertility defects of pollen from p5cs1 p5cs2/P5CS2 mutants, transmission of mutant alleles through pollen was tested in two ways. First, the number of progeny inheriting a dominant sulfadiazine resistance marker linked to p5cs2 was determined. Second, the number of p5cs2/p5cs2 embryos was determined. A ratio of resistant to susceptible plantlets close to 50%, and the absence of aborted embryos were consistent with the hypothesis that the male gametophyte carrying both p5cs1 and p5cs2 alleles is rarely transmitted to the offspring. In addition, in reciprocal crosses with wild type, about 50% of the p5cs2 mutant alleles were transmitted to the sporophytic generation when p5cs1 p5cs2/P5CS2 was used as a female, while less than 1% of the p5cs2 alleles could be transmitted to the outcrossed progeny when p5cs1 p5cs2/P5CS2 was used as a male. Morphological and functional analysis of mutant pollen revealed a population of small, degenerated, and unviable pollen grains, indicating that the mutant homozygous for p5cs1 and heterozygous for p5cs2 is impaired in pollen development, and suggesting a role for proline in male gametophyte development. Consistent with these findings, we found that pollen from p5cs1 homozygous mutants, display defects similar to, but less pronounced than pollen from p5cs1 p5cs2/P5CS2 mutants. Finally, we show that pollen from p5cs1 p5cs2/P5CS2 plants contains less proline than wild type and that exogenous proline supplied from the beginning of another development can partially complement both morphological and functional pollen defects

  7. A Thermodynamic Model for Predicting Mineral Reactivity in Supercritical Carbon Dioxide: I. Phase Behavior of Carbon Dioxide - Water - Chloride Salt Systems Across the H2O-Rich to the CO2-Rich Regions

    SciTech Connect

    Springer, Ronald D.; Wang, Zheming; Anderko, Andre; Wang, Peiming; Felmy, Andrew R.

    2012-09-05

    Phase equilibria in mixtures containing carbon dioxide, water, and chloride salts have been investigated using a combination of solubility measurements and thermodynamic modeling. The solubility of water in the CO2-rich phase of ternary mixtures of CO2, H2O and NaCl or CaCl2 was determined, using near infrared spectroscopy, at 90 atm and 40 to 100 °C. These measurements fill a gap in the experimental database for CO2 water salt systems, for which phase composition data have been available only for the H2O-rich phases. A thermodynamic model for CO2 water salt systems has been constructed on the basis of the previously developed Mixed-Solvent Electrolyte (MSE) framework, which is capable of modeling aqueous solutions over broad ranges of temperature and pressure, is valid to high electrolyte concentrations, treats mixed-phase systems (with both scCO2 and water present) and can predict the thermodynamic properties of dry and partially water-saturated supercritical CO2 over broad ranges of temperature and pressure. Within the MSE framework the standard-state properties are calculated from the Helgeson-Kirkham-Flowers equation of state whereas the excess Gibbs energy includes a long-range electrostatic interaction term expressed by a Pitzer-Debye-Hückel equation, a virial coefficient-type term for interactions between ions and a short-range term for interactions involving neutral molecules. The parameters of the MSE model have been evaluated using literature data for both the H2O-rich and CO2-rich phases in the CO2 - H2O binary and for the H2O-rich phase in the CO2 - H2O - NaCl / KCl / CaCl2 / MgCl2 ternary and multicompontent systems. The model accurately represents the properties of these systems at temperatures from 0°C to 300 °C and pressures up to ~4000 atm. Further, the solubilities of H2O in CO2-rich phases that are predicted by the model are in agreement with the new measurements for the CO2 - H2O - NaCl and CO2 - H2O - CaCl2 systems. Thus, the model can be

  8. Proline biosynthesis is required for endoplasmic reticulum stress tolerance in Saccharomyces cerevisiae.

    PubMed

    Liang, Xinwen; Dickman, Martin B; Becker, Donald F

    2014-10-03

    The amino acid proline is uniquely involved in cellular processes that underlie stress response in a variety of organisms. Proline is known to minimize protein aggregation, but a detailed study of how proline impacts cell survival during accumulation of misfolded proteins in the endoplasmic reticulum (ER) has not been performed. To address this we examined in Saccharomyces cerevisiae the effect of knocking out the PRO1, PRO2, and PRO3 genes responsible for proline biosynthesis. The null mutants pro1, pro2, and pro3 were shown to have increased sensitivity to ER stress relative to wild-type cells, which could be restored by proline or the corresponding genetic complementation. Of these mutants, pro3 was the most sensitive to tunicamycin and was rescued by anaerobic growth conditions or reduced thiol reagents. The pro3 mutant cells have higher intracellular reactive oxygen species, total glutathione, and a NADP(+)/NADPH ratio than wild-type cells under limiting proline conditions. Depletion of proline biosynthesis also inhibits the unfolded protein response (UPR) indicating proline protection involves the UPR. To more broadly test the role of proline in ER stress, increased proline biosynthesis was shown to partially rescue the ER stress sensitivity of a hog1 null mutant in which the high osmolality pathway is disrupted.

  9. Remelting of refractory inclusions in the chondrule-forming regions: Evidence from chondrule-bearing type C calcium-aluminum-rich inclusions from Allende

    NASA Astrophysics Data System (ADS)

    Krot, Alexander N.; Yurimoto, Hisayoshi; Hutcheon, Ian D.; Chaussidon, Marc; MacPherson, Glenn J.; Paque, Julie

    2007-08-01

    We describe the mineralogy, petrology, oxygen, and magnesium isotope compositions of three coarse-grained, igneous, anorthite-rich (type C) Ca-Al-rich inclusions (CAIs) (ABC, TS26, and 93) that are associated with ferromagnesian chondrule-like silicate materials from the CV carbonaceous chondrite Allende. The CAIs consist of lath-shaped anorthite (An99), Cr-bearing Al-Ti-diopside (Al and Ti contents are highly variable), spinel, and highly åkermanitic and Na-rich melilite (Åk63-74, 0.4-0.6 wt% Na2O). TS26 and 93 lack Wark-Lovering rim layers; ABC is a CAI fragment missing the outermost part. The peripheral portions of TS26 and ABC are enriched in SiO2 and depleted in TiO2 and Al2O3 compared to their cores and contain relict ferromagnesian chondrule fragments composed of forsteritic olivine (Fa6-8) and low-Ca pyroxene/pigeonite (Fs1Wo1-9). The relict grains are corroded by Al-Ti-diopside of the host CAIs and surrounded by haloes of augite (Fs0.5Wo30-42). The outer portion of CAI 93 enriched in spinel is overgrown by coarse-grained pigeonite (Fs0.5-2Wo5-17), augite (Fs0.5Wo38-42), and anorthitic plagioclase (An84). Relict olivine and low-Ca pyroxene/pigeonite in ABC and TS26, and the pigeonite-augite rim around 93 are 16O-poor (Δ17O ˜ -1‰ to -8‰). Spinel and Al-Ti-diopside in cores of CAIs ABC, TS26, and 93 are 16O-enriched (Δ17O down to -20‰), whereas Al-Ti-diopside in the outer zones, as well as melilite and anorthite, are 16O-depleted to various degrees (Δ17O = -11‰ to 2‰). In contrast to typical Allende CAIs that have the canonical initial 26Al/27Al ratio of ˜5 × 10-5 ABC, 93, and TS26 are 26Al-poor with (26Al/27Al)0 ratios of (4.7 ± 1.4) × 10-6 (1.5 ± 1.8) × 10-6 <1.2 × 10-6 respectively. We conclude that ABC, TS26, and 93 experienced remelting with addition of ferromagnesian chondrule silicates and incomplete oxygen isotopic exchange in an 16O-poor gaseous reservoir, probably in the chondrule-forming region. This melting episode could

  10. An N-terminal glycine-rich sequence contributes to retrovirus trimer of hairpins stability

    SciTech Connect

    Wilson, Kirilee A.; Maerz, Anne L.; Baer, Severine; Drummer, Heidi E.; Poumbourios, Pantelis . E-mail: apoumbourios@burnet.edu.au

    2007-08-10

    Retroviral transmembrane proteins (TMs) contain a glycine-rich segment linking the N-terminal fusion peptide and coiled coil core. Previously, we reported that the glycine-rich segment (Met-326-Ser-337) of the human T-cell leukemia virus type 1 (HTLV-1) TM, gp21, is a determinant of membrane fusion function [K.A. Wilson, S. Baer, A.L. Maerz, M. Alizon, P. Poumbourios, The conserved glycine-rich segment linking the N-terminal fusion peptide to the coiled coil of human T-cell leukemia virus type 1 transmembrane glycoprotein gp21 is a determinant of membrane fusion function, J. Virol. 79 (2005) 4533-4539]. Here we show that the reduced fusion activity of an I334A mutant correlated with a decrease in stability of the gp21 trimer of hairpins conformation, in the context of a maltose-binding protein-gp21 chimera. The stabilizing influence of Ile-334 required the C-terminal membrane-proximal sequence Trp-431-Ser-436. Proline substitution of four of five Gly residues altered gp21 trimer of hairpins stability. Our data indicate that flexibility within and hydrophobic interactions mediated by this region are determinants of gp21 stability and membrane fusion function.

  11. Proline accumulation and metabolism-related genes expression profiles in Kosteletzkya virginica seedlings under salt stress

    PubMed Central

    Wang, Hongyan; Tang, Xiaoli; Wang, Honglei; Shao, Hong-Bo

    2015-01-01

    Proline accumulation is a common response to salt stress in many plants. Salt stress also increased proline concentration in roots, stems, and leaves of Kosteletzkya virginica seedling treated with 300 mM NaCl for 24 h and reached 3.75-, 4.76-, and 6.83-fold higher than controls. Further study on proline content in leaves under salt stress showed that proline content increased with increasing NaCl concentrations or time. The proline level peaked at 300 mM NaCl for 24 h and reached more than sixfold higher than control, but at 400 mM NaCl for 24 h proline content fell back slightly along with wilting symptom. To explore the cause behind proline accumulation, we first cloned full length genes related to proline metabolism including KvP5CS1, KvOAT, KvPDH, and KvProT from K. virginica and investigated their expression profiles. The results revealed that the expressions of KvP5CS1 and KvProT were sharply up-regulated by salt stress and the expression of KvOAT showed a slight increase with increasing salt concentrations or time, while the expression of KvPDH was not changed much and slightly decreased before 12 h and then returned to the original level. As the key enzyme genes for proline biosynthesis, the up-regulated expression of KvP5CS1 played a more important role than KvOAT for proline accumulation in leaves under salt stress. The low expression of KvPDH for proline catabolism also made a contribution to proline accumulation before 12 h. PMID:26483809

  12. Sources of superoxide/H2O2 during mitochondrial proline oxidation.

    PubMed

    Goncalves, Renata L S; Rothschild, Daniel E; Quinlan, Casey L; Scott, Gary K; Benz, Christopher C; Brand, Martin D

    2014-01-01

    p53 Inducible gene 6 (PIG6) encodes mitochondrial proline dehydrogenase (PRODH) and is up-regulated several fold upon p53 activation. Proline dehydrogenase is proposed to generate radicals that contribute to cancer cell apoptosis. However, there are at least 10 mitochondrial sites that can produce superoxide and/or H2O2, and it is unclear whether proline dehydrogenase generates these species directly, or instead drives production by other sites. Amongst six cancer cell lines, ZR75-30 human breast cancer cells had the highest basal proline dehydrogenase levels, and mitochondria isolated from ZR75-30 cells consumed oxygen and produced H2O2 with proline as sole substrate. Insects use proline oxidation to fuel flight, and mitochondria isolated from Drosophila melanogaster were even more active with proline as sole substrate than ZR75-30 mitochondria. Using mitochondria from these two models we identified the sites involved in formation of superoxide/H2O2 during proline oxidation. In mitochondria from Drosophila the main sites were respiratory complexes I and II. In mitochondria from ZR75-30 breast cancer cells the main sites were complex I and the oxoglutarate dehydrogenase complex. Even with combinations of substrates and respiratory chain inhibitors designed to minimize the contributions of other sites and maximize any superoxide/H2O2 production from proline dehydrogenase itself, there was no significant direct contribution of proline dehydrogenase to the observed H2O2 production. Thus proline oxidation by proline dehydrogenase drives superoxide/H2O2 production, but it does so mainly or exclusively by providing anaplerotic carbon for other mitochondrial dehydrogenases and not by producing superoxide/H2O2 directly.

  13. Determination of proline in honey: comparison between official methods, optimization and validation of the analytical methodology.

    PubMed

    Truzzi, Cristina; Annibaldi, Anna; Illuminati, Silvia; Finale, Carolina; Scarponi, Giuseppe

    2014-05-01

    The study compares official spectrophotometric methods for the determination of proline content in honey - those of the International Honey Commission (IHC) and the Association of Official Analytical Chemists (AOAC) - with the original Ough method. Results show that the extra time-consuming treatment stages added by the IHC method with respect to the Ough method are pointless. We demonstrate that the AOACs method proves to be the best in terms of accuracy and time saving. The optimized waiting time for the absorbance recording is set at 35min from the removal of reaction tubes from the boiling bath used in the sample treatment. The optimized method was validated in the matrix: linearity up to 1800mgL(-1), limit of detection 20mgL(-1), limit of quantification 61mgL(-1). The method was applied to 43 unifloral honey samples from the Marche region, Italy.

  14. Regulation of proline metabolism in mycobacteria and its role in carbon metabolism under hypoxia.

    PubMed

    Berney, Michael; Weimar, Marion R; Heikal, Adam; Cook, Gregory M

    2012-05-01

    Genes with a role in proline metabolism are strongly expressed when mycobacterial cells are exposed to nutrient starvation and hypoxia. Here we show that proline metabolism in mycobacteria is mediated by the monofunctional enzymes Δ(1) -pyrroline-5-carboxylate dehydrogenase (PruA) and proline dehydrogenase (PruB). Proline metabolism was controlled by a unique membrane-associated DNA-binding protein PruC. Under hypoxia, addition of proline led to higher biomass production than in the absence of proline despite excess carbon and nitrogen. To identify the mechanism responsible for this enhanced growth, microarray analysis of wild-type Mycobacterium smegmatis versus pruC mutant was performed. Expression of the DNA repair machinery and glyoxalases was increased in the pruC mutant. Glyoxalases are proposed to degrade methylglyoxal, a toxic metabolite produced by various bacteria due to an imbalance in intermediary metabolism, suggesting the pruC mutant was under methylglyoxal stress. Consistent with this notion, pruB and pruC mutants were hypersensitive to methylglyoxal. Δ(1) -pyrroline-5-carboxylate is reported to react with methylglyoxal to form non-toxic 2-acetyl-1-pyrroline, thus providing a link between proline metabolism and methylglyoxal detoxification. In support of this mechanism, we show that proline metabolism protects mycobacterial cells from methylglyoxal toxicity and that functional proline dehydrogenase, but not Δ(1) -pyrroline-5-carboxylate dehydrogenase, is essential for this protective effect.

  15. Safety of L-proline as a stabilizer for immunoglobulin products.

    PubMed

    Hagan, John B; Wasserman, Richard L; Baggish, Jeffrey S; Spycher, Martin O; Berger, Melvin; Shashi, Vandana; Lohrmann, Emanuel; Sullivan, Kathleen E

    2012-02-01

    Privigen(®) (immune globulin intravenous [human], 10% liquid) and Hizentra(®) (immune globulin subcutaneous [human], 20% liquid) are stabilized by proline. The clinical implications of administering proline-containing immunoglobulin products to patients with defects of proline metabolism have not been addressed; Privigen and Hizentra are contraindicated in these patients. Some patients with chromosome 22q11.2 deletion syndrome have elevated proline levels; however, only 3-4% of patients also have an immunodeficiency that requires IgG therapy. This review summarizes the evidence related to the safety and pharmacokinetics of proline assessed in Privigen and Hizentra preclinical and clinical studies, and subsequent implications for patients with defects in proline metabolism. Clinical data indicate that proline does not accumulate after Privigen or Hizentra treatment and is not associated with adverse events. There is no evidence to suggest that patients with defects of proline metabolism would be affected by transient elevations in plasma proline following Privigen and/or Hizentra treatment.

  16. Relationship between proline and Hg2+-induced oxidative stress in a tolerant rice mutant.

    PubMed

    Wang, Feijuan; Zeng, Bin; Sun, Zongxiu; Zhu, Cheng

    2009-05-01

    There has been little agreement regarding the mechanism by which proline reduces heavy metal stress. The present work examines the relationship between Hg(2+)-induced oxidative stress and proline accumulation in rice and explores the possible mechanisms through which proline protects against Hg(2+) stress. The effect of proline on alleviation of Hg(2+) toxicity was studied by spectrophotography and enzymatic methods. Hg(2+) induced oxidative stress in rice by increasing lipid peroxidation. Pretreatment of the rice with 2 mM proline for 12 h profoundly alleviated Hg(2+)-induced lipid peroxidation and minimized H(2)O(2) accumulation. Proline pretreatment significantly reduced (p < 0.01) the Hg(2+) content in rice leaves. A comparison of the effects of proline pretreatment on H(2)O(2) accumulation by Hg(2+) and aminotrazole suggested that proline protected cells from Hg(2+)-induced oxidative stress by scavenging reactive oxygen species. The present work demonstrates a protective effect of proline on Hg(2+) toxicity through detoxifying reactive oxygen species, rather than chelating metal ions or maintaining the water balance under Hg(2+) stress.

  17. Nitrogen availability regulates proline and ethylene production and alleviates salinity stress in mustard (Brassica juncea).

    PubMed

    Iqbal, Noushina; Umar, Shahid; Khan, Nafees A

    2015-04-15

    Proline content and ethylene production have been shown to be involved in salt tolerance mechanisms in plants. To assess the role of nitrogen (N) in the protection of photosynthesis under salt stress, the effect of N (0, 5, 10, 20 mM) on proline and ethylene was studied in mustard (Brassica juncea). Sufficient N (10 mM) optimized proline production under non-saline conditions through an increase in proline-metabolizing enzymes, leading to osmotic balance and protection of photosynthesis through optimal ethylene production. Excess N (20 mM), in the absence of salt stress, inhibited photosynthesis and caused higher ethylene evolution but lower proline production compared to sufficient N. In contrast, under salt stress with an increased demand for N, excess N optimized ethylene production, which regulates the proline content resulting in recovered photosynthesis. The effect of excess N on photosynthesis under salt stress was further substantiated by the application of the ethylene biosynthesis inhibitor, 1-aminoethoxy vinylglycine (AVG), which inhibited proline production and photosynthesis. Without salt stress, AVG promoted photosynthesis in plants receiving excess N by inhibiting stress ethylene production. The results suggest that a regulatory interaction exists between ethylene, proline and N for salt tolerance. Nitrogen differentially regulates proline production and ethylene formation to alleviate the adverse effect of salinity on photosynthesis in mustard.

  18. Expression of beta-keratin mRNAs and proline uptake in epidermal cells of growing scales and pad lamellae of gecko lizards.

    PubMed

    Alibardi, Lorenzo; Toni, Mattia; Dalla Valle, Luisa

    2007-07-01

    Beta-keratins form a large part of the proteins contained in the hard beta layer of reptilian scales. The expression of genes encoding glycine-proline-rich beta-keratins in normal and regenerating epidermis of two species of gecko lizards has been studied by in situ hybridization. The probes localize mRNAs in differentiating oberhautchen and beta cells of growing scales and in modified scales, termed pad lamellae, on the digits of gecko lizards. In situ localization at the ultrastructural level shows clusters of gold particles in the cytoplasm among beta-keratin filaments of oberhautchen and beta cells. They are also present in the differentiating elongation or setae of oberhautchen cells present in pad lamellae. Setae allow geckos to adhere and climb vertical surfaces. Oberhautchen and beta cells also incorporate tritiated proline. The fine localization of the beta-keratin mRNAs and the uptake of proline confirms the biomolecular data that identified glycine-proline-rich beta-keratin in differentiating beta cells of gecko epidermis. The present study also shows the presence of differentiating and metabolically active cells in both inner and outer oberhautchen/beta cells at the base of the outer setae localized at the tip of pad lamellae. The addition of new beta and alpha cells to the corneous layer near the tip of the outer setae explains the anterior movement of the setae along the apical free-margin of pad lamellae. The rapid replacement of setae ensures the continuous usage of the gecko's adhesive devices, the pad lamellae, during most of their active life.

  19. Synthesis, spectral characterization and biological studies of some organotin(IV) complexes of L-proline, trans-hydroxy- L-proline and L-glutamine

    NASA Astrophysics Data System (ADS)

    Nath, Mala; Jairath, Ruchi; Eng, George; Song, Xueqing; Kumar, Ashok

    2005-12-01

    New organotin(IV) complexes of the general formula R 3Sn(L) (where R = Me, n-Bu and HL = L-proline; R = Me, Ph and HL = trans-hydroxy- L-proline and L-glutamine) and R 2Sn(L) 2 (where R = n-Bu, Ph and HL = L-proline; R = Ph, HL = trans-hydroxy- L-proline) have been synthesized by the reaction of R nSnCl 4- n (where n = 2 or 3) with sodium salt of the amino acid (HL). n-Bu 2Sn(Pro) 2 was synthesized by the reaction of n-Bu 2SnO with L-proline under azeotropic removal of water. The bonding and coordination behavior in these complexes have been discussed on the basis of IR and 119Sn Mössbauer spectroscopic studies in the solid-state. Their coordination behavior in solution has been discussed with the help of multinuclear ( 1H, 13C and 119Sn) NMR spectral studies. The 119Sn Mössbauer and IR studies indicate that L-proline and trans-hydroxy- L-proline show similar coordination behavior towards organotin(IV) compounds. Pentacoordinate trigonal-bipyramidal and hexacoordinate octahedral structures, respectively, have been proposed for the tri- and diorganotin(IV) complexes of L-proline and trans-hydroxy- L-proline, in which the carboxylate group acts as bidentate group. L-Glutamine shows different coordination behavior towards organotin(IV) compounds, it acts as monoanionic bidentate ligand coordinating through carboxylate and amino group. The triorganotin(IV) complexes of L-glutamine have been proposed to have trigonal-bipyramidal environment around tin. The newly synthesized complexes have been tested for their antiinflammatory and cardiovascular activities. Their LD 50 values are >1000 mg kg -1.

  20. Growth of plutons by incremental emplacement of sheets in crystal-rich host: Evidence from Miocene intrusions of the Colorado River region, Nevada, USA

    USGS Publications Warehouse

    Miller, C.F.; Furbish, D.J.; Walker, B.A.; Claiborne, L.L.; Koteas, G.C.; Bleick, H.A.; Miller, J.S.

    2011-01-01

    Growing evidence supports the notion that plutons are constructed incrementally, commonly over long periods of time, yet field evidence for the multiple injections that seem to be required is commonly sparse or absent. Timescales of up to several million years, among other arguments, indicate that the dominant volume does not remain largely molten, yet if growing plutons are constructed from rapidly solidifying increments it is unlikely that intrusive contacts would escape notice. A model wherein magma increments are emplaced into melt-bearing but crystal-rich host, rather than either solid or crystal-poor material, provides a plausible explanation for this apparent conundrum. A partially solidified intrusion undoubtedly comprises zones with contrasting melt fraction and therefore strength. Depending on whether these zones behave elastically or ductilely in response to dike emplacement, intruding magma may spread to form sheets by either of two mechanisms. If the melt-bearing host is elastic on the relevant timescale, magma spreads rather than continuing to propagate upward, where it encounters a zone of higher rigidity (higher crystal fraction). Similarly, if the dike at first ascends through rigid, melt-poor material and then encounters a zone that is weak enough (poor enough in crystals) to respond ductilely, the ascending material will also spread because the dike tip ceases to propagate as in rigid material. We propose that ascending magma is thus in essence trapped, by either mechanism, within relatively crystal-poor zones. Contacts will commonly be obscure from the start because the contrast between intruding material (crystal-poorer magma) and host (crystal-richer material) is subtle, and they may be obscured even further by subsequent destabilization of the crystal-melt framework. Field evidence and zircon zoning stratigraphy in plutons of the Colorado River region of southern Nevada support the hypothesis that emplacement of magma replenishments into a

  1. Conformational preferences of substituted prolines in the collagen triple helix.

    PubMed

    Mooney, Sean D; Kollman, Peter A; Klein, Teri E

    2002-07-05

    Researchers have recently questioned the role hydroxylated prolines play in stabilizing the collagen triple helix. To address these issues, we have developed new molecular mechanics parameters for the simulation of peptides containing 4(R)-fluoroproline (Flp), 4(R)-hydroxyproline (Hyp), and 4(R)-aminoproline (Amp). Simulations of peptides based on these parameters can be used to determine the components that stabilize hydroxyproline over proline in the triple helix. The dihedrals F-C-C-N, O-C-C-N, and N-C-C-N were built using a N-beta-ethyl amide model. One nanosecond simulations were performed on the trimers [(Pro-Pro-Gly)(10)](3), [(Pro-Hyp-Gly)(10)](3), [(Pro-Amp-Gly)(10)](3), [(Pro-Amp(1+)-Gly)(10)](3), and [(Pro-Flp-Gly)(10)](3) in explicit solvent. The results of our simulations suggest that pyrrolidine ring conformation is mediated by the strength of the gauche effect and classical electrostatic interactions.

  2. A HuD-ZBP1 ribonucleoprotein complex localizes GAP-43 mRNA into axons through its 3' untranslated region AU-rich regulatory element.

    PubMed

    Yoo, Soonmoon; Kim, Hak H; Kim, Paul; Donnelly, Christopher J; Kalinski, Ashley L; Vuppalanchi, Deepika; Park, Michael; Lee, Seung J; Merianda, Tanuja T; Perrone-Bizzozero, Nora I; Twiss, Jeffery L

    2013-09-01

    Localized translation of axonal mRNAs contributes to developmental and regenerative axon growth. Although untranslated regions (UTRs) of many different axonal mRNAs appear to drive their localization, there has been no consensus RNA structure responsible for this localization. We recently showed that limited expression of ZBP1 protein restricts axonal localization of both β-actin and GAP-43 mRNAs. β-actin 3'UTR has a defined element for interaction with ZBP1, but GAP-43 mRNA shows no homology to this RNA sequence. Here, we show that an AU-rich regulatory element (ARE) in GAP-43's 3'UTR is necessary and sufficient for its axonal localization. Axonal GAP-43 mRNA levels increase after in vivo injury, and GAP-43 mRNA shows an increased half-life in regenerating axons. GAP-43 mRNA interacts with both HuD and ZBP1, and HuD and ZBP1 co-immunoprecipitate in an RNA-dependent fashion. Reporter mRNA with the GAP-43 ARE competes with endogenous β-actin mRNA for axonal localization and decreases axon length and branching similar to the β-actin 3'UTR competing with endogenous GAP-43 mRNA. Conversely, over-expressing GAP-43 coding sequence with its 3'UTR ARE increases axonal elongation and this effect is lost when just the ARE is deleted from GAP-43's 3'UTR. We have recently found that over-expression of GAP-43 using an axonally targeted construct with the 3'UTRs of GAP-43 promoted elongating growth of axons, while restricting the mRNA to the cell body with the 3'UTR of γ-actin had minimal effect on axon length. In this study, we show that the ARE in GAP-43's 3'UTR is responsible for localization of GAP-43 mRNA into axons and is sufficient for GAP-43 protein's role in elongating axonal growth.

  3. Proline metabolism increases katG expression and oxidative stress resistance in Escherichia coli.

    PubMed

    Zhang, Lu; Alfano, James R; Becker, Donald F

    2015-02-01

    The oxidation of l-proline to glutamate in Gram-negative bacteria is catalyzed by the proline utilization A (PutA) flavoenzyme, which contains proline dehydrogenase (PRODH) and Δ(1)-pyrroline-5-carboxylate (P5C) dehydrogenase domains in a single polypeptide. Previous studies have suggested that aside from providing energy, proline metabolism influences oxidative stress resistance in different organisms. To explore this potential role and the mechanism, we characterized the oxidative stress resistance of wild-type and putA mutant strains of Escherichia coli. Initial stress assays revealed that the putA mutant strain was significantly more sensitive to oxidative stress than the parental wild-type strain. Expression of PutA in the putA mutant strain restored oxidative stress resistance, confirming that depletion of PutA was responsible for the oxidative stress phenotype. Treatment of wild-type cells with proline significantly increased hydroperoxidase I (encoded by katG) expression and activity. Furthermore, the ΔkatG strain failed to respond to proline, indicating a critical role for hydroperoxidase I in the mechanism of proline protection. The global regulator OxyR activates the expression of katG along with several other genes involved in oxidative stress defense. In addition to katG, proline increased the expression of grxA (glutaredoxin 1) and trxC (thioredoxin 2) of the OxyR regulon, implicating OxyR in proline protection. Proline oxidative metabolism was shown to generate hydrogen peroxide, indicating that proline increases oxidative stress tolerance in E. coli via a preadaptive effect involving endogenous hydrogen peroxide production and enhanced catalase-peroxidase activity.

  4. Enhanced thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 by rational engineering of a glycine to proline mutation.

    PubMed

    Tian, Jian; Wang, Ping; Gao, Shan; Chu, Xiaoyu; Wu, Ningfeng; Fan, Yunliu

    2010-12-01

    Protein thermostability can be increased by some glycine to proline mutations in a target protein. However, not all glycine to proline mutations can improve protein thermostability, and this method is suitable only at carefully selected mutation sites that can accommodate structural stabilization. In this study, homology modeling and molecular dynamics simulations were used to select appropriate glycine to proline mutations to improve protein thermostability, and the effect of the selected mutations was proved by the experiments. The structure of methyl parathion hydrolase (MPH) from Ochrobactrum sp. M231 (Ochr-MPH) was constructed by homology modeling, and molecular dynamics simulations were performed on the modeled structure. A profile of the root mean square fluctuations of Ochr-MPH was calculated at the nanosecond timescale, and an eight-amino acid loop region (residues 186-193) was identified as having high conformational fluctuation. The two glycines nearest to this region were selected as mutation targets that might affect protein flexibility in the vicinity. The structures and conformational fluctuations of two single mutants (G194P and G198P) and one double mutant (G194P/G198P) were modeled and analyzed using molecular dynamics simulations. The results predicted that the mutant G194P had the decreased conformational fluctuation in the loop region and might increase the thermostability of Ochr-MPH. The thermostability and kinetic behavior of the wild-type and three mutant enzymes were measured. The results were consistent with the computational predictions, and the mutant G194P was found to have higher thermostability than the wild-type enzyme.

  5. N-Acetylated Proline-Glycine-Proline Accelerates Cutaneous Wound Healing and Neovascularization by Human Endothelial Progenitor Cells

    PubMed Central

    Kwon, Yang Woo; Heo, Soon Chul; Lee, Tae Wook; Park, Gyu Tae; Yoon, Jung Won; Jang, Il Ho; Kim, Seung-Chul; Ko, Hyun-Chang; Ryu, Youngjae; Kang, Hyeona; Ha, Chang Man; Lee, Sang Chul; Kim, Jae Ho

    2017-01-01

    Human endothelial progenitor cells (hEPCs) are promising therapeutic resources for wound repair through stimulating neovascularization. However, the hEPCs-based cell therapy has been hampered by poor engraftment of transplanted cells. In this study, we explored the effects of N-acetylated Proline-Glycine-Proline (Ac-PGP), a degradation product of collagen, on hEPC-mediated cutaneous wound healing and neovascularization. Treatment of hEPCs with Ac-PGP increased migration, proliferation, and tube-forming activity of hEPCs in vitro. Knockdown of CXCR2 expression in hEPCs abrogated the stimulatory effects of Ac-PGP on migration and tube formation. In a cutaneous wound healing model of rats and mice, topical application of Ac-PGP accelerated cutaneous wound healing with promotion of neovascularization. The positive effects of Ac-PGP on wound healing and neovascularization were blocked in CXCR2 knockout mice. In nude mice, the individual application of Ac-PGP treatment or hEPC injection accelerated wound healing by increasing neovascularization. Moreover, the combination of Ac-PGP treatment and hEPC injection further stimulated wound healing and neovascularization. Topical administration of Ac-PGP onto wound bed stimulated migration and engraftment of transplanted hEPCs into cutaneous dermal wounds. Therefore, these results suggest novel applications of Ac-PGP in promoting wound healing and augmenting the therapeutic efficacy of hEPCs. PMID:28230162

  6. Sonochemical asymmetric hydrogenation of isophorone on proline modified Pd/Al2O3 catalysts.

    PubMed

    Mhadgut, Shilpa C; Bucsi, Imre; Török, Marianna; Török, Béla

    2004-04-21

    The sonochemical asymmetric hydrogenation of isophorone (3,3,5-trimethyl-2-cyclohexenone) by proline-modified Pd/Al2O3 catalysts is described; presonication of a commercial Pd/Al2O3-proline catalytic system resulted in highly enhanced enantioselectivities (up to 85% ee).

  7. Localization of two post-proline cleaving peptidases in the midgut of Tenebrio molitor larvae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two soluble post-proline cleaving peptidase activities, PPCP1 and PPCP2, were demonstrated in the midgut of Tenebrio molitor larvae with the substrate benzyloxycarbonyl-L-alanyl-L-proline p-nitroanilide. Both activities were serine peptidases. PPCP1 was active in acidic buffers, with maximum activit...

  8. Proline induces calcium-mediated oxidative burst and salicylic acid signaling.

    PubMed

    Chen, Jiugeng; Zhang, Yueqin; Wang, Cuiping; Lü, Weitao; Jin, Jing Bo; Hua, Xuejun

    2011-05-01

    Although free proline accumulation is a well-documented phenomenon in many plants in response to a variety of environmental stresses, and is proposed to play protective roles, high intracellular proline content, by either exogenous application or endogenous over-production, in the absence of stresses, is found to be inhibitory to plant growth. We have shown here that exogenous application of proline significantly induced intracellular Ca(2+) accumulation in tobacco and calcium-dependent ROS production in Arabidopsis seedlings, which subsequently enhanced salicylic acid (SA) synthesis and PR genes expression. This suggested that proline can promote a reaction similar to hypersensitive response during pathogen infection. Other amino acids, such as glutamate, but not arginine and phenylalanine, were also found to be capable of inducing PR gene expression. In addition, proline at concentration as low as 0.5 mM could induce PR gene expression. However, proline could not induce the expression of PDF1.2 gene, the marker gene for jasmonic acid signaling pathway. Furthermore, proline-induced SA production is mediated by NDR1-dependent signaling pathway, but not that mediated by PAD4. Our data provide evidences that exogenous proline, and probably some other amino acids can specifically induce SA signaling and defense response.

  9. Hydrogen sulfide and proline cooperate to alleviate cadmium stress in foxtail millet seedlings.

    PubMed

    Tian, Baohua; Qiao, Zengjie; Zhang, Liping; Li, Hua; Pei, Yanxi

    2016-12-01

    Hydrogen sulfide (H2S) and some functional amino acids in crops have been involved in the defense system against heavy-metal pollution. Here we report the relationships and functions of H2S and proline to cadmium (Cd) stress. Sodium hydrosulfide (NaHS) pretreatment decreased the electrolytic leakage and the malondialdehyde and hydrogen peroxide contents while enhancing photosynthesis in Cd-treated seedlings. Furthermore, pretreatment with NaHS markedly exacerbated Cd-induced alterations in proline content, the activities of proline-5-carboxylate reductase (P5CR) and proline dehydrogenase (PDH), and the transcript levels of P5CR and PDH. When endogenous H2S was scavenged or inhibited by various H2S modulators, the Cd-induced increase in endogenous proline was weakened. Combined pretreatment with H2S and proline was moderately higher in the Cd-stressed growth status, stomata movements and oxidative damage of seedlings compared to a single treatment with H2S or proline. These results suggest that H2S and proline cooperate to alleviate Cd-damage in foxtail millet.

  10. Proteomic analysis reveals diverse proline hydroxylation-mediated oxygen-sensing cellular pathways in cancer cells

    PubMed Central

    Liu, Bing; Gao, Yankun; Ruan, Hai-Bin; Chen, Yue

    2016-01-01

    Proline hydroxylation is a critical cellular mechanism regulating oxygen-response pathways in tumor initiation and progression. Yet, its substrate diversity and functions remain largely unknown. Here, we report a system-wide analysis to characterize proline hydroxylation substrates in cancer cells using an immunoaffinity-purification assisted proteomics strategy. We identified 562 sites from 272 proteins in HeLa cells. Bioinformatic analysis revealed that proline hydroxylation substrates are significantly enriched with mRNA processing and stress-response cellular pathways with canonical and diverse flanking sequence motifs. Structural analysis indicates a significant enrichment of proline hydroxylation participating in the secondary structure of substrate proteins. Our study identified and validated Brd4, a key transcription factor, as a novel proline hydroxylation substrate. Functional analysis showed that the inhibition of proline hydroxylation pathway significantly reduced the proline hydroxylation abundance on Brd4 and affected Brd4-mediated transcriptional activity as well as cell proliferation in AML leukemia cells. Taken together, our study identified a broad regulatory role of proline hydroxylation in cellular oxygen-sensing pathways and revealed potentially new targets that dynamically respond to hypoxia microenvironment in tumor cells. PMID:27764789

  11. Proline biosynthesis augments tumor cell growth and aerobic glycolysis: involvement of pyridine nucleotides.

    PubMed

    Liu, Wei; Hancock, Chad N; Fischer, Joseph W; Harman, Meredith; Phang, James M

    2015-11-24

    The metabolism of the nonessential amino acid proline contributes to tumor metabolic reprogramming. Previously we showed that MYC increases proline biosynthesis (PB) from glutamine. Here we show MYC increases the expression of the enzymes in PB at both protein and mRNA levels. Blockade of PB decreases tumor cell growth and energy production. Addition of Δ(1)-pyrroline-5-carboxylate (P5C) or proline reverses the effects of P5C synthase knockdown but not P5C reductases knockdown. Importantly, the reversal effect of proline was blocked by concomitant proline dehydrogenase/oxidase (PRODH/POX) knockdown. These findings suggest that the important regulatory contribution of PB to tumor growth derives from metabolic cycling between proline and P5C rather than product proline or intermediate P5C. We further document the critical role of PB in maintaining pyridine nucleotide levels by connecting the proline cycle to glycolysis and to the oxidative arm of the pentose phosphate pathway. These findings establish a novel function of PB in tumorigenesis, linking the reprogramming of glucose, glutamine and pyridine nucleotides, and may provide a novel target for antitumor therapy.

  12. A Low T, High RH, and Potentially Life-Friendly Environment Within the Martian Salt-Rich Subsurface in Equatorial Regions

    NASA Astrophysics Data System (ADS)

    Wang, A.; Zheng, M. P.; Kong, F. J.; Ling, Z. C.; Kong, W. G.; Sobron, P.; Jolliff, B. L.

    2011-03-01

    Hydrated sulfates found in the subsurface at Gusev and at saline playa on the Tibet Plateau imply a high RH environment, supported by lab studies. A high-RH, salt-rich subsurface can accommodate organisms, e.g., halophiles in the Tibet saline playa.

  13. Solvent and conformation dependence of amide I vibrations in peptides and proteins containing proline.

    PubMed

    Roy, Santanu; Lessing, Joshua; Meisl, Georg; Ganim, Ziad; Tokmakoff, Andrei; Knoester, Jasper; Jansen, Thomas L C

    2011-12-21

    We present a mixed quantum-classical model for studying the amide I vibrational dynamics (predominantly CO stretching) in peptides and proteins containing proline. There are existing models developed for determining frequencies of and couplings between the secondary amide units. However, these are not applicable to proline because this amino acid has a tertiary amide unit. Therefore, a new parametrization is required for infrared-spectroscopic studies of proteins that contain proline, such as collagen, the most abundant protein in humans and animals. Here, we construct the electrostatic and dihedral maps accounting for solvent and conformation effects on frequency and coupling for the proline unit. We examine the quality and the applicability of these maps by carrying out spectral simulations of a number of peptides with proline in D(2)O and compare with experimental observations.

  14. Evaluation of potential cationic probes for the detection of proline and betaine.

    PubMed

    Kalsoom, Umme; Breadmore, Michael C; Guijt, Rosanne M; Boyce, Mary C

    2014-12-01

    Osmoregulants are the substances that help plants to tolerate environmental extremes such as salinity and drought. Proline and betaine are two of the most commonly studied osmoregulants. An indirect UV CE method has been developed for simultaneous determination of these osmoregulants. A variety of reported probes and compounds were examined as potential probes for the indirect detection of proline and betaine. Mobility and UV-absorption properties highlighted sulfanilamide as a potential probe for indirect analysis of proline and betaine. Using 5 mM sulfanilamide at pH 2.2 with UV detection at 254 nm, proline and betaine were separated in less than 15 min. The LODs for proline and betaine were 11.6 and 28.3 μM, respectively. The developed method was successfully applied to quantification of these two osmoregulants in spinach and beetroot samples.

  15. Effect of l-Proline on Sake Brewing and Ethanol Stress in Saccharomyces cerevisiae

    PubMed Central

    Takagi, Hiroshi; Takaoka, Miki; Kawaguchi, Akari; Kubo, Yoshito

    2005-01-01

    During the fermentation of sake, cells of Saccharomyces cerevisiae are exposed to high concentrations of ethanol, thereby damaging the cell membrane and functional proteins. l-Proline protects yeast cells from damage caused by freezing or oxidative stress. In this study, we evaluated the role of intracellular l-proline in cells of S. cerevisiae grown under ethanol stress. An l-proline-accumulating laboratory strain carries a mutant allele of PRO1, pro1D154N, which encodes the Asp154Asn mutant γ-glutamyl kinase. This mutation increases the activity of γ-glutamyl kinase and γ-glutamyl phosphate reductase, which catalyze the first two steps of l-proline synthesis and which together may form a complex in vivo. When cultured in liquid medium in the presence of 9% and 18% ethanol under static conditions, the cell viability of the l-proline-accumulating laboratory strain is greater than the cell viability of the parent strain. This result suggests that intracellular accumulation of l-proline may confer tolerance to ethanol stress. We constructed a novel sake yeast strain by disrupting the PUT1 gene, which is required for l-proline utilization, and replacing the wild-type PRO1 allele with the pro1D154N allele. The resultant strain accumulated l-proline and was more tolerant to ethanol stress than was the control strain. We used the strain that could accumulate l-proline to brew sake containing five times more l-proline than what is found in sake brewed with the control strain, without affecting the fermentation profiles. PMID:16332860

  16. Synthesis, Release, and Recapture of Compatible Solute Proline by Osmotically Stressed Bacillus subtilis Cells

    PubMed Central

    Hoffmann, Tamara; von Blohn, Carsten; Stanek, Agnieszka; Moses, Susanne; Barzantny, Helena

    2012-01-01

    Bacillus subtilis synthesizes large amounts of the compatible solute proline as a cellular defense against high osmolarity to ensure a physiologically appropriate level of hydration of the cytoplasm and turgor. It also imports proline for this purpose via the osmotically inducible OpuE transport system. Unexpectedly, an opuE mutant was at a strong growth disadvantage in high-salinity minimal media lacking proline. Appreciable amounts of proline were detected in the culture supernatant of the opuE mutant strain, and they rose concomitantly with increases in the external salinity. We found that the intracellular proline pool of severely salinity-stressed cells of the opuE mutant was considerably lower than that of its opuE+ parent strain. This loss of proline into the medium and the resulting decrease in the intracellular proline content provide a rational explanation for the observed salt-sensitive growth phenotype of cells lacking OpuE. None of the known MscL- and MscS-type mechanosensitive channels of B. subtilis participated in the release of proline under permanently imposed high-salinity growth conditions. The data reported here show that the OpuE transporter not only possesses the previously reported role for the scavenging of exogenously provided proline as an osmoprotectant but also functions as a physiologically highly important recapturing device for proline that is synthesized de novo and subsequently released by salt-stressed B. subtilis cells. The wider implications of our findings for the retention of compatible solutes by osmotically challenged microorganisms and the roles of uptake systems for compatible solutes are considered. PMID:22685134

  17. Identification of highly deformed even-even nuclei in the neutron- and proton-rich regions of the nuclear chart from the B(E2)↑ and E2 predictions in the generalized differential equation model

    NASA Astrophysics Data System (ADS)

    Nayak, R. C.; Pattnaik, S.

    2015-11-01

    We identify here the possible occurrence of large deformations in the neutron- and proton-rich (n-rich and p-rich) regions of the nuclear chart from extensive predictions of the values of the reduced quadrupole transition probability B(E2)↑ for the transition from the ground state to the first 2+ state and the corresponding excitation energy E2 of even-even nuclei in the recently developed generalized differential equation (GDE) model exclusively meant for these physical quantities. This is made possible from our analysis of the predicted values of these two physical quantities and the corresponding deformation parameters derived from them such as the quadrupole deformation β2, the ratio of β2 to the Weisskopf single-particle β2(sp) and the intrinsic electric quadrupole moment Q0, calculated for a large number of both known as well as hitherto unknown even-even isotopes of oxygen to fermium (0 to FM; Z = 8-100). Our critical analysis of the resulting data convincingly support possible existence of large collectivity for the nuclides 30,32Ne,34Mg, 60Ti, 42,62,64Cr,50,68Fe, 52,72Ni, 72,70,96Kr,74,76Sr,78,80,106,108Zr, 82,84,110,112Mo, 140Te,144Xe, 148Ba,122Ce, 128,156Nd,130,132,158,160Sm and 138,162,164,166Gd, whose values of β2 are found to exceed 0.3 and even 0.4 in some cases. Our findings of large deformations in the exotic n-rich regions support the existence of another “island of inversion” in the heavy-mass region possibly caused by breaking of the N = 70 subshell closure.

  18. Unraveling Δ1-Pyrroline-5-Carboxylate-Proline Cycle in Plants by Uncoupled Expression of Proline Oxidation Enzymes*

    PubMed Central

    Miller, Gad; Honig, Arik; Stein, Hanan; Suzuki, Nobuhiro; Mittler, Ron; Zilberstein, Aviah

    2009-01-01

    The two-step oxidation of proline in all eukaryotes is performed at the inner mitochondrial membrane by the consecutive action of proline dehydrogenase (ProDH) that produces Δ1-pyrroline-5-carboxylate (P5C) and P5C dehydrogenase (P5CDH) that oxidizes P5C to glutamate. This catabolic route is down-regulated in plants during osmotic stress, allowing free Pro accumulation. We show here that overexpression of MsProDH in tobacco and Arabidopsis or impairment of P5C oxidation in the Arabidopsis p5cdh mutant did not change the cellular Pro to P5C ratio under ambient and osmotic stress conditions, indicating that P5C excess was reduced to Pro in a mitochondrial-cytosolic cycle. This cycle, involving ProDH and P5C reductase, exists in animal cells and now demonstrated in plants. As a part of the cycle, Pro oxidation by the ProDH-FAD complex delivers electrons to the electron transport chain. Hyperactivity of the cycle, e.g. when an excess of exogenous l-Pro is provided, generates mitochondrial reactive oxygen species (ROS) by delivering electrons to O2, as demonstrated by the mitochondria-specific MitoSox staining of superoxide ions. Lack of P5CDH activity led to higher ROS production under dark and light conditions in the presence of Pro excess, as well as rendered plants hypersensitive to heat stress. Balancing mitochondrial ROS production during increased Pro oxidation is therefore critical for avoiding Pro-related toxic effects. Hence, normal oxidation of P5C to Glu by P5CDH is key to prevent P5C-Pro intensive cycling and avoid ROS production from electron run-off. PMID:19635803

  19. Genes encoding osmoregulatory proline/glycine betaine transporters and the proline catabolic system are present and expressed in diverse clinical Escherichia coli isolates.

    PubMed

    Culham, D E; Emmerson, K S; Lasby, B; Mamelak, D; Steer, B A; Gyles, C L; Villarejo, M; Wood, J M

    1994-05-01

    Sixty-three clinical isolates identified as Escherichia coli, 30 from the human urinary tract and 33 derived from other human origins, were screened for proline/glycine betaine transporters similar to those that support proline catabolism and proline- or glycine betaine-based osmoregulation in E. coli K-12. Both molecular (DNA- and protein-based) analyses and physiological tests were performed. All tests were calibrated with E. coli K-12 derivatives from which genetic loci putP (encoding a proline transporter required for proline catabolism), proP, and (or) proU (loci encoding osmoregulatory proline/glycine betaine transporters) had been deleted. All clinical isolates showed both enhanced sensitivity to the toxic proline analogue azetidine-2-carboxylate on media of high osmolality and growth stimulation by glycine betaine in an artificial urine preparation of high osmolality. DNA sequences similar to the putP, proP, and proU loci of E. coli K-12 were detected by DNA amplification and (or) hybridization and protein specifically reactive with antibodies raised against the ProX protein of E. coli K-12 (a ProU constituent) was detected by western blotting in over 95% of the isolates. Two anomalous isolates were reclassified as non-E. coli on the basis of the API 20E series of tests. A protein immunochemically cross-reactive with the ProP protein of E. coli K-12 was also expressed by the clinical isolates. Since all three transporters were ubiquitous, no particular correlation between clinical origin and PutP, ProP, or ProU activity was observed. These data suggest that the transporters encoded in loci putP, proP, and proU perform housekeeping functions essential for the survival of E. coli cells in diverse habitats.

  20. The Metal-Rich Universe

    NASA Astrophysics Data System (ADS)

    Israelian, Garik; Meynet, Georges

    2012-10-01

    Preface; Part I. Abundances in the Galaxy: Field Stars: 1. Metal-rich stars and stellar populations: A brief history and new results; 2. The metal-rich nature of stars with planets; 3. Solar chemical peculiarities; 4. Kinematics of metal-rich stars with and without planets; 5. Elemental abundance trends in the metal-rich thin and thick disks; 6. Metal-rich massive stars - how metal-rich are they?; 7. Hercules stream stars and the metal-rich thick disk; 8. Abundance survey of the galactic thick disk; Part II. Abundances in the Galaxy: Galactic Stars in Clusters, Bulges and Centre: 9. Galactic open clusters with super solar metallicities; 10. Old and very metal-rich open clusters in the BOCCE project; 11. Massive stars vs. nebular abundances in the Orion nebula; 12. Abundance surveys of metal-rich bulge stars; 13. Metal abundances in the galactic center; 14. Light elements in the galactic bulge; 15. Metallicity and ages of selected G-K giants; Part III. Observations - Abundances in Extragalactic Contexts: 16. Stellar abundances of early-type galaxies and galactic spheroids: Evidence for metal-rich stars; 17. Measuring chemical abundances in extragalactic metal-rich HII regions; 18. On the maximum oxygen abundance in metal-rich spiral galaxies; 19. Starbursts and their contribution to metal enrichment; 20. High metallicities at high redshifts; 21. Evolution of dust and elemental abundances in quasar DLAs and GRB afterglows as a function of cosmic time; 22. Dust, metals and diffuse interstellar bands in damped Lyman Alpha systems; 23. Tracing metallicities in the Universe with the James Webb Space Telescope; Part IV. Stellar Populations and Mass Functions: 24. The stellar initial mass function of metal-rich populations; 25. IMF effects on the metallicity and colour evolution of disk galaxies; 26. The metallicity of circumnuclear star forming regions; 27. The stellar population of bulges; 28. The metallicity distribution of the stars in elliptical galaxies; 29. Wolf

  1. Effect of water stress on proline accumulation of genetically modified potatoes (Solanum tuberosum L.) generating fructans.

    PubMed

    Knipp, Gabriele; Honermeier, Bernd

    2006-03-01

    This study aimed to investigate the effect of water stress on the accumulation of proline in the leaves of transgenic potato lines generating fructans. The results of the series of bifactorial experiments in 2002 and 2003 indicated an increase of the proline level in leaves of all potato lines examined under water deficit. In addition, an increase in proline concentration during plant development was observed. The proline content was related to leaf water potential and relative water content (RWC), which indicates that proline could be involved in osmoregulation of potato plants under the experimental conditions. Surprisingly, under water deficit, the proline level was lowest in most of the transgenic SST/FFT-lines, which generate fructan molecules with a high degree of polymerization. Therefore, a pleiotropic effect can not be excluded as the reason for the divergence in behavior of these transgenic lines. The present results suggest that the modification of carbohydrate metabolism, especially the high content of soluble carbohydrates, may affect water stress-induced proline accumulation.

  2. Proline auxotrophy in Sinorhizobium meliloti results in a plant-specific symbiotic phenotype.

    PubMed

    diCenzo, George C; Zamani, Maryam; Cowie, Alison; Finan, Turlough M

    2015-12-01

    In order to effectively manipulate rhizobium-legume symbioses for our benefit, it is crucial to first gain a complete understanding of the underlying genetics and metabolism. Studies with rhizobium auxotrophs have provided insight into the requirement for amino acid biosynthesis during the symbiosis; however, a paucity of available L-proline auxotrophs has limited our understanding of the role of L-proline biosynthesis. Here, we examined the symbiotic phenotypes of a recently described Sinorhizobium meliloti L-proline auxotroph. Proline auxotrophy was observed to result in a host-plant-specific phenotype. The S. meliloti auxotroph displayed reduced symbiotic capability with alfalfa (Medicago sativa) due to a decrease in nodule mass formed and therefore a reduction in nitrogen fixed per plant. However, the proline auxotroph formed nodules on white sweet clover (Melilotus alba) that failed to fix nitrogen. The rate of white sweet clover nodulation by the auxotroph was slightly delayed, but the final number of nodules per plant was not impacted. Examination of white sweet clover nodules by confocal microscopy and transmission electron microscopy revealed the presence of the S. meliloti proline auxotroph cells within the host legume cells, but few differentiated bacteroids were identified compared with the bacteroid-filled plant cells of WT nodules. Overall, these results indicated that L-proline biosynthesis is a general requirement for a fully effective nitrogen-fixing symbiosis, likely due to a transient requirement during bacteroid differentiation.

  3. The relationship of proline content and metabolism on the productivity of maize plants

    PubMed Central

    Špoljarević, Marija; Agić, Dejan; Lisjak, Miroslav; Gumze, Andrej; Wilson, Ian D; Hancock, John T

    2011-01-01

    The free proline content in maize ear-leaves, silk and pollen were analyzed in field grown plants which had matured to the pollination stage. Using maize hybrids PR34F02, PR35P12 and PR36B08 field trials were set up at two locations in eastern Croatia in two different years. Two enzymes of proline metabolism were analyzed in the same leaf samples and specific activities of synthetase (P5CS) and proline dehydrogenase (PDH). Plant productivity was evaluated at harvest by the estimation of total and fully developed grain number per ear and per plant, the mean single grain mass, and the mass of grain per plant. The year in which the plants were grown had a very significant effect on the free proline content in the leaf and pollen, as well as on the enzyme activities assayed. The differences between the plants from the two localities were very significant in all tested parameters of plant grain productivity. There was a significant genotype effect on proline content and P5CS total activity in leaf and on all the productivity parameters. Some of the correlations established suggest that the rate of proline synthesis and degradation in maize ear-leaf at pollination might contribute to the final grain production of the maize plant. Multiple regression analyses was used to further analyze the relationship between proline and grain productivity, but it is clear that future work should include other environmental conditions, plant species and organs such as roots. PMID:21415600

  4. Chemical Tools To Decipher Regulation of Phosphatases by Proline Isomerization on Eukaryotic RNA Polymerase II.

    PubMed

    Mayfield, Joshua E; Fan, Shuang; Wei, Shuo; Zhang, Mengmeng; Li, Bing; Ellington, Andrew D; Etzkorn, Felicia A; Zhang, Yan Jessie

    2015-10-16

    Proline isomerization greatly impacts biological signaling but is subtle and difficult to detect in proteins. We characterize this poorly understood regulatory mechanism for RNA polymerase II carboxyl terminal domain (CTD) phosphorylation state using novel, direct, and quantitative chemical tools. We determine the proline isomeric preference of three CTD phosphatases: Ssu72 as cis-proline specific, Scp1 and Fcp1 as strongly trans-preferred. Due to this inherent characteristic, these phosphatases respond differently to enzymes that catalyze the isomerization of proline, like Ess1/Pin1. We demonstrate that this selective regulation of RNA polymerase II phosphorylation state exists within human cells, consistent with in vitro assays. These results support a model in which, instead of a global enhancement of downstream enzymatic activities, proline isomerases selectively boost the activity of a subset of CTD regulatory factors specific for cis-proline. This leads to diversified phosphorylation states of CTD in vitro and in cells. We provide the chemical tools to investigate proline isomerization and its ability to selectively enhance signaling in transcription and other biological contexts.

  5. Protective effect of green tea extract against proline-induced oxidative damage in the rat kidney.

    PubMed

    Delwing-Dal Magro, Débora; Roecker, Roberto; Junges, Gustavo M; Rodrigues, André F; Delwing-de Lima, Daniela; da Cruz, José G P; Wyse, Angela T S; Pitz, Heloisa S; Zeni, Ana L B

    2016-10-01

    We investigated, in vivo (acute and chronic), the effects of proline on thiobarbituric acid-reactive substances (TBA-RS) and on the activities of antioxidant enzymes such as catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in renal tissues (cortex and medulla) of rats. For acute administration, 29-day-old rats received a single subcutaneous injection of proline (18.2μmol/g body weight) or an equivalent volume of 0.9% saline solution and were sacrificed 1h later. For chronic treatment, proline was injected subcutaneously in the rats twice a day from the 6th to the 28th day of age, and the animals were killed 12h after the last injection. The results showed that acute administration of proline enhanced CAT, SOD and GSH-Px activities, as well as, TBARS in the cortex and decreased CAT activity in the medulla, while chronic treatment increased the activities of SOD in the cortex and increased CAT, SOD and GSH-Px in the medulla of rats. Furthermore, the green tea extract treatment for one week or from the 6th to the 28th day of age prevented the alterations caused by acute and chronic, respectively, proline administration. Herein, we demonstrated that proline alters antioxidant defenses and induces lipid peroxidation in the kidney of rats and the green tea extract was capable to counteract the proline-induced alterations.

  6. Proline Dehydrogenase Regulates Redox State and Respiratory Metabolism in Trypanosoma cruzi

    PubMed Central

    Paes, Lisvane Silva; Suárez Mantilla, Brian; Zimbres, Flávia Menezes; Pral, Elisabeth Mieko Furusho; Diogo de Melo, Patrícia; Tahara, Erich B.; Kowaltowski, Alicia J.; Elias, Maria Carolina; Silber, Ariel Mariano

    2013-01-01

    Over the past three decades, L-proline has become recognized as an important metabolite for trypanosomatids. It is involved in a number of key processes, including energy metabolism, resistance to oxidative and nutritional stress and osmoregulation. In addition, this amino acid supports critical parasite life cycle processes by acting as an energy source, thus enabling host-cell invasion by the parasite and subsequent parasite differentiation. In this paper, we demonstrate that L-proline is oxidized to Δ1-pyrroline-5-carboxylate (P5C) by the enzyme proline dehydrogenase (TcPRODH, E.C. 1.5.99.8) localized in Trypanosoma cruzi mitochondria. When expressed in its active form in Escherichia coli, TcPRODH exhibits a Km of 16.58±1.69 µM and a Vmax of 66±2 nmol/min mg. Furthermore, we demonstrate that TcPRODH is a FAD-dependent dimeric state protein. TcPRODH mRNA and protein expression are strongly upregulated in the intracellular epimastigote, a stage which requires an external supply of proline. In addition, when Saccharomyces cerevisiae null mutants for this gene (PUT1) were complemented with the TcPRODH gene, diminished free intracellular proline levels and an enhanced sensitivity to oxidative stress in comparison to the null mutant were observed, supporting the hypothesis that free proline accumulation constitutes a defense against oxidative imbalance. Finally, we show that proline oxidation increases cytochrome c oxidase activity in mitochondrial vesicles. Overall, these results demonstrate that TcPRODH is involved in proline-dependant cytoprotection during periods of oxidative imbalance and also shed light on the participation of proline in energy metabolism, which drives critical processes of the T. cruzi life cycle. PMID:23894476

  7. Proline Accumulation and Its Implication in Cold Tolerance of Regenerable Maize Callus 1

    PubMed Central

    Duncan, David R.; Widholm, Jack M.

    1987-01-01

    Embryogenic callus of maize (Zea mays L.) inbreds B37wx, H99, H993H95, Mo17, and Pa91 accumulated proline to levels 2.1 to 2.5 times that of control callus when subjected to mannitol-induced water stress, cool temperatures (19°C) and abscisic acid (ABA). A combination of 0.53 molar mannitol plus 0.1 millimolar ABA induced a proline accumulation to about 4.5 times that of control callus, equivalent to approximately 0.18 millimoles proline per gram fresh weight of callus. Proline accumulation was directly related to the level of mannitol in the medium. Levels of ABA greater than 1.0 micromolar were required in the medium to induce proline accumulation comparable to that induced by mannitol. Mannitol and ABA levels that induced maximum accumulation of proline also inhibited callus growth and increased tolerance to cold. Proline (12 millimolar) added to the culture media also increased the tolerance of callus to 4°C. The increased cold tolerance induced by the combination of mannitol and ABA has permitted the storage of the maize inbreds A632, A634Ht, B37wx, C103DTrf, Fr27rhm, H99, Pa91, Va35, and W117Ht at 4°C for 90 days which is more than double the typical survival time of callus. These studies show that proline and conditions which induce proline accumulation increase the cold tolerance of regenerable maize callus. Images Fig. 1 Fig. 5 PMID:16665311

  8. Involvement of Phosphatidylinositol 3-kinase in the regulation of proline catabolism in Arabidopsis thaliana

    PubMed Central

    Leprince, Anne-Sophie; Magalhaes, Nelly; De Vos, Delphine; Bordenave, Marianne; Crilat, Emilie; Clément, Gilles; Meyer, Christian; Munnik, Teun; Savouré, Arnould

    2015-01-01

    Plant adaptation to abiotic stresses such as drought and salinity involves complex regulatory processes. Deciphering the signaling components that are involved in stress signal transduction and cellular responses is of importance to understand how plants cope with salt stress. Accumulation of osmolytes such as proline is considered to participate in the osmotic adjustment of plant cells to salinity. Proline accumulation results from a tight regulation between its biosynthesis and catabolism. Lipid signal components such as phospholipases C and D have previously been shown to be involved in the regulation of proline metabolism in Arabidopsis thaliana. In this study, we demonstrate that proline metabolism is also regulated by class-III Phosphatidylinositol 3-kinase (PI3K), VPS34, which catalyses the formation of phosphatidylinositol 3-phosphate (PI3P) from phosphatidylinositol. Using pharmacological and biochemical approaches, we show that the PI3K inhibitor, LY294002, affects PI3P levels in vivo and that it triggers a decrease in proline accumulation in response to salt treatment of A. thaliana seedlings. The lower proline accumulation is correlated with a lower transcript level of Pyrroline-5-carboxylate synthetase 1 (P5CS1) biosynthetic enzyme and higher transcript and protein levels of Proline dehydrogenase 1 (ProDH1), a key-enzyme in proline catabolism. We also found that the ProDH1 expression is induced in a pi3k-hemizygous mutant, further demonstrating that PI3K is involved in the regulation of proline catabolism through transcriptional regulation of ProDH1. A broader metabolomic analysis indicates that LY294002 also reduced other metabolites, such as hydrophobic and aromatic amino acids and sugars like raffinose. PMID:25628629

  9. Plant-derived compatible solutes proline betaine and betonicine confer enhanced osmotic and temperature stress tolerance to Bacillus subtilis.

    PubMed

    Bashir, Abdallah; Hoffmann, Tamara; Kempf, Bettina; Xie, Xiulan; Smits, Sander H J; Bremer, Erhard

    2014-10-01

    L-Proline is a widely used compatible solute and is employed by Bacillus subtilis, through both synthesis and uptake, as an osmostress protectant. Here, we assessed the stress-protective potential of the plant-derived L-proline derivatives N-methyl-L-proline, L-proline betaine (stachydrine), trans-4-L-hydroxproline and trans-4-hydroxy-L-proline betaine (betonicine) for cells challenged by high salinity or extremes in growth temperature. l-Proline betaine and betonicine conferred salt stress protection, but trans-4-L-hydroxyproline and N-methyl-L-proline was unable to do so. Except for L-proline, none of these compounds served as a nutrient for B. subtilis. L-Proline betaine was a considerably better osmostress protectant than betonicine, and its import strongly reduced the l-proline pool produced by B. subtilis under osmotic stress conditions, whereas a supply of betonicine affected the L-proline pool only modestly. Both compounds downregulated the transcription of the osmotically inducible opuA operon, albeit to different extents. Mutant studies revealed that L-proline betaine was taken up via the ATP-binding cassette transporters OpuA and OpuC, and the betaine-choline-carnitine-transporter-type carrier OpuD; betonicine was imported only through OpuA and OpuC. L-Proline betaine and betonicine also served as temperature stress protectants. A striking difference between these chemically closely related compounds was observed: L-proline betaine was an excellent cold stress protectant, but did not provide heat stress protection, whereas the reverse was true for betonicine. Both compounds were primarily imported in temperature-challenged cells via the high-capacity OpuA transporter. We developed an in silico model for the OpuAC-betonicine complex based on the crystal structure of the OpuAC solute receptor complexed with L-proline betaine.

  10. A contemporary approach for treatment planning of horizontally resorbed alveolar ridge: Ridge split technique with simultaneous implant placement using platelet rich fibrin membrane application in mandibular anterior region.

    PubMed

    Parthiban, Prathahini S; Lakshmi, R Vijaya; Mahendra, Jaideep; Sreekumar, K; Namasivayam, Ambalavanan

    2017-01-01

    Treatment of edentulous sites with horizontal atrophy represents a clinical situation in which the positioning of endosseous implants might be complex or sometimes impossible without a staged regenerative approach. This case report presents management of horizontally deficient mandibular anterior ridge with a contemporary approach to treatment planning and application of platelet-rich fibrin membrane for ridge split technique and simultaneous implant placement. Implants in anterior mandibular area are considered to be most predictable, stable, with high success rate and patients' satisfaction with implant esthetics. In contrast to traditional ridge augmentation techniques, ridge splitting allows for immediate implant placement following surgery and eradicates the possible morbidity from a second surgical site.

  11. Proline Residues as Switches in Conformational Changes Leading to Amyloid Fibril Formation

    PubMed Central

    Taler-Verčič, Ajda; Hasanbašić, Samra; Berbić, Selma; Stoka, Veronika; Turk, Dušan; Žerovnik, Eva

    2017-01-01

    Here we discuss studies of the structure, folding, oligomerization and amyloid fibril formation of several proline mutants of human stefin B, which is a protein inhibitor of lysosomal cysteine cathepsins and a member of the cystatin family. The structurally important prolines in stefin B are responsible for the slow folding phases and facilitate domain swapping (Pro 74) and loop swapping (Pro 79). Moreover, our findings are compared to β2-microglobulin, a protein involved in dialysis-related amyloidosis. The assessment of the contribution of proline residues to the process of amyloid fibril formation may shed new light on the critical molecular events involved in conformational disorders. PMID:28272335

  12. Proline iminopeptidase PepI overexpressing Lactobacillus casei as an adjunct starter in Edam cheese

    PubMed Central

    Navidghasemizad, Sahar; Takala, Timo M; Alatossava, Tapani; Saris, Per EJ

    2013-01-01

    In this study the growth of genetically modified Lactobacillus casei LAB6, overexpressing proline iminopeptidase PepI and its capacity to increase free proline was investigated during ripening of Edam cheese. The strain successfully survived 12 weeks of ripening period in cheese. The food-grade plasmid pLEB604, carrying the pepI gene, was stable, and PepI enzyme was active in LAB6 cells isolated at different stages of the ripening process. However, HPLC analyses indicated that Lb. casei LAB6 could not increase the amount of free proline in ripened cheese. PMID:23851577

  13. Proline Residues as Switches in Conformational Changes Leading to Amyloid Fibril Formation.

    PubMed

    Taler-Verčič, Ajda; Hasanbašić, Samra; Berbić, Selma; Stoka, Veronika; Turk, Dušan; Žerovnik, Eva

    2017-03-07

    Here we discuss studies of the structure, folding, oligomerization and amyloid fibril formation of several proline mutants of human stefin B, which is a protein inhibitor of lysosomal cysteine cathepsins and a member of the cystatin family. The structurally important prolines in stefin B are responsible for the slow folding phases and facilitate domain swapping (Pro 74) and loop swapping (Pro 79). Moreover, our findings are compared to β₂-microglobulin, a protein involved in dialysis-related amyloidosis. The assessment of the contribution of proline residues to the process of amyloid fibril formation may shed new light on the critical molecular events involved in conformational disorders.

  14. Thermal unfolding of the N-terminal region of p53 monitored by circular dichroism spectroscopy.

    PubMed

    Schaub, Leasha J; Campbell, James C; Whitten, Steven T

    2012-11-01

    It has been estimated that 30% of eukaryotic protein and 70% of transcription factors are intrinsically disordered (ID). The biochemical significance of proteins that lack stable tertiary structure, however, is not clearly understood, largely owing to an inability to assign well-defined structures to specific biological tasks. In an attempt to investigate the structural character of ID protein, we have measured the circular dichroism spectrum of the N-terminal region of p53 over a range of temperatures and solution conditions. p53 is a well-studied transcription factor that has a proline-rich N-terminal ID region containing two activation domains. High proline content is a property commonly associated with ID, and thus p53 may be a good model system for investigating the biochemical importance of ID. The spectra presented here suggest that the N-terminal region of p53 may adopt an ordered structure under physiological conditions and that this structure can be thermally unfolded in an apparent two-state manner. The midpoint temperature for this thermal unfolding of the N-terminal region of p53 was at the near-physiological temperature of 39°C, suggesting the possibility of a physiological role for the observed structural equilibrium.

  15. The study of neutron-rich nuclei production in the region of the closed shell N=126 in the multi-nucleon transfer reaction 136Xe+208Pb

    NASA Astrophysics Data System (ADS)

    Novikov, K.; Harca, I. M.; Kozulin, E. M.; Dmitriev, S.; Itkis, J.; Knyazheva, G.; Loktev, T.; Corradi, L.; Valiente-Dobon, J.; Fioretto, E.; Montanari, D.; Stefanini, A. M.; Vardaci, E.; Quero, D.; Montagnoli, G.; Scarlassara, F.; Strano, E.; Pollarolo, G.; Piot, J.; Mijatović, T.; Szilner, S.; Ackermann, D.; Chubarian, G.; Trzaska, W. H.

    2016-04-01

    The unexplored area of heavy neutron rich nuclei is extremely important for nuclear astrophysics investigations and, in particular, for the understanding of the r-process of astrophysical nucleogenesis. For the production of heavy neutron rich nuclei located along the neutron closed shell N=126 (probably the last "waiting point" in the r-process of nucleosynthesis) the low-energy multi-nucleon transfer reaction 136Xe+208Pb at Elab=870MeV was explored. Due to the stabilizing effect of the closed neutron shells in both nuclei, N=82 and N=126, and the rather favorable proton transfer from lead to xenon, the light fragments formed in this process are well bound and the Q-value of the reaction is nearly zero. Measurements were performed with the PRISMA spectrometer in coincidence with an additional time-of-flight (ToF) arm on the +20 beam line of the PIAVE-ALPI accelerator in Legnaro, Italy. The PRISMA spectrometer allows identification of the A, Z and velocity of the projectile-like fragments (PLF), while the second arm gives access to the target-like fragments (TLF). Details on the experimental setup and preliminary results are reported.

  16. Boron-rich mud volcanoes of the Black Sea region: modern analogues to ancient sea-floor tourmalinites associated with Sullivan-type Pb-Zn deposits?

    USGS Publications Warehouse

    Slack, J.F.; Turner, R.J.W.; Ware, P.L.G.

    1998-01-01

    Large submarine mud volcanoes in the abyssal part of the Black Sea south of the Crimean Peninsula are similar in many respects to synsedimentary mud volcanoes in the Mesoproterozoic Belt-Purcell basin. One of the Belt-Purcell mud volcanoes directly underlies the giant Sullivan Pb-Zn-Ag deposit in southeastern British Columbia. Footwall rocks to the Sullivan deposit comprise variably tourmalinized siltstone, conglomerate, and related fragmental rock; local thin pyrrhotite-rich and spessartine-quartz beds are interpreted as Fe and Fe-Mn exhalites, respectively. Analogous Fe- and Mn-rich sediments occur near the abyssal Black Sea mud volcanoes. Massive pyrite crusts and associated carbonate chimneys discovered in relatively shallow waters (~200 m depth) west of the Crimean Peninsula indicate an active sea-floor-hydrothermal system. Subaerial mud volcanoes on the Kerch and Taman Peninsulas (~100 km north of the abyssal mud volcanoes) contain saline thermal waters that locally have very high B contents (to 915 mg/L). These data suggest that tourmalinites might be forming in or near submarine Black Sea mud volcanoes, where potential may also exist for Sullivan-type Pb-Zn mineralization.

  17. Amyloidogenesis Abolished by Proline Substitutions but Enhanced by Lipid Binding

    PubMed Central

    Jiang, Ping; Xu, Weixin; Mu, Yuguang

    2009-01-01

    The influence of lipid molecules on the aggregation of a highly amyloidogenic segment of human islet amyloid polypeptide, hIAPP20–29, and the corresponding sequence from rat has been studied by all-atom replica exchange molecular dynamics (REMD) simulations with explicit solvent model. hIAPP20–29 fragments aggregate into partially ordered β-sheet oligomers and then undergo large conformational reorganization and convert into parallel/antiparallel β-sheet oligomers in mixed in-register and out-of-register patterns. The hydrophobic interaction between lipid tails and residues at positions 23–25 is found to stabilize the ordered β-sheet structure, indicating a catalysis role of lipid molecules in hIAPP20–29 self-assembly. The rat IAPP variants with three proline residues maintain unstructured micelle-like oligomers, which is consistent with non-amyloidogenic behavior observed in experimental studies. Our study provides the atomic resolution descriptions of the catalytic function of lipid molecules on the aggregation of IAPP peptides. PMID:19360098

  18. Conformational profile of a proline-arginine hybrid

    PubMed Central

    Revilla-López, Guillermo; Jiménez, Ana I.; Cativiela, Carlos; Nussinov, Ruth; Alemán, Carlos; Zanuy, David

    2010-01-01

    The intrinsic conformational preferences of a new non-proteinogenic amino acid have been explored by computational methods. This tailored molecule, named (βPro)Arg, is conceived as a replacement for arginine in bioactive peptides when the stabilization of folded turn-like conformations is required. The new residue features a proline skeleton that bears the guanidilated side chain of arginine at the Cβ position of the five-membered pyrrolidine ring, either in a cis or a trans orientation with respect to the carboxylic acid. The conformational profile of the N-acetyl-N'-methylamide derivatives of the cis and trans isomers of (βPro)Arg has been examined in the gas phase and in solution by B3LYP/6–31+G(d,p) calculations and molecular dynamics simulations. The main conformational features of both isomers represent a balance between geometric restrictions imposed by the five-membered pyrrolidine ring and the ability of the guanidilated side chain to interact with the backbone through hydrogen-bonds. Thus, both cis and trans (βPro)Arg exhibit a preference for the αL conformation as a consequence of the interactions established between the guanidinium moiety and the main-chain amide groups. PMID:20886854

  19. A spectral line list for water isotopologues in the 1100-4100 cm-1 region for application to CO2-rich planetary atmospheres

    NASA Astrophysics Data System (ADS)

    Gamache, Robert R.; Farese, Michaella; Renaud, Candice L.

    2016-08-01

    A line list of transitions for seven isotopologues of water vapor in the 1100-4100 wavenumber range has been constructed for application to atmospheres rich in CO2. The quantum transitions for H216O, H218O, H217O, HD16O, HD18O, and HD17O are from the 2012 HITRAN database (Rothman et al., 2013) and those for D216O are from the ab initio line list of Shirin et al. (2008). The database was constructed using the best available measured parameters and augmented with theoretical calculations. The half-width, γ, its temperature dependence, n, and the line shift, δ, are for CO2 as the colliding partner or self-collisions and are determined using the Modified Complex Robert-Bonamy (MCRB) theory. The resulting line list contains 282 557 transitions and is available, in the HITRAN 2012 format, from the corresponding author or from the supplemental information of the journal.

  20. Sodium and proline accumulation as osmoregulators in tolerance of sugar beet genotypes to salinity.

    PubMed

    Pakniyat, H; Armion, M

    2007-11-15

    Twenty eight sugar beet genotypes were analysed for their tolerance at 3 NaCl levels (0, 3000 and 6000 mg NaCl kg(-1) soil) and Na+, K+, Na+/K+ and free proline were measured from the leaf samples. Results showed that increasing salinity level caused an increase in Na+, Na+/K+ and proline, but a decrease in K+ content of leaf samples (p < or = 0.01). As compared to non-tolerant genotypes, tolerant ones accumulated more Na+ and Na+/K+ and proline and less K+. It seems that Na+ and proline accumulation in shoots are effective mechanisms for osmotic pressure adjustment and plant tolerance to salinity, a mechanism commonly seen in sugar beet ancestors.

  1. The thermodynamic activity of proline in ternary solutions of different water potentials.

    PubMed

    Pahlich, E; Stadermann, T

    1984-06-01

    The particular colligative properties of proline caused us to investigate the thermodynamic activity of this amino acid in detail. The dependence of the activity coefficients γ of proline (γ = thermodynamic activity/molality) on the pH of the solutions, the composition of the solution and the water potential has been measured. The results show that the activity coefficient of proline varies according to the solute milieu. The most pronounced alterations of the activity coefficient could be observed in polyethylene glycol solutions in contrast to KCl- and saccharose solutions where the effect was less distinct. The results described provide a basis for discussing water stress induced metabolic alterations in terms of thermodynamic entities. Changed rates of proline metabolizing sequences and changed ratios of the vacuole/extravacuole distribution of this amino acid in stressed and un-stressed plants may partially be explained by thermodynamic causes.

  2. Growth, biochemical modifications and proline metabolism in Helianthus annuus L. as induced by drought stress.

    PubMed

    Manivannan, P; Jaleel, C Abdul; Sankar, B; Kishorekumar, A; Somasundaram, R; Lakshmanan, G M A; Panneerselvam, R

    2007-10-01

    In the present investigation, two watering treatments, viz., 100% and 60% field capacity (FC) were used to understand the effects of water deficit on early growth, biomass allocation, pigment and biochemical constituents and proline metabolism of five varieties of Sunflower (Helianthus annuus L.) plants. We found that there was a significant difference in early growth, dry matter accumulation, pigment, biochemical constituents and proline metabolism among the five varieties. The root length, shoot length, total leaf area, fresh and dry weight, chlorophyll a, b, total chlorophyll and carotenoid were significantly reduced under water stress treatments. Water stress increased the proline, free amino acid and glycinebetaine contents along with increased activity of gamma-glutamyl kinase but the activity of proline oxidase reduced as a consequence of water stress.

  3. Isolation of cis-3-Amino-l-Proline from Cultured Mycelia of Morchella esculenta Fr.

    PubMed

    Moriguchi, M; Sada, S; Hatanaka, S

    1979-11-01

    cis-3-Amino-l-proline, identified once as a nonprotein amino acid from the fruiting bodies of Morchella esculenta Fr., was isolated also from the growth medium and cultured mycelia of the same fungus.

  4. Proline transport by brush-border membrane vesicles of lobster antennal glands

    SciTech Connect

    Behnke, R.D.; Wong, R.K.; Huse, S.M.; Reshkin, S.J.; Ahearn, G.A. )

    1990-02-01

    Purified brush-border membrane vesicles (BBMV) of lobster antennal gland labyrinth and bladder were separately formed by a magnesium precipitation technique. L-(3H)proline uptake was stimulated by a transmembrane NaCl gradient (outside (o) greater than inside (i)) to a greater extent in BBMV from labyrinth than those from the bladder. Detailed study of the labyrinth proline-transport processes revealed a specific dependence on NaCl, with negligible stimulatory effects by NaSCN, Na-gluconate, or KCl. A transmembrane proton gradient (o greater than i) was without stimulatory effect on proline transport. A transmembrane potential difference alone, in the presence of equilibrated NaCl and L-(3H)proline, led to net influx of the labeled amino acid, suggesting that the uptake process was electrogenic and capable of bringing about the net transfer of positive charge to the vesicle interior. Although a transmembrane Na gradient alone, in the presence of equilibrated Cl and L-(3H)proline, was able to bring about the net influx of the amino acid, a transmembrane Cl gradient alone under Na- and L-(3H)proline-equilibrated conditions was not, suggesting that only the Na gradient could energize the carrier process through cotransport, while the anion served an essential activating role. Proline influx by these vesicles occurred by the combination of at least one saturable Michaelis-Menten carrier system (apparent Kt = 0.37 mM; apparent JM = 1.19 nmol.mg protein-1.10 s-1) and apparent diffusion (P = 0.33 nmol.mg protein-1.10 s-1.mM-1). Static head analysis of the transport process suggested a cotransport stoichiometry of 2 Na:1 proline with essential activation by Cl ion.

  5. Proline accumulation in lemongrass (Cymbopogon flexuosus Stapf.) due to heavy metal stress.

    PubMed

    Handique, G K; Handique, A K

    2009-03-01

    Toxic heavy metals viz. lead, mercury and cadmium induced differential accumulation of proline in lemongrass (Cymbopogon flexuosus Stapf.) grown in soil amended with 50, 100, 200, 350 and 500 mg kg(-1) of the metals have been studied. Proline accumulation was found to be metal specific, organ specific and linear dose dependant. Further, proline accumulation following short term exposure (two months after transplantation) was higher than long term exposure (nine months after transplantation). Proline accumulation following short term exposure was 2.032 to 3.839 micro moles g(-1) for cadmium (50-200 mg kg(-1)); the corresponding range for mercury was 1.968 to 5.670 micro moles g(-1) and 0.830 to 4.567 micro moles g(-1) for lead (50-500 mg kg(-1) for mercury and lead). Proline accumulation was consistently higher in young tender leaf than old leaf, irrespective of the metal or duration of exposure. For cadmium treatment proline level was 2.032 to 3.839 micro moles g(-1) for young leaves while the corresponding value for old leaf was 1.728 to 2.396 micro moles g(-1) following short term exposure. The same trend was observed for the other two metals and duration of exposure. For control set proline accumulation in root was 0.425 micro moles g(-1) as against 0.805 and 0.533 micro moles g(-1) in young and old leaves respectively indicating that proline accumulation in root are lower than leaves, under both normal and stressed condition.

  6. Proline Can Have Opposite Effects on Fast and Slow Protein Folding Phases

    PubMed Central

    Osváth, Szabolcs; Gruebele, Martin

    2003-01-01

    Proline isomerization is well known to cause additional slow phases during protein refolding. We address a new question: does the presence of prolines significantly affect the very fast kinetics that lead to the formation of folding intermediates? We examined both the very slow (10–100 min) and very fast (4 μs–2.5 ms) folding kinetics of the two-domain enzyme yeast phosphoglycerate kinase by temperature-jump relaxation. Phosphoglycerate kinase contains a conserved cis-proline in position 204, in addition to several trans-prolines. Native cis-prolines have the largest effect on folding kinetics because the unfolded state favors trans isomerization, so we compared the kinetics of a P204H mutant with the wild-type as a proof of principle. The presence of Pro-204 causes an additional slow phase upon refolding from the cold denatured state, as reported in the literature. Contrary to this, the fast folding events are sped up in the presence of the cis-proline, probably by restriction of the conformational space accessible to the molecule. The wild-type and Pro204His mutant would be excellent models for off-lattice simulations probing the effects of conformational restriction on short timescales. PMID:12885665

  7. Cadmium toxicity-induced proline accumulation is coupled to iron depletion.

    PubMed

    Sharmila, P; Kumari, P Kusuma; Singh, Kavita; Prasad, N V S R K; Pardha-Saradhi, P

    2017-03-01

    Investigations were conducted to elucidate the key factor behind Cd(2+)-toxicity-induced proline accumulation in Indian mustard (Brassica juncea) by raising seedlings, independently in distilled water (DW) and mineral growth medium (MGM) in the presence of 0-500 μM CdCl2. Invariably, Cd(2+)-induced toxicity, measured in terms of growth, was significantly more prominent in seedlings raised in DW than those raised in MGM. Cd(2+) brought about a significant reduction in growth and photosystem II activity with a concomitant increase in proline levels, in a concentration-dependent manner. Interestingly, the level of iron in shoots of seedlings decreased proportionately with increase in Cd(2+) toxicity. Cd(2+)-promoted proline accumulation was significantly higher in seedlings raised in DW than those raised in MGM. Depletion of essential cations (viz. Ca(2+), Mg(2+), K(+), and Fe(2+)) from MGM one at a time revealed that depletion of Fe(2+) leads to maximal proline accumulation under Cd(2+) toxicity. Interestingly, proline level in seedlings raised under Cd(2+) toxicity in DW supplemented with Fe(2+) was similar to that recorded in seedlings raised in MGM. Our results convincingly demonstrated that Cd(2+)-induced iron deficiency promotes proline accumulation.

  8. Stereochemical Sequence Ion Selectivity: Proline versus Pipecolic-acid-containing Protonated Peptides

    NASA Astrophysics Data System (ADS)

    Abutokaikah, Maha T.; Guan, Shanshan; Bythell, Benjamin J.

    2017-01-01

    Substitution of proline by pipecolic acid, the six-membered ring congener of proline, results in vastly different tandem mass spectra. The well-known proline effect is eliminated and amide bond cleavage C-terminal to pipecolic acid dominates instead. Why do these two ostensibly similar residues produce dramatically differing spectra? Recent evidence indicates that the proton affinities of these residues are similar, so are unlikely to explain the result [Raulfs et al., J. Am. Soc. Mass Spectrom. 25, 1705-1715 (2014)]. An additional hypothesis based on increased flexibility was also advocated. Here, we provide a computational investigation of the "pipecolic acid effect," to test this and other hypotheses to determine if theory can shed additional light on this fascinating result. Our calculations provide evidence for both the increased flexibility of pipecolic-acid-containing peptides, and structural changes in the transition structures necessary to produce the sequence ions. The most striking computational finding is inversion of the stereochemistry of the transition structures leading to "proline effect"-type amide bond fragmentation between the proline/pipecolic acid-congeners: R (proline) to S (pipecolic acid). Additionally, our calculations predict substantial stabilization of the amide bond cleavage barriers for the pipecolic acid congeners by reduction in deleterious steric interactions and provide evidence for the importance of experimental energy regime in rationalizing the spectra.

  9. One- and two-dimensional polymers from proline and calcium bromide.

    PubMed

    Lamberts, Kevin; Şerb, Mihaela-Diana; Englert, Ulli

    2015-04-01

    Reactions of calcium bromide with enantiopure and racemic proline in aqueous solution lead to two solids in which the zwitterionic amino acid acts as a bridging ligand between neighbouring cations. Depending on the chirality of the amino acid, topologically very different products are obtained. With racemic proline, bromide acts as a simple uncoordinated counter-anion for the cationic heterochiral chains in catena-poly[[aquacalcium(II)]-μ-aqua-μ3-DL-proline-μ2-DL-proline], {[Ca(C5H9NO2)2(H2O)2]Br2}n. In agreement with chemical intuition, only carboxylate and aqua O atoms coordinate the alkaline earth cation in a low-symmetry arrangement. In contrast, L-proline affords the two-dimensional network poly[dibromidobis(μ2-L-proline)calcium(II)], [CaBr2(C5H9NO2)2]n, with an unexpected CaBr2 unit in a more regular coordination sphere.

  10. Inhibition of serine and proline racemases by substrate-product analogues.

    PubMed

    Harty, Matthew; Nagar, Mitesh; Atkinson, Logan; Legay, Christina M; Derksen, Darren J; Bearne, Stephen L

    2014-01-01

    d-Amino acids can play important roles as specific biosynthetic building blocks required by organisms or act as regulatory molecules. Consequently, amino acid racemases that catalyze the formation of d-amino acids are potential therapeutic targets. Serine racemase catalyzes the reversible formation of d-serine (a modulator of neurotransmission) from l-serine, while proline racemase (an essential enzymatic and mitogenic protein in trypanosomes) catalyzes the reversible conversion of l-proline to d-proline. We show the substrate-product analogue α-(hydroxymethyl)serine is a modest, linear mixed-type inhibitor of serine racemase from Schizosaccharomyces pombe (Ki=167±21mM, Ki'=661±81mM, cf. Km=19±2mM). The bicyclic substrate-product analogue of proline, 7-azabicyclo[2.2.1]heptan-7-ium-1-carboxylate is a weak inhibitor of proline racemase from Clostridium sticklandii, giving only 29% inhibition at 142.5mM. However, the more flexible bicyclic substrate-product analogue tetrahydro-1H-pyrrolizine-7a(5H)-carboxylate is a noncompetitive inhibitor of proline racemase from C. sticklandii (Ki=111±15mM, cf. Km=5.7±0.5mM). These results suggest that substrate-product analogue inhibitors of racemases may only be effective when the active site is capacious and/or plastic, or when the inhibitor is sufficiently flexible.

  11. Quest for Cells Responsible for Age-related Increase of Salivary Glycine and Proline.

    PubMed

    Hino, Shunsuke; Nishiyama, Akira; Matsuta, Tomohiko; Horie, Norio; Shimoyama, Tetsuo; Tanaka, Shoji; Sakagami, Hiroshi

    2016-01-01

    We have previously reported that salivary glycine and proline levels are increased to nearly butanoate level in elderly people. In order to identify the source of glycine and proline, we performed high-performance liquid chromatography analysis of amino acid production to a total of seven oral cells before and after stimulation with inflammation inducers. We found that production of amino acids (per a given number of cells) by normal oral mesenchymal cells (gingival fibroblast, pulp cell, periodontal ligament fibroblast) was approximately three-fold that of oral squamous cell carcinoma cell lines (HSC-2, HSC-3, HSC-4, Ca9-22), and that production of glycine and especially proline by all these seven cells was much lower than that of glutamine and glutamic acid. Treatment of three oral mesenchymal cells with interleukin (IL)-1β or lipopoly-saccharide (LPS) reproducibly increased the production of glutamic acid and glutamine, but not that of glycine and proline. Glycine and proline only marginally stimulated the IL-8 production by IL-1β-stimulated gingival fibroblast, whereas glycine dose-dependently inhibited the nitric oxide production by lipopolysaccharide-stimulated mouse macrophage-like RAW264.7 cells. These data demonstrated that normal oral mesenchymal cells are not the major source of glycine and proline that accumulates in the saliva of aged people, suggesting the involvement of the deregulation of collagen metabolism during aging.

  12. Hypoxia may increase rat insulin mRNA levels by promoting binding of the polypyrimidine tract-binding protein (PTB) to the pyrimidine-rich insulin mRNA 3'-untranslated region.

    PubMed Central

    Tillmar, Linda; Welsh, Nils

    2002-01-01

    BACKGROUND: Recent reports identify the 3'-UTR of insulin mRNA as crucial for control of insulin messenger stability. This region contains a pyrimidine-rich sequence, which is similar to the hypoxia-responsive mRNA-stabilizing element of tyrosine hydroxylase. This study aimed to determine whether hypoxia affects insulin mRNA levels. MATERIALS AND METHODS: Rat islets were incubated at normoxic or hypoxic conditions and with or without hydrogen peroxide and a nitric oxide donor. Insulin mRNA was determined by Northern hybridization. Islet homogenates were used for electrophoretic mobility shift assay with an RNA-oligonucleotide, corresponding to the pyrimidine-rich sequence of the 3'-UTR of rat insulin I mRNA. The expression of reporter gene mRNA, in islets transfected with reporter gene constructs containing the wild-type or mutated insulin mRNA pyrimidine-rich sequences, was measured by semiquantitive RT-PCR. RESULTS: Insulin mRNA was increased in response to hypoxia. This was paralleled by increased binding of the polypyrimidine tract-binding protein (PTB) to the pyrimidine-rich sequence of the 3'-UTR of insulin mRNA, which was counteracted by hydrogen peroxide. The reporter gene mRNA level containing the wild-type binding site was not increased in response to hypoxia, but mutation of the site resulted in a destabilization of the mRNA. CONCLUSIONS: The complete understanding of different diabetic conditions requires the elucidation of mechanisms that control insulin gene expression. Our data show that hypoxia may increase insulin mRNA levels by promoting the binding of PTB to the insulin mRNA 3'-UTR. Hydrogen peroxide abolishes the hypoxic effect indicating involvement of reactive oxygen species and/or the redox potential in the oxygen-signaling pathway. PMID:12359957

  13. Conformation of a group 2 late embryogenesis abundant protein from soybean. Evidence of poly (L-proline)-type II structure.

    PubMed

    Soulages, Jose L; Kim, Kangmin; Arrese, Estela L; Walters, Christina; Cushman, John C

    2003-03-01

    Late embryogenesis abundant (LEA) proteins are members of a large group of hydrophilic, glycine-rich proteins found in plants, algae, fungi, and bacteria known collectively as hydrophilins that are preferentially expressed in response to dehydration or hyperosmotic stress. Group 2 LEA (dehydrins or responsive to abscisic acid) proteins are postulated to stabilize macromolecules against damage by freezing, dehydration, ionic, or osmotic stress. However, the structural and physicochemical properties of group 2 LEA proteins that account for such functions remain unknown. We have analyzed the structural properties of a recombinant form of a soybean (Glycine max) group 2 LEA (rGmDHN1). Differential scanning calorimetry of purified rGmDHN1 demonstrated that the protein does not display a cooperative unfolding transition upon heating. Ultraviolet absorption and circular dichroism spectroscopy revealed that the protein is in a largely hydrated and unstructured conformation in solution. However, ultraviolet absorption and circular dichroism measurements collected at different temperatures showed that the protein exists in equilibrium between two extended conformational states: unordered and left-handed extended helical or poly (L-proline)-type II structures. It is estimated that 27% of the residues of rGmDHN1 adopt or poly (L-proline)-type II-like helical conformation at 12 degrees C. The content of extended helix gradually decreases to 15% as the temperature is increased to 80 degrees C. Studies of the conformation of the protein in solution in the presence of liposomes, trifluoroethanol, and sodium dodecyl sulfate indicated that rGmDHN1 has a very low intrinsic ability to adopt alpha-helical structure and to interact with phospholipid bilayers through amphipathic alpha-helices. The ability of the protein to remain in a highly extended conformation at low temperatures could constitute the basis of the functional role of GmDHN1 in the prevention of freezing, desiccation

  14. A conserved proline residue in Dothideomycete Avr4 effector proteins is required to trigger a Cf-4-dependent hypersensitive response.

    PubMed

    Mesarich, Carl H; Stergiopoulos, Ioannis; Beenen, Henriek G; Cordovez, Viviane; Guo, Yanan; Karimi Jashni, Mansoor; Bradshaw, Rosie E; de Wit, Pierre J G M

    2016-01-01

    CfAvr4, a chitin-binding effector protein produced by the Dothideomycete tomato pathogen Cladosporium fulvum, protects the cell wall of this fungus against hydrolysis by secreted host chitinases during infection. However, in the presence of the Cf-4 immune receptor of tomato, CfAvr4 triggers a hypersensitive response (HR), which renders the pathogen avirulent. Recently, several orthologues of CfAvr4 have been identified from phylogenetically closely related species of Dothideomycete fungi. Of these, DsAvr4 from Dothistroma septosporum also triggers a Cf-4-dependent HR, but CaAvr4 and CbAvr4 from Cercospora apii and Cercospora beticola, respectively, do not. All, however, bind chitin. To identify the region(s) and specific amino acid residue(s) of CfAvr4 and DsAvr4 required to trigger a Cf-4-dependent HR, chimeric and mutant proteins, in which specific protein regions or single amino acid residues, respectively, were exchanged between CfAvr4 and CaAvr4 or DsAvr4 and CbAvr4, were tested for their ability to trigger an HR in Nicotiana benthamiana plants transgenic for the Cf-4 immune receptor gene. Based on this approach, a single region common to CfAvr4 and DsAvr4 was determined to carry a conserved proline residue necessary for the elicitation of this HR. In support of this result, a Cf-4-dependent HR was triggered by mutant CaAvr4 and CbAvr4 proteins carrying an arginine-to-proline substitution at this position. This study provides the first step in deciphering how Avr4 orthologues from different Dothideomycete fungi trigger a Cf-4-dependent HR.

  15. Decreased cysteine and proline synthesis in parenterally fed, premature infants.

    PubMed

    Miller, R G; Jahoor, F; Jaksic, T

    1995-07-01

    Little is known about the amino acid (AA) biosynthetic capacity and requirements of premature infants. This study assessed the synthesis of seven biochemically nonessential AA from a universal precursor, glucose, in stable, parenterally fed, premature neonates. Seven infants (six boys, one girl) were studied at a mean age of 6.3 +/- 0.6 (SEM) days; mean gestational age was 29.7 +/- 1.3 (SEM) weeks, and mean birth weight was 1,222.8 +/- 176.5 (SEM) grams. All infants were parenterally fed a mixture of 7.5% to 12.5% dextrose and 2.2% Trophamine, with or without lipid. Mean caloric intake was 93 +/- 8.4 (SEM) kcal/kg/d, and total AA intake was standardized at 2.86 g/kg/d AA, plus supplemental cysteine (30 mg/g AA/d). Each infant received a 4-hour continuous, unprimed intravenous infusion of a stable isotope tracer of D(-)[U13C] glucose (200 mg/kg). Blood samples were obtained before and at the end of the infusion. Conversion of the glucose tracer into seven biochemically nonessential AA (cysteine [Cys], proline [Pro], aspartate [Asp], serine [Ser], glutamate [Glu], alanine [Ala], and glycine [Gly]) was assessed by measuring their isotopic enrichment in plasma, using gas chromatography/mass spectrometry (GC/MS), and expressed as mole percent excess (MPE) (mean +/- SEM). The isotopic enrichment of plasma glucose was also measured using GC/MS. Free plasma AA concentrations (mean +/- SD) were measured using an automated amino acid analyzer. Mean MPE for M + 1, M + 2 and M + 3 Cys, and for M + 1 and M + 3 Pro were not significantly different from 0; M + 2 Pro barely achieved statistical significance (P = .048).(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Diversification of the plant-specific hybrid glycine-rich protein (HyGRP) genes in cereals

    PubMed Central

    Fujino, Kenji; Obara, Mari; Sato, Koji

    2014-01-01

    Plant-specific hybrid proline- or glycine-rich proteins (HyP/GRPs) are involved in diverse gene functions including plant development and responses to biotic and abiotic stresses. The quantitative trait locus, qLTG3-1, enhances seed germination in rice under low-temperature conditions and encodes a member with a glycine-rich motif of the HyP/GRP family. The function of this gene may be related to the weakening of tissue covering the embryo during seed germination. In the present study, the diversification of the HyP/GRP gene family was elucidated in rice based on phylogenetic relationships and gene expression levels. At least 21 members of the HyP/GRP family have been identified in the rice genome and clustered in five regions on four chromosomes by tandem and chromosomal duplications. Of these, OsHyPRP05 (qLTG3-1) and its paralogous gene, OsHyPRP21, had a glycine-rich motif. Furthermore, orthologous genes with a glycine-rich motif and the HyP/GRP gene family were detected in four genome-sequenced monocots: 12 in barley, 10 in Brachypodium, 20 in maize, and 28 in sorghum, using a BLAST search of qLTG3-1 as the query. All members of the HyP/GRP family in these five species were classified into seven main groups, which were clustered together in these species. These results suggested that the HyP/GRP gene family was formed in the ancestral genome before the divergence of these species. The collinearity of chromosomal regions around qLTG3-1 and its orthologous genes were conserved among rice, Brachypodium, sorghum, and maize, indicating that qLTG3-1 and orthologous genes conserve gene function during seed germination. PMID:25309566

  17. Paneth Cell-Rich Regions Separated by a Cluster of Lgr5+ Cells Initiate Crypt Fission in the Intestinal Stem Cell Niche.

    PubMed

    Langlands, Alistair J; Almet, Axel A; Appleton, Paul L; Newton, Ian P; Osborne, James M; Näthke, Inke S

    2016-06-01

    The crypts of the intestinal epithelium house the stem cells that ensure the continual renewal of the epithelial cells that line the intestinal tract. Crypt number increases by a process called crypt fission, the division of a single crypt into two daughter crypts. Fission drives normal tissue growth and maintenance. Correspondingly, it becomes less frequent in adulthood. Importantly, fission is reactivated to drive adenoma growth. The mechanisms governing fission are poorly understood. However, only by knowing how normal fission operates can cancer-associated changes be elucidated. We studied normal fission in tissue in three dimensions using high-resolution imaging and used intestinal organoids to identify underlying mechanisms. We discovered that both the number and relative position of Paneth cells and Lgr5+ cells are important for fission. Furthermore, the higher stiffness and increased adhesion of Paneth cells are involved in determining the site of fission. Formation of a cluster of Lgr5+ cells between at least two Paneth-cell-rich domains establishes the site for the upward invagination that initiates fission.

  18. Sequencing of a QTL-rich region of the Theobroma cacao genome using pooled BACs and the identification of trait specific candidate genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: BAC-based physical maps provide for sequencing across an entire genome or selected sub-genome regions of biological interest. Using the minimum tiling path as a guide, it is possible to select specific BAC clones from prioritized genome sections such as a genetically defined QTL interv...

  19. Comparative Analysis of the Mitochondrial Genomes of Callitettixini Spittlebugs (Hemiptera: Cercopidae) Confirms the Overall High Evolutionary Speed of the AT-Rich Region but Reveals the Presence of Short Conservative Elements at the Tribal Level

    PubMed Central

    Liu, Jie; Bu, Cuiping; Wipfler, Benjamin; Liang, Aiping

    2014-01-01

    The present study compares the mitochondrial genomes of five species of the spittlebug tribe Callitettixini (Hemiptera: Cercopoidea: Cercopidae) from eastern Asia. All genomes of the five species sequenced are circular double-stranded DNA molecules and range from 15,222 to 15,637 bp in length. They contain 22 tRNA genes, 13 protein coding genes (PCGs) and 2 rRNA genes and share the putative ancestral gene arrangement of insects. The PCGs show an extreme bias of nucleotide and amino acid composition. Significant differences of the substitution rates among the different genes as well as the different codon position of each PCG are revealed by the comparative evolutionary analyses. The substitution speeds of the first and second codon position of different PCGs are negatively correlated with their GC content. Among the five species, the AT-rich region features great differences in length and pattern and generally shows a 2–5 times higher substitution rate than the fastest PCG in the mitochondrial genome, atp8. Despite the significant variability in length, short conservative segments were identified in the AT-rich region within Callitettixini, although absent from the other groups of the spittlebug superfamily Cercopoidea. PMID:25285442

  20. Metallophytes in biotopes polluted by waste dumps rich in Zn-Pb, Cd (Olkusz region) - review of previous and planned research

    NASA Astrophysics Data System (ADS)

    Rożek, Dorota

    2013-09-01

    The aim of that publication was the presentation of previous and planned research concerning selected vascular plants and soils near Olkusz (Southern Poland). The extremely high concentration of heavy metals in soils from that region was caused by the natural geochemical aureoles of dispersed metals (due to weathering of Zn-Pb-Fe ore sulphides) and mining and processing of shallowly occurring metalliferous deposits (containing Ag-Pb and Zn-Pb ores) since XII century. The condition of stress in metals, shortage of water and some plant nutrition led to formation of some adaptable vegetation features by plants growing in that region. Some species called metallophytes have been already detailed investigated. Moreover some geochemical and pedological research of soil have been already done. However the conditions of habitat of pioneering species such as Koeleria glauca and Corynephorus canescens are not still recognized.

  1. Proline Adsorption on TiO2(1 1 0) Single Crystal Surface: A Study by High Resolution Photoelectron Spectroscopy

    SciTech Connect

    Fleming,G.; Adib, K.; Rodriguez, J.; Barteau, M.; Idriss, H.

    2007-01-01

    The surface chemistry and binding of dl-proline were investigated on the oxidised (stoichiometric) and reduced (sub-stoichiometric) TiO2(1 1 0) single crystal surfaces. TiO2 was chosen as the substrate as it best represents the surface of a biomedical implant, which bio-molecules interact with during the healing of bone/teeth fractures (molecular recognition). High resolution X-ray photoelectron spectroscopy (HR-XPS) studies of the C1s and N1s regions revealed that dl-proline is present in two forms (dissociated and zwitterionic) on the oxidised TiO2 surface. On TiO2(1 1 0) surfaces reduced by Ar+ sputtering, a significant increase in the amount of zwitterionic proline at the surface was detected when compared with the oxidised surface. Study of the temperature effect showed that in both cases the zwitterionic structure was the less stable structure. The reason for its relative instability appears to be thermodynamic.

  2. A Solution NMR Investigation into the Murine Amelogenin Splice-Variant LRAP (Leucine-Rich Amelogenin Protein).

    SciTech Connect

    Buchko, Garry W.; Tarasevich, Barbara J.; Roberts, Jacky; Snead, Malcolm L.; Shaw, Wendy J.

    2010-09-01

    Amelogenins are the dominant proteins present in ameloblasts during the early stages of enamel biomineralization, making up >90% of the matrix protein. Along with the full-length protein there are several splice-variant isoforms of amelogenin present including LRAP (Leucine-Rich Amelogenin Protein), a protein that consists of the first 33 and the last 26 residues of full-length amelogenin. Using solution-state NMR spectroscopy we have assigned the 1H-15N HSQC spectrum of murine LRAP (rp(H)LRAP) in 2% acetic acid at pH 3.0 by making extensive use of previous chemical shift assignments for full-length murine amelogenin (rp(H)M180). This correlation was possible because LRAP, like the full-length protein, is intrinsically disordered under these solution conditions. The major difference between the 1H-15N HSQC spectra of rp(H)M180 and rp(H)LRAP was an additional set of amide resonances for each of the seven non-proline residues between S12* and Y12 at the N-terminus of rp(H)LRAP indicating that the N-terminal region of LRAP exists in two different conformations. Analysis of the proline carbon chemical shifts suggest that the molecular basis for the two states is not a cis-trans isomerization of one or more of the proline residues in the N-terminal region and is likely due to a slow exchange process. As observed with rp(H)M180, residue specific changes in molecular dynamics, manifested by the reduction in intensity and disappearance of 1H-15N HSQC cross peaks, were observed with the addition of NaCl to rp(H)LRAP. These perturbations may signal early events governing supramolecular self-assembly of rp(H)LRAP into nanospheres. However, the different pattern of 1H-15N HSQC cross peak perturbation between rp(H)LRAP and rp(H)M180 in high salt suggest that the termini may behave differently in their respective nanospheres, and perhaps, these differences account for the cell signaling properties attributable to LRAP but not the full-length protein.

  3. A solution NMR investigation into the murine amelogenin splice-variant LRAP (Leucine-Rich Amelogenin Protein).

    PubMed

    Buchko, Garry W; Tarasevich, Barbara J; Roberts, Jacky; Snead, Malcolm L; Shaw, Wendy J

    2010-09-01

    Amelogenins are the dominant proteins present in ameloblasts during the early stages of enamel biomineralization, making up >90% of the matrix protein. Along with the full-length protein there are several splice-variant isoforms of amelogenin present including LRAP (Leucine-Rich Amelogenin Protein), a protein that consists of the first 33 and the last 26 residues of full-length amelogenin. Using solution-state NMR spectroscopy we have assigned the (1)H-(15)N HSQC spectrum of murine LRAP (rp(H)LRAP) in 2% acetic acid at pH 3.0 by making extensive use of previous chemical shift assignments for full-length murine amelogenin (rp(H)M180). This correlation was possible because LRAP, like the full-length protein, is intrinsically disordered under these solution conditions. The major difference between the (1)H-(15)N HSQC spectra of rp(H)M180 and rp(H)LRAP was an additional set of amide resonances for each of the seven non-proline residues between S12 and Y12 near the N-terminus of rp(H)LRAP indicating that the N-terminal region of LRAP exists in two different conformations. Analysis of the proline carbon chemical shifts suggests that the molecular basis for the two states is not a cis-trans isomerization of one or more of the proline residues in the N-terminal region. Starting from 2% acetic acid, where rp(H)LRAP was monomeric in solution, NaCl addition effected residue specific changes in molecular dynamics manifested by the reduction in intensity and disappearance of (1)H-(15)N HSQC cross peaks. As observed for the full-length protein, these perturbations may signal early events governing supramolecular self-assembly of rp(H)LRAP into nanospheres. However, the different patterns of (1)H-(15)N HSQC cross peak perturbation between rp(H)LRAP and rp(H)M180 in high salt suggest that the termini may behave differently in their respective nanospheres, and perhaps, these differences contribute to the cell signaling properties attributable to LRAP but not to the full

  4. A solution NMR investigation into the murine amelogenin splice-variant LRAP (Leucine-Rich Amelogenin Protein)

    PubMed Central

    Buchko, Garry W.; Tarasevich, Barbara J.; Roberts, Jacky; Snead, Malcolm L.; Shaw, Wendy J.

    2010-01-01

    Amelogenins are the dominant proteins present in ameloblasts during the early stages of enamel biomineralization, making up >90% of the matrix protein. Along with the full-length protein there are several splice-variant isoforms of amelogenin present including LRAP (Leucine-Rich Amelogenin Protein), a protein that consists of the first 33 and the last 26 residues of full-length amelogenin. Using solution-state NMR spectroscopy we have assigned the 1H-15N HSQC spectrum of murine LRAP (rp(H)LRAP) in 2% acetic acid at pH 3.0 by making extensive use of previous chemical shift assignments for full-length murine amelogenin (rp(H)M180). This correlation was possible because LRAP, like the full-length protein, is intrinsically disordered under these solution conditions. The major difference between the 1H-15N HSQC spectra of rp(H)M180 and rp(H)LRAP was an additional set of amide resonances for each of the seven non-proline residues between S12* and Y12 at the N-terminus of rp(H)LRAP indicating that the N-terminal region of LRAP exists in two different conformations. Analysis of the proline carbon chemical shifts suggest that the molecular basis for the two states is not a cis-trans isomerization of one or more of the proline residues in the N-terminal region. Starting from 2% acetic acid, where rp(H)LRAP was monomeric in solution, NaCl addition effected residue specific changes in molecular dynamics manifested by the reduction in intensity and disappearance of 1H-15N HSQC cross peaks. As observed for the full length protein, these perturbations may signal early events governing supramolecular self-assembly of rp(H)LRAP into nanospheres. However, the different pattern of 1H-15N HSQC cross peak perturbation between rp(H)LRAP and rp(H)M180 in high salt suggest that the termini may behave differently in their respective nanospheres, and perhaps, these differences contribute to the cell signaling properties attributable to LRAP but not the full-length protein. PMID:20304108

  5. Proline Metabolism is Essential for Trypanosoma brucei brucei Survival in the Tsetse Vector

    PubMed Central

    Mantilla, Brian S.; Dyer, Naomi A.; Biran, Marc; Bringaud, Frédéric; Lehane, Michael J.; Acosta-Serrano, Alvaro

    2017-01-01

    Adaptation to different nutritional environments is essential for life cycle completion by all Trypanosoma brucei sub-species. In the tsetse fly vector, L-proline is among the most abundant amino acids and is mainly used by the fly for lactation and to fuel flight muscle. The procyclic (insect) stage of T. b. brucei uses L-proline as its main carbon source, relying on an efficient catabolic pathway to convert it to glutamate, and then to succinate, acetate and alanine as the main secreted end products. Here we investigated the essentiality of an undisrupted proline catabolic pathway in T. b. brucei by studying mitochondrial Δ1-pyrroline-5-carboxylate dehydrogenase (TbP5CDH), which catalyzes the irreversible conversion of gamma-glutamate semialdehyde (γGS) into L-glutamate and NADH. In addition, we provided evidence for the absence of a functional proline biosynthetic pathway. TbP5CDH expression is developmentally regulated in the insect stages of the parasite, but absent in bloodstream forms grown in vitro. RNAi down-regulation of TbP5CDH severely affected the growth of procyclic trypanosomes in vitro in the absence of glucose, and altered the metabolic flux when proline was the sole carbon source. Furthermore, TbP5CDH knocked-down cells exhibited alterations in the mitochondrial inner membrane potential (ΔΨm), respiratory control ratio and ATP production. Also, changes in the proline-glutamate oxidative capacity slightly affected the surface expression of the major surface glycoprotein EP-procyclin. In the tsetse, TbP5CDH knocked-down cells were impaired and thus unable to colonize the fly’s midgut, probably due to the lack of glucose between bloodmeals. Altogether, our data show that the regulated expression of the proline metabolism pathway in T. b. brucei allows this parasite to adapt to the nutritional environment of the tsetse midgut. PMID:28114403

  6. An integrated NIR and TIR approach to the plagiocase-rich regions on the Moon using M3 and Diviner data

    NASA Astrophysics Data System (ADS)

    Donaldson Hanna, K. L.; Pieters, C. M.; Mustard, J. F.; Cheek, L. C.; Wyatt, M. B.; Thomas, I. R.; Bowles, N. E.; Greenhagen, B. T.; Lucey, P. G.; Paige, D. A.

    2011-10-01

    Recent near infrared (NIR) observations from SELENE [1], [2] and M3 [3] have uniquely identified Fe-bearing crystalline plagioclase regions on the Moon. These results are significant because they validate earlier NIR observations [4], [5] as well as characterize the widespread distribution of crystalline plagioclase across the lunar surface. The identification of Fe-bearing crystalline plagioclase in the NIR comes from a broad absorption band at approximately 1.3 μm due to electronic transitions of Fe2+ in the crystal structure. While previous NIR lab studies of plagioclase have suggested that the band depth and center position of the 1.3 μm feature may vary with Fe and An content [6], [7], the relationship between NIR spectral properties of plagioclase and its composition (An#) has yet to be quantified. Regions of nearly pure crystalline plagioclase (<5% olivine and pyroxene) as identified in NIR spectra are ideal areas to investigate the utility of thermal infrared (TIR) Diviner data to constrain plagioclase compositions. Diviner has three spectral bands near the 8 μm region chosen specifically to measure the peak of the Christiansen Feature (CF), an emissivity maximum indicative of composition [8]. New lab measurements of varying compositions of the plagioclase solid solution series demonstrate that the CF position is diagnostic of composition and linearly related to An# even when measured under a simulated lunar environment [9]. Thus an integrated NIR and TIR approach will enable plagioclase compositions to be mapped across the lunar surface and is significant for identifying rock types (e.g. ferroan anorthosites versus Alkali-suite rocks) and may ultimately constrain their method of formation (e. g. magma ocean crystallization or plutons).

  7. Structural Properties and Stereochemically Distinct Folding Preferences of 4,5-cis and trans-Methano-L-Proline Oligomers: The Shortest Crystalline PPII-Type Helical Proline-Derived Tetramer.

    PubMed

    Berger, Gilles; Vilchis-Reyes, Miguel; Hanessian, Stephen

    2015-11-02

    The synthesis, structural properties, and folding patterns of a series of L-proline methanologues represented by cis- and trans-4,5-methano-L-proline amides and their oligomers are reported as revealed by X-ray crystallography, circular dichroism measurements, and DFT calculations. We disclose the first example of a crystalline tetrameric proline congener to exhibit a polyproline II helical conformation. Experimental evidence of PPII-type helical arrangement (both in solution and in the solid state) of cis-4,5-methano-L-proline oligomers is supported by theoretical calculations reflecting the extent of n→π* stabilization of the trans-amide conformation.

  8. The internal region leucine-rich repeat 6 of decorin interacts with low density lipoprotein receptor-related protein-1, modulates transforming growth factor (TGF)-β-dependent signaling, and inhibits TGF-β-dependent fibrotic response in skeletal muscles.

    PubMed

    Cabello-Verrugio, Claudio; Santander, Cristian; Cofré, Catalina; Acuña, Maria José; Melo, Francisco; Brandan, Enrique

    2012-02-24

    Decorin is a small proteoglycan, composed of 12 leucine-rich repeats (LRRs) that modulates the activity of transforming growth factor type β (TGF-β) and other growth factors, and thereby influences proliferation and differentiation in a wide array of physiological and pathological processes, such as fibrosis, in several tissues and organs. Previously we described two novel modulators of the TGF-β-dependent signaling pathway: LDL receptor-related protein (LRP-1) and decorin. Here we have determined the regions in decorin that are responsible for interaction with LRP-1 and are involved in TGF-β-dependent binding and signaling. Specifically, we used decorin deletion mutants, as well as peptides derived from internal LRR regions, to determine the LRRs responsible for these decorin functions. Our results indicate that LRR6 and LRR5 participate in the interaction with LRP-1 and TGF-β as well as in its dependent signaling. Furthermore, the internal region (LRR6i), composed of 11 amino acids, is responsible for decorin binding to LRP-1 and subsequent TGF-β-dependent signaling. Furthermore, using an in vivo approach, we also demonstrate that the LRR6 region of decorin can inhibit TGF-β mediated action in response to skeletal muscle injury.

  9. The Internal Region Leucine-rich Repeat 6 of Decorin Interacts with Low Density Lipoprotein Receptor-related Protein-1, Modulates Transforming Growth Factor (TGF)-β-dependent Signaling, and Inhibits TGF-β-dependent Fibrotic Response in Skeletal Muscles*

    PubMed Central

    Cabello-Verrugio, Claudio; Santander, Cristian; Cofré, Catalina; Acuña, Maria José; Melo, Francisco; Brandan, Enrique

    2012-01-01

    Decorin is a small proteoglycan, composed of 12 leucine-rich repeats (LRRs) that modulates the activity of transforming growth factor type β (TGF-β) and other growth factors, and thereby influences proliferation and differentiation in a wide array of physiological and pathological processes, such as fibrosis, in several tissues and organs. Previously we described two novel modulators of the TGF-β-dependent signaling pathway: LDL receptor-related protein (LRP-1) and decorin. Here we have determined the regions in decorin that are responsible for interaction with LRP-1 and are involved in TGF-β-dependent binding and signaling. Specifically, we used decorin deletion mutants, as well as peptides derived from internal LRR regions, to determine the LRRs responsible for these decorin functions. Our results indicate that LRR6 and LRR5 participate in the interaction with LRP-1 and TGF-β as well as in its dependent signaling. Furthermore, the internal region (LRR6i), composed of 11 amino acids, is responsible for decorin binding to LRP-1 and subsequent TGF-β-dependent signaling. Furthermore, using an in vivo approach, we also demonstrate that the LRR6 region of decorin can inhibit TGF-β mediated action in response to skeletal muscle injury. PMID:22203668

  10. H3.3 demarcates GC-rich coding and subtelomeric regions and serves as potential memory mark for virulence gene expression in Plasmodium falciparum

    PubMed Central

    Fraschka, Sabine Anne-Kristin; Henderson, Rob Wilhelmus Maria; Bártfai, Richárd

    2016-01-01

    Histones, by packaging and organizing the DNA into chromatin, serve as essential building blocks for eukaryotic life. The basic structure of the chromatin is established by four canonical histones (H2A, H2B, H3 and H4), while histone variants are more commonly utilized to alter the properties of specific chromatin domains. H3.3, a variant of histone H3, was found to have diverse localization patterns and functions across species but has been rather poorly studied in protists. Here we present the first genome-wide analysis of H3.3 in the malaria-causing, apicomplexan parasite, P. falciparum, which revealed a complex occupancy profile consisting of conserved and parasite-specific features. In contrast to other histone variants, PfH3.3 primarily demarcates euchromatic coding and subtelomeric repetitive sequences. Stable occupancy of PfH3.3 in these regions is largely uncoupled from the transcriptional activity and appears to be primarily dependent on the GC-content of the underlying DNA. Importantly, PfH3.3 specifically marks the promoter region of an active and poised, but not inactive antigenic variation (var) gene, thereby potentially contributing to immune evasion. Collectively, our data suggest that PfH3.3, together with other histone variants, indexes the P. falciparum genome to functionally distinct domains and contribute to a key survival strategy of this deadly pathogen. PMID:27555062

  11. The Smad3 linker region contains a transcriptional activation domain.

    PubMed

    Wang, Guannan; Long, Jianyin; Matsuura, Isao; He, Dongming; Liu, Fang

    2005-02-15

    Transforming growth factor-beta (TGF-beta)/Smads regulate a wide variety of biological responses through transcriptional regulation of target genes. Smad3 plays a key role in TGF-beta/Smad-mediated transcriptional responses. Here, we show that the proline-rich linker region of Smad3 contains a transcriptional activation domain. When the linker region is fused to a heterologous DNA-binding domain, it activates transcription. We show that the linker region physically interacts with p300. The adenovirus E1a protein, which binds to p300, inhibits the transcriptional activity of the linker region, and overexpression of p300 can rescue the linker-mediated transcriptional activation. In contrast, an adenovirus E1a mutant, which cannot bind to p300, does not inhibit the linker-mediated transcription. The native Smad3 protein lacking the linker region is unable to mediate TGF-beta transcriptional activation responses, although it can be phosphorylated by the TGF-beta receptor at the C-terminal tail and has a significantly increased ability to form a heteromeric complex with Smad4. We show further that the linker region and the C-terminal domain of Smad3 synergize for transcriptional activation in the presence of TGF-beta. Thus our findings uncover an important function of the Smad3 linker region in Smad-mediated transcriptional control.

  12. UV Photodissociation of Proline-containing Peptide Ions: Insights from Molecular Dynamics

    NASA Astrophysics Data System (ADS)

    Girod, Marion; Sanader, Zeljka; Vojkovic, Marin; Antoine, Rodolphe; MacAleese, Luke; Lemoine, Jérôme; Bonacic-Koutecky, Vlasta; Dugourd, Philippe

    2015-03-01

    UV photodissociation of proline-containing peptide ions leads to unusual product ions. In this paper, we report laser-induced dissociation of a series of proline-containing peptides at 213 nm. We observe specific fragmentation pathways corresponding to the formation of (y-2), (a + 2) and (b + 2) fragment ions. This was not observed at 266 nm or for peptides which do not contain proline residues. In order to obtain insights into the fragmentation dynamics at 213 nm, a small peptide (RPK for arginine-proline-lysine) was studied both theoretically and experimentally. Calculations of absorption spectra and non-adiabatic molecular dynamics (MD) were made. Second and third excited singlet states, S2 and S3, lie close to 213 nm. Non-adiabatic MD simulation starting from S2 and S3 shows that these transitions are followed by C-C and C-N bond activation close to the proline residue. After this first relaxation step, consecutive rearrangements and proton transfers are required to produce unusual (y-2), (a + 2) and (b + 2) fragment ions. These fragmentation mechanisms were confirmed by H/D exchange experiments.

  13. Use of prolines for improving growth and other properties of plants and algae

    DOEpatents

    Unkefer, Pat J.; Knight, Thomas J.; Martinez, Rodolfo A.

    2004-12-14

    Increasing the concentration of prolines, such as 2-hydroxy-5-oxoproline, in the foliar portions of plants has been shown to cause an increase in carbon dioxide fixation, growth rate, dry weight, nutritional value (amino acids), nodulation and nitrogen fixation, photosynthetically derived chemical energy, and resistance to insect pests over the same properties for wild type plants. This can be accomplished in four ways: (1) the application of a solution of the proline directly to the foliar portions of the plant by spraying these portions; (2) applying a solution of the proline to the plant roots; (3) genetically engineering the plant and screening to produce lines that over-express glutamine synthetase in the leaves which gives rise to increased concentration of the metabolite, 2-hydroxy-5-oxoproline (this proline is also known as 2-oxoglutaramate); and (4) impairing the glutamine synthetase activity in the plant roots which causes increased glutamine synthetase activity in the leaves which gives rise to increased concentration of 2-hydroxy-5-oxoproline. Prolines have also been found to induce similar effects in algae.

  14. Use of prolines for improving growth and other properties of plants and algae

    DOEpatents

    Unkefer, Pat J.; Knight, Thomas J.; Martinez, Rodolfo A.

    2003-04-29

    Increasing the concentration of prolines such as 2-hydroxy-5-oxoproline, in the foliar portions of plants has been shown to cause an increase in carbon dioxide fixation, growth rate, dry weight, nutritional value (amino acids), nodulation and nitrogen fixation, photosynthetically derived chemical energy, and resistance to insect pests over the same properties for wild type plants. This can be accomplished in four ways: (1) the application of a solution of the proline directly to the foliar portions of the plant by spraying these portions; (2) applying a solution of the proline to the plant roots; (3) genetically engineering the plant and screening to produce lines that overexpress glutamine synthetase in the leaves which gives rise to increased concentration of the metabolite, 2-hydroxy-5-oxoproline (this proline is also known as 2-oxoglutaramnate); and (4) impairing the glutamine synthetase activity in the plant roots which causes increased glutamine synthetase activity in the leaves which gives rise to increased concentration of 2-hydroxy-5-oxoproline. Prolines have also been found to induce similar effects in algae.

  15. Use of prolines for improving growth and other properties of plants and algae

    DOEpatents

    Unkefer, Pat J.; Knight, Thomas J.; Martinez, Rodolfo A.

    2003-07-15

    Increasing the concentration of prolines, such as 2-hydroxy-5-oxoproline, in the foliar portions of plants has been shown to cause an increase in carbon dioxide fixation, growth rate, dry weight, nutritional value (amino acids), nodulation and nitrogen fixation, photosynthetically derived chemical energy, and resistance to insect pests over the same properties for wild type plants. This can be accomplished in four ways: (1) the application of a solution of the proline directly to the foliar portions of the plant by spraying these portions; (2) applying a solution of the proline to the plant roots; (3) genetically engineering the plant and screening to produce lines that over-express glutamine synthetase in the leaves which gives rise to increased concentration of the metabolite, 2-hydroxy-5-oxoproline (this proline is also known as 2-oxoglutaramate); and (4) impairing the glutamine synthetase activity in the plant roots which causes increased glutamine synthetase activity in the leaves which gives rise to increased concentration of 2-hydroxy-5-oxoproline. Prolines have also been found to induce similar effects in algae.

  16. HR-MAS NMR metabolomics of 'Swingle' citrumelo rootstock genetically modified to overproduce proline.

    PubMed

    de Oliveira, Caroline S; Carlos, Eduardo F; Vieira, Luiz G E; Lião, Luciano M; Alcantara, Glaucia B

    2014-08-01

    The accumulation of proline is a typical physiological response to abiotic stresses in higher plants. 'Swingle' citrumelo, an important rootstock for citrus production, has been modified with a mutated Δ(1)-pyrroline-5-carboxylate synthetase gene (VaP5CSF129A) linked to the cauliflower mosaic virus 35S promoter to induce the overproduction of free proline. This paper presents a comparative metabolomic study of nontransgenic versus transgenic 'Swingle' citrumelo plants with high endogenous proline. (1)H high-resolution magic angle spinning nuclear magnetic resonance spectroscopy and multivariate analysis showed significant differences in some metabolites between the nontransgenic and transgenic leaves and roots. The overproduction of proline has reduced the sucrose content in transgenic leaves, revealing a metabolic cost for these plants. In roots, the high level of free proline acts for the adjustment of cation-anion balance, causing the reduction of acetic acid content. The same sucrose level in roots indicates that they can be considered as sucrose sink. Similar behavior may be waited for fruits produced on transgenic rootstock.

  17. Mycorrhizas alter sucrose and proline metabolism in trifoliate orange exposed to drought stress

    PubMed Central

    Wu, Hui-Hui; Zou, Ying-Ning; Rahman, Mohammed Mahabubur; Ni, Qiu-Dan; Wu, Qiang-Sheng

    2017-01-01

    Arbuscular mycorrhizal fungi (AMF) can enhance drought tolerance in plants, whereas little is known regarding AMF contribution to sucrose and proline metabolisms under drought stress (DS). In this study, Funneliformis mosseae and Paraglomus occultum were inoculated into trifoliate orange (Poncirus trifoliata) under well watered and DS. Although the 71-days DS notably (P < 0.05) inhibited mycorrhizal colonization, AMF seedlings showed significantly (P < 0.05) higher plant growth performance and leaf relative water content, regardless of soil water status. AMF inoculation significantly (P < 0.05) increased leaf sucrose, glucose and fructose concentration under DS, accompanied with a significant increase of leaf sucrose phosphate synthase, neutral invertase, and net activity of sucrose-metabolized enzymes and a decrease in leaf acid invertase and sucrose synthase activity. AMF inoculation produced no change in leaf ornithine-δ-aminotransferase activity, but significantly (P < 0.05) increased leaf proline dehydrogenase activity and significantly (P < 0.05) decreased leaf both Δ1-pyrroline-5-carboxylate reductase and Δ1-pyrroline-5-carboxylate synthetase activity, resulting in lower proline accumulation in AMF plants under DS. Our results therefore suggest that AMF strongly altered leaf sucrose and proline metabolism through regulating sucrose- and proline-metabolized enzyme activities, which is important for osmotic adjustment of the host plant. PMID:28181575

  18. Elongation Factor 2 Kinase Is Regulated by Proline Hydroxylation and Protects Cells during Hypoxia.

    PubMed

    Moore, Claire E J; Mikolajek, Halina; Regufe da Mota, Sergio; Wang, Xuemin; Kenney, Justin W; Werner, Jörn M; Proud, Christopher G

    2015-05-01

    Protein synthesis, especially translation elongation, requires large amounts of energy, which is often generated by oxidative metabolism. Elongation is controlled by phosphorylation of eukaryotic elongation factor 2 (eEF2), which inhibits its activity and is catalyzed by eEF2 kinase (eEF2K), a calcium/calmodulin-dependent α-kinase. Hypoxia causes the activation of eEF2K and induces eEF2 phosphorylation independently of previously known inputs into eEF2K. Here, we show that eEF2K is subject to hydroxylation on proline-98. Proline hydroxylation is catalyzed by proline hydroxylases, oxygen-dependent enzymes which are inactivated during hypoxia. Pharmacological inhibition of proline hydroxylases also stimulates eEF2 phosphorylation. Pro98 lies in a universally conserved linker between the calmodulin-binding and catalytic domains of eEF2K. Its hydroxylation partially impairs the binding of calmodulin to eEF2K and markedly limits the calmodulin-stimulated activity of eEF2K. Neuronal cells depend on oxygen, and eEF2K helps to protect them from hypoxia. eEF2K is the first example of a protein directly involved in a major energy-consuming process to be regulated by proline hydroxylation. Since eEF2K is cytoprotective during hypoxia and other conditions of nutrient insufficiency, it may be a valuable target for therapy of poorly vascularized solid tumors.

  19. Tomato QM-like protein protects Saccharomyces cerevisiae cells against oxidative stress by regulating intracellular proline levels.

    PubMed

    Chen, Changbin; Wanduragala, Srimevan; Becker, Donald F; Dickman, Martin B

    2006-06-01

    Exogenous proline can protect cells of Saccharomyces cerevisiae from oxidative stress. We altered intracellular proline levels by overexpressing the proline dehydrogenase gene (PUT1) of S. cerevisiae. Put1p performs the first enzymatic step of proline degradation in S. cerevisiae. Overexpression of Put1p results in low proline levels and hypersensitivity to oxidants, such as hydrogen peroxide and paraquat. A put1-disrupted yeast mutant deficient in Put1p activity has increased protection from oxidative stress and increased proline levels. Following a conditional life/death screen in yeast, we identified a tomato (Lycopersicon esculentum) gene encoding a QM-like protein (tQM) and found that stable expression of tQM in the Put1p-overexpressing strain conferred protection against oxidative damage from H2O2, paraquat, and heat. This protection was correlated with reactive oxygen species (ROS) reduction and increased proline accumulation. A yeast two-hybrid system assay was used to show that tQM physically interacts with Put1p in yeast, suggesting that tQM is directly involved in modulating proline levels. tQM also can rescue yeast from the lethality mediated by the mammalian proapoptotic protein Bax, through the inhibition of ROS generation. Our results suggest that tQM is a component of various stress response pathways and may function in proline-mediated stress tolerance in plants.

  20. Anomalous Seismic Velocity Drop in Iron and Biotite Rich Amphibolite to Granulite Facies Transitional Rocks from Deccan Volcanic Covered 1993 Killari Earthquake Region, Maharashtra (India): a Case Study

    NASA Astrophysics Data System (ADS)

    Pandey, O. P.; Tripathi, Priyanka; Vedanti, Nimisha; Srinivasa Sarma, D.

    2016-07-01

    65 Ma Deccan Volcanic Province of western India forms one of the largest flood basaltic eruptions on the surface of the earth. The nature of the concealed crust below this earthquake prone region, which is marked by several low velocity zones at different depths has hardly been understood. These low velocity zones have been invariably interpreted as fluid-filled zones, genetically connected to earthquake nucleation. While carrying out detailed geological and petrophysical studies on the Late Archean basement cores, obtained from a 617 m deep KLR-1 borehole, drilled in the epicentral zone of 1993 Killari earthquake region of the southern Deccan Volcanic Province, we came across several instances where we observed remarkable drop in measured P-wave velocity in a number of high density cores. We provide detailed petrographic and geological data on 11 such anomalous samples which belong to mid-crustal amphibolite to granulite facies transitional rocks. They are associated with a mean P-wave velocity of 6.02 km/s (range 5.82-6.22 km/s) conforming to granitic upper crust, but in contrast have a high mean density of 2.91 g/cm3 (range 2.75-3.08 g/cm3), which characterise mid to lower crust. This velocity drop, which is as much as 15 % in some cores, is primarily attributed to FeOT enrichment (up to about 23 wt%) during the course of mantle-fluid driven retrogressive metasomatic reactions, caused by exhumation of deep-seated mafic rocks. Presence of Iron content (mainly magnetite), widely seen as opaques in thin sections of the rocks, seems to have resulted into sharp increase in density, as well as mean atomic weight. Our study indicates that the measured V p is inversely related to FeOT content as well as mean atomic weight of the rock.

  1. The effect of a proline residue on the rate of growth and the space group of alpha-spectrin SH3-domain crystals.

    PubMed

    Cámara-Artigas, Ana; Andújar-Sánchez, Monserrat; Ortiz-Salmerón, Emilia; Cuadri, Celia; Casares, Salvador

    2009-12-01

    alpha-Spectrin SH3-domain (Spc-SH3) crystallization is characterized by very fast growth of the crystals in the presence of ammonium sulfate as a precipitant agent. The origin of this behaviour can be attributed to the presence of a proline residue that participates in a crystal contact mimicking the binding of proline-rich sequences to SH3 domains. This residue, Pro20, is located in the RT loop and is the main contact in one of the interfaces present in the orthorhombic Spc-SH3 crystal structures. In order to understand the molecular interactions that are responsible for the very fast crystal growth of the wild-type (WT) Spc-SH3 crystals, the crystal structure of a triple mutant in which the residues Ser19-Pro20-Arg21 in the RT loop have been replaced by Gly19-Asp20-Ser21 (GDS Spc-SH3 mutant) has been solved. The removal of the critical proline residue results in slower nucleation of the Spc-SH3 crystals and a different arrangement of the protein molecules in the unit cell, leading to a crystal that belongs to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 42.231, c = 93.655 A, and that diffracts to 1.45 A resolution. For both WT Spc-SH3 and the GDS mutant, light-scattering experiments showed that a dimer was formed in solution within a few minutes of the addition of 2 M ammonium sulfate at pH 6.5 and allowed the proposal of a mechanism for the nucleation and crystal growth of Spc-SH3 in which the Pro20 residue plays a key role in the rate of crystal growth.

  2. Two subsites in the binding domain of the acetylcholine receptor: an aromatic subsite and a proline subsite.

    PubMed

    Kachalsky, S G; Jensen, B S; Barchan, D; Fuchs, S

    1995-11-07

    The ligand binding site of the nicotinic acetylcholine receptor (AcChoR) is localized in the alpha-subunit within a domain containing the tandem Cys-192 and -193. By analyzing the binding-site region of AcChoR from animal species that are resistant to alpha-neurotoxins, we have previously shown that four residues in this region, at positions 187, 189, 194, and 197, differ between animals sensitive (e.g., mouse) and resistant (e.g., mongoose and snake) to alpha-bungarotoxin (alpha-BTX). In the present study, we performed site-directed mutagenesis on a fragment of the mongoose AcChoR alpha-subunit (residues 122-205) and exchanged residues 187, 189, 194, and 197, either alone or in combination, with those present in the mouse alpha-subunit sequence. Only the mongoose fragment in which all four residues were mutated to the mouse ones exhibited alpha-BTX binding similar to that of the mouse fragment. The mongoose double mutation in which Leu-194 and His-197 were replaced with proline residues, which are present at these positions in the mouse AcChoR and in all other toxin binders, bound alpha-BTX to approximately 60% of the level of binding exhibited by the mouse fragment. In addition, replacement of either Pro-194 or -197 in the mouse fragment with serine and histidine, respectively, markedly decreased alpha-BTX binding. All other mutations resulted in no or just a small increase in alpha-BTX binding. These results have led us to propose two subsites in the binding domain for alpha-BTX: the proline subsite, which includes Pro-194 and -197 and is critical for alpha-BTX binding, and the aromatic subsite, which includes amino acid residues 187 and 189 and determines the extent of alpha-BTX binding.

  3. Two subsites in the binding domain of the acetylcholine receptor: an aromatic subsite and a proline subsite.

    PubMed Central

    Kachalsky, S G; Jensen, B S; Barchan, D; Fuchs, S

    1995-01-01

    The ligand binding site of the nicotinic acetylcholine receptor (AcChoR) is localized in the alpha-subunit within a domain containing the tandem Cys-192 and -193. By analyzing the binding-site region of AcChoR from animal species that are resistant to alpha-neurotoxins, we have previously shown that four residues in this region, at positions 187, 189, 194, and 197, differ between animals sensitive (e.g., mouse) and resistant (e.g., mongoose and snake) to alpha-bungarotoxin (alpha-BTX). In the present study, we performed site-directed mutagenesis on a fragment of the mongoose AcChoR alpha-subunit (residues 122-205) and exchanged residues 187, 189, 194, and 197, either alone or in combination, with those present in the mouse alpha-subunit sequence. Only the mongoose fragment in which all four residues were mutated to the mouse ones exhibited alpha-BTX binding similar to that of the mouse fragment. The mongoose double mutation in which Leu-194 and His-197 were replaced with proline residues, which are present at these positions in the mouse AcChoR and in all other toxin binders, bound alpha-BTX to approximately 60% of the level of binding exhibited by the mouse fragment. In addition, replacement of either Pro-194 or -197 in the mouse fragment with serine and histidine, respectively, markedly decreased alpha-BTX binding. All other mutations resulted in no or just a small increase in alpha-BTX binding. These results have led us to propose two subsites in the binding domain for alpha-BTX: the proline subsite, which includes Pro-194 and -197 and is critical for alpha-BTX binding, and the aromatic subsite, which includes amino acid residues 187 and 189 and determines the extent of alpha-BTX binding. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7479887

  4. Electrochemical investigation of cellular uptake of quantum dots decorated with a proline-rich cell penetrating peptide.

    PubMed

    Marín, Sergio; Pujals, Sílvia; Giralt, Ernest; Merkoçi, Arben

    2011-02-16

    The use of square wave voltammetry to monitor the cellular uptake, in HeLa cells, of quantum dots (QD) decorated with sweet arrow peptide (SAP) is reported. A SAP derivative containing an additional N-terminal cysteine residue (C-SAP) was synthesized using the solid-phase method and conjugated to QDs. The obtained results show that QDs-SAP either interact with the extracellular cell membrane matrix or translocate the bilayer. The first situation, membrane adsorption, is probably a transient state before cellular uptake. Both confocal microscopy and SWV results support the detection of this cellular internalization process. The developed electrochemical investigation technique can provide valuable insights into the study of peptide-mediated delivery, as well as the design and development of nanoparticle probes for intracellular imaging, diagnostic, and therapeutic applications. In addition, the described electrochemical interrogation is low cost, is easy to use, and offers future interest for diagnostics including cell analysis.

  5. Methods for Elucidating the Mechanism of Action of Proline-Rich and Other Non-lytic Antimicrobial Peptides.

    PubMed

    Benincasa, Monica; Runti, Giulia; Mardirossian, Mario; Gennaro, Renato; Scocchi, Marco

    2017-01-01

    A distinct group of antimicrobial peptides kills bacteria by interfering with internal cellular functions and without concurrent lytic effects on cell membranes. Here we describe some methods to investigate the mechanisms of action of these antimicrobial peptides. They include assays to detect the possible temporal separation between membrane permeabilization and bacterial killing events, to assess the capacity of antimicrobial peptides to cross the bacterial membranes and reside in the cytoplasm, and later to inhibit vital cell functions such as DNA transcription and protein translation.

  6. Induction of unique structural changes in guanine-rich DNA regions by the triazoloacridone C-1305, a topoisomerase II inhibitor with antitumor activities

    PubMed Central

    Lemke, Krzysztof; Wojciechowski, Marcin; Laine, William; Bailly, Christian; Colson, Pierre; Baginski, Maciej; Larsen, Annette K.; Skladanowski, Andrzej

    2005-01-01

    We recently reported that the antitumor triazoloacridone, compound C-1305, is a topoisomerase II poison with unusual properties. In this study we characterize the DNA interactions of C-1305 in vitro, in comparison with other topoisomerase II inhibitors. Our results show that C-1305 binds to DNA by intercalation and possesses higher affinity for GC- than AT-DNA as revealed by surface plasmon resonance studies. Chemical probing with DEPC indicated that C-1305 induces structural perturbations in DNA regions with three adjacent guanine residues. Importantly, this effect was highly specific for C-1305 since none of the other 22 DNA interacting drugs tested was able to induce similar structural changes in DNA. Compound C-1305 induced stronger structural changes in guanine triplets at higher pH which suggested that protonation/deprotonation of the drug is important for this drug-specific effect. Molecular modeling analysis predicts that the zwitterionic form of C-1305 intercalates within the guanine triplet, resulting in widening of both DNA grooves and aligning of the triazole ring with the N7 atoms of guanines. Our results show that C-1305 binds to DNA and induces very specific and unusual structural changes in guanine triplets which likely plays an important role in the cytotoxic and antitumor activity of this unique compound. PMID:16254080