C-Terminal Tyrosine Residue Modifications Modulate the Protective Phosphorylation of Serine 129 of α-Synuclein in a Yeast Model of Parkinson's Disease.

Science.gov (United States)

Kleinknecht, Alexandra; Popova, Blagovesta; Lázaro, Diana F; Pinho, Raquel; Valerius, Oliver; Outeiro, Tiago F; Braus, Gerhard H

2016-06-01

Parkinson´s disease (PD) is characterized by the presence of proteinaceous inclusions called Lewy bodies that are mainly composed of α-synuclein (αSyn). Elevated levels of oxidative or nitrative stresses have been implicated in αSyn related toxicity. Phosphorylation of αSyn on serine 129 (S129) modulates autophagic clearance of inclusions and is prominently found in Lewy bodies. The neighboring tyrosine residues Y125, Y133 and Y136 are phosphorylation and nitration sites. Using a yeast model of PD, we found that Y133 is required for protective S129 phosphorylation and for S129-independent proteasome clearance. αSyn can be nitrated and form stable covalent dimers originating from covalent crosslinking of two tyrosine residues. Nitrated tyrosine residues, but not di-tyrosine-crosslinked dimers, contributed to αSyn cytotoxicity and aggregation. Analysis of tyrosine residues involved in nitration and crosslinking revealed that the C-terminus, rather than the N-terminus of αSyn, is modified by nitration and di-tyrosine formation. The nitration level of wild-type αSyn was higher compared to that of A30P mutant that is non-toxic in yeast. A30P formed more dimers than wild-type αSyn, suggesting that dimer formation represents a cellular detoxification pathway in yeast. Deletion of the yeast flavohemoglobin gene YHB1 resulted in an increase of cellular nitrative stress and cytotoxicity leading to enhanced aggregation of A30P αSyn. Yhb1 protected yeast from A30P-induced mitochondrial fragmentation and peroxynitrite-induced nitrative stress. Strikingly, overexpression of neuroglobin, the human homolog of YHB1, protected against αSyn inclusion formation in mammalian cells. In total, our data suggest that C-terminal Y133 plays a major role in αSyn aggregate clearance by supporting the protective S129 phosphorylation for autophagy and by promoting proteasome clearance. C-terminal tyrosine nitration increases pathogenicity and can only be partially detoxified by �

Molecular dissection of the C-terminal regulatory domain of the plant plasma membrane H+-ATPase AHA2: Mapping of residues that when altered give rise to an activated enzyme

DEFF Research Database (Denmark)

Axelsen, K.B.; Venema, K.; Jah, T.

1999-01-01

in an extension of the C-terminus unique to plant H+-ATPases, Alteration of residues in both regions led to increased binding of yeast 14-3-3 protein to the plasma membrane of transformed cells. Taken together, our data suggest that modification of residues in two regions of the C-terminal regulatory domain......The plasma membrane H+-ATPase is a proton pump belonging to the P-type ATPase superfamily and is important for nutrient acquisition in plants, The H+-ATPase is controlled by an autoinhibitory C-terminal regulatory domain and is activated by 14-3-3 proteins which bind to this part of the enzyme......+-ATPase. The enzymes were characterized by their ability to promote growth in acidic conditions and to promote H+ extrusion from intact cells, both of which are measures of plasma membrane H+-ATPase activity, and were also characterized with respect to kinetic properties such as affinity for H+ and ATP. Residues...

Gamow-Jordan vectors and non-reducible density operators from higher-order S-matrix poles

International Nuclear Information System (INIS)

Bohm, A.; Loewe, M.; Maxson, S.; Patuleanu, P.; Puentmann, C.; Gadella, M.

1997-01-01

In analogy to Gamow vectors that are obtained from first-order resonance poles of the S-matrix, one can also define higher-order Gamow vectors which are derived from higher-order poles of the S-matrix. An S-matrix pole of r-th order at z R =E R -iΓ/2 leads to r generalized eigenvectors of order k=0,1,hor-ellipsis,r-1, which are also Jordan vectors of degree (k+1) with generalized eigenvalue (E R -iΓ/2). The Gamow-Jordan vectors are elements of a generalized complex eigenvector expansion, whose form suggests the definition of a state operator (density matrix) for the microphysical decaying state of this higher-order pole. This microphysical state is a mixture of non-reducible components. In spite of the fact that the k-th order Gamow-Jordan vectors has the polynomial time-dependence which one always associates with higher-order poles, the microphysical state obeys a purely exponential decay law. copyright 1997 American Institute of Physics

Characterization of a digestive carboxypeptidase from the insect pest corn earworm (Helicoverpa armigera) with novel specificity towards C-terminal glutamate residues.

Science.gov (United States)

Bown, David P; Gatehouse, John A

2004-05-01

Carboxypeptidases were purified from guts of larvae of corn earworm (Helicoverpa armigera), a lepidopteran crop pest, by affinity chromatography on immobilized potato carboxypeptidase inhibitor, and characterized by N-terminal sequencing. A larval gut cDNA library was screened using probes based on these protein sequences. cDNA HaCA42 encoded a carboxypeptidase with sequence similarity to enzymes of clan MC [Barrett, A. J., Rawlings, N. D. & Woessner, J. F. (1998) Handbook of Proteolytic Enzymes. Academic Press, London.], but with a novel predicted specificity towards C-terminal acidic residues. This carboxypeptidase was expressed as a recombinant proprotein in the yeast Pichia pastoris. The expressed protein could be activated by treatment with bovine trypsin; degradation of bound pro-region, rather than cleavage of pro-region from mature protein, was the rate-limiting step in activation. Activated HaCA42 carboxypeptidase hydrolysed a synthetic substrate for glutamate carboxypeptidases (FAEE, C-terminal Glu), but did not hydrolyse substrates for carboxypeptidase A or B (FAPP or FAAK, C-terminal Phe or Lys) or methotrexate, cleaved by clan MH glutamate carboxypeptidases. The enzyme was highly specific for C-terminal glutamate in peptide substrates, with slow hydrolysis of C-terminal aspartate also observed. Glutamate carboxypeptidase activity was present in larval gut extract from H. armigera. The HaCA42 protein is the first glutamate-specific metallocarboxypeptidase from clan MC to be identified and characterized. The genome of Drosophila melanogaster contains genes encoding enzymes with similar sequences and predicted specificity, and a cDNA encoding a similar enzyme has been isolated from gut tissue in tsetse fly. We suggest that digestive carboxypeptidases with sequence similarity to the classical mammalian enzymes, but with specificity towards C-terminal glutamate, are widely distributed in insects.

Substitutions of PrP N-terminal histidine residues modulate scrapie disease pathogenesis and incubation time in transgenic mice.

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Eigenbrod, Sabina; Frick, Petra; Bertsch, Uwe; Mitteregger-Kretzschmar, Gerda; Mielke, Janina; Maringer, Marko; Piening, Niklas; Hepp, Alexander; Daude, Nathalie; Windl, Otto; Levin, Johannes; Giese, Armin; Sakthivelu, Vignesh; Tatzelt, Jörg; Kretzschmar, Hans; Westaway, David

2017-01-01

Prion diseases have been linked to impaired copper homeostasis and copper induced-oxidative damage to the brain. Divalent metal ions, such as Cu2+ and Zn2+, bind to cellular prion protein (PrPC) at octapeptide repeat (OR) and non-OR sites within the N-terminal half of the protein but information on the impact of such binding on conversion to the misfolded isoform often derives from studies using either OR and non-OR peptides or bacterially-expressed recombinant PrP. Here we created new transgenic mouse lines expressing PrP with disrupted copper binding sites within all four histidine-containing OR's (sites 1-4, H60G, H68G, H76G, H84G, "TetraH>G" allele) or at site 5 (composed of residues His-95 and His-110; "H95G" allele) and monitored the formation of misfolded PrP in vivo. Novel transgenic mice expressing PrP(TetraH>G) at levels comparable to wild-type (wt) controls were susceptible to mouse-adapted scrapie strain RML but showed significantly prolonged incubation times. In contrast, amino acid replacement at residue 95 accelerated disease progression in corresponding PrP(H95G) mice. Neuropathological lesions in terminally ill transgenic mice were similar to scrapie-infected wt controls, but less severe. The pattern of PrPSc deposition, however, was not synaptic as seen in wt animals, but instead dense globular plaque-like accumulations of PrPSc in TgPrP(TetraH>G) mice and diffuse PrPSc deposition in (TgPrP(H95G) mice), were observed throughout all brain sections. We conclude that OR and site 5 histidine substitutions have divergent phenotypic impacts and that cis interactions between the OR region and the site 5 region modulate pathogenic outcomes by affecting the PrP globular domain.

Modeling the Influence of Diffusion-Controlled Reactions and Residual Termination and Deactivation on the Rate and Control of Bulk ATRP at High Conversions

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Ali Mohammad Rabea

2015-04-01

Full Text Available In high-conversion atom transfer radical polymerization (ATRP, all the reactions, such as radical termination, radical deactivation, dormant chain activation, monomer propagation, etc. could become diffusion controlled sooner or later, depending on relative diffusivities of the involved reacting species. These diffusion-controlled reactions directly affect the rate of polymerization and the control of polymer molecular weight. A model is developed to investigate the influence of diffusion-controlled reactions on the high conversion ATRP kinetics. Model simulation reveals that diffusion-controlled termination slightly increases the rate, but it is the diffusion-controlled deactivation that causes auto-acceleration in the rate (“gel effect” and loss of control. At high conversions, radical chains are “trapped” because of high molecular weight. However, radical centers can still migrate through (1 radical deactivation–activation cycles and (2 monomer propagation, which introduce “residual termination” reactions. It is found that the “residual termination” does not have much influence on the polymerization kinetics. The migration of radical centers through propagation can however facilitate catalytic deactivation of radicals, which improves the control of polymer molecular weight to some extent. Dormant chain activation and monomer propagation also become diffusion controlled and finally stop the polymerization when the system approaches its glass state.

Fragment-Specific Fixation Versus Volar Locking Plates in Primarily Nonreducible or Secondarily Redisplaced Distal Radius Fractures: A Randomized Controlled Study.

Science.gov (United States)

Landgren, Marcus; Abramo, Antonio; Geijer, Mats; Kopylov, Philippe; Tägil, Magnus

2017-03-01

To compare the patient-reported, clinical, and radiographic outcome of 2 methods of internal fixation in distal radius fractures. Fifty patients, mean age 56 years (range, 21-69 years) with primarily nonreducible or secondarily redisplaced distal radius fractures were randomized to open reduction internal fixation using volar locking plates (n = 25) or fragment-specific fixation (n = 25). The patients were assessed on grip strength, range of motion, patient-reported outcome (Quick Disabilities of the Arm, Shoulder, and Hand), pain (visual analog scale), health-related quality of life (Short Form-12 [SF-12]), and radiographic evaluation. Grip strength at 12 months was the primary outcome measure. At 12 months, no difference was found in grip strength, which was 90% of the uninjured side in the volar plate group and 87% in the fragment-specific fixation group. No differences were found in range of motion and the median Quick Disabilities of the Arm, Shoulder, and Hand score was 5 in both groups. The overall complication rate was significant, 21% in the volar locking plate group, compared with 52% in the fragment-specific group. In treatment of primarily nonreducible or secondarily redisplaced distal radius fractures, volar locking plates and fragment-specific fixation both achieve good and similar patient-reported outcomes, although more complications were recorded in the fragment-specific group. Therapeutic II. Copyright © 2017 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.

Feline Immunodeficiency Virus Vif N-Terminal Residues Selectively Counteract Feline APOBEC3s.

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Gu, Qinyong; Zhang, Zeli; Cano Ortiz, Lucía; Franco, Ana Cláudia; Häussinger, Dieter; Münk, Carsten

2016-12-01

Feline immunodeficiency virus (FIV) Vif protein counteracts feline APOBEC3s (FcaA3s) restriction factors by inducing their proteasomal degradation. The functional domains in FIV Vif for interaction with FcaA3s are poorly understood. Here, we have identified several motifs in FIV Vif that are important for selective degradation of different FcaA3s. Cats (Felis catus) express three types of A3s: single-domain A3Z2, single-domain A3Z3, and double-domain A3Z2Z3. We proposed that FIV Vif would selectively interact with the Z2 and the Z3 A3s. Indeed, we identified two N-terminal Vif motifs (12LF13 and 18GG19) that specifically interacted with the FcaA3Z2 protein but not with A3Z3. In contrast, the exclusive degradation of FcaA3Z3 was regulated by a region of three residues (M24, L25, and I27). Only a FIV Vif carrying a combination of mutations from both interaction sites lost the capacity to degrade and counteract FcaA3Z2Z3. However, alterations in the specific A3s interaction sites did not affect the cellular localization of the FIV Vif protein and binding to feline A3s. Pulldown experiments demonstrated that the A3 binding region localized to FIV Vif residues 50 to 80, outside the specific A3 interaction domain. Finally, we found that the Vif sites specific to individual A3s are conserved in several FIV lineages of domestic cat and nondomestic cats, while being absent in the FIV Vif of pumas. Our data support a complex model of multiple Vif-A3 interactions in which the specific region for selective A3 counteraction is discrete from a general A3 binding domain. Both human immunodeficiency virus (HIV) and feline immunodeficiency virus (FIV) Vif proteins counteract their host's APOBEC3 restriction factors. However, these two Vif proteins have limited sequence homology. The molecular interaction between FIV Vif and feline APOBEC3s are not well understood. Here, we identified N-terminal FIV Vif sites that regulate the selective interaction of Vif with either feline APOBEC3Z

Order through disorder: hyper-mobile C-terminal residues stabilize the folded state of a helical peptide. a molecular dynamics study.

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Kalliopi K Patapati

Full Text Available Conventional wisdom has it that the presence of disordered regions in the three-dimensional structures of polypeptides not only does not contribute significantly to the thermodynamic stability of their folded state, but, on the contrary, that the presence of disorder leads to a decrease of the corresponding proteins' stability. We have performed extensive 3.4 µs long folding simulations (in explicit solvent and with full electrostatics of an undecamer peptide of experimentally known helical structure, both with and without its disordered (four residue long C-terminal tail. Our simulations clearly indicate that the presence of the apparently disordered (in structural terms C-terminal tail, increases the thermodynamic stability of the peptide's folded (helical state. These results show that at least for the case of relatively short peptides, the interplay between thermodynamic stability and the apparent structural stability can be rather subtle, with even disordered regions contributing significantly to the stability of the folded state. Our results have clear implications for the understanding of peptide energetics and the design of foldable peptides.

Obstetric outcomes in reduced and non-reduced twin pregnancies. A single hospital experience

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UmmKulthoum E. AlShelaly

2015-09-01

Full Text Available Objectives: To compare pregnancy outcomes between high-order multiple pregnancies resulting from assisted reproductive technology (ART reduced to twins and non-reduced pregnancies, and to evaluate indications for using ART. Methods: This is a descriptive retrospective review of women with high-order multiple pregnancies reduced to twin carried out at the Department of Obstetrics & Gynecology, King Faisal Specialist Hospital & Research Center, Riyadh, Saudi Arabia between December 2010 and December 2013. The control group consisted of subjects with twin pregnancies who received their fertility treatment at the same hospital during the same period. Results: One hundred and twelve women were included in this study. Of women reaching fetal viability, significantly more women delivered before the thirtieth week in the study group (50% versus 12%, p<0.004. Miscarriage/delivery prior to fetal viability, chorioamnionitis, and preterm premature rupture of membranes were statistically higher in the study group. A total of 83% of the miscarriages in the study group were in women carrying 4 or more fetuses initially, and 50% of women in the study group were multiparous with no clear indication for fertility treatment. Conclusion: Although fetal reduction is a safe procedure, it is associated with complications. Primary prevention of high-order multiple pregnancy is recommended.

Distributional map of the terminal and sub-terminal sugar residues of the glycoconjugates in the prepubertal and postpubertal testis of a subject affected by complete androgen insensitivity syndrome (Morris's syndrome): lectin histochemical study.

Science.gov (United States)

Gheri, G; Vannelli, G B; Marini, M; Zappoli Thyrion, G D; Gheri, R G; Sgambati, E

2004-01-01

In the present research we have investigated the distribution of the sugar residues of the glycoconjugates in the prepubertal and postpubertal testes of a subject with Morris's syndrome (CAIS, Complete Androgen Insensitivity Syndrome). For this purpose a battery of six horseradish peroxidase-conjugated lectins was used (SBA, PNA, WGA, ConA, LTA and UEAI). We have obtained a complete distributional map of the terminal and sub-terminal oligosaccharides in the tunica albuginea, interstitial tissue, lamina propria of the seminiferous tubules, Leydig cells, Sertoli cells, spermatogonia, mastocytes and endothelial cells. Furthermore the present study has shown that a large amount of sugar residues were detectable in the prepubertal and postpubertal testes but that some differences exist with particular regard to the Sertoli cells. The Sertoli cells and the Leydig cells of the retained prepubertal testis of the patient affected by Morris's syndrome were characterized by the presence of alpha-L-fucose, which was absent in the retained prepubertal testis of the normal subjects. Comparing the results on the postpubertal testis with those obtained on the same aged testis of healthy subjects we have demonstrated that alpha-L-fucose in the Sertoli and Leydig cells and D-galactose-N-acetyl-D-galactosamine in the Leydig cells are a unique feature of the subject affected by Morris's syndrome. D-galactose (ss1,3)-N-acetyl-D-galactosamine and sialic acid, which are present in the Leydig cells of the normal testis were never observed in the same cells of the postpubertal testis of the CAIS patient.

Lysine as helix C-capping residue in a synthetic peptide.

Science.gov (United States)

Esposito, G; Dhanapal, B; Dumy, P; Varma, V; Mutter, M; Bodenhausen, G

1997-01-01

The structure of the synthetic peptide CH3CO(Leu-Ser-Leu-Leu-Leu-Ser-Leu)3Lys-NH2 in trifluoroethanol/water 60/40 (volume ratio) was characterized by two-dimensional nmr spectroscopy. The peptide, closely related to the amphiphilic helix models designed by W. F. De-Grado and co-workers to mimic protein ion channels [(1988) Science, Vol. 240, p. 1177-1181], folds into a regular helix spanning residues 1-20. Evidence for a helix C-terminal capping conformation, involving the terminal lysine residue, was observed from Overhauser effects and checked for consistency by restrained molecular dynamics simulations. The side-chain amino group of Lys22 forms a hydrogen bond with the carbonyl of Leu18, and the distorted helical geometry of the terminal dipeptide allows the inclusion of a water bridge between the backbone NH of the Lys22 residue and the carbonyls of Leu19 and Ser20.

Amino-terminal residues of ΔNp63, mutated in ectodermal dysplasia, are required for its transcriptional activity.

Science.gov (United States)

Lena, Anna Maria; Duca, Sara; Novelli, Flavia; Melino, Sonia; Annicchiarico-Petruzzelli, Margherita; Melino, Gerry; Candi, Eleonora

2015-11-13

p63, a member of the p53 family, is a crucial transcription factor for epithelial development and skin homeostasis. Heterozygous mutations in TP63 gene have been associated with human ectodermal dysplasia disorders. Most of these TP63 mutations are missense mutations causing amino acidic substitutions at p63 DNA binding or SAM domains that reduce or abolish the transcriptional activity of mutants p63. A significant number of mutants, however, resides in part of the p63 protein that apparently do not affect DNA binding and/or transcriptional activity, such as the N-terminal domain. Here, we characterize five p63 mutations at the 5' end of TP63 gene aiming to understand the pathogenesis of the diseases and to uncover the role of ΔNp63α N-terminus residues in determining its transactivation potential. Copyright © 2015 Elsevier Inc. All rights reserved.

The N-terminal tail of hERG contains an amphipathic α-helix that regulates channel deactivation.

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Chai Ann Ng

Full Text Available The cytoplasmic N-terminal domain of the human ether-a-go-go related gene (hERG K+ channel is critical for the slow deactivation kinetics of the channel. However, the mechanism(s by which the N-terminal domain regulates deactivation remains to be determined. Here we show that the solution NMR structure of the N-terminal 135 residues of hERG contains a previously described Per-Arnt-Sim (PAS domain (residues 26-135 as well as an amphipathic α-helix (residues 13-23 and an initial unstructured segment (residues 2-9. Deletion of residues 2-25, only the unstructured segment (residues 2-9 or replacement of the α-helix with a flexible linker all result in enhanced rates of deactivation. Thus, both the initial flexible segment and the α-helix are required but neither is sufficient to confer slow deactivation kinetics. Alanine scanning mutagenesis identified R5 and G6 in the initial flexible segment as critical for slow deactivation. Alanine mutants in the helical region had less dramatic phenotypes. We propose that the PAS domain is bound close to the central core of the channel and that the N-terminal α-helix ensures that the flexible tail is correctly orientated for interaction with the activation gating machinery to stabilize the open state of the channel.

Sites of termination of in vitro DNA synthesis on psoralen phototreated single-stranded templates

International Nuclear Information System (INIS)

Piette, J.; Hearst, J.

1985-01-01

Single-stranded DNA has been photochemically induced to react with 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and used as substrate for DNA replication with E. coli DNA polymerase I large fragment. By using the dideoxy sequencing procedure, it is possible to map the termination sites on the template photoreacted with HMT. These sites occur at the nucleotides preceding each thymine residue (and a few cytosine residues), emphasizing the fact that in a single-stranded stretch of DNA, HMT reacts with each thymine residue without any specificity regarding the flanking base sequence of the thymine residues. In addition, termination of DNA synthesis due to psoralen-adducted thymine is not influenced by the efficiency of the 3'-5' exonuclease proof-reading activity of the DNA polymerase. (author)

Structural and dynamic properties of the C-terminal region of the Escherichia coli RNA chaperone Hfq: integrative experimental and computational studies.

Science.gov (United States)

Wen, Bin; Wang, Weiwei; Zhang, Jiahai; Gong, Qingguo; Shi, Yunyu; Wu, Jihui; Zhang, Zhiyong

2017-08-09

In Escherichia coli, hexameric Hfq is an important RNA chaperone that facilitates small RNA-mediated post-transcriptional regulation. The Hfq monomer consists of an evolutionarily conserved Sm domain (residues 1-65) and a flexible C-terminal region (residues 66-102). It has been recognized that the existence of the C-terminal region is important for the function of Hfq, but its detailed structural and dynamic properties remain elusive due to its disordered nature. In this work, using integrative experimental techniques, such as nuclear magnetic resonance spectroscopy and small-angle X-ray scattering, as well as multi-scale computational simulations, new insights into the structure and dynamics of the C-terminal region in the context of the Hfq hexamer are provided. Although the C-terminal region is intrinsically disordered, some residues (83-86) are motionally restricted. The hexameric core may affect the secondary structure propensity of the C-terminal region, due to transient interactions between them. The residues at the rim and the proximal side of the core have significantly more transient contacts with the C-terminal region than those residues at the distal side, which may facilitate the function of the C-terminal region in the release of double-stranded RNAs and the cycling of small non-coding RNAs. Structure ensembles constructed by fitting the experimental data also support that the C-terminal region prefers to locate at the proximal side. From multi-scale simulations, we propose that the C-terminal region may play a dual role of steric effect (especially at the proximal side) and recruitment (at the both sides) in the binding process of RNA substrates. Interestingly, we have found that these motionally restricted residues may serve as important binding sites for the incoming RNAs that is probably driven by favorable electrostatic interactions. These integrative studies may aid in our understanding of the functional role of the C-terminal region of Hfq.

Structure of antigenetic determinants in the amino-terminal region of bovine fibrinogen Aα chain

International Nuclear Information System (INIS)

Tanswell, P.; Reiter, H.; Timpl, R.

1978-01-01

A radioimmunoassay was developed for peptide F-CB1α from the amino of bovine fibrinogen Aα chain, isolated after reduction and carboxymethylation of the multichain disulfide-linked cyanogen bromide peptide F-CB1. Seven out of twelve different rabbit antisera produced against fibrinogen, peptide F-CB1 or Aα chain showed distinct binding to 125 I-labelled F-CB1α. Thrombin cleavage of F-CB1α yielded two fragments: fibrinopeptide A (residues 1-19) and the carboxy-terminal fragment Th2 (residues 20-54). Antisera could be classified into three groups according to whether they recognized antigenic determinants on fibrinopeptide A, on peptide Th2 or as they showed diminished reactions with both fragments. Only little or no cross-reaction was observed with the amino-terminal cyanogen bromide peptides of Bβ and γ chain. Proteolytic fragments of fibrinopeptide A were isolated and tested for inhibitory activity with two antisera. One antiserum contained anitbodies binding selectively to the amino-terminal sequence (residues 4-11) and did not cross-react with human fibrinopeptide A. Another antiserum showed a specific binding restricted to the carboxy-terminal sequence (residues 11-18) and cross-reacted completely with human fibrinopeptide A. These results correlate well with the primary structures of the two fibrinopeptides. The antigenic activity of the peptide fragment Th2 was localized on a 15-residue tryptic peptide derived from the central portion of the sequence. These and further data indicate that at least six different antigenic determinants are present in peptide F-CB1α. (orig.) 891 AJ [de

Comparison of the outcomes between reduced and nonreduced triplet pregnancies achieved by Assisted Reproductive Technology.

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Shiva, Marzieh; Mohammadi Yeganeh, Ladan; Mirzaagha, Elaheh; Chehrazi, Mohammad; Bagheri Lankarani, Narges

2014-10-01

In recent years, the significant increase in multiple pregnancies as a result of assisted reproductive technology (ART) has introduced the concept of multifetal reduction techniques. However, it is still unclear whether there are significant advantages of using this technique. To compare the outcomes of triplet pregnancies achieved by ART managed expectantly with those receiving fetal reduction interventions. In this retrospective study of 115 triplet pregnancies, 57 pregnancies were reduced to twins while 58 were managed expectantly. The fetal loss rate before 24 weeks did not differ between reduced and nonreduced pregnancies (12.3% vs 12.1%). However, the results of those using fetal reduction techniques showed a lower incidence of preterm labour (26.3% vs 50%, P = 0.009), higher mean gestational age at delivery (35.1 ± 2.6 vs 32.4 ± 3.6 weeks, P = 0.002) and higher mean birthweights compared with the control group (2188 ± 547 vs 1674 ± 546 g, P fetal loss. Additionally, there was a lower perinatal mortality, higher live birth rate and lower NICU admission. © 2014 The Royal Australian and New Zealand College of Obstetricians and Gynaecologists.

The arginine residue within the C-terminal active core of Bombyx mori pheromone biosynthesis-activating neuropeptide (PBAN is essential for receptor binding and activation

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Takeshi eKawai

2012-03-01

Full Text Available In most lepidopteran insects, the biosynthesis of sex pheromones is regulated by pheromone biosynthesis activating neuropeptide (PBAN. Bombyx mori PBAN (BomPBAN consists of 33 amino acid residues and contains a C-terminus FSPRLamide motif as the active core. Among neuropeptides containing the FXPRLamide motif, the arginine (Arg, R residue two positions from the C-terminus is highly conserved across several neuropeptides, which can be designated as RXamide peptides. The purpose of this study was to reveal the role of the Arg residue in the BomPBAN active core. We synthesized a ten-residue peptide corresponding to the C-terminal part of BomPBAN with a series of point mutants at the 2nd position (ie, Arg from the C-terminus, termed the C2 position, and measured their efficacy in stimulating Ca2+ influx in insect cells concomitantly expressing a fluorescent PBAN receptor chimera (PBANR-EGFP and loaded with the fluorescent Ca2+ indicator, Fura Red-AM. PBAN analogs with the C2 position replaced with alanine (Ala, A, aspartic acid (Asp, D, serine (Ser, S or L-2-aminooctanoic acid (Aoc decreased PBAN-like activity. RC2A (SKTRYFSPALamide and RC2D (SKTRYFSPDLamide had the lowest activity and could not inhibit the activity of PBAN C10 (SKTRYFSPRLamide. We also prepared Rhodamine Red-labeled PBAN analogs of the mutants and examined their ability to bind PBANR. In contrast to 100 nM Rhodamine Red-PBAN C10, none of the mutants at the same concentration exhibited PBANR binding. Taken together, our results demonstrate that the C2 Arg residue in BomPBAN is essential for PBANR binding and activation.

Activation of human acid sphingomyelinase through modification or deletion of C-terminal cysteine.

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Qiu, Huawei; Edmunds, Tim; Baker-Malcolm, Jennifer; Karey, Kenneth P; Estes, Scott; Schwarz, Cordula; Hughes, Heather; Van Patten, Scott M

2003-08-29

One form of Niemann-Pick disease is caused by a deficiency in the enzymatic activity of acid sphingomyelinase. During efforts to develop an enzyme replacement therapy based on a recombinant form of human acid sphingomyelinase (rhASM), purified preparations of the recombinant enzyme were found to have substantially increased specific activity if cell harvest media were stored for several weeks at -20 degrees C prior to purification. This increase in activity was found to correlate with the loss of the single free thiol on rhASM, suggesting the involvement of a cysteine residue. It was demonstrated that a variety of chemical modifications of the free cysteine on rhASM all result in substantial activation of the enzyme, and the modified cysteine responsible for this activation was shown to be the C-terminal residue (Cys629). Activation was also achieved by copper-promoted dimerization of rhASM (via cysteine) and by C-terminal truncation using carboxypeptidase Y. The role of the C-terminal cysteine in activation was confirmed by creating mutant forms of rhASM in which this residue was either deleted or replaced by a serine, with both forms having substantially higher specific activity than wild-type rhASM. These results indicate that purified rhASM can be activated in vitro by loss of the free thiol on the C-terminal cysteine via chemical modification, dimerization, or deletion of this amino acid residue. This method of activation is similar to the cysteine switch mechanism described previously for matrix metalloproteinases and could represent a means of posttranslational regulation of ASM activity in vivo.

Interaction of C-terminal truncated human alphaA-crystallins with target proteins.

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Anbarasu Kumarasamy

2008-09-01

Full Text Available Significant portion of alphaA-crystallin in human lenses exists as C-terminal residues cleaved at residues 172, 168, and 162. Chaperone activity, determined with alcohol dehydrogenase (ADH and betaL-crystallin as target proteins, was increased in alphaA(1-172 and decreased in alphaA(1-168 and alphaA(1-162. The purpose of this study was to show whether the absence of the C-terminal residues influences protein-protein interactions with target proteins.Our hypothesis is that the chaperone-target protein binding kinetics, otherwise termed subunit exchange rates, are expected to reflect the changes in chaperone activity. To study this, we have relied on fluorescence resonance energy transfer (FRET utilizing amine specific and cysteine specific fluorescent probes. The subunit exchange rate (k for ADH and alphaA(1-172 was nearly the same as that of ADH and alphaA-wt, alphaA(1-168 had lower and alphaA(1-162 had the lowest k values. When betaL-crystallin was used as the target protein, alphaA(1-172 had slightly higher k value than alphaA-wt and alphaA(1-168 and alphaA(1-162 had lower k values. As expected from earlier studies, the chaperone activity of alphaA(1-172 was slightly better than that of alphaA-wt, the chaperone activity of alphaA(1-168 was similar to that of alphaA-wt and alphaA(1-162 had substantially decreased chaperone activity.Cleavage of eleven C-terminal residues including Arg-163 and the C-terminal flexible arm significantly affects the interaction with target proteins. The predominantly hydrophilic flexible arm appears to be needed to keep the chaperone-target protein complex soluble.

Two distinct binding modes define the interaction of Brox with the C-terminal tails of CHMP5 and CHMP4B.

Science.gov (United States)

Mu, Ruiling; Dussupt, Vincent; Jiang, Jiansheng; Sette, Paola; Rudd, Victoria; Chuenchor, Watchalee; Bello, Nana F; Bouamr, Fadila; Xiao, Tsan Sam

2012-05-09

Interactions of the CHMP protein carboxyl terminal tails with effector proteins play important roles in retroviral budding, cytokinesis, and multivesicular body biogenesis. Here we demonstrate that hydrophobic residues at the CHMP4B C-terminal amphipathic α helix bind a concave surface of Brox, a mammalian paralog of Alix. Unexpectedly, CHMP5 was also found to bind Brox and specifically recruit endogenous Brox to detergent-resistant membrane fractions through its C-terminal 20 residues. Instead of an α helix, the CHMP5 C-terminal tail adopts a tandem β-hairpin structure that binds Brox at the same site as CHMP4B. Additional Brox:CHMP5 interface is furnished by a unique CHMP5 hydrophobic pocket engaging the Brox residue Y348 that is not conserved among the Bro1 domains. Our studies thus unveil a β-hairpin conformation of the CHMP5 protein C-terminal tail, and provide insights into the overlapping but distinct binding profiles of ESCRT-III and the Bro1 domain proteins. Copyright © 2012 Elsevier Ltd. All rights reserved.

Three C-terminal residues from the sulphonylurea receptor contribute to the functional coupling between the KATP channel subunits SUR2A and Kir6.2

Science.gov (United States)

Dupuis, Julien P; Revilloud, Jean; Moreau, Christophe J; Vivaudou, Michel

2008-01-01

Cardiac ATP-sensitive potassium (KATP) channels are metabolic sensors formed by the association of the inward rectifier potassium channel Kir6.2 and the sulphonylurea receptor SUR2A. SUR2A adjusts channel gating as a function of intracellular ATP and ADP and is the target of pharmaceutical openers and blockers which, respectively, up- and down-regulate Kir6.2. In an effort to understand how effector binding to SUR2A translates into Kir6.2 gating modulation, we examined the role of a 65-residue SUR2A fragment linking transmembrane domain TMD2 and nucleotide-binding domain NBD2 that has been shown to interact with Kir6.2. This fragment of SUR2A was replaced by the equivalent residues of its close homologue, the multidrug resistance protein MRP1. The chimeric construct was expressed in Xenopus oocytes and characterized using the patch-clamp technique. We found that activation by MgADP and synthetic openers was greatly attenuated although apparent affinities were unchanged. Further chimeragenetic and mutagenetic studies showed that mutation of three residues, E1305, I1310 and L1313 (rat numbering), was sufficient to confer this defective phenotype. The same mutations had no effects on channel block by the sulphonylurea glibenclamide or by ATP, suggesting a role for these residues in activatory – but not inhibitory – transduction processes. These results indicate that, within the KATP channel complex, the proximal C-terminal of SUR2A is a critical link between ligand binding to SUR2A and Kir6.2 up-regulation. PMID:18450778

Dimeric structure of the N-terminal domain of PriB protein from Thermoanaerobacter tengcongensis solved ab initio

Energy Technology Data Exchange (ETDEWEB)

Liebschner, Dorothee [National Cancer Institute, Argonne National Laboratory, Argonne, IL 60439 (United States); Brzezinski, Krzysztof [National Cancer Institute, Argonne National Laboratory, Argonne, IL 60439 (United States); University of Bialystok, 15-399 Bialystok (Poland); Dauter, Miroslawa [Argonne National Laboratory, Argonne, IL 60439 (United States); Dauter, Zbigniew, E-mail: dauter@anl.gov [National Cancer Institute, Argonne National Laboratory, Argonne, IL 60439 (United States); Nowak, Marta; Kur, Józef; Olszewski, Marcin, E-mail: dauter@anl.gov [Gdansk University of Technology, 80-952 Gdansk (Poland); National Cancer Institute, Argonne National Laboratory, Argonne, IL 60439 (United States)

2012-12-01

The N-terminal domain of the PriB protein from the thermophilic bacterium T. tengcongensis (TtePriB) was expressed and its crystal structure has been solved at the atomic resolution of 1.09 Å by direct methods. PriB is one of the components of the bacterial primosome, which catalyzes the reactivation of stalled replication forks at sites of DNA damage. The N-terminal domain of the PriB protein from the thermophilic bacterium Thermoanaerobacter tengcongensis (TtePriB) was expressed and its crystal structure was solved at the atomic resolution of 1.09 Å by direct methods. The protein chain, which encompasses the first 104 residues of the full 220-residue protein, adopts the characteristic oligonucleotide/oligosaccharide-binding (OB) structure consisting of a five-stranded β-barrel filled with hydrophobic residues and equipped with four loops extending from the barrel. In the crystal two protomers dimerize, forming a six-stranded antiparallel β-sheet. The structure of the N-terminal OB domain of T. tengcongensis shows significant differences compared with mesophile PriBs. While in all other known structures of PriB a dimer is formed by two identical OB domains in separate chains, TtePriB contains two consecutive OB domains in one chain. However, sequence comparison of both the N-terminal and the C-terminal domains of TtePriB suggests that they have analogous structures and that the natural protein possesses a structure similar to a dimer of two N-terminal domains.

Multiple roles of genome-attached bacteriophage terminal proteins

International Nuclear Information System (INIS)

Redrejo-Rodríguez, Modesto; Salas, Margarita

2014-01-01

Protein-primed replication constitutes a generalized mechanism to initiate DNA or RNA synthesis in linear genomes, including viruses, gram-positive bacteria, linear plasmids and mobile elements. By this mechanism a specific amino acid primes replication and becomes covalently linked to the genome ends. Despite the fact that TPs lack sequence homology, they share a similar structural arrangement, with the priming residue in the C-terminal half of the protein and an accumulation of positively charged residues at the N-terminal end. In addition, various bacteriophage TPs have been shown to have DNA-binding capacity that targets TPs and their attached genomes to the host nucleoid. Furthermore, a number of bacteriophage TPs from different viral families and with diverse hosts also contain putative nuclear localization signals and localize in the eukaryotic nucleus, which could lead to the transport of the attached DNA. This suggests a possible role of bacteriophage TPs in prokaryote-to-eukaryote horizontal gene transfer. - Highlights: • Protein-primed genome replication constitutes a strategy to initiate DNA or RNA synthesis in linear genomes. • Bacteriophage terminal proteins (TPs) are covalently attached to viral genomes by their primary function priming DNA replication. • TPs are also DNA-binding proteins and target phage genomes to the host nucleoid. • TPs can also localize in the eukaryotic nucleus and may have a role in phage-mediated interkingdom gene transfer

Multiple roles of genome-attached bacteriophage terminal proteins

Energy Technology Data Exchange (ETDEWEB)

Redrejo-Rodríguez, Modesto; Salas, Margarita, E-mail: msalas@cbm.csic.es

2014-11-15

Protein-primed replication constitutes a generalized mechanism to initiate DNA or RNA synthesis in linear genomes, including viruses, gram-positive bacteria, linear plasmids and mobile elements. By this mechanism a specific amino acid primes replication and becomes covalently linked to the genome ends. Despite the fact that TPs lack sequence homology, they share a similar structural arrangement, with the priming residue in the C-terminal half of the protein and an accumulation of positively charged residues at the N-terminal end. In addition, various bacteriophage TPs have been shown to have DNA-binding capacity that targets TPs and their attached genomes to the host nucleoid. Furthermore, a number of bacteriophage TPs from different viral families and with diverse hosts also contain putative nuclear localization signals and localize in the eukaryotic nucleus, which could lead to the transport of the attached DNA. This suggests a possible role of bacteriophage TPs in prokaryote-to-eukaryote horizontal gene transfer. - Highlights: • Protein-primed genome replication constitutes a strategy to initiate DNA or RNA synthesis in linear genomes. • Bacteriophage terminal proteins (TPs) are covalently attached to viral genomes by their primary function priming DNA replication. • TPs are also DNA-binding proteins and target phage genomes to the host nucleoid. • TPs can also localize in the eukaryotic nucleus and may have a role in phage-mediated interkingdom gene transfer.

Dissipation and residue of bifenthrin in wheat under field conditions.

Science.gov (United States)

You, Xiangwei; Jiang, Naiwen; Liu, Fengmao; Liu, Congyun; Wang, Suli

2013-02-01

Field trials were carried out to investigate the dissipation and residue levels of bifenthrin in wheat. After extraction with acetonitrile, the samples were cleaned up by dispersive solid-phase extraction and detected by gas chromatography-mass spectrometry. The half-lives of bifenthrin in wheat seedlings ranged from 2.4 to 10.5 days. At harvest time, the terminal residues of bifenthrin were below the maximum residue limit (0.5 mg/kg) set by Codex Alimentarius Committee or European Union in wheat grain, which suggested that the use of this pesticide was safe for humans. However, the relatively high residue levels of bifenthrin in wheat straw should be paid attention to.

Properties of catalase-peroxidase lacking its C-terminal domain

International Nuclear Information System (INIS)

Baker, Ruletha D.; Cook, Carma O.; Goodwin, Douglas C.

2004-01-01

Catalase-peroxidases have a two-domain structure. The N-terminal domain contains the bifunctional active site, but the function of the C-terminal domain is unknown. We produced catalase-peroxidase containing only its N-terminal domain (KatG Nterm ). Removal of the C-terminal domain did not result in unexpected changes in secondary structure as evaluated by CD, but KatG Nterm had neither catalase nor peroxidase activity. Partial recovery of both activities was achieved by incubating KatG Nterm with the separately expressed and isolated KatG C-terminal domain. Spectroscopic measurements revealed a shift in heme environment from a mixture of high-spin species (wtKatG) to exclusively hexacoordinate, low-spin (KatG Nterm ). Moreover, a >1000-fold lower k on for CN - binding was observed for KatG Nterm . EPR spectra for KatG Nterm and the results of site-specific substitution of active site histidines suggested that the distal histidine was the sixth ligand. Thus, one important role for the C-terminal domain may be to support the architecture of the active site, preventing heme ligation by this catalytically essential residue

Light microscopic detection of sugar residues in glycoconjugates of salivary glands and the pancreas with lectin-horseradish peroxidase conjugates. I. Mouse.

Science.gov (United States)

Schulte, B A; Spicer, S S

1983-12-01

Mouse salivary glands and pancreases were stained with a battery of ten horseradish peroxidase-conjugated lectins. Lectin staining revealed striking differences in the structure of oligosaccharides of stored intracellular secretory glycoproteins and glycoconjugates associated with the surface of epithelial cells lining excretory ducts. The percentage of acinar cells containing terminal alpha-N-acetylgalactosamine residues varied greatly in submandibular glands of 30 male mice, but all submandibular acinar cells contained oligosaccharides with terminal sialic acid and penultimate beta-galactose residues. The last named dimer was abundant in secretory glycoprotein of all mucous acinar cells in murine sublingual glands and an additional 20-50% of these cells in all glands contained terminal N-acetylglucosamine residues. In contrast, terminal alpha-N-acetylgalactosamine was abundant in sublingual serous demilune secretions. Serous acinar cells in the exorbital lacrimal gland, posterior lingual gland, parotid gland and pancreas exhibited a staining pattern unique to each organ. In contrast, the apical cytoplasm and surface of striated duct epithelial cells in the submandibular, sublingual, parotid and exorbital lacrimal gland stained similarly. A comparison of staining with conjugated lectins reported biochemically to have very similar carbohydrate binding specificity has revealed some remarkable differences in their reactivity, suggesting different binding specificity for the same terminal sugars having different glycosidic linkages or with different penultimate sugar residues.

Structure of bacteriophage T4 fibritin: a segmented coiled coil and the role of the C-terminal domain.

Science.gov (United States)

Tao, Y; Strelkov, S V; Mesyanzhinov, V V; Rossmann, M G

1997-06-15

Oligomeric coiled-coil motifs are found in numerous protein structures; among them is fibritin, a structural protein of bacteriophage T4, which belongs to a class of chaperones that catalyze a specific phage-assembly process. Fibritin promotes the assembly of the long tail fibers and their subsequent attachment to the tail baseplate; it is also a sensing device that controls the retraction of the long tail fibers in adverse environments and, thus, prevents infection. The structure of fibritin had been predicted from sequence and biochemical analyses to be mainly a triple-helical coiled coil. The determination of its structure at atomic resolution was expected to give insights into the assembly process and biological function of fibritin, and the properties of modified coiled-coil structures in general. The three-dimensional structure of fibritin E, a deletion mutant of wild-type fibritin, was determined to 2.2 A resolution by X-ray crystallography. Three identical subunits of 119 amino acid residues form a trimeric parallel coiled-coil domain and a small globular C-terminal domain about a crystallographic threefold axis. The coiled-coil domain is divided into three segments that are separated by insertion loops. The C-terminal domain, which consists of 30 residues from each subunit, contains a beta-propeller-like structure with a hydrophobic interior. The residues within the C-terminal domain make extensive hydrophobic and some polar intersubunit interactions. This is consistent with the C-terminal domain being important for the correct assembly of fibritin, as shown earlier by mutational studies. Tight interactions between the C-terminal residues of adjacent subunits counteract the latent instability that is suggested by the structural properties of the coiled-coil segments. Trimerization is likely to begin with the formation of the C-terminal domain which subsequently initiates the assembly of the coiled coil. The interplay between the stabilizing effect of the C-terminal

Effect of N-Terminal Acylation on the Activity of Myostatin Inhibitory Peptides.

Science.gov (United States)

Takayama, Kentaro; Nakamura, Akari; Rentier, Cédric; Mino, Yusaku; Asari, Tomo; Saga, Yusuke; Taguchi, Akihiro; Yakushiji, Fumika; Hayashi, Yoshio

2016-04-19

Inhibition of myostatin, which negatively regulates skeletal muscle growth, is a promising strategy for the treatment of muscle atrophic disorders, such as muscular dystrophy, cachexia and sarcopenia. Recently, we identified peptide A (H-WRQNTRYSRIEAIKIQILSKLRL-NH2 ), the 23-amino-acid minimum myostatin inhibitory peptide derived from mouse myostatin prodomain, and highlighted the importance of its N-terminal tryptophan residue for the effective inhibition. In this study, we synthesized a series of acylated peptide derivatives focused on the tryptophan residue to develop potent myostatin inhibitors. As a result of the investigation, a more potent derivative of peptide A was successfully identified in which the N-terminal tryptophan residue is replaced with a 2-naphthyloxyacetyl moiety to give an inhibitory peptide three times (1.19±0.11 μm) more potent than parent peptide A (3.53±0.25 μm). This peptide could prove useful as a new starting point for the development of improved inhibitory peptides. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification and characterization of a core fucosidase from the bacterium Elizabethkingia meningoseptica.

Science.gov (United States)

Li, Tiansheng; Li, Mengjie; Hou, Linlin; Guo, Yameng; Wang, Lei; Sun, Guiqin; Chen, Li

2018-01-26

All reported α-l-fucosidases catalyze the removal of nonreducing terminal l-fucoses from oligosaccharides or their conjugates, while having no capacity to hydrolyze core fucoses in glycoproteins directly. Here, we identified an α-fucosidase from the bacterium Elizabethkingia meningoseptica with catalytic activity against core α-1,3-fucosylated substrates, and we named it core fucosidase I (cFase I). Using site-specific mutational analysis, we found that three acidic residues (Asp-242, Glu-302, and Glu-315) in the predicted active pocket are critical for cFase I activity, with Asp-242 and Glu-315 acting as a pair of classic nucleophile and acid/base residues and Glu-302 acting in an as yet undefined role. These findings suggest a catalytic mechanism for cFase I that is different from known α-fucosidase catalytic models. In summary, cFase I exhibits glycosidase activity that removes core α-1,3-fucoses from substrates, suggesting cFase I as a new tool for glycobiology, especially for studies of proteins with core fucosylation. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

The C-Terminal O-S Acyl Shift Pathway under Acidic Condition to Propose Peptide-Thioesters

Directory of Open Access Journals (Sweden)

Bo Mi Kim

2016-11-01

Full Text Available Peptide-thioester is a pivotal intermediate for peptide ligation and N-, C-terminal cyclization. In this study, desired pathway and the side products of two C-terminal handles, hydroxyethylthiol (HET and hydroxypropylthiol (HPT are described in different conditions as well as kinetic studies. In addition, a new mechanism of C-terminal residue racemization is proposed on the basis of differentiation of products derived from the two C-terminal handles in preparing peptide thioesters through an acid-catalyzed tandem thiol switch, first by an intramolecular O-S acyl shift, and then by an intermolecular S-S exchange.

Synthetic and mechanistic insight into nosylation of glycine residues

DEFF Research Database (Denmark)

Stuhr-Hansen, Nicolai; Sølling, Theis Ivan; Strømgaard, Kristian

2013-01-01

The Fukuyama-Mitsunobu alkylation procedure is widely used to introduce alkyl substituents to amino groups in general and N-alkylation of peptides in particular. Here we have investigated the procedure in detail for N-alkylation of peptides with N-terminal glycine residues, based on the observati...

Antibodies to Both Terminal and Internal B-Cell Epitopes of Francisella tularensis O-Polysaccharide Produced by Patients with Tularemia

Science.gov (United States)

Lu, Zhaohua; Perkins, Hillary M.

2014-01-01

Francisella tularensis, the Gram-negative bacterium that causes tularemia, is considered a potential bioterrorism threat due to its low infectivity dose and the high morbidity and mortality from respiratory disease. We previously characterized two mouse monoclonal antibodies (MAbs) specific for the O-polysaccharide (O antigen [OAg]) of F. tularensis lipopolysaccharide (LPS): Ab63, which targets a terminal epitope at the nonreducing end of OAg, and Ab52, which targets a repeating internal OAg epitope. These two MAbs were protective in a mouse model of respiratory tularemia. To determine whether these epitope types are also targeted by humans, we tested the ability of each of 18 blood serum samples from 11 tularemia patients to inhibit the binding of Ab63 or Ab52 to F. tularensis LPS in a competition enzyme-linked immunosorbent assay (ELISA). Although all serum samples had Ab63- and Ab52-inhibitory activities, the ratios of Ab63 to Ab52 inhibitory potencies varied 75-fold. However, the variation was only 2.3-fold for sequential serum samples from the same patient, indicating different distributions of terminal- versus internal-binding antibodies in different individuals. Western blot analysis using class-specific anti-human Ig secondary antibodies showed that both terminal- and internal-binding OAg antibodies were of the IgG, IgM, and IgA isotypes. These results support the use of a mouse model to discover protective B-cell epitopes for tularemia vaccines or prophylactic/therapeutic antibodies, and they present a general strategy for interrogating the antibody responses of patients and vaccinees to microbial carbohydrate epitopes that have been characterized in experimental animals. PMID:24351753

Dual N- and C-terminal helices are required for endoplasmic reticulum and lipid droplet association of alcohol acetyltransferases in Saccharomyces cerevisiae.

Directory of Open Access Journals (Sweden)

Jyun-Liang Lin

Full Text Available In the yeast Saccharomyces cerevisiae two alcohol acetyltransferases (AATases, Atf1 and Atf2, condense short chain alcohols with acetyl-CoA to produce volatile acetate esters. Such esters are, in large part, responsible for the distinctive flavors and aromas of fermented beverages including beer, wine, and sake. Atf1 and Atf2 localize to the endoplasmic reticulum (ER and Atf1 is known to localize to lipid droplets (LDs. The mechanism and function of these localizations are unknown. Here, we investigate potential mechanisms of Atf1 and Atf2 membrane association. Segments of the N- and C-terminal domains of Atf1 (residues 24-41 and 508-525, respectively are predicted to be amphipathic helices. Truncations of these helices revealed that the terminal domains are essential for ER and LD association. Moreover, mutations of the basic or hydrophobic residues in the N-terminal helix and hydrophobic residues in the C-terminal helix disrupted ER association and subsequent sorting from the ER to LDs. Similar amphipathic helices are found at both ends of Atf2, enabling ER and LD association. As was the case with Atf1, mutations to the N- and C-terminal helices of Atf2 prevented membrane association. Sequence comparison of the AATases from Saccharomyces, non-Saccharomyces yeast (K. lactis and P. anomala and fruits species (C. melo and S. lycopersicum showed that only AATases from Saccharomyces evolved terminal amphipathic helices. Heterologous expression of these orthologs in S. cerevisiae revealed that the absence of terminal amphipathic helices eliminates LD association. Combined, the results of this study suggest a common mechanism of membrane association for AATases via dual N- and C-terminal amphipathic helices.

A study of the alterability of terminal residues stabilized by a mineral-binder-based solidification/stabilization process; Etude de l`alterabilite de residus ultimes stabilises par un procede de solidification, stabilisation a base de liants mineraux

Energy Technology Data Exchange (ETDEWEB)

Humez, N.; Prost, R. [Institut National de Recherches Agronomiques (INRA), 78 - Versailles (France). Unite de Science du Sol; Piketty, L.; Schnuriger, B.; Benchara, A.; Pichat, P. [Centre de Solidification, Stabilisation et Stockage, Sarp Industries, 78 - Limay (France)

1997-12-31

A new laboratory approach has been developed for the study of the long term behaviour of stabilized terminal residues, and is based on the experimental method for the study of rock alteration. The method consists in examining the migration of elements that are present in the solution contained in the pores, and studying the structural modifications which result from the dissolution of certain constituents, the transformation and the neo-formation of other ones in the material. The method allows for the evaluation, in a short time, of the alterability and perenniality of stabilized terminal wastes. Results show that the long term alterability of solidified/stabilized samples that have been processed using the ECOFIX method, is low

X-ray crystal structure of the N-terminal region of Moloney murine leukemia virus integrase and its implications for viral DNA recognition: N-Terminal Region of M-MuLV Integrase

Energy Technology Data Exchange (ETDEWEB)

Guan, Rongjin [Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Aiyer, Sriram [Department of Pharmacology, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Cote, Marie L. [Department of Biochemistry, Robert Wood Johnson Medical School, UMDNJ, Piscataway New Jersey 08854; Xiao, Rong [Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Jiang, Mei [Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Acton, Thomas B. [Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Roth, Monica J. [Department of Pharmacology, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Montelione, Gaetano T. [Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Department of Biochemistry, Robert Wood Johnson Medical School, UMDNJ, Piscataway New Jersey 08854

2017-02-03

The retroviral integrase (IN) carries out the integration of a dsDNA copy of the viral genome into the host DNA, an essential step for viral replication. All IN proteins have three general domains, the N-terminal domain (NTD), the catalytic core domain, and the C-terminal domain. The NTD includes an HHCC zinc finger-like motif, which is conserved in all retroviral IN proteins. Two crystal structures of Moloney murine leukemia virus (M-MuLV) IN N-terminal region (NTR) constructs that both include an N-terminal extension domain (NED, residues 1–44) and an HHCC zinc-finger NTD (residues 45–105), in two crystal forms are reported. The structures of IN NTR constructs encoding residues 1–105 (NTR1–105) and 8–105 (NTR8–105) were determined at 2.7 and 2.15 Å resolution, respectively and belong to different space groups. While both crystal forms have similar protomer structures, NTR1–105 packs as a dimer and NTR8–105 packs as a tetramer in the asymmetric unit. The structure of the NED consists of three anti-parallel β-strands and an α-helix, similar to the NED of prototype foamy virus (PFV) IN. These three β-strands form an extended β-sheet with another β-strand in the HHCC Zn2+ binding domain, which is a unique structural feature for the M-MuLV IN. The HHCC Zn2+ binding domain structure is similar to that in HIV and PFV INs, with variations within the loop regions. Differences between the PFV and MLV IN NEDs localize at regions identified to interact with the PFV LTR and are compared with established biochemical and virological data for M-MuLV. Proteins 2017; 85:647–656.

Quantitative analysis of glycosaminoglycans, chondroitin/dermatan sulfate, hyaluronic acid, heparan sulfate, and keratan sulfate by liquid chromatography-electrospray ionization-tandem mass spectrometry.

Science.gov (United States)

Osago, Harumi; Shibata, Tomoko; Hara, Nobumasa; Kuwata, Suguru; Kono, Michihaya; Uchio, Yuji; Tsuchiya, Mikako

2014-12-15

We developed a method using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) with a selected reaction monitoring (SRM) mode for simultaneous quantitative analysis of glycosaminoglycans (GAGs). Using one-shot analysis with our MS/MS method, we demonstrated the simultaneous quantification of a total of 23 variously sulfated disaccharides of four GAG classes (8 chondroitin/dermatan sulfates, 1 hyaluronic acid, 12 heparan sulfates, and 2 keratan sulfates) with a sensitivity of less than 0.5 pmol within 20 min. We showed the differences in the composition of GAG classes and the sulfation patterns between porcine articular cartilage and yellow ligament. In addition to the internal disaccharides described above, some saccharides derived from the nonreducing terminal were detected simultaneously. The simultaneous quantification of both internal and nonreducing terminal saccharides could be useful to estimate the chain length of GAGs. This method would help to establish comprehensive "GAGomic" analysis of biological tissues. Copyright © 2014 Elsevier Inc. All rights reserved.

Persistence of malathion residues in stored wheat

International Nuclear Information System (INIS)

Farghaly, M.; Zayed, S.M.A.D.

1990-01-01

The persistence of succinate- 14 C-malathion in stored wheat was investigated under local conditions during a storage period of 32 weeks. The insecticide penetrated readily into the seed and up to 16% of the applied dose was found to be bound after 32 weeks in storage. Total terminal residues declined to 9.3 and 21.0 mg/kg from initially applied doses of 12.2 and 24.4 mg/kg respectively. A small percentage of malaoxon was detected only during the early weeks after treatment (3-5%). Malathion was the major constituent of the extractable residues. In addition, seven degradation products were detected and identified. (author). 6 refs, 2 figs, 2 tabs

Assessment of radioactive residues arising from radiolabel instability in a multiple dose tissue distribution study in rats

International Nuclear Information System (INIS)

Slatter, J.G.; Sams, J.P.; Easter, J.A.

2003-01-01

Our study objectives were to quantitatively determine the effect of radiolabel instability on terminal phase radioactive tissue residues in a multiple dose tissue distribution study, to quantitatively compare tissue residue artifacts (non drug-related radioactivity) from two chemically-distinct radiolabel locations, and to conduct a definitive multiple dose tissue distribution study using the better of the two radiolabeled compounds. We compared the excretion and tissue distribution in rats of [ 14 C]linezolid, radiolabeled in two different locations, after 7 consecutive once daily [ 14 C] oral doses. The radiolabels were in the acetamide (two carbon) and oxazolidinone (isolated carbon) functional groups. Terminal phase tissue residue and excretion data were compared to data from rats dosed orally with [ 14 C]sodium acetate. Drug-related radioactivity was excreted rapidly over 24 h. After a single dose, the acetamide and oxazolidinone radiolabel sites both gave 3% of dose as exhaled 14 CO 2 . After 7 daily [ 14 C] oral doses, terminal phase radioactive tissue residues were higher from the acetamide radiolabel, relative to the oxazolidinone radiolabel, and were primarily not drug-related. In the definitive tissue distribution study, low concentrations of drug-related radioactivity in skin and thyroid were observed. We conclude that although small amounts of radiolabel instability do not significantly affect single dose tissue radioactivity C max and area under the curve (AUC), artifacts arising from radiolabel instability can prolong the apparent terminal phase half life and complicate study data interpretation. When possible, it is always preferable to use a completely stable radiolabel site. (author)

Assessment of radioactive residues arising from radiolabel instability in a multiple dose tissue distribution study in rats

Energy Technology Data Exchange (ETDEWEB)

Slatter, J.G. [Pharmacia Corp., Peapack, NJ (United States); Sams, J.P.; Easter, J.A. [Pharmacia Corp., Kalamazoo, MI (United States)] [and others

2003-05-01

Our study objectives were to quantitatively determine the effect of radiolabel instability on terminal phase radioactive tissue residues in a multiple dose tissue distribution study, to quantitatively compare tissue residue artifacts (non drug-related radioactivity) from two chemically-distinct radiolabel locations, and to conduct a definitive multiple dose tissue distribution study using the better of the two radiolabeled compounds. We compared the excretion and tissue distribution in rats of [{sup 14}C]linezolid, radiolabeled in two different locations, after 7 consecutive once daily [{sup 14}C] oral doses. The radiolabels were in the acetamide (two carbon) and oxazolidinone (isolated carbon) functional groups. Terminal phase tissue residue and excretion data were compared to data from rats dosed orally with [{sup 14}C]sodium acetate. Drug-related radioactivity was excreted rapidly over 24 h. After a single dose, the acetamide and oxazolidinone radiolabel sites both gave 3% of dose as exhaled {sup 14}CO{sub 2}. After 7 daily [{sup 14}C] oral doses, terminal phase radioactive tissue residues were higher from the acetamide radiolabel, relative to the oxazolidinone radiolabel, and were primarily not drug-related. In the definitive tissue distribution study, low concentrations of drug-related radioactivity in skin and thyroid were observed. We conclude that although small amounts of radiolabel instability do not significantly affect single dose tissue radioactivity C{sub max} and area under the curve (AUC), artifacts arising from radiolabel instability can prolong the apparent terminal phase half life and complicate study data interpretation. When possible, it is always preferable to use a completely stable radiolabel site. (author)

Analysis of proteolytic processes and enzymatic activities in the generation of huntingtin n-terminal fragments in an HEK293 cell model.

Directory of Open Access Journals (Sweden)

Andrew T N Tebbenkamp

Full Text Available N-terminal fragments of mutant huntingtin (htt that terminate between residues 90-115, termed cleavage product A or 1 (cp-A/1, form intracellular and intranuclear inclusion bodies in the brains of patients with Huntington's disease (HD. These fragments appear to be proteolytic products of the full-length protein. Here, we use an HEK293 cell culture model to investigate huntingtin proteolytic processing; previous studies of these cells have demonstrated cleavage of htt to cp-A/1 like htt fragments.Recombinant N-terminal htt fragments, terminating at residue 171 (also referred to as cp-B/2 like, were efficiently cleaved to produce cp-A/1 whereas fragments representing endogenous caspase, calpain, and metalloproteinase cleavage products, terminating between residues 400-600, were inefficiently cleaved. Using cysteine-labeling techniques and antibody binding mapping, we localized the C-terminus of the cp-A/1 fragments produced by HEK293 cells to sequences minimally limited by cysteine 105 and an antibody epitope composed of residues 115-124. A combination of genetic and pharmacologic approaches to inhibit potential proteases, including γ-secretase and calpain, proved ineffective in preventing production of cp-A/1.Our findings indicate that HEK293 cells express a protease that is capable of efficiently cleaving cp-B/2 like fragments of htt with normal or expanded glutamine repeats. For reasons that remain unclear, this protease cleaves longer htt fragments, with normal or expanded glutamine expansions, much less efficiently. The protease in HEK293 cells that is capable of generating a cp-A/1 like htt fragment may be a novel protease with a high preference for a cp-B/2-like htt fragment as substrate.

Mapping the residues of protein kinase CK2 implicated in substrate recognition

DEFF Research Database (Denmark)

Sarno, S; Boldyreff, B; Marin, O

1995-01-01

, hampering the calculation of kinetic parameters. In contrast 3 mutants (K74-77A, K79R80K83A and R191,195K198A) phosphorylated the peptide with reduced efficiency accounted for by increased Km and decreased Vmax values. By using derivatives of the RRRADDSDDDDD peptide in which individual aspartyl residues......, respectively. These data support the conclusion that the basic residues present in the p+1 loop of CK2 alpha specifically recognize the acidic determinant adjacent to the C-terminal side of serine, while the specificity determinants located more down-stream are variably recognized by different residues...

Structure predictions of two Bauhinia variegata lectins reveal patterns of C-terminal properties in single chain legume lectins.

Science.gov (United States)

Moreira, Gustavo M S G; Conceição, Fabricio R; McBride, Alan J A; Pinto, Luciano da S

2013-01-01

Bauhinia variegata lectins (BVL-I and BVL-II) are single chain lectins isolated from the plant Bauhinia variegata. Single chain lectins undergo post-translational processing on its N-terminal and C-terminal regions, which determines their physiological targeting, carbohydrate binding activity and pattern of quaternary association. These two lectins are isoforms, BVL-I being highly glycosylated, and thus far, it has not been possible to determine their structures. The present study used prediction and validation algorithms to elucidate the likely structures of BVL-I and -II. The program Bhageerath-H was chosen from among three different structure prediction programs due to its better overall reliability. In order to predict the C-terminal region cleavage sites, other lectins known to have this modification were analysed and three rules were created: (1) the first amino acid of the excised peptide is small or hydrophobic; (2) the cleavage occurs after an acid, polar, or hydrophobic residue, but not after a basic one; and (3) the cleavage spot is located 5-8 residues after a conserved Leu amino acid. These rules predicted that BVL-I and -II would have fifteen C-terminal residues cleaved, and this was confirmed experimentally by Edman degradation sequencing of BVL-I. Furthermore, the C-terminal analyses predicted that only BVL-II underwent α-helical folding in this region, similar to that seen in SBA and DBL. Conversely, BVL-I and -II contained four conserved regions of a GS-I association, providing evidence of a previously undescribed X4+unusual oligomerisation between the truncated BVL-I and the intact BVL-II. This is the first report on the structural analysis of lectins from Bauhinia spp. and therefore is important for the characterisation C-terminal cleavage and patterns of quaternary association of single chain lectins.

The C-terminal domain of the bacteriophage T4 terminase docks on the prohead portal clip region during DNA packaging

Science.gov (United States)

Dixit, Aparna Banerjee; Ray, Krishanu; Thomas, Julie A.; Black, Lindsay W.

2013-01-01

Bacteriophage ATP-based packaging motors translocate DNA into a pre-formed prohead through a dodecameric portal ring channel to high density. We investigated portal–terminase docking interactions at specifically localized residues within a terminase-interaction region (aa279–316) in the phage T4 portal protein gp20 equated to the clip domain of the SPP1 portal crystal structure by 3D modeling. Within this region, three residues allowed A to C mutations whereas three others did not, consistent with informatics analyses showing the tolerated residues are not strongly conserved evolutionarily. About 7.5 nm was calculated by FCS-FRET studies employing maleimide Alexa488 dye labeled A316C proheads and gp17 CT-ReAsH supporting previous work docking the C-terminal end of the T4 terminase (gp17) closer to the N-terminal GFP-labeled portal (gp20) than the N-terminal end of the terminase. Such a terminase–portal orientation fits better to a proposed “DNA crunching” compression packaging motor and to portal determined DNA headful cutting. PMID:24074593

Structure of the Escherichia coli RNA polymerase α subunit C-terminal domain

International Nuclear Information System (INIS)

Lara-González, Samuel; Birktoft, Jens J.; Lawson, Catherine L.

2010-01-01

The crystal structure of the dimethyllysine derivative of the E. coli RNA polymerase α subunit C-terminal domain is reported at 2.0 Å resolution. The α subunit C-terminal domain (αCTD) of RNA polymerase (RNAP) is a key element in transcription activation in Escherichia coli, possessing determinants responsible for the interaction of RNAP with DNA and with transcription factors. Here, the crystal structure of E. coli αCTD (α subunit residues 245–329) determined to 2.0 Å resolution is reported. Crystals were obtained after reductive methylation of the recombinantly expressed domain. The crystals belonged to space group P2 1 and possessed both pseudo-translational symmetry and pseudo-merohedral twinning. The refined coordinate model (R factor = 0.193, R free = 0.236) has improved geometry compared with prior lower resolution determinations of the αCTD structure [Jeon et al. (1995 ▶), Science, 270, 1495–1497; Benoff et al. (2002 ▶), Science, 297, 1562–1566]. An extensive dimerization interface formed primarily by N- and C-terminal residues is also observed. The new coordinates will facilitate the improved modeling of αCTD-containing multi-component complexes visualized at lower resolution using X-ray crystallography and electron-microscopy reconstruction

Teichuronic acid reducing terminal N-acetylglucosamine residue linked by phosphodiester to peptidoglycan of Micrococcus luteus

International Nuclear Information System (INIS)

Gassner, G.T.; Dickie, J.P.; Hamerski, D.A.; Magnuson, J.K.; Anderson, J.S.

1990-01-01

Teichuronic acid-peptidoglycan complex isolated from Micrococcus luteus cells by lysozyme digestion in osmotically stabilized medium was treated with mild acid to cleave the linkage joining teichuronic acid to peptidoglycan. This labile linkage was shown to be the phosphodiester which joins N-acetylglucosamine, the residue located at the reducing end of the teichuronic acid, through its anomeric hydroxyl group to a 6-phosphomuramic acid, a residue of the glycan strand of peptidoglycan. 31 P nuclear magnetic resonance spectroscopy of the lysozyme digest of cell walls demonstrated the presence of a phosphodiester which was converted to a phosphomonoester by the conditions which released teichuronic acid from cell walls. Reduction of acid-liberated reducing end groups by NaB 3 H 4 followed by complete acid hydrolysis yielded [ 3 H] glucosaminitol from the true reducing end residue of teichuronic acid and [ 3 H]glucitol from the sites of fragmentation of teichuronic acid. The amount of N-acetylglucosamine detected was approximately stoichiometric with the amount of phosphate in the complex. Partial fragmentation of teichuronic acid provides an explanation of the previous erroneous identification of the reducing end residue

Copper(II) Binding Sites in N-Terminally Acetylated α-Synuclein: A Theoretical Rationalization.

Science.gov (United States)

Ramis, Rafael; Ortega-Castro, Joaquín; Vilanova, Bartolomé; Adrover, Miquel; Frau, Juan

2017-08-03

The interactions between N-terminally acetylated α-synuclein and Cu(II) at several binding sites have been studied with DFT calculations, specifically with the M06 hybrid functional and the ωB97X-D DFT-D functional. In previous experimental studies, Cu(II) was shown to bind several α-synuclein residues, including Met1-Asp2 and His50, forming square planar coordination complexes. Also, it was determined that a low-affinity binding site exists in the C-terminal domain, centered on Asp121. However, in the N-terminally acetylated protein, present in vivo, the Met1 site is blocked. In this work, we simplify the representation of the protein by modeling each experimentally found binding site as a complex between an N-terminally acetylated α-synuclein dipeptide (or several independent residues) and a Cu(II) cation, and compare the results with a number of additional, structurally analogous sites not experimentally found. This way of representing the binding sites, although extremely simple, allows us to reproduce experimental results and to provide a theoretical rationale to explain the preference of Cu(II) for certain sites, as well as explicit geometrical structures for the complexes formed. These results are important to understand the interactions between α-synuclein and Cu(II), one of the factors inducing structural changes in the protein and leading to aggregated forms of it which may play a role in neurodegeneration.

Characterization of a novel ß-galactosidase from Bifidobacterium adolescentis DSM 20083 active towards transgalactooligosaccharides

NARCIS (Netherlands)

Laere, van K.M.J.; Abee, T.; Schols, H.A.; Beldman, G.; Voragen, A.G.J.

2000-01-01

This paper reports on the effects of both reducing and nonreducing transgalactooligosaccharides (TOS) comprising 2 to 8 residues on the growth of Bifidobacterium adolescentis DSM 20083 and on the production of a novel -galactosidase (-Gal II). In cells grown on TOS, in addition to the

Statistical Characterization of the Charge State and Residue Dependence of Low-Energy CID Peptide Dissociation Patterns

International Nuclear Information System (INIS)

Huang, Yingying; Triscari, Joseph M.; Tseng, George C.; Pasa-Tolic, Ljiljana; Lipton, Mary S.; Smith, Richard D.; Wysocki, Vicki H.

2005-01-01

Data mining was performed on 28 330 unique peptide tandem mass spectra for which sequences were assigned with high confidence. By dividing the spectra into different sets based on structural features and charge states of the corresponding peptides, chemical interactions involved in promoting specific cleavage patterns in gas-phase peptides were characterized. Pairwise fragmentation maps describing cleavages at all Xxx-Zzz residue combinations for b and y ions reveal that the difference in basicity between Arg and Lys results in different dissociation patterns for singly charged Arg- and Lys-ending tryptic peptides. While one dominant protonation form (proton localized) exists for Arg-ending peptides, a heterogeneous population of different protonated forms or more facile interconversion of protonated forms (proton partially mobile) exists for Lys-ending peptides. Cleavage C-terminal to acidic residues dominates spectra from peptides that have a localized proton and cleavage N-terminal to Pro dominates those that have a mobile or partially mobile proton. When Pro is absent from peptides that have a mobile or partially mobile proton, cleavage at each peptide bond becomes much more prominent. Whether the above patterns can be found in b ions, y ions, or both depends on the location of the proton holder(s). Enhanced cleavages C-terminal to branched aliphatic residues (Ile, Val, Leu) are observed in both b and y ions from peptides that have a mobile proton, as well as in y ions from peptides that have a partially mobile proton; enhanced cleavages N-terminal to these residues are observed in b ions from peptides that have a partially mobile proton. Statistical tools have been designed to visualize the fragmentation maps and measure the similarity between them. The pairwise cleavage patterns observed expand our knowledge of peptide gas-phase fragmentation behaviors and should be useful in algorithm development that employs improved models to predict fragment ion

C-terminal substitution of MDM2 interacting peptides modulates binding affinity by distinctive mechanisms.

Directory of Open Access Journals (Sweden)

Christopher J Brown

Full Text Available The complex between the proteins MDM2 and p53 is a promising drug target for cancer therapy. The residues 19-26 of p53 have been biochemically and structurally demonstrated to be a most critical region to maintain the association of MDM2 and p53. Variation of the amino acid sequence in this range obviously alters the binding affinity. Surprisingly, suitable substitutions contiguous to this region of the p53 peptides can yield tightly binding peptides. The peptide variants may differ by a single residue that vary little in their structural conformations and yet are characterized by large differences in their binding affinities. In this study a systematic analysis into the role of single C-terminal mutations of a 12 residue fragment of the p53 transactivation domain (TD and an equivalent phage optimized peptide (12/1 were undertaken to elucidate their mechanistic and thermodynamic differences in interacting with the N-terminal of MDM2. The experimental results together with atomistically detailed dynamics simulations provide insight into the principles that govern peptide design protocols with regard to protein-protein interactions and peptidomimetic design.

Interaction of the N-terminal segment of pulmonary surfactant protein SP-C with interfacial phospholipid films

DEFF Research Database (Denmark)

Plasencia, Inés; Keough, Kevin M W; Perez-Gil, Jesus

2005-01-01

Pulmonary surfactant protein SP-C is a 35-residue polypeptide composed of a hydrophobic transmembrane alpha-helix and a polycationic, palmitoylated-cysteine containing N-terminal segment. This segment is likely the only structural motif the protein projects out of the bilayer in which SP-C is ins......Pulmonary surfactant protein SP-C is a 35-residue polypeptide composed of a hydrophobic transmembrane alpha-helix and a polycationic, palmitoylated-cysteine containing N-terminal segment. This segment is likely the only structural motif the protein projects out of the bilayer in which SP...... or anionic phospholipid monolayers. The peptide expands the pi-A compression isotherms of interfacial phospholipid/peptide films, and perturbs the lipid packing of phospholipid films during compression-driven liquid-expanded to liquid-condensed lateral transitions, as observed by epifluorescence microscopy....... These results demonstrate that the sequence of the SP-C N-terminal region has intrinsic ability to interact with, insert into, and perturb the structure of zwitterionic and anionic phospholipid films, even in the absence of the palmitic chains attached to this segment in the native protein. This effect has been...

Purification, crystallization and preliminary X-ray diffraction analysis of the C-terminal fragment of the MvfR protein from Pseudomonas aeruginosa

International Nuclear Information System (INIS)

Kefala, Katerina; Kotsifaki, Dina; Providaki, Mary; Kapetaniou, Evangelia G.; Rahme, Lawrence; Kokkinidis, Michael

2012-01-01

MvfRC87, a 242-residue C-terminal segment of the LysR-type transcriptional regulator MvfR, was produced in Escherichia coli, purified and crystallized. The LysR-type transcriptional regulator MvfR plays a critical role in Pseudomonas aeruginosa pathogenicity via the transcriptional regulation of multiple quorum-sensing-regulated virulence factors. The protein also controls pathogenic type VI secretion loci. MvfRC87, a 242-residue C-terminal segment of MvfR, was produced in Escherichia coli, purified and crystallized. X-ray diffraction data were collected using synchrotron radiation and crystallographic parameters were determined

Involvement of the N-terminal part of cyclophilin B in the interaction with specific Jurkat T-cell binding sites.

Science.gov (United States)

Mariller, C; Haendler, B; Allain, F; Denys, A; Spik, G

1996-07-15

Cyclophilin B (CyPB) is secreted in biological fluids such as blood or milk and binds to a specific receptor present on the human lymphoblastic cell line Jurkat and on human peripheral blood lymphocytes. This study was intended to specify the areas of CyPB that are involved in the interaction with the receptor. A synthetic peptide corresponding to the first 24 N-terminal amino acid residues of CyPB was shown to specifically recognize the receptor. Moreover, modification of Arg18 of CyPB by p-hydroxyphenlglyoxal led to a dramatic loss of affinity for the receptor. However, when this residue was replaced by an alanine residue using site-directed mutagenesis, no modification of the binding properties was found, suggesting that Arg18 is not directly involved but is sufficiently close to the interaction site to interfere with the binding when modified. Competitive binding experiments using a chimaeric protein made up of the 24 N-terminal amino acid residues of CyPB fused to the cyclophilin A core sequence confirmed the involvement of this region of CyPB in receptor binding.

N-terminal protein processing: A comparative proteogenomic analysis

Energy Technology Data Exchange (ETDEWEB)

Bonissone, Stefano; Gupta, Nitin; Romine, Margaret F.; Bradshaw, Ralph A.; Pevzner, Pavel A.

2013-01-01

N-Terminal Methionine Excision (NME) is a universally conserved mechanism with the same specificity across all life forms that removes the first Methionine in proteins when the second residue is Gly, Ala, Ser, Cys, Thr, Pro, or Val. In spite of its necessity for proper cell functioning, the functional role of NME remains unclear. In 1988, Arfin and Bradshaw connected NME with the N-end protein degradation rule and postulated that the role of NME is to expose the stabilizing residues with the goal to resist protein degradation. While this explanation (that treats 7 stabilizing residues in the same manner) has become the de facto dogma of NME, comparative proteogenomics analysis of NME tells a different story. We suggest that the primary role of NME is to expose only two (rather than seven) amino acids Ala and Ser for post-translational modifications (e.g., acetylation) rather than to regulate protein degradation. We argue that, contrary to the existing view, NME is not crucially important for proteins with 5 other stabilizing residue at the 2nd positions that are merely bystanders (their function is not affected by NME) that become exposed to NME because their sizes are comparable or smaller than the size of Ala and Ser.

PrP N-terminal domain triggers PrPSc-like aggregation of Dpl

International Nuclear Information System (INIS)

Erlich, Paul; Cesbron, Jean-Yves; Lemaire-Vieille, Catherine; Curt, Aurelie; Andrieu, Jean-Pierre; Schoehn, Guy; Jamin, Marc; Gagnon, Jean

2008-01-01

Transmissible spongiform encephalopathies are fatal neurodegenerative disorders thought to be transmitted by self-perpetuating conformational conversion of a neuronal membrane glycoprotein (PrP C , for 'cellular prion protein') into an abnormal state (PrP Sc , for 'scrapie prion protein'). Doppel (Dpl) is a protein that shares significant biochemical and structural homology with PrP C . In contrast to its homologue PrP C , Dpl is unable to participate in prion disease progression or to achieve an abnormal PrP Sc -like state. We have constructed a chimeric mouse protein, composed of the N-terminal domain of PrP C (residues 23-125) and the C-terminal part of Dpl (residues 58-157). This chimeric protein displays PrP-like biochemical and structural features; when incubated in presence of NaCl, the α-helical monomer forms soluble β-sheet-rich oligomers which acquire partial resistance to pepsin proteolysis in vitro, as do PrP oligomers. Moreover, the presence of aggregates akin to protofibrils is observed in soluble oligomeric species by electron microscopy

Completion of decommissioning: Monitoring for site release and license termination

International Nuclear Information System (INIS)

Boing, L.E.

1997-01-01

To request termination of a license upon completion of dismantling or decommissioning activities, documenting any residual radioactivity to show that the levels are environmentally acceptable will be necessary. When the regulators approve the decommissioning plan, they establish what the release criteria for the decommissioned site will be at the time of the site release and license termination. The criteria are numeric guidelines for direct radiation in soils and on surfaces. If the regulatory body finds that the measured on-site values are below the guidelines, the site will be acceptable for unrestricted release (no radiological controls or future use). If areas are found above those values, more decontamination or cleanup of these areas may be required unless the regulatory body grants an exemption

Docking Studies of Binding of Ethambutol to the C-Terminal Domain of the Arabinosyltransferase from Mycobacterium tuberculosis

Directory of Open Access Journals (Sweden)

Guillermo Salgado-Moran

2013-01-01

Full Text Available The binding of ethambutol to the C-terminal domain of the arabinosyltransferase from Mycobacterium tuberculosis was studied. The analysis was performed using an in silico approach in order to find out, by docking calculations and energy descriptors, the conformer of Ethambutol that forms the most stable complex with the C-terminal domain of arabinosyltransferase. The complex shows that location of the Ethambutol coincides with the cocrystallization ligand position and that amino acid residues ASH1051, ASN740, ASP1052, and ARG1055 should be critical in the binding of Ethambutol to C-terminal domain EmbC.

Oxidation of myosin by haem proteins generates myosin radicals and protein cross-links

DEFF Research Database (Denmark)

Lametsch, Marianne Lund; Luxford, Catherine; Skibsted, Leif Horsfelt

2008-01-01

of thiyl and tyrosyl radicals is consistent with the observed consumption of cysteine and tyrosine residues, the detection of di-tyrosine by HPLC and the detection of both reducible (disulfide bond) and non-reducible cross-links between myosin molecules by SDS/PAGE. The time course of radical formation...

Binding of transcription termination protein nun to nascent RNA and template DNA.

Science.gov (United States)

Watnick, R S; Gottesman, M E

1999-12-17

The amino-terminal arginine-rich motif of coliphage HK022 Nun binds phage lambda nascent transcript, whereas the carboxyl-terminal domain interacts with RNA polymerase (RNAP) and blocks transcription elongation. RNA binding is inhibited by zinc (Zn2+) and stimulated by Escherichia coli NusA. To study these interactions, the Nun carboxyl terminus was extended by a cysteine residue conjugated to a photochemical cross-linker. The carboxyl terminus contacted NusA and made Zn2+-dependent intramolecular contacts. When Nun was added to a paused transcription elongation complex, it cross-linked to the DNA template. Nun may arrest transcription by anchoring RNAP to DNA.

Multiple native-like conformations trapped via self-association-induced hydrophobic collapse of the 33-residue beta-sheet domain from platelet factor 4.

OpenAIRE

Ilyina, E; Mayo, K H

1995-01-01

Native platelet factor 4 (PF4) (70 residues) has a hydrophobic three-stranded anti-parallel beta-sheet domain on to which is folded an amphipathic C-terminal alpha-helix and an aperiodic N-terminal domain. The 33-amino acid beta-sheet domain from PF4 (residues 23-55) has been synthesized and studied by c.d. and n.m.r. At 10 degrees C and low concentration, peptide 23-55 appears to exist in aqueous solution in a random-coil distribution of highly flexible conformational states. Some preferred ...

A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain

Science.gov (United States)

Harper, J. F.; Hong, B.; Hwang, I.; Guo, H. Q.; Stoddard, R.; Huang, J. F.; Palmgren, M. G.; Sze, H.; Evans, M. L. (Principal Investigator)

1998-01-01

To study transporters involved in regulating intracellular Ca2+, we isolated a full-length cDNA encoding a Ca2+-ATPase from a model plant, Arabidopsis, and named it ACA2 (Arabidopsis Ca2+-ATPase, isoform 2). ACA2p is most similar to a "plasma membrane-type" Ca2+-ATPase, but is smaller (110 kDa), contains a unique N-terminal domain, and is missing a long C-terminal calmodulin-binding regulatory domain. In addition, ACA2p is localized to an endomembrane system and not the plasma membrane, as shown by aqueous-two phase fractionation of microsomal membranes. ACA2p was expressed in yeast as both a full-length protein (ACA2-1p) and an N-terminal truncation mutant (ACA2-2p; Delta residues 2-80). Only the truncation mutant restored the growth on Ca2+-depleted medium of a yeast mutant defective in both endogenous Ca2+ pumps, PMR1 and PMC1. Although basal Ca2+-ATPase activity of the full-length protein was low, it was stimulated 5-fold by calmodulin (50% activation around 30 nM). In contrast, the truncated pump was fully active and insensitive to calmodulin. A calmodulin-binding sequence was identified within the first 36 residues of the N-terminal domain, as shown by calmodulin gel overlays on fusion proteins. Thus, ACA2 encodes a novel calmodulin-regulated Ca2+-ATPase distinguished by a unique N-terminal regulatory domain and a non-plasma membrane localization.

Maximizing Selective Cleavages at Aspartic Acid and Proline Residues for the Identification of Intact Proteins

Science.gov (United States)

Foreman, David J.; Dziekonski, Eric T.; McLuckey, Scott A.

2018-04-01

A new approach for the identification of intact proteins has been developed that relies on the generation of relatively few abundant products from specific cleavage sites. This strategy is intended to complement standard approaches that seek to generate many fragments relatively non-selectively. Specifically, this strategy seeks to maximize selective cleavage at aspartic acid and proline residues via collisional activation of precursor ions formed via electrospray ionization (ESI) under denaturing conditions. A statistical analysis of the SWISS-PROT database was used to predict the number of arginine residues for a given intact protein mass and predict a m/z range where the protein carries a similar charge to the number of arginine residues thereby enhancing cleavage at aspartic acid residues by limiting proton mobility. Cleavage at aspartic acid residues is predicted to be most favorable in the m/z range of 1500-2500, a range higher than that normally generated by ESI at low pH. Gas-phase proton transfer ion/ion reactions are therefore used for precursor ion concentration from relatively high charge states followed by ion isolation and subsequent generation of precursor ions within the optimal m/z range via a second proton transfer reaction step. It is shown that the majority of product ion abundance is concentrated into cleavages C-terminal to aspartic acid residues and N-terminal to proline residues for ions generated by this process. Implementation of a scoring system that weights both ion fragment type and ion fragment area demonstrated identification of standard proteins, ranging in mass from 8.5 to 29.0 kDa. [Figure not available: see fulltext.

Functional interaction between the N- and C-terminal domains of murine leukemia virus surface envelope protein

International Nuclear Information System (INIS)

Lu, C.-W.; Roth, Monica J.

2003-01-01

A series of murine leukemia viruses (MuLVs) with chimeric envelope proteins (Env) was generated to map functional interactions between the N- and the C-terminal domains of surface proteins (SU). All these chimeras have the 4070A amphotropic receptor-binding region flanked by various lengths of Moloney ecotropic N- and C-terminal Env. A charged residue, E49 (E16 on the mature protein), was identified at the N-terminals of Moloney MuLV SU that is important for the interaction with the C-terminal domain of the SU. The region that interacts with E49 was localized between junction 4 (R265 of M-MuLV Env) and junction 6 (L374 of M-MuLV Env) of SU. Sequencing the viable chimeric Env virus populations identified residues within the SU protein that improved the replication kinetics of the input chimeric Env viruses. Mutations in the C-domain of SU (G387E/R, L435I, L442P) were found to improve chimera IV4, which displayed a delayed onset of replication. The replication of AE6, containing a chimeric junction in the SU C-terminus, was improved by mutations in the N-domain (N40H, E80K), the proline-rich region (Q252R), or the transmembrane protein (L538N). Altogether, these observations provide insights into the structural elements required for Env function

The carboxy-terminal tail or the intracellular loop 3 is required for β-arrestin-dependent internalization of a mammalian type II GnRH receptor.

Science.gov (United States)

Madziva, Michael T; Mkhize, Nonhlanhla N; Flanagan, Colleen A; Katz, Arieh A

2015-08-15

The type II GnRH receptor (GnRH-R2) in contrast to mammalian type I GnRH receptor (GnRH-R1) has a cytosolic carboxy-terminal tail. We investigated the role of β-arrestin 1 in GnRH-R2-mediated signalling and mapped the regions in GnRH-R2 required for recruitment of β-arrestin, employing internalization assays. We show that GnRH-R2 activation of ERK is dependent on β-arrestin and protein kinase C. Appending the tail of GnRH-R2 to GnRH-R1 enabled GRK- and β-arrestin-dependent internalization of the chimaeric receptor. Surprisingly, carboxy-terminally truncated GnRH-R2 retained β-arrestin and GRK-dependent internalization, suggesting that β-arrestin interacts with additional elements of GnRH-R2. Mutating serine and threonine or basic residues of intracellular loop 3 did not abolish β-arrestin 1-dependent internalization but a receptor lacking these basic residues and the carboxy-terminus showed no β-arrestin 1-dependent internalization. Our results suggest that basic residues at the amino-terminal end of intracellular loop 3 or the carboxy-terminal tail are required for β-arrestin dependent internalization. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Adenovirus fibre shaft sequences fold into the native triple beta-spiral fold when N-terminally fused to the bacteriophage T4 fibritin foldon trimerisation motif.

Science.gov (United States)

Papanikolopoulou, Katerina; Teixeira, Susana; Belrhali, Hassan; Forsyth, V Trevor; Mitraki, Anna; van Raaij, Mark J

2004-09-03

Adenovirus fibres are trimeric proteins that consist of a globular C-terminal domain, a central fibrous shaft and an N-terminal part that attaches to the viral capsid. In the presence of the globular C-terminal domain, which is necessary for correct trimerisation, the shaft segment adopts a triple beta-spiral conformation. We have replaced the head of the fibre by the trimerisation domain of the bacteriophage T4 fibritin, the foldon. Two different fusion constructs were made and crystallised, one with an eight amino acid residue linker and one with a linker of only two residues. X-ray crystallographic studies of both fusion proteins shows that residues 319-391 of the adenovirus type 2 fibre shaft fold into a triple beta-spiral fold indistinguishable from the native structure, although this is now resolved at a higher resolution of 1.9 A. The foldon residues 458-483 also adopt their natural structure. The intervening linkers are not well ordered in the crystal structures. This work shows that the shaft sequences retain their capacity to fold into their native beta-spiral fibrous fold when fused to a foreign C-terminal trimerisation motif. It provides a structural basis to artificially trimerise longer adenovirus shaft segments and segments from other trimeric beta-structured fibre proteins. Such artificial fibrous constructs, amenable to crystallisation and solution studies, can offer tractable model systems for the study of beta-fibrous structure. They can also prove useful for gene therapy and fibre engineering applications.

Conformation of dehydropentapeptides containing four achiral amino acid residues – controlling the role of L-valine

Directory of Open Access Journals (Sweden)

Michał Jewgiński

2014-03-01

Full Text Available Structural studies of pentapeptides containing an achiral block, built from two dehydroamino acid residues (ΔZPhe and ΔAla and two glycines, as well as one chiral L-Val residue were performed using NMR spectroscopy. The key role of the L-Val residue in the generation of the secondary structure of peptides is discussed. The obtained results suggest that the strongest influence on the conformation of peptides arises from a valine residue inserted at the C-terminal position. The most ordered conformation was found for peptide Boc-Gly-ΔAla-Gly-ΔZPhe-Val-OMe (3, which adopts a right-handed helical conformation.

The unique N-terminal zinc finger of synaptotagmin-like protein 4 reveals FYVE structure.

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Miyamoto, Kazuhide; Nakatani, Arisa; Saito, Kazuki

2017-12-01

Synaptotagmin-like protein 4 (Slp4), expressed in human platelets, is associated with dense granule release. Slp4 is comprised of the N-terminal zinc finger, Slp homology domain, and C2 domains. We synthesized a compact construct (the Slp4N peptide) corresponding to the Slp4 N-terminal zinc finger. Herein, we have determined the solution structure of the Slp4N peptide by nuclear magnetic resonance (NMR). Furthermore, experimental, chemical modification of Cys residues revealed that the Slp4N peptide binds two zinc atoms to mediate proper folding. NMR data showed that eight Cys residues coordinate zinc atoms in a cross-brace fashion. The Simple Modular Architecture Research Tool database predicted the structure of Slp4N as a RING finger. However, the actual structure of the Slp4N peptide adopts a unique C 4 C 4 -type FYVE fold and is distinct from a RING fold. To create an artificial RING finger (ARF) with specific ubiquitin-conjugating enzyme (E2)-binding capability, cross-brace structures with eight zinc-ligating residues are needed as the scaffold. The cross-brace structure of the Slp4N peptide could be utilized as the scaffold for the design of ARFs. © 2017 The Protein Society.

Structures of the Gasdermin D C-Terminal Domains Reveal Mechanisms of Autoinhibition.

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Liu, Zhonghua; Wang, Chuanping; Rathkey, Joseph K; Yang, Jie; Dubyak, George R; Abbott, Derek W; Xiao, Tsan Sam

2018-05-01

Pyroptosis is an inflammatory form of programmed cell death that plays important roles in immune protection against infections and in inflammatory disorders. Gasdermin D (GSDMD) is an executor of pyroptosis upon cleavage by caspases-1/4/5/11 following canonical and noncanonical inflammasome activation. GSDMD N-terminal domain assembles membrane pores to induce cytolysis, whereas its C-terminal domain inhibits cell death through intramolecular association with the N domain. The molecular mechanisms of autoinhibition for GSDMD are poorly characterized. Here we report the crystal structures of the human and murine GSDMD C-terminal domains, which differ from those of the full-length murine GSDMA3 and the human GSDMB C-terminal domain. Mutations of GSDMD C-domain residues predicted to locate at its interface with the N-domain enhanced pyroptosis. Our results suggest that GSDMDs may employ a distinct mode of intramolecular domain interaction and autoinhibition, which may be relevant to its unique role in pyroptosis downstream of inflammasome activation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Microstructural observations on the terminal penetration of long rod projectile

OpenAIRE

Krushna Kumbhar; P. Ponguru Senthil; A.K. Gogia

2017-01-01

Present study focuses on the terminal penetration of tungsten heavy alloy (WHA) long rod penetrator impacted against armour steel at an impact velocity of 1600 m/s. The residual penetrator and armour steel target recovered after the ballistic test have been characterized using optical microscope, scanning electron microscope (SEM) and electron probe micro analyzer (EPMA). Metallurgical changes in target steel and WHA remnant have been analysed. Large shear stresses and shear localization have...

Manual for conducting radiological surveys in support of license termination

International Nuclear Information System (INIS)

Berger, J.D.

1992-06-01

This document describes a process for conducting radiological surveys during decommissioning, to demonstrate that residual radioactive material satisfies criteria established by the U.S. Nuclear Regulatory Commission (NRC) for termination of a license. The Manual describes procedures for design and conduct of surveys in a manner which will provide a high degree of assurance that NRC guidelines and conditions have been satisfied. The Manual also describes methods for documenting the survey findings in a final report to the NRC. This Manual updates information contained in NUREG/CR-2082, Monitoring for Compliance with Decommissioning Termination Survey Criteria, (ORNL 1981). It incorporates statistical approaches to survey design and data interpretation used by the Environmental Protection Agency for evaluation of hazardous materials sites under Superfund (CERCLA). Quality assurance is emphasized throughout. (author)

Effect of roller/crimper designs in terminating rye cover crop in small-scale conservation systems

Science.gov (United States)

In recent years, use of cover crops in no-till organic production systems has steadily increased. When cover crops are terminated at an appropriate growth stage, the unincorporated residue mulch protects the soil from erosion, runoff, soil compaction, and weed pressure, and conserves soil water. In ...

Roles of the C-terminal domains of human dihydrodiol dehydrogenase isoforms in the binding of substrates and modulators: probing with chimaeric enzymes.

Science.gov (United States)

Matsuura, K; Hara, A; Deyashiki, Y; Iwasa, H; Kume, T; Ishikura, S; Shiraishi, H; Katagiri, Y

1998-01-01

Human liver dihydrodiol dehydrogenase (DD; EC 1.3.1.20) exists in isoforms (DD1, DD2 and DD4) composed of 323 amino acids. DD1 and DD2 share 98% amino acid sequence identity, but show lower identities (approx. 83%) with DD4, in which a marked difference is seen in the C-terminal ten amino acids. DD4 exhibits unique catalytic properties, such as the ability to oxidize both (R)- and (S)-alicyclic alcohols equally, high dehydrogenase activity for bile acids, potent inhibition by steroidal anti-inflammatory drugs and activation by sulphobromophthalein and clofibric acid derivatives. In this study, we have prepared chimaeric enzymes, in which we exchanged the C-terminal 39 residues between the two enzymes. Compared with DD1, CDD1-4 (DD1 with the C-terminal sequence of DD4) had increased kcat/Km values for 3alpha-hydroxy-5beta-androstanes and bile acids of 3-9-fold and decreased values for the other substrates by 5-100-fold. It also became highly sensitive to DD4 inhibitors such as phenolphthalein and hexoestrol. Another chimaeric enzyme, CDD4-1 (DD4 with the C-terminal sequence of DD1), showed the same (S)-stereospecificity for the alicyclic alcohols as DD1, had decreased kcat/Km values for bile acids with 7beta- or 12alpha-hydroxy groups by more than 120-fold and was resistant to inhibition by betamethasone. In addition, the activation effects of sulphobromophthalein and bezafibrate decreased or disappeared for CDD4-1. The recombinant DD4 with the His314-->Pro (the corresponding residue of DD1) mutation showed intermediate changes in the properties between those of wild-type DD4 and CDD4-1. The results indicate that the binding of substrates, inhibitors and activators to the enzymes is controlled by residues in their C-terminal domains; multiple residues co-ordinately act as determinants for substrate specificity and inhibitor sensitivity. PMID:9820821

GBNV encoded movement protein (NSm) remodels ER network via C-terminal coiled coil domain

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Singh, Pratibha; Savithri, H.S., E-mail: bchss@biochem.iisc.ernet.in

2015-08-15

Plant viruses exploit the host machinery for targeting the viral genome–movement protein complex to plasmodesmata (PD). The mechanism by which the non-structural protein m (NSm) of Groundnut bud necrosis virus (GBNV) is targeted to PD was investigated using Agrobacterium mediated transient expression of NSm and its fusion proteins in Nicotiana benthamiana. GFP:NSm formed punctuate structures that colocalized with mCherry:plasmodesmata localized protein 1a (PDLP 1a) confirming that GBNV NSm localizes to PD. Unlike in other movement proteins, the C-terminal coiled coil domain of GBNV NSm was shown to be involved in the localization of NSm to PD, as deletion of this domain resulted in the cytoplasmic localization of NSm. Treatment with Brefeldin A demonstrated the role of ER in targeting GFP NSm to PD. Furthermore, mCherry:NSm co-localized with ER–GFP (endoplasmic reticulum targeting peptide (HDEL peptide fused with GFP). Co-expression of NSm with ER–GFP showed that the ER-network was transformed into vesicles indicating that NSm interacts with ER and remodels it. Mutations in the conserved hydrophobic region of NSm (residues 130–138) did not abolish the formation of vesicles. Additionally, the conserved prolines at positions 140 and 142 were found to be essential for targeting the vesicles to the cell membrane. Further, systematic deletion of amino acid residues from N- and C-terminus demonstrated that N-terminal 203 amino acids are dispensable for the vesicle formation. On the other hand, the C-terminal coiled coil domain when expressed alone could also form vesicles. These results suggest that GBNV NSm remodels the ER network by forming vesicles via its interaction through the C-terminal coiled coil domain. Interestingly, NSm interacts with NP in vitro and coexpression of these two proteins in planta resulted in the relocalization of NP to PD and this relocalization was abolished when the N-terminal unfolded region of NSm was deleted. Thus, the NSm

Structure discrimination for the C-terminal domain of Escherichia coli trigger factor in solution

International Nuclear Information System (INIS)

Yao Yong; Bhabha, Gira; Kroon, Gerard; Landes, Mindy; Dyson, H. Jane

2008-01-01

NMR measurements can give important information on solution structure, without the necessity for a full-scale solution structure determination. The C-terminal protein binding domain of the ribosome-associated chaperone protein trigger factor is composed of non-contiguous parts of the polypeptide chain, with an interpolated prolyl isomerase domain. A construct of the C-terminal domain of Escherichia coli trigger factor containing residues 113-149 and 247-432, joined by a Gly-Ser-Gly-Ser linker, is well folded and gives excellent NMR spectra in solution. We have used NMR measurements on this construct, and on a longer construct that includes the prolyl isomerase domain, to distinguish between two possible structures for the C-terminal domain of trigger factor, and to assess the behavior of the trigger factor C-terminal domain in solution. Two X-ray crystal structures, of intact trigger factor from E. coli (Ferbitz et al., Nature 431:590-596, 2004), and of a truncated trigger factor from Vibrio cholerae (Ludlam et al., Proc Natl Acad Sci USA 101:13436-13441, 2004) showed significant differences in the structure of the C-terminal domain, such that the two structures could not be superimposed. We show using NMR chemical shifts and long range nuclear Overhauser effects that the secondary and tertiary structure of the E. coli C-terminal domain in solution is consistent with the crystal structure of the E. coli trigger factor and not with the V. cholerae protein. Given the similarity of the amino acid sequences of the E. coli and V. cholerae proteins, it appears likely that the structure of the V. cholerae protein has been distorted as a result of truncation of a 44-amino acid segment at the C-terminus. Analysis of residual dipolar coupling measurements shows that the overall topology of the solution structure is completely inconsistent with both structures. Dynamics analysis of the C-terminal domain using T 1 , T 2 and heteronuclear NOE parameters show that the protein is

Dual role of the carboxyl-terminal region of pig liver L-kynurenine 3-monooxygenase: mitochondrial-targeting signal and enzymatic activity.

Science.gov (United States)

Hirai, Kumiko; Kuroyanagi, Hidehito; Tatebayashi, Yoshitaka; Hayashi, Yoshitaka; Hirabayashi-Takahashi, Kanako; Saito, Kuniaki; Haga, Seiich; Uemura, Tomihiko; Izumi, Susumu

2010-12-01

l-kynurenine 3-monooxygenase (KMO) is an NAD(P)H-dependent flavin monooxygenase that catalyses the hydroxylation of l-kynurenine to 3-hydroxykynurenine, and is localized as an oligomer in the mitochondrial outer membrane. In the human brain, KMO may play an important role in the formation of two neurotoxins, 3-hydroxykynurenine and quinolinic acid, both of which provoke severe neurodegenerative diseases. In mosquitos, it plays a role in the formation both of eye pigment and of an exflagellation-inducing factor (xanthurenic acid). Here, we present evidence that the C-terminal region of pig liver KMO plays a dual role. First, it is required for the enzymatic activity. Second, it functions as a mitochondrial targeting signal as seen in monoamine oxidase B (MAO B) or outer membrane cytochrome b(5). The first role was shown by the comparison of the enzymatic activity of two mutants (C-terminally FLAG-tagged KMO and carboxyl-terminal truncation form, KMOΔC50) with that of the wild-type enzyme expressed in COS-7 cells. The second role was demonstrated with fluorescence microscopy by the comparison of the intracellular localization of the wild-type, three carboxyl-terminal truncated forms (ΔC20, ΔC30 and ΔC50), C-terminally FLAG-tagged wild-type and a mutant KMO, where two arginine residues, Arg461-Arg462, were replaced with Ser residues.

Dynamics of termination during in vitro replication of ultraviolet-irradiated DNA with DNA polymerase III holoenzyme of Escherichia coli

International Nuclear Information System (INIS)

Shwartz, H.; Livneh, Z.

1987-01-01

During in vitro replication of UV-irradiated single-stranded DNA with Escherichia coli DNA polymerase III holoenzyme termination frequently occurs at pyrimidine photodimers. The termination stage is dynamic and characterized by at least three different events: repeated dissociation-reinitiation cycles of the polymerase at the blocked termini; extensive hydrolysis of ATP to ADP and inorganic phosphate; turnover of dNTPs into dNMP. The reinitiation events are nonproductive and are not followed by further elongation. The turnover of dNTPs into dNMPs is likely to result from repeated cycles of insertion of dNMP residues opposite the blocking lesions followed by their excision by the 3'----5' exonucleolytic activity of the polymerase. Although all dNTPs are turned over, there is a preference for dATP, indicating that DNA polymerase III holoenzyme has a preference for inserting a dAMP residue opposite blocking pyrimidine photodimers. We suggest that the inability of the polymerase to bypass photodimers during termination is due to the formation of defective initiation-like complexes with reduced stability at the blocked termini

Rapid Mass Spectrometric Analysis of a Novel Fucoidan, Extracted from the Brown Alga Coccophora langsdorfii

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Stanislav D. Anastyuk

2014-01-01

Full Text Available The novel highly sulfated (35% fucoidan fraction Cf2 , which contained, along with fucose, galactose and traces of xylose and uronic acids was purified from the brown alga Coccophora langsdorfii. Its structural features were predominantly determined (in comparison with fragments of known structure by a rapid mass spectrometric investigation of the low-molecular-weight fragments, obtained by “mild” (5 mg/mL and “exhaustive” (maximal concentration autohydrolysis. Tandem matrix-assisted laser desorption/ionization mass spectra (MALDI-TOF/TOFMS of fucooligosaccharides with even degree of polymerization (DP, obtained by “mild” autohydrolysis, were the same as that observed for fucoidan from Fucus evanescens, which have a backbone of alternating (1 → 3- and (1 → 4 linked sulfated at C-2 and sometimes at C-4 of 3-linked α-L-Fucp residues. Fragmentation patterns of oligosaccharides with odd DP indicated sulfation at C-2 and at C-4 of (1 → 3 linked α-L-Fucp residues on the reducing terminus. Minor sulfation at C-3 was also suggested. The “exhaustive” autohydrolysis allowed us to observe the “mixed” oligosaccharides, built up of fucose/xylose and fucose/galactose. Xylose residues were found to occupy both the reducing and nonreducing termini of FucXyl disaccharides. Nonreducing galactose residues as part of GalFuc disaccharides were found to be linked, possibly, by 2-type of linkage to fucose residues and were found to be sulfated, most likely, at position C-2.

Investigation of the structural preference and flexibility of the loop residues in amyloid fibrils of the HET-s prion.

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Dolenc, Jožica; Meier, Beat H; Rusu, Victor H; van Gunsteren, Wilfred F

2016-02-17

The structural variability of a 16-residue loop (residues 246-261) which is in part disordered and connects two layers of the β-solenoid formed by the prion-form of HET-s and its prion domain HET-s(218-289) is investigated using molecular dynamics computer simulation. A system of three HET-s(218-289) molecules in a β-sheet structure as in the fibril is simulated in aqueous solution. The trajectory structures appear to be consistent with the Cα chemical shift data obtained. In order to delineate the influence of the β-sheet core of the fibril upon the structural variability of the loop, the latter is also simulated without the β-sheet core, but with its N- and C-terminal residues restrained at their positions in the fibril. The analysis of the trajectories shows that the structural variability of the loop is restricted by the β-sheet core, least at its N-terminal end and most in the middle of the trimer.

Covalent Bonding of Pyrrolobenzodiazepines (PBDs) to Terminal Guanine Residues within Duplex and Hairpin DNA Fragments

Science.gov (United States)

Mantaj, Julia; Jackson, Paul J. M.; Karu, Kersti; Rahman, Khondaker M.; Thurston, David E.

2016-01-01

Pyrrolobenzodiazepines (PBDs) are covalent-binding DNA-interactive agents with growing importance as payloads in Antibody Drug Conjugates (ADCs). Until now, PBDs were thought to covalently bond to C2-NH2 groups of guanines in the DNA-minor groove across a three-base-pair recognition sequence. Using HPLC/MS methodology with designed hairpin and duplex oligonucleotides, we have now demonstrated that the PBD Dimer SJG-136 and the C8-conjugated PBD Monomer GWL-78 can covalently bond to a terminal guanine of DNA, with the PBD skeleton spanning only two base pairs. Control experiments with the non-C8-conjugated anthramycin along with molecular dynamics simulations suggest that the C8-substituent of a PBD Monomer, or one-half of a PBD Dimer, may provide stability for the adduct. This observation highlights the importance of PBD C8-substituents, and also suggests that PBDs may bind to terminal guanines within stretches of DNA in cells, thus representing a potentially novel mechanism of action at the end of DNA strand breaks. PMID:27055050

NMR structure of the N-terminal domain of the replication initiator protein DnaA

Energy Technology Data Exchange (ETDEWEB)

Wemmer, David E.; Lowery, Thomas J.; Pelton, Jeffrey G.; Chandonia, John-Marc; Kim, Rosalind; Yokota, Hisao; Wemmer, David E.

2007-08-07

DnaA is an essential component in the initiation of bacterial chromosomal replication. DnaA binds to a series of 9 base pair repeats leading to oligomerization, recruitment of the DnaBC helicase, and the assembly of the replication fork machinery. The structure of the N-terminal domain (residues 1-100) of DnaA from Mycoplasma genitalium was determined by NMR spectroscopy. The backbone r.m.s.d. for the first 86 residues was 0.6 +/- 0.2 Angstrom based on 742 NOE, 50 hydrogen bond, 46 backbone angle, and 88 residual dipolar coupling restraints. Ultracentrifugation studies revealed that the domain is monomeric in solution. Features on the protein surface include a hydrophobic cleft flanked by several negative residues on one side, and positive residues on the other. A negatively charged ridge is present on the opposite face of the protein. These surfaces may be important sites of interaction with other proteins involved in the replication process. Together, the structure and NMR assignments should facilitate the design of new experiments to probe the protein-protein interactions essential for the initiation of DNA replication.

A study of the influence of charged residues on β-hairpin formation by nuclear magnetic resonance and molecular dynamics.

Science.gov (United States)

Makowska, Joanna; Zmudzińska, Wioletta; Uber, Dorota; Chmurzyński, Lech

2014-12-01

Chain reversals are often nucleation sites in protein folding. The β-hairpins of FBP28 WW domain and IgG are stable and have been proved to initiate the folding and are, therefore, suitable for studying the influence of charged residues on β-hairpin conformation. In this paper, we carried out NMR examination of the conformations in solution of two fragments from the FPB28 protein (PDB code: 1E0L) (N-terminal part) namely KTADGKT-NH2 (1E0L 12-18, D7) and YKTADGKTY-NH2 (1E0L 11-19, D9), one from the B3 domain of the protein G (PDB code: 1IGD), namely DDATKT-NH2 (1IGD 51-56) (Dag1), and three variants of Dag1 peptide: DVATKT-NH2 (Dag2), OVATKT-NH2 (Dag3) and KVATKT-NH2 (Dag4), respectively, in which the original charged residue were replaced with non-polar residues or modified charged residues. It was found that both the D7 and D9 peptides form a large fraction bent conformations. However, no hydrophobic contacts between the terminal Tyr residues of D9 occur, which suggests that the presence of a pair of like-charged residues stabilizes chain reversal. Conversely, only the Dag1 and Dag2 peptides exhibit some chain reversal; replacing the second aspartic-acid residue with a valine and the first one with a basic residue results in a nearly extended conformation. These results suggest that basic residues farther away in sequence can result in stabilization of chain reversal owing to screening of the non-polar core. Conversely, smaller distance in sequence prohibits this screening, while the presence oppositely-charged residues can stabilize a turn because of salt-bridge formation.

3' terminal diversity of MRP RNA and other human noncoding RNAs revealed by deep sequencing.

Science.gov (United States)

Goldfarb, Katherine C; Cech, Thomas R

2013-09-21

Post-transcriptional 3' end processing is a key component of RNA regulation. The abundant and essential RNA subunit of RNase MRP has been proposed to function in three distinct cellular compartments and therefore may utilize this mode of regulation. Here we employ 3' RACE coupled with high-throughput sequencing to characterize the 3' terminal sequences of human MRP RNA and other noncoding RNAs that form RNP complexes. The 3' terminal sequence of MRP RNA from HEK293T cells has a distinctive distribution of genomically encoded termini (including an assortment of U residues) with a portion of these selectively tagged by oligo(A) tails. This profile contrasts with the relatively homogenous 3' terminus of an in vitro transcribed MRP RNA control and the differing 3' terminal profiles of U3 snoRNA, RNase P RNA, and telomerase RNA (hTR). 3' RACE coupled with deep sequencing provides a valuable framework for the functional characterization of 3' terminal sequences of noncoding RNAs.

Mode of inhibition of HIV-1 Integrase by a C-terminal domain-specific monoclonal antibody*

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Merkel George

2006-06-01

Full Text Available Abstract Background To further our understanding of the structure and function of HIV-1 integrase (IN we developed and characterized a library of monoclonal antibodies (mAbs directed against this protein. One of these antibodies, mAb33, which is specific for the C-terminal domain, was found to inhibit HIV-1 IN processing activity in vitro; a corresponding Fv fragment was able to inhibit HIV-1 integration in vivo. Our subsequent studies, using heteronuclear nuclear magnetic resonance spectroscopy, identified six solvent accessible residues on the surface of the C-terminal domain that were immobilized upon binding of the antibody, which were proposed to comprise the epitope. Here we test this hypothesis by measuring the affinity of mAb33 to HIV-1 proteins that contain Ala substitutions in each of these positions. To gain additional insight into the mode of inhibition we also measured the DNA binding capacity and enzymatic activities of the Ala substituted proteins. Results We found that Ala substitution of any one of five of the putative epitope residues, F223, R224, Y226, I267, and I268, caused a decrease in the affinity of the mAb33 for HIV-1 IN, confirming the prediction from NMR data. Although IN derivatives with Ala substitutions in or near the mAb33 epitope exhibited decreased enzymatic activity, none of the epitope substitutions compromised DNA binding to full length HIV-1 IN, as measured by surface plasmon resonance spectroscopy. Two of these derivatives, IN (I276A and IN (I267A/I268A, exhibited both increased DNA binding affinity and uncharacteristic dissociation kinetics; these proteins also exhibited non-specific nuclease activity. Results from these investigations are discussed in the context of current models for how the C-terminal domain interacts with substrate DNA. Conclusion It is unlikely that inhibition of HIV-1 IN activity by mAb33 is caused by direct interaction with residues that are essential for substrate binding. Rather

On the terminal homologation of physiologically active peptides as a means of increasing stability in human serum--neurotensin, opiorphin, B27-KK10 epitope, NPY.

Science.gov (United States)

Seebach, Dieter; Lukaszuk, Aneta; Patora-Komisarska, Krystyna; Podwysocka, Dominika; Gardiner, James; Ebert, Marc-Olivier; Reubi, Jean Claude; Cescato, Renzo; Waser, Beatrice; Gmeiner, Peter; Hübner, Harald; Rougeot, Catherine

2011-05-01

The terminal homologation by CH(2) insertion into the peptides mentioned in the title is described. This involves replacement of the N-terminal amino acid residue by a β(2) - and of the C-terminal amino acid residue by a β(3) -homo-amino acid moiety (β(2) hXaa and β(3) hXaa, resp.; Fig. 1). In this way, the structure of the peptide chain from the N-terminal to the C-terminal stereogenic center is identical, and the modified peptide is protected against cleavage by exopeptidases (Figs. 2 and 3). Neurotensin (NT; 1) and its C-terminal fragment NT(8-13) are ligands of the G-protein-coupled receptors (GPCR) NT1, NT2, NT3, and NT analogs are promising tools to be used in cancer diagnostics and therapy. The affinities of homologated NT analogs, 2b-2e, for NT1 and NT2 receptors were determined by using cell homogenates and tumor tissues (Table 1); in the latter experiments, the affinities for the NT1 receptor are more or less the same as those of NT (0.5-1.3 vs. 0.6 nM). At the same time, one of the homologated NT analogs, 2c, survives in human plasma for 7 days at 37° (Fig. 6). An NMR analysis of NT(8-13) (Tables 2 and 4, and Fig. 8) reveals that this N-terminal NT fragment folds to a turn in CD(3) OH. - In the case of the human analgesic opiorphin (3a), a pentapeptide, and of the HIV-derived B27-KK10 (4a), a decapeptide, terminal homologation (→3b and 4b, resp.) led to a 7- and 70-fold half-life increase in plasma (Fig. 9). With N-terminally homologated NPY, 5c, we were not able to determine serum stability; the peptide consisting of 36 amino acid residues is subject to cleavage by endopetidases. Three of the homologated compounds, 2b, 2c, and 5c, were shown to be agonists (Fig. 7 and 11). A comparison of terminal homologation with other stability-increasing terminal modifications of peptides is performed (Fig. 5), and possible applications of the neurotensin analogs, described herein, are discussed. Copyright © 2011 Verlag Helvetica Chimica

The C-terminal random coil region tunes the Ca²⁺-binding affinity of S100A4 through conformational activation.

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Annette Duelli

Full Text Available S100A4 interacts with many binding partners upon Ca2+ activation and is strongly associated with increased metastasis formation. In order to understand the role of the C-terminal random coil for the protein function we examined how small angle X-ray scattering of the wild-type S100A4 and its C-terminal deletion mutant (residues 1-88, Δ13 changes upon Ca2+ binding. We found that the scattering intensity of wild-type S100A4 changes substantially in the 0.15-0.25 Å-1 q-range whereas a similar change is not visible in the C-terminus deleted mutant. Ensemble optimization SAXS modeling indicates that the entire C-terminus is extended when Ca2+ is bound. Pulsed field gradient NMR measurements provide further support as the hydrodynamic radius in the wild-type protein increases upon Ca2+ binding while the radius of Δ13 mutant does not change. Molecular dynamics simulations provide a rational explanation of the structural transition: the positively charged C-terminal residues associate with the negatively charged residues of the Ca2+-free EF-hands and these interactions loosen up considerably upon Ca2+-binding. As a consequence the Δ13 mutant has increased Ca2+ affinity and is constantly loaded at Ca2+ concentration ranges typically present in cells. The activation of the entire C-terminal random coil may play a role in mediating interaction with selected partner proteins of S100A4.

Structure of the EMMPRIN N-terminal domain 1: Dimerization via [beta]-strand swapping

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Luo, Jinquan; Teplyakov, Alexey; Obmolova, Galina; Malia, Thomas; Wu, Sheng-Jiun; Beil, Eric; Baker, Audrey; Swencki-Underwood, Bethany; Zhao, Yonghong; Sprenkle, Justin; Dixon, Ken; Sweet, Raymond; Gilliland, Gary L.; (Centocor)

2010-09-27

Extracellular matrix metalloproteinase inducer (EMMPRIN), also known as Hab18G, CD147, Basigin, M6, and neurothelin, is a membrane glycoprotein expressed on the surface of various cell types and many cancer cells. EMMPRIN stimulates adjacent fibroblasts and tumor cells to produce matrix metalloproteinases and plays an important role in tumor invasion and metastasis, angiogenesis, spermatogensis and fertilization, cell-cell adhesion and communication, and other biological processes (reviewed in Ref. 1 and references therein). It was demonstrated that the EMMPRIN extracellular domain (ECD), which structurally belongs to the IgG superfamily, can form homo-oligomers in a cis dependent manner and the N-terminal domain 1 (residues 22-101) was necessary and sufficient to mediate this interaction. The crystal structure of the ECD of recombinant human EMMPRIN (Hab18G/CD147) expressed in E. coli was reported at 2.8 {angstrom} resolution (Yu et al. 2008). The construct consists of residues 22-205 of the mature protein and has both an N-terminal IgC2 domain (ND1, residues 22-101) and a C-terminal IgC2 domain (ND2, residues 107-205). The two domains are joined by a five amino acid residue linker that constitutes a flexible hinge between the two domains. The crystal form has four copies of the molecule in the asymmetric unit, each of which has a different inter-domain angle that varies from 121{sup o} to 144{sup o}. The two domains each have a conserved disulfide bridge and both are comprised of two {beta}-sheets formed by strands EBA and GFCC, and DEBA and AGFCC for ND1 and ND2, respectively. Based on the crystal packing in this structure, the authors proposed that lateral packing between the two IgG domains of EMMPRIN ECD represents a potential mechanism for cell adhesion. Here we report the 2.0-{angstrom} crystal structure of the N-terminal domain of EMMPRIN ECD (ND1) expressed in mammalian cells. The overall structure of the domain is very similar to that in the full length

Flexible xxx-asp/asn and gly-xxx residues of equine cytochrome C in matrix-assisted laser desorption/ionization in-source decay mass spectrometry.

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Takayama, Mitsuo

2012-01-01

The backbone flexibility of a protein has been studied from the standpoint of the susceptibility of amino acid residues to in-source decay (ISD) in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Residues more susceptible to MALDI-ISD, namely Xxx-Asp/Asn and Gly-Xxx, were identified from the discontinuous intense peak of c'-ions originating from specific cleavage at N-Cα bonds of the backbone of equine cytochrome c. The identity of the residues susceptible to ISD was consistent with the known flexible backbone amides as estimated by hydrogen/deuterium exchange (HDX) experiments. The identity of these flexible amino acid residues (Asp, Asn, and Gly) is consistent with the fact that these residues are preferred in flexible secondary structure free from intramolecular hydrogen-bonded structures such as α-helix and β-sheet. The MALDI-ISD spectrum of equine cytochrome c gave not only intense N-terminal side c'-ions originating from N-Cα bond cleavage at Xxx-Asp/Asn and Gly-Xxx residues, but also C-terminal side complement z'-ions originating from the same cleavage sites. The present study implies that MALDI-ISD can give information about backbone flexibility of proteins, comparable with the protection factors estimated by HDX.

Flexible Xxx–Asp/Asn and Gly–Xxx Residues of Equine Cytochrome c in Matrix-Assisted Laser Desorption/Ionization In-Source Decay Mass Spectrometry

Science.gov (United States)

Takayama, Mitsuo

2012-01-01

The backbone flexibility of a protein has been studied from the standpoint of the susceptibility of amino acid residues to in-source decay (ISD) in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Residues more susceptible to MALDI-ISD, namely Xxx–Asp/Asn and Gly–Xxx, were identified from the discontinuous intense peak of c′-ions originating from specific cleavage at N–Cα bonds of the backbone of equine cytochrome c. The identity of the residues susceptible to ISD was consistent with the known flexible backbone amides as estimated by hydrogen/deuterium exchange (HDX) experiments. The identity of these flexible amino acid residues (Asp, Asn, and Gly) is consistent with the fact that these residues are preferred in flexible secondary structure free from intramolecular hydrogen-bonded structures such as α-helix and β-sheet. The MALDI-ISD spectrum of equine cytochrome c gave not only intense N-terminal side c′-ions originating from N–Cα bond cleavage at Xxx–Asp/Asn and Gly–Xxx residues, but also C-terminal side complement z′-ions originating from the same cleavage sites. The present study implies that MALDI-ISD can give information about backbone flexibility of proteins, comparable with the protection factors estimated by HDX. PMID:24349908

Radiotracer studies of fungicide residues in food plants

International Nuclear Information System (INIS)

1990-04-01

Agricultural fungicides are chemicals used on seeds, crops and in soils throughout the growing season. Fungicide treatments may lead to various levels of chemical residues in food commodities. Primary emphasis has been placed on ethylenebisdithiocarbamates (EBDCs), an important group of agrofungicides used in preparations for spraying or dusting major crops such as apples, pears, broccoli, cabbages, egg plants, cauliflower, grapes, lettuce, peppers, celery, cucumbers and tomatoes. Treatments with EBDCs result in terminal residues containing ethylenthiourea (ETU). This is a toxicologically significant decomposition product which has attracted considerable attention in recent years due to indications of its potential goitrogenic and carcinogenic properties. In recognition of the need for a coordinated examination of ETU levels in food, particularly under tropical conditions, the program of radiotracer techniques as a tool for studying fungicide residue problems on food was initiated in 1984. In current studies, three EBDCs, maneb, zineb and mancozeb from different manufacturers in different countries were analysed. This report describes the model protocols (Annexes I, II and III) as they were set up for determination of residues in commodities and soil, using radiotracer and conventional chromatographic techniques . In the 16 papers presented in this report C 14 -labelled EBDCs are determined in plants, vegetables, and soils, before and after cooking, as a function of time and of other agricultural parameters. Refs, figs and tabs

Highly potent antimicrobial peptides from N-terminal membrane-binding region of E. coli MreB.

Science.gov (United States)

Saikia, Karabi; Sravani, Yalavarthi Durga; Ramakrishnan, Vibin; Chaudhary, Nitin

2017-02-23

Microbial pathogenesis is a serious health concern. The threat escalates as the existing conventional antimicrobials are losing their efficacy against the evolving pathogens. Peptides hold promise to be developed into next-generation antibiotics. Antimicrobial peptides adopt amphipathic structures that could selectively bind to and disrupt the microbial membranes. Interaction of proteins with membranes is central to all living systems and we reasoned that the membrane-binding domains in microbial proteins could be developed into efficient antimicrobials. This is an interesting approach as self-like sequences could elude the microbial strategies of degrading the antimicrobial peptides, one of the mechanisms of showing resistance to antimicrobials. We selected the 9-residue-long membrane-binding region of E. coli MreB protein. The 9-residue peptide (C-terminal amide) and its N-terminal acetylated analog displayed broad-spectrum activity, killing Gram-negative bacteria, Gram-positive bacteria, and fungi. Extension with a tryptophan residue at the N-terminus drastically improved the activity of the peptides with lethal concentrations ≤10 μM against all the organisms tested. The tryptophan-extended peptides caused complete killing of C. albicans as well as gentamicin and methicillin resistant S. aureus at 5 μM concentration. Lipid-binding studies and electron microscopic analyses of the peptide-treated microbes suggest membrane disruption as the mechanism of killing.

Kaposi's sarcoma herpesvirus C-terminal LANA concentrates at pericentromeric and peri-telomeric regions of a subset of mitotic chromosomes

International Nuclear Information System (INIS)

Kelley-Clarke, Brenna; Ballestas, Mary E.; Komatsu, Takashi; Kaye, Kenneth M.

2007-01-01

The Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) tethers KSHV terminal repeat (TR) DNA to mitotic chromosomes to efficiently segregate episomes to progeny nuclei. LANA contains N- and C-terminal chromosome binding regions. We now show that C-terminal LANA preferentially concentrates to paired dots at pericentromeric and peri-telomeric regions of a subset of mitotic chromosomes through residues 996-1139. Deletions within C-terminal LANA abolished both self-association and chromosome binding, consistent with a requirement for self-association to bind chromosomes. A deletion abolishing TR DNA binding did not affect chromosome targeting, indicating LANA's localization is not due to binding its recognition sequence in chromosomal DNA. LANA distributed similarly on human and non-human mitotic chromosomes. These results are consistent with C-terminal LANA interacting with a cell factor that concentrates at pericentromeric and peri-telomeric regions of mitotic chromosomes

Estimation of the basicity of the donor strength of terminal groups in cationic polymethine dyes

Science.gov (United States)

Kachkovsky, Alexey; Obernikhina, Nataliya; Prostota, Yaroslav; Naumenko, Antonina; Melnyk, Dmitriy; Yashchuk, Valeriy

2018-02-01

The well-known conception of the basicity of the terminal groups in the cationic polymethine dyes showing their donor properties is examined (considered) in detail. The various approachs are proposed to quantitative quantum-chemical estimation of a donor strength of the terminal groups in cationic polymethine dyes: shift of the frontier levels upon introducing terminal residues in comparison with unsybstituted polymethine cation; transferring of the electron density from the terminal groups to the polymethine chain and hence manifested itself as a redistribution of total positive charge between molecular fragments; changes of the charge alternation at carbon atoms along the chain. All approach correlate between them and agree with the concept of the basicity as a capability of terminal heterocycles to show its donor properties in the polymethine dyes. The results of the fulfilled calculations of numerous examples are presented; the proposed parameters point correctly the tendency in the change donor strength upon varying of the chemical constitution: the dimension of cycle, introducing of various heteroatoms, linear or angular annelating by benzene ring; as well as direct to take into consideration the existence of local levels.

The N-terminal 33 amino acid domain of Siva-1 is sufficient for nuclear localization

Energy Technology Data Exchange (ETDEWEB)

Chen, J.Y.; Yang, L.X. [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou (China); Huang, Z.F. [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou (China); Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou (China); Key Laboratory of Tropical Diseases Control, Sun Yat-sen University, Ministry of Education in China, Guangzhou (China)

2013-12-02

Siva-1 induces apoptosis in multiple pathological processes and plays an important role in the suppression of tumor metastasis, protein degradation, and other functions. Although many studies have demonstrated that Siva-1 functions in the cytoplasm, a few have found that Siva-1 can relocate to the nucleus. In this study, we found that the first 33 amino acid residues of Siva-1 are required for its nuclear localization. Further study demonstrated that the green fluorescent protein can be imported into the nucleus after fusion with these 33 amino acid residues. Other Siva-1 regions and domains showed less effect on Siva-1 nuclear localization. By site-mutagenesis of all of these 33 amino acid residues, we found that mutants of the first 1-18 amino acids affected Siva-1 nuclear compartmentalization but could not complete this localization independently. In summary, we demonstrated that the N-terminal 33 amino acid residues were sufficient for Siva-1 nuclear localization, but the mechanism of this translocation needs additional investigation.

The N-terminal 33 amino acid domain of Siva-1 is sufficient for nuclear localization

International Nuclear Information System (INIS)

Chen, J.Y.; Yang, L.X.; Huang, Z.F.

2013-01-01

Siva-1 induces apoptosis in multiple pathological processes and plays an important role in the suppression of tumor metastasis, protein degradation, and other functions. Although many studies have demonstrated that Siva-1 functions in the cytoplasm, a few have found that Siva-1 can relocate to the nucleus. In this study, we found that the first 33 amino acid residues of Siva-1 are required for its nuclear localization. Further study demonstrated that the green fluorescent protein can be imported into the nucleus after fusion with these 33 amino acid residues. Other Siva-1 regions and domains showed less effect on Siva-1 nuclear localization. By site-mutagenesis of all of these 33 amino acid residues, we found that mutants of the first 1-18 amino acids affected Siva-1 nuclear compartmentalization but could not complete this localization independently. In summary, we demonstrated that the N-terminal 33 amino acid residues were sufficient for Siva-1 nuclear localization, but the mechanism of this translocation needs additional investigation

AQP4 plasma membrane trafficking or channel gating is not significantly modulated by phosphorylation at C-terminal serine residues

DEFF Research Database (Denmark)

Assentoft, Mette; Larsen, Brian R; Olesen, Emma T B

2014-01-01

heterologous expression in Xenopus laevis oocytes (along with serine-to-aspartate mutants of the same residues to mimic a phosphorylation). None of the mutant AQP4 constructs displayed alterations in the unit water permeability. Thus phosphorylation of six different serine residues in the COOH terminus of AQP4....... Phosphorylation of aquaporins can regulate plasma membrane localization and, possibly, the unit water permeability via gating of the AQP channel itself. In vivo phosphorylation of six serine residues in the COOH terminus of AQP4 has been detected by mass spectrometry: Ser(276), Ser(285), Ser(315), Ser(316), Ser...

RESIDUAL VALUE RISK IN AUTOMOTIVE OPERATING LEASE CONTRACTS

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Ivan Tot

2017-01-01

Full Text Available The subject of the research in this paper are automotive operating lease contracts in the Croatian business practice. The provisions of the general terms and conditions for operating lease contracts of the Croatian leasing companies are being analysed, particulary those relating to the rights and obligations of the parties to the contract after the operating lease contract was terminated and the motor vehicle returned to the lessor. The existence of three contractual models of the automotive operating lease contract in the Croatian business practice is established, which vary with regard to the assignment and the distribution of the residual value risk. Those contractual models are being compared with the two most common contractual models of automotive lease contracts in the Austrian and German business practice: the lease contract with the excess mileage adjustment and the lease contract with the terminal rent adjustment. On the basis of the results of this comparison, applicability of the legal solutions, developed in the Austrian and German jurisprudence and legal literature regarding the lease contract with the excess mileage adjustment and the lease contract with the terminal rent adjustment, to the automotive operating lease contract within the framework of Croatian law, is being examinded.

The first N-terminal unprotected (Gly-Aib)n peptide: H-Gly-Aib-Gly-Aib-OtBu.

Science.gov (United States)

Gessmann, Renate; Brückner, Hans; Petratos, Kyriacos

2015-12-01

Glycine (Gly) is incorporated in roughly half of all known peptaibiotic (nonribosomally biosynthesized antibiotic peptides of fungal origin) sequences and is the residue with the greatest conformational flexibility. The conformational space of Aib (α-aminoisobutyric acid) is severely restricted by the second methyl group attached to the Cα atom. Most of the crystal structures containing Aib are N-terminal protected. Deprotection of the N- or C-terminus of peptides may alter the hydrogen-bonding scheme and/or the structure and may facilitate crystallization. The structure reported here for glycyl-α-aminoisobutyrylglycyl-α-aminoisobutyric acid tert-butyl ester, C16H30N4O5, describes the first N-terminal-unprotected (Gly-Aib)n peptide. The achiral peptide could form an intramolecular hydrogen bond between the C=O group of Gly1 and the N-H group of Aib4. This hydrogen bond is found in all tetrapeptides and N-terminal-protected tripeptides containing Aib, apart from one exception. In the present work, this hydrogen bond is not observed (N...O = 5.88 Å). Instead, every molecule is hydrogen bonded to six other symmetry-related molecules with a total of eight hydrogen bonds per molecule. The backbone conformation starts in the right-handed helical region (and the left-handed helical region for the inverted molecule) and reverses the screw sense in the last two residues.

78 FR 23331 - Ballard Terminal Railroad Company, L.L.C.-Lease Exemption-Line of Eastside Community Rail, LLC

Science.gov (United States)

2013-04-18

... residual common carrier rights and obligations, including the right to reinstitute rail service, over the... resume providing common carrier rail service over this trackage. These filings will be addressed by the... Railroad Company, L.L.C.--Lease Exemption--Line of Eastside Community Rail, LLC Ballard Terminal Railroad...

Bonds broken and formed during the mixed-linkage glucan : xyloglucan endotransglucosylase reaction catalysed by Equisetum hetero-trans-β-glucanase.

Science.gov (United States)

Simmons, Thomas J; Fry, Stephen C

2017-03-08

Mixed-linkage glucan∶xyloglucan endotransglucosylase (MXE) is one of the three activities of the recently characterised hetero-trans-β-glucanase (HTG), which among land plants is known only from Equisetum species. The biochemical details of the MXE reaction were incompletely understood - details that would promote understanding of MXE's role in vivo and enable its full technological exploitation. We investigated HTG's site of attack on one of its donor substrates, mixed-linkage (1→3),(1→4)-β-d-glucan (MLG), with radioactive oligosaccharides of xyloglucan as the acceptor substrate. Comparing three different MLG preparations, we showed that the enzyme favours those with a high content of cellotetraose blocks. The reaction products were analysed by enzymic digestion, thin-layer chromatography (TLC), high-pressure liquid chromatography (HPLC) and gel-permeation chromatography (GPC). Equisetum HTG consistently cleaved the MLG at the third consecutive β-(1→4)-bond following (towards the reducing terminus) a β-(1→3)-bond. It then formed a β-(1→4)-bond between the MLG and the non-reducing terminal glucose residue of the xyloglucan oligosaccharide, consistent with its xyloglucan endotransglucosylase/hydrolase subfamily membership. Using size-homogeneous barley MLG as the donor substrate, we showed that HTG does not favour any particular region of the MLG chain relative to the polysaccharide's reducing and non-reducing termini; rather, it selects its target cellotetraosyl unit stochastically along the MLG molecule. This work improves our understanding of how enzymes can exhibit promiscuous substrate specificities and provides the foundations to explore strategies for engineering novel substrate specificities into transglycanases. © 2017 The Author(s).

Neurospora tryptophan synthase: N-terminal analysis and the sequence of the pyridoxal phosphate active site peptide

International Nuclear Information System (INIS)

Pratt, M.L.; Hsu, P.Y.; DeMoss, J.A.

1986-01-01

Tryptophan synthase (TS), which catalyzes the final step of tryptophan biosynthesis, is a multifunctional protein requiring pyridoxal phosphate (B6P) for two of its three distinct enzyme activities. TS from Neurospora has a blocked N-terminal, is a homodimer of 150 KDa and binds one mole of B6P per mole of subunit. The authors shown the N-terminal residue to be acyl-serine. The B6P-active site of holoenzyme was labelled by reduction of the B6P-Schiff base with [ 3 H]-NaBH 4 , and resulted in a proportionate loss of activity in the two B6P-requiring reactions. SDS-polyacrylamide gel electrophoresis of CNBr-generated peptides showed the labelled, active site peptide to be 6 KDa. The sequence of this peptide, purified to apparent homogeneity by a combination of C-18 reversed phase and TSK gel filtration HPLC is: gly-arg-pro-gly-gln-leu-his-lys-ala-glu-arg-leu-thr-glu-tyr-ala-gly-gly-ala-gln-ile-xxx-leu-lys-arg-glu-asp-leu-asn-his-xxx-gly-xxx-his-/sub ***/-ile-asn-asn-ala-leu. Although four residues (xxx, /sub ***/) are unidentified, this peptide is minimally 78% homologous with the corresponding peptide from yeast TS, in which residue (/sub ***/) is the lysine that binds B6P

Terminal velocities for a large sample of O stars, B supergiants, and Wolf-Rayet stars

International Nuclear Information System (INIS)

Prinja, R.K.; Barlow, M.J.; Howarth, I.D.

1990-01-01

It is argued that easily measured, reliable estimates of terminal velocities for early-type stars are provided by the central velocity asymptotically approached by narrow absorption features and by the violet limit of zero residual intensity in saturated P Cygni profiles. These estimators are used to determine terminal velocities, v(infinity), for 181 O stars, 70 early B supergiants, and 35 Wolf-Rayet stars. For OB stars, the values are typically 15-20 percent smaller than the extreme violet edge velocities, v(edge), while for WR stars v(infinity) = 0.76 v(edge) on average. New mass-loss rates for WR stars which are thermal radio emitters are given, taking into account the new terminal velocities and recent revisions to estimates of distances and to the mean nuclear mass per electron. The relationships between v(infinity), the surface escape velocities, and effective temperatures are examined. 67 refs

Evolution of Chemical Diversity in a Group of Non-Reduced Polyketide Gene Clusters: Using Phylogenetics to Inform the Search for Novel Fungal Natural Products

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Kurt Throckmorton

2015-09-01

Full Text Available Fungal polyketides are a diverse class of natural products, or secondary metabolites (SMs, with a wide range of bioactivities often associated with toxicity. Here, we focus on a group of non-reducing polyketide synthases (NR-PKSs in the fungal phylum Ascomycota that lack a thioesterase domain for product release, group V. Although widespread in ascomycete taxa, this group of NR-PKSs is notably absent in the mycotoxigenic genus Fusarium and, surprisingly, found in genera not known for their secondary metabolite production (e.g., the mycorrhizal genus Oidiodendron, the powdery mildew genus Blumeria, and the causative agent of white-nose syndrome in bats, Pseudogymnoascus destructans. This group of NR-PKSs, in association with the other enzymes encoded by their gene clusters, produces a variety of different chemical classes including naphthacenediones, anthraquinones, benzophenones, grisandienes, and diphenyl ethers. We discuss the modification of and transitions between these chemical classes, the requisite enzymes, and the evolution of the SM gene clusters that encode them. Integrating this information, we predict the likely products of related but uncharacterized SM clusters, and we speculate upon the utility of these classes of SMs as virulence factors or chemical defenses to various plant, animal, and insect pathogens, as well as mutualistic fungi.

Cytochemical localization of small intestinal glycoconjugates by lectin histochemistry in controls and subjects with cystic fibrosis.

Science.gov (United States)

Jacobs, L R; De Fontes, D; Cox, K L

1983-05-01

Human mucosal glycoconjugates were examined in normal small intestinal biopsies from five control subjects using six different fluorescein-conjugated lectins: Triticum vulgare agglutinin (WGA), Ulex europaeus agglutinin I (UEA1), Ricinus communis agglutinin I (RCA1), glycin max-soy bean agglutinin (SBA), Dolichus biflorus agglutinin (DBA), and Arachis hypogaea peanut agglutinin (PNA). These plant agglutinins bind to specific nonreducing end-terminal carbohydrate residues. Only the lectins derived from WGA, which produced the strongest staining, and UEA1 consistently bound to both intestinal goblet cell mucin and epithelial cell microvillar membranes. The intensity of lectin binding was greatest in the upper villus and diminished down towards the crypt, being weakest in the crypt base. Similar histochemical studies carried out on small bowel biopsies from five patients with cystic fibrosis revealed no major qualitative differences between the intestinal glycoconjugates in normal subjects and those with cystic fibrosis. These results suggest that glycoconjugate biosynthesis of human intestinal goblet cell mucin and epithelial cell membranes may be complete and hence full differentiation achieved only when these cells have migrated out of the crypt and onto the villus.

Structural analyses and immunomodulatory properties of fructo-oligosaccharides from onion (Allium cepa).

Science.gov (United States)

Kumar, V Prasanna; Prashanth, K V Harish; Venkatesh, Y P

2015-03-06

Onion (Allium cepa) is an immune-boosting food rich in fructans. The major aim of this study is to characterize and investigate the immunomodulatory properties of onion fructo-oligosaccharides (FOS). FOS was isolated from onion bulbs by hot 80% ethanol extraction (yield: ∼4.5 g/100 g fw) followed by gel permeation chromatography. NMR of onion FOS revealed unusual β-D-Glc terminal residue at the non-reducing end. TLC and ESI-MS analyses showed that onion FOS ranged from trisaccharides to hexasaccharides. Onion FOS (50 μg/mL) significantly increased (∼3-fold) the proliferation of mouse splenocytes/thymocytes vs. control. Further, onion FOS enhanced (∼2.5-fold) the production of nitric oxide by peritoneal exudates cells (PECs) from Wistar rats; intracellular free radicals production and phagocytic activity of isolated murine PECs were also augmented. Our structural and in vitro results indicate that onion FOS comprising of tri- to hexasaccharide units belongs to inulin-type fructans, and possess immunostimulatory activities towards murine lymphocytes and macrophages. Copyright © 2014 Elsevier Ltd. All rights reserved.

Amino-terminal extension present in the methionine aminopeptidase type 1c of Mycobacterium tuberculosis is indispensible for its activity

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Kumaran Sangaralingam

2011-07-01

Full Text Available Abstract Background Methionine aminopeptidase (MetAP is a ubiquitous enzyme in both prokaryotes and eukaryotes, which catalyzes co-translational removal of N-terminal methionine from elongating polypeptide chains during protein synthesis. It specifically removes the terminal methionine in all organisms, if the penultimate residue is non-bulky and uncharged. The MetAP action for exclusion of N-terminal methionine is mandatory in 50-70% of nascent proteins. Such an activity is required for proper sub cellular localization, additional processing and eventually for the degradation of proteins. Results We cloned genes encoding two such metalloproteases (MtMetAP1a and MtMetAP1c present in Mycobacterium tuberculosis and expressed them as histidine-tagged proteins in Escherichia coli. Although they have different substrate preferences, for Met-Ala-Ser, we found, MtMetAP1c had significantly high enzyme turnover rate as opposed to MtMetAP1a. Circular dichroism spectroscopic studies as well as monitoring of enzyme activity indicated high temperature stability (up to 50°C of MtMetAP1a compared to that of the MtMetAP1c. Modelling of MtMetAP1a based on MtMetAP1c crystal structure revealed the distinct spatial arrangements of identical active site amino acid residues and their mutations affected the enzymatic activities of both the proteins. Strikingly, we observed that 40 amino acid long N-terminal extension of MtMetAP1c, compared to its other family members, contributes towards the activity and stability of this enzyme, which has never been reported for any methionine aminopeptidase. Furthermore, mutational analysis revealed that Val-18 and Pro-19 of MtMetAP1c are crucial for its enzymatic activity. Consistent with this observation, molecular dynamic simulation studies of wild-type and these variants strongly suggest their involvement in maintaining active site conformation of MtMetAP1c. Conclusion Our findings unequivocally emphasized that N-terminal

Two-terminal video coding.

Science.gov (United States)

Yang, Yang; Stanković, Vladimir; Xiong, Zixiang; Zhao, Wei

2009-03-01

Following recent works on the rate region of the quadratic Gaussian two-terminal source coding problem and limit-approaching code designs, this paper examines multiterminal source coding of two correlated, i.e., stereo, video sequences to save the sum rate over independent coding of both sequences. Two multiterminal video coding schemes are proposed. In the first scheme, the left sequence of the stereo pair is coded by H.264/AVC and used at the joint decoder to facilitate Wyner-Ziv coding of the right video sequence. The first I-frame of the right sequence is successively coded by H.264/AVC Intracoding and Wyner-Ziv coding. An efficient stereo matching algorithm based on loopy belief propagation is then adopted at the decoder to produce pixel-level disparity maps between the corresponding frames of the two decoded video sequences on the fly. Based on the disparity maps, side information for both motion vectors and motion-compensated residual frames of the right sequence are generated at the decoder before Wyner-Ziv encoding. In the second scheme, source splitting is employed on top of classic and Wyner-Ziv coding for compression of both I-frames to allow flexible rate allocation between the two sequences. Experiments with both schemes on stereo video sequences using H.264/AVC, LDPC codes for Slepian-Wolf coding of the motion vectors, and scalar quantization in conjunction with LDPC codes for Wyner-Ziv coding of the residual coefficients give a slightly lower sum rate than separate H.264/AVC coding of both sequences at the same video quality.

Identification of succinimide sites in proteins by N-terminal sequence analysis after alkaline hydroxylamine cleavage.

Science.gov (United States)

Kwong, M. Y.; Harris, R. J.

1994-01-01

Under favorable conditions, Asp or Asn residues can undergo rearrangement to a succinimide (cyclic imide), which may also serve as an intermediate for deamidation and/or isoaspartate formation. Direct identification of such succinimides by peptide mapping is hampered by their lability at neutral and alkaline pH. We determined that incubation in 2 M hydroxylamine, 0.2 M Tris buffer, pH 9, for 2 h at 45 degrees C will specifically cleave on the C-terminal side of succinimides without cleavage at Asn-Gly bonds; yields are typically approximately 50%. N-terminal sequence analysis can then be used to identify an internal sequence generated by cleavage of the succinimide, hence identifying the succinimide site. PMID:8142891

Characterization of C-terminally engineered laccases.

Science.gov (United States)

Liu, Yingli; Cusano, Angela Maria; Wallace, Erin C; Mekmouche, Yasmina; Ullah, Sana; Robert, Viviane; Tron, Thierry

2014-08-01

Extremities of proteins are potent sites for functionalization. Carboxy terminus variants of the Trametes sp. strain C30 LAC3 laccase were generated and produced in Saccharomyces cerevisiae. A variant deleted of the last 13 residues (CΔ) and its 6 His tagged counterpart (CΔ6H) were found active enzymes. The production of CΔ6H resulted in the synthesis of a unusually high proportion of highly glycosylated forms of the enzyme therefore allowing the additional purification of a hyper-glycosylated form of CΔ6H noted CΔ6Hh. Properties of CΔ, CΔ6H and CΔ6Hh were compared. Globally, LAC3 catalytic efficiency was moderately affected by terminal modifications except in CΔ for which the kcat/KM ratio decreased 4 fold (with syringaldazine as substrate) and 10 fold (with ABTS as substrate) respectively. The catalytic parameters kcat and KM of CΔ6H and CΔ6Hh were found to be strictly comparable revealing that over glycosylation does not affect the enzyme catalytic efficiency. To the contrary, in vitro deglycosylation of laccase drastically reduced its activity. So, despite a complex glycosylated pattern observed for some of the variant enzymes, terminal sequences of laccases appear to be appropriate sites for the functionalization/immobilization of laccase. Copyright © 2014 Elsevier B.V. All rights reserved.

The EBNA-2 N-Terminal Transactivation Domain Folds into a Dimeric Structure Required for Target Gene Activation.

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Anders Friberg

2015-05-01

Full Text Available Epstein-Barr virus (EBV is a γ-herpesvirus that may cause infectious mononucleosis in young adults. In addition, epidemiological and molecular evidence links EBV to the pathogenesis of lymphoid and epithelial malignancies. EBV has the unique ability to transform resting B cells into permanently proliferating, latently infected lymphoblastoid cell lines. Epstein-Barr virus nuclear antigen 2 (EBNA-2 is a key regulator of viral and cellular gene expression for this transformation process. The N-terminal region of EBNA-2 comprising residues 1-58 appears to mediate multiple molecular functions including self-association and transactivation. However, it remains to be determined if the N-terminus of EBNA-2 directly provides these functions or if these activities merely depend on the dimerization involving the N-terminal domain. To address this issue, we determined the three-dimensional structure of the EBNA-2 N-terminal dimerization (END domain by heteronuclear NMR-spectroscopy. The END domain monomer comprises a small fold of four β-strands and an α-helix which form a parallel dimer by interaction of two β-strands from each protomer. A structure-guided mutational analysis showed that hydrophobic residues in the dimer interface are required for self-association in vitro. Importantly, these interface mutants also displayed severely impaired self-association and transactivation in vivo. Moreover, mutations of solvent-exposed residues or deletion of the α-helix do not impair dimerization but strongly affect the functional activity, suggesting that the EBNA-2 dimer presents a surface that mediates functionally important intra- and/or intermolecular interactions. Our study shows that the END domain is a novel dimerization fold that is essential for functional activity. Since this specific fold is a unique feature of EBNA-2 it might provide a novel target for anti-viral therapeutics.

3′ terminal diversity of MRP RNA and other human noncoding RNAs revealed by deep sequencing

Science.gov (United States)

2013-01-01

Background Post-transcriptional 3′ end processing is a key component of RNA regulation. The abundant and essential RNA subunit of RNase MRP has been proposed to function in three distinct cellular compartments and therefore may utilize this mode of regulation. Here we employ 3′ RACE coupled with high-throughput sequencing to characterize the 3′ terminal sequences of human MRP RNA and other noncoding RNAs that form RNP complexes. Results The 3′ terminal sequence of MRP RNA from HEK293T cells has a distinctive distribution of genomically encoded termini (including an assortment of U residues) with a portion of these selectively tagged by oligo(A) tails. This profile contrasts with the relatively homogenous 3′ terminus of an in vitro transcribed MRP RNA control and the differing 3′ terminal profiles of U3 snoRNA, RNase P RNA, and telomerase RNA (hTR). Conclusions 3′ RACE coupled with deep sequencing provides a valuable framework for the functional characterization of 3′ terminal sequences of noncoding RNAs. PMID:24053768

Residual nilpotence and residual solubility of groups

International Nuclear Information System (INIS)

Mikhailov, R V

2005-01-01

The properties of the residual nilpotence and the residual solubility of groups are studied. The main objects under investigation are the class of residually nilpotent groups such that each central extension of these groups is also residually nilpotent and the class of residually soluble groups such that each Abelian extension of these groups is residually soluble. Various examples of groups not belonging to these classes are constructed by homological methods and methods of the theory of modules over group rings. Several applications of the theory under consideration are presented and problems concerning the residual nilpotence of one-relator groups are considered.

Structural and functional importance of transmembrane domain 3 (TM3) in the aspartate:alanine antiporter AspT: topology and function of the residues of TM3 and oligomerization of AspT.

Science.gov (United States)

Nanatani, Kei; Maloney, Peter C; Abe, Keietsu

2009-04-01

AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of l-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of l-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes l-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a

Structural and Functional Importance of Transmembrane Domain 3 (TM3) in the Aspartate:Alanine Antiporter AspT: Topology and Function of the Residues of TM3 and Oligomerization of AspT▿

Science.gov (United States)

Nanatani, Kei; Maloney, Peter C.; Abe, Keietsu

2009-01-01

AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of l-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of l-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes l-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a

Specificity of N-terminal methionyl peptidase: analysis by site-directed mutagenesis

International Nuclear Information System (INIS)

Kasper, T.J.; Boissel, J.P.; Bunn, H.F.

1987-01-01

The start site of eukaryotic translation is normally an AUG codon. The corresponding N-terminal methionine is most often removed when the nascent chain reaches about 30 residues. Data from a survey of 1764 eukaryotic protein sequences suggest that the residue adjacent to the initiator Met determines Met cleavage. In order to investigate the mechanism of this reaction, the authors have prepared oligonucleotide-directed mutants of human β-globin from gapped heteroduplexes of a T3/T7 plasmid containing a globin cDNA clone. To date, the authors have produced mutants encoding for 15 of 19 possible amino acid replacements at position 1 in the β-globin chain. These mutants have been confirmed by dideoxy sequencing, transcribed in vitro, and translated in a rabbit reticulocyte lysate in the presence of 35 S-methionine. Labeled translation products were then isolated by cation exchange HPLC, and tryptic peptides were analyzed by RP-HPLC. Thus far, this structural analysis has shown that for β-1 Val, Ala, and Ser, the initiator Met is cleaved, whereas for β-1 Lys, Met, Glu, Trp, Asn, Tyr, and Glu, initiator Met is retained. For β-1 Leu initiator Met is cleaved with a frequency of about 50%. These results are consistent with the data obtained from the previous survey. The expression of site-directed mutants in a cell-free system can also be used to investigate other N-terminal processing events, such as acetylation and myristylation

4-alkyl-L-(Dehydro)proline biosynthesis in actinobacteria involves N-terminal nucleophile-hydrolase activity of γ-glutamyltranspeptidase homolog for C-C bond cleavage

Science.gov (United States)

Zhong, Guannan; Zhao, Qunfei; Zhang, Qinglin; Liu, Wen

2017-07-01

γ-Glutamyltranspeptidases (γ-GTs), ubiquitous in glutathione metabolism for γ-glutamyl transfer/hydrolysis, are N-terminal nucleophile (Ntn)-hydrolase fold proteins that share an autoproteolytic process for self-activation. γ-GT homologues are widely present in Gram-positive actinobacteria where their Ntn-hydrolase activities, however, are not involved in glutathione metabolism. Herein, we demonstrate that the formation of 4-Alkyl-L-(dehydro)proline (ALDP) residues, the non-proteinogenic α-amino acids that serve as vital components of many bioactive metabolites found in actinobacteria, involves unprecedented Ntn-hydrolase activity of γ-GT homologue for C-C bond cleavage. The related enzymes share a key Thr residue, which acts as an internal nucleophile for protein hydrolysis and then as a newly released N-terminal nucleophile for carboxylate side-chain processing likely through the generation of an oxalyl-Thr enzyme intermediate. These findings provide mechanistic insights into the biosynthesis of various ALDP residues/associated natural products, highlight the versatile functions of Ntn-hydrolase fold proteins, and particularly generate interest in thus far less-appreciated γ-GT homologues in actinobacteria.

The pH-sensitive structure of the C-terminal domain of voltage-gated proton channel and the thermodynamic characteristics of Zn{sup 2+} binding to this domain

Energy Technology Data Exchange (ETDEWEB)

Zhao, Qing; Li, Chuanyong; Li, Shu Jie, E-mail: shujieli@nankai.edu.cn

2015-01-02

Highlights: • The α-helical content of the C-terminus is decreased with a pH increase. • The thermostability of the C-terminus is decreased with a pH increase. • Zn{sup 2+} binds to His{sup 244} and His{sup 266} residues within the C-terminal domain. • The binding of Zn{sup 2+} to His{sup 244} residue is an endothermic heat reaction. • The binding of Zn{sup 2+} to His{sup 266} residue is an exothermic heat reaction. - Abstract: The voltage-gated proton channel Hv1 is strongly sensitive to Zn{sup 2+}. The H{sup +} conduction is decreased at a high concentration of Zn{sup 2+} and Hv1 channel closing is slowed by the internal application of Zn{sup 2+}. Although the recent studies demonstrated that Zn{sup 2+} interacts with the intracellular C-terminal domain, the binding sites and details of the interaction remain unknown. Here, we studied the pH-dependent structural stability of the intracellular C-terminal domain of human Hv1 and showed that Zn{sup 2+} binds to His{sup 244} and His{sup 266} residues. The thermodynamics signature of Zn{sup 2+} binding to the two sites was investigated by isothermal titration calorimetry. The binding of Zn{sup 2+} to His{sup 244} (mutant H266A) and His{sup 266} (mutant H244A) were an endothermic heat reaction and an exothermic heat reaction, respectively.

Carboxyl-terminal multi-site phosphorylation regulates internalization and desensitization of the human sst2 somatostatin receptor.

Science.gov (United States)

Lehmann, Andreas; Kliewer, Andrea; Schütz, Dagmar; Nagel, Falko; Stumm, Ralf; Schulz, Stefan

2014-04-25

The somatostatin receptor 2 (sst2) is the pharmacological target of somatostatin analogs that are widely used in the diagnosis and treatment of human neuroendocrine tumors. We have recently shown that the stable somatostatin analogs octreotide and pasireotide (SOM230) stimulate distinct patterns of sst2 receptor phosphorylation and internalization. Like somatostatin, octreotide promotes the phosphorylation of at least six carboxyl-terminal serine and threonine residues namely S341, S343, T353, T354, T356 and T359, which in turn leads to a robust receptor endocytosis. Unlike somatostatin, pasireotide stimulates a selective phosphorylation of S341 and S343 of the human sst2 receptor followed by a partial receptor internalization. Here, we show that exchange of S341 and S343 by alanine is sufficient to block pasireotide-driven internalization, whereas mutation of T353, T354, T356 and T359 to alanine is required to strongly inhibited both octreotide- and somatostatin-induced internalization. Yet, combined mutation of T353, T354, T356 and T359 is not sufficient to prevent somatostatin-driven β-arrestin mobilization and receptor desensitization. Replacement of all fourteen carboxyl-terminal serine and threonine residues by alanine completely abrogates sst2 receptor internalization and β-arrestin mobilization in HEK293 cells. Together, our findings demonstrate for the first time that agonist-selective sst2 receptor internalization is regulated by multi-site phosphorylation of its carboxyl-terminal tail. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Terminal separation plant for collecting petroleum and by-product gas

Energy Technology Data Exchange (ETDEWEB)

Marinin, N S; Shcherbina, V E; Burma, A I

1966-06-08

A separation plant at a transportation terminal, for collecting petroleum and by-product gas, consists of 1 or 2 vessels with gas separating device, automatic control devices, demulsifier distributors, a mixer for mixing hot water with the demulsified residue and raw crude oil stream, an apparatus for supplying oil-in-water emulsion under a water cushion, and 2 separating partitions which are located at the end of the vessel. In order to fully use the volume of the vessel, one partition does not touch the bottom, while the other does not touch the top of the tank.

Structure of the human histone chaperone FACT Spt16 N-terminal domain

Energy Technology Data Exchange (ETDEWEB)

Marcianò, G.; Huang, D. T., E-mail: d.huang@beatson.gla.ac.uk [Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow G61 1BD, Scotland (United Kingdom)

2016-01-22

The Spt16–SSRP1 heterodimer is a histone chaperone that plays an important role in regulating chromatin assembly. Here, a crystal structure of the N-terminal domain of human Spt16 is presented and it is shown that this domain may contribute to histone binding. The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of human Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding.

Structure of the human histone chaperone FACT Spt16 N-terminal domain

International Nuclear Information System (INIS)

Marcianò, G.; Huang, D. T.

2016-01-01

The Spt16–SSRP1 heterodimer is a histone chaperone that plays an important role in regulating chromatin assembly. Here, a crystal structure of the N-terminal domain of human Spt16 is presented and it is shown that this domain may contribute to histone binding. The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of human Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding

Evolutionary analysis of a novel zinc ribbon in the N-terminal region of threonine synthase.

Science.gov (United States)

Kaur, Gurmeet; Subramanian, Srikrishna

2017-10-18

Threonine synthase (TS) catalyzes the terminal reaction in the biosynthetic pathway of threonine and requires pyridoxal phosphate as a cofactor. TSs share a common catalytic domain with other fold type II PALP dependent enzymes. TSs are broadly grouped into two classes based on their sequence, quaternary structure, and enzyme regulation. We report the presence of a novel zinc ribbon domain in the N-terminal region preceding the catalytic core in TS. The zinc ribbon domain is present in TSs belonging to both classes. Our sequence analysis reveals that archaeal TSs possess all zinc chelating residues to bind a metal ion that are lacking in the structurally characterized homologs. Phylogenetic analysis suggests that TSs with an N-terminal zinc ribbon likely represents the ancestral state of the enzyme while TSs without a zinc ribbon must have diverged later in specific lineages. The zinc ribbon and its N- and C-terminal extensions are important for enzyme stability, activity and regulation. It is likely that the zinc ribbon domain is involved in higher order oligomerization or mediating interactions with other biomolecules leading to formation of larger metabolic complexes.

Stability assessment on a library scale: a rapid method for the evaluation of the commutability and insertion of residues in C-terminal loops of the CH3 domains of IgG1-Fc.

Science.gov (United States)

Hasenhindl, Christoph; Traxlmayr, Michael W; Wozniak-Knopp, Gordana; Jones, Phil C; Stadlmayr, Gerhard; Rüker, Florian; Obinger, Christian

2013-10-01

Antigen-binding Fc fragments (Fcab) are generated by engineering the C-terminal loop regions in the CH3 domain of human immunoglobulin G class 1-crystallizable fragment (IgG1-Fc). For an optimum library design with high percentage of well-folded clones for efficient binder selection, information about the correlation between primary structure and stability is needed. Here, we present a rapid method that allows determination of the overall stability of whole libraries of IgG1-Fc on the surface of yeast by flow cytometry. Libraries of IgG1-Fc mutants with distinct regions in AB-, CD- and EF-loops of the CH3 domains randomized or carrying therein insertions of five additional residues were constructed, incubated at increasing temperatures and probed for residual binding of generic Fc ligands. Calculated temperatures of half-maximal irreversible denaturation of the libraries gave a clear hierarchy of tolerance to randomization of distinct loop positions. Experimental data were evaluated by a computational approach and are discussed with respect to the structure of IgG1-Fc and variation in sequence and length of these loops in homologous Fc proteins. Generally, the described method allows for quick assessment of the effects of randomization of distinct regions on the foldability and stability of a yeast-displayed protein library.

Osteogenic cell differentiation on H-terminated and O-terminated nanocrystalline diamond films

Directory of Open Access Journals (Sweden)

Liskova J

2015-01-01

Full Text Available Jana Liskova,1 Oleg Babchenko,2 Marian Varga,2 Alexander Kromka,2 Daniel Hadraba,1 Zdenek Svindrych,1 Zuzana Burdikova,1 Lucie Bacakova1 1Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic; 2Institute of Physics, Academy of Sciences of the Czech Republic, Prague, Czech Republic Abstract: Nanocrystalline diamond (NCD films are promising materials for bone implant coatings because of their biocompatibility, chemical resistance, and mechanical hardness. Moreover, NCD wettability can be tailored by grafting specific atoms. The NCD films used in this study were grown on silicon substrates by microwave plasma-enhanced chemical vapor deposition and grafted by hydrogen atoms (H-termination or oxygen atoms (O-termination. Human osteoblast-like Saos-2 cells were used for biological studies on H-terminated and O-terminated NCD films. The adhesion, growth, and subsequent differentiation of the osteoblasts on NCD films were examined, and the extracellular matrix production and composition were quantified. The osteoblasts that had been cultivated on the O-terminated NCD films exhibited a higher growth rate than those grown on the H-terminated NCD films. The mature collagen fibers were detected in Saos-2 cells on both the H-terminated and O-terminated NCD films; however, the quantity of total collagen in the extracellular matrix was higher on the O-terminated NCD films, as were the amounts of calcium deposition and alkaline phosphatase activity. Nevertheless, the expression of genes for osteogenic markers – type I collagen, alkaline phosphatase, and osteocalcin – was either comparable on the H-terminated and O-terminated films or even lower on the O-terminated films. In conclusion, the higher wettability of the O-terminated NCD films is promising for adhesion and growth of osteoblasts. In addition, the O-terminated surface also seems to support the deposition of extracellular matrix proteins and extracellular matrix

F5C: A variant of Faugère’s F5 algorithm with reduced Gröbner bases

OpenAIRE

Eder, Christian; Perry, John

2010-01-01

Faugere's F5 algorithm computes a Groebner basis incrementally, by computing a sequence of (non-reduced) Groebner bases. The authors describe a variant of F5, called F5C, that replaces each intermediate Groebner basis with its reduced Groebner basis. As a result, F5C considers fewer polynomials and performs substantially fewer polynomial reductions, so that it terminates more quickly. We also provide a generalization of Faugere's characterization theorem for Groebner bases.

Organizational Relationship Termination Competence

DEFF Research Database (Denmark)

Ritter, Thomas; Geersbro, Jens

2011-01-01

termination are found to significantly affect a firm's relationship termination competence. The findings suggest that managers should regard termination as a legitimate option in customer relationship management. In order to decrease the number of unwanted customers, managers must accept termination......Most firms are involved in a number of customer relationships that drain the firm's resources. However, many firms are hesitant to address this problem. This paper investigates customer relationship termination at the organizational level. We develop and analyze the organizational dimensions...... of organizational termination in order to improve our understanding of the management of termination. The impact of these termination dimensions on the percentage of unwanted customers is developed and tested using PLS on data gathered from a cross-sectional survey of more than 800 sales representatives. We find...

Kitimat LNG terminal

International Nuclear Information System (INIS)

Schmaltz, I.; Boulton, R.

2007-01-01

Kitimat Liquefied Natural Gas (LNG) terminal is a terminal development company owned by Galveston LNG, a privately owned Canadian energy development company. This presentation provided information on Kitimat LNG with particular reference to its terminal located in Bish Cove on the Douglas Channel in British Columbia. This LNG terminal is reported to be the only fully permitted regasification terminal on the west coast of Canada and the United States. The presentation addressed market fundamentals including several graphs, such as world natural gas proved reserves in 2006; LNG supplements to Canadian gas supplies; global LNG demand for 2005-2020; average annual United States LNG imports; and global LNG liquefaction projects. Other market fundamentals were described, including that Kitimat is the only other approved terminal aside from the Costa Azul terminal in Mexico; Kitimat is the only west coast LNG import terminal that connects to midwest and eastern North American markets through existing gas pipelines; LNG producers are looking for destination diversification; and markets and marketers are looking for supply diversification. The authors noted that by 2010, western Canadian gas demand will exceed Californian demand. Other topics that were discussed in the presentation included Canadian natural gas field receipts; unadjusted bitumen production outlook; oil sands gas demand; forward basis fundamentals; and the commercial drivers of the Kitimat LNG terminal. The presentation also discussed the pacific trail pipelines, a partnership between Galveston LNG and Pacific Northern Gas to develop the natural gas transmission line from Kitimat to Summit. The presentation concluded with a discussion of the benefits of Kitimat LNG terminal such as providing access to the largest natural gas markets in the world via major gas transmission lines with spare capacity. figs

Differential expression and localization of glycosidic residues in in vitro- and in vivo-matured cumulus-oocyte complexes in equine and porcine species.

Science.gov (United States)

Accogli, Gianluca; Douet, Cécile; Ambruosi, Barbara; Martino, Nicola Antonio; Uranio, Manuel Filioli; Deleuze, Stefan; Dell'Aquila, Maria Elena; Desantis, Salvatore; Goudet, Ghylène

2014-12-01

Glycoprotein oligosaccharides play major roles during reproduction, yet their function in gamete interactions is not fully elucidated. Identification and comparison of the glycan pattern in cumulus-oocyte complexes (COCs) from species with different efficiencies of in vitro spermatozoa penetration through the zona pellucida (ZP) could help clarify how oligosaccharides affect gamete interactions. We compared the expression and localization of 12 glycosidic residues in equine and porcine in vitro-matured (IVM) and preovulatory COCs by means of lectin histochemistry. The COCs glycan pattern differed between animals and COC source (IVM versus preovulatory). Among the 12 carbohydrate residues investigated, the IVM COCs from these two species shared: (a) sialo- and βN-acetylgalactosamine (GalNAc)-terminating glycans in the ZP; (b) sialylated and fucosylated glycans in cumulus cells; and (c) GalNAc and N-acetylglucosamine (GlcNAc) glycans in the ooplasm. Differences in the preovulatory COCs of the two species included: (a) sialoglycans and GlcNAc terminating glycans in the equine ZP versus terminal GalNAc and internal GlcNAc in the porcine ZP; (b) terminal galactosides in equine cumulus cells versus terminal GlcNAc and fucose in porcine cohorts; and (c) fucose in the mare ooplasm versus lactosamine and internal GlcNAc in porcine oocyte cytoplasm. Furthermore, equine and porcine cumulus cells and oocytes contributed differently to the synthesis of ZP glycoproteins. These results could be attributed to the different in vitro fertilization efficiencies between these two divergent, large-animal models. © 2014 Wiley Periodicals, Inc.

Structural characterization of glucosylated lactose derivatives synthesized by the Lactobacillus reuteri GtfA and Gtf180 glucansucrase enzymes.

Science.gov (United States)

Pham, Hien T T; Dijkhuizen, Lubbert; van Leeuwen, Sander S

2017-09-08

Glucansucrase enzymes from lactic acid bacteria are receiving strong interest because of their wide range of gluco-oligosaccharide and polysaccharide products from sucrose, some of which have prebiotic potential. Glucansucrases GtfA and Gtf180 from Lactobacillus reuteri strains are known to convert sucrose into α-glucans with different types of linkages, but also to use other molecules as acceptor substrates. Here we report that incubation of (N-terminally truncated versions of) these enzymes with lactose plus sucrose resulted in synthesis of at least 5 glucosylated lactose products of a degree of polymerization (DP) of 3-4. Only glucansucrase Gtf180-ΔN also produced larger lactose-based oligosaccharides (up to DP9). Structural characterization of the glucosylated lactose products DP3-4 revealed glycosidic bonds other than (α1→4)/(α1→6) typical for GtfA-ΔN and (α1→3)/(α1→6) typical for Gtf180-ΔN: Both GtfA-ΔN and Gtf180-ΔN now introduced a glucosyl residue (α1→3)- or (α1→4)-linked to the non-reducing galactose unit of lactose. Both enzymes also were able to introduce a glucosyl residue (α1→2)-linked to the reducing glucose unit of lactose. These lactose derived oligosaccharides potentially are interesting prebiotic compounds. Copyright © 2017 Elsevier Ltd. All rights reserved.

Decay accelerating factor (DAF) is anchored to membranes by a C-terminal glycolipid

International Nuclear Information System (INIS)

Medof, M.E.; Haas, R.; Walter, E.I.; Rosenberry, T.L.

1986-01-01

Purified 70 kDa membrane (m) DAF incorporates into cells when added in vitro. A 2 kDa smaller DAF form which functions extrinsically like C4bp but is unable to incorporate can be isolated from urine (u). Because of common deficits of mDAF and acetylcholinesterase (AChE) in erythrocytes (E) of patients with paroxysmal nocturnal hemoglobinuria (PNH), mDAF was analyzed for a O-terminal glycolipid membrane anchor similar to that in E AChE. Incubation of E with phosphatidylinositol-specific phospholipase C, an enzyme which cleaves a similar glycolipid anchor in trypanosome variant surface glycoproteins (mfVSGs), released 20% of the DAF antigen. The released DAF species resembled uDAF in size, extrinsic model of C4b2a decay, and lack of hydrophobicity. Reductive radiomethylation of mDAF with [ 14 C]HCHO and NaCNBH 3 revealed ethanolamine and glucosamine in proportions similar to those in the E AChE glycolipid anchor. Papain cleavage of radiomethylated mDAF released the labeled ethanolamine and glucosamine in small O-terminal fragments from the residual DAF that retained N-terminal Asp. Following labeling of the anchors of mDAF and E AChE with the lipophilic photoreagent 3-trifluoromethyl-3-(m-[ 125 I]iodophenyl)diazirine, cleavage at the glucosamine residue by deamination quantitatively released the label from both proteins. Biosynthetic labeling of Hela cells with [ 3 H]ethanolamine resulted in rapid 3 H incorporation into both 48 kDa proDAF and 70 kDa mDAF. These data indicate that mDAF is anchored by a glycolipid similar to that in E AChE, mfVSGs and Thy-1 antigen and raise the possibility that a defect in the assembly or attachment of this structure could account for the deficits of mDAF and E AChE in PNH

Structure, Orientation and Dynamics of the C-Terminal Hexapeptide of LRAP Determined Using Solid State NMR

International Nuclear Information System (INIS)

Shaw, Wendy J.; Ferris, Kim F.

2008-01-01

Amelogenin is the predominant protein found during enamel development and has been shown to be essential to proper enamel formation. LRAP is a naturally occurring splice variant that preserves the charged N- and C-termini of full length amelogenin, regions thought to be crucial in interacting with hydroxyapatite. Particularly, the highly charged C-terminal hexapeptide (KREEVD) is thought to be the region most intimately interacting with HAP. We investigated the structure of this charged region, as well as the proximity to the surface and the mobility of two of the residues. We found the structure to be consistent with a random coil or more extended structure, as has been found for more internalized residues in the C-terminus. The backbone K 54 ( 13 C(prime)), V 58 ( 13 C(prime)) and V 58 ( 15 N) were all found to be very close to the surface of HAP, ∼ 6.0 (angstrom), suggesting a strong interaction and emphasizing the importance of these residues in interacting with HAP. However, both ends of the hexapeptide, at residues K 54 and V 58 , experience significant mobility under hydrated conditions, implying that another portion of the protein helps to stabilize the strong LRAP-HAP interaction. Interestingly, the backbone of the C-terminal third of the protein is consistently 6.0 (angstrom) from the HAP surface, suggesting that this region of the protein is laying flat on the surface with no 3-dimensional folding. The combination of these features, i.e., a random coil structure, a significant mobility and a lack of three-dimensional folding in this region of the protein may be important in a functional role, allowing the C-terminus to effectively interact with HAP while at the same time allowing maximum crystal inhibition

Growth hormone receptor C-terminal domains required for growth hormone-induced intracellular free Ca2+ oscillations and gene transcription

DEFF Research Database (Denmark)

Billestrup, N; Bouchelouche, P; Allevato, G

1995-01-01

of varying frequency and amplitude. GH-induced transcription of the serine protease inhibitor 2.1 gene required the same C-terminal 52-amino acid domain of the receptor as for Ca2+ signaling. Mutation of the four proline residues in the conserved box 1 region of the GHR, which is responsible for binding...

Dissipation and residue of clothianidin in granules and pesticide fertilizers used in cabbage and soil under field conditions.

Science.gov (United States)

Zhang, P W; Wang, S Y; Huang, C L; Fu, J T; Huang, R L; Li, Z H; Zhang, Z X

2018-01-01

The single application of 0.5 % clothianidin granules, a novel formulation, was used to control pests in vegetables under a high dose. In this article, residues of clothianidin in cabbage and soil samples under field conditions from Guangzhou, Nanning, and Qianjiang were determined by HPLC. The terminal residues of clothianidin in cabbage were less than the limit of detection (clothianidin residual, clothianidin granules and fertilizers of chicken manure, urea, and organic fertilizer were mixed into different pesticide fertilizers through their normal field using dosage and evaluate residual influence of clothianidin in different formula. After analysis of variance of the effect factors, the effect of different pesticide types on half-life was not significant, but the effect of sample types was significant. Clothianidin granules and pesticide fertilizers could be safely applied in cabbage under a single high-dose administration.

Structure of a tropomyosin N-terminal fragment at 0.98 Å resolution

International Nuclear Information System (INIS)

Meshcheryakov, Vladimir A.; Krieger, Inna; Kostyukova, Alla S.; Samatey, Fadel A.

2011-01-01

The crystal structure of the N-terminal fragment of the short nonmuscle α-tropomyosin has been determined at a resolution of 0.98 Å. Tropomyosin (TM) is an elongated two-chain protein that binds along actin filaments. Important binding sites are localized in the N-terminus of tropomyosin. The structure of the N-terminus of the long muscle α-TM has been solved by both NMR and X-ray crystallography. Only the NMR structure of the N-terminus of the short nonmuscle α-TM is available. Here, the crystal structure of the N-terminus of the short nonmuscle α-TM (αTm1bZip) at a resolution of 0.98 Å is reported, which was solved from crystals belonging to space group P3 1 with unit-cell parameters a = b = 33.00, c = 52.03 Å, α = β = 90, γ = 120°. The first five N-terminal residues are flexible and residues 6–35 form an α-helical coiled coil. The overall fold and the secondary structure of the crystal structure of αTM1bZip are highly similar to the NMR structure and the atomic coordinates of the corresponding C α atoms between the two structures superimpose with a root-mean-square deviation of 0.60 Å. The crystal structure validates the NMR structure, with the positions of the side chains being determined precisely in our structure

Structure of a tropomyosin N-terminal fragment at 0.98 Å resolution

Energy Technology Data Exchange (ETDEWEB)

Meshcheryakov, Vladimir A. [Okinawa Institute of Science and Technology, Okinawa (Japan); Krieger, Inna [Texas A& M University, College Station, Texas (United States); Kostyukova, Alla S. [Robert Wood Johnson Medical School, Piscataway, New Jersey (United States); Samatey, Fadel A., E-mail: f.a.samatey@oist.jp [Okinawa Institute of Science and Technology, Okinawa (Japan)

2011-09-01

The crystal structure of the N-terminal fragment of the short nonmuscle α-tropomyosin has been determined at a resolution of 0.98 Å. Tropomyosin (TM) is an elongated two-chain protein that binds along actin filaments. Important binding sites are localized in the N-terminus of tropomyosin. The structure of the N-terminus of the long muscle α-TM has been solved by both NMR and X-ray crystallography. Only the NMR structure of the N-terminus of the short nonmuscle α-TM is available. Here, the crystal structure of the N-terminus of the short nonmuscle α-TM (αTm1bZip) at a resolution of 0.98 Å is reported, which was solved from crystals belonging to space group P3{sub 1} with unit-cell parameters a = b = 33.00, c = 52.03 Å, α = β = 90, γ = 120°. The first five N-terminal residues are flexible and residues 6–35 form an α-helical coiled coil. The overall fold and the secondary structure of the crystal structure of αTM1bZip are highly similar to the NMR structure and the atomic coordinates of the corresponding C{sup α} atoms between the two structures superimpose with a root-mean-square deviation of 0.60 Å. The crystal structure validates the NMR structure, with the positions of the side chains being determined precisely in our structure.

Critical structural and functional roles for the N-terminal insertion sequence in surfactant protein B analogs.

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Frans J Walther

2010-01-01

Full Text Available Surfactant protein B (SP-B; 79 residues belongs to the saposin protein superfamily, and plays functional roles in lung surfactant. The disulfide cross-linked, N- and C-terminal domains of SP-B have been theoretically predicted to fold as charged, amphipathic helices, suggesting their participation in surfactant activities. Earlier structural studies with Mini-B, a disulfide-linked construct based on the N- and C-terminal regions of SP-B (i.e., approximately residues 8-25 and 63-78, confirmed that these neighboring domains are helical; moreover, Mini-B retains critical in vitro and in vivo surfactant functions of the native protein. Here, we perform similar analyses on a Super Mini-B construct that has native SP-B residues (1-7 attached to the N-terminus of Mini-B, to test whether the N-terminal sequence is also involved in surfactant activity.FTIR spectra of Mini-B and Super Mini-B in either lipids or lipid-mimics indicated that these peptides share similar conformations, with primary alpha-helix and secondary beta-sheet and loop-turns. Gel electrophoresis demonstrated that Super Mini-B was dimeric in SDS detergent-polyacrylamide, while Mini-B was monomeric. Surface plasmon resonance (SPR, predictive aggregation algorithms, and molecular dynamics (MD and docking simulations further suggested a preliminary model for dimeric Super Mini-B, in which monomers self-associate to form a dimer peptide with a "saposin-like" fold. Similar to native SP-B, both Mini-B and Super Mini-B exhibit in vitro activity with spread films showing near-zero minimum surface tension during cycling using captive bubble surfactometry. In vivo, Super Mini-B demonstrates oxygenation and dynamic compliance that are greater than Mini-B and compare favorably to full-length SP-B.Super Mini-B shows enhanced surfactant activity, probably due to the self-assembly of monomer peptide into dimer Super Mini-B that mimics the functions and putative structure of native SP-B.

Modulation of procaspase-7 self-activation by PEST amino acid residues of the N-terminal prodomain and intersubunit linker.

Science.gov (United States)

Alves, Juliano; Garay-Malpartida, Miguel; Occhiucci, João M; Belizário, José E

2017-12-01

Procaspase-7 zymogen polypeptide is composed of a short prodomain, a large subunit (p20), and a small subunit (p10) connected to an intersubunit linker. Caspase-7 is activated by an initiator caspase-8 and -9, or by autocatalysis after specific cleavage at IQAD 198 ↓S located at the intersubunit linker. Previously, we identified that PEST regions made of amino acid residues Pro (P), Glu (E), Asp (D), Ser (S), Thr (T), Asn (N), and Gln (Q) are conserved flanking amino acid residues in the cleavage sites within a prodomain and intersubunit linker of all caspase family members. Here we tested the impact of alanine substitution of PEST amino acid residues on procaspase-7 proteolytic self-activation directly in Escherichia coli. The p20 and p10 subunit cleavage were significantly delayed in double caspase-7 mutants in the prodomain (N18A/P26A) and intersubunit linker (S199A/P201A), compared with the wild-type caspase-7. The S199A/P201A mutants effectively inhibited the p10 small subunit cleavage. However, the mutations did not change the kinetic parameters (k cat /K M ) and optimal tetrapeptide specificity (DEVD) of the purified mutant enzymes. The results suggest a role of PEST-amino acid residues in the molecular mechanism for prodomain and intersubunit cleavage and caspase-7 self-activation.

Structural Insight into the Critical Role of the N-Terminal Region in the Catalytic Activity of Dual-Specificity Phosphatase 26.

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Eun-Young Won

Full Text Available Human dual-specificity phosphatase 26 (DUSP26 is a novel target for anticancer therapy because its dephosphorylation of the p53 tumor suppressor regulates the apoptosis of cancer cells. DUSP26 inhibition results in neuroblastoma cell cytotoxicity through p53-mediated apoptosis. Despite the previous structural studies of DUSP26 catalytic domain (residues 61-211, DUSP26-C, the high-resolution structure of its catalytically active form has not been resolved. In this study, we determined the crystal structure of a catalytically active form of DUSP26 (residues 39-211, DUSP26-N with an additional N-terminal region at 2.0 Å resolution. Unlike the C-terminal domain-swapped dimeric structure of DUSP26-C, the DUSP26-N (C152S monomer adopts a fold-back conformation of the C-terminal α8-helix and has an additional α1-helix in the N-terminal region. Consistent with the canonically active conformation of its protein tyrosine phosphate-binding loop (PTP loop observed in the structure, the phosphatase assay results demonstrated that DUSP26-N has significantly higher catalytic activity than DUSP26-C. Furthermore, size exclusion chromatography-multiangle laser scattering (SEC-MALS measurements showed that DUSP26-N (C152S exists as a monomer in solution. Notably, the crystal structure of DUSP26-N (C152S revealed that the N-terminal region of DUSP26-N (C152S serves a scaffolding role by positioning the surrounding α7-α8 loop for interaction with the PTP-loop through formation of an extensive hydrogen bond network, which seems to be critical in making the PTP-loop conformation competent for phosphatase activity. Our study provides the first high-resolution structure of a catalytically active form of DUSP26, which will contribute to the structure-based rational design of novel DUSP26-targeting anticancer therapeutics.

Removal of a C-terminal serine residue proximal to the inter-chain disulfide bond of a human IgG1 lambda light chain mediates enhanced antibody stability and antibody dependent cell-mediated cytotoxicity

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Shen, Yang; Zeng, Lin; Zhu, Aiping; Blanc, Tim; Patel, Dipa; Pennello, Anthony; Bari, Amtul; Ng, Stanley; Persaud, Kris; Kang, Yun (Kenneth); Balderes, Paul; Surguladze, David; Hindi, Sagit; Zhou, Qinwei; Ludwig, Dale L.; Snavely, Marshall

2013-01-01

Optimization of biophysical properties is a critical success factor for the developability of monoclonal antibodies with potential therapeutic applications. The inter-domain disulfide bond between light chain (Lc) and heavy chain (Hc) in human IgG1 lends structural support for antibody scaffold stability, optimal antigen binding, and normal Fc function. Recently, human IgG1λ has been suggested to exhibit significantly greater susceptibility to reduction of the inter Lc-Hc disulfide bond relative to the same disulfide bond in human IgG1κ. To understand the molecular basis for this observed difference in stability, the sequence and structure of human IgG1λ and human IgG1κ were compared. Based on this Lc comparison, three single mutations were made in the λ Lc proximal to the cysteine residue, which forms a disulfide bond with the Hc. We determined that deletion of S214 (dS) improved resistance of the association between Lc and Hc to thermal stress. In addition, deletion of this terminal serine from the Lc of IgG1λ provided further benefit, including an increase in stability at elevated pH, increased yield from transient transfection, and improved in vitro antibody dependent cell-mediated cytotoxicity (ADCC). These observations support the conclusion that the presence of the terminal serine of the λ Lc creates a weaker inter-chain disulfide bond between the Lc and Hc, leading to slightly reduced stability and a potential compromise in IgG1λ function. Our data from a human IgG1λ provide a basis for further investigation of the effects of deleting terminal serine from λLc on the stability and function of other human IgG1λ antibodies. PMID:23567210

New OprM structure highlighting the nature of the N-terminal anchor

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Laura eMONLEZUN

2015-07-01

Full Text Available Among the different mechanisms used by bacteria to resist antibiotics, active efflux plays a major role. In gram-negative bacteria, active efflux is carried out by tripartite efflux pumps that form a macromolecular assembly spanning both membranes of the cellular wall. At the outer membrane level, a well-conserved Outer Membrane Factor (OMF protein acts as an exit duct, but its sequence varies greatly among different species. The OMFs share a similar tri-dimensional structure that includes a beta-barrel pore domain that stabilizes the channel within the membrane. In addition, OMFs are often subjected to different N-terminal post-translational modifications, such as an acylation with a lipid. The role of additional N-terminal anchors is all the more intriguing since it is not always required among the OMFs family. Understanding this optional post-translational modification could open new research lines in the field of antibiotics resistance. In E. coli, it has been shown that CusC is modified with a tri-acylated lipid, whereas TolC does not show any. In the case of OprM from Pseudomonas aeruginosa, the N-terminal modification remains a matter of debate, therefore, we used several approaches to investigate this issue. As definitive evidence, we present a new X ray structure at 3.8Å resolution that was solved in a new space group, making it possible to model the N-terminal residue as a palmitylated cysteine.

Interaction between the C-terminal domains of measles virus nucleoprotein and phosphoprotein: a tight complex implying one binding site.

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Blocquel, David; Habchi, Johnny; Costanzo, Stéphanie; Doizy, Anthony; Oglesbee, Michael; Longhi, Sonia

2012-10-01

The intrinsically disordered C-terminal domain (N(TAIL) ) of the measles virus (MeV) nucleoprotein undergoes α-helical folding upon binding to the C-terminal X domain (XD) of the phosphoprotein. The N(TAIL) region involved in binding coupled to folding has been mapped to a conserved region (Box2) encompassing residues 489-506. In the previous studies published in this journal, we obtained experimental evidence supporting a K(D) for the N(TAIL) -XD binding reaction in the nM range and also showed that an additional N(TAIL) region (Box3, aa 517-525) plays a role in binding to XD. In striking contrast with these data, studies published in this journal by Kingston and coworkers pointed out a much less stable complex (K(D) in the μM range) and supported lack of involvement of Box3 in complex formation. The objective of this study was to critically re-evaluate the role of Box3 in N(TAIL) -XD binding. Since our previous studies relied on N(TAIL) -truncated forms possessing an irrelevant Flag sequence appended at their C-terminus, we, herein, generated an N(TAIL) devoid of Box3 and any additional C-terminal residues, as well as a form encompassing only residues 482-525. We then used isothermal titration calorimetry to characterize the binding reactions between XD and these N(TAIL) forms. Results effectively argue for the presence of a single XD-binding site located within Box2, in agreement with the results by Kingston et al., while providing clear experimental support for a high-affinity complex. Altogether, the present data provide mechanistic insights into the replicative machinery of MeV and clarify a hitherto highly debated point. Copyright © 2012 The Protein Society.

Quadratic residues and non-residues selected topics

CERN Document Server

Wright, Steve

2016-01-01

This book offers an account of the classical theory of quadratic residues and non-residues with the goal of using that theory as a lens through which to view the development of some of the fundamental methods employed in modern elementary, algebraic, and analytic number theory. The first three chapters present some basic facts and the history of quadratic residues and non-residues and discuss various proofs of the Law of Quadratic Reciprosity in depth, with an emphasis on the six proofs that Gauss published. The remaining seven chapters explore some interesting applications of the Law of Quadratic Reciprocity, prove some results concerning the distribution and arithmetic structure of quadratic residues and non-residues, provide a detailed proof of Dirichlet’s Class-Number Formula, and discuss the question of whether quadratic residues are randomly distributed. The text is a valuable resource for graduate and advanced undergraduate students as well as for mathematicians interested in number theory.

Selective effects of charge on G protein activation by FSH-receptor residues 551-555 and 650-653.

Science.gov (United States)

Grasso, P; Deziel, M R; Reichert, L E

1995-01-01

Two cytosolic regions of the rat testicular FSH receptor (FSHR), residues 533-555 and 645-653, have been identified as G protein-coupling domains. We localized the activity in these domains to their C-terminal sequences, residues 551-555 (KIAKR, net charge +3) and 650-653 (RKSH, net charge +3), and examined the effects of charge on G protein activation by the C-terminal peptides, using synthetic analogs containing additions, through alanine (A) linkages, of arginine (R, +), histidine (H, +) or both. RA-KIAKR (net charge +4) mimicked the effect of FSHR-(551-555) on guanine nucleotide exchange in rat testis membranes, but reduced its ability to inhibit FSH-stimulated estradiol biosynthesis in cultured rat Sertoli cells. Further increasing net charge by the addition of H (HARA-KIAKR, net charge +5) increased guanosine 5'-triphosphate (GTP) binding, but eliminated FSHR-(551-555) effects on FSH-stimulated steroidogenesis. HA-RKSH (net charge +4) significantly inhibited guanine nucleotide exchange in rat testis membranes, but stimulated basal and potentiated FSH-induced estradiol biosynthesis in cultured rat Sertoli cells. Addition of two H residues (HAHA-RKSH, net charge +5) restored GTP binding and further potentiated basal and FSH-stimulated steroidogenesis. These results suggest that positive charges in G protein-coupling domains of the FSHR play a role in modulating G protein activation and postbinding effects of FSH, such as steroidogenesis.

Crystal Structure of the Carboxy-Terminal Region of the Bacteriophage T4 Proximal Long Tail Fiber Protein Gp34

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Meritxell Granell

2017-06-01

Full Text Available Long tail fibers of bacteriophage T4 are formed by proteins gp34, gp35, gp36, and gp37, with gp34 located at the phage-proximal end and gp37 at the phage-distal, receptor-binding end. We have solved the structure of the carboxy-terminal region of gp34, consisting of amino acids 894–1289, by single-wavelength anomalous diffraction and extended the structure to amino acids 744–1289 using data collected from crystals containing longer gp34-fragments. The structure reveals three repeats of a mixed α-β fibrous domain in residues 744 to 877. A triple-helical neck connects to an extended triple β-helix domain (amino acids 900–1127 punctuated by two β-prism domains. Next, a β-prism domain decorated with short helices and extended β-helices is present (residues 1146–1238, while the C-terminal end is capped with another short β-helical region and three β-hairpins. The structure provides insight into the stability of the fibrous gp34 protein.

Investigation of functional aspects of the N-terminal region of elongation factor Tu from Escherichia coli using a protein engineering approach

DEFF Research Database (Denmark)

Laurberg, M; Mansilla, Francisco; Clark, Brian F. C.

1998-01-01

The function of the N-terminal region of elongation factor Tu is still unexplained. Until recently, it has not been visible in electron density maps from x-ray crystallography studies, but the presence of several well conserved basic residues suggest that this part of the molecule is of structural...

A terminal-labelling microcytotoxity assay with 125I-iododeoxyuridine as a label for target cells

International Nuclear Information System (INIS)

Stirrat, G.M.

1976-01-01

The development of a terminal-labelling microcytotoxicity assay is described in which target cells (fetal fibroblasts) were labelled with 125 I-iododeoxyuridine after effector (lymphoid) cells had been incubated with them for 24 h. The time-course for the development of cell-mediated cytotoxicity was assessed following allogeneic skin grafting. 'Non-specific' cytotoxicity detracts from the sensitivity of all microcytotoxicity assays and the terminal-labelling assay using 125 I is no exception. The non-specific effects can be reduced but not eliminated by the removal of adherent cells. The optimum target cell/effector cell ratio would seem to be between 1:100 and 1:250. Residual lymph node cells did not appear to incorporate enough label to affect the test results. In vivo correlates of in vitro findings are still not easy to determine

Amino-terminal sequence of glycoprotein D of herpes simplex virus types 1 and 2

International Nuclear Information System (INIS)

Eisenberg, R.J.; Long, D.; Hogue-Angeletti, R.; Cohen, G.H.

1984-01-01

Glycoprotein D (gD) of herpes simplex virus is a structural component of the virion envelope which stimulates production of high titers of herpes simplex virus type-common neutralizing antibody. The authors caried out automated N-terminal amino acid sequencing studies on radiolabeled preparations of gD-1 (gD of herpes simplex virus type 1) and gD-2 (gD of herpes simplex virus type 2). Although some differences were noted, particularly in the methionine and alanine profiles for gD-1 and gD-2, the amino acid sequence of a number of the first 30 residues of the amino terminus of gD-1 and gD-2 appears to be quite similar. For both proteins, the first residue is a lysine. When we compared out sequence data for gD-1 with those predicted by nucleic acid sequencing, the two sequences could be aligned (with one exception) starting at residue 26 (lysine) of the predicted sequence. Thus, the first 25 amino acids of the predicted sequence are absent from the polypeptides isolated from infected cells

Tor forms a dimer through an N-terminal helical solenoid with a complex topology

Science.gov (United States)

Baretić, Domagoj; Berndt, Alex; Ohashi, Yohei; Johnson, Christopher M.; Williams, Roger L.

2016-04-01

The target of rapamycin (Tor) is a Ser/Thr protein kinase that regulates a range of anabolic and catabolic processes. Tor is present in two complexes, TORC1 and TORC2, in which the Tor-Lst8 heterodimer forms a common sub-complex. We have determined the cryo-electron microscopy (EM) structure of Tor bound to Lst8. Two Tor-Lst8 heterodimers assemble further into a dyad-symmetry dimer mediated by Tor-Tor interactions. The first 1,300 residues of Tor form a HEAT repeat-containing α-solenoid with four distinct segments: a highly curved 800-residue N-terminal 'spiral', followed by a 400-residue low-curvature 'bridge' and an extended `railing' running along the bridge leading to the 'cap' that links to FAT region. This complex topology was verified by domain insertions and offers a new interpretation of the mTORC1 structure. The spiral of one TOR interacts with the bridge of another, which together form a joint platform for the Regulatory Associated Protein of TOR (RAPTOR) regulatory subunit.

Basic residues in the 74-83 and 191-198 segments of protein kinase CK2 catalytic subunit are implicated in negative but not in positive regulation by the beta-subunit

DEFF Research Database (Denmark)

Sarno, S; Vaglio, P; Marin, O

1997-01-01

by the beta-subunit many fold more than that of alpha wild type, while extrastimulation by beta mutant D55L56E57A, observable with alpha wild type, is abolished with these mutants. These data support the conclusion that down regulation by the acidic residues clustered in the N-terminal moiety of beta...... is mediated by basic residues in the 74-83 and in the 191-198 sequences of the alpha-subunit. These are also implicated in substrate recognition consistent with the concept that the N-terminal acidic region of the beta subunit operates as a pseudosubstrate. In contrast, another CK2alpha mutant, V66A, is more...

The thyroxine-binding site of human apolipoprotein-A-I: Location in the N-terminal domain

International Nuclear Information System (INIS)

Benvenga, S.; Cahnmann, H.J.; Robbins, J.

1991-01-01

We tested the ability of nine monoclonal antibodies (MAb) against human apolipoprotein-A-I (apoA-I), the 28.3-kDa major apoprotein of high density lipoproteins (HDL), to inhibit its photoaffinity labeling with [125I]T4. Two forms were evaluated: isolated lipid-free apoA-I (Sigma or Calbiochem) and lipid-complexed apoA-I [HDL2, (density, 1.063-1.125 g/ml) and HDL3 (density, 1.125-1.210 g/ml)]. After labeling with 0.5 nM [125I]T4 in the presence of MAb or normal mouse IgG, the products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent densitometric quantitation of radioactivity associated with the 28.3-kDa band. Group I MAbs, namely those having epitopes in the N-terminal portion of apoA-I, include MAb 16 (epitopes at residues 1-16), 4 and 14 (residues 1-86), and 18 (residues 98-105); group II includes MAbs 7,10, 15, and 17 (epitopes at residues 87-148); group III includes MAb 9 (residues 149-243). All group I MAbs inhibited [125I]T4 binding to isolated apoA-I with this order of potency: MAb 16 greater than MAb 14 greater than MAb 4 greater than MAb 18. In the case of lipid-associated apoA-I, the pattern of hierarchy was variable, presumably related to the known markedly polydisperse nature of HDL, but a constant feature, in contrast to the case of isolated apoA-I, was that MAb 4 was more potent than MAb 14. Group II MAbs gave less than 3% inhibition in both isolated and lipid-complexed apoA-I. Group III MAb 9 either failed to inhibit or gave 18-27% inhibition (one preparation each of HDL2 and HDL3). We conclude that the T4 site of apoA-I is in the N-terminal domain of apoA-I, closer to the epitope for MAb 16 than to that for MAb 18, and that conformational changes occurring when apoA-I is associated with lipids in the HDL particle alter the spatial relationship between some epitopes and the T4 site

Natural monomeric form of fetal bovine serum acetylcholinesterase lacks the C-terminal tetramerization domain.

Science.gov (United States)

Saxena, Ashima; Hur, Regina S; Luo, Chunyuan; Doctor, Bhupendra P

2003-12-30

Acetylcholinesterase isolated from fetal bovine serum (FBS AChE) was previously characterized as a globular tetrameric form. Analysis of purified preparations of FBS AChE by gel permeation chromatography revealed the presence of a stable, catalytically active, monomeric form of this enzyme. The two forms could be distinguished from each other based on their molecular weight, hydrodynamic properties, kinetic properties, thermal stability, and the type of glycans they carry. No differences between the two forms were observed for the binding of classical inhibitors such as edrophonium and propidium or inhibitors that are current or potential drugs for the treatment of Alzheimer's disease such as (-) huperzine A and E2020; tacrine inhibited the monomeric form 2-3-fold more potently than the tetrameric form. Sequencing of peptides obtained from an in-gel tryptic digest of the monomer and tetramer by tandem mass spectrometry indicated that the tetramer consists of 583 amino acid residues corresponding to the mature form of the enzyme, whereas the monomer consists of 543-547 amino acid residues. The subunit molecular weight of the protein component of the monomer (major species) was determined to be 59 414 Da and that of the tetramer as 64 239 Da. The N-terminal of the monomer and the tetramer was Glu, suggesting that the monomer is not a result of truncation at the N-terminal. The only differences detected were at the C-terminus. The tetramer yielded the expected C-terminus, CSDL, whereas the C-terminus of the monomer yielded a mixture of peptides, of which LLSATDTLD was the most abundant. These results suggest that monomeric FBS AChE is trimmed at the C-terminus, and the results are consistent with the involvement of C-terminal amino acids in the assembly of monomers into tetramers.

Fine tuning of the catalytic activity of colicin E7 nuclease domain by systematic N-terminal mutations

Czech Academy of Sciences Publication Activity Database

Németh, E.; Körtvélyesi, T.; Thulstrup, P. W.; Christensen, H. E. M.; Kožíšek, Milan; Nagata, K.; Czene, A.; Gyurcsik, B.

2014-01-01

Roč. 23, č. 8 (2014), s. 1113-1122 ISSN 0961-8368 Grant - others:Seventh Framework Programme of the European Union(XE) FP7-312284; OPPC(CZ) CZ.2.16/3.1.00/24016 Institutional support: RVO:61388963 Keywords : DNA cleavage * flow linear dichroism * isothermal calorimetry * positively charged N-terminal residues * Zn2+ binding Subject RIV: CE - Biochemistry Impact factor: 2.854, year: 2014

The two C-terminal tyrosines stabilize occluded Na/K pump conformations containing Na or K ions.

Science.gov (United States)

Vedovato, Natascia; Gadsby, David C

2010-07-01

Interactions of the three transported Na ions with the Na/K pump remain incompletely understood. Na/K pump crystal structures show that the extended C terminus of the Na,K-adenosine triphosphatase (ATPase) alpha subunit directly contacts transmembrane helices. Deletion of the last five residues (KETYY in almost all Na/K pumps) markedly lowered the apparent affinity for Na activation of pump phosphorylation from ATP, a reflection of cytoplasmic Na affinity for forming the occluded E1P(Na3) conformation. ATPase assays further suggested that C-terminal truncations also interfere with low affinity Na interactions, which are attributable to extracellular effects. Because extracellular Na ions traverse part of the membrane's electric field to reach their binding sites in the Na/K pump, their movements generate currents that can be monitored with high resolution. We report here electrical measurements to examine how Na/K pump interactions with extracellular Na ions are influenced by C-terminal truncations. We deleted the last two (YY) or five (KESYY) residues in Xenopus laevis alpha1 Na/K pumps made ouabain resistant by either of two kinds of point mutations and measured their currents as 10-mM ouabain-sensitive currents in Xenopus oocytes after silencing endogenous Xenopus Na/K pumps with 1 microM ouabain. We found the low affinity inhibitory influence of extracellular Na on outward Na/K pump current at negative voltages to be impaired in all of the C-terminally truncated pumps. Correspondingly, voltage jump-induced transient charge movements that reflect pump interactions with extracellular Na ions were strongly shifted to more negative potentials; this signals a several-fold reduction of the apparent affinity for extracellular Na in the truncated pumps. Parallel lowering of Na affinity on both sides of the membrane argues that the C-terminal contacts provide important stabilization of the occluded E1P(Na3) conformation, regardless of the route of Na ion entry into the

Residues and dissipation of kresoxim methyl in apple under field condition.

Science.gov (United States)

Malhat, Farag; Kamel, Essam; Saber, Ayman; Hassan, Ehab; Youssef, Ahmed; Almaz, Monir; Hassan, Ayman; Fayz, Abd El-Salam

2013-09-01

The dissipation and residual levels of kresoxim methyl in apple under field condition were determined by using HPLC-DAD with QuEChERS method. At fortification levels of 0.05, 0.1, 0.5 and 1.0 mg kg(-1) in apple, it was shown that recoveries were ranged from 91.1% to 96.9% with coefficient variation of the method (CV%) for repeatability ranged from 1.27% to 4.77%. The limit of quantification (LOQ) of the method was 0.05 mg kg(-1). The dissipation rates of kresoxim methyl were described by using first-order kinetics and its half-life, as they are ranged from 4.58 to 4.77 days in apple. The terminal residues of kresoxim methyl were below the FAO/WHO maximum residue limit (MRL, 0.2 mg kg(-1)) in apple when measured 14 days after the final application, which suggested that the use of this fungicide was safe for humans. This study would help in providing the basic information for developing regulation to guard a safe use of kresoxim methyl in apple orchard and to prevent health problem from consumers. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effect of leaching residual methyl methacrylate concentrations on in vitro cytotoxicity of heat polymerized denture base acrylic resin processed with different polymerization cycles

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Canan Bural

2011-08-01

Full Text Available OBJECTIVES: Residual methyl methacrylate (MMA may leach from the acrylic resin denture bases and have adverse effects on the oral mucosa. This in vitro study evaluated and correlated the effect of the leaching residual MMA concentrations ([MMA]r on in vitro cytotoxicity of L-929 fibroblasts. MATERIAL AND METHODS: A total of 144 heat-polymerized acrylic resin specimens were fabricated using 4 different polymerization cycles: (1 at 74ºC for 9 h, (2 at 74ºC for 9 h and terminal boiling (at 100ºC for 30 min, (3 at 74ºC for 9 h and terminal boiling for 3 h, (4 at 74ºC for 30 min and terminal boiling for 30 min. Specimens were eluted in a complete cell culture medium at 37ºC for 1, 2, 5 and 7 days. [MMA]r in eluates was measured using high-performance liquid chromatography. In vitro cytotoxicity of eluates on L-929 fibroblasts was evaluated by means of cell proliferation using a tetrazolium salt XTT (sodium 3´-[1-phenyl-aminocarbonyl-3,4-tetrazolium]bis(4-methoxy-6-nitrobenzenesulphonic acid assay. Differences in [MMA]r of eluates and cell proliferation values between polymerization cycles were statistically analyzed by Kruskal-Wallis, Friedman and Dunn's multiple comparison tests. The correlation between [MMA]r of eluates and cell proliferation was analyzed by Pearson's correlation test (p<0.05. RESULTS: [MMA]r was significantly (p<0.001 higher in eluates of specimens polymerized with cycle without terminal boiling after elution of 1 and 2 days. Cell proliferation values for all cycles were significantly (p<0.01 lower in eluates of 1 day than those of 2 days. The correlation between [MMA]r and cell proliferation values was negative after all elution periods, showing significance (p<0.05 for elution of 1 and 2 days. MMA continued to leach from acrylic resin throughout 7 days and leaching concentrations markedly reduced after elution of 1 and 2 days. CONCLUSION: Due to reduction of leaching residual MMA concentrations, use of terminal boiling in

CONTAINER TERMINALS IN EUROPE

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Bart W. WIEGMANS

2001-01-01

Full Text Available This paper aims to address the linkage between logistics (in particular, the management of marketing channel flows and transport markets, while also the interaction between these two markets and intermodal container terminals is analysed. The marketing channel theory is used to describe all relevant actors and flows that run through marketing channels, starting with customer needs and ending with customer satisfaction. Porter's theory of competitive advantages is used to review competitive forces in both markets. Finally, a competitor analysis is performed for the logistics and transport market. These theories are applied so as to be able to determine the competitive position of intermodal container terminals with a view to the management of marketing channel flows and the physical transport of freight flows. Hence, the central question of this paper is: Which markets are served by intermodal container terminals and with whom are they competing? At present, neither the maritime container terminals nor the continental container terminals appear to have a significant influence in the logistics service market; they concentrate mainly on the physical movement of containers (transshipment. Furthermore, maritime container terminals and continental container terminals are not dominant players in the transport service market. Our conclusion is that continental terminals are predominantly competing with unimodal road transport, with neighbouring continental terminals and with barge transport companies.

Gas-Phase Hydrogen-Deuterium Exchange Labeling of Select Peptide Ion Conformer Types: a Per-Residue Kinetics Analysis.

Science.gov (United States)

Khakinejad, Mahdiar; Kondalaji, Samaneh Ghassabi; Tafreshian, Amirmahdi; Valentine, Stephen J

2015-07-01

The per-residue, gas-phase hydrogen deuterium exchange (HDX) kinetics for individual amino acid residues on selected ion conformer types of the model peptide KKDDDDDIIKIIK have been examined using ion mobility spectrometry (IMS) and HDX-tandem mass spectrometry (MS/MS) techniques. The [M + 4H](4+) ions exhibit two major conformer types with collision cross sections of 418 Å(2) and 446 Å(2); the [M + 3H](3+) ions also yield two different conformer types having collision cross sections of 340 Å(2) and 367 Å(2). Kinetics plots of HDX for individual amino acid residues reveal fast- and slow-exchanging hydrogens. The contributions of each amino acid residue to the overall conformer type rate constant have been estimated. For this peptide, N- and C-terminal K residues exhibit the greatest contributions for all ion conformer types. Interior D and I residues show decreased contributions. Several charge state trends are observed. On average, the D residues of the [M + 3H](3+) ions show faster HDX rate contributions compared with [M + 4H](4+) ions. In contrast the interior I8 and I9 residues show increased accessibility to exchange for the more elongated [M + 4H](4+) ion conformer type. The contribution of each residue to the overall uptake rate showed a good correlation with a residue hydrogen accessibility score model calculated using a distance from charge site and initial incorporation site for nominal structures obtained from molecular dynamic simulations (MDS).

The differences in heparin binding for the C-terminal basic-sequence-rich peptides of HPV-16 and HPV-18 capsid protein L1

International Nuclear Information System (INIS)

Sun Jian; Yu Jisheng; Yu Zhiwu; Zha Xiao; Wu Yuqing

2012-01-01

Graphial abstract: The differences in heparin binding for the C-terminal basic-sequence-rich peptides of HPV-16 and HPV-18 capsid protein L1. Highlights: ► Several driving forces contribute to the interaction between heparin and peptides. ► C-terminal of HPV L1 is a potential candidate for the attachment to host cells. ► The C-terminal peptides of HPV-16 and -18 L1 have different heparin-binding. ► The different heparin-binding provides an explanation for the distinct prevalences. - Abstract: The high-risk types of human papillomaviruses (HPV) HPV-16 and -18 are the predominant types associated with cervical cancer. HPV-16 and -18 account for about 50% and 20%, respectively, of cervical cancers worldwide. While the reason and molecular mechanism of the distinct prevalence and distributions between them remain poorly understood, the binding affinity of cell surface receptor with capsid proteins, especially L1, may be involved. We examined heparin binding with two synthetic peptides corresponding to the 14 amino acid C-terminal peptides of HPV-16 and -18 L1 with the goal of comparing the equivalent residues in different HPV types. Using isothermal titration calorimetry (ITC) and static right-angle light scattering (SLS), we determined the binding constant K, reaction enthalpy ΔH, and other thermodynamic parameters in the interaction. Especially, we assessed the role of specific residues in binding with heparin by comparing the NMR spectra of free and heparin-bound peptides.

Mutated but Not Deleted Ovine PrP(C) N-Terminal Polybasic Region Strongly Interferes with Prion Propagation in Transgenic Mice.

Science.gov (United States)

Khalifé, Manal; Reine, Fabienne; Paquet-Fifield, Sophie; Castille, Johan; Herzog, Laetitia; Vilotte, Marthe; Moudjou, Mohammed; Moazami-Goudarzi, Katayoun; Makhzami, Samira; Passet, Bruno; Andréoletti, Olivier; Vilette, Didier; Laude, Hubert; Béringue, Vincent; Vilotte, Jean-Luc

2016-02-01

Mammalian prions are proteinaceous infectious agents composed of misfolded assemblies of the host-encoded, cellular prion protein (PrP). Physiologically, the N-terminal polybasic region of residues 23 to 31 of PrP has been shown to be involved in its endocytic trafficking and interactions with glycosaminoglycans or putative ectodomains of membrane-associated proteins. Several recent reports also describe this PrP region as important for the toxicity of mutant prion proteins and the efficiency of prion propagation, both in vitro and in vivo. The question remains as to whether the latter observations made with mouse PrP and mouse prions would be relevant to other PrP species/prion strain combinations given the dramatic impact on prion susceptibility of minimal amino acid substitutions and structural variations in PrP. Here, we report that transgenic mouse lines expressing ovine PrP with a deletion of residues 23 to 26 (KKRP) or mutated in this N-terminal region (KQHPH instead of KKRPK) exhibited a variable, strain-dependent susceptibility to prion infection with regard to the proportion of affected mice and disease tempo relative to findings in their wild-type counterparts. Deletion has no major effect on 127S scrapie prion pathogenesis, whereas mutation increased by almost 3-fold the survival time of the mice. Deletion marginally affected the incubation time of scrapie LA19K and ovine bovine spongiform encephalopathy (BSE) prions, whereas mutation caused apparent resistance to disease. Recent reports suggested that the N-terminal polybasic region of the prion protein could be a therapeutic target to prevent prion propagation or toxic signaling associated with more common neurodegenerative diseases such as Alzheimer's disease. Mutating or deleting this region in ovine PrP completes the data previously obtained with the mouse protein by identifying the key amino acid residues involved. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Interaction of N-terminal peptide analogues of the Na+,K+-ATPase with membranes.

Science.gov (United States)

Nguyen, Khoa; Garcia, Alvaro; Sani, Marc-Antoine; Diaz, Dil; Dubey, Vikas; Clayton, Daniel; Dal Poggetto, Giovanni; Cornelius, Flemming; Payne, Richard J; Separovic, Frances; Khandelia, Himanshu; Clarke, Ronald J

2018-06-01

The Na + ,K + -ATPase, which is present in the plasma membrane of all animal cells, plays a crucial role in maintaining the Na + and K + electrochemical potential gradients across the membrane. Recent studies have suggested that the N-terminus of the protein's catalytic α-subunit is involved in an electrostatic interaction with the surrounding membrane, which controls the protein's conformational equilibrium. However, because the N-terminus could not yet be resolved in any X-ray crystal structures, little information about this interaction is so far available. In measurements utilising poly-l-lysine as a model of the protein's lysine-rich N-terminus and using lipid vesicles of defined composition, here we have identified the most likely origin of the interaction as one between positively charged lysine residues of the N-terminus and negatively charged headgroups of phospholipids (notably phosphatidylserine) in the surrounding membrane. Furthermore, to isolate which segments of the N-terminus could be involved in membrane binding, we chemically synthesized N-terminal fragments of various lengths. Based on a combination of results from RH421 UV/visible absorbance measurements and solid-state 31 P and 2 H NMR using these N-terminal fragments as well as MD simulations it appears that the membrane interaction arises from lysine residues prior to the conserved LKKE motif of the N-terminus. The MD simulations indicate that the strength of the interaction varies significantly between different enzyme conformations. Copyright © 2018 Elsevier B.V. All rights reserved.

Two-dimensional ultrasonography in of medical abortion in the harem of residual experience%二维超声对药物流产后宫内残留的体会

Institute of Scientific and Technical Information of China (English)

原占国

2015-01-01

In recent years, medical abortion in wide clinical application, especially the menopause acuities were 49 days of pregnancy termination. But many patients use easy cause intrauterine residue after medical abortion. Now statistics from May 2009 to May 2011 among 420 patients with medical abortion of 50 cases of intrauterine residue analysis is as follows.

Visual communication and terminal equipment

International Nuclear Information System (INIS)

Kang, Cheol Hui

1988-06-01

This book is divided two parts about visual communication and terminal equipment. The first part introduces visual communication, which deals with foundation of visual communication, technique of visual communication, equipment of visual communication, a facsimile and pictorial image system. The second part contains terminal equipment such as telephone, terminal equipment for data transmission on constitution and constituent of terminal equipment for data transmission, input device and output device, terminal device and up-to-date terminal device.

Visual communication and terminal equipment

Energy Technology Data Exchange (ETDEWEB)

Kang, Cheol Hui

1988-06-15

This book is divided two parts about visual communication and terminal equipment. The first part introduces visual communication, which deals with foundation of visual communication, technique of visual communication, equipment of visual communication, a facsimile and pictorial image system. The second part contains terminal equipment such as telephone, terminal equipment for data transmission on constitution and constituent of terminal equipment for data transmission, input device and output device, terminal device and up-to-date terminal device.

Dissipation and Residues of Dichlorprop-P and Bentazone in Wheat-Field Ecosystem

Directory of Open Access Journals (Sweden)

Xiaoxiao Feng

2016-05-01

Full Text Available Dichlorprop-P and bentazone have been widely used in the prevention and control of weeds in wheat field ecosystems. There is a concern that pesticide residues and metabolites remain on or in the wheat. Thus, the study of the determination and monitoring of their residues in wheat has important significance. A rapid, simple and reliable QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe method was modified, developed and validated for the determination of dichlorprop-P, bentazone and its metabolites (6-hydroxy-bentazone and 8-hydroxy-bentazone in wheat (wheat plants, wheat straw and grains of wheat using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS. The average recoveries of this method ranged from 72.9% to 108.7%, and the limits of quantification (LOQs were 2.5–12 μg/kg. The dissipation and final residue of four compounds in three provinces (Shandong, Jiangsu and Heilongjiang in China were studied. The trial results showed that the half-lives of dichlorprop-P and bentazone were 1.9–2.5 days and 0.5–2.4 days in wheat plants, respectively. The terminal residues in grains of wheat and wheat straw at harvest were all much below the maximum residue limit (MRL of 0.2 mg/kg for dichlorprop-P and 0.1 mg/kg for bentazone established by the European Union (EU, Regulation No. 396/2005.

A non-catalytic histidine residue influences the function of the metalloprotease of Listeria monocytogenes.

Science.gov (United States)

Forster, Brian M; Bitar, Alan Pavinski; Marquis, Hélène

2014-01-01

Mpl, a thermolysin-like metalloprotease, and PC-PLC, a phospholipase C, are synthesized as proenzymes by the intracellular bacterial pathogen Listeria monocytogenes. During intracellular growth, L. monocytogenes is temporarily confined in a membrane-bound vacuole whose acidification leads to Mpl autolysis and Mpl-mediated cleavage of the PC-PLC N-terminal propeptide. Mpl maturation also leads to the secretion of both Mpl and PC-PLC across the bacterial cell wall. Previously, we identified negatively charged and uncharged amino acid residues within the N terminus of the PC-PLC propeptide that influence the ability of Mpl to mediate the maturation of PC-PLC, suggesting that these residues promote the interaction of the PC-PLC propeptide with Mpl. In the present study, we identified a non-catalytic histidine residue (H226) that influences Mpl secretion across the cell wall and its ability to process PC-PLC. Our results suggest that a positive charge at position 226 is required for Mpl functions other than autolysis. Based on the charge requirement at this position, we hypothesize that this residue contributes to the interaction of Mpl with the PC-PLC propeptide.

Transglycosylation reactions of Bacillus stearothermophilus maltogenic amylase with acarbose and various acceptors

International Nuclear Information System (INIS)

Hwa Park, K.; Jeong Kim, M.; Seob Lee, H.; Kim, D.; Soo Han, N.; Robyt, J.F.

1998-01-01

It was observed that Bacillus stearothermophilus maltogenic amylase cleaved the first glycosidic bond of acarbose to produce glucose and a pseudotrisaccharide (PTS) that was transferred to C-6 of the glucose to give an α-(1-6) glycosidic linkage and the formation of isoacarbose. The addition of a number of different carbohydrates to the digest gave transfer products in which PTS was primarily attached α-(1-6) to d-glucose, d-mannose, d-galactose, and methyl α-d-glucopyranoside. With d-fructopyranose and d-xylopyranose, PTS was linked α-(1-5) and α-(1-4), respectively. PTS was primarily transferred to C-6 of the nonreducing residue of maltose, cellobiose, lactose, and gentiobiose. Lesser amounts of α-(1-3) and/or α-(1-4) transfer products were also observed for these carbohydrate acceptors. The major transfer product to sucrose gave PTS linked α-(1-4) to the glucose residue. α,α-Trehalose gave two major products with PTS linked α-(1-6) and α-(1-4). Maltitol gave two major products with PTS linked α-(1-6) and α-(1-4) to the glucopyranose residue. Raffinose gave two major products with PTS linked α-(1-6) and α-(1-4) to the d-galactopyranose residue. Maltotriose gave two major products with PTS linked α-(1-6) and α-(1-4) to the nonreducing end glucopyranose residue. Xylitol gave PTS linked α-(1-5) as the major product and d-glucitol gave PTS linked α-(1-6) as the only product. The structures of the transfer products were determined using thin layer-chromatography, high-performance ion chromatography, enzyme hydrolysis, methylation analysis and 13 C NMR spectroscopy. The best acceptor was gentiobiose, followed closely by maltose and cellobiose, and the weakest acceptor was d-glucitol. (Copyright (c) 1998 Elsevier Science B.V., Amsterdam. All rights reserved.)

Transglycosylation reactions of Bacillus stearothermophilus maltogenic amylase with acarbose and various acceptors

Energy Technology Data Exchange (ETDEWEB)

Hwa Park, K; Jeong Kim, M; Seob Lee, H; Kim, D [Department of Food Science and Technology and Research Center for New Bio-Materials in Agriculture, Seoul National University, Suwon (Korea, Republic of); Soo Han, N; Robyt, J F [Laboratory for Carbohydrate Chemistry and Enzymology, Department of Biochemistry and Biophysics, Iowa State University, Ames, IA (United States)

1998-12-15

It was observed that Bacillus stearothermophilus maltogenic amylase cleaved the first glycosidic bond of acarbose to produce glucose and a pseudotrisaccharide (PTS) that was transferred to C-6 of the glucose to give an {alpha}-(1-6) glycosidic linkage and the formation of isoacarbose. The addition of a number of different carbohydrates to the digest gave transfer products in which PTS was primarily attached {alpha}-(1-6) to d-glucose, d-mannose, d-galactose, and methyl {alpha}-d-glucopyranoside. With d-fructopyranose and d-xylopyranose, PTS was linked {alpha}-(1-5) and {alpha}-(1-4), respectively. PTS was primarily transferred to C-6 of the nonreducing residue of maltose, cellobiose, lactose, and gentiobiose. Lesser amounts of {alpha}-(1-3) and/or {alpha}-(1-4) transfer products were also observed for these carbohydrate acceptors. The major transfer product to sucrose gave PTS linked {alpha}-(1-4) to the glucose residue. {alpha},{alpha}-Trehalose gave two major products with PTS linked {alpha}-(1-6) and {alpha}-(1-4). Maltitol gave two major products with PTS linked {alpha}-(1-6) and {alpha}-(1-4) to the glucopyranose residue. Raffinose gave two major products with PTS linked {alpha}-(1-6) and {alpha}-(1-4) to the d-galactopyranose residue. Maltotriose gave two major products with PTS linked {alpha}-(1-6) and {alpha}-(1-4) to the nonreducing end glucopyranose residue. Xylitol gave PTS linked {alpha}-(1-5) as the major product and d-glucitol gave PTS linked {alpha}-(1-6) as the only product. The structures of the transfer products were determined using thin layer-chromatography, high-performance ion chromatography, enzyme hydrolysis, methylation analysis and {sup 13}C NMR spectroscopy. The best acceptor was gentiobiose, followed closely by maltose and cellobiose, and the weakest acceptor was d-glucitol. (Copyright (c) 1998 Elsevier Science B.V., Amsterdam. All rights reserved.)

Recombinant production of peptide C-terminal α-amides using an engineered intein

DEFF Research Database (Denmark)

Albertsen, Louise; Shaw, Allan C; Norrild, Jens Chr.

2013-01-01

is that they contain a C-terminal that is α-amidated, and this amidation is crucial for biological function. A challenge is to generate such peptides by recombinant means and particularly in a production scale. Here, we have examined an intein-mediated approach to generate a PYY derivative in a larger scale. Initially......, we experienced challenges with hydrolysis of the intein fusion protein, which was reduced by a T3C mutation in the intein. Subsequently, we further engineered the intein to decrease the absolute size and improve the relative yield of the PYY derivative, which was achieved by substituting 54 residues...

Computational comparison of β-mannosidases of animals, humans, microbes, and plants

OpenAIRE

SAMRA, Zahoor Qadir; ATHAR, Muhammad Amin

2011-01-01

The b-mannosidase (MANB) enzyme is involved in removing mannose residue from the nonreducing end, and its impaired activity leads to b-mannosidosis. MANB amino acid sequences of humans, other mammals, plants, fungi, and bacteria were compared to determine their similarities, differences, and predicted 3D structures. Our cloned MANB DNA sequence showed a 99% similarity to a previously reported human MANB DNA sequence but 16 nucleotide differences were observed, showing the polymorphic nature o...

Computational comparison of β-mannosidases of animals, humans, microbes, and plants

OpenAIRE

SAMRA, Zahoor Qadir; ATHAR, Muhammad Amin

2014-01-01

The b-mannosidase (MANB) enzyme is involved in removing mannose residue from the nonreducing end, and its impaired activity leads to b-mannosidosis. MANB amino acid sequences of humans, other mammals, plants, fungi, and bacteria were compared to determine their similarities, differences, and predicted 3D structures. Our cloned MANB DNA sequence showed a 99% similarity to a previously reported human MANB DNA sequence but 16 nucleotide differences were observed, showing the polymorphic nature o...

N-terminal truncation enables crystallization of the receptor-binding domain of the FedF bacterial adhesin

Energy Technology Data Exchange (ETDEWEB)

De Kerpel, Maia; Van Molle, Inge [Department of Ultrastructure, Vrije Universiteit Brussel (VUB), Flanders Interuniversity Institute for Biotechnology (VIB), Pleinlaan 2, 1050 Brussels (Belgium); Brys, Lea [Department of Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), Flanders Interuniversity Institute for Biotechnology (VIB), Pleinlaan 2, 1050 Brussels (Belgium); Wyns, Lode; De Greve, Henri; Bouckaert, Julie, E-mail: bouckaej@vub.ac.be [Department of Ultrastructure, Vrije Universiteit Brussel (VUB), Flanders Interuniversity Institute for Biotechnology (VIB), Pleinlaan 2, 1050 Brussels (Belgium)

2006-12-01

The N-terminal receptor-binding domain of the FedF adhesin from enterotoxigenic E. coli has been crystallized. This required the deletion of its first 14 residues, which are also cleaved off naturally. FedF is the two-domain tip adhesin of F18 fimbriae from enterotoxigenic Escherichia coli. Bacterial adherence, mediated by the N-terminal receptor-binding domain of FedF to carbohydrate receptors on intestinal microvilli, causes diarrhoea and oedema disease in newly weaned piglets and induces the secretion of Shiga toxins. A truncate containing only the receptor-binding domain of FedF was found to be further cleaved at its N-terminus. Reconstruction of this N-terminal truncate rendered FedF amenable to crystallization, resulting in crystals with space group P2{sub 1}2{sub 1}2{sub 1} and unit-cell parameters a = 36.20, b = 74.64, c = 99.03 Å that diffracted to beyond 2 Å resolution. The binding specificity of FedF was screened for on a glycan array, exposing 264 glycoconjugates, to identify specific receptors for cocrystallization with FedF.

Crystal Structure of the N-terminal Domain of the Group B Streptococcus Alpha C Protein

Energy Technology Data Exchange (ETDEWEB)

Auperin,T.; Bolduc, G.; Baron, M.; Heroux, A.; Filman, D.; Madoff, L.; Hogle, J.

2005-01-01

Group B Streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis, and meningitis among neonates and an important cause of morbidity among pregnant women and immunocompromised adults. Invasive diseases due to GBS are attributed to the ability of the pathogen to translocate across human epithelial surfaces. The alpha C protein (ACP) has been identified as an invasin that plays a role in internalization and translocation of GBS across epithelial cells. The soluble N-terminal domain of ACP (NtACP) blocks the internalization of GBS. We determined the 1.86-{angstrom} resolution crystal structure of NtACP comprising residues Ser{sup 52} through Leu{sup 225} of the full-length ACP. NtACP has two domains, an N-terminal {beta}-sandwich and a C-terminal three-helix bundle. Structural and topological alignments reveal that the {beta}-sandwich shares structural elements with the type III fibronectin fold (FnIII), but includes structural elaborations that make it unique. We have identified a potential integrin-binding motif consisting of Lys-Thr-Asp{sup 146}, Arg{sup 110}, and Asp{sup 118}. A similar arrangement of charged residues has been described in other invasins. ACP shows a heparin binding activity that requires NtACP. We propose a possible heparin-binding site, including one surface of the three-helix bundle, and nearby portions of the sandwich and repeat domains. We have validated this prediction using assays of the heparin binding and cell-adhesion properties of engineered fragments of ACP. This is the first crystal structure of a member of the highly conserved Gram-positive surface alpha-like protein family, and it will enable the internalization mechanism of GBS to be dissected at the atomic level.

N-terminal peptides from unprocessed prion proteins enter cells by macropinocytosis

International Nuclear Information System (INIS)

Magzoub, Mazin; Sandgren, Staffan; Lundberg, Pontus; Oglecka, Kamila; Lilja, Johanna; Wittrup, Anders; Goeran Eriksson, L.E.; Langel, Ulo; Belting, Mattias; Graeslund, Astrid

2006-01-01

A peptide derived from the N-terminus of the unprocessed bovine prion protein (bPrPp), incorporating the hydrophobic signal sequence (residues 1-24) and a basic domain (KKRPKP, residues 25-30), internalizes into mammalian cells, even when coupled to a sizeable cargo, and therefore functions as a cell-penetrating peptide (CPP). Confocal microscopy and co-localization studies indicate that the internalization of bPrPp is mainly through macropinocytosis, a fluid-phase endocytosis process, initiated by binding to cell-surface proteoglycans. Electron microscopy studies show internalized bPrPp-DNA-gold complexes residing in endosomal vesicles. bPrPp induces expression of a complexed luciferase-encoding DNA plasmid, demonstrating the peptide's ability to transport the cargo across the endosomal membrane and into the cytosol and nucleus. The novel CPP activity of the unprocessed N-terminal domain of PrP could be important for the retrotranslocation of partly processed PrP and for PrP trafficking inside or between cells, with implications for the infectivity associated with prion diseases

Antecedents of Customer Relationship Termination

DEFF Research Database (Denmark)

Geersbro, Jens; Ritter, Thomas

To end business relationships, or to more actively terminate relationships, has long been acknowledged as part of customer relationship management. However, compared to other elements such as initiation and maintenance of relationships, little is known about the termination of business...... relationships as a managerial task. This paper contributes by (1) developing a conceptualization of relationship termination competence and (2) analyzing its antecedents. The empirical results identify termination acceptance, definition non-customers, organizational relationship termination routines......, and motivation as significant antecedents. Because of this, managers need to develop their organizations in order to use relationship termination as a vital strategy....

Violaxanthin de-epoxidase disulphides and their role in activity and thermal stability.

Science.gov (United States)

Hallin, Erik Ingmar; Guo, Kuo; Åkerlund, Hans-Erik

2015-05-01

Violaxanthin de-epoxidase (VDE) catalyses the conversion of violaxanthin to zeaxanthin at the lumen side of the thylakoids during exposure to intense light. VDE consists of a cysteine-rich N-terminal domain, a lipocalin-like domain and a negatively charged C-terminal domain. That the cysteines are important for the activity of VDE is well known, but in what way is less understood. In this study, wild-type spinach VDE was expressed in E. coli as inclusion bodies, refolded and purified to give a highly active and homogenous preparation. The metal content (Fe, Cu, Ni, Mn, Co and Zn) was lower than 1 mol% excluding a metal-binding function of the cysteines. To investigate which of the 13 cysteines that could be important for the function of VDE, we constructed mutants where the cysteines were replaced by serines, one by one. For 12 out of 13 mutants the activity dropped by more than 99.9%. A quantification of free cysteines showed that only the most N-terminal of these cysteines was in reduced form in the native VDE. A disulphide pattern in VDE of C9-C27, C14-C21, C33-C50, C37-C46, C65-C72 and C118-C284 was obtained after digestion of VDE with thermolysin followed by mass spectroscopy analysis of reduced versus non-reduced samples. The residual activity found for the mutants showed a variation that was consistent with the results obtained from mass spectroscopy. Reduction of the disulphides resulted in loss of a rigid structure and a decrease in thermal stability of 15 °C.

Mutations at the S1 sites of methionine aminopeptidases from Escherichia coli and Homo sapiens reveal the residues critical for substrate specificity.

Science.gov (United States)

Li, Jing-Ya; Cui, Yong-Mei; Chen, Ling-Ling; Gu, Min; Li, Jia; Nan, Fa-Jun; Ye, Qi-Zhuang

2004-05-14

Methionine aminopeptidase (MetAP) catalyzes the removal of methionine from newly synthesized polypeptides. MetAP carries out this cleavage with high precision, and Met is the only natural amino acid residue at the N terminus that is accepted, although type I and type II MetAPs use two different sets of residues to form the hydrophobic S1 site. Characteristics of the S1 binding pocket in type I MetAP were investigated by systematic mutation of each of the seven S1 residues in Escherichia coli MetAP type I (EcMetAP1) and human MetAP type I (HsMetAP1). We found that Tyr-65 and Trp-221 in EcMetAP1, as well as the corresponding residues Phe-197 and Trp-352 in HsMetAP1, were essential for the hydrolysis of a thiopeptolide substrate, Met-S-Gly-Phe. Mutation of Phe-191 to Ala in HsMetAP1 caused inactivity in contrast to the full activity of EcMetAP1(Y62A), which may suggest a subtle difference between the two type I enzymes. The more striking finding is that mutation of Cys-70 in EcMetAP1 or Cys-202 in HsMetAP1 opens up the S1 pocket. The thiopeptolides Leu-S-Gly-Phe and Phe-S-Gly-Phe, with previously unacceptable Leu or Phe as the N-terminal residue, became efficient substrates of EcMetAP1(C70A) and HsMetAP1(C202A). The relaxed specificity shown in these S1 site mutants for the N-terminal residues was confirmed by hydrolysis of peptide substrates and inhibition by reaction products. The structural features at the enzyme active site will be useful information for designing specific MetAP inhibitors for therapeutic applications.

Flanking signal and mature peptide residues influence signal peptide cleavage

Directory of Open Access Journals (Sweden)

Ranganathan Shoba

2008-12-01

Full Text Available Abstract Background Signal peptides (SPs mediate the targeting of secretory precursor proteins to the correct subcellular compartments in prokaryotes and eukaryotes. Identifying these transient peptides is crucial to the medical, food and beverage and biotechnology industries yet our understanding of these peptides remains limited. This paper examines the most common type of signal peptides cleavable by the endoprotease signal peptidase I (SPase I, and the residues flanking the cleavage sites of three groups of signal peptide sequences, namely (i eukaryotes (Euk (ii Gram-positive (Gram+ bacteria, and (iii Gram-negative (Gram- bacteria. Results In this study, 2352 secretory peptide sequences from a variety of organisms with amino-terminal SPs are extracted from the manually curated SPdb database for analysis based on physicochemical properties such as pI, aliphatic index, GRAVY score, hydrophobicity, net charge and position-specific residue preferences. Our findings show that the three groups share several similarities in general, but they display distinctive features upon examination in terms of their amino acid compositions and frequencies, and various physico-chemical properties. Thus, analysis or prediction of their sequences should be separated and treated as distinct groups. Conclusion We conclude that the peptide segment recognized by SPase I extends to the start of the mature protein to a limited extent, upon our survey of the amino acid residues surrounding the cleavage processing site. These flanking residues possibly influence the cleavage processing and contribute to non-canonical cleavage sites. Our findings are applicable in defining more accurate prediction tools for recognition and identification of cleavage site of SPs.

Crystal structure studies of NADP{sup +} dependent isocitrate dehydrogenase from Thermus thermophilus exhibiting a novel terminal domain

Energy Technology Data Exchange (ETDEWEB)

Kumar, S.M. [Department of Studies in Physics, University of Mysore, Mysore 570 006 (India); Pampa, K.J. [Department of Studies in Microbiology, University of Mysore, Mysore 570 006 (India); Manjula, M. [Department of Studies in Physics, University of Mysore, Mysore 570 006 (India); Abdoh, M.M.M. [Department of Physics, Faculty of Science, An-Najah National University, Nablus, West Bank, Palestine (Country Unknown); Kunishima, Naoki [Advanced Protein Crystallography Research Group, RIKEN SPring-8 Center, Harima Institute, Hyogo 679-5148 (Japan); Lokanath, N.K., E-mail: lokanath@physics.uni-mysore.ac.in [Department of Studies in Physics, University of Mysore, Mysore 570 006 (India)

2014-06-20

Highlights: • We determined the structure of isocitrate dehydrogenase with citrate and cofactor. • The structure reveals a unique novel terminal domain involved in dimerization. • Clasp domain shows significant difference, and catalytic residues are conserved. • Oligomerization of the enzyme is quantized with subunit-subunit interactions. • Novel domain of this enzyme is classified as subfamily of the type IV. - Abstract: NADP{sup +} dependent isocitrate dehydrogenase (IDH) is an enzyme catalyzing oxidative decarboxylation of isocitrate into oxalosuccinate (intermediate) and finally the product α-ketoglutarate. The crystal structure of Thermus thermophilus isocitrate dehydrogenase (TtIDH) ternary complex with citrate and cofactor NADP{sup +} was determined using X-ray diffraction method to a resolution of 1.80 Å. The overall fold of this protein was resolved into large domain, small domain and a clasp domain. The monomeric structure reveals a novel terminal domain involved in dimerization, very unique and novel domain when compared to other IDH’s. And, small domain and clasp domain showing significant differences when compared to other IDH’s of the same sub-family. The structure of TtIDH reveals the absence of helix at the clasp domain, which is mainly involved in oligomerization in other IDH’s. Also, helices/beta sheets are absent in the small domain, when compared to other IDH’s of the same sub family. The overall TtIDH structure exhibits closed conformation with catalytic triad residues, Tyr144-Asp248-Lys191 are conserved. Oligomerization of the protein is quantized using interface area and subunit–subunit interactions between protomers. Overall, the TtIDH structure with novel terminal domain may be categorized as a first structure of subfamily of type IV.

Rebaselining of the plutonium residue elimination project at Rocky Flats Environmental Technology Site

International Nuclear Information System (INIS)

Sailor, W.C.; Catlett, D.S.; Burns, T.P.

1997-01-01

Systems Engineering and Value Engineering principles were put into practice in rebaselining the Pu Residue Stabilization and Elimination Project at the Rocky Flats Environmental Technology Site. Tradeoff studies were conducted as to how to best rebaseline the system under the new Safeguards Termination Limits (STSs) issued by the Department of Energy. Through the use of a computerized database, the means by which Stakeholder values and other high-level requirements have been included in the tradeoff studies were documented. 13 refs., 2 figs., 1 tab

Rebaselining of the plutonium residue elimination project at Rocky Flats Environmental Technology Site

Energy Technology Data Exchange (ETDEWEB)

Sailor, W.C.; Catlett, D.S.; Burns, T.P. [and others

1997-03-01

Systems Engineering and Value Engineering principles were put into practice in rebaselining the Pu Residue Stabilization and Elimination Project at the Rocky Flats Environmental Technology Site. Tradeoff studies were conducted as to how to best rebaseline the system under the new Safeguards Termination Limits (STSs) issued by the Department of Energy. Through the use of a computerized database, the means by which Stakeholder values and other high-level requirements have been included in the tradeoff studies were documented. 13 refs., 2 figs., 1 tab.

Characterization of glutamate decarboxylase from Lactobacillus plantarum and its C-terminal function for the pH dependence of activity.

Science.gov (United States)

Shin, Sun-Mi; Kim, Hana; Joo, Yunhye; Lee, Sang-Jae; Lee, Yong-Jik; Lee, Sang Jun; Lee, Dong-Woo

2014-12-17

The gadB gene encoding glutamate decarboxylase (GAD) from Lactobacillus plantarum was cloned and expressed in Escherichia coli. The recombinant enzyme exhibited maximal activity at 40 °C and pH 5.0. The 3D model structure of L. plantarum GAD proposed that its C-terminal region (Ile454-Thr468) may play an important role in the pH dependence of catalysis. Accordingly, C-terminally truncated (Δ3 and Δ11 residues) mutants were generated and their enzyme activities compared with that of the wild-type enzyme at different pH values. Unlike the wild-type GAD, the mutants showed pronounced catalytic activity in a broad pH range of 4.0-8.0, suggesting that the C-terminal region is involved in the pH dependence of GAD activity. Therefore, this study may provide effective target regions for engineering pH dependence of GAD activity, thereby meeting industrial demands for the production of γ-aminobutyrate in a broad range of pH values.

Identifying SARS-CoV membrane protein amino acid residues linked to virus-like particle assembly.

Directory of Open Access Journals (Sweden)

Ying-Tzu Tseng

Full Text Available Severe acute respiratory syndrome coronavirus (SARS-CoV membrane (M proteins are capable of self-assembly and release in the form of membrane-enveloped vesicles, and of forming virus-like particles (VLPs when coexpressed with SARS-CoV nucleocapsid (N protein. According to previous deletion analyses, M self-assembly involves multiple M sequence regions. To identify important M amino acid residues for VLP assembly, we coexpressed N with multiple M mutants containing substitution mutations at the amino-terminal ectodomain, carboxyl-terminal endodomain, or transmembrane segments. Our results indicate that a dileucine motif in the endodomain tail (218LL219 is required for efficient N packaging into VLPs. Results from cross-linking VLP analyses suggest that the cysteine residues 63, 85 and 158 are not in close proximity to the M dimer interface. We noted a significant reduction in M secretion due to serine replacement for C158, but not for C63 or C85. Further analysis suggests that C158 is involved in M-N interaction. In addition to mutations of the highly conserved 107-SWWSFNPE-114 motif, substitutions at codons W19, W57, P58, W91, Y94 or F95 all resulted in significantly reduced VLP yields, largely due to defective M secretion. VLP production was not significantly affected by a tryptophan replacement of Y94 or F95 or a phenylalanine replacement of W19, W57 or W91. Combined, these results indicate the involvement of specific M amino acids during SARS-CoV virus assembly, and suggest that aromatic residue retention at specific positions is critical for M function in terms of directing virus assembly.

LNG TERMINAL SAFE OPERATION MANAGEMENT

Directory of Open Access Journals (Sweden)

Andrzej ADAMKIEWICZ

2012-07-01

Full Text Available This article presents the significance of LNG terminal safety issues in natural gas sea transport. It shows particular requirements for LNG transmission installations resulting from the specific properties of LNG. Out of the multi‐layer critical safety areas comprising structural elements of the terminal safety system, possibilities to decrease the risk of emergency occurrence on LNG terminals have been selected. Tasks performed by the LNG terminal, together with its own personnel and the outside one, have been defined. General theses for LNG terminal safety have been formulated.

Identification of residues in the heme domain of soluble guanylyl cyclase that are important for basal and stimulated catalytic activity.

Directory of Open Access Journals (Sweden)

Padmamalini Baskaran

Full Text Available Nitric oxide signals through activation of soluble guanylyl cyclase (sGC, a heme-containing heterodimer. NO binds to the heme domain located in the N-terminal part of the β subunit of sGC resulting in increased production of cGMP in the catalytic domain located at the C-terminal part of sGC. Little is known about the mechanism by which the NO signaling is propagated from the receptor domain (heme domain to the effector domain (catalytic domain, in particular events subsequent to the breakage of the bond between the heme iron and Histidine 105 (H105 of the β subunit. Our modeling of the heme-binding domain as well as previous homologous heme domain structures in different states point to two regions that could be critical for propagation of the NO activation signal. Structure-based mutational analysis of these regions revealed that residues T110 and R116 in the αF helix-β1 strand, and residues I41 and R40 in the αB-αC loop mediate propagation of activation between the heme domain and the catalytic domain. Biochemical analysis of these heme mutants allows refinement of the map of the residues that are critical for heme stability and propagation of the NO/YC-1 activation signal in sGC.

Highly efficient residue-selective labeling with isotope-labeled Ile, Leu, and Val using a new auxotrophic E. coli strain

International Nuclear Information System (INIS)

Miyanoiri, Yohei; Ishida, Yojiro; Takeda, Mitsuhiro; Terauchi, Tsutomu; Inouye, Masayori; Kainosho, Masatsune

2016-01-01

We recently developed a practical protocol for preparing proteins bearing stereo-selectively 13 C-methyl labeled leucines and valines, instead of the commonly used 13 C-methyl labeled precursors for these amino acids, by E. coli cellular expression. Using this protocol, proteins with any combinations of isotope-labeled or unlabeled Leu and Val residues were prepared, including some that could not be prepared by the precursor methods. However, there is still room for improvement in the labeling efficiencies for Val residues, using the methods with labeled precursors or Val itself. This is due to the fact that the biosynthesis of Val could not be sufficiently suppressed, even by the addition of large amounts of Val or its precursors. In this study, we completely solved this problem by using a mutant strain derived from E. coli BL21(DE3), in which the metabolic pathways depending on two enzymes, dihydroxy acid dehydratase and β-isopropylmalate dehydrogenase, are completely aborted by deleting the ilvD and leuB genes, which respectively encode these enzymes. The ΔilvD E. coli mutant terminates the conversion from α,β-dihydroxyisovalerate to α-ketoisovalerate, and the conversion from α,β-dihydroxy-α-methylvalerate to α-keto-β-methylvalerate, which produce the preceding precursors for Val and Ile, respectively. By the further deletion of the leuB gene, the conversion from Val to Leu was also fully terminated. Taking advantage of the double-deletion mutant, ΔilvDΔleuB E. coli BL21(DE3), an efficient and residue-selective labeling method with various isotope-labeled Ile, Leu, and Val residues was established.

Highly efficient residue-selective labeling with isotope-labeled Ile, Leu, and Val using a new auxotrophic E. coli strain

Energy Technology Data Exchange (ETDEWEB)

Miyanoiri, Yohei [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan); Ishida, Yojiro [Rutgers University-Robert Wood Johnson Medical School, Center for Advanced Biotechnology and Medicine (United States); Takeda, Mitsuhiro [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan); Terauchi, Tsutomu [Tokyo Metropolitan University, Graduate School of Science and Engineering (Japan); Inouye, Masayori [Rutgers University-Robert Wood Johnson Medical School, Center for Advanced Biotechnology and Medicine (United States); Kainosho, Masatsune, E-mail: kainosho@tmu.ac.jp [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan)

2016-06-15

We recently developed a practical protocol for preparing proteins bearing stereo-selectively {sup 13}C-methyl labeled leucines and valines, instead of the commonly used {sup 13}C-methyl labeled precursors for these amino acids, by E. coli cellular expression. Using this protocol, proteins with any combinations of isotope-labeled or unlabeled Leu and Val residues were prepared, including some that could not be prepared by the precursor methods. However, there is still room for improvement in the labeling efficiencies for Val residues, using the methods with labeled precursors or Val itself. This is due to the fact that the biosynthesis of Val could not be sufficiently suppressed, even by the addition of large amounts of Val or its precursors. In this study, we completely solved this problem by using a mutant strain derived from E. coli BL21(DE3), in which the metabolic pathways depending on two enzymes, dihydroxy acid dehydratase and β-isopropylmalate dehydrogenase, are completely aborted by deleting the ilvD and leuB genes, which respectively encode these enzymes. The ΔilvD E. coli mutant terminates the conversion from α,β-dihydroxyisovalerate to α-ketoisovalerate, and the conversion from α,β-dihydroxy-α-methylvalerate to α-keto-β-methylvalerate, which produce the preceding precursors for Val and Ile, respectively. By the further deletion of the leuB gene, the conversion from Val to Leu was also fully terminated. Taking advantage of the double-deletion mutant, ΔilvDΔleuB E. coli BL21(DE3), an efficient and residue-selective labeling method with various isotope-labeled Ile, Leu, and Val residues was established.

Processivity and Subcellular Localization of Glycogen Synthase Depend on a Non-catalytic High Affinity Glycogen-binding Site*

OpenAIRE

Díaz, Adelaida; Martínez-Pons, Carlos; Fita, Ignacio; Ferrer, Juan C.; Guinovart, Joan J.

2011-01-01

Glycogen synthase, a central enzyme in glucose metabolism, catalyzes the successive addition of α-1,4-linked glucose residues to the non-reducing end of a growing glycogen molecule. A non-catalytic glycogen-binding site, identified by x-ray crystallography on the surface of the glycogen synthase from the archaeon Pyrococcus abyssi, has been found to be functionally conserved in the eukaryotic enzymes. The disruption of this binding site in both the archaeal and the human muscle glycogen synth...

Nonleaking battery terminals.

Science.gov (United States)

Snider, W. E.; Nagle, W. J.

1972-01-01

Three different terminals were designed for usage in a 40 ampere/hour silver zinc battery which has a 45% KOH by weight electrolyte in a plastic battery case. Life tests, including thermal cycling, electrical charge and discharge for up to three years duration, were conducted on these three different terminal designs. Tests for creep rate and tensile strength were conducted on the polyphenylene oxide plastic battery cases. Some cases were unused and others containing KOH electrolyte were placed on life tests. The design and testing of nonleaking battery terminals for use with a KOH electrolyte in a plastic case are considered.

Residual stresses

International Nuclear Information System (INIS)

Sahotra, I.M.

2006-01-01

The principal effect of unloading a material strained into the plastic range is to create a permanent set (plastic deformation), which if restricted somehow, gives rise to a system of self-balancing within the same member or reaction balanced by other members of the structure., known as residual stresses. These stresses stay there as locked-in stresses, in the body or a part of it in the absence of any external loading. Residual stresses are induced during hot-rolling and welding differential cooling, cold-forming and extruding: cold straightening and spot heating, fabrication and forced fitting of components constraining the structure to a particular geometry. The areas which cool more quickly develop residual compressive stresses, while the slower cooling areas develop residual tensile stresses, and a self-balancing or reaction balanced system of residual stresses is formed. The phenomenon of residual stresses is the most challenging in its application in surface modification techniques determining endurance mechanism against fracture and fatigue failures. This paper discusses the mechanism of residual stresses, that how the residual stresses are fanned and what their behavior is under the action of external forces. Such as in the case of a circular bar under limit torque, rectangular beam under limt moment, reclaiming of shafts welds and peening etc. (author)

Microstructural observations on the terminal penetration of long rod projectile

Directory of Open Access Journals (Sweden)

Krushna Kumbhar

2017-12-01

Full Text Available Present study focuses on the terminal penetration of tungsten heavy alloy (WHA long rod penetrator impacted against armour steel at an impact velocity of 1600 m/s. The residual penetrator and armour steel target recovered after the ballistic test have been characterized using optical microscope, scanning electron microscope (SEM and electron probe micro analyzer (EPMA. Metallurgical changes in target steel and WHA remnant have been analysed. Large shear stresses and shear localization have resulted in local failure and formation of erosion products. Severe plastic deformation acts as precursor for formation of adiabatic shear band (ASB induced cracks in target steel. Recovered WHA penetrator remnant also exhibits severe plastic deformation forming localized shear bands, ASB induced cracks and shock induced cracks.

In vitro and in vivo mapping of the Prunus necrotic ringspot virus coat protein C-terminal dimerization domain by bimolecular fluorescence complementation.

Science.gov (United States)

Aparicio, Frederic; Sánchez-Navarro, Jesús A; Pallás, Vicente

2006-06-01

Interactions between viral proteins are critical for virus viability. Bimolecular fluorescent complementation (BiFC) technique determines protein interactions in real-time under almost normal physiological conditions. The coat protein (CP) of Prunus necrotic ringspot virus is required for multiple functions in its replication cycle. In this study, the region involved in CP dimerization has been mapped by BiFC in both bacteria and plant tissue. Full-length and C-terminal deleted forms of the CP gene were fused in-frame to the N- and C-terminal fragments of the yellow fluorescent protein. The BiFC analysis showed that a domain located between residues 9 and 27 from the C-end plays a critical role in dimerization. The importance of this C-terminal region in dimer formation and the applicability of the BiFC technique to analyse viral protein interactions are discussed.

Dynamics of Linker Residues Modulate the Nucleic Acid Binding Properties of the HIV-1 Nucleocapsid Protein Zinc Fingers

Science.gov (United States)

Zargarian, Loussiné; Tisné, Carine; Barraud, Pierre; Xu, Xiaoqian; Morellet, Nelly; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

2014-01-01

The HIV-1 nucleocapsid protein (NC) is a small basic protein containing two zinc fingers (ZF) separated by a short linker. It is involved in several steps of the replication cycle and acts as a nucleic acid chaperone protein in facilitating nucleic acid strand transfers occurring during reverse transcription. Recent analysis of three-dimensional structures of NC-nucleic acids complexes established a new property: the unpaired guanines targeted by NC are more often inserted in the C-terminal zinc finger (ZF2) than in the N-terminal zinc finger (ZF1). Although previous NMR dynamic studies were performed with NC, the dynamic behavior of the linker residues connecting the two ZF domains remains unclear. This prompted us to investigate the dynamic behavior of the linker residues. Here, we collected 15N NMR relaxation data and used for the first time data at several fields to probe the protein dynamics. The analysis at two fields allows us to detect a slow motion occurring between the two domains around a hinge located in the linker at the G35 position. However, the amplitude of motion appears limited in our conditions. In addition, we showed that the neighboring linker residues R29, A30, P31, R32, K33 displayed restricted motion and numerous contacts with residues of ZF1. Our results are fully consistent with a model in which the ZF1-linker contacts prevent the ZF1 domain to interact with unpaired guanines, whereas the ZF2 domain is more accessible and competent to interact with unpaired guanines. In contrast, ZF1 with its large hydrophobic plateau is able to destabilize the double-stranded regions adjacent to the guanines bound by ZF2. The linker residues and the internal dynamics of NC regulate therefore the different functions of the two zinc fingers that are required for an optimal chaperone activity. PMID:25029439

Conversion of functionally undefined homopentameric protein PbaA into a proteasome activator by mutational modification of its C-terminal segment conformation.

Science.gov (United States)

Yagi-Utsumi, Maho; Sikdar, Arunima; Kozai, Toshiya; Inoue, Rintaro; Sugiyama, Masaaki; Uchihashi, Takayuki; Yagi, Hirokazu; Satoh, Tadashi; Kato, Koichi

2018-01-01

Recent bioinformatic analyses identified proteasome assembly chaperone-like proteins, PbaA and PbaB, in archaea. PbaB forms a homotetramer and functions as a proteasome activator, whereas PbaA does not interact with the proteasome despite the presence of an apparent C-terminal proteasome activation motif. We revealed that PbaA forms a homopentamer predominantly in the closed conformation with its C-terminal segments packed against the core domains, in contrast to the PbaB homotetramer with projecting C-terminal segments. This prompted us to create a novel proteasome activator based on a well-characterized structural framework. We constructed a panel of chimeric proteins comprising the homopentameric scaffold of PbaA and C-terminal segment of PbaB and subjected them to proteasome-activating assays as well as small-angle X-ray scattering and high-speed atomic force microscopy. The results indicated that the open conformation and consequent proteasome activation activity could be enhanced by replacement of the crystallographically disordered C-terminal segment of PbaA with the corresponding disordered segment of PbaB. Moreover, these effects can be produced just by incorporating two glutamate residues into the disordered C-terminal segment of PbaA, probably due to electrostatic repulsion among the negatively charged segments. Thus, we successfully endowed a functionally undefined protein with proteasome-activating activity by modifying its C-terminal segment. © The Author(s) 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Residues 240-250 in the C-terminus of the Pirh2 protein complement the function of the RING domain in self-ubiquitination of the Pirh2 protein.

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Rami Abou Zeinab

Full Text Available Pirh2 is a p53 inducible gene that encodes a RING-H2 domain and is proposed to be a main regulator of p53 protein, thus fine tuning the DNA damage response. Pirh2 interacts physically with p53 and promotes its MDM2-independent ubiquitination and subsequent degradation as well as participates in an auto-regulatory feedback loop that controls p53 function. Pirh2 also self-ubiquitinates. Interestingly, Pirh2 is overexpressed in a wide range of human tumors. In this study, we investigated the domains and residues essential for Pirh2 self-ubiquitination. Deletions were made in each of the three major domains of Pirh2: the N-terminal domain (NTD, Ring domain (RING, and C-terminal domain (CTD. The effects of these deletions on Pirh2 self-ubiquitination were then assessed using in vitro ubiquitination assays. Our results demonstrate that the RING domain is essential, but not sufficient, for Pirh2 self-ubiquitination and that residues 240-250 of the C-terminal domain are also essential. Our results demonstrate that Pirh2 mediated p53 polyubiquitination occurs mainly through the K48 residue of ubiquitin in vitro. Our data further our understanding of the mechanism of Pirh2 self-ubiquitination and may help identify valuable therapeutic targets that play roles in reducing the effects of the overexpression of Pirh2, thus maximizing p53's response to DNA damage.

Intrinsic terminators in Mycoplasma hyopneumoniae transcription.

Science.gov (United States)

Fritsch, Tiago Ebert; Siqueira, Franciele Maboni; Schrank, Irene Silveira

2015-04-08

Mycoplasma hyopneumoniae, an important pathogen of swine, exhibits a low guanine and cytosine (GC) content genome. M. hyopneumoniae genome is organised in long transcriptional units and promoter sequences have been mapped upstream of all transcription units. These analysis provided insights into the gene organisation and transcription initiation at the genome scale. However, the presence of transcriptional terminator sequences in the M. hyopneumoniae genome is poorly understood. In silico analyses demonstrated the presence of putative terminators in 82% of the 33 monocistronic units (mCs) and in 74% of the 116 polycistronic units (pCs) considering different classes of terminators. The functional activity of 23 intrinsic terminators was confirmed by RT-PCR and qPCR. Analysis of all terminators found by three software algorithms, combined with experimental results, allowed us to propose a pattern of RNA hairpin formation during the termination process and to predict the location of terminators in the M. hyopneumoniae genome sequence. The stem-loop structures of intrinsic terminators of mycoplasma diverge from the pattern of terminators found in other bacteria due the low content of guanine and cytosine. In M. hyopneumoniae, transcription can end after a transcriptional unit and before its terminator sequence and can also continue past the terminator sequence with RNA polymerases gradually releasing the RNA.

C-terminal sequences of hsp70 and hsp90 as non-specific anchors for tetratricopeptide repeat (TPR) proteins.

Science.gov (United States)

Ramsey, Andrew J; Russell, Lance C; Chinkers, Michael

2009-10-12

Steroid-hormone-receptor maturation is a multi-step process that involves several TPR (tetratricopeptide repeat) proteins that bind to the maturation complex via the C-termini of hsp70 (heat-shock protein 70) and hsp90 (heat-shock protein 90). We produced a random T7 peptide library to investigate the roles played by the C-termini of the two heat-shock proteins in the TPR-hsp interactions. Surprisingly, phages with the MEEVD sequence, found at the C-terminus of hsp90, were not recovered from our biopanning experiments. However, two groups of phages were isolated that bound relatively tightly to HsPP5 (Homo sapiens protein phosphatase 5) TPR. Multiple copies of phages with a C-terminal sequence of LFG were isolated. These phages bound specifically to the TPR domain of HsPP5, although mutation studies produced no evidence that they bound to the domain's hsp90-binding groove. However, the most abundant family obtained in the initial screen had an aspartate residue at the C-terminus. Two members of this family with a C-terminal sequence of VD appeared to bind with approximately the same affinity as the hsp90 C-12 control. A second generation pseudo-random phage library produced a large number of phages with an LD C-terminus. These sequences acted as hsp70 analogues and had relatively low affinities for hsp90-specific TPR domains. Unfortunately, we failed to identify residues near hsp90's C-terminus that impart binding specificity to individual hsp90-TPR interactions. The results suggest that the C-terminal sequences of hsp70 and hsp90 act primarily as non-specific anchors for TPR proteins.

Asymmetry in the burial of hydrophobic residues along the histone chains of Eukarya, Archaea and a transcription factor

Directory of Open Access Journals (Sweden)

Silverman B David

2005-10-01

Full Text Available Abstract Background The histone fold is a common structural motif of proteins involved in the chromatin packaging of DNA and in transcription regulation. This single chain fold is stabilized by either homo- or hetero-dimer formation in archaea and eukarya. X-ray structures at atomic resolution have shown the eukaryotic nucleosome core particle to consist of a central tetramer of two bound H3-H4 dimers flanked by two H2A-H2B dimers. The c-terminal region of the H3 histone fold involved in coupling the two eukaryotic dimers of the tetramer, through a four-fold helical bundle, had previously been shown to be a region of reduced burial of hydrophobic residues within the dimers, and thereby provide a rationale for the observed reduced stability of the H3-H4 dimer compared with that of the H2A-H2B dimer. Furthermore, comparison between eukaryal and archaeal histones had suggested that this asymmetry in the distribution of hydrophobic residues along the H3 histone chains could be due to selective evolution that enhanced the coupling between the eukaryotic dimers of the tetramer. Results and discussion The present work describes calculations utilizing the X-ray structures at atomic resolution of a hyperthermophile from Methanopyrus kandleri (HMk and a eukaryotic transcription factor from Drosophila melanogaster (DRm, that are structurally homologous to the eukaryotic (H3-H42 tetramer. The results for several other related structures are also described. Reduced burial of hydrophobic residues, at the homologous H3 c-terminal regions of these structures, is found to parallel the burial at the c-terminal regions of the H3 histones and is, thereby, expected to affect dimer stability and the processes involving histone structural rearrangement. Significantly different sequence homology between the two histones of the HMk doublet with other archaeal sequences is observed, and how this might have occurred during selection to enhance tetramer stability is

Residual stresses

International Nuclear Information System (INIS)

Macherauch, E.

1978-01-01

Residual stresses are stresses which exist in a material without the influence of external powers and moments. They come into existence when the volume of a material constantly changes its form as a consequence of mechanical, thermal, and/or chemical processes and is hindered by neighbouring volumes. Bodies with residual stress are in mechanical balance. These residual stresses can be manifested by means of all mechanical interventions disturbing this balance. Acoustical, optical, radiological, and magnetical methods involving material changes caused by residual stress can also serve for determining residual stress. Residual stresses have an ambivalent character. In technical practice, they are feared and liked at the same time. They cause trouble because they can be the cause for unexpected behaviour of construction elements. They are feared since they can cause failure, in the worst case with catastrophical consequences. They are appreciated, on the other hand, because, in many cases, they can contribute to improvements of the material behaviour under certain circumstances. But they are especially liked for their giving convenient and (this is most important) mostly uncontrollable explanations. For only in very few cases we have enough knowledge and possibilities for the objective evaluation of residual stresses. (orig.) [de

New tyrosinase inhibitory decapeptide: Molecular insights into the role of tyrosine residues.

Science.gov (United States)

Ochiai, Akihito; Tanaka, Seiya; Imai, Yuta; Yoshida, Hisashi; Kanaoka, Takumi; Tanaka, Takaaki; Taniguchi, Masayuki

2016-06-01

Tyrosinase, a rate-limiting enzyme in melanin biosynthesis, catalyzes the hydroxylation of l-tyrosine to 3,4-dihydroxy-l-phenylalanine (l-dopa) (monophenolase reaction) and the subsequent oxidation of l-dopa to l-dopaquinone (diphenolase reaction). Thus, tyrosinase inhibitors have been proposed as skin-lightening agents; however, many of the existing inhibitors cannot be widely used in the cosmetic industry due to their high cytotoxicity and instability. On the other hand, some tyrosinase inhibitory peptides have been reported as safe. In this study, we found that the peptide TH10, which has a similar sequence to the characterized inhibitory peptide P4, strongly inhibits the monophenolase reaction with a half-maximal inhibitory concentration of 102 μM. Seven of the ten amino acid residues in TH10 were identical to P4; however, TH10 possesses one N-terminal tyrosine, whereas P4 contains three tyrosine residues located at its N-terminus, center, and C-terminus. Subsequent analysis using sequence-shuffled variants indicated that the tyrosine residues located at the N-terminus and center of P4 have little to no contribution to its inhibitory activity. Furthermore, docking simulation analysis of these peptides with mushroom tyrosinase demonstrated that the active tyrosine residue was positioned close to copper ions, suggesting that TH10 and P4 bind to tyrosinase as a substrate analogue. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

LNG TERMINAL SAFE OPERATION MANAGEMENT

OpenAIRE

Andrzej ADAMKIEWICZ; Włodzimierz KAMIŃSKI

2012-01-01

This article presents the significance of LNG terminal safety issues in natural gas sea transport. It shows particular requirements for LNG transmission installations resulting from the specific properties of LNG. Out of the multi‐layer critical safety areas comprising structural elements of the terminal safety system, possibilities to decrease the risk of emergency occurrence on LNG terminals have been selected. Tasks performed by the LNG terminal, together with its own personnel and the out...

Cloning, expression, purification, crystallization and preliminary X-ray studies of the C-terminal domain of Rv3262 (FbiB) from Mycobacterium tuberculosis

International Nuclear Information System (INIS)

Rehan, Aisyah M.; Bashiri, Ghader; Paterson, Neil G.; Baker, Edward N.; Squire, Christopher J.

2011-01-01

The C-terminal domain of FbiB, a bifunctional protein that is essential for the biosynthesis of cofactor F 420 in M. tuberculosis, has been expressed, purified and crystallized. The crystals diffracted to 2.0 Å resolution and were suitable for structure determination. During cofactor F 420 biosynthesis, the enzyme F 420 -γ-glutamyl ligase (FbiB) catalyzes the addition of γ-linked l-glutamate residues to form polyglutamylated F 420 derivatives. In Mycobacterium tuberculosis, Rv3262 (FbiB) consists of two domains: an N-terminal domain from the F 420 ligase superfamily and a C-terminal domain with sequence similarity to nitro-FMN reductase superfamily proteins. To characterize the role of the C-terminal domain of FbiB in polyglutamyl ligation, it has been purified and crystallized in an apo form. The crystals diffracted to 2.0 Å resolution using a synchrotron source and belonged to the tetragonal space group P4 1 2 1 2 (or P4 3 2 1 2), with unit-cell parameters a = b = 136.6, c = 101.7 Å, α = β = γ = 90°

Evaluation of residue-residue contact predictions in CASP9

KAUST Repository

Monastyrskyy, Bohdan

2011-01-01

This work presents the results of the assessment of the intramolecular residue-residue contact predictions submitted to CASP9. The methodology for the assessment does not differ from that used in previous CASPs, with two basic evaluation measures being the precision in recognizing contacts and the difference between the distribution of distances in the subset of predicted contact pairs versus all pairs of residues in the structure. The emphasis is placed on the prediction of long-range contacts (i.e., contacts between residues separated by at least 24 residues along sequence) in target proteins that cannot be easily modeled by homology. Although there is considerable activity in the field, the current analysis reports no discernable progress since CASP8.

Termination unit

Energy Technology Data Exchange (ETDEWEB)

Traeholt, Chresten; Willen, Dag; Roden, Mark; Tolbert, Jerry C.; Lindsay, David; Fisher, Paul W.; Nielsen, Carsten Thidemann

2016-05-03

Cable end section comprises end-parts of N electrical phases/neutral, and a thermally-insulation envelope comprising cooling fluid. The end-parts each comprises a conductor and are arranged with phase 1 innermost, N outermost surrounded by the neutral, electrical insulation being between phases and N and neutral. The end-parts comprise contacting surfaces located sequentially along the longitudinal extension of the end-section. A termination unit has an insulating envelope connected to a cryostat, special parts at both ends comprising an adapter piece at the cable interface and a closing end-piece terminating the envelope in the end-section. The special parts houses an inlet and/or outlet for cooling fluid. The space between an inner wall of the envelope and a central opening of the cable is filled with cooling fluid. The special part at the end connecting to the cryostat houses an inlet or outlet, splitting cooling flow into cable annular flow and termination annular flow.

Molecular interactions and residues involved in force generation in the T4 viral DNA packaging motor.

Science.gov (United States)

Migliori, Amy D; Smith, Douglas E; Arya, Gaurav

2014-12-12

Many viruses utilize molecular motors to package their genomes into preformed capsids. A striking feature of these motors is their ability to generate large forces to drive DNA translocation against entropic, electrostatic, and bending forces resisting DNA confinement. A model based on recently resolved structures of the bacteriophage T4 motor protein gp17 suggests that this motor generates large forces by undergoing a conformational change from an extended to a compact state. This transition is proposed to be driven by electrostatic interactions between complementarily charged residues across the interface between the N- and C-terminal domains of gp17. Here we use atomistic molecular dynamics simulations to investigate in detail the molecular interactions and residues involved in such a compaction transition of gp17. We find that although electrostatic interactions between charged residues contribute significantly to the overall free energy change of compaction, interactions mediated by the uncharged residues are equally if not more important. We identify five charged residues and six uncharged residues at the interface that play a dominant role in the compaction transition and also reveal salt bridging, van der Waals, and solvent hydrogen-bonding interactions mediated by these residues in stabilizing the compact form of gp17. The formation of a salt bridge between Glu309 and Arg494 is found to be particularly crucial, consistent with experiments showing complete abrogation in packaging upon Glu309Lys mutation. The computed contributions of several other residues are also found to correlate well with single-molecule measurements of impairments in DNA translocation activity caused by site-directed mutations. Copyright © 2014 Elsevier Ltd. All rights reserved.

Hydroxyl Radical-Mediated Novel Modification of Peptides: N-Terminal Cyclization through the Formation of α-Ketoamide.

Science.gov (United States)

Lee, Seon Hwa; Kyung, Hyunsook; Yokota, Ryo; Goto, Takaaki; Oe, Tomoyuki

2015-01-20

The hydroxyl radical-mediated oxidation of peptides and proteins constitutes a large group of post-translational modifications that can result in structural and functional changes. These oxidations can lead to hydroxylation, sulfoxidation, or carbonylation of certain amino acid residues and cleavage of peptide bonds. In addition, hydroxyl radicals can convert the N-terminus of peptides to an α-ketoamide via abstraction of the N-terminal α-hydrogen and hydrolysis of the ketimine intermediate. In the present study, we identified N-terminal cyclization as a novel modification mediated by a hydroxyl radical. The reaction of angiotensin (Ang) II (DRVYIHPF) and the hydroxyl radical generated by the Cu(II)/ascorbic acid (AA) system or UV/hydrogen peroxide system produced N-terminal cyclized-Ang II (Ang C) and pyruvamide-Ang II (Ang P, CH3COCONH-RVYIHPF). The structure of Ang C was confirmed by mass spectrometry and comparison to an authentic standard. The subsequent incubation of isolated Ang P in the presence of Cu(II)/AA revealed that Ang P was the direct precursor of Ang C. The proposed mechanism involves the formation of a nitrogen-centered (aminyl) radical, which cyclizes to form a five-membered ring containing the alkoxy radical. The subsequent β-scission reaction of the alkoxyl radical results in the cleavage of the terminal CH3CO group. The initial aminyl radical can be stabilized by chelation to the Cu(II) ions. The affinity of Ang C toward the Ang II type 1 receptor was significantly lower than that of Ang II or Ang P. Ang C was not further metabolized by aminopeptidase A, which converts Ang II to Ang III. Hydroxyl radical-mediated N-terminal cyclization was also observed in other Ang peptides containing N-terminal alanine, arginine, valine, and amyloid β 1-11 (DAEFRHDSGYE).

C-terminal region of herpes simplex virus ICP8 protein needed for intranuclear localization

International Nuclear Information System (INIS)

Taylor, Travis J; Knipe, David M.

2003-01-01

The herpes simplex virus single-stranded DNA-binding protein, ICP8, localizes initially to structures in the nucleus called prereplicative sites. As replication proceeds, these sites mature into large globular structures called replication compartments. The details of what signals or proteins are involved in the redistribution of viral and cellular proteins within the nucleus between prereplicative sites and replication compartments are poorly understood; however, we showed previously that the dominant-negative d105 ICP8 does not localize to prereplicative sites and prevents the localization of other viral proteins to prereplicative sites (J. Virol. 74 (2000) 10122). Within the residues deleted in d105 (1083 to 1168), we identified a region between amino acid residues 1080 and 1135 that was predicted by computer models to contain two α-helices, one with considerable amphipathic nature. We used site-specific and random mutagenesis techniques to identify residues or structures within this region that are required for proper ICP8 localization within the nucleus. Proline substitutions in the predicted helix generated ICP8 molecules that did not localize to prereplicative sites and acted as dominant-negative inhibitors. Other substitutions that altered the charged residues in the predicted α-helix to alanine or leucine residues had little or no effect on ICP8 intranuclear localization. The predicted α-helix was dispensable for the interaction of ICP8 with the U L 9 origin-binding protein. We propose that this C-terminal α-helix is required for localization of ICP8 to prereplicative sites by binding viral or cellular factors that target or retain ICP8 at specific intranuclear sites

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kbkw Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kmyl Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

krbg Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kril Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

ksus Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

padq Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kbil Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

krfd Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kdug Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

ktix Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kcod Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kslk Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kgfl Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kguc Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kmlu Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kbff Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

ksmn Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kdro Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kmce Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

ktpa Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kmot Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kcre Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

klws Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kotm Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

khqm Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kabr Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

klal Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kelp Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kecg Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

khbg Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kpbf Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

konp Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

pkwa Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

ktvf Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

paga Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

khks Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kdsm Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kpsm Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kgrb Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kgmu Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

papg Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kbgm Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

pamc Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

klrd Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

ksan Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

patk Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kowb Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

klru Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kfxe Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kjct Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kcrg Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

paaq Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kaex Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

klbx Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kmia Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kpit Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kcrw Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

paen Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kast Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kuin Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kmht Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

krsw Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kbpi Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kcys Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kflo Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kphx Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

pakn Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

pabt Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

krdg Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

khdn Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kjac Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

The N-terminal domain of APJ, a CNS-based coreceptor for HIV-1, is essential for its receptor function and coreceptor activity

International Nuclear Information System (INIS)

Zhou Naiming; Zhang Xiaoling; Fan Xuejun; Argyris, Elias; Fang Jianhua; Acheampong, Edward; DuBois, Garrett C.; Pomerantz, Roger J.

2003-01-01

The human APJ, a G protein-coupled seven-transmembrane receptor, has been found to be dramatically expressed in the human central nervous system (CNS) and also to serve as a coreceptor for the entry of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV). Studies with animal models suggested that APJ and its natural ligand, apelin, play an important role in the central control of body fluid homeostasis, and in regulation of blood pressure and cardiac contractility. In this study, we characterize the structural and functional determinants of the N-terminal domain of APJ in interactions with its natural ligand and HIV-1 envelope glycoprotein. We demonstrate that the second 10 residues of the N-terminal domain of APJ are critical for association with apelin, while the first 20 amino acids play an important role in supporting cell-cell fusion mediated by HIV-1 gp120. With site-directed mutagenesis, we have identified that the negatively charged amino acid residues Glu20 and Asp23 are involved in receptor and coreceptor functions, but residues Tyr10 and Tyr11 substantially contribute to coreceptor function for both T-tropic (CXCR4) and dual-tropic (CXCR4 and CCR5) HIV-1 isolates. Thus, this study provides potentially important information for further characterizing APJ-apelin functions in vitro and in vivo and designing small molecules for treatment of HIV-1 infection in the CNS

Conformational effects of a common codon 751 polymorphism on the C-terminal domain of the xeroderma pigmentosum D protein

Directory of Open Access Journals (Sweden)

Monaco Regina

2009-01-01

Full Text Available Aim: The xeroderma pigmentosum D (XPD protein is a DNA helicase involved in the repair of DNA damage, including nucleotide excision repair (NER and transcription-coupled repair (TCR. The C-terminal domain of XPD has been implicated in interactions with other components of the TFIIH complex, and it is also the site of a common genetic polymorphism in XPD at amino acid residue 751 (Lys->Gln. Some evidence suggests that this polymorphism may alter DNA repair capacity and increase cancer risk. The aim of this study was to investigate whether these effects could be attributable to conformational changes in XPD induced by the polymorphism. Materials and Methods: Molecular dynamics techniques were used to predict the structure of the wild-type and polymorphic forms of the C-terminal domain of XPD and differences in structure produced by the polymorphic substitution were determined. Results: The results indicate that, although the general configuration of both proteins is similar, the substitution produces a significant conformational change immediately N-terminal to the site of the polymorphism. Conclusion: These results provide support for the hypothesis that this polymorphism in XPD could affect DNA repair capability, and hence cancer risk, by altering the structure of the C-terminal domain.

Ports and Terminals : Planning and Functional Design

NARCIS (Netherlands)

Groenveld, R.; Velsink, H.

1993-01-01

1. Maritime transport, means and commodities 3. Principles of integrated port planning 4. Planning and design of a port's water areas 5. Port terminals - introduction 6. Conventional general cargo terminals 7. Container terminals 8. Oil & liquid gas terminals 9. Dry bulk cargo terminals 10. Fishery

The critical main-chain length for helix formation in water: determined in a peptide series with alternating Aib and Ala residues exclusively and detected with ECD spectroscopy.

Science.gov (United States)

Longo, Edoardo; Moretto, Alessandro; Formaggio, Fernando; Toniolo, Claudio

2011-10-01

Critical main-chain length for peptide helix formation in the crystal (solid) state and in organic solvents has been already reported. In this short communication, we describe our results aiming at assessing the aforementioned parameter in water solution. To this goal, we synthesized step-by-step by solution procedures a complete series of N-terminally acetylated, C-terminally methoxylated oligopeptides, characterized only by alternating Aib and Ala residues, from the dimer to the nonamer level. All these compounds were investigated by electronic circular dichroism in the far-UV region in water solution as a function of chemical structure, namely presence/absence of an ester moiety or a negative charge at the C-terminus, and temperature. We find that the critical main-chain lengths for 3(10)- and α-helices, although still formed to a limited extent, in aqueous solution are six and eight residues, respectively. © 2011 Wiley-Liss, Inc.

A cluster of aspartic residues in the extracellular loop II of PAR 4 is important for thrombin interaction and activation of platelets.

Science.gov (United States)

Sánchez Centellas, Daniel; Gudlur, Sushanth; Vicente-Carrillo, Alejandro; Ramström, Sofia; Lindahl, Tomas L

2017-06-01

Thrombin activates platelets via proteolytic cleavage of protease-activated receptors (PARs) 1 and 4. The two PARs have distinct but complementary roles. The mechanisms responsible for PAR1 activation by thrombin have been extensively studied. However, much less is known regarding thrombin activation of PAR4, especially the potential involvement of regions of PAR4 other than the N-terminal, which is bound to the catalytic site of thrombin. We have studied PAR4 in S. cerevisiae strain MMY12, an expression system in which the GPCR receptors are connected to a Lac Z reporter gene resulting in increased β-galactosidase activity. This approach was used to assess PAR4 mutants to evaluate the contribution of different aspartic residues in facilitating PAR4 activation. Furthermore, peptides mimicking parts of the PAR4 N-terminal and the second extracellular loop (ECLII) were tested for their ability to inhibit platelet activation by thrombin. Binding of these peptides to γ-thrombin was studied by monitoring the decrease in tryptophan fluorescence intensity of thrombin. We conclude that not only the N-terminal but also the electronegative aspartic residues D224, D230 and D235 (located in ECLII) are be important for PAR4 binding to thrombin. We further suggest that they play a role for the tethered ligand binding to the receptor, as mutations also affected activation in response to a PAR4-activating peptide mimicking the new N-terminal formed after cleavage. This agrees with previous results on PAR1 and thrombin binding. We suggest that the ECLII of PAR4 could be a potential target for antithrombotic drug development. Copyright © 2017 Elsevier Ltd. All rights reserved.

Determination of bromothalonil residues and degradation in apple and soil by QuEChERS and GC-MS/MS.

Science.gov (United States)

Liu, Huijun; Guo, Baoyuan; Wang, Huili; Li, Jianzhong; Zheng, Lin

2014-04-01

The dissipation and residues of bromothalonil in apple and soil under field condition were analyzed by QuEChERS (quick, easy, cheap, effective, rugged and safe) combined with gas chromatography-mass spectrometer method. The recoveries were ranged from 80.8 % to 106 % with coefficient variation for repeatability ranged from 3.08 % to 7.09 % at fortification levels of 0.02, 0.04 and 0.2 mg/kg in apple and soil. The limit of quantification of the method was 0.31 μg/kg. The dissipation rates of bromothalonil followed the first-order kinetics and the half-lives were from 3.61 to 3.98 days in apple and from 4.65 to 9.29 days in soil. In apple, the terminal residues of bromothalonil were below the China maximum residue limit (0.2 mg/kg) after 7 days of application. This work contributed to provide the basic information for a safe usage of bromothalonil in apple orchard and preventing health problem from consumers in China.

The N-Terminal GYPSY Motif Is Required for Pilin-Specific Sortase SrtC1 Functionality in Lactobacillus rhamnosus Strain GG.

Directory of Open Access Journals (Sweden)

François P Douillard

Full Text Available Predominantly identified in pathogenic Gram-positive bacteria, sortase-dependent pili are also found in commensal species, such as the probiotic-marketed strain Lactobacillus rhamnosus strain GG. Pili are typically associated with host colonization, immune signalling and biofilm formation. Comparative analysis of the N-terminal domains of pilin-specific sortases from various piliated Gram-positive bacteria identified a conserved motif, called GYPSY, within the signal sequence. We investigated the function and role of the GYPSY residues by directed mutagenesis in homologous (rod-shaped and heterologous (coccoid-shaped expression systems for pilus formation. Substitutions of some of the GYPSY residues, and more specifically the proline residue, were found to have a direct impact on the degree of piliation of Lb. rhamnosus GG. The present findings uncover a new signalling element involved in the functionality of pilin-specific sortases controlling the pilus biogenesis of Lb. rhamnosus GG and related piliated Gram-positive species.

The N-Terminal GYPSY Motif Is Required for Pilin-Specific Sortase SrtC1 Functionality in Lactobacillus rhamnosus Strain GG

Science.gov (United States)

Douillard, François P.; Rasinkangas, Pia; Bhattacharjee, Arnab; Palva, Airi; de Vos, Willem M.

2016-01-01

Predominantly identified in pathogenic Gram-positive bacteria, sortase-dependent pili are also found in commensal species, such as the probiotic-marketed strain Lactobacillus rhamnosus strain GG. Pili are typically associated with host colonization, immune signalling and biofilm formation. Comparative analysis of the N-terminal domains of pilin-specific sortases from various piliated Gram-positive bacteria identified a conserved motif, called GYPSY, within the signal sequence. We investigated the function and role of the GYPSY residues by directed mutagenesis in homologous (rod-shaped) and heterologous (coccoid-shaped) expression systems for pilus formation. Substitutions of some of the GYPSY residues, and more specifically the proline residue, were found to have a direct impact on the degree of piliation of Lb. rhamnosus GG. The present findings uncover a new signalling element involved in the functionality of pilin-specific sortases controlling the pilus biogenesis of Lb. rhamnosus GG and related piliated Gram-positive species. PMID:27070897

Tandem Terminal Ion Source

International Nuclear Information System (INIS)

Harper, G.C.; Lindner, C.E.; Myers, A.W.; Wechel, T.D. van

2000-01-01

OAK-B135 Tandem Terminal Ion Source. The terminal ion source (TIS) was used in several experiments during this reporting period, all for the 7 Be(γ) 8 B experiment. Most of the runs used 1 H + at terminal voltages from 0.3 MV to 1.5 MV. One of the runs used 2 H + at terminal voltage of 1.4 MV. The other run used 4 He + at a terminal voltage of 1.37 MV. The list of experiments run with the TIS to date is given in table 1 below. The tank was opened four times for unscheduled source repairs. On one occasion the tank was opened to replace the einzel lens power supply which had failed. The 10 kV unit was replaced with a 15 kV unit. The second time the tank was opened to repair the extractor supply which was damaged by a tank spark. On the next occasion the tank was opened to replace a source canal which had sputtered away. Finally, the tank was opened to replace the discharge bottle which had been coated with aluminum sputtered from the exit canal

Tandem Terminal Ion Source

International Nuclear Information System (INIS)

None

2000-01-01

OAK-B135 Tandem Terminal Ion Source. The terminal ion source (TIS) was used in several experiments during this reporting period, all for the(sup 7)Be((gamma))(sup 8)B experiment. Most of the runs used(sup 1)H(sup+) at terminal voltages from 0.3 MV to 1.5 MV. One of the runs used(sup 2)H(sup+) at terminal voltage of 1.4 MV. The other run used(sup 4)He(sup+) at a terminal voltage of 1.37 MV. The list of experiments run with the TIS to date is given in table 1 below. The tank was opened four times for unscheduled source repairs. On one occasion the tank was opened to replace the einzel lens power supply which had failed. The 10 kV unit was replaced with a 15 kV unit. The second time the tank was opened to repair the extractor supply which was damaged by a tank spark. On the next occasion the tank was opened to replace a source canal which had sputtered away. Finally, the tank was opened to replace the discharge bottle which had been coated with aluminum sputtered from the exit canal

Stereochemical determinants of C-terminal specificity in PDZ peptide-binding domains: a novel contribution of the carboxylate-binding loop.

Science.gov (United States)

Amacher, Jeanine F; Cushing, Patrick R; Bahl, Christopher D; Beck, Tobias; Madden, Dean R

2013-02-15

PDZ (PSD-95/Dlg/ZO-1) binding domains often serve as cellular traffic engineers, controlling the localization and activity of a wide variety of binding partners. As a result, they play important roles in both physiological and pathological processes. However, PDZ binding specificities overlap, allowing multiple PDZ proteins to mediate distinct effects on shared binding partners. For example, several PDZ domains bind the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), an epithelial ion channel mutated in CF. Among these binding partners, the CFTR-associated ligand (CAL) facilitates post-maturational degradation of the channel and is thus a potential therapeutic target. Using iterative optimization, we previously developed a selective CAL inhibitor peptide (iCAL36). Here, we investigate the stereochemical basis of iCAL36 specificity. The crystal structure of iCAL36 in complex with the CAL PDZ domain reveals stereochemical interactions distributed along the peptide-binding cleft, despite the apparent degeneracy of the CAL binding motif. A critical selectivity determinant that distinguishes CAL from other CFTR-binding PDZ domains is the accommodation of an isoleucine residue at the C-terminal position (P(0)), a characteristic shared with the Tax-interacting protein-1. Comparison of the structures of these two PDZ domains in complex with ligands containing P(0) Leu or Ile residues reveals two distinct modes of accommodation for β-branched C-terminal side chains. Access to each mode is controlled by distinct residues in the carboxylate-binding loop. These studies provide new insights into the primary sequence determinants of binding motifs, which in turn control the scope and evolution of PDZ interactomes.

Kajian Kinerja Terminal Batu Ampar Kota Balikpapan

Directory of Open Access Journals (Sweden)

Randha Alief Chikita

2018-01-01

Full Text Available Batu Ampar Terminal Balikpapan is the only type A passenger terminal in Balikpapan City. The purpose of this study is to determine the operational performance of the terminal at this time and also to determine the level of service in the terminal. The results of this study indicate that for FIFO queue discipline analysis on AKDP and AKAP bus lines, it is known that ρ <1 means that there is currently no queue in the terminal. For the analysis of terminal facilities it is known that there are still some terminal facilities that are not yet available from the main facilities and supporting facilities, therefore the need for additional facilities in order to meet the standard of passenger terminal type A. In the next 15 years analysis for traffic intensity value is approaching 1 which means in the future will cause the queue in the terminal, so it is necessary for the improvement of terminal performance. For the service performance with IPA method there are 35 variables that there are 7 variables that enter in quadrant I. In the next step to do analysis to know the priority of handling by using QFD method. Keywords: Batu Ampar Terminal Balikpapan, IPA, terminal performance, QFD

25 CFR 11.1114 - Termination.

Science.gov (United States)

2010-04-01

... Minor-in-Need-of-Care Procedure § 11.1114 Termination. (a) Parental rights to a child may be terminated... rights if, following efforts to prevent or eliminate the need to remove the minor, it finds such efforts... alternatives: (1) If parental rights to a child are terminated, the children's court shall place the minor in a...

St. James marine terminal facility description

Energy Technology Data Exchange (ETDEWEB)

1993-10-01

The US Department of Energy (DOE) currently owns and operates a marine terminal on the west bank of the Mississippi River at St. James, Louisiana. The St. James facility was constructed by the Department to provide marine services associated with the fill and drawdown of the Strategic Petroleum Reserve (SPR) crude oil storage facilities located at Bayou Choctaw and Weeks Island, Louisiana. Although strategic to the mission of the SPR in the event of a national emergency, the St. James terminal is situated such that it has a high potential to also serve the commercial industry`s needs for crude oil terminalling and storage. The St. James terminal is located approximately 45 miles west of New Orleans and 30 miles southeast of Baton Rouge, and approximately 160 miles upstream from the mouth of the Mississippi River. Construction of the St. James terminal was initiated in 1978 and was completed in 1980. Since then, the terminal has received and transferred over 125 million barrels of crude oil to the SPR sites for storage. For crude oil distribution, the St. James terminal was connected to the neighboring LOCAP terminal by a 0.1 mile 36-inch pipeline in 1981 and to the Capline terminal by a 0.5 mile 30-inch pipeline in 1988. The terminal also has a 30-inch pipeline connection to the Koch oil terminal which was used for initial fill purposes; however, this pipeline has been disconnected and is currently inactive. A complete description of the St. James terminal facilities, operational capabilities, operational certifications, and future Government requirements are presented in Sections 2, 3, 4, and 5 respectively.

Highly conserved small subunit residues influence rubisco large subunit catalysis.

Science.gov (United States)

Genkov, Todor; Spreitzer, Robert J

2009-10-30

The chloroplast enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO(2) fixation. With a deeper understanding of its structure-function relationships and competitive inhibition by O(2), it may be possible to engineer an increase in agricultural productivity and renewable energy. The chloroplast-encoded large subunits form the active site, but the nuclear-encoded small subunits can also influence catalytic efficiency and CO(2)/O(2) specificity. To further define the role of the small subunit in Rubisco function, the 10 most conserved residues in all small subunits were substituted with alanine by transformation of a Chlamydomonas reinhardtii mutant that lacks the small subunit gene family. All the mutant strains were able to grow photosynthetically, indicating that none of the residues is essential for function. Three of the substitutions have little or no effect (S16A, P19A, and E92A), one primarily affects holoenzyme stability (L18A), and the remainder affect catalysis with or without some level of associated structural instability (Y32A, E43A, W73A, L78A, P79A, and F81A). Y32A and E43A cause decreases in CO(2)/O(2) specificity. Based on the x-ray crystal structure of Chlamydomonas Rubisco, all but one (Glu-92) of the conserved residues are in contact with large subunits and cluster near the amino- or carboxyl-terminal ends of large subunit alpha-helix 8, which is a structural element of the alpha/beta-barrel active site. Small subunit residues Glu-43 and Trp-73 identify a possible structural connection between active site alpha-helix 8 and the highly variable small subunit loop between beta-strands A and B, which can also influence Rubisco CO(2)/O(2) specificity.

Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease Npro

International Nuclear Information System (INIS)

Gottipati, Keerthi; Acholi, Sudheer; Ruggli, Nicolas; Choi, Kyung H.

2014-01-01

Pestivirus N pro is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N pro blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N pro' s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N pro -GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N pro proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N pro' s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N pro does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N pro' s autoproteolysis is studied using N pro -GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N pro prefers small amino acids with non-branched beta carbons at the P1 position

Site-directed fluorescence labeling reveals a revised N-terminal membrane topology and functional periplasmic residues in the Escherichia coli cell division protein FtsK.

Science.gov (United States)

Berezuk, Alison M; Goodyear, Mara; Khursigara, Cezar M

2014-08-22

In Escherichia coli, FtsK is a large integral membrane protein that coordinates chromosome segregation and cell division. The N-terminal domain of FtsK (FtsKN) is essential for division, and the C terminus (FtsKC) is a well characterized DNA translocase. Although the function of FtsKN is unknown, it is suggested that FtsK acts as a checkpoint to ensure DNA is properly segregated before septation. This may occur through modulation of protein interactions between FtsKN and other division proteins in both the periplasm and cytoplasm; thus, a clear understanding of how FtsKN is positioned in the membrane is required to characterize these interactions. The membrane topology of FtsKN was initially determined using site-directed reporter fusions; however, questions regarding this topology persist. Here, we report a revised membrane topology generated by site-directed fluorescence labeling. The revised topology confirms the presence of four transmembrane segments and reveals a newly identified periplasmic loop between the third and fourth transmembrane domains. Within this loop, four residues were identified that, when mutated, resulted in the appearance of cellular voids. High resolution transmission electron microscopy of these voids showed asymmetric division of the cytoplasm in the absence of outer membrane invagination or visible cell wall ingrowth. This uncoupling reveals a novel role for FtsK in linking cell envelope septation events and yields further evidence for FtsK as a critical checkpoint of cell division. The revised topology of FtsKN also provides an important platform for future studies on essential interactions required for this process. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The N-terminal tropomyosin- and actin-binding sites are important for leiomodin 2's function.

Science.gov (United States)

Ly, Thu; Moroz, Natalia; Pappas, Christopher T; Novak, Stefanie M; Tolkatchev, Dmitri; Wooldridge, Dayton; Mayfield, Rachel M; Helms, Gregory; Gregorio, Carol C; Kostyukova, Alla S

2016-08-15

Leiomodin is a potent actin nucleator related to tropomodulin, a capping protein localized at the pointed end of the thin filaments. Mutations in leiomodin-3 are associated with lethal nemaline myopathy in humans, and leiomodin-2-knockout mice present with dilated cardiomyopathy. The arrangement of the N-terminal actin- and tropomyosin-binding sites in leiomodin is contradictory and functionally not well understood. Using one-dimensional nuclear magnetic resonance and the pointed-end actin polymerization assay, we find that leiomodin-2, a major cardiac isoform, has an N-terminal actin-binding site located within residues 43-90. Moreover, for the first time, we obtain evidence that there are additional interactions with actin within residues 124-201. Here we establish that leiomodin interacts with only one tropomyosin molecule, and this is the only site of interaction between leiomodin and tropomyosin. Introduction of mutations in both actin- and tropomyosin-binding sites of leiomodin affected its localization at the pointed ends of the thin filaments in cardiomyocytes. On the basis of our new findings, we propose a model in which leiomodin regulates actin poly-merization dynamics in myocytes by acting as a leaky cap at thin filament pointed ends. © 2016 Ly, Moroz, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Structural investigation of a C-terminal EphA2 receptor mutant: Does mutation affect the structure and interaction properties of the Sam domain?

Science.gov (United States)

Mercurio, Flavia A; Costantini, Susan; Di Natale, Concetta; Pirone, Luciano; Guariniello, Stefano; Scognamiglio, Pasqualina L; Marasco, Daniela; Pedone, Emilia M; Leone, Marilisa

2017-09-01

Ephrin A2 receptor (EphA2) plays a key role in cancer, it is up-regulated in several types of tumors and the process of ligand-induced receptor endocytosis, followed by degradation, is considered as a potential path to diminish tumor malignancy. Protein modulators of this mechanism are recruited at the cytosolic Sterile alpha motif (Sam) domain of EphA2 (EphA2-Sam) through heterotypic Sam-Sam associations. These interactions engage the C-terminal helix of EphA2 and close loop regions (the so called End Helix side). In addition, several studies report on destabilizing mutations in EphA2 related to cataract formation and located in/or close to the Sam domain. Herein, we analyzed from a structural point of view, one of these mutants characterized by the insertion of a novel 39 residue long polypeptide at the C-terminus of EphA2-Sam. A 3D structural model was built by computational methods and revealed partial disorder in the acquired C-terminal tail and a few residues participating in an α-helix and two short β-strands. We investigated by CD and NMR studies the conformational properties in solution of two peptides encompassing the whole C-terminal tail and its predicted helical region, respectively. NMR binding experiments demonstrated that these peptides do not interact relevantly with either EphA2-Sam or its interactor Ship2-Sam. Molecular dynamics (MD) simulations further indicated that the EphA2 mutant could be represented only through a conformational ensemble and that the C-terminal tail should not largely wrap the EphA2-Sam End-Helix interface and affect binding to other Sam domains. Copyright © 2017 Elsevier B.V. All rights reserved.

Aging and the shape of cognitive change before death: terminal decline or terminal drop?

Science.gov (United States)

MacDonald, Stuart W S; Hultsch, David F; Dixon, Roger A

2011-05-01

Relative to typical age-related cognitive decrements, the terms "terminal decline" and "terminal drop" refer to the phenomenon of increased cognitive decline in proximity to death. Given that these terms are not necessarily synonymous, we examined the important theoretical distinction between the two alternative trajectories or shapes of changes they imply. We used 12-year (5-wave) data from the Victoria Longitudinal Study to directly test whether pre-death cognitive decrements follow a terminal decline (generally gradual) or a terminal drop (more abrupt) shape. Pre-death trajectories of cognitive decline for n=265 decedents (Mage = 72.67 years, SD = 6.44) were examined separately for 5 key cognitive constructs (verbal speed, working memory, episodic memory, semantic memory, and crystallized ability). Several classes of linear mixed models evaluated whether cognitive decline increased per additional year closer to death. Findings indicated that the shape of pre-death cognitive change was predominantly characterized by decline that is steeper as compared with typical aging-related change, but still best described as slow and steady decline, especially as compared with precipitous drop. The present findings suggest that terminal decline and terminal drop trajectories may not be mutually exclusive but could rather reflect distinct developmental trajectories within the same individual.

42 CFR 489.53 - Termination by CMS.

Science.gov (United States)

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false Termination by CMS. 489.53 Section 489.53 Public... Reinstatement After Termination § 489.53 Termination by CMS. (a) Basis for termination of agreement with any provider. CMS may terminate the agreement with any provider if CMS finds that any of the following failings...

Lazy Productivity via Termination

NARCIS (Netherlands)

Endrullis, J.; Hendriks, R.D.A.

2011-01-01

We present a procedure for transforming strongly sequential constructor-based term rewriting systems (TRSs) into context-sensitive TRSs in such a way that productivity of the input system is equivalent to termination of the output system. Thereby automated termination provers become available for

Conserved residues in the coiled-coil pocket of human immunodeficiency virus type 1 gp41 are essential for viral replication and interhelical interaction

International Nuclear Information System (INIS)

Mo Hongmei; Konstantinidis, Alex K.; Stewart, Kent D.; Dekhtyar, Tatyana; Ng, Teresa; Swift, Kerry; Matayoshi, Edmund D.; Kati, Warren; Kohlbrenner, William; Molla, Akhteruzzaman

2004-01-01

The human immunodeficiency virus type 1 (HIV-1) gp41 plays an important role in mediating the fusion of HIV with host cells. During the fusion process, three N-terminal helices and three C-terminal helices pack in an anti-parallel direction to form a six-helix bundle. X-ray crystallographic analysis of the gp41 core demonstrated that within each coiled-coil interface, there is a deep and large pocket, formed by a cluster of residues in the N-helix coiled-coil. In this report, we systematically analyzed the role of seven conserved residues that are either lining or packing this pocket on the infectivity and interhelical interaction using novel approaches. Our results show that residues L568, V570, W571, and K574 of the N-helix that are lining the side chain and right wall of the pocket are important for establishing a productive infection. Mutations V570A and W571A completely abolished replication, while replication of the L568A and K574A mutants was significantly attenuated relative to wild type. Similarly, residues W628, W631, and I635 of the C-helix that insert into the pocket are essential for infectivity. The impaired infectivity of these seven mutants is in part attributed to the loss in binding affinity of the interhelical interaction. Molecular modeling of the crystal structure of the coiled-coil further shows that alanine substitution of those residues disrupts the hydrophobic interaction between the N- and C-helix. These results suggest that the conserved residues in the coiled-coil domain play a key role in HIV infection and this coiled-coil pocket is a good target for development of inhibitors against HIV. In addition, our data indicate that the novel fluorescence polarization assay described in this study could be valuable in screening for inhibitors that block the interhelical interaction and HIV entry

Nontraumatic terminal ileal perforation

Directory of Open Access Journals (Sweden)

Wani Rauf A

2006-03-01

Full Text Available Abstract Background There is still confusion and controversy over the diagnosis and optimal surgical treatment of non traumatic terminal ileal perforation-a cause of obscure peritonitis. Methods This study was a prospective study aimed at evaluating the clinical profile, etiology and optimal surgical management of patients with nontraumatic terminal ileal perforation. Results There were 79 cases of nontraumatic terminal ileal perforation; the causes for perforation were enteric fever(62%, nonspecific inflammation(26%, obstruction(6%, tuberculosis(4% and radiation enteritis (1%. Simple closure of the perforation (49% and end to side ileotransverse anastomosis(42% were the mainstay of the surgical management. Conclusion Terminal ileal perforation should be suspected in all cases of peritonitis especially in developing countries and surgical treatment should be optimized taking various accounts like etiology, delay in surgery and operative findings into consideration to reduce the incidence of deadly complications like fecal fistula.

COMPARATIVE STUDY OF THE EFFECTS OF DETONATION NANODIAMONDS WITH VARIED PROPERTIES ON FUNCTIONAL STATE OF BRAIN NERVE TERMINALS

Directory of Open Access Journals (Sweden)

M. A. Galkin

2016-12-01

Full Text Available The aim of the study was to compare the effects of detonation nanodiamond preparations from different batches cleaned from impurities by diverse methods of chemical treatment on the membrane potential and glutamate transport characteristics of rat brain nerve terminals. The size of nanodiamond particles vary from 10–20 nm to 10 μm. There are carbonyl, hydroxyl and carboxyl functional groups on the surface of the particles. Physical-chemical properties such as a magnetic susceptibility and the amount of incombustible residue in samples of detonation nanodia-mond vary depending on the synthesis regime and the method of chemical cleaning of the product and therefore, the neuroactive properties of nanodiamonds from different batches can be different. It was shown by dynamic light scattering analysis that nanodiamond preparations from different batches treated by diverse technologies of chemical treatment had varied average size of particles and distribution of particles by size. Nanodiamond preparations from different batches changed the plasma membrane potential and caused membrane depolarization of nerve terminals. Analysis of the effects of nanodiamonds on transporter-mediated L-[14C]glutamate uptake by nerve terminals also revealed that all studied nanodiamond preparations decreased abovementioned parameter. Therefore, detonation nanodiamonds from different batches have similar principal effects on functional state of nerve terminals, however variability in their physical and chemical properties is associated with diverse strength of these effects.

The heparin-binding site in tetranectin is located in the N-terminal region and binding does not involve the carbohydrate recognition domain.

Science.gov (United States)

Lorentsen, R H; Graversen, J H; Caterer, N R; Thogersen, H C; Etzerodt, M

2000-04-01

Tetranectin is a homotrimeric plasma and extracellular-matrix protein that binds plasminogen and complex sulphated polysaccharides including heparin. In terms of primary and tertiary structure, tetranectin is related to the collectin family of Ca(2+)-binding C-type lectins. Tetranectin is encoded in three exons. Exon 3 encodes the carbohydrate recognition domain, which binds to kringle 4 in plasminogen at low levels of Ca(2+). Exon 2 encodes an alpha-helix, which is necessary and sufficient to govern the trimerization of tetranectin by assembling into a triple-helical coiled-coil structural element. Here we show that the heparin-binding site in tetranectin resides not in the carbohydrate recognition domain but within the N-terminal region, comprising the 16 amino acid residues encoded by exon 1. In particular, the lysine residues in the decapeptide segment KPKKIVNAKK (tetranectin residues 6-15) are shown to be of primary importance in heparin binding.

Characterization of an extensin-modifying metalloprotease: N-terminal processing and substrate cleavage pattern of Pectobacterium carotovorum Prt1.

Science.gov (United States)

Feng, Tao; Nyffenegger, Christian; Højrup, Peter; Vidal-Melgosa, Silvia; Yan, Kok-Phen; Fangel, Jonatan Ulrik; Meyer, Anne S; Kirpekar, Finn; Willats, William G; Mikkelsen, Jørn D

2014-12-01

Compared to other plant cell wall-degrading enzymes, proteases are less well understood. In this study, the extracellular metalloprotease Prt1 from Pectobacterium carotovorum (formerly Erwinia carotovora) was expressed in Escherichia coli and characterized with respect to N-terminal processing, thermal stability, substrate targets, and cleavage patterns. Prt1 is an autoprocessing protease with an N-terminal signal pre-peptide and a pro-peptide which has to be removed in order to activate the protease. The sequential cleavage of the N-terminus was confirmed by mass spectrometry (MS) fingerprinting and N-terminus analysis. The optimal reaction conditions for the activity of Prt1 on azocasein were at pH 6.0, 50 °C. At these reaction conditions, K M was 1.81 mg/mL and k cat was 1.82 × 10(7) U M(-1). The enzyme was relatively stable at 50 °C with a half-life of 20 min. Ethylenediaminetetraacetic acid (EDTA) treatment abolished activity; Zn(2+) addition caused regain of the activity, but Zn(2+)addition decreased the thermal stability of the Prt1 enzyme presumably as a result of increased proteolytic autolysis. In addition to casein, the enzyme catalyzed degradation of collagen, potato lectin, and plant extensin. Analysis of the cleavage pattern of different substrates after treatment with Prt1 indicated that the protease had a substrate cleavage preference for proline in substrate residue position P1 followed by a hydrophobic residue in residue position P1' at the cleavage point. The activity of Prt1 against plant cell wall structural proteins suggests that this enzyme might become an important new addition to the toolbox of cell-wall-degrading enzymes for biomass processing.

Optimised ExpTime Tableaux for ℋℐ over Finite Residuated Lattices

Directory of Open Access Journals (Sweden)

Jian Huang

2014-01-01

Full Text Available This study proposes to adopt a novel tableau reasoning algorithm for the description logic ℋℐ with semantics based on a finite residuated De Morgan lattice. The syntax, semantics, and logical properties of this logic are given, and a sound, complete, and terminating tableaux algorithm for deciding fuzzy ABox consistency and concept satisfiability problem with respect to TBox is presented. Moreover, based on extended and/or completion-forest with a series of sound optimization technique for checking satisfiability with respect to a TBox in the logic, a new optimized ExpTime (complexity-optimal tableau decision procedure is presented here. The experimental evaluation indicates that the optimization techniques we considered result in improved efficiency significantly.

The practice of terminal discharge.

Science.gov (United States)

Radha Krishna, Lalit Kumar; Murugam, Vengadasalam; Quah, Daniel Song Chiek

2017-01-01

'Terminal discharges' are carried out in Singapore for patients who wish to die at home. However, if due diligence is not exercised, parallels may be drawn with euthanasia. We present a theoretical discussion beginning with the definition of terminal discharges and the reasons why they are carried out in Singapore. By considering the intention behind terminal discharges and utilising a multidisciplinary team to deliberate on the clinical, social and ethical intricacies with a patient- and context-specific approach, euthanasia is avoided. It is hoped that this will provide a platform for professionals in palliative medicine to negotiate challenging issues when arranging a terminal discharge, so as to avoid the pitfall of committing euthanasia in a country such as Singapore where euthanasia is illegal. It is hoped that a set of guidelines for terminal discharges may someday be realised to assist professionals in Singapore and around the world.

Identification of C-terminal hydrophobic residues important for dimerization and all known functions of ParB of Pseudomonas aeruginosa.

NARCIS (Netherlands)

Mierzejewska, J.; Bartosik, A.A.; Macioszek, M.; Plochocka, D.; Thomas, C.M.G.; Jagura-Burdzy, G.

2012-01-01

The ParB protein of Pseudomonas aeruginosa is important for growth, cell division, nucleoid segregation and different types of motility. To further understand its function we have demonstrated a vital role of the hydrophobic residues in the C terminus of ParB(P.a.). By in silico modelling of the

Differential binding properties of Gal/GalNAc specific lectins available for characterization of glycoreceptors.

Science.gov (United States)

Wu, A M; Song, S C; Sugii, S; Herp, A

1997-01-01

Differentiating the binding properties of applied lectins should facilitate the selection of lectins for characterization of glycoreceptors on the cell surface. Based on the binding specificities studied by inhibition assays of lectin-glycan interactions, over twenty Gal and/or GalNAc specific lectins have been divided into eight groups according to their specificity for structural units (lectin determinants), which are the disaccharide as all or part of the determinants and of GalNAc alpha 1-->Ser (Thr) of the peptide chain. A scheme of codes for lectin determinants is illustrated as follows: (1) F (GalNAc alpha 1-->3GalNAc), Forssman specific disaccharide--Dolichos biflorus (DBL), Helix pomatia (HPL) and Wistaria floribunda (WFL) lectins. (2) A (GalNAc alpha 1-->3 Gal), blood group A specific disaccharide--Codium fragile subspecies tomentosoides (CFT), Soy bean (SBL), Vicia villosa-A4 (VVL-A4), and Wistaria floribunda (WFL) lectins. (3) Tn (GalNAc alpha 1-->Ser (Thr) of the protein core)--Vicia villosa B4 (VVL-B4), Salvia sclarea (SSL), Maclura pomifera (MPL), Bauhinia purpurea alba (BPL) and Artocarpus integrifolia (Jacalin, AIL). (4) T (Gal beta 1-->3GalNAc), the mucin type sugar sequences on the human erythrocyte membrane(T alpha), T antigen or the disaccharides at the terminal nonreducing end of gangliosides (T beta)--Peanut (PNA), Bauhinia purpurea alba (BPL), Maclura pomifera (MPL), Sophora japonica (SJL), Artocarpus lakoocha (Artocarpin) lectins and Abrus precatorius agglutinin (APA).(5) I and II (Gal beta 1-->3(4)GlcNAc)--the disaccharide residue at the nonreducing end of the carbohydrate chains derived from either N- or O-glycosidic linkage--Ricinus communis agglutinin (RCA1), Datura stramonium (TAL, Thorn apple), Erythrina cristagalli (ECL, Coral tree), and Geodia cydonium (GCL). (6) B (Gal alpha 1-->3Gal), human blood group B specific disaccharide--Griffonia(Banderiaea) simplicifolia B4 (GSI-B4). (7) E (Gal alpha 1-->4Gal), receptors for pathogenic E

Polymorphisms at Amino Acid Residues 141 and 154 Influence Conformational Variation in Ovine PrP

Science.gov (United States)

Yang, Sujeong; Thackray, Alana M.; Hopkins, Lee; Monie, Tom P.; Burke, David F.; Bujdoso, Raymond

2014-01-01

Polymorphisms in ovine PrP at amino acid residues 141 and 154 are associated with susceptibility to ovine prion disease: Leu141Arg154 with classical scrapie and Phe141Arg154 and Leu141His154 with atypical scrapie. Classical scrapie is naturally transmissible between sheep, whereas this may not be the case with atypical scrapie. Critical amino acid residues will determine the range or stability of structural changes within the ovine prion protein or its functional interaction with potential cofactors, during conversion of PrPC to PrPSc in these different forms of scrapie disease. Here we computationally identified that regions of ovine PrP, including those near amino acid residues 141 and 154, displayed more conservation than expected based on local structural environment. Molecular dynamics simulations showed these conserved regions of ovine PrP displayed genotypic differences in conformational repertoire and amino acid side-chain interactions. Significantly, Leu141Arg154 PrP adopted an extended beta sheet arrangement in the N-terminal palindromic region more frequently than the Phe141Arg154 and Leu141His154 variants. We supported these computational observations experimentally using circular dichroism spectroscopy and immunobiochemical studies on ovine recombinant PrP. Collectively, our observations show amino acid residues 141 and 154 influence secondary structure and conformational change in ovine PrP that may correlate with different forms of scrapie. PMID:25126555

Termination of cycle rewriting

NARCIS (Netherlands)

Zantema, H.; König, B.; Bruggink, H.J.S.; Dowek, G.

2014-01-01

String rewriting can not only be applied on strings, but also on cycles and even on general graphs. In this paper we investigate termination of string rewriting applied on cycles, shortly denoted as cycle rewriting, which is a strictly stronger requirement than termination on strings. Most

78 FR 5717 - Safety Zone; Military Ocean Terminal Concord Safety Zone, Suisun Bay, Military Ocean Terminal...

Science.gov (United States)

2013-01-28

...-AA00 Safety Zone; Military Ocean Terminal Concord Safety Zone, Suisun Bay, Military Ocean Terminal... Guard is establishing a safety zone in the navigable waters of Suisun Bay near Military Ocean Terminal Concord, CA in support of military onload and offload operations. This safety zone is established to...

The formation of a native-like structure containing eight conserved hydrophobic residues is rate limiting in two-state protein folding of ACBP

DEFF Research Database (Denmark)

Kragelund, Birthe Brandt; Osmark, Peter; Neergaard, Thomas B.

1999-01-01

The acyl-coenzyme A-binding proteins (ACBPs) contain 26 highly conserved sequence positions. The majority of these have been mutated in the bovine protein, and their influence on the rate of two-state folding and unfolding has been measured. The results identify eight sequence positions, out of 24...... probed, that are critical for fast productive folding. The residues are all hydrophobic and located in the interface between the N- and C-terminal helices. The results suggest that one specific site dominated by conserved hydrophobic residues forms the structure of the productive rate-determining folding...... step and that a sequential framework model can describe the protein folding reaction....

Novel Insights into Structure-Activity Relationships of N-Terminally Modified PACE4 Inhibitors.

Science.gov (United States)

Kwiatkowska, Anna; Couture, Frédéric; Levesque, Christine; Ly, Kévin; Beauchemin, Sophie; Desjardins, Roxane; Neugebauer, Witold; Dory, Yves L; Day, Robert

2016-02-04

PACE4 plays important roles in prostate cancer cell proliferation. The inhibition of this enzyme has been shown to slow prostate cancer progression and is emerging as a promising therapeutic strategy. In previous work, we developed a highly potent and selective PACE4 inhibitor, the multi-Leu (ML) peptide, an octapeptide with the sequence Ac-LLLLRVKR-NH2 . Here, with the objective of developing a useful compound for in vivo administration, we investigate the effect of N-terminal modifications. The inhibitory activity, toxicity, stability, and cell penetration properties of the resulting analogues were studied and compared to the unmodified inhibitor. Our results show that the incorporation of a polyethylene glycol (PEG) moiety leads to a loss of antiproliferative activity, whereas the attachment of a lipid chain preserves or improves it. However, the lipidated peptides are significantly more toxic when compared with their unmodified counterparts. Therefore, the best results were achieved not by the N-terminal extension but by the protection of both ends with the d-Leu residue and 4-amidinobenzylamide, which yielded the most stable inhibitor, with an excellent activity and toxicity profile. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Residue behavior and risk assessment of mixed formulation of imidacloprid and chlorfenapyr in chieh-qua under field conditions.

Science.gov (United States)

Huang, Jian Xiang; Liu, Cong Yun; Lu, Da Hai; Chen, Jia Jia; Deng, Yi Cai; Wang, Fu Hua

2015-10-01

A simple and rapid method based on high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed for the simultaneous determination of imidacloprid and chlorfenapyr residues in chieh-qua. Field trials were designed to investigate the dissipation and terminal residue behavior of the mixed formulation of imidacloprid and chlorfenapyr in chieh-qua in Guangzhou and Nanning areas. Risk assessment was performed by calculating the risk quotient (RQ) values. The developed analytical method exhibited recoveries of 89.9-110.3% with relative standard deviations (RSDs) of 2.8-12.5% at the spiked levels of 0.01, 0.10, and 1.00 mg/kg. The limit of detection (LOD) was 0.003 mg/kg, and the limit of quantification (LOQ) was 0.01 mg/kg for both imidacloprid and chlorfenapyr. It was found that the half-lives of imidacloprid in chieh-qua under field conditions were 3.3 and 3.5 days in Guangzhou and Nanning at a dose of 180 g ai/ha, while the half-lives of chlorfenapyr were 3.3 and 2.6 days, respectively. The terminal residues of imidacloprid and chlorfenapyr were from 0.01 to 0.21 mg/kg and from 0.01 to 0.46 mg/kg, respectively. Results of dietary exposure assessment showed that the RQ values were much lower than 1, indicating that the risk of imidacloprid and chlorfenapyr applied in chieh-qua was negligible to human health under recommended dosage and good agricultural practices. The proposed study would provide guidance for safe and reasonable use of imidacloprid and chlorfenapyr in chieh-qua cultivation in China.

A survey of residual analysis and a new test of residual trend.

Science.gov (United States)

McDowell, J J; Calvin, Olivia L; Klapes, Bryan

2016-05-01

A survey of residual analysis in behavior-analytic research reveals that existing methods are problematic in one way or another. A new test for residual trends is proposed that avoids the problematic features of the existing methods. It entails fitting cubic polynomials to sets of residuals and comparing their effect sizes to those that would be expected if the sets of residuals were random. To this end, sampling distributions of effect sizes for fits of a cubic polynomial to random data were obtained by generating sets of random standardized residuals of various sizes, n. A cubic polynomial was then fitted to each set of residuals and its effect size was calculated. This yielded a sampling distribution of effect sizes for each n. To test for a residual trend in experimental data, the median effect size of cubic-polynomial fits to sets of experimental residuals can be compared to the median of the corresponding sampling distribution of effect sizes for random residuals using a sign test. An example from the literature, which entailed comparing mathematical and computational models of continuous choice, is used to illustrate the utility of the test. © 2016 Society for the Experimental Analysis of Behavior.

Critical residues in the PMEL/Pmel17 N-terminus direct the hierarchical assembly of melanosomal fibrils

Science.gov (United States)

Leonhardt, Ralf M.; Vigneron, Nathalie; Hee, Jia Shee; Graham, Morven; Cresswell, Peter

2013-01-01

PMEL (also called Pmel17 or gp100) is a melanocyte/melanoma-specific glycoprotein that plays a critical role in melanosome development by forming a fibrillar amyloid matrix in the organelle for melanin deposition. Although ultimately not a component of mature fibrils, the PMEL N-terminal region (NTR) is essential for their formation. By mutational analysis we establish a high-resolution map of this domain in which sequence elements and functionally critical residues are assigned. We show that the NTR functions in cis to drive the aggregation of the downstream polycystic kidney disease (PKD) domain into a melanosomal core matrix. This is essential to promote in trans the stabilization and terminal proteolytic maturation of the repeat (RPT) domain–containing MαC units, precursors of the second fibrillogenic fragment. We conclude that during melanosome biogenesis the NTR controls the hierarchical assembly of melanosomal fibrils. PMID:23389629

The catalytic chain of human complement subcomponent C1r. Purification and N-terminal amino acid sequences of the major cyanogen bromide-cleavage fragments.

Science.gov (United States)

Arlaud, G J; Gagnon, J; Porter, R R

1982-01-01

1. The a- and b-chains of reduced and alkylated human complement subcomponent C1r were separated by high-pressure gel-permeation chromatography and isolated in good yield and in pure form. 2. CNBr cleavage of C1r b-chain yielded eight major peptides, which were purified by gel filtration and high-pressure reversed-phase chromatography. As determined from the sum of their amino acid compositions, these peptides accounted for a minimum molecular weight of 28 000, close to the value 29 100 calculated from the whole b-chain. 3. N-Terminal sequence determinations of C1r b-chain and its CNBr-cleavage peptides allowed the identification of about two-thirds of the amino acids of C1r b-chain. From our results, and on the basis of homology with other serine proteinases, an alignment of the eight CNBr-cleavage peptides from C1r b-chain is proposed. 4. The residues forming the 'charge-relay' system of the active site of serine proteinases (His-57, Asp-102 and Ser-195 in the chymotrypsinogen numbering) are found in the corresponding regions of C1r b-chain, and the amino acid sequence around these residues has been determined. 5. The N-terminal sequence of C1r b-chain has been extended to residue 60 and reveals that C1r b-chain lacks the 'histidine loop', a disulphide bond that is present in all other known serine proteinases.

Dissipation kinetics of emamectin benzoate and lufenuron residues in cabbage grown under field conditions.

Science.gov (United States)

Dong, Bizhang; Zhao, Qing; Hu, Jiye

2015-12-01

Residue analysis of emamectin benzoate and lufenuron in cabbage matrices and soil was developed using a quick, easy, cheap, effective, rugged, and safe (QuEChERS) method and ultra high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The samples were extracted with 1% acetic acid in acetonitrile (v/v) or 1% acetic acid in acetonitrile/water (5:1, v/v) and cleaned up by dispersive solid-phase extraction. Mean recoveries and relative standard deviations (RSDs) in all samples ranged 87.8-100.0 % and 3.6-12.6% for emamectin benzoate and 87.8-104.8 % and 6.2-11.5% for lufenuron, respectively. The validated method was used to evaluate the dissipation rate of emamectin benzoate and lufenuron in cabbage and soil as well as the residual levels in harvested cabbage and soil at different preharvest intervals (PHI). The half-lives of emamectin benzoate and lufenuron were 1.08-2.70 and 1.74-5.04 days in cabbage, and 1.42-4.01 and 0.94-6.18 days in soil, respectively. The terminal residues were below the China maximum residue limits (MRLs) at 3 days for emamectin benzoate (0.1 mg kg(-1)) and European Union MRLs at 5 days for lufenuron (0.5 mg kg(-1)), which suggested that 5 days could be recommended as the PHI for the commercial formulation of emamectin benzoate and lufenuron application in the Chinese cabbage field.

Structural basis for new pattern of conserved amino acid residues related to chitin-binding in the antifungal peptide from the coconut rhinoceros beetle Oryctes rhinoceros.

Science.gov (United States)

Hemmi, Hikaru; Ishibashi, Jun; Tomie, Tetsuya; Yamakawa, Minoru

2003-06-20

Scarabaecin isolated from hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros is a 36-residue polypeptide that has antifungal activity. The solution structure of scarabaecin has been determined from twodimensional 1H NMR spectroscopic data and hybrid distance geometry-simulated annealing protocol calculation. Based on 492 interproton and 10 hydrogen-bonding distance restraints and 36 dihedral angle restraints, we obtained 20 structures. The average backbone root-mean-square deviation for residues 4-35 is 0.728 +/- 0.217 A from the mean structure. The solution structure consists of a two-stranded antiparallel beta-sheet connected by a type-I beta-turn after a short helical turn. All secondary structures and a conserved disulfide bond are located in the C-terminal half of the peptide, residues 18-36. Overall folding is stabilized by a combination of a disulfide bond, seven hydrogen bonds, and numerous hydrophobic interactions. The structural motif of the C-terminal half shares a significant tertiary structural similarity with chitin-binding domains of plant and invertebrate chitin-binding proteins, even though scarabaecin has no overall sequence similarity to other peptide/polypeptides including chitin-binding proteins. The length of its primary structure, the number of disulfide bonds, and the pattern of conserved functional residues binding to chitin in scarabaecin differ from those of chitin-binding proteins in other invertebrates and plants, suggesting that scarabaecin does not share a common ancestor with them. These results are thought to provide further strong experimental evidence to the hypothesis that chitin-binding proteins of invertebrates and plants are correlated by a convergent evolution process.

46 CFR 525.2 - Terminal schedules.

Science.gov (United States)

2010-10-01

... domestic law and international conventions and agreements adopted by the United States; such terminal... their own negligence, or that impose upon others the obligation to indemnify or hold-harmless the terminals from liability for their own negligence. (2) Enforcement of terminal schedules. Any schedule that...

The N-terminal region of the dopamine D2 receptor, a rhodopsin-like GPCR, regulates correct integration into the plasma membrane and endocytic routes

Science.gov (United States)

Cho, DI; Min, C; Jung, KS; Cheong, SY; Zheng, M; Cheong, SJ; Oak, MH; Cheong, JH; Lee, BK; Kim, KM

2012-01-01

BACKGROUND AND PURPOSE Functional roles of the N-terminal region of rhodopsin-like GPCR family remain unclear. Using dopamine D2 and D3 receptors as a model system, we probed the roles of the N-terminal region in the signalling, intracellular trafficking of receptor proteins, and explored the critical factors that determine the functionality of the N-terminal region. EXPERIMENTAL APPROACH The N-terminal region of the D2 receptor was gradually shortened or switched with that of the D3 receptor or a non-specific sequence (FLAG), or potential N-terminal glycosylation sites were mutated. Effects of these manipulations on surface expression, internalization, post-endocytic behaviours and signalling were determined. KEY RESULTS Shortening the N-terminal region of the D2 receptor enhanced receptor internalization and impaired surface expression and signalling; ligand binding, desensitization and down-regulation were not affected but their association with a particular microdomain, caveolae, was disrupted. Replacement of critical residues within the N-terminal region with the FLAG epitope failed to restore surface expression but partially restored the altered internalization and signalling. When the N-terminal regions were switched between D2 and D3 receptors, cell surface expression pattern of each receptor was switched. Mutations of potential N-terminal glycosylation sites inhibited surface expression but enhanced internalization of D2 receptors. CONCLUSIONS AND IMPLICATIONS Shortening of N-terminus or mutation of glycosylation sites located within the N-terminus enhanced receptor internalization but impaired the surface expression of D2 receptors. The N-terminal region of the D2 receptor, in a sequence-specific manner, controls the receptor's conformation and integration into the plasma membrane, which determine its subcellular localization, intracellular trafficking and signalling properties. PMID:22117524

Solution structure of the C-terminal X domain of the measles virus phosphoprotein and interaction with the intrinsically disordered C-terminal domain of the nucleoprotein.

Science.gov (United States)

Gely, Stéphane; Lowry, David F; Bernard, Cédric; Jensen, Malene R; Blackledge, Martin; Costanzo, Stéphanie; Bourhis, Jean-Marie; Darbon, Hervé; Daughdrill, Gary; Longhi, Sonia

2010-01-01

In this report, the solution structure of the nucleocapsid-binding domain of the measles virus phosphoprotein (XD, aa 459-507) is described. A dynamic description of the interaction between XD and the disordered C-terminal domain of the nucleocapsid protein, (N(TAIL), aa 401-525), is also presented. XD is an all alpha protein consisting of a three-helix bundle with an up-down-up arrangement of the helices. The solution structure of XD is very similar to the crystal structures of both the free and bound form of XD. One exception is the presence of a highly dynamic loop encompassing XD residues 489-491, which is involved in the embedding of the alpha-helical XD-binding region of N(TAIL). Secondary chemical shift values for full-length N(TAIL) were used to define the precise boundaries of a transient helical segment that coincides with the XD-binding domain, thus shedding light on the pre-recognition state of N(TAIL). Titration experiments with unlabeled XD showed that the transient alpha-helical conformation of N(TAIL) is stabilized upon binding. Lineshape analysis of NMR resonances revealed that residues 483-506 of N(TAIL) are in intermediate exchange with XD, while the 475-482 and 507-525 regions are in fast exchange. The N(TAIL) resonance behavior in the titration experiments is consistent with a complex binding model with more than two states.

Concept Layout Model of Transportation Terminals

Directory of Open Access Journals (Sweden)

Li-ya Yao

2012-01-01

Full Text Available Transportation terminal is the key node in transport systems. Efficient terminals can improve operation of passenger transportation networks, adjust the layout of public transportation networks, provide a passenger guidance system, and regulate the development of commercial forms, as well as optimize the assembly and distribution of modern logistic modes, among others. This study aims to clarify the relationship between the function and the structure of transportation terminals and establish the function layout design. The mapping mechanism of demand, function, and structure was analyzed, and a quantitative relationship between function and structure was obtained from a design perspective. Passenger demand and terminal structure were decomposed into several demand units and structural elements following the principle of reverse engineering. The relationship maps between these two kinds of elements were then analyzed. Function-oriented concept layout model of transportation terminals was established using the previous method. Thus, a technique in planning and design of transportation structures was proposed. Meaningful results were obtained from the optimization of transportation terminal facilities, which guide the design of the functional layout of transportation terminals and improve the development of urban passenger transportation systems.

Oligosaccharides Derived from Sucrose

Science.gov (United States)

Monsan, Pierre F.; Ouarné, Francois

Sucrose is a non-reducing disaccharide, consisting of an α-D-glucopyranosyl residue and a β-D-fructofuranosyl residue linked covalently by their respective anomeric carbons (α-D-glucopyranosyl-1,2-β-D-fructofuranoside). It is not just a simple disaccharide, among others: in fact, the energy of its glycosidic bond is higher than that of a usual glycosidic bond. It is equal to 27.6 kJ/mol, which is similar to the energy of a nucleotide-sugar bond as in UDP-glucose or ADP-glucose. This means that sucrose is a protected and activated form of D-glucose (as well as of D-fructose), which plays a key role in the metabolism of plants, for a wide variety of synthesis reactions.

RNA Binding of T-cell Intracellular Antigen-1 (TIA-1) C-terminal RNA Recognition Motif Is Modified by pH Conditions*

Science.gov (United States)

Cruz-Gallardo, Isabel; Aroca, Ángeles; Persson, Cecilia; Karlsson, B. Göran; Díaz-Moreno, Irene

2013-01-01

T-cell intracellular antigen-1 (TIA-1) is a DNA/RNA-binding protein that regulates critical events in cell physiology by the regulation of pre-mRNA splicing and mRNA translation. TIA-1 is composed of three RNA recognition motifs (RRMs) and a glutamine-rich domain and binds to uridine-rich RNA sequences through its C-terminal RRM2 and RRM3 domains. Here, we show that RNA binding mediated by either isolated RRM3 or the RRM23 construct is controlled by slight environmental pH changes due to the protonation/deprotonation of TIA-1 RRM3 histidine residues. The auxiliary role of the C-terminal RRM3 domain in TIA-1 RNA recognition is poorly understood, and this work provides insight into its binding mechanisms. PMID:23902765

Mobile Termination and Mobile Penetration

OpenAIRE

Hurkens, Sjaak; Jeon, Doh-Shin

2009-01-01

In this paper, we study how access pricing affects network competition when subscription demand is elastic and each network uses non-linear prices and can apply termination-based price discrimination. In the case of a fixed per minute termination charge, we find that a reduction of the termination charge below cost has two oppos- ing effects: it softens competition but helps to internalize network externalities. The former reduces mobile penetration while the latter boosts it. We find that fi...

Mobile termination and mobile penetration

OpenAIRE

Hurkens, Sjaak

2009-01-01

In this paper, we study how access pricing affects network competition when subscription demand is elastic and each network uses non-linear prices and can apply termination-based price discrimination. In the case of a fixed per minute termination charge, we find that a reduction of the termination charge below cost has two opposing effects: it softens competition but helps to internalize network externalities. The former reduces mobile penetration while the latter boosts it. We find that firm...

Role of four conserved aspartic acid residues of EF-loops in the metal ion binding and in the self-assembly of ciliate Euplotes octocarinatus centrin.

Science.gov (United States)

Liu, Wen; Duan, Lian; Sun, Tijian; Yang, Binsheng

2016-12-01

Ciliate Euplotes octocarinatus centrin (EoCen) is an EF-hand calcium-binding protein closely related to the prototypical calcium sensor protein calmodulin. Four mutants (D37K, D73K, D110K and D146K) were created firstly to elucidate the importance of the first aspartic acid residues (Asp37, Asp73, Asp110 and Asp146) in the beginning of the four EF-loops of EoCen. Aromatic-sensitized Tb 3+ fluorescence indicates that the aspartic acid residues are very important for the metal-binding of EoCen, except for Asp73 (in EF-loop II). Resonance light scattering (RLS) measurements for different metal ions (Ca 2+ and Tb 3+ ) binding proteins suggest that the order of four conserved aspartic acid residues for contributing to the self-assembly of EoCen is Asp37 > Asp146 > Asp110 > Asp73. Cross-linking experiment also exhibits that Asp37 and Asp146 play critical role in the self-assembly of EoCen. Asp37, in site I, which is located in the N-terminal domain, plays the most important role in the metal ion-dependent self-assembly of EoCen, and there is cooperativity between N-terminal and C-terminal domain (especially the site IV). In addition, the dependence of Tb 3+ induced self-assembly of EoCen and the mutants on various factors, including ionic strength and pH, were characterized using RLS. Finally, 2-p-toluidinylnaphthalene-6-sulfonate (TNS) binding, ionic strength and pH control experiments indicate that in the process of EoCen self-assembly, molecular interactions are mediated by both electrostatic and hydrophobic forces, and the hydrophobic interaction has the important status.

49 CFR 374.309 - Terminal facilities.

Science.gov (United States)

2010-10-01

... 49 Transportation 5 2010-10-01 2010-10-01 false Terminal facilities. 374.309 Section 374.309... REGULATIONS Adequacy of Intercity Motor Common Carrier Passenger Service § 374.309 Terminal facilities. (a... attendants and be regularly patrolled. (b) Outside facilities. At terminals and stations that are closed when...

Prematurely terminated slug tests

International Nuclear Information System (INIS)

Karasaki, K.

1990-07-01

A solution of the well response to a prematurely terminated slug test (PTST) is presented. The advantages of a PTST over conventional slug tests are discussed. A systematized procedure of a PTST is proposed, where a slug test is terminated in the midpoint of the flow point, and the subsequent shut-in data is recorded and analyzed. This method requires a downhole shut-in device and a pressure transducer, which is no more than the conventional deep-well slug testing. As opposed to slug tests, which are ineffective when a skin is present, more accurate estimate of formation permeability can be made using a PTST. Premature termination also shortens the test duration considerably. Because in most cases no more information is gained by completing a slug test to the end, the author recommends that conventional slug tests be replaced by the premature termination technique. This study is part of an investigation of the feasibility of geologic isolation of nuclear wastes being carried out by the US Department of Energy and the National Cooperative for the Storage of Radioactive Waste of Switzerland

Role of the Cationic C-Terminal Segment of Melittin on Membrane Fragmentation.

Science.gov (United States)

Therrien, Alexandre; Fournier, Alain; Lafleur, Michel

2016-05-05

The widespread distribution of cationic antimicrobial peptides capable of membrane fragmentation in nature underlines their importance to living organisms. In the present work, we determined the impact of the electrostatic interactions associated with the cationic C-terminal segment of melittin, a 26-amino acid peptide from bee venom (net charge +6), on its binding to model membranes and on the resulting fragmentation. In order to detail the role played by the C-terminal charges, we prepared a melittin analogue for which the four cationic amino acids in positions 21-24 were substituted with the polar residue citrulline, providing a peptide with the same length and amphiphilicity but with a lower net charge (+2). We compared the peptide bilayer affinity and the membrane fragmentation for bilayers prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine (DPPS) mixtures. It is shown that neutralization of the C-terminal considerably increased melittin affinity for zwitterionic membranes. The unfavorable contribution associated with transferring the cationic C-terminal in a less polar environment was reduced, leaving the hydrophobic interactions, which drive the peptide insertion in bilayers, with limited counterbalancing interactions. The presence of negatively charged lipids (DPPS) in bilayers increased melittin binding by introducing attractive electrostatic interactions, the augmentation being, as expected, greater for native melittin than for its citrullinated analogue. The membrane fragmentation power of the peptide was shown to be controlled by electrostatic interactions and could be modulated by the charge carried by both the membrane and the lytic peptide. The analysis of the lipid composition of the extracted fragments from DPPC/DPPS bilayers revealed no lipid specificity. It is proposed that extended phase separations are more susceptible to lead to the extraction of a lipid species in a specific manner

Involvement of C-Terminal Histidines in Soybean PM1 Protein Oligomerization and Cu2+ Binding.

Science.gov (United States)

Liu, Guobao; Liu, Ke; Gao, Yang; Zheng, Yizhi

2017-06-01

Late embryogenesis abundant (LEA) proteins are widely distributed among plant species, where they contribute to abiotic stress tolerance. LEA proteins can be classified into seven groups according to conserved sequence motifs. The PM1 protein from soybean, which belongs to the Pfam LEA_1 group, has been shown previously to be at least partially natively unfolded, to bind metal ions and potentially to stabilize proteins and membranes. Here, we investigated the role of the PM1 C-terminal domain and in particular the multiple histidine residues in this half of the protein. We constructed recombinant plasmids expressing full-length PM1 and two truncated forms, PM1-N and PM1-C, which represent the N- and C-terminal halves of the protein, respectively. Immunoblotting and cross-linking experiments showed that full-length PM1 forms oligomers and high molecular weight (HMW) complexes in vitro and in vivo, while PM1-C, but not PM1-N, also formed oligomers and HMW complexes in vitro. When the histidine residues in PM1 and PM1-C were chemically modified, oligomerization was abolished, suggesting that histidines play a key role in this process. Furthermore, we demonstrated that high Cu2+ concentrations promote oligomerization and induce PM1 and PM1-C to form HMW complexes. Therefore, we speculate that PM1 proteins not only maintain ion homeostasis in the cytoplasm, but also potentially stabilize and protect other proteins during abiotic stress by forming a large, oligomeric molecular shield around biological targets. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

9 CFR 3.91 - Terminal facilities.

Science.gov (United States)

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Terminal facilities. 3.91 Section 3.91... Primates 2 Transportation Standards § 3.91 Terminal facilities. (a) Placement. Any persons subject to the... with inanimate cargo or with other animals in animal holding areas of terminal facilities. Nonhuman...

9 CFR 3.18 - Terminal facilities.

Science.gov (United States)

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Terminal facilities. 3.18 Section 3.18... Cats 1 Transportation Standards § 3.18 Terminal facilities. (a) Placement. Any person subject to the... inanimate cargo in animal holding areas of terminal facilities. (b) Cleaning, sanitization, and pest control...

Prokaryote community dynamics in anaerobic co-digestion of swine manure, rice straw and industrial clay residuals.

Science.gov (United States)

Jiménez, Janet; Theuerl, Susanne; Bergmann, Ingo; Klocke, Michael; Guerra, Gilda; Romero-Romero, Osvaldo

The aim of this study was to analyze the effect of the addition of rice straw and clay residuals on the prokaryote methane-producing community structure in a semi-continuously stirred tank reactor fed with swine manure. Molecular techniques, including terminal restriction fragment length polymorphism and a comparative nucleotide sequence analyses of the prokaryotic 16S rRNA genes, were performed. The results showed a positive effect of clay addition on methane yield during the co-digestion of swine manure and rice straw. At the digestion of swine manure, the bacterial phylum Firmicutes and the archaeal family Methanosarcinaceae, particularly Methanosarcina species, were predominant. During the co-digestion of swine manure and rice straw the microbial community changed, and with the addition of clay residual, the phylum Bacteroidetes predominated. The new nutritional conditions resulted in a shift in the archaeal family Methanosarcinaceae community as acetoclastic Methanosaeta species became dominant.

Nuclear import of influenza B virus nucleoprotein: Involvement of an N-terminal nuclear localization signal and a cleavage-protection motif

International Nuclear Information System (INIS)

Wanitchang, Asawin; Narkpuk, Jaraspim; Jongkaewwattana, Anan

2013-01-01

The nucleoprotein of influenza B virus (BNP) shares several characteristics with its influenza A virus counterpart (ANP), including localization in the host's nucleus. However, while the nuclear localization signal(s) (NLS) of ANP are well characterized, little is known about those of BNP. In this study, we showed that the fusion protein bearing the BNP N-terminus fused with GFP (N70–GFP) is exclusively nuclear, and identified a highly conserved KRXR motif spanning residues 44–47 as a putative NLS. In addition, we demonstrated that residues 3–15 of BNP, though not an NLS, are also crucial for nuclear import. Results from mutational analyses of N70–GFP and the full-length BNP suggest that this region may be required for protection of the N-terminus from proteolytic cleavage. Altogether, we propose that the N-terminal region of BNP contains the NLS and cleavage-protection motif, which together drive its nuclear localization. - Highlights: • The N-terminal region of BNP is required for nuclear accumulation. • The conserved motif at position 44–47 is a putative nuclear localization signal. • The first 15 amino acids of BNP may function as a cleavage-protection motif. • BNP may get access to the nucleus via a mechanism distinct from ANP

Nuclear import of influenza B virus nucleoprotein: Involvement of an N-terminal nuclear localization signal and a cleavage-protection motif

Energy Technology Data Exchange (ETDEWEB)

Wanitchang, Asawin; Narkpuk, Jaraspim; Jongkaewwattana, Anan, E-mail: anan.jon@biotec.or.th

2013-08-15

The nucleoprotein of influenza B virus (BNP) shares several characteristics with its influenza A virus counterpart (ANP), including localization in the host's nucleus. However, while the nuclear localization signal(s) (NLS) of ANP are well characterized, little is known about those of BNP. In this study, we showed that the fusion protein bearing the BNP N-terminus fused with GFP (N70–GFP) is exclusively nuclear, and identified a highly conserved KRXR motif spanning residues 44–47 as a putative NLS. In addition, we demonstrated that residues 3–15 of BNP, though not an NLS, are also crucial for nuclear import. Results from mutational analyses of N70–GFP and the full-length BNP suggest that this region may be required for protection of the N-terminus from proteolytic cleavage. Altogether, we propose that the N-terminal region of BNP contains the NLS and cleavage-protection motif, which together drive its nuclear localization. - Highlights: • The N-terminal region of BNP is required for nuclear accumulation. • The conserved motif at position 44–47 is a putative nuclear localization signal. • The first 15 amino acids of BNP may function as a cleavage-protection motif. • BNP may get access to the nucleus via a mechanism distinct from ANP.

7 CFR 1469.25 - Contract violations and termination.

Science.gov (United States)

2010-01-01

... termination without delay. (c) If NRCS terminates a contract due to breach of contract, the participant will... terminates a contract due to breach of contract, or the participant voluntarily terminates the contract... 7 Agriculture 10 2010-01-01 2010-01-01 false Contract violations and termination. 1469.25 Section...

Compact Termination for Structural Soft-goods

Science.gov (United States)

Wilkes, Robert, Jr.

2013-01-01

Glass fiber is unique in its ability to withstand atomic oxygen and ultraviolet radiation in-space environments. However, glass fiber is also difficult to terminate by traditional methods without decreasing its strength significantly. Glass fiber products are especially sensitive to bend radius, and do not work very well with traditional 'sewn loop on pin' type connections. As with most composites, getting applied loads from a metallic structure into the webbing without stress concentrations is the key to a successful design. A potted end termination has been shown in some preliminary work to out-perform traditional termination methods. It was proposed to conduct a series of tensile tests on structural webbing or cord to determine the optimum potting geometry, and to then be able to estimate a weight and volume savings over traditional sewn-overa- pin connections. During the course of the investigation into potted end terminations for glass fiber webbing, a new and innovative connection was developed that has lower weight, reduced fabrication time, and superior thermal tolerance over the metallic end terminations that were to be optimized in the original proposal. This end termination essentially transitions the flexible glass fiber webbing into a rigid fiberglass termination, which can be bolted/fastened with traditional methods

9 CFR 3.40 - Terminal facilities.

Science.gov (United States)

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Terminal facilities. 3.40 Section 3.40... Pigs and Hamsters Transportation Standards § 3.40 Terminal facilities. No person subject to the Animal... animal holding areas of a terminal facility where shipments of live guinea pigs or hamsters are...

9 CFR 3.65 - Terminal facilities.

Science.gov (United States)

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Terminal facilities. 3.65 Section 3.65... Transportation Standards § 3.65 Terminal facilities. No person subject to the Animal Welfare regulations shall commingle shipments of live rabbits with inanimate cargo. All animal holding areas of a terminal facility...

Evaluation of residue-residue contact predictions in CASP9

KAUST Repository

Monastyrskyy, Bohdan; Fidelis, Krzysztof; Tramontano, Anna; Kryshtafovych, Andriy

2011-01-01

This work presents the results of the assessment of the intramolecular residue-residue contact predictions submitted to CASP9. The methodology for the assessment does not differ from that used in previous CASPs, with two basic evaluation measures

Battery system including batteries that have a plurality of positive terminals and a plurality of negative terminals

Science.gov (United States)

Dougherty, Thomas J; Symanski, James S; Kuempers, Joerg A; Miles, Ronald C; Hansen, Scott A; Smith, Nels R; Taghikhani, Majid; Mrotek, Edward N; Andrew, Michael G

2014-01-21

A lithium battery for use in a vehicle includes a container, a plurality of positive terminals extending from a first end of the lithium battery, and a plurality of negative terminals extending from a second end of the lithium battery. The plurality of positive terminals are provided in a first configuration and the plurality of negative terminals are provided in a second configuration, the first configuration differing from the second configuration. A battery system for use in a vehicle may include a plurality of electrically connected lithium cells or batteries.

Termination of plastic-clad fiber

International Nuclear Information System (INIS)

Nance, W.R.

1982-03-01

Optical waveguides are ideal in a nuclear weapon environment because of their resistance to electromagnetic interference. Of the fibers on today's market, plastic-clad silica (PCS) is the most radiation resistant and therfore the best choice. Because terminating PCS is complex, this paper attemps to address the major problems associated with these terminations including selecting the proper connector and optimizing the terminating procedures. The sources of losses in the connectors are summarized and typical loss values are given for four connectors which were tested

Effect of Ge surface termination on oxidation behavior

Science.gov (United States)

Lee, Younghwan; Park, Kibyung; Cho, Yong Soo; Lim, Sangwoo

2008-09-01

Sulfur-termination was formed on the Ge(1 0 0) surface using (NH 4) 2S solution. Formation of Ge-S and the oxidation of the S-terminated Ge surface were monitored with multiple internal reflection Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. In the 0.5, 5, or 20% (NH 4) 2S solution, H-termination on the Ge(1 0 0) surface was substituted with S-termination in 1 min. When the S-terminated Ge(1 0 0) surface was exposed in air ambient, the oxidation was retarded for about 3600 min. The preservation time of the oxide layer up to one monolayer of S-terminated Ge(1 0 0) surface was about 120 times longer than for the H-terminated Ge(1 0 0) surface. However, the oxidation of S-terminated Ge(1 0 0) surface drastically increased after the threshold time. There was no significant difference in threshold time between S-terminations formed in 0.5, 5, and 20% (NH 4) 2S solutions. With the surface oxidation, desorption of S on the Ge surface was observed. The desorption behavior of sulfur on the S-terminated Ge(1 0 0) surface was independent of the concentration of the (NH 4) 2S solution that forms S-termination. Non-ideal S-termination on Ge surfaces may be related to drastic oxidation of the Ge surface. Finally, with the desulfurization on the S-terminated Ge(1 0 0) surface, oxide growth is accelerated.

Interactions of photoactive DNAs with terminal deoxynucleotidyl transferase: Identification of peptides in the DNA binding domain

International Nuclear Information System (INIS)

Farrar, Y.J.K.; Evans, R.K.; Beach, C.M.; Coleman, M.S.

1991-01-01

Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the DNA binding site by a photoactive DNA substrate (hetero-40-mer duplex containing eight 5-azido-dUMP residues at one 3' end). Under optimal photolabeling conditions, 27-40% of the DNA was covalently cross-linked to terminal transferase. The specificity of the DNA and protein interaction was demonstrated by protection of photolabeling at the DNA binding domain with natural DNA substrates. In order to recover high yields of modified peptides from limited amounts of starting material, protein modified with 32 P-labeled photoactive DNA and digested with trypsin was extracted 4 times with phenol followed by gel filtration chromatography. All peptides not cross-linked to DNA were extracted into the phenol phase while the photolyzed DNA and the covalently cross-linked peptides remained in the aqueous phase. The 32 P-containing peptide-DNA fraction was subjected to amino acid sequence analysis. Two sequences, Asp 221 -Lys 231 (peptide B8) and Cys 234 -Lys 249 (peptide B10), present in similar yield, were identified. Structure predictions placed the two peptides in an α-helical array of 39 angstrom which would accommodate a DNA helix span of 11 nucleotides. These peptides share sequence similarity with a region in DNA polymerase β that has been implicated in the binding of DNA template

Analysis of the Varicella-Zoster Virus IE62 N-Terminal Acidic Transactivating Domain and Its Interaction with the Human Mediator Complex▿

OpenAIRE

Yamamoto, Shinobu; Eletsky, Alexander; Szyperski, Thomas; Hay, John; Ruyechan, William T.

2009-01-01

The varicella-zoster virus major transactivator, IE62, contains a potent N-terminal acidic transcriptional activation domain (TAD). Our experiments revealed that the minimal IE62 TAD encompasses amino acids (aa) 19 to 67. We showed that the minimal TAD interacts with the human Mediator complex. Site-specific mutations revealed residues throughout the minimal TAD that are important for both activation and Mediator interaction. The TAD interacts directly with aa 402 to 590 of the MED25 subunit,...

Inter-machine comparison of the termination phase and energy conversion in tokamak disruptions with runaway current plateau formation and implications for ITER

International Nuclear Information System (INIS)

Martín-Solís, J.R.; Loarte, A.; Hollmann, E.M.; Esposito, B.; Riccardo, V.

2014-01-01

The termination of the current and the loss of runaway electrons following runaway current plateau formation during disruptions have been investigated in the JET, DIII-D and FTU tokamaks. Substantial conversion of magnetic energy into runaway kinetic energy, up to ∼10 times the initial plateau runaway kinetic energy, has been inferred for the slowest current terminations. Both modelling and experiment suggest that, in present devices, the efficiency of conversion into runaway kinetic energy is determined to a great extent by the characteristic runaway loss time, τ diff , and the resistive time of the residual ohmic plasma after the disruption, τ res , increasing with the ratio τ diff /τ res . It is predicted that, in large future devices such as ITER, the generation of runaways by the avalanche mechanism will play an important role, particularly for slow runaway discharge terminations, increasing substantially the amount of energy deposited by the runaways onto the plasma-facing components by the conversion of magnetic energy of the runaway plasma into runaway kinetic energy. Estimates of the power fluxes on the beryllium plasma-facing components during runaway termination in ITER indicate that for runaway currents of up to 2 MA no melting of the components is expected. For larger runaway currents, minimization of the effects of runaway impact on the first wall requires a reduction in the kinetic energy of the runaway beam before termination and, in addition, high plasma density n e and low ohmic plasma resistance (long τ res ) to prevent large conversion of magnetic into runaway kinetic energy during slow current terminations. (paper)

Toxicity and residual efficacy of chlorantraniliprole, spinetoram, and emamectin benzoate to obliquebanded leafroller (Lepidoptera: Tortricidae).

Science.gov (United States)

Sial, Ashfaq A; Brunner, Jay F

2010-08-01

Studies were conducted to determine the residual toxicity of spinetoram, chlorantraniliprole, and emamectin benzoate to obliquebanded leafroller, Choristoneura rosaceana (Harris) (Lepidoptera: Tortricidae). Larvae were exposed to apple (Malus spp.) foliage collected at different intervals after an airblast sprayer application at the manufacturer-recommended field rate and half the field rate. A mortality of 100% was recorded at field rate applications of spinetoram, chlorantraniliprole, and emamectin benzoate through 59, 38, and 10 d after treatment (DAT), respectively. Significantly less foliage was consumed by C. rosaceana larvae surviving in the emamectin, chlorantraniliprole, and spinetoram treatments compared with those exposed to untreated foliage. Third-instar C. rosaceana exposed to fresh residues on terminal foliage showed 100% mortality after 5-d exposure to spinetoram residues and after 10-d exposure to chlorantraniliprole and emamectin benzoate. The effects of larval movement from foliage with fresh residues was examined by transferring neonate larvae from foliage treated with spinetoram, chlorantraniliprole, or emamectin benzoate to untreated foliage after various exposure intervals. An exposure of 1, 3, and 6 d was required for spinetoram, chlorantraniliprole, and emamectin benzoate to cause 100% mortality at the field rate, respectively. The higher the concentration of chlorantraniliprole and emamectin benzoate, the less exposure time was necessary to cause high levels of mortality in C. rosaceana neonates. Our results indicate that these novel insecticides are highly toxic to C. rosaceana larvae. Implications of these results for C. rosaceana management programs are discussed.

Role of conserved cysteine residues in Herbaspirillum seropedicae NifA activity.

Science.gov (United States)

Oliveira, Marco A S; Baura, Valter A; Aquino, Bruno; Huergo, Luciano F; Kadowaki, Marco A S; Chubatsu, Leda S; Souza, Emanuel M; Dixon, Ray; Pedrosa, Fábio O; Wassem, Roseli; Monteiro, Rose A

2009-01-01

Herbaspirillum seropedicae is an endophytic diazotrophic bacterium that associates with economically important crops. NifA protein, the transcriptional activator of nif genes in H. seropedicae, binds to nif promoters and, together with RNA polymerase-sigma(54) holoenzyme, catalyzes the formation of open complexes to allow transcription initiation. The activity of H. seropedicae NifA is controlled by ammonium and oxygen levels, but the mechanisms of such control are unknown. Oxygen sensitivity is attributed to a conserved motif of cysteine residues in NifA that spans the central AAA+ domain and the interdomain linker that connects the AAA+ domain to the C-terminal DNA binding domain. Here we mutagenized this conserved motif of cysteines and assayed the activity of mutant proteins in vivo. We also purified the mutant variants of NifA and tested their capacity to bind to the nifB promoter region. Chimeric proteins between H. seropedicae NifA, an oxygen-sensitive protein, and Azotobacter vinelandii NifA, an oxygen-tolerant protein, were constructed and showed that the oxygen response is conferred by the central AAA+ and C-terminal DNA binding domains of H. seropedicae NifA. We conclude that the conserved cysteine motif is essential for NifA activity, although single cysteine-to-serine mutants are still competent at binding DNA.

Mechanisms of DNA replication termination.

Science.gov (United States)

Dewar, James M; Walter, Johannes C

2017-08-01

Genome duplication is carried out by pairs of replication forks that assemble at origins of replication and then move in opposite directions. DNA replication ends when converging replication forks meet. During this process, which is known as replication termination, DNA synthesis is completed, the replication machinery is disassembled and daughter molecules are resolved. In this Review, we outline the steps that are likely to be common to replication termination in most organisms, namely, fork convergence, synthesis completion, replisome disassembly and decatenation. We briefly review the mechanism of termination in the bacterium Escherichia coli and in simian virus 40 (SV40) and also focus on recent advances in eukaryotic replication termination. In particular, we discuss the recently discovered E3 ubiquitin ligases that control replisome disassembly in yeast and higher eukaryotes, and how their activity is regulated to avoid genome instability.

The disordered C-terminal domain of human DNA glycosylase NEIL1 contributes to its stability via intramolecular interactions.

Science.gov (United States)

Hegde, Muralidhar L; Tsutakawa, Susan E; Hegde, Pavana M; Holthauzen, Luis Marcelo F; Li, Jing; Oezguen, Numan; Hilser, Vincent J; Tainer, John A; Mitra, Sankar

2013-07-10

NEIL1 [Nei (endonuclease VIII)-like protein 1], one of the five mammalian DNA glycosylases that excise oxidized DNA base lesions in the human genome to initiate base excision repair, contains an intrinsically disordered C-terminal domain (CTD; ~100 residues), not conserved in its Escherichia coli prototype Nei. Although dispensable for NEIL1's lesion excision and AP lyase activities, this segment is required for efficient in vivo enzymatic activity and may provide an interaction interface for many of NEIL1's interactions with other base excision repair proteins. Here, we show that the CTD interacts with the folded domain in native NEIL1 containing 389 residues. The CTD is poised for local folding in an ordered structure that is induced in the purified fragment by osmolytes. Furthermore, deletion of the disordered tail lacking both Tyr and Trp residues causes a red shift in NEIL1's intrinsic Trp-specific fluorescence, indicating a more solvent-exposed environment for the Trp residues in the truncated protein, which also exhibits reduced stability compared to the native enzyme. These observations are consistent with stabilization of the native NEIL1 structure via intramolecular, mostly electrostatic, interactions that were disrupted by mutating a positively charged (Lys-rich) cluster of residues (amino acids 355-360) near the C-terminus. Small-angle X-ray scattering (SAXS) analysis confirms the flexibility and dynamic nature of NEIL1's CTD, a feature that may be critical to providing specificity for NEIL1's multiple, functional interactions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Radiotracer studies of agrochemical residues in meat, milk and related products of livestock and poultry

International Nuclear Information System (INIS)

1987-01-01

The joint FAO/IAEA programme was initiated in 1981 and terminates with this report. The specific objectives of the programme have been to evaluate the magnitude, fate and significance of agricultural chemical residues in edible tissues and by-products of livestock and poultry, aided by radiotracer techniques. It was anticipated that the data arising from studies conducted under this programme would be useful in assessing the toxicological significance of studied chemicals to exposed animals and to humans who may consume potentially polluted meat, milk or eggs. A separate abstract was prepared for each of the 13 papers in this report

Rigid Residue Scan Simulations Systematically Reveal Residue Entropic Roles in Protein Allostery.

Directory of Open Access Journals (Sweden)

Robert Kalescky

2016-04-01

Full Text Available Intra-protein information is transmitted over distances via allosteric processes. This ubiquitous protein process allows for protein function changes due to ligand binding events. Understanding protein allostery is essential to understanding protein functions. In this study, allostery in the second PDZ domain (PDZ2 in the human PTP1E protein is examined as model system to advance a recently developed rigid residue scan method combining with configurational entropy calculation and principal component analysis. The contributions from individual residues to whole-protein dynamics and allostery were systematically assessed via rigid body simulations of both unbound and ligand-bound states of the protein. The entropic contributions of individual residues to whole-protein dynamics were evaluated based on covariance-based correlation analysis of all simulations. The changes of overall protein entropy when individual residues being held rigid support that the rigidity/flexibility equilibrium in protein structure is governed by the La Châtelier's principle of chemical equilibrium. Key residues of PDZ2 allostery were identified with good agreement with NMR studies of the same protein bound to the same peptide. On the other hand, the change of entropic contribution from each residue upon perturbation revealed intrinsic differences among all the residues. The quasi-harmonic and principal component analyses of simulations without rigid residue perturbation showed a coherent allosteric mode from unbound and bound states, respectively. The projection of simulations with rigid residue perturbation onto coherent allosteric modes demonstrated the intrinsic shifting of ensemble distributions supporting the population-shift theory of protein allostery. Overall, the study presented here provides a robust and systematic approach to estimate the contribution of individual residue internal motion to overall protein dynamics and allostery.

Biochemical Characterization of Mycobacterium smegmatis RnhC (MSMEG_4305), a Bifunctional Enzyme Composed of Autonomous N-Terminal Type I RNase H and C-Terminal Acid Phosphatase Domains.

Science.gov (United States)

Jacewicz, Agata; Shuman, Stewart

2015-08-01

Mycobacterium smegmatis encodes several DNA repair polymerases that are adept at incorporating ribonucleotides, which raises questions about how ribonucleotides in DNA are sensed and removed. RNase H enzymes, of which M. smegmatis encodes four, are strong candidates for a surveillance role. Here, we interrogate the biochemical activity and nucleic acid substrate specificity of M. smegmatis RnhC, a bifunctional RNase H and acid phosphatase. We report that (i) the RnhC nuclease is stringently specific for RNA:DNA hybrid duplexes; (ii) RnhC does not selectively recognize and cleave DNA-RNA or RNA-DNA junctions in duplex nucleic acid; (iii) RnhC cannot incise an embedded monoribonucleotide or diribonucleotide in duplex DNA; (iv) RnhC can incise tracts of 4 or more ribonucleotides embedded in duplex DNA, leaving two or more residual ribonucleotides at the cleaved 3'-OH end and at least one or two ribonucleotides on the 5'-PO4 end; (v) the RNase H activity is inherent in an autonomous 140-amino-acid (aa) N-terminal domain of RnhC; and (vi) the C-terminal 211-aa domain of RnhC is an autonomous acid phosphatase. The cleavage specificity of RnhC is clearly distinct from that of Escherichia coli RNase H2, which selectively incises at an RNA-DNA junction. Thus, we classify RnhC as a type I RNase H. The properties of RnhC are consistent with a role in Okazaki fragment RNA primer removal or in surveillance of oligoribonucleotide tracts embedded in DNA but not in excision repair of single misincorporated ribonucleotides. RNase H enzymes help cleanse the genome of ribonucleotides that are present either as ribotracts (e.g., RNA primers) or as single ribonucleotides embedded in duplex DNA. Mycobacterium smegmatis encodes four RNase H proteins, including RnhC, which is characterized in this study. The nucleic acid substrate and cleavage site specificities of RnhC are consistent with a role in initiating the removal of ribotracts but not in single-ribonucleotide surveillance. Rnh

What is a "good enough" termination?

Science.gov (United States)

Gabbard, Glen O

2009-06-01

In Freud's technique papers, he failed to develop a systematic approach to termination. Much of the existing literature is based on psychoanalytic mythologies about the way patients are expected to end analysis. The models described in the literature are often starkly at odds with what one sees in clinical practice. A wish for idealized versions of termination underlies much of what has been written, and we need to shift to a conceptual model involving "good enough" termination. A number of different endings to psychoanalysis may, in the long run, lead to productive outcomes; these models are examined, as are various approaches to the dilemmas presented at the time of termination.

Trauma and termination.

Science.gov (United States)

Ferraro, F

1995-02-01

The author suggests a particular reading of the thesis put forward by Freud in 'Analysis terminable and interminable' that an effective and more definitive conclusion may be expected in analyses of cases with traumatic aetiology. This reading shifts the emphasis from the patient's history to the possibility of its crystallising in focal nuclei emerging within the analytic relationship under the pressure of the termination. The revival of separation anxieties which cannot be worked through, and their crystallisation in precipitating traumatic events, may give rise to decisive psychic work allowing the analysis to be brought to a conclusion. Two case histories are presented to show how the end of the analysis assumes the form of a new trauma, which reactivates in the present, traumatic anxieties from the patient's own infantile history. In the first case a premature birth and in the second a miscarriage, originally experienced as isolated automatic events without time or history, are relived in the terminal phase as vicissitudes of the transference, so that new meaning can be assigned to them and they can be withdrawn from the somatic cycle of repetition. The powerful tendency to act out and the intense countertransference pressure on the analyst are discussed in the light of the specificities of this phase, which is crucial to the success of the analysis. This leads to a re-examination, in the concluding notes, of some theoretical questions inherent in the problem of the termination and, in particular, to a discussion of the ambiguous concept of a natural ending.

Preparation and Analysis of N-Terminal Chemokine Receptor Sulfopeptides Using Tyrosylprotein Sulfotransferase Enzymes.

Science.gov (United States)

Seibert, Christoph; Sanfiz, Anthony; Sakmar, Thomas P; Veldkamp, Christopher T

2016-01-01

In most chemokine receptors, one or multiple tyrosine residues have been identified within the receptor N-terminal domain that are, at least partially, modified by posttranslational tyrosine sulfation. For example, tyrosine sulfation has been demonstrated for Tyr-3, -10, -14, and -15 of CCR5, for Tyr-3, -14, and -15 of CCR8, and for Tyr-7, -12, and -21 of CXCR4. While there is evidence for several chemokine receptors that tyrosine sulfation is required for optimal interaction with the chemokine ligands, the precise role of tyrosine sulfation for chemokine receptor function remains unclear. Furthermore, the function of the chemokine receptor N-terminal domain in chemokine binding and receptor activation is also not well understood. Sulfotyrosine peptides corresponding to the chemokine receptor N-termini are valuable tools to address these important questions both in structural and functional studies. However, due to the lability of the sulfotyrosine modification, these peptides are difficult to obtain using standard peptide chemistry methods. In this chapter, we provide methods to prepare sulfotyrosine peptides by enzymatic in vitro sulfation of peptides using purified recombinant tyrosylprotein sulfotransferase (TPST) enzymes. In addition, we also discuss alternative approaches for the generation of sulfotyrosine peptides and methods for sulfopeptide analysis. © 2016 Elsevier Inc. All rights reserved.

N-Terminal Domains in Two-Domain Proteins Are Biased to Be Shorter and Predicted to Fold Faster Than Their C-Terminal Counterparts

Directory of Open Access Journals (Sweden)

Etai Jacob

2013-04-01

Full Text Available Computational analysis of proteomes in all kingdoms of life reveals a strong tendency for N-terminal domains in two-domain proteins to have shorter sequences than their neighboring C-terminal domains. Given that folding rates are affected by chain length, we asked whether the tendency for N-terminal domains to be shorter than their neighboring C-terminal domains reflects selection for faster-folding N-terminal domains. Calculations of absolute contact order, another predictor of folding rate, provide additional evidence that N-terminal domains tend to fold faster than their neighboring C-terminal domains. A possible explanation for this bias, which is more pronounced in prokaryotes than in eukaryotes, is that faster folding of N-terminal domains reduces the risk for protein aggregation during folding by preventing formation of nonnative interdomain interactions. This explanation is supported by our finding that two-domain proteins with a shorter N-terminal domain are much more abundant than those with a shorter C-terminal domain.

Efficiency analysis of container ports and terminals

OpenAIRE

Liu, Q.

2010-01-01

In the past two decades the steady growth of seaborne trade has resulted in the increase of container ships, container ports and their terminals. The structure of the shipping market is, moreover, continuously evolving. On the carrier side, shipping companies form consortia and alliances; on the port side, global terminal operators and dedicated container terminals are emerging. The aim of this research is to evaluate the efficiency of container ports and terminals and to study...

Strategic technology alliance termination : an empirical investigation

NARCIS (Netherlands)

Sadowski, B.M.; Duysters, G.M.

2008-01-01

There is growing consensus that overall alliance termination rates are high. However, despite this track record of termination and despite unsurpassed growth rates of strategic technology alliances, little is known about the reasons for their termination. Typically strategic alliances have been

Restoration of environments with radioactive residues. Papers and discussions. Proceedings of an international symposium

International Nuclear Information System (INIS)

2000-01-01

After the use of radioactive substances or the application of nuclear energy, radioactive residues may remain in the environment and may give rise to radiation exposure of people. Radioactive residues can originate from several sources. They may be remnants following the termination of a practice such as the decommissioning of a nuclear power plant. Occasionally, residues can derive from the accumulation of radionuclides from normal discharges to the environment of radioactive effluents from practices. Most commonly, they are the result of human activities that have been performed in the past without proper regard for the current international radiation protection requirements. They can also be the consequence of accidents that have released radioactive material to the environment. Radioactive residues are also generated by nature, e.g. in natural deposits of radioactive material found in the earth. Spring waters convey these materials. No matter what the cause of radioactive residues may be, there is the question of whether any restoration of the affected environment is required and, if so, to what extent it should be performed. The International Symposium on Restoration of Environments with Radioactive Residues was devoted to discussing these questions and to fostering the exchange of information on this subject. The Symposium explored five main topics: global overview of the problem; restoration principles and criteria; case studies of actual radiological assessments; a critical analysis of the case studies; and the role of public participation in the decision aiding and decision making processes. The Programme Committee of the Symposium accepted a number of contributed papers and posters for consideration at the Symposium, and these were published shortly before the Symposium. In early 2000, the IAEA released as Working Material a draft, unedited compilation of the present Proceedings Series publication. The present publication, which constitutes the record of

Terminal addition in a cellular world.

Science.gov (United States)

Torday, J S; Miller, William B

2018-07-01

Recent advances in our understanding of evolutionary development permit a reframed appraisal of Terminal Addition as a continuous historical process of cellular-environmental complementarity. Within this frame of reference, evolutionary terminal additions can be identified as environmental induction of episodic adjustments to cell-cell signaling patterns that yield the cellular-molecular pathways that lead to differing developmental forms. Phenotypes derive, thereby, through cellular mutualistic/competitive niche constructions in reciprocating responsiveness to environmental stresses and epigenetic impacts. In such terms, Terminal Addition flows according to a logic of cellular needs confronting environmental challenges over space-time. A reconciliation of evolutionary development and Terminal Addition can be achieved through a combined focus on cell-cell signaling, molecular phylogenies and a broader understanding of epigenetic phenomena among eukaryotic organisms. When understood in this manner, Terminal Addition has an important role in evolutionary development, and chronic disease might be considered as a form of 'reverse evolution' of the self-same processes. Copyright © 2017. Published by Elsevier Ltd.

Concept Layout Model of Transportation Terminals

OpenAIRE

Yao, Li-ya; Sun, Li-shan; Wang, Wu-hong; Xiong, Hui

2012-01-01

Transportation terminal is the key node in transport systems. Efficient terminals can improve operation of passenger transportation networks, adjust the layout of public transportation networks, provide a passenger guidance system, and regulate the development of commercial forms, as well as optimize the assembly and distribution of modern logistic modes, among others. This study aims to clarify the relationship between the function and the structure of transportation terminals and establish ...

14 CFR 272.12 - Termination.

Science.gov (United States)

2010-01-01

... Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION (AVIATION PROCEEDINGS) ECONOMIC REGULATIONS ESSENTIAL AIR SERVICE TO THE FREELY ASSOCIATED STATES § 272.12 Termination. These provisions shall terminate on October 1, 1998, unless the program of essential air service to the Federated States of...

Transmembrane signalling at the epidermal growth factor receptor. Positive regulation by the C-terminal phosphotyrosine residues

DEFF Research Database (Denmark)

Magni, M; Pandiella, A; Helin, K

1991-01-01

a positive role in the regulation of transmembrane signalling at the EGF receptor. The stepwise decrease in signal generation observed in single, double and triple point mutants suggest that the role of phosphotyrosine residues is not in the participation in specific amino acid sequences, but rather...... in the double and the triple mutants. In the latter mutant, expression of the EGF-receptor-activated lipolytic enzyme phospholipase C gamma was unchanged, whereas its tyrosine phosphorylation induced by the growth factor was lowered to approx. 25% of that in the controls. In all of the cell clones employed......, the accumulation of inositol phosphates induced by treatment with fetal calf serum varied only slightly, whereas the same effect induced by EGF was consistently lowered in those lines expressing mutated receptors. This decrease was moderate for those receptors missing only the distal tyrosine (point and deletion...

Residual gas analysis

International Nuclear Information System (INIS)

Berecz, I.

1982-01-01

Determination of the residual gas composition in vacuum systems by a special mass spectrometric method was presented. The quadrupole mass spectrometer (QMS) and its application in thin film technology was discussed. Results, partial pressure versus time curves as well as the line spectra of the residual gases in case of the vaporization of a Ti-Pd-Au alloy were demonstrated together with the possible construction schemes of QMS residual gas analysers. (Sz.J.)

Assessing the Availability of Wood Residues and Residue Markets in Virginia

OpenAIRE

Alderman, Delton R. Jr.

1998-01-01

A statewide mail survey of primary and secondary wood product manufacturers was undertaken to quantify the production and consumption of wood residues in Virginia. Two hundred and sixty-six wood product manufacturers responded to the study and they provided information on the production, consumption, markets, income or disposal costs, and disposal methods of wood residues. Hardwood and pine sawmills produce approximately 66 percent of Virginia's wood residues. Virginia's wood product man...

Identification of a direct interaction between residue 19 in the helical portion of calcitonin and the amino-terminal domain of the calcitonin receptor from photoaffinity cross-linking and mutational studies

International Nuclear Information System (INIS)

Pham, V.; Wade, J.; McDowall, S.G.; Quiza, M.; Sexton, P.M.

2001-01-01

Full text: Calcitonins (CTs) are 32 amino acid hormones with both peripheral and central actions mediated via specific cell surface receptors, which belong to the superfamily of class II G-protein coupled receptors. Chimeric receptor and mutational data suggested that the helical portion (residues 8-22) of salmon CT (sCT) is important for high affinity binding to the amino-terminal extracellular domain of the human CT receptor (hCTR). In this study, we have developed photoactive sCT analogues [Arg 11, 18 , Bpa 19 ]sCT and [Arg 11 , 18 , Bpa 19 ]sCT(8-32) that incorporate a photolabile Bpa (p-benzoyl-L-phenylalanine) into position 19 of the helical domain of the ligand and used this to determine a specific receptor fragment proximate to it. These analogues saturably bound to the CTR with high affinity (IC 50 = 3 nM) which was similar to that of the natural sCT and its antagonist (IC 50 = 2 nM and 20 nM, respectively). Upon photolysis, radioiodinated 125 I-[Arg 11, 18 , Bpa 19 ]sCT and 125 I-[Arg 11,18 , Bpa 19 ]sCT(8-32) efficiently and specifically cross-linked to hCTR stably expressed in baby hamster kidney cells (Hollexl cells, ∼ 800,000 receptors per cell), generating a single radiolabeled band of ∼ 72-kDa on SDS/PAGE autoradiography. To identify the 'contact domain' within CTR involved in binding of 125 I-[Arg1 1 , 18 , Bpa 19 ]sCT and 125 I-[Arg 11, 18 , Bpa 19 ]sCT(8-32), the radiolabeled band containing the ligand-receptor conjugate was subjected to chemical and enzymatic cleavage. Cyanogen bromide cleavage of the native receptor yielded a radiolabeled fragment of apparent Mr ∼ 31-kDa that shifted to Mr ∼ 14 kDa after deglycosylation. This receptor domain corresponded to amino acids 59-134 of the hCTR, located at the amino-terminal extracellular region of the receptor. These results provide the first direct demonstration of a contact domain between calcitonin and its receptor, and will contribute towards the modelling of CT-CTR interface. Copyright

Terminal structure

Science.gov (United States)

Schmidt, Frank [Langenhagen, DE; Allais, Arnaud [Hannover, DE; Mirebeau, Pierre [Villebon sur Yvette, FR; Ganhungu, Francois [Vieux-Reng, FR; Lallouet, Nicolas [Saint Martin Boulogne, FR

2009-10-20

A terminal structure (2) for a superconducting cable (1) is described. It consists of a conductor (2a) and an insulator (2b) that surrounds the conductor (2a), wherein the superconducting cable (1) has a core with a superconducting conductor (5) and a layer of insulation that surrounds the conductor (5), and wherein the core is arranged in such a way that it can move longitudinally in a cryostat. The conductor (2a) of the terminal structure (2) is electrically connected with the superconducting conductor (5) or with a normal conductor (6) that is connected with the superconducting conductor (5) by means of a tubular part (7) made of an electrically conductive material, wherein the superconducting conductor (5) or the normal conductor (6) can slide in the part (7) in the direction of the superconductor.

43 CFR 29.13 - Termination.

Science.gov (United States)

2010-10-01

... Termination. Upon termination of operations of the Pipeline, the full disposition of all claims, and the... shall request that Congress provide for final disposition of the Fund. If Congress at any time establishes a comprehensive oil pollution liability fund which supersedes or repeals the Fund, the Fund assets...

7 CFR 983.89 - Termination.

Science.gov (United States)

2010-01-01

..., ARIZONA, AND NEW MEXICO Miscellaneous Provisions § 983.89 Termination. (a) The Secretary may at any time..., That such majority has, during such representative period, produced for market more than fifty percent of the volume of such pistachios produced for market, but such termination shall be announced at...